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High Grade CANNABIS BIBLIOGRAPHY SORTED AND ALPHABETIZED BY SUBJECT

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
This is an attempt to make the posted articles easier to find and use. I am still interested in additional posts anyone that thinks they can help by posting really great reference articles, here are a list of the subjects I use.
I am sure I, and you, will find additional subjects, please suggest any subjects that should be added, try and include a DOI number or if no DOI a http:// link to download any paper, I will then post in the right subject posted in alphabetical order .

With a DOI number you can get any science paper by going to https://sci-hub.se/ and pasting in the DOI number.
If the link above does not work type sci-hub into google and use the https://sci-hub.se/ link that pops up, it works, then paste in the DOI. A few papers do not have a working DOI, I tried to find a http:// link for those papers. Recently some journals block the DOI of newly published papers for the first year or two, in that case I try to find a http:// that has the Pdf, if not I will publish what info I have and post FIND Pdf on the bottom of the abstract. If anyone sees a DOI or http:// that does not yield a Pdf please let me know and send me a link to the actual Pdf so I can post it for all.

I also welcome discussion of any paper posted, good or bad, share what you think so others can expand their understanding of Cannabis.
I will post a * before the title of the top 5-10 papers in each subject, these are must reads if you have interest in that Cannabis subject.
I will try and add all of the abstracts to all of the articles I can.
If anyone finds a http link or Doi number that does not work, please tell me so I can attempt to fix it.
If anyone has suggestions for additional subjects for any specific paper to be posted in, please tell me, I will add the paper to both subjects.
The only real disadvantage of this way of displaying the Biblography is if people want to see just the latest additions to this Biblography, so maybe I should first list new additions to the
CANNABIS BIBLIOGRAPHY SORTED AND ALPHABETIZED BY SUBJECT with their titles in red for a few months then make it black like the rest? I will do that.
I hate posting abstracts of papers without a free Pdf or DOI link to download the paper, but I do anyway just in case folks see a paper they are want now and are willing to pay for a new paper I do not have a free link for yet. If you know a link for the missing Pdf PM me so I can add it.

IC Archaeology/History....................... ................PAGE 1, POST 1

IC Breeding Cannabis...................... .....................PAGE 1, POST 3

IC Cannabinoids.................. .............................. ..PAGE 1, POST 8

IC Cannabinoid Receptors...,,,,.............. ..............PAGE 1, POST 9

IC Cannabis Analysis...................... ........................PAGE 1, POST 11

IC Cannabis Art........................... ..........................PAGE 1, POST 12
IC Cannabis Botany........................ .......................PAGE 1, POST 12
IC Cannabis Collectables.................. ......................PAGE 1, POST 12
IC Cannabis Plant Growth Hormones...................... .PAGE 1, POST 12
IC Cannabis R&D......................... .. ........................ PAGE 1, POST 12

IC Cannabis Seeds......................... .........................PAGE 1, POST 13
IC Cannabis Seed Oil........................... ....................PAGE 1, POST 13
IC Classification/Taxonomy...................... ................PAGE 1, POST 13

IC DNA.............................. .............................. .......PAGE 1, POST 14

IC Drug Cannabis...................... .............................. .PAGE 2, POST 16
IC Ethnobotany................... .............................. .......PAGE 2, POST 16
IC Flavonoids.................... .............................. .....PAGE 2 POST 16

IC Hemp/Cannabis Cultivation................... ............PAGE 2, POST 17

IC Hemp Processing
.............................. ................PAGE 2, POST 18

IC Interviews Cannabis People........................ ......PAGE 2, POST 19
IC In Vitro......................... .............................. ......PAGE 2, POST 19
IC Legal......................... .............................. ...........PAGE 2, POST 19

IC Medical Cannabis/Endocannabinoids Part 1 A-D......PAGE 2, POST 20

IC Medical Cannabis/Endocannabinoids Part 2 E-Z......PAGE 2, POST 21

IC Pest and Disease....................... .......................PAGE 2, POST 22

IC Terpenes .............................. ..........................PAGE 2, POST 24

IC Trichomes .............................. .........................PAGE 2, POST 26


I have posted maybe 2000+ articles, almost organized into 27 categories, all on 2 pages of this post Pg1 & Pg 2

IC Archaeology/History

*A fragrant grave - revealing the mummified remains of a 17th-century bishop
Per Lagerås, National Historical Museums, Sweden
Current World Archaeology · March 2016 Issue 76
https://www.world-archaeology.com/fe...entury-bishop/
Bishop Peder Winstrup died in December 1679, aged 74, and was buried beneath Lund Cathedral. When, in 2014, it was decided that his coffin should be removed from the crypt, a team of archaeologists took the opportunity to look inside. What they discovered surprised everyone: his clothes, his skin, and his hair were so perfectly preserved that he looked almost as if he were sleeping rather than having been dead for more than three centuries. But another shock awaited the team: the bishop was not alone. Secreted at his feet was the tiny body of a human foetus, probably a still-born baby. Winstrup and the baby lay on a bed of well-preserved plants, its pillows stuffed with herbs. Could these be the reason the remains looked so fresh? An interdisciplinary research team, directed by Per Karsten, Historical Museum at Lund University, was rapidly assembled to investigate.

"ICE MAIDEN"
A mummy unearthed from the pastures of heaven
January 1994 National geographic 186(4):80-103
N. Polosmak
https://nationalgeographicbackissues.. .ober-1994.html
https://siberiantimes.com/science/ca...ique-mri-scan/
https://www.pbs.org/wgbh/nova/transc...7siberian.html

American Weed: A History of Cannabis Cultivation in the United States
Nick Johnson
Introduction: Marijuana Menaces the Midway
EchoGéo 48(48) July 2019
DOI: 10.4000/echogeo.17650
//www.researchgate.net/publication/334452841_American_Weed_A_Hist ory_of_Cannabis_Cultivation_in _the_United_States
In the summer of 1929, Reefer Madness descended upon the Windy City. In late April, the Illinois house of representatives had passed a bill to ban “loco-weed,” a plant whose “Mexican form” was “marijuana,” a “narcotic” (Brown, 1929a)1 Two months later, as the bill languished in the senate, the Chicago Tribune ran an article and accompanying backpage photo on marijuana, attempting to spur the legislature into action. The paper claimed that the “dangerous, habit forming drug” had been “introduced a dozen years ago or so by Mexican laborers” and was now spreading across the city, ensnaring “thousands of workingmen,” “youths and girls,” as well as “school children.” (Chicago Tribune, 1929a). In the photo, two dark-skinned men with sun hats are crouched next to some cannabis plants “in the southern part of the city,” “gathering marijuana” while the “legislature delays action” (Chicago Tribune, 1929b; Falck, 2010, p. 80-81). The newspaper clearly intended the photo to be visual proof of marijuana’s “Mexican” origins, as well as a swipe at the legislature for stalling while devious foreigners harvested a dangerous drug. The accompanying article claimed that cannabis “seeds” were “brought by Mexicans” and “planted in tiny patches near the box car homes of the laborers.” But if Mexicans were blamed for the drug’s introduction, the rest of the article made clear that they could hardly be held responsible for its spread. In addition to naming two “alleged sellers of marijuana cigarets” as “Harry Johnson” and “Richard Drake,” the report also claimed that marijuana smoking was widespread “in South Chicago, in Blue Island, in Kensington, and other outlying districts, and it can be purchased in restaurants, drug stores, and poolrooms” – all of which were not exclusively the domain of Mexicans (Chicago Tribune, 1929a). Nature, too, helped the “loco weed”

An Archaeological and Historical Account of Cannabis in China
Hui-Lin Li
October 1973 Economic Botany 28(4):437-448
DOI: 10.1007/BF02862859
From a historical vantage, Cannabis has been found in China since Neolithic times, about 6,000 years ago, with a continuous record of cultivation down to the present. This record stands unique in comparison to those of other regions in Asia, and it strongly indicates the plant to be indigenous. New archeological finds in recent years considerably substantiate and extend its early history. The very scattered references in historical literature are in need of organization and analysis. These records are assembled here, followed by some notes on the possible routes of early diffusion of the plant in relation to its usage.

Ancient Cannabis Burial Shroud in a Central Eurasian Cemetery
HONGEN JIANG, LONG WANG, MARK D. MERLIN, ROBERT C. CLARKE,
YAN PAN, YONG ZHANG, GUOQIANG XIAO, AND XIAOLIAN DING
Economic Botany, Vol 70(X), 2016, 9 pp.
DOI: 10.1007/s12231-016-9351-1
An extraordinary cache of ancient, well-preserved Cannabis plant remains was recently discovered in a tomb in the Jiayi cemetery of Turpan, NW China. Radiometric dating of this tomb and the archeobotanical remains it contained indicate that they are approximately 2800–2400 years old. Both morphological and anatomical features support the identification of the plant remains as Cannabis. Research discussed in this paper describes 13 nearly whole plants of Cannabis that appear to have been locally produced and purposefully arranged and used as a burial shroud which was placed upon a male corpse. This unique discovery provides new insight into the ritualistic use of Cannabis in prehistoric Central Eurasia. Furthermore, the fragmented infructescences of Cannabis discovered in other tombs of the Jiayi cemetery, together with similar Cannabis remains recovered from coeval tombs in the ancient Turpan cemetery along with those found in the Altai Mountains region, reveal that Cannabis was used by the local Central Eurasian people for ritual and/or medicinal purposes in the first millennium before the Christian era.

Ancient usage of cannabis
Aaron Clauset, Kollen Post
Science 364(6445):1043.8-1044 June 2019
DOI: 10.1126/science.364.6445.1043-h
The cultivation of cannabis extends back into distant prehistory. During excavations at a cemetery on the Pamir Plateau on the border of China and Tajikistan, Ren et al. found cannabinoid oils in wooden braziers. The finding is consistent with psychoactive cannabis use in burial rituals as early as 500 BCE. The cannabis featured high levels of THC (tetrahydrocannabinol)—higher than in wild varieties of the plant. Artifacts recovered from the burials as well as isotopic evidence from human remains suggest a high degree of cultural and economic exchange with neighboring peoples. Thus, people in the region may have been engaged in the hybridization of disparate populations of cannabis plants for the purpose of increasing their potency.

*A new insight into Cannabis sativa (Cannabaceae) utilization from 2500-year-old Yanghai Tombs, Xinjiang, China
Hong-En Jiang, Xiao Li, You-Xing Zhao, David K.Ferguson, Francis Hueber, Subir Bera, Yu-Fei Wang, Liang-Cheng Zhao, Chang-Jiang Liu, Cheng-Sen Li
Journal of Ethnopharmacology Volume 108, Issue 3, 6 December 2006, Pages 414-422
doi: 10.1016/j.jep.2006.05.034
A cache of shoots, leaves and fruits dated by 14C at 2500 years B.P. were unearthed in the Yanghai Tombs, Turpan District in Xinjiang, China. By comparing the morphological and anatomical characteristics of the plant remains found in the tomb and specimens of modern plants, it is shown that the remains belong toCannabis. Based on the shamanistic background of the deceased man and ancient customs, it is assumed that the Cannabis was utilized for ritual/medicinal purposes.

A PHYSIOLOGICALLY ACTIVE PRINCIPLE FROM CANNABIS SATIVA (MARIHUANA)
HAAGEN-SMIT, A. J., WAWRA, C. Z., KOEPFLI, J. B., ALLES, G. A., FEIGEN, G. A., & PRATER, A. N.
Science, 91(2373), 602–603. (1940).
doi:10.1126/science.91.2373.602
While it has long been known that the physiological activity of Cannabis sativa (marihuana or hashish) is associated with its contained resins, no physiologically active crystalline material has heretofore been isolated.
We report in this note the isolation of such a substance. The hydrocarbon nonacosane and an oily product termed canabinol were first isolated by Wood, Spivey and Easterfie1d In 1938 Bergel, Todd and Work reported the preparation of a crystallilie p-nitro benzoate of cannabinol which could be used to separate the cannabinol from the oil by chromatographic adsorption methods. Recently an oily product which was named cannabidiol was isolated by Adams, Hunt and Clark. None of these well-defined products has exhibited the characteristic physiological activities that are shown by the crude drug though canabinol was found to be quite toxic. Reviews of the earlier work on the chemistry of Cannabis have been published by Walton and by Blatt.
Work on the separation of physiologically active fractions from alcoholic extracts of Cannabis sativa has been in progress for the past year in our laboratories. The extracts of Minnesota wild hemp used for the work were generously supplied by the Narcotics Laboratory, United States Treasury Department, and we are indebted to Nessrs. H. J. Anslinger and H. J. Wollner for their collaboration which made this work possible. The alcohol extract of the crude drug was diluted with water to yield a seventy per cent, alcohol solution, and this vas partitioned into petroleum ether. Salt forming compounds were extracted and then colored substances were largely removed by adsorption on zinc carbonate. The resultant resinous material was fractionally precipitated from methanol with water and there was obtained a physiologically active fraction of about one twentieth the weight of the crude resin material. This purified product 1~as fractionally distilled under 0.005 mm pressure, with the most active fraction distilling at 128O-135O C. This fraction is a red-colored oil which shows typical activity in dogs following an oral dose of 1.0 mg per kg. By cooling a solution of this oil in a methanol-acetic acid mixture, some crystalline material was obtained. This was then recrystallized several times from methanol to yield colorless needles melting at 128O-129O C.

Archaeological Evidence for the Tradition of Psychoactive Plant Use in the Old World.
Merlin, M. D.
Economic Botany, 57(3), 295–323. (2003).
doi:10.1663/0013-0001(2003)057[0295:aeftto]2.0.co;2
We live in an age when a divine vision is dismissed as an hallucination, and desire to experience a direct communication with god is often interpreted as a sign of mental illness. Nevertheless, some scholars and scientists assert that such visions and communications are fundamentally derived from an ancient and ongoing cultural tradition. The hypothesis presented here suggests that humans have a very ancient tradition involving the use of mind-altering experiences to produce profound, more or less spiritual and cultural understanding. Much evidence for the early use of Cannabis for fiber, food, medicine, ceremony and recreation can be gleaned from ancient written records (Fig. 12, 13, 14, and 15). However, the archae ological record for early use of Cannabis is much less extensive. Nevertheless, the macrofossil, pollen, and indirect material evidence from prehistory are substantial.

Archaeobotanical evidence of the use of medicinal cannabis in a secular context unearthed from south China
Yunjun Bai , Ming Jiang , Tao Xie , Chao Jiang , Man Gu , Xinying Zhou , Xue Yan , Yuan Yuan , Luqi Huang
J Ethnopharmacol. 2021 Jul 15;275:114114.
doi: 10.1016/j.jep.2021.114114
https://www.researchgate.net/publica...om_South_China
Ethnopharmacological relevance: As one of the first plants used by ancient people, cannabis has been used for medicinal purposes for thousands of years. The long history of medicinal cannabis use contrasts with the paucity of archaeobotanical records. Moreover, physical evidence of medicinal cannabis use in a secular context is much rarer than evidence of medicinal cannabis use in religious or ritual activities, which impedes our understanding of the history of medicinal cannabis use.
Aim of the study: This study aims to provide archaeobotanical evidence of medicinal cannabis use and analyse the specific medicinal usage of cannabis in a secular context in ancient times.
Materials and methods: Plant remains were collected from the Laoguanshan Cemetery of the Han Dynasty in Chengdu, South China, with the archaeological flotation process and were identified based on morphological and anatomical characteristics. The examination of the medicinal significance of the remains relied on the investigation of the documentation on unearthed medical bamboo slips, the diseases of the tomb occupants, the cemetery's cultural background and Chinese historical records.
Results: The botanical remains were accurately identified as cannabis. More than 120 thousand fruits were found, which represents the largest amount of cannabis fruit remains that have been statistically analysed from any cemetery in the world thus far. The cannabis fruits are suspected to have been used for medical purposes in a secular context and were most likely used to stop severe bleeding of the uterus and treat lumbago and/or arthralgia.
Conclusions: The cannabis fruit remains reported here likely represent the first physical evidence of medicinal cannabis use for the treatment of metrorrhagia, severe lumbago, and/or arthralgia. This study emphasizes the importance of the evidence of the diseases suffered by the occupants of the tomb in determining the medicinal use of cannabis in a secular context and contributes to a comprehensive understanding of the ancient history of medicinal cannabis.

Breaking the begging bowl: morals, drugs, and madness in the fate of the Muslim faqīr.
Green, N.
South Asian History and Culture, 5(2), 226–245.(2014).
doi:10.1080/19472498.2014.883761
This article follows a set of developments that transformed the meaning and value of begging as a religious pursuit in colonial India. Focusing on the Muslim faqīrs, the article argues that missionaries, colonial officials, and physicians joined together in a moral and then medical critique of the faqīrs as venerated idlers and sanctified drug users. The moral dimensions of the critique were then taken up by Muslim and Hindu reformists. Positioned at the centre of an immoral nexus, for their British critics the faqīrs were key to the spread of drug abuse and in turn insanity among their followers. For Indian reformists and then nationalists, this nexus also connected the faqīrs to the moral, economic, and physical weakening of the nation. In both of these critical visions, the begging mendicant was seen as an actively harmful figure whose misdeeds ranged from promoting the inversed morality of an anti-work ethic to peddling the evils of drug addiction and rousing the riotous masses on holy days. By drawing on a range of missionary, medical and Muslim reformist texts, the article shows how from around 1870 the discourses of Islamic reform and Indian nationalism gradually joined forces with the medical and moral discipline of empire such that by the 1920s the faqīrs had gained an assembly of powerful enemies. In this way, the colonial period is seen as a crucial period of transition in the meanings of begging and drug use that would leave the venerated mendicants of former times disempowered in post-colonial South Asia.

Cannabinoid pharmacology: the first 66 years.
Pertwee, R. G.
British Journal of Pharmacology, 147(S1), S163–S171. (2009).
doi:10.1038/sj.bjp.0706406
Research into the pharmacology of individual cannabinoids that began in the 1940s, several decades after the presence of a cannabinoid was first detected in cannabis, is concisely reviewed. Also described is how this pharmacological research led to the discovery of cannabinoid CB1 and CB2 receptors and of endogenous ligands for these receptors, to the development of CB1- and CB2-selective agonists and antagonists and to the realization that the endogenous cannabinoid systemhas significant roles in both health and disease, and that drugs which mimic, augment or block the actions of endogenously released cannabinoids must have important therapeutic applications. Some goals for future research are identified.

Cannabinoid research in the 2010s
Mauro Maccarrone and Steve PH Alexander
bph_1930 2409..2410
DOI:10.1111/j.1476-5381.2012.01930.x
Cannabis sativa is possibly the plant with the longest history of cultivation by man (Russo, 2007). It has long been exploited for its fibre; as a biomass converter, it has exceptional utility. For most people, however, there is the association of cannabis with ‘recreational drugs’, which has lead to the profusion of names associated with the plant and extracts thereof (marijuana,hashish, bhang, weed, grass, etc.). The ‘modern’ scientific era of cannabis research was prompted by the discovery of the major psychoactive ingredient in cannabis extracts (Gaoni and Mechoulam, 1964). This was, of course, D9-tetrahydrocannabinol or THC. Raphael Mechoulam has numerous publications, filled with seminal observations, including the identification of the two ‘best’ candidates for endogenous cannabinoid molecules: anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (Mechoulam et al., 1995). He has become something of an icon in the cannabis field, with this issue of BJP containing a series of original articles prompted by a symposium held in Jerusalem in November 2010 to celebrate his 80th birthday. The first issue, entitled ‘Cannabinoids in Biology and Medicine’, containing primarily reviews, was published in August 2011 (https://onlinelibrary.wiley.com/doi/...sue-7/issuetoc). Current research incannabinoid-related areas is vibrant, with the added focus of TRPV1 ion channels, PPAR nuclear receptors and the ‘orphan’ G-protein coupled receptors, GPR18, GPR55 and GPR119, as molecular targets of cannabinoids and cannabinoid-like molecules. Furthermore, the identification of endogenous agonists at cannabinoid receptors which lead to the demonstration of multiple routes for synthesis and transformation of these endocannabinoids has added to the molecular targets available for potential exploitation.

Cannabis
Chris S Duvall
February 2015
Publisher: Botanical Series, Reaktion Books, London
https://www.researchgate.net/publica...65399_Cannabis
Cannabis, one of humanity’s first domesticated plants, has been utilized for spiritual, therapeutic, recreational, and even punitive reasons for thousands of years. Humans have excellent practical knowledge of Cannabis uses, yet limited understanding of its sociocultural consequences, past or present due to its widespread prohibition. In Cannabis, Chris Duvall explores the cultural history and geography of humanity’s most widely distributed crop, which supplies both hemp and marijuana. This book provides a global view of the plant, with coverage of little-studied regions including Africa and Australia. This book focuses on the plant’s currently most valuable product, the psychoactive drug marijuana. Cannabis also covers the history of hemp and its use as a fiber source for ropes and textiles; as a source of edible hempseeds; and as a source of industrial oil for paints and fuel. This book does not advocate either the prohibition or legalisation of the drug but challenges received wisdom on both sides of the debate. Cannabis explores and analyses a wide range of sources to provide a better understanding of its current prohibition, as well as of the diversity of human–Cannabis relationships across the globe. This, the author argues, is necessary to redress the oversimplistic portrayals of marijuana and hemp that dominate discourse on the subject, and ultimately to improve how the crop is managed worldwide. This highly accessible, richly illustrated volume is an essential read given rapidly evolving debates about prohibition, and in light of changes in the legality of marijuana in Uruguay, some U.S. states, and other jurisdictions worldwide

Cannabis and Frankincense at the Judahite shrine of Arad
Eran Ariea , Baruch Rosenb and Dvory Namdarc
TEL AVIV Vol. 47, 2020, 5–28
DOI: 10.1080/03344355.2020.1732046
Two limestone monoliths, interpreted as altars, were found in the Judahite shrine at Tel Arad. Unidentified dark material preserved on their upper surfaces was submitted for organic residue analysis at two unrelated laboratories that used similar established extraction methods. On the smaller altar, residues of cannabinoids such as ?9 -teterahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) were detected, along with an assortment of terpenes and terpenoids, suggesting that cannabis inflorescences had been burnt on it. Organic residues attributed to animal dung were also found, suggesting that the cannabis resin had been mixed with dung to enable mild heating. The larger altar contained an assemblage of indicative triterpenes such as boswellic acid and norursatriene, which derives from frankincense. The additional presence of animal fat?in related compounds such as testosterone, androstene and cholesterol?suggests that resin was mixed with it to facilitate evaporation. These well-preserved residues shed new light on the use of 8th century Arad altars and on incense offerings in Judah during the Iron Age.

Cannabis and hemp in the Ottoman Empire
ERHAN AFYONCU
https://www.dailysabah.com/feature/2...ottoman-empire
Turkey is looking to revive cannabis cultivation in order to begin using it in industry. Though the Ottoman Empire had a huge hemp industry, the plant has not been cultivated in Anatolia for decades
In Turkish, the cannabis plant is also known as "kendir," which is also the name of the fiber made out of it. The fiber and seeds of the cannabis plant are used to make a variety of different products around the world. It is prohibited in many countries because of its miscellaneous use as a drug. However, industrial hemp is used in the production of many things, including fabric, yarn, naval materials, cosmetics, vehicle frames, soap and cellulose. It was commonly grown during the Ottoman era.

Cannabis and Medicinal Research History of Medical Cannabis in Sri Lanka
Weliange W S,
J Neurol Neurophysiol 2018, Volume 9
Conference: Cannabis and Medicinal Research, Japan
DOI: 10.4172/2155-9562-C9-085
Island of Sri Lanka was occupied by Homo sapiens since 40,000 years ago. They separated into Yaksha, Naga and Deva tribes. A Yaksha king called Ravana ruled Sri Lanka around 10,000 BC and established methods of medical practices. In 509 BC a team led by Indian prince Vijaya arrived and started agriculture civilization. In 341 AD King Buddadasa wrote a medical pharmacopeia; Sarartha Sangrahaya in which he medicinal values of Cannabis were described. Since then medical books described Cannabis as an important herbal ingredient. These books include Yogarnavaya and Prayagorathnavaliya (1232), Vaidyacintamani – wish-fulfilling gem of Medicine (1707), Glossary of Synonyms of Medicinal Plants (1798), Yogasekaraya (1894), Kolavidiya (1900), Es Veda Potha (Opthalmic treatments) (1908). Saraswathi Nigantuwa (1918), Sri Sarangadara Samhitha (1929), Chemistry and Pharmacology of Indian and Sri Lankan Medicinal Plants (1935), Sri Lankan Ayurvedic Pharmacopeia (1937), Senehe shathakam (1940), Thel beheth potha (book of Medicinal Oils) in 1954, Atheesara Chikithsawa (Diareal treatments) in 1962, Encyclopedia of Medicinal Plants (1963), Purana Rahas thel beheth Potha (Ancient secretes of Medicinal Oils) in 1969, Desheeya Vaidya Sabdakoshaya (Dictionary of Indigenous Medicine) in 1970, Go Rathnaya (Treating Cows) in 1980, Desheeya Guli Kalka Sagaraya (Edible Medicines) in 1999. In 2015 Minister of Health, Nutrition & Indigenous Medicine Dr. Rajitha Dissanayake initiated promotion of Cannabis for Indigenous Medicine. In 2017 a comprehensive book about Cannabis was written by W.S. Weliange. Government interference is necessary to educate people about the overall importance of Cannabis for health, society and the Nature.

Cannabis and Tobacco in Precolonial and Colonial Africa
Chris S. Duvall
Oxford Research Encyclopedia of African History (2017)
10.1093/acrefore/9780190277734.013.44
Cannabis and tobacco have longstanding roles in African societies. Despite botanical and pharmacological dissimilarities, it is worthwhile to consider tobacco and cannabis together because they have been for centuries the most commonly and widely smoked drug plants. Cannabis, the source of marijuana and hashish, was introduced to eastern Africa from southern Asia, and dispersed widely within Africa mostly after 1500. In sub-Saharan Africa, cannabis was taken into ethnobotanies that included pipe smoking, a practice invented in Africa; in Asia, it had been consumed orally. Smoking significantly changes the drug pharmacologically, and the African innovation of smoking cannabis initiated the now-global practice. Africans developed diverse cultures of cannabis use, including Central African practices that circulated widely in the Atlantic world via slave trading. Tobacco was introduced to Africa from the Americas in the late 1500s. It gained rapid, widespread popularity, and Africans developed distinctive modes of tobacco production and use. Primary sources on these plants are predominantly from European observers, which limits historical knowledge because Europeans strongly favored tobacco and were mostly ignorant or disdainful of African cannabis uses. Both plants have for centuries been important subsistence crops. Tobacco was traded across the continent beginning in the 1600s; cannabis was less valuable but widely exchanged by the same century, and probably earlier. Both plants became cash crops under colonial regimes. Tobacco helped sustain mercantilist and slave-trade economies, became a focus of colonial and postcolonial economic development efforts, and remains economically important. Cannabis was outlawed across most of the continent by 1920. Africans resisted its prohibition, and cannabis production remains economically significant despite its continued illegality.

Cannabis condemned: the proscription of Indian hemp.
Kendell, R.
Addiction, 98(2), 143–151.(2003).
doi:10.1046/j.1360-0443.2003.00273.x
Aims To find out how cannabis came to be subject to international narcotics legislation. Method Examination of the records of the 1925 League of Nations’ Second Opium Conference, of the 1894 Report of the Indian Hemp Drugs Commission and other contemporary documents. Findings Although cannabis (Indian hemp) was not on the agenda of the Second Opium Conference, a claim by the Egyptian delegation that it was as dangerous as opium, and should therefore be subject to the same international controls, was supported by several other countries. No formal evidence was produced and conference delegates had not been briefed about cannabis. The only objections came from Britain and other colonial powers. They did not dispute the claim that cannabis was comparable to opium, but they did want to avoid a commitment to eliminating its use in their Asian and African territories.

*Cannabis, Evolution and Ethnobotany
Robert C Clarke and Mark Merlin
University of California Press
https://magicgreenery.com/download/i...Merlin2013.pdf remove the s from https if needed
Cannabis is one of the world’s most useful plant groups. It has been a part of human culture for thousands of years beginning in
Eurasia, and today it is associated with people in almost all parts of the world. Although Cannabis is most often thought of as a
“drug plant,” its use for a huge number of other purposes including fiber, food, paper, medicine, and so on is almost
unparalleled, ranking it with the coconut palm and bamboos. Cannabis is truly a remarkable genus of multipurpose plants with
extensive and complicated histories. A fully comprehensive, documented history of Cannabis’s evolution and its widespread,
diverse use by humans has never been published. This book is an attempt to accomplish that task. The evolution of Cannabis and
the great variety of human-Cannabis relationships are presented here in greater depth than ever before. How this project
developed and progressed is an interesting story in itself.
The coauthors have worked earnestly over the past 15 years or more to produce this book; however, they began to focus their
scholarly and scientific interests on Cannabis well before their collaboration started in 1996. Both Mark Merlin and Robert
Clarke first initiated their research on Cannabis while enrolled in undergraduate programs in the University of California system
at their respective campuses decades ago. Their independent and joint field work involving the genus necessitated extensive
travel across several continents to countless libraries and museums, complemented by innumerable interviews in regions where
Cannabis has either ancient roots or only a relatively modern history of cultivation and use. This volume represents the better
part of two scholarly careers spent following the historic trail of both the evolutionary biology and ethnobotanical heritage of
Cannabis.

Cannabis in Ancient Central Eurasian Burials
Mark D. Merlin and Robert C. Clarke
In ANCIENT PSYCHOACTIVE SUBSTANCES
Edited by Scott M. Fitzpatrick
https://d2r6h7ytneza1l.cloudfront.ne.. .ck_Excerpt.pdf
Over the vast time span within which humans have known and used Cannabis for many purposes, it has been heralded as one of our supreme resources and cursed as one of our utmost burdens. Today the consumption of mind-altering Cannabis plant material for recreational or medicinal reasons is widely known. However, the original and early use of psychoactive Cannabis may have been principally for ritualistic religious purposes (for a comprehensive discussion of the evolutionary biology and ethnobotanical history of the genus Cannabis, see Clarke and Merlin 2013; also see Duvall 2015; Small 2015). The natural origin area of Cannabis was most likely the central steppe and forest zones of Eurasia. Early modern humans probably first encountered and utilized one or more of the products of this annual, herbaceous genus in its native biogeographical range. Remarkable early twentiethand twenty-first-century discoveries of archaeobotanical remains in ancient burials confirm the nonfood and nonfiber use of Cannabis in Central Eurasia at least by the first millennium BCE. In these cases, Cannabis appears to have been used for mind-altering ceremonial, purification, or therapeutic purposes. This chapter focuses on the presence and putative uses of psychoactive Cannabis in ancient burials that are well over two thousand years old and found in southeastern Russia and western China.

Cannabis in Asia: its center of origin and early cultivation, based on a synthesis of subfossil pollen and archaeobotanical studies
John M. McPartland,· William Hegman ,· Tengwen Long
Vegetation History and Archaeobotany
doi:10.1007/s00334-019-00731-8
Biogeographers assign the Cannabis centre of origin to “Central Asia”, mostly based on wild-type plant distribution data. We sought greater precision by adding new data: 155 fossil pollen studies (FPSs) in Asia. Many FPSs assign pollen of either Cannabis or Humulus (C –H ) to collective names (e.g. Cannabis/Humulus or Cannabaceae). To dissect these aggregate data,
we used ecological proxies. C –H pollen in a steppe assemblage (with Poaceae, Artemisia, Chenopodiaceae) was identified as wild-type Cannabis. C –H pollen in a forest assemblage (Alnus, Salix, Quercus, Robinia , Juglans) was identified as Humulus . C –H pollen curves that upsurged alongside crop pollen were identified as cultivated hemp. Subfossil seeds (fruits) at archaeological sites also served as evidence of cultivation. All sites were mapped using geographic information system software. The oldest C –H pollen consistent with Cannabis dated to 19.6 ago (Ma), in northwestern China. However, Cannabis and Humulus diverged 27.8 Ma, estimated by a molecular clock analysis. We bridged the temporal gap between the divergence date and the oldest pollen by mapping the earliest appearance of Artemisia . These data converge on the northeastern Tibetan Plateau, which we deduce as the Cannabis centre of origin, in the general vicinity of Qinghai Lake. This co-localizes with the first steppe community that evolved in Asia. From there, Cannabis first dispersed west (Europe by 6 Ma) then east (eastern
China by 1.2 Ma). Cannabis pollen in India appeared by 32.6 thousand years (ka) ago. The earliest archaeological evidence was found in Japan, 10,000 bce , followed by China.

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https://www.academia.edu/27477304/Cannabis_in_Form_Information_o n_Cannabis?auto=download&email _work_card=download-paper
General Information on Cannabis 150 Pg
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Cannabis Indica in 19th-Century Psychiatry.
CARLSON, E. T.
American Journal of Psychiatry, 131(9), 1004–1007. (1974).
doi:10.1176/ajp.131.9.1004
The author presents a stuck ofthe history and usage of cannabis indicus (the 19th-century pharmacological term referring to the plant we today call cannabis sativa indica). His review ofthe drug’s physiological and psychological effects reveals that most ofthe effects reported in the 1960s were known to writers ofthe 19th century, when the drug was alternate/v considered a curefor and a cause ofinsanity.

Cannabis in Eurasia: origin of human use and Bronze Age trans-continental connections
Tengwen Long, Mayke Wagner, Dieter Demske, Pavel E. Tarasov
Vegetation History and Archaeobotany · June 2016
DOI: 10.1007/s00334-016-0579-6
A systematic review of archaeological and palaeoenvironmental records of cannabis (fibres, pollen, achenes and imprints of achenes) reveals its complex history in Eurasia. A multiregional origin of human use of the plant is proposed, considering the more or less contemporaneous appearance of cannabis records in two distal parts (Europe and East Asia) of the continent. A marked increase in cannabis achene records from East Asia between ca. 5,000 and 4,000 cal bp might be associated with the establishment of a trans-Eurasian exchange/migration network through the steppe zone, influenced by the more intensive exploitation of cannabis achenes popular in Eastern Europe pastoralist communities. The role of the Hexi Corridor region as a hub for an East Asian spread of domesticated plants, animals and cultural elements originally from Southwest Asia and Europe is highlighted. More systematic, interdisciplinary and well-dated data, especially from South Russia and Central Asia, are necessary to address the unresolved issues in understanding the complex history of human cannabis utilisation.

Cannabis in India: Ancient lore and modern medicine
Ethan Budd Russo
In book: Cannabinoids as Therapeutics March 2006
DOI: 10.1007/3-7643-7358-X_1
India is a land steeped in faith and mysticism. Ayurveda, combining the Sanskrit words for life and knowledge, is a system of medicine intertwined inextricably with these traits. That a core of belief combined with empirical experimentation could produce a viable medical regimen still widely practiced after well over 3000 years is astounding to Western physicians. Cannabis was similarly bound to faith and mysticism in India in the past, in the Hindu and Islamic traditions, as well as in numerous other minority religions [1]. Merlin recently explained it well [2], “with the powerful tools of modern science and human imagination, our understanding of our deep-rooted desire to experience ecstasy in the original sense of the word (to break the mind free from the body and communicate with the ‘gods’ or the ancestors) will become clear with time”. This chapter will seek to examine the medical claims for cannabis of the past, and place them in a contemporary light given current pharmacological knowledge. Ayurveda is based on a conceptual medical system that seeks to balance three functional elements, called doshas, that the human body is composed of, and are commonly represented as Vata or Vayu (ether or air), Pitta (fire and water) and Kapha (phlegm or water and earth). Nadkarni [3] has rejected these simple relationships in favor of more abstract assignations

Cannabis is indigenous to Europe and cultivation began during the Copper or Bronze age: a probabilistic synthesis of fossil pollen studies
John M. McPartland, · Geoffrey W. Guy · William Hegman
Vegetation History and Archaeobotany (2018) 27:635–648
doi:10.1007/s00334-018-0678-7
Conventional wisdom states Cannabis sativa originated in Asia and its dispersal to Europe depended upon human transport. Various Neolithic or Bronze age groups have been named as pioneer cultivators. These theses were tested by examining fossil pollen studies (FPSs), obtained from the European Pollen Database. Many FPSs report Cannabis or Humulus (C /H ) with collective names (e.g. Cannabis /Humulus or Cannabaceae). To dissect these aggregate data, we used ecological proxies to differentiate C /H pollen, as follows: unknown C /H pollen that appeared in a pollen assemblage suggestive of steppe (Poaceae, Artemisia , Chenopodiaceae) we interpreted as wild-type Cannabis. C /H pollen in a mesophytic forest assemblage (Alnus, Salix, Populus ) we interpreted as Humulus. C /H pollen curves that upsurged and appeared de novo alongside crop pollen grains we interpreted as cultivated hemp. FPSs were mapped and compared to the territories of archaeological cultures. We analysed 479 FPSs from the Holocene/Late Glacial, plus 36 FPSs from older strata. The results showed C /H pollen consistent with wild-type C. sativa in steppe and dry tundra landscapes throughout Europe during the early Holocene, Late Glacial, and previous glaciations. During the warm and wet Holocene Climactic Optimum, forests replaced steppe, and Humulus dominated. Cannabis retreated to steppe refugia. C /H pollen consistent with cultivated hemp first appeared in the Pontic-Caspian steppe refugium. GIS mapping linked cultivation with the Copper age Varna/Gumelni?a culture, and the Bronze age Yamnaya and Terramara cultures. An Iron age steppe culture, the Scythians, likely introduced hemp cultivation to Celtic and Proto-Slavic cultures.

Cannabis Prohibition in Egypt, 1880–1939: From Local Ban to League of Nations Diplomacy
Liat Kozma
Middle Eastern Studies (2011) Vol. 47, No. 3, 443–460,
DOI: 10.1080/00263206.2011.553890
German criminologist Sebastian Scheer recently challenged the North American focus of drug policy research. Most US and non-US scholarship, he argued, explains drug prohibition, and especially cannabis prohibition, in terms of American domestic policy and the 1937 American Marihuana Tax Act. The history of international cannabis prohibition is often narrated as an extension of the prohibition era, of American relations with its southern neighbours and with its Mexican immigrants. International prohibition, however, dates back to 1925 and to the League of Nations’ Second Opium Convention, in which the US did not play a leading role at all. The role of Italy, South Africa, Egypt and Turkey in international cannabis prohibition, he claimed, is largely overlooked.1 Focusing on Egypt, this article thus fills a gap in drug policy literature. James Mills’ Cannabis Britanica confronted the North American bias of cannabis scholarship by focusing on the Indian case. In one of his chapters, and then in a more recent article, Mills examined League of Nations debates, and thus Egypt’s role in international prohibition. Mills’ argument, in a nutshell, is that the Egyptian delegation’s uncompromising support of prohibition was a direct consequence of British imperial interests in the 1920s. It was also colonial medical doctors’ reports regarding the connection between cannabis and insanity that convinced the Egyptians that hashish was indeed dangerous. The Egyptian stand on cannabis, he claims, had no precedent in Egypt’s international diplomacy in the late nineteenth or early twentieth centuries.2 Relying, as he does, solely on British and League of Nations’ documentation, Mills presents only a partial picture of how cannabis prohibition was conceived of in Egypt itself, sometimes in conflict and sometimes in dialogue with colonial assumptions and policies. Like Ronen Shamir and Daphna Hacker, in their discussion of the 1894 Indian Hemp Commission, I maintain here that elite notions of class distinctions and civilizing of the lower classes were at the heart of indigenous debates on cannabis consumption and prohibition.3 This article, then, queries Mills’ conclusions by going back almost five decades and examining policies, elite discourses and colonial debates within Egypt.

Cannabis sativa (Cannabaceae) in ancient clay plaster of Ellora Caves, India
M. Singh, M. M. Sardesai
Current science 110(5):884-891 March 2016
DOI: 10.18520/cs/v110/i5/884-891
The present research trend is to explore sustainable construction materials having least environmental impact that also encapsulate in terms of our natural resources. The present communication discusses the use of raw hemp as an organic additive in the clay plaster of the 6th century AD Buddhist Caves of Ellora, a World Heritage Site. Cannabis sativa L. admixed in the clay plaster has been identified using scanning electron microscope, Fourier transform infrared spectroscopy and stereomicroscopic studies and the results are compared with fresh specimens. The study indicates that many valuable properties of hemp were known to the ancient Indians in the 6th century AD.

Cannabis utilization and diffusion patterns in prehistoric Europe: a critical analysis of archaeological evidence
John McPartland, William Hegman
November 2017 Vegetation History and Archaeobotany 27(Suppl 4)
DOI: 10.1007/s00334-017-0646-7
Archaeological evidence of Cannabis sativa is comprised of textiles, cordage, fibre and seeds, or pottery impressions of those materials, as well as pseudoliths and phytoliths (pollen is not addressed here). Previous summaries of this evidence connect hemp with Bronze and Iron Age cultures in Europe. This study improves upon earlier summaries by: (1) accessing a larger database; (2) relying on original studies instead of secondary sources; (3) stratifying evidence by its relative robustness or validity. We coupled digital text-searching engines with internet archives of machine-readable texts, augmented by citation tracking of retrieved articles. The database was large, so we limited retrieval to studies that predated 27 bce for west-central Europe, and pre-ce 400 for eastern Europe. Validity of evidence was scaled, from less robust (e.g., pottery impressions of fibre) to more robust (e.g. microscopic analysis of seeds). Archaeological sites were mapped using ArcGIS 10.3. The search retrieved 136 studies, a yield four-fold greater than previous summaries when parsed to our geographic/time constraints. Only 12.5% of studies came from secondary literature. No robust evidence supports claims of Neolithic hemp usage. One Copper Age site in southeastern Europe shows robust evidence (from the Gumelni?a-Varna culture). More robust evidence appears during the Bronze Age in southeastern Europe (Yamnaya and Catacomb cultures). An Iron Age steppe culture, the Scythians, likely introduced hemp cultivation to Celtic, Slavic and Finno-Ugric cultures.The results correlate with a recent palynology study of fossil pollen in Europe. We discuss possible autochthonous domestication of Cannabis in Europe.

Cannabis utilization and diffusion patterns in prehistoric Europe: a critical analysis of archeological evidence
John M. McPartland, William Hegman
Online Resource 1, Extended methods
• accuracy and precision of various dating methods
https://www.researchgate.net/publica...pdf/references
The robustness (validity) of inferences made from archaeobotanical evidence
• textiles, cordage
• fiber
• phytoliths
• pseudoliths
• seeds
• pottery impressions of fiber or seeds
• wood charcoal
• phytochemical and genetic approaches

CONCEPTUAL REVIEW ON VIJAYA (CANNABIS SATIVA LINN.): A FORGOTTEN AMBROSIA
G.Siva Ram et al /
Int. J. Res. Ayurveda Pharm. 9 (2), 2018
DOI: 10.7897/2277-4343.09228
Vijaya (Cannabis sativa Linn.) is associated with spiritual and medicinal aspect of Indian cultural heritage. Currently it is a controlled substance placed
under Schedule-1 in the United Nations Convention on Psychotropic Substances due to its abusive & narcotic nature. Ayurveda, the science of life has details regarding Vijaya identification, cultivation techniques, collection method, purification process, formulations, indications, antidotes, specific dietetics, etc. According to Ayurveda Vijaya, an analeptic herb is originated from amrita (ambrosia) & having the properties of Visha (poison) requires Sodhana (purification) to be utilized as a medicament. Purification helps to detoxify the unwanted qualities & also improves the therapeutic value.
Approximately fifty diseases have been cited in Ayurvedic classics which can be treated with single or compound preparations of Vijaya. Bioavailability
& efficacy is very quick as it is an ushna virya (hot potency) herb having Tikshna (sharp), Vyavayi (bioavailability even before digestion) & Yogavahi
(synergistic action) properties. In this twenty first century research in the therapeutic aspects of Cannabis sativa lead to an increase in the awareness &
knowledge of the ‘medical Cannabis’ among the scientific community. Revalidation of the medicinal evidence of Vijaya present in the ancient
Ayurvedic literature provides scope for more refined research.

Cultivation and manufacture of linen and hemp in New Spain, 1777-1800 (IN SPANISH)
Written by Ramón María Serrera Contreras
https://books.google.es/books?id=p9U...20grua&f=false

DEFORESTATION, CANNABIS CULTIVATION AND SCHWINGMOOR FORMATION AT CORS LLYN (LLYN MIRE), CENTRAL WALES
CN French, PD Moore –
New Phytologist, 1986 - Wiley Online Library
https://nph.onlinelibrary.wiley.com/...1986.tb00823.x
DOIL 10.1111/j.1469-8137.1986.tb00823.x
Find PDF
Pollen and macrofossil diagrams from the schwingmoor sediments of Llyn Mire are presented. Prior to the formation of the schwingmoor, Cannabis sativa was cultivated around Llyn lake, first to the east and later to the west of the basin. Despite Cannabis cultivation so close to Llyn, there is no evidence to suggest that the lake was used for retting purposes. A period of woodland clearance occurred immediately after the Cannabis cultivation and this process may have been responsible for the changes which led to schwingmoor formation in the basin. It is suggested that increased soil erosion into the lake, together with some eutrophication, encouraged the colonization of the lake surface by a floating mat of vegetation. The schwingmoor was formed in historic times by a floating carpet of such taxa as Carex rostrata, Sphagnum recurvum and Sphagnum section Subsecunda. The mat was initiated in the western part of the basin.
The use made of Cannabis/Humulus pollen density as a marker horizon in these studies demanded that these pollen taxa should be adequately separated, and the large numbers of grains of this type present in the sediments permitted the employment of numerical methods based on pore protrusion, which are described here.

Early phytocannabinoid chemistry to endocannabinoids and beyond
Raphael Mechoulam, Lumír O. Hanuš, Roger Pertwee, Allyn C. Howlett
Nature Reviews Neuroscience volume 15, pages 757–764 (2014)
DOI: 10.1038/nrn3811
Isolation and structure elucidation of most of the major cannabinoid constituents — including ?9-tetrahydrocannabinol (?9-THC), which is the principal psychoactive molecule in Cannabis sativa — was achieved in the 1960s and 1970s. It was followed by the identification of two cannabinoid receptors in the 1980s and the early 1990s and by the identification of the endocannabinoids shortly thereafter. There have since been considerable advances in our understanding of the endocannabinoid system and its function in the brain, which reveal potential therapeutic targets for a wide range of brain disorders.

In Japanese but easy to translate with Google translate
Fossil hemp fruits in the earliest Jomon period from the Okinoshima site, Chiba Prefecture, Japan
Makiko Kobayashi Arata Momohara, Susumu Okitsu, Seiichi Yanagisawa and Tozo Okamoto
https://hisbot.jp/journalfiles/1601/1601_011-018.pdf
Fruits of Cannabis sativa were found from the sediment of the earliest Jomon period at the Okinoshima site, Tateyama City, Chiba Prefecture, central Japan. The fruit of Cannabis sativa can be distinguished from that of Humulus by the size and morphology. The stones of Humulus lupulus and H. scandens have a circlular lateral view and a circular or heart-shaped attachment on the top, and lacks a shallow hollow at the base. The stone of Humulus lupulus is smaller than that of Cannabis sativa. The fruits obtained from the Okinoshima site and those of Cannabis sativa both have an ovoid lateral view, a round knob on the top instead of a heart-shaped attachment, and a shallow hollow at the base. Thus the fruits of the Okinoshima site were identified as Cannabis sativa, the oldest record of its fruits in the world, although its fibers have been found from the sediments of the incipient Jomon period at the Torihama shell midden, Fukui Prefecture. Contrary to fossil fibers that can be imported from outside Japan, existence of fossil fruits implies that Cannabis sativa grew in Japan in the early Jomon period. Hemp may have been cultivated and utilized around the site

From schizophrenia to sainthood – Tajuddin Fakir
Amruta Huddar, Tasneem Raja, Sanjeev Jain, Swaran P. Singh
Asian Journal of Psychiatry xxx (xxxx) xxx
DOI: 10.1016/j.ajp.2020.102465
This paper discusses the case of Tajuddin, a patient in the mental asylum of Nagpur towards the end of the 19th century. Discussions are based on hospital records and annual reports and relevant literature review of the life and times of Tajuddin. Hospital and associated records indicate that Tajuddin was thought to be suffering from cannabis psychosis. He was released from the British army for inappropriate behaviour and was admitted to the Nagpur Mental Asylum, currently a Regional Mental Hospital. During his inpatient stay Tajuddin was believed to have special powers and was considered a saint. The hospital, its staff and patients continue to pay homage to Tajuddin to this day. Religious Trusts established in his name extend all the way up to Mecca. His followers include high ranking officers and Bollywood celebrities. Tajuddin was a charismatic leader, despite suffering from what currently may be considered schizophrenia. His case reflects a curious contradiction of the cultural understandings of psychosis and the shifting sands on which psychiatry’s diagnostic foundations are built.

*Frozen Tombs of Siberia: The Pazyryk Burials of Iron Age Horsemen.
Rudenko, Sergei Ivanovich (1970).
University of California Press. ISBN 0520013956.
https://historyandsoon.wordpress.com.. .zyryk-burials/
https://sci-hub.tw/10.1525/aa.1973.75.4.02a01350
https://issuu.com/horsebackarchers/d...the_altai_moun

Giorgio Samorini Network
https://samorini.it/
Presentation This web-site wants to bring cognitive contributions and instruments for the study of the phenomenological field of the psychoactive drugs; a field of research known as Science of Drugs.
This web-site does not deal with the problematic aspects of the drugs, nor is interested on the apologetical aspects related to their modern use, but reports a bulk of scientific data which concern the drugs as phenomenon, in their relationship with the living beings, especially the human beings.
The editor of this web-site is an independent researcher specialized on the ethnobotany and the anthropology of drugs, author of specialized books and writings in Italian and in other languages (see his bibliography).
Although many texts of this site are in Italian, English titles are everywhere present, and this double-language is applied to the texts of different places of the site; furthermore, the wide section “Documentation” contains documents in many languages. All the translations from the other languages vs. Italian are edited by the author of the site.
The data here presented are referenced by specific bibliographies located at the bottom of each page, and integrated in the section “Documentation”.

HASHISH IN ISLAM 9TH TO 18TH CENTURY
GABRIEL G. NAHAS,
Bull. N.Y. Acad. Med.
Vol. 58, No. 9, December 1982
https://www.ncbi.nlm.nih.gov/pmc/art...00095-0056.pdf
CANNABIS was used as an intoxicant @hang) in India and Iran as far back as 1000 B.C.1,2 It was adopted in the Moslem Middle East 1,800 years later, two centuries after the death of the prophet Mohammed. Indeed, during his life time (A.D. 570-632), the use of cannabis preparations (known in the Middle East as hashish, which means "grass" in Arabic) was unknown. This might be the reason why the prophet did not explicitly forbid in the holy Koran intoxication by cannabis, although he proscribed that induced by fermented beverages (alcohol, wine, beer). There is no evidence that the Arabs became familiar with the intoxicating properties of hashish before the ninth century. At that time, they had already conquered Iraq and Syria and swept eastward to the border of Persia and Central Asia and westward through Asia Minor, North Africa, and Spain. (It was in 752 that the relentless Muslim expansion was halted at Poitiers by the Frankish king Charles Martel.) In the ninth century, well after the establishment in A.D. 750 of the splendid Abasside caliphate in Bagdad, noted for its universities, Arab scholars translated the Greek texts of Dioscorides and Galen, and became familiar with the medicinal properties of cannabis. One physician of the early 10th century, Ibn Wahshiyah, warned of possible complications resulting from use of hashish. In his book, On Poisons, he claimed that the plant extract might cause death when mixed with other drugs. Another physician, the Persian born al-Rhazes, counselled against over-prescribing cannabis.' Traders travelling to Persia from India and Central Asia also may have spread knowledge of the plant's medicinal properties. the use of hashish as an intoxicant surfaced in Islam. Called hashish instead of bhang, the Hindu designation, it was first consumed by members of religious Persian and Iraqui sects located at the eastern periphery of the Islamic empire which bordered the central steppes where the plant had its origins. And there was little cultural opposition at first because the holy Koran, which formulates in detail all of the rules of daily Muslim living, does not forbid explicitly the consumption of cannabis, although it proscribes the use of fermented beverages. And around A.D. 1000 the Fatima King al-Hakim issued an edict prohibiting the sale of alcohol throughout Syria and Egypt,3 but did not ban cannabis.

Hashish in Morocco and Lebanon A comparative study
Kenza Afsahi Salem Darwich
International Journal of Drug Policy (2016),
doi:10.1016/j.drugpo.2016.02.024
Maghreb and Middle East have a long history of consumption, production and marketing of cannabis. Over the past 12 centuries, migration, trade and different spiritual practices and trends have led to the expansion of cannabis markets. This long period is marked by stages and rifts caused by foreign interference, a worldwide prohibition of cannabis at the beginning of the 20th century and increased global demand in the 1960s. In the 1960s and the 1970s,
global cannabis production increased, particularly in developing countries. Morocco and Lebanon became major producers of hashish for export to markets in West and Central Europe, Africa and the Middle East. The two countries have cultural and religious commonalities and similar socio-economic conditions in cannabis production areas. However, there are also some important differences in the way crop growing areas have developed in
the Rif and in the Beqaa. Unlike in Morocco where cannabis is a traditional and locally consumed crop, cannabis consumption in Lebanon remains marginal. By using a comparative approach, this paper aims to evaluate changes in production in the traditional areas of cannabis cultivation in the Rif and the Beqaa and to better understand the role that these countries play in current trends in the global cultivation and consumption of cannabis

Hemp in ancient rope and fabric from the Christmas Cave in Israel: Talmudic background and DNA sequence identification
Terence M. Murphy, Nahum Ben-Yehuda , R.E. Taylor, John R. Southon
Journal of Archaeological Science 38 (2011) 2579e2588
doi:10.1016/j.jas.2011.05.004
The “Christmas Cave”, a cave in the Qidron Valley near the Dead Sea and Qumran, has yielded a complex collection of plant-derived rope and fabric artifacts. Using polymerase chain reaction (PCR) to amplify DNA of the samples, we estimated the sizes and determined restriction patterns and base sequences of chloroplast genes, primarily rbcL (gene for the large subunit of ribulose bisphosphate carboxylase). DNA was successfully extracted from all samples, but was limited to sizes of approximately 200e300 base pairs. As expected, the DNA extracted from the samples was identified as coming primarily from flax (Linum usitatissamum L.), but two samples had a significant fraction, and all samples had at least a trace, of hemp (Cannabis sativa L.) DNA. Artifacts from the Christmas Cave were thought to date from Roman times, but it was thought possible that some could be much older. Accelerator mass spectrometry (AMS)-based 14C dating confirmed that the samples contained representatives from both the Roman and Chalcolithic periods. This paper provides a synthesis of DNA, isotope, and literary analysis to illuminate
textile history at the Christmas Cave site.

Hemp Production in Italy
Stefano Amaducci
June 2005 Journal of Industrial Hemp 10(1):109-115
DOI: 10.1300/J237v10n01_09
After a short history of hemp in Italy, this article lists the events that have brought back the cultivation of this fibre crop in Italy in recent years. The cultivation technique used for baby hemp is briefly described and the preliminary results of its processing are given. Baby hemp is a hemp crop grown at high plant densities (400-500 plants m2) that is chemically desiccated when the height of 120-140 cm is reached and that is harvested with the machines used for flax. Advantages, problems and possible solutions for this technique are presented

HIGH POINTS: AN HISTORICAL GEOGRAPHY OF CANNABIS
BARNEY WARF
Geographical Review (2014) Volume 104, Issue 4 414-438
DOI: 10.1111/j.1931-0846.2014.12038.x
Cannabis, including hemp and its psychoactive counterpart, has a long but
largely overlooked historical geography. Situating the topic within varied perspectives such as world-systems theory, Foucauldian biopolitics, and the moral economy of drugs, this paper charts its diffusion over several millennia, noting the contingent and uneven ways in which it was enveloped within varying social and political circumstances. Following a brief theorization, it explores the plant’s early uses in East and South Asia, its shift to the Middle East, and resultant popularity in the Arab world and Africa. Next, it turns to its expansion under colonialism, including deliberate cultivation by Portuguese and British authorities in the New World as part of the construction of a pacified labor force. The fifth section offers an overview of cannabis’s contested history in the United States, in which a series of early 20th-century moral panics led to its demonization; later, the drug enjoyed gradual liberalization. Keywords: cannabis, marijuana, hemp, drugs, moral geographies

History of Cannabis and Its Preparations in Saga, Science, and Sobriquet
Ethan B. Russo
CHEMISTRY & BIODIVERSITY – Vol. 4 (8):1614-48 (2007)
DOI: 10.1002/cbdv.200790144
Cannabis sativa L. is possibly one of the oldest plants cultivated by man, but has remained a source of controversy throughout its history.Whether pariah or panacea, this most versatile botanical has provided a mirror to medicine and has pointed the way in the last two decades toward a host of medical challenges from analgesia to weight loss through the discovery of its myriad biochemical attributes and the endocannabinoid system wherein many of its components operate. This study surveys the history of cannabis, its genetics and preparations. A review of cannabis usage in Ancient Egypt will serve as an archetype, while examining first mentions from various Old World cultures and their pertinence for contemporary scientific investigation. Cannabis historians of the past have provided promising clues to potential treatments for a wide array of currently puzzling medical syndromes including chronic pain, spasticity, cancer, seizure disorders, nausea, anorexia, and infectious disease that remain challenges for 21st century medicine. Information gleaned from the history of cannabis administration in its various forms may provide useful points of departure for research into novel delivery techniques and standardization of cannabis-based medicines that will allow their prescription for treatment of these intractable medical conditions.

Histories of Cannabis Use and Control in Nigeria, 1927–1967
Gernot Klantschnig
In: Drugs in Africa, Histories and Ethnographies of Use, Trade, and Control
Charles Ambler; Neil Carrier; Gernot Klantschnig
DOI: 10.1057/9781137321916_4
Much of the available research on illegal drugs, such as cannabis, heroin,
or cocaine, has shown a weak understanding of the drugs’ historical roots
in Africa and the domestic meanings of these substances and their control.
This has been a result of a lack of openly available sources on these substances and also because much of this work has been conducted by international control agencies or researchers working closely with them and hence research has often served an immediate policy purpose rather than a better historical understanding of drugs.
A few recent studies have started to alleviate these shortcomings. Explicitly challenging these ahistorical views, Emmanuel Akyeampong and Stephen Ellis have begun to sketch the longer history of West Africa’s role in the trade in cannabis, heroin, and cocaine dating it back to the beginning of the twentieth century. A few other authors have begun work on other illegal or quasi-legal substances in other parts of the continent. These new studies have been most successful at highlighting the historical dynamics of drugs and their control when they have tied their analyses to the more developed literature on the social history of alcohol—an explicit aim of many of the contributions in this volume as well

A mummy unearthed from the pastures of heaven
October 1994 National geographic 186(4):80-103
N. Polosmak
https://nationalgeographicbackissues.. .ober-1994.html
Locked in an icy burial chamber beneath the Siberian steppes for 2, 400 years, a Pazyryk gentle- woman comes to light along with possessions chosen for eternity.

ICE MUMMIES: SIBERIAN ICE MAIDEN
https://www.pbs.org/wgbh/nova/transc...7siberian.html
Tonight on NOVA, sacrificial horses guard her tomb. Gold and silk adorn her body. For 24 centuries, she was frozen in time. Was she a priestess? A warrior chief? NOVA unearths the secrets of "The Siberian Ice Maiden".
The thunder of hooves on the Siberian steppes echoes a legendary past. Mounted tribes once ruled these high plateaus, where towering stone monuments reach toward the heavens. Golden treasures from these days are rare and enigmatic. But new finds cast light on a culture cloaked in mystery: Sacrificed animals, valued possessions, and a startling emissary from this age of warriors—a 2400 year old woman frozen in time. But this Ice Maiden will not be left to rest. Removed from the grave, her body has traveled half way around the world to be displayed and admired. Now, she is returning to Siberia, back to the scientist who discovered her, and who hopes to learn more of the Ice Maiden's secrets.
When Natalia Polosmak found this woman and the wealth of artifacts buried with her, it was celebrated as an archeological triumph. But now, taken from her tomb, the body has sparked passion and controversy—among both scientists and the people of her homeland.
Few archaeologists have ventured into the rugged Altay Mountains. But in 1993, Natalia Polosmak was determined to reach the Ukok Plateau. In a remote part of Asia where four countries converge, she was drawn by tales of an ancient people called the Pazyryk.

Iconic 2,500 year old Siberian princess 'died from breast cancer', reveals MRI scan
https://siberiantimes.com/science/ca...ique-mri-scan/
Studies of the mummified Ukok 'princess' - named after the permafrost plateau in the Altai Mountains where her remains were found - have already brought extraordinary advances in our understanding of the rich and ingenious Pazyryk culture.
The tattoos on her skin are works of great skill and artistry, while her fashion and beauty secrets - from items found in her burial chamber which even included a 'cosmetics bag' - allow her impressive looks to be recreated more than two millennia after her death.
Now Siberian scientists have discerned more about the likely circumstances of her demise, but also of her life, use of cannabis, and why she was regarded as a woman of singular importance to her mountain people.
Her use of drugs to cope with the symptoms of her illnesses evidently gave her 'an altered state of mind', leading her kinsmen to the belief that she could communicate with the spirits, the experts believe.
The MRI, conducted in Novosibirsk by eminent academics Andrey Letyagin and Andrey Savelov, showed that the 'princess' suffered from osteomyelitis, an infection of the bone or bone marrow, from childhood or adolescence

Identification of Cannabis Fiber from the Astana Cemeteries, Xinjiang, China, with Reference to Its Unique Decorative Utilization
TAO CHEN, SHUWEN YAO, MARK MERLIN, HUIJUAN MAI, ZHENWEI QIU, YAOWU HU, BO WANG, CHANGSUI WANG,
AND HONGEN JIANG
March 2014 Economic Botany 68(1)
DOI: 10.1007/s12231-014-9261-z
In the Turpan District of Xinjiang, China, large numbers of ancient clay figurines, with representations including equestrians, animals, and actors, have been excavated from the Astana Cemeteries and date from about the 3rd to the 9th centuries C.E. Based on visual inspection, the tails of some of the figurines representing horses are made of plant fibers. Fourier Transform Infrared (FTIR) spectroscopy, light microscope examination, and drying–twist tests demonstrated that these fibers were extracted from one or more stalks of hemp (Cannabis) plants. This is a unique report of the utilization of Cannabis bast fibers for figurine decoration in ancient Turpan.

INDIAN HEMP DRUGS AND INSANITY.
G. F. W. Ewens,
THE Indian Medical Gazette NOVEMBER, 1904.
https://www.ncbi.nlm.nih.gov/pmc/art...71518-0021.pdf
the Superintendent of the Central Lunatic Asylum, Lahore, raises again the question which ten years ago was very much debated among medical men in India.
It will be observed that, as a result of his experience while in charge of the large asylum at Lahore, Capt. Evvens is of opinion (1) that there is a form of mental disease which seems to have a direct relation to the excessive use of hemp drugs, as "a definite effect following a definite cause (2) it has a " definite train of symptoms of a fairly regular character."
Those of us who were in India ten years ago will remember the Report of the Indian Hemp Drugs Commission.


Instruction to sow, cultivate and benefit flax and hemp in New Spain (IN SPANISH)
Written by Miguel de la Grua Talamanca and Branciforte
https://books.google.es/books?id=98p...20grua&f=false

Man and Cannabis in Africa: A Study of Diffusion
Brian M. du Toit
African Economic History No. 1 (Spring, 1976), pp. 17-35
https://www.jstor.org/stable/4617576...n_tab_contents
The past decade has seen an awakening of research interests regarding psychoactive and hallucinogenic drugs. While the new world is particulary rich in these natural products, no drug has seen a wide a distribution nor as universal an appeal as Cannabis. This hallucinogen is known by different local refferents but the most widely distributed is marijuana in the United States and Latin America, and hemp or Indian hemp in many of the Anglophone areas of the world. While it has near universial distrabution, it is nonetheless to the old world we must look for its origin and original acceptance.
Cannabis was originally cultivated as a fiber plant and only its leaves were used in the pharmacopeia of different peoples. Linnaeus classified it as a simple species Cannabis sativa, but recent research indicates there may well be several species. At this stage we are not concerned with this botanical question but intend to focus on the scocial use and diffusion of the plant through Africa.
In this paper we will examine in turn the historical, sociological, and linguistic evidence relating to the Cannabis plant in Africa. Then after a brief review of current hypotheses regarding the diffusion of Cannabis, we will propose a more encompassing to account for its spread in sub-Saharan Africa.

Marijuana Australiana.
John Lawrence Jiggens
https://eprints.qut.edu.au/15949/1/J...ens_Thesis.pdf
The word 'marijuana' was introduced to Australia by the US Bureau of Narcotics via the Diggers newspaper, Smith's Weekly, in 1938. Marijuana was said to be 'a new drug that maddens victims' and it was sensationally described as an 'evil sex drug'. The resulting tabloid furore saw the plant cannabis sativa banned in Australia, even though cannabis had been a well-known and widely used drug in Australia for many decades.
In 1964, a massive infestation of wild cannabis was found growing along a stretch of the Hunter River between Singleton and Maitland in New South Wales. The explosion in Australian marijuana use began there.It was fuelled after 1967 by US soldiers on rest and recreation leave from Vietnam. It was the Baby-Boomer young who were turning on. Pot smoking was overwhelmingly associated with the generation born in the decade after the Second World War. As the conflict over the Vietnam War raged in Australia,it provoked intense generational conflict between the Baby-Boomers and older generations. Just as in the US, pot was adopted by Australian Baby-Boomers as their symbol; and, as in the US, the attack on pot users served as code for an attack on the young, the Left, and the alternative.
In 1976,the 'War on Drugs ' began in earnest in Australia with paramilitary attacks on the hippie colonies at Cedar Bay in Queensland and! Tuntable Falls in New South Wales.Itwas a time of increasing US style prohibition characterised by 'tough-on-drugs' right-wing rhetoric, police crackdowns, numerous murders, and a marijuana drought followed quickly by a heroin plague; in short by a massive worsening of 'the drug problem'. During this decade, organised crime moved into the pot scene and the price of pot skyrocketed, reaching $450 an ounce in 1988. Thanks to the Americanisation of drugs policy, the black market made 'a killing'.
In Marijuana Australiana I argue that the 'War on Drugs' developed -not for health reasons -but for :reasons of social control; as a domestic counter-revolution against the Whitlamite, Baby-Boomer generation by older Nix.onite Drug War warriors like Queensland Premier, Bjelke-Petersen. It was a misuse of drugs policy which greatly worsened drug problems, bringing with it American-style organised crime.
As the subtitle suggests, Marijuana Australiana relies significantly on 'alternative' sources,and I trawl the waters of popular culture,. looking for songs, posters,comics and underground magazines to produce an 'underground' history of cannabis in Australia. This 'pop' approach is balanced with a hard-edged, quantitative analysis of the size of the marijuana market, the movement of price, and the seizure figures in the section called 'History By Numbers'. As Alfred McCoy notes, we need to understand drugs as commodities. It is only through a detailed understanding of the drug trade that the deeper secrets of this underground world can be revealed. In this section,I present an economic history of the cannabis market and formulate three laws of the market

Truely great book
*Marihuana - The First Twelve Thousand Years
Ernest L Abel
January 1980 Publisher: Plenum Press
DOI: 10.1007/978-1-4899-2189-5
Introduction
Of all the plants men have ever grown, none has been praised and denounced as often as marihuana (Cannabis sativa). Throughout the ages, marihuana has been extolled as one of man's greatest benefactors and cursed as one of his greatest scourges. Marihuana is undoubtedly a herb that has been many things to many people. Armies and navies have used it to make war, men and women to make love. Hunters and fishermen have snared the most ferocious creatures, from the tiger to the shark, in its herculean weave. Fashion designers have dressed the most elegant women in its supple knit. Hangmen have snapped the necks of thieves and murderers with its fiber. Obstetricians have eased the pain of childbirth with its leaves. Farmers have crushed its seeds and used the oil within to light their lamps. Mourners have thrown its seeds into blazing fires and have had their sorrow transformed into blissful ecstasy by the fumes that filled the air.
Marihuana has been known by many names: hemp, hashish, dagga, bhang, loco weed, grass-the list is endless. Formally christened Cannabis sativa in 1753 by Carl Linnaeus, marihuana is one of nature's hardiest specimens. It needs little care to thrive. One need not talk to it, sing to it, or play soothing tranquil Brahms lullabies to coax it to grow. It is as vigorous as a weed. It is ubiquitous. It fluorishes under nearly every possible climatic condition.
It sprouts from the earth not meekly, not cautiously in suspense of where it is and what it may find, but defiantly, arrogantly, confident that whatever the conditions it has the stamina to survive. It is not a magnanimous herb. Plants unfortunate enough to fall in the shade of its serrated leaflets will find that marihuana does not share its sunlight. It wants it all. Marihuana also does not like to share its territory. It encroaches on its neighbors. Its roots gobble up all the nutrients in the soil, and like a vampire it sucks the life blood from the earth.
Marihuana is a very rapidly growing plant, attaining a usual height of three to twenty feet at maturity. Five hundred years ago, the French author Rabelais wrote that it was "sown at the first coming of the swallows and pulled out of the ground when the cicadies began to get hoarse."
Marihuana is dioecious, which means that there are sexually distinct male and female plants. At one time, farmers believed that only the females produced the intoxicating hashish resin. Now it is known that both sexes produce this gummy secretion. The male, however, manufactures less resin and produces flowers earlier than the female. To prevent a pollinating marriage, cannabis growers destroy these males as soon as they are detected. Had he known of this age-old custom, Freud might have written an insightful treatise on the symbolism of this bit of agricultural castration.
The intoxicating resin is secreted by glandular hairs located around the flowers and to a certain extent in the lower portions of the plant. The actual substance in the resin responsible for the plant's inebriating effects is a chemical called delta-9-tetrahydrocannabinol (~ THC). In very hot climates, as in India and North Africa, so much resin is produced that the plant appears to be covered with a sticky dew even as it bakes under the parching rays of the hot sun. This resin serves as a protective shield preventing loss of water from the plant to the dry air. And of course, the more resin, the more A 9 -THC likely to be present. Cannabis seeds are brownish and rather hard. When pressed, they yield a yellowish-green oil once used to make soap, lamp oil, paint, and varnish. Bird fanciers claim that hemp seed stimulates birds to develop superior plumage. While the seeds contain far less -THC than the leaves or flowers, the chemical is still present. Although there are no reports of any birds flying into trees or houses after feasting on a meal of cannabis seeds, it was by burning these seeds and inhaling the fumes given off that some ancient societies first experienced cannabis's intoxicating powers.
The stem of the plant is square and hollow and covered with strong fibers. The first step in removing these fibers is called retting and involves soaking the stems so that partial decomposition occurs. This disengages the nonfibrous tissue. The stem is then bent so that the fibers can separate. Once separated, they can be stripped away and spun into thread or twisted into cordage and rope.
Cannabis will grow under most conditions that will support life. It is inherently indestructible. Long after other species of plants have disappeared because of drought, infestation, or climatic changes, cannabis will still exist. Cannabis is one of nature's best examples of survival of the fittest. Depending on the conditions under which it grows, cannabis will either produce more resin or more fiber. When raised in hot, dry climates, resin is produced in great quantities and fiber quality is poor. In countries with mild, humid weather, less resin is produced and the fiber is stronger and more durable. It is because of these climate-related characteristics that most Europeans knew very little of the intoxicating properties of the cannabis plant until the nineteenth century when hashish was imported from India and the Arab countries. Prior to this time, cannabis was merely a valuable source of fiber and seed oil to most Europeans, nothing more. In India, Persia, and the Arab countries, the main value of the plant resided in its inebriating resin. People in these countries were also among the first to use cannabis fiber to make nets and ropes. But the sticky covering on the plant was what they valued most, especially where alcohol was proscribed by religious doctrine. Depending on his personal interests, the cannabis farmer could increase his yield of fiber or resin by various measures. To produce a plant with a better fiber, he grew his plants very close to one another. This reduced the amount of sunlight falling on individual plants and
promoted the growth of long stems and fibers. To obtain more resin, he sowed his seeds farther apart. This gave each plant more sunlight and forced the plant to secrete more resin in order to keep itself from drying out. But regardless of whether he was after the fiber or the resin, male plants were always destroyed before they could court the females, since the production of seeds by the female invariably reduced the quality of fiber and resin.
Cannabis was harvested by various methods. If the fiber were primarily of interest, the stems would be cut fairly close to the ground with a specially designed sickle with the blade set at right angles to the handle. Harvesting the resin was a different matter. People who grew cannabis for personal pleasure simply snipped some leaves whenever the desire moved them. In countries such as Nepal where cannabis became part of the agricultural economy, the resin was gathered more systematically but in a less sanitary fashion: after the female plants were ripe with their sticky coverings, workers were hired to run naked through the cannabis fields. As they brushed against the plants, a certain amount of resin would adhere to their bodies. At the end of each run they would scrape the sticky resin from their bodies and start again. Since cannabis resin and water do not mix very well, the perspiration from their sweating bodies did not find its way into these scrapings. (The same cannot be
said for whatever else was on their bodies.) After the harvesting was through, the scrapings were shaped into bricks and readied for market. Buyers were rarely finicky about anything other than how pleasureable was the intoxication they felt when they consumed their purchases.

New fossil evidence for the past cultivation and processing of hemp (Cannabis sativa) in Eastern England
R. H. W. BRADSHAW, P. COXON, J. R. A. GREIG, A. R. HALL
November 1981 New Phytologist
doi: 10.1111/j.1469-8137.1981.tb02331.x
Fossil records of Cannabiaceae pollen and Cannabis achenes from Flandrian deposits at three sites in eastern England are presented as further evidence for the past cultivation of this crop. It is suggested that retting of hemp to extract fibre was carried out at each of the sites.

NORTH INDIA: THE CANNABIS SANCTUARY
https://www.indianlandraceexchange.c.. .bis-sanctuary/
Introduction - India has known about cannabis from the very dawn of the civilizations at the indus valley and has since used the plant for important life saving applications such as medicine, food, and even shelter. Inked in the ancient vedas by the Indian scholars are some of the earliest documented accounts of cannabis used as a medicine that goes back to 2000 BCE, and even the people who invaded India couldn’t stay too far from this plant,e.g. the Portuguese who captured Goa in 1510, quickly learned how cannabis played an important role in Indian culture, day to day applications and especially about the narcotic effects of the plant. A glimpse of which can be found in the Notes by Garcia de orta
O bangue é formado por folhas secas e hastes tentras de cânhamo (Canabis sativa, Lin.) que se fumam o mascam e que embriaga como o ópio.

Translation: Bangue (Bhang) is made of dry leaves and tender stemps of hemp, which they smoke or Garcia de orta A botanist who wrote about foreign cannabis colonies of Portugal in 1534 in his work "Glossário luso-asiatico". chew, and it intoxicates like opium Even during the British colonial rule in India which lasted over 200 years, in 1894 an extensive study was conducted and a report was published by an indo-british team known as Indian hemp drugs commission, which pinned down the Physical, psychological and socio-cultural effects of cannabis in india.
The plant itself is virtually found in every direction you go to in India However North India is widely cited as the biggest charas producing region,featuring the notorious and the elusive highland himalayan villages, spreading all the way from Kashmir to Himachal and down to the China border in Uttarakhand. Any kind of enquiry concerning cannabis in India is incomplete without understanding the role of north indian cannabis regions, such as - Jammu and Kashmir, himachal pradesh, haryana, punjab, uttar pradesh, uttrakhand and rajasthan.Although it is to be noted duely that punjab and haryana being flat lands amongst the listed states doesn't have any rigorous cultivation or large feral fields of cannabis anymore just like the capital state New Delhi where an exponentially rising population has already taken a severe toll on the nature and some of the regional cultivars from these areas have either vanished or are on the verge of being lost forever.

On the Natural History, Action, and Uses of Indian Hemp
Mon J Med Sci. 1851 Jul; 4(19): 26–45.
Alexander Christison, M.D., Edin.1
Indian hemp has been long known in India, Persia, and other Eastern countries as a medicinal and intoxicating agent, but was little known to Europeans until it was brought prominently into notice by Dr O'Shaughnessy of Calcutta, in the year 1839. [On Indian Hemp, &c.; Calcutta, 1839.]
The ancients were almost ignorant of its virtues. The Greek physicians, as we are told by Dioscorides, were acquainted with the emollient properties of the seeds of hemp ; but they seem to have been wholly unaware of the narcotic virtues of the plant (Diosc. iv. civ.). Galen and his contemporaries were not much more informed on the subject; for that author merely speaks of its seeds being sometimes used as a whet after supper, to create a de'sire for wine, but condemns the practice, because, when used freely, they heat the system and cause determination towards the head (Opera, ii. 53, Edit. Basiliae, 1549 ; De Facultatibus Alimentorum, c. xlix.). It is alleged, however, that hemp was known at an early period to the Chinese. In a communication to the Academie des Sciences in 1849, extracts are produced from a Chinese work, showing that so far back as a.d. 220, a Chinese physician, named Hoa-Tho, pro- duced insensibility in his patients by means of a preparation of hemp, and that operations were then performed without pain to the patient (Stanislas Julien, inComptes Rendus,&c., 1849, p. 197). This statement would, however, require further confirmation. There seems little doubt that in the year 600 the Hindoos were in the habit of employing hemp ; and that it has been in constant use ever since as a means of allaying pain, and more particularly as an intoxicating agent, among the inhabitants of the East.

ON THE PREPARATIONS OF THE INDIAN HEMP, OR GUJNJAH,* (Cannabis Indica)
W. B. O'SIAUGHNESSY,
https://www.ncbi.nlm.nih.gov/pmc/art...00864-0001.pdf
The narcotic effects of hemp are popularly known in the South of Africa, South America, Turkey, Egypt, Asia Minor, India, and the adjacent territories of the Malays, Burmese, and Siamese. In all these countries hemp is used in various forms, by the dissipated and depraved, as the ready agent of a pleasing intoxication. In the popular medicine of these nations, we find it extensively employed for a multitude of affections, especially those in which spasm or neuralgic pain are the prominent symptoms. But in Western Europe its use, either as a stimulant or as a remedy, is equally unknown. With the exception of the trial, as a frolic, of the Egyptian " hasheesh," by a few youths in Marseilles, and of the clinical use of the wine of hemp by Hahnemann, as shown in a sub. sequent extract, I have been unable to trace any notice of the employment of this drug in Europe. Much difference of opinion exists on the question, whether the hemp so abundant in Europe, even in high northern latitudes, is identical in specific characters with the hemp of Asia Minor and India. The extraordinary symptoms produced by the latter depend on a resinous secretion with which it abounds, and which seems totally absent in the European kind. The closest physical resemblance or even identity exists between both plants; difference of climate seems to me more than sufficient to account for the absence of the resinous secretion, and consequent want of narcotic power in that indigenous in colder countries.

Origin, Early History, Cultivation, and Characteristics of the Traditional Varieties of Moroccan Cannabis sativa L.
Fatima Bachir, Mohamed Eddouks, Mohamed Arahou, and Mohammed Fekhaoui
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0020
Background: Cannabis has been cultivated and used for centuries in the north Moroccan Rif (local name is kif). However, its history is poorly known and the date of its first introduction and dispersal in Morocco is still difficult to be precise.
Aim: The purpose of the present work is to review the literature on the origin, history, and cultivation of Cannabis in Morocco, as well as data on the morphological, genetic, and phytochemical characteristics of local cultivated varieties.
Discussion: Considering the importance of preserving the fragile environment of the Rif and the future development of the Moroccan medical Cannabis market, which will require authentication of the raw material, the use of local strains which are well adapted to the particular environment of the Rif is highly recommended. However, there is no document that summarizes and clarifies the nomenclature and the characteristics of local Moroccan Cannabis. In addition, the recent adoption by Rif growers of improved hybrid cultivars is obliterating the traits and peculiarities of Moroccan Cannabis through genetic introgression.
Conclusion: Summarizing and discussing the data from the literature on the characteristics of local Moroccan Cannabis varieties may be useful for their identification and the localization of the areas of the Rif region where their cultivation is still practiced.
Find PDF, I have

Physical evidence for the antiquity of Cannabis sativa L.
Fleming, M. P., and R. C. Clarke
Journal of the International Hemp Association 5(2): 80-95. 9 1998.
https://www.internationalhempassocia.. ./jiha5208.html
Cannabis has been an important economic crop plant for six millennia. Its uses for fiber, food, oil, medicine, and as a recreational/religious drug have been prevalent throughout this period. Recent palynological research into the agricultural and environmental history of Cannabis has produced curves for Cannabaceae pollen at a number of sites in Europe and America. Additional archaeological remains and written records provide evidence for both Old and New World occurrences. This paper discusses the origin, domestication and migration of hemp as a crop plant as documented by palynological and archaeological evidence. In addition, the comparative morphology of Cannabis andHumulus pollen grains is described, and the problems of interpreting Cannabaceae pollen in the stratigraphic record are discussed.

Phytochemical and genetic analyses of ancient cannabis from Central Asia
Ethan B. Russo, Hong-En Jiang, Xiao Li, Alan Sutton, Andrea Carboni, Francesca del Bianco, Giuseppe Mandolino, David J. Potter, You-Xing Zhao, Subir Bera, Yong-Bing Zhang, En-Guo Lü, David K. Ferguson, Francis Hueber, Liang-Cheng Zhao, Chang-Jiang Liu, Yu-Fei Wang, and Cheng-Sen
J Exp Bot. 2008 Nov; 59(15): 4171–4182.
DOI: 10.1093/jxb/ern260
The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.

Preliminary report on a mid-19th century Cannabis pollen peak in NE Spain: Historical context and potential chronological significance
Valentí Rull and Teresa Vegas-Vilarrúbia
The Holocene
2014, Vol. 24(10) 1378–1383
DOI: 10.1177/0959683614540964
Cannabis sativa L. (hemp) was introduced in Europe from Asia Minor during classical Greek and Roman times. Since then, hemp pollen abundances between 10% and 80% in Late Holocene sediments have been considered indicative of the local or regional cultivation and/or retting of this plant. In this paper, a unique pollen peak of >60% recorded in Lake Montcortès (pre-Pyrenean foothills, NE Spain) is evaluated as a potential chronostratigraphic marker. Previously, this pollen peak was dated to ad 1757 using a depth–age model based on AMS 14C dates from sedimentary macrofossils, but a recent calibration of the model using varve counting has refined the date of the hemp pollen maximum to ad 1839. This date coincides with an outstanding socio-political shift from feudalism to liberalism in Spain and the corresponding dismantling of the royal navy, the main consumer of hemp fibre. These events produced a well-documented decline in Cannabis cultivation across the Iberian Peninsula. The sharpness of the Cannabis pollen peak, its accurate dating using annual varves and its almost exact coincidence with outstanding and widespread historical events suggest that this palynological landmark could be used as a chronostratigraphic marker for recent centuries. This possibility, as well as the geographical extent of this potential datum, should be confirmed with further studies.

P U R P L E and GOLD over THOUSANDS of YEARS
NATALIA POLOSMAK
HYPOTHESES AND FACTS Archaeology DISCOVERY OF SIBERIA
Science first hand : 30 Jan 2005 , Discovery of SIBERIA , volume 4, N1
https://scfh.ru/en/papers/purple-and...ands-of-years/
The first burial mounds with “frozen” tombs were discovered by V. V. Radlov (Katanda burial mound and a burial mound at the Berel burial site of the Pazyryk culture, Kazakhstan) in 1865. He was the first to find clothes preserved in ice. One of the most remarkable of his finds — the so-called Katanda caftan — is now exhibited at the State Historical Museum (Moscow). The collection of this museum has another unique find from the Katanda burial, a “tailcoat” of sable fur covered with Chinese silk (this garment is called a “tailcoat” because it is made in this style)

Recent palaeoenvironmental evidence for the processing of hemp (Cannabis sativa L.) in eastern England during the medieval period
Benjamin R. Gearey, M. Jane Bunting, Harry Kenward
Medieval Archaeology · January 2005
DOI: 10.1179/007660905X54125
Hemp (Cannabis sativa L.) — whose origins as a domesticated plant probably lie in C. Asia — has been cultivated in England since at least a.d. 800 (and before this perhaps in the Roman Period), mainly for its fibre, which was used to make sails, ropes, fishing nets and clothes, as well as for the oil from hempseed. Hemp cultivation may have reached a peak during the early 16th century, when Henry VIII decreed that increased hemp production was required to supply the expanding navy.33 Evidence for the locations where
the crop was cultivated and processed is available in several different forms, including written evidence in parish records and government reports, place-name evidence (e.g. Hempholme and some instances of Hempstead), and features on old maps, such as Hempisfield (hemp field).

Report of the Indian Hemp Drugs Commission, 1893-94: A Critical Review
Oriana Josseau Kalant
The International Journal of the Addictions, 7(1), pp. 77-96, 1972
https://sci-hub.tw/10.3109/10826087209026763
Despite various attempts by the British authorities to regulate the use of hemp drugs in India, the matter had been left largely in the hands of provincial governments until the end of the 19th century. Laws and practices therefore varied widely from one region to another. In response to questions in the British Parliament, a Commission was set up in 1893 to examine the situation in Bengal, but on the initiative of the Governor General the scope of the inquiry was broadened to include the whole of British India. The present review consists of a summary of those sections of the Report which are of most interest to present day readers, followed by a critical appraisal of the Report and of its relevance to modern issues connected with cannabis use

Results of molecular analysis of an archaeological hemp(Cannabis sativa L.) DNA sample from North West China
Ashutosh Mukherjee, Satyesh Chandra Roy, S. De Bera, Hong-En Jiang, Xiao Li, Cheng-Sen Li, Subir Bera.
Genetic Resources and Crop Evolution 55(4):481-485 · June 2008
DOI: 10.1007/s10722-008-9343-9
"Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C.sativa sample showed that C.sativa races might have been introduced into China from the European–Siberian center of diversity."


Sedimentary cannabinol tracks the history of hemp retting
Marlène Lavrieux, Jérémy Jacob, Jean-Robert Disnar, Jean-Gabriel Bréheret, Claude Le Milbeau,Yannick Miras, and Valérie Andrieu-Ponel
Geology 41(7):751-754 July 2013
doi:10.1130/G34073.1 |
Hemp (Cannabis sp.) has been a fundamental plant for the development of human societies. Its fibers have long been used for textiles and rope making, which requires prior stem retting. This process is essential for extracting fi bers from the stem of the plant, but can adversely affect the quality of surface waters. The history of human activities related to hemp (its domestication, spread, and processing) is frequently reconstructed from seeds and pollen detected in archaeological sites or in sedimentary archives, but this method does not always make it possible to ascertain whether retting took place. Hemp is also known to contain phytocannabinoids, a type of chemicals that is specific to the plant. Here we report on the detection of one of these chemicals, cannabinol (CBN), preserved in a sediment record from a lake in the French Massif Central covering the past 1800 yr. The presence of this molecule in the sedimentary record is related to retting. Analysis of the evolution of CBN concentrations shows that hemp retting was a significant activity in the area until ca. A.D. 1850. These findings, supported by pollen analyses and historical data, show that this novel sedimentary tracer can help to better constrain past impacts of human activities on the environment

Siberian Princess reveals her 2,500 year old tattoos
The Siberian Times
https://siberiantimes.com/culture/ot...r-old-tattoos/
The ancient mummy of a mysterious young woman, known as the Ukok Princess, is finally returning home to the Altai Republic this month.
She is to be kept in a special mausoleum at the Republican National Museum in capital Gorno-Altaisk, where eventually she will be displayed in a glass sarcophagus to tourists.
For the past 19 years, since her discovery, she was kept mainly at a scientific institute in Novosibirsk, apart from a period in Moscow when her remains were treated by the same scientists who preserve the body of Soviet founder Vladimir Lenin.
To mark the move 'home', The Siberian Times has obtained intricate drawings of her remarkable tattoos, and those of two men, possibly warriors, buried near her on the remote Ukok Plateau, now a UNESCO world cultural and natural heritage site, some 2,500 metres up in the Altai Mountains in a border region close to frontiers of Russia with Mongolia, China and Kazakhstan.
Reconstruction of a warrior's tattoos, who was discovered on the same plateau as the 'Princess'. All drawings of tattoos, here and below, were made by Elena Shumakova, Institute of Archeology and Ethnography, Siberian Branch of Russian Academy of Science
They are all believed to be Pazyryk people - a nomadic people described in the 5th century BC by the Greek historian Herodotus - and the colourful body artwork is seen as the best preserved and most elaborate ancient tattoos anywhere in the world

Some Historical Aspects of Marijuana.
Winek, C. L.
Clinical Toxicology, 10(2), 243–253.(1977).
doi:10.3109/1556365770898796
Marijuana, by whatever species of Cannabis it is called, has been known to science and medicine for almost 5000 years. It is reported to have been contained in the Chinese Emperor Nuna' s Herbal dated 2737 B.C. It was not given the name Cannabis sativa L. until Linnaeus named it in 1753 A.D. In searching some of the older Materia Medica texts and journals, one finds reference to the medicinal uses of the plant and its products. In the 1868 edition (3rd) of Materia Medica for the Use of Students (Published by Lindsay & Blakiston, Philadelphia) by John B. Biddle, M.D. of the Jefferson Medical College of Philadelphia the following is given on pp. 66-67:

Spiritual Benefit from Cannabis
Frederick J. Heide, Tai Chang, Natalie Porter, Eric Edelson, Joseph C. Walloch
Journal of Psychoactive Drugs
DOI: 10.1080/02791072.2021.1941443
Like many mind-altering plants, cannabis has been part of spiritual practices for thousands of years. It has deep roots in Hinduism, Islam, Rastafarianism, and indigenous traditions in Asia, Africa, and elsewhere. Yet almost no attention has been given to how contemporary adults employ it spiritually. A sample of 1087 participants (mean age = 38.9) completed an online survey assessing their use of cannabis and other substances, as well as spiritual and psychological characteristics. Spiritual benefit from cannabis was reported by 66.1% of the sample, and 5.5% reported it had sometimes been a spiritual hindrance. A MANOVA showed that those who reported spiritual benefit differed significantly from those who did not on several outcome variables, and a post hoc descriptive discriminant analysis revealed that expansiveness motivation, non-theistic daily spiritual experience, meditation frequency, and two mindfulness facets contributed most to differentiating the two groups. The majority of the sample (63%) was free of cannabis use disorder. Compared to disordered groups, the non-disordered group was significantly older and scored lower on experiential avoidance, psychological distress, and several motives for use. Results suggest that spiritual motives for cannabis use may be widespread. Implications for future research on spiritual use of cannabis are discussed.
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Taming Cannabis: Drugs and Empire in Nineteenth-Century France
David A. Guba
Book, Amazon
Despite having the highest rates of cannabis use in the continent, France enforces the most repressive laws against the drug in all of Europe. Perhaps surprisingly, France was once the epicentre of a global movement to medicalize cannabis, specifically hashish, in the treatment of disease. In Taming Cannabis David Guba examines how nineteenth-century French authorities routinely blamed hashish consumption, especially among Muslim North Africans, for behaviour deemed violent and threatening to the social order. This association of hashish with violence became the primary impetus for French pharmacists and physicians to tame the drug and deploy it in the homeopathic treatment of mental illness and epidemic disease during the 1830s and 1840s. Initially heralded as a wonder drug capable of curing insanity,
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The Ancient Cultivation of Hemp
H. Godwin
Antiquity Volume 41, Issue 161 March 1967 , pp. 42-49 doi:10.1017/S0003598X00038928
THE discovery in a long pollen diagram from East Anglia of a substantial curve for a pollen-grain referable to Cannabis sativa, L., the Indian hemp, raised the hope that we might, through palynology, have the means of tracing the history of cultivation of this important and sinister economic plant in England and in Western Europe. It was clearly essential that pollen-analytic evidence should be related fully to existing historical and archaeological knowledge, and aided by a notice in this journal (ANTIQUITY, 1964, 287), and by the notable kindness of a great many academic colleagues, I have put together a condensed historical account of the plant in antiquity as preface to a description of the pollen-analytic data.

The oldest archeological data evidencing the relationship of Homo sapiens with psychoactive plants: A worldwide overview
GIORGIO SAMORINI
Journal of Psychedelic Studies
DOI: 10.1556/2054.2019.008
Modern sophisticated archeometric instruments are increasingly capable of detecting the presence of psychoactive plant sources in archeological contexts, testifying the antiquity of humanity’s search for altered states of consciousness.
The purpose of this article is to provide a general picture of these findings, covering the main psychoactive plant sources of the world, and identifying the most ancient dates so far evidenced by archeology. This review is based on the archeological literature identifying the presence of psychoactive plant sources, relying on original research documents. The research produced two main results: (a) a systematization of the types of archeological evidence that testify the relationship between Homo sapiens and these psychoactive sources, subdivided into direct evidence (i.e., material findings, chemical, and genetic) and indirect evidence (i.e., anthropophysical, iconographic, literary, and paraphernalia); and (b) producing a list of the earliest known dates of the relationship of H. sapiens with the main psychoactive plant sources. There appears to be a general diffusion of the use of plant drugs from at least the Neolithic period (for the Old World) and the pre-Formative period (for the Americas). These dates should not to be understood as the first use of these materials, instead they refer to the oldest dates currently determined by either direct or indirect archeological evidence. Several of these dates are likely to be modified back in time by future excavations and finds

The Origin and Use of Cannabis in Eastern Asia
HuI-LIN LI
ECONOMIC BOTANY 28: 293-301. July-September, 1974.
DOI:10.1007/BF02861426
Cannabis sativa is one of man's oldest cultivated plants. Botanically it is distinct
from all other plants and readily recognized. Yet among individual plants it is
extremely variable. It now grows spontaneously in great abundance and ubiquity.
While most botanists consider the plant monotypic, some regard it as consisting of more than one species and a number of varieties, and so propose several different systems of classification. The systematics of this plant still awaits classification by further botanical studies. Cannabis is generally believed to be an Asiatic plant. There is no concerted agreement among botanists as to where the plant originally grew wild and where its cultivation first began. Estimates range within the wide span of temperate Asia from the Caucasus Mountains and the Caspian Sea through western and central Asia to eastern Asia. There is no easy way to distinguish between wild and spontaneous or adventitious, and semi-cultivated or cultivated plants. Therefore, much remains to be done in determining the geographical origin of the plant. These difficulties in classification and origin arise from the long and close association of Cannabis with man. Man has caused its extreme variations and wide dispersion. It will no longer suffice to study the plant itself alone. The influence of man must be considered side by side with the botanical facts in order to unveil the complex nature of this plant.ature of this plant.

The origins of cannabis smoking: Chemical residue evidence from the first millennium BCE in the Pamirs
Meng Ren, Zihua Tang, Xinhua Wu, Robert Spengler, Hongen Jiang, Yimin Yang, and Nicole Boivin
Science Advances 12 Jun 2019:
Vol. 5, no. 6, eaaw1391
DOI: 10.1126/sciadv.aaw1391
Cannabis is one of the oldest cultivated plants in East Asia, grown for grain and fiber as well as for recreational,
medical, and ritual purposes. It is one of the most widely used psychoactive drugs in the world today, but little is
known about its early psychoactive use or when plants under cultivation evolved the phenotypical trait of
increased specialized compound production. The archaeological evidence for ritualized consumption of cannabis
is limited and contentious. Here, we present some of the earliest directly dated and scientifically verified evidence
for ritual cannabis smoking. This phytochemical analysis indicates that cannabis plants were burned in wooden
braziers during mortuary ceremonies at the Jirzankal Cemetery (ca. 500 BCE) in the eastern Pamirs region. This
suggests cannabis was smoked as part of ritual and/or religious activities in western China by at least 2500 years
ago and that the cannabis plants produced high levels of psychoactive compounds.

*The Pharmacohistory of Cannabis Sativa
Raphael Mechoulam
In book: Cannabinoids as Therapeutic Agents June 2019
DOI: 10.1201/9780429260667-1
https://www.researchgate.net/publica...annabis_Sativa
Cannabis sativa L. was one of the first plants to be used by man for fiber, food, medicine, and in social and religious rituals. Several names for cannabis were used, mostly associated with the term azallu. The Scythians were a tribe of violent warriors who ruled the Crimea and, at different times, parts of southern Russia, the Balkans, Anatolia, and the Middle East around 700 B.C. The Middle Ages in Europe stayed their course, as regarded medical and not-somedical use of cannabis, till the 19th century. Cannabis was part of the religious lore of the Aryans, a nomad tribe, which invaded India from the north circa 2000 B.C. The Indians had a much better understanding of cannabis than the Europeans. In various parts of India cannabis was used for a large number of diseases and to improve the physical and mental states of the user. The use in leprosy in China deserves further investigation.

The Religious and Medicinal Uses of Cannabisin China, India and Tibet.
Touw, M.
Journal of Psychoactive Drugs, 13(1), 23–34.(1981).
doi:10.1080/02791072.1981.10471447
The earliest trace of cannabis use is an archaeological find of hemp textile in China dating fro m 4000 B.C. (Li & Lin 1974 ). Hemp thread and rope fro m 3000 B.C. have also been found in Chinese-occupied Turkestan (Fisher 19 75 ). The Rh-Ya (50 0 B.C., but pointing back many centuries earlier) mention s its use for fiber (Bouquet 1950), as do the Shih-Ching (l Oth-7th centuries B.C.), the Li-Chi (l 00 B.C.) an d the Chou Li (c. 200 H.C.) (Li & Lin 1974). A grain crop was obtained fro m the achene as well, according to the latter three classics ( Li & Lin 1974 ), though the earliest archaeological evidence of this use found to date is fro m the Han Dynasty (206 B.C.-220 A.D.). Cannabis grain was not merely an auxiliary crop, for in ancient times it was counted as one of t he "five grains," together with rice, barley, millet and soy beans (Li 197 5). To this day a large seeded variety of hemp grows in the far northeast of China, which may well be a relict of its use for grain. Alt ho ugh cannabis ceased to be an important food in China just before the beginning of the Christian era due to the introduction of new crops (Keng 1974), it is still a source of cooking oil and grain in parts of Nepal.

‘Tis in our nature: taking the human-cannabis relationship seriously in health science and public policy.
Aggarwal, S. K.
Frontiers in Psychiatry, 4. (2013).
doi:10.3389/fpsyt.2013.00006
To find clearheaded scientific perspective on cannabis use through the prevailing thick smokescreen requires recognizing just what sort of smoke obscures our better understanding. In the United States, in large part, the smokescreen is made up of culture war-charged political rhetoric and obstructionism from those in positions of authority setting up a prejudicial ideological framing for cannabis use. National leaders throughout the twentieth century have taken opportunities afforded by high office or its pursuit to publicly opine on the dangers of cannabis, such as when then-Presidential candidate Ronald Reagan famously stated in 1980 that “leading medical researchers are coming to the conclusion that marijuana, pot, grass, whatever you want to call it, is probably the most dangerous drug in the United States and we haven’t begun to find out all of the ill-effects. But they are permanent ill-effects. The loss of memory, for example Grass (1999).” Not only is such rhetoric overly simplistic, it also obscures and distorts pre-existing facts. In this particular case, Reagan’s statement obscures the fact that the American Medical Association testified in 1937 on record to Congress that, after nearly 100 years of professional experience in Western medical practice with over 2000 prescribable marketed cannabis preparations (Antique Cannabis Museum, 2012), practitioners found that cannabis had an irreplaceable therapeutic role as an aid in the remembering of old and long-forgotten memories in psychotherapy patients (U.S. Congress, 1937). When in office, Reagan’s first drug czar, Carlton Turner, blamed cannabis use for young people’s involvement in “anti-military, anti-nuclear power, anti-big business, anti-authority demonstrations” (Schlosser, 1997), all dissenting positions toward government initiatives. Such clear scapegoating rhetoric has roots in the government’s racialized Reefer Madness campaign of the 1930s which linked cannabis use in Blacks, Latinos, jazz musicians, and juvenile delinquents to racial miscegenation and homicidal mania

Upper Palaeolithic fibre technology: interlaced woven finds from Pavlov I, Czech Republic, c. 26,000 years ago.
Adovasio, J. M., Soffer, O., & Klíma, B.
Antiquity, 70(269), 526–534.(1996).
doi:10.1017/s0003598x0008368x
The later Palaeolithic sites of Moravia, the region of the Czech Republic west of Prague and north of Vienna, continue to provide remarkable new materials. To the art mobilier for which Dolni V6stonice and Pavlov have been celebrated, there has recently been added the technologies of groundstone and ceramics - and now woven materials, interlaced basketry or textiles, again of a kind one expects only from a quite later era.

The Question of Cannabis: Cannabis Bibliography
Written by United Nations. Commission on Narcotic Drugs 1965
https://books.google.es/books?id=xXr...page&q&f=false
1860 article BIB

If any articles fit into more than one subject I will just post it in both, in this case I will put it in Classification/Taxonomy but if people also think it should go in others I can do that, I have maybe 1,000+ papers to sort and categorize by subject so I am trying to keep it easy for my self. If anyone has suggestions for additional subjects on any specific paper please tell me. ANY NEWLY ADDED PAPER I WILL POST THE TITLE IN RED FOR AT LEAST 3 MONTHS TO MAKE IT EASIER TO SEE WHAT HAS RECENTLY BEEN ADDED TO THE BIBLIOGRAPHY
 
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clearheaded

Active member
wonderful idea sam! thanks for doing this! perhaps a "best of" or "sam select" top 3 or 5 in each category? or perhaps just overall must read etc.


I did add a red * to the best 5 in each subject-SamS
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Breeding Cannabis

A Belated Green Revolution for Cannabis: Virtual Genetic Resources to Fast-Track Cultivar Development
Matthew T. Welling, Tim Shapter, Terry J. Rose, Lei Liu, Rhia Stanger, and Graham J. King,
July 2016Frontiers in Plant Science 7
DOI: 10.3389/fpls.2016.01113
Cannabis is a predominantly diecious phenotypically diverse domesticated genus with few if any extant natural populations. International narcotics conventions and associated legislation have constrained the establishment, characterization, and use of Cannabis genetic resource collections. This has resulted in the underutilization of genepool variability in cultivar development and has limited the inclusion of secondary genepools associated with genetic improvement strategies of the Green Revolution. The structured screening of ex situ germplasm and the exploitation of locally-adapted intraspecific traits is expected to facilitate the genetic improvement of Cannabis. However, limited attempts have been made to establish the full extent of genetic resources available for pre-breeding. We present a thorough critical review of Cannabis ex situ genetic resources, and discuss recommendations for conservation, pre-breeding characterization, and genetic analysis that will underpin future cultivar development. We consider East Asian germplasm to be a priority for conservation based on the prolonged historical cultivation of Cannabis in this region over a range of latitudes, along with the apparent high levels of genetic diversity and relatively low representation in published genetic resource collections. Seed cryopreservation could improve conservation by reducing hybridization and genetic drift that may occur during Cannabis germplasm regeneration. Given the unique legal status of Cannabis, we propose the establishment of a global virtual core collection based on the collation of consistent and comprehensive provenance meta-data and the adoption of high-throughput DNA sequencing technologies. This would enable representative core collections to be used for systematic phenotyping, and so underpin breeding strategies for the genetic improvement of Cannabis.

A chemotaxonomic analysis of terpenoid variation in Cannabis
Karl W. Hillig
Biochemical Systematics and Ecology 32 (2004) 875–891
doi:10.1016/j.bse.2004.04.004
To determine whether the terpenoid composition of the essential oil of Cannabis is useful for chemotaxonomic discrimination, extracts of pistillate inflorescences of 162 greenhouse grown plants of diverse origin were analyzed by gas chromatography. Peak area ratios of 48 compounds were subjected to multivariate analysis and the results interpreted with respect to
geographic origin and taxonomic affiliation. A canonical analysis in which the plants were pre-assigned to C. sativa or C. indica based on previous genetic, morphological, and chemotaxonomic studies resulted in 91% correct assignment of the plants to their pre-assigned species. A scatterplot on the first two principal component axes shows that plants of accessions from Afghanistan assigned to the wide-leaflet drug biotype (an infraspecific taxon of unspecified rank) of C. indica group apart from the other putative taxa. The essential oil of these plants usually had relatively high ratios of guaiol, isomers of eudesmol, and other unidentified compounds. Plants assigned to the narrow-leaflet drug biotype of C. indica tended to have relatively high ratios of trans-b-farnesene. Cultivars of the two drug biotypes may exhibit distinctive medicinal properties due to significant differences in terpenoid composition.

A new technology for predicting the fiber content in hemp bast
Deng Gang, Ding Ming liang, Cheng Xia, Liu Fei Hu
PeerJ Preprints (2017)
doi.: 10.7287/peerj.preprints.3458v1
Hemp (Cannabis sativa L.) is a traditional fiber crop, which is becoming one of the most important industrial fibers, with a promising future in many fields. To accelerate the breeding of hemp cultivars with increased fiber content, it is important to establish a preflowering method that can be used to predict the potential fiber content in hemp bast. This study investigated the correlation between fiber content in the stem bast portion and in the entire stem. In addition, the variation in the bast fiber content during the sampling period was studied in three hemp cultivars. It was clear that the bast fiber content in hemp stems was determined up to 40 d before emergence of the staminate buds. The fiber content of the bast (in a sample piece 30cm long and covering one-quarter of the stem girth, sampled at two-fifths of the plant height from the soil, 20 d before the staminate buds emerged) was shown to be representative of the fiber content of the entire stem. In conclusion, this new method would allow breeders to select the hemp plants for high bast fiber content during the early to middle growth periods, before the male buds emerged, potentially accelerating the genetic improvement of fiber content in industrial hemp

Not Cannabis specific
*A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce
Charles J. Underwood, Kitty Vijverberg, Diana Rigola, Shunsuke Okamoto, Carla Oplaat, Rik H. M. Op den Camp, Tatyana Radoeva, Stephen E. Schauer, Joke Fierens, Kim Jansen, Sandra Mansveld, Marco Busscher, Wei Xiong, Erwin Datema, Koen Nijbroek, Evert-Jan Blom, Ross Bicknell, Andrew Catanach, Sylvia Erasmuson, Christopher Winefield, Arjen J. van Tunen, Marcel Prins, M. Eric Schranz & Peter J. van Dijk
Nature Genetics (2022)
https://www.nature.com/articles/s41588-021-00984-y
Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelionPAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.
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Agricultural innovations in Morocco’s cannabis industry.
Chouvy, P.-A., & Macfarlane, J.
International Journal of Drug Policy, 58, 85–91.(2018).
doi:10.1016/j.drugpo.2018.04.013
Background: Cannabis cultivation in Morocco’s mountainous Rif region is undergoing its most profound development since mass production of hashish began in the early 1980s. The adoption of high-yielding varieties of cannabis, modern agricultural practices, and modern hashish production techniques began in the mid–2000s and accelerated after the mid-2010s, with the result that more potent and varied cannabis derivatives are now being produced and that increased quantities of highly potent hashish are seized in Europe. Methods: This research was initiated to answer a simple research question: how, and to what extent, is the illegal cannabis industry modernizing in Morocco, now that we know that it explains the THC increase in hashish seized in Europe? To answer this question in the context of a lack of literature and quantitative data, empirical fieldwork was undertaken in cannabis fields and hashish-producing farms in Morocco in July and October 2017. A mostly qualitative approach to data collection was employed through participatory rural appraisals (discussions, interviews, direct observations). As such, this work builds predominantly upon primary research. Results: Fieldwork showed that, subsequent to very localized, experimental beginnings in the early 2000s, the progressive and varied adoption of agricultural innovations at the cultivation and production stages has spread throughout the Rif during the 2010s. Interviews and direct observations conducted in the field indicate that the ongoing adoption of modern farming techniques has enabled the production of high-quality hashish and potent modern extracts. The still ongoing modernization and professionalization of the Moroccan cannabis industry is a testimony of the country’s leading position in global hashish production. Conclusion: What the future holds for Moroccan cannabis growers is difficult to predict. How legalization processes manifest themselves in Moroccan and European policies, and how upcoming developments will affect the social, economic, political and ecological stability of the region, remains largely unknown. However, the spread of cannabis cultivation in the Rif is clearly pushing

Not Cannabis specific
*Apoptosis gene EI24, compositions, and methods of use
https://patents.google.com/patent/US5843659A/en
https://patentimages.storage.googleapi s.com/72/63/11/675686448e1963/US5843659.pdf
Disclosed is the isolation and characterization of EI24, a novel gene whose 2.4 kb mRNA is induced following etoposide treatment. Induction of EI24 mRNA by etoposide required expression of wild-type p53. Overexpression of functional p53 was sufficient to induce expression of the EI24 mRNA. The EI24 mRNA was also induced in a p53-dependent manner by ionizing irradiation of primary murine thymocytes. The invention is thus directed to an isolated EI24 protein, nucleotide sequences coding for and regulating expression of the protein, antibodies directed against the protein, and recombinant vectors and host cells containing the genetic sequences coding for and regulating the expression of the protein sequence. The invention is also directed to genomic DNA, cDNA, and RNA encoding the EI24 protein sequence and to corresponding antisense RNA sequences. Antibodies can be used to detect EI24 in biological specimens, including, for example, human tissue samples. The present invention is further directed to methods of treating degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death. The present invention is further directed to methods for diagnosing degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death, as well as methods for monitoring the progress of such degenerative disorders.

Not Cannabis Specific
Artificially induced polyploidization in Humulus lupulus L. and its effect on morphological and chemical traits
Anna Trojak-Goluch and Urszula Skomra
Breeding Science 63: 393–399 (2013)
doi:10.1270/jsbbs.63.393
https://www.jstage.jst.go.jp/article.../_pdf/-char/en
Chemically induced polyploids were obtained by the colchicine treatment of shoot tips of Humulus lupulus L. ‘Sybilla’. Flow cytometry revealed that most of the treatments resulted in the production of tetraploids. The highest number of tetraploids was obtained when explants were immersed in 0.05% colchicine for 48 h. A field experiment was conducted to compare diploid and tetraploid plants and assess the effect of genome polyploidization on the morphological and chemical characteristics. Tetraploids showed significant differences in relation to diploids. They had thinner and shorter shoots. The influence of chromosome doubling was also reflected in the length, width and area of leaves. The length of female flowers in the tetraploids was significantly shorter than that observed in diploids. Tetraploids produced a diverse number of lupuline glands that were almost twice as large as those observed in diploids. The most distinct effect of genome polyploidization was a significant increase in the weight of cones and spindles. Contents of major chemical constituents of hop cones was little affected by ploidy level. Total essential oils were significantly lower than those in diploids. However there was a significant increase in the proportion of humulene, caryophyllene and farnesene, oils desired by the brewing industry.

Not Cannabis Specific
BREEDING AND GENETICS Development of an Improved Method of Mitotic Metaphase Chromosome Preparation Compatible for Fluorescence in situ Hybridization in Cotton
Ryan J. Andres and Vasu Kuraparthy
The Journal of Cotton Science 17:149–156 (2013)
https://www.researchgate.net/publica...tion_in_Cotton
Molecular cytogenetic techniques, especially Fluorescence In situ Hybridization (FISH) and Genomic In situ Hybridization (GISH), are excellent tools to study the structure and function of chromosomes, polyploidy, aneuploidy, alien gene introgression and genome evolution and physical mapping of genes. For many applications, in situ hybridizations require reliable and efficient methods of chromosome preparation with well preserved and dispersed chromo-somes and little or no cell wall debris. However, such protocols have not yet been published for cotton mitotic chromosome preparations. In the current study, an improved method for preparing mitotic metaphase chromosomes of tetraploid cotton was developed. Root tips were collected from lab-grown three-day-old cotton seedlings and pre-treated with high-pressure nitrous oxide (N 2 O). Following fixation with acetic acid, root tip meristems were removed, enzymatically digested, gently macerated, and then processed to create suspensions of protoplasts in acetic acid and methanol. Suspended protoplasts were then dropped onto a glass slide in order to distribute the protoplasts and spread the chromosomes. Using this approach, acceptable mitotic indexes and high-quality chromosomal spreads with no cell wall debris were obtained. This drop method of preparing chromosome spreads was tested for its compatibility for in situ hybridization using rDNA as probe in FISH. Additionally, varying pre-treatment times with N 2 O were investigated. Our results show that the combination of a 95-minute N 2 O pre-treatment, a 55-minute enzymatic degradation, and the "drop method" efficiently produce cotton mitotic metaphase

Breeding Distinct Industrial Hemp Varieties for the 21st Century
Seth Crawford, Ph.D.
https://www.youtube.com/watch?v=lNDP...z0DPbFWwlMJDqk
Industrial scale production of non-psychoactive cannabinoids began in earnest in the US in 2015 under Section 7606 of the 2014 Farm Bill. Seed for planting was initially scarce due to DEA prohibitions on importation and a lack of natively developed cultivars. Our company began identifying specific breeding strategies to produce agronomically advanced cultivars of hybrid cannabis that still remain federally compliant as “industrial hemp”. Since 2016, these breeding programs have led to the creation of the first (a) high content type III (CBD) day neutral varieties (“autoflowering”), (b) predictably “early” flowering type III photoperiod varieties, (c) high content type IV (CBG) dominant day neutral varieties, and (d) predictably “early” flowering type IV photoperiod varieties. In each circumstance, the varieties developed are 100% female (verified using Medicinal Genomics’ youPCR colorimetric screen), with only 1 in 4000 plants sporting a male phenotype in the field. In 2018, our seed was used to plant over 4000 acres across the US. Our most recent experimental results demonstrate that pure type IV (CBG) varieties can produce CBG to THC ratios of over 300:1, which enables raw oil extractions below the federal 0.3% THC requirement.

Breeding of industrial hemp with a high content of cannabigerol by the case of ‘Vik 2020’ cultivar Plant Varieties Studying and Protection 17(2):105-112 (2021) DOI: 10.21498/2518-1017.17.2.2021.236514
https://www.researchgate.net/publica...'_cultivar
To create an industrial hemp variety of the Central European ecological and geographical type with a high cannabigerol content and universal application. Methods. Breeding (self-pollination, creation of artificial populations, selection), field, biochemical (thin-layer and gas-liquid chromatography of cannabinoid compounds), instrumental and technological assessment of fibre quality, and statistical methods. Results. Variety ‘Vik 2020’ was obtained as a result of creation of artificial populations. The plants are characterized by higher content of cannabigerol (1.034 ± 0.0323%), and almost zero of other secondary metabolites, such as cannabidivarin, cannabidiol, cannabichromene and psychotropic tetrahydrocannabinol (0.003 ± 0.0011; 0.018 ± 0.0080; 0.012 ± 0.0027, and 0.005 ± 0.0012%, respectively). The t rait of cannabigerol content is quite stable within the population and is not correlated with the trait of tetrahydrocannabinol content (r = -0.23). TLC showed that cannabigerol accumulated mainly in the form of cannabigerolic acid and to a lesser extent as a neutral compound, which is consistent with the theory that this substance is a precursor for the synthesis of other cannabinoids. According to the results of the competitive variety test, when growing to obtain fibre and seeds, the variety features short height, specifically significantly lower total (206.4 cm) and technical stem length (135.6 cm) compared to the standard variety, significantly higher inflorescence length (70.8 cm), which determine the formation of the significant yield of biomass suitable for pharmaceutical use and high seed yield (0.98 t/ha). The yield of total fibre was the same as in the standard variety (29.0%), but its quality and technological value for primary processing were higher. The variety had a homogeneous sex structure, resistance to bioltic and abiotic environmental factors. Plants reached biological maturity in 116 days (BBCH 89). This cultivar is recommended for obtaining seeds, quality fiber and potentially cannabigerol (on condition of changes in legislation). Conclusions. The efficiency of using self-pollinating lines in breeding with their subsequent combining into a synthetic population and improving selection was proved by the case of a new variety of industrial hemp ‘Vik 2020’, characterized by an increased content of cannabigerol and the absence of psychotropic properties

Not Cannabis specific
Breeding of triploid common hop cultivars (Humulus lupulus L.)
Anna Trojak-Goluch, Urszula Skomra
Polish Journal of Agronomy 2018, 34, 3–10
doi: 10.26114/pja.iung.357.2018.34.01
Genome polyploidisation plays a special role in the progress of crop improvement in agriculture. Duplication of the entire genome is associated with significant phenotypic changes in plants, which most often lead to an increase in production at an unchanged level of input. Triploid hop genotypes are distinguished from diploids by their higher yielding potential, increased alpha-acid content and absence of seeds. For this reason,
triploid hop cones are an extremely useful raw material for the brewing industry. Studies on the polyploidisation of hop genomes were initiated by Dark in 1948. In the 1950s, American researchers Neve and Farrar made an important contribution to hop triploid breeding. A significant improvement in yield per unit area and in the quality of hop raw material was brought about by the release of aromatic triploid cultivars: Willamette and Columbia to hop farmers by Haunold et al. in 1977. The development of a method for the induction of tetraploid hops using colchicine in in vitro cultures has resulted in a number of valuable high alpha as well as aromatic triploid hop cultivars being obtained in New Zealand. As a result of the breeding work carried out in Slovenia in the 1990s, an array of triploid cultivars was obtained, the introduction of which resulted in a significant increase in the cultivation area of aromatic cultivars in this country. Currently, breeding work aimed at obtaining super alpha and aromatic triploid hop cultivars is being carried out in Poland at the Institute of Soil Science and Plant Cultivation – State Research Institute.

Not Cannabis specific
Breeding Triploid Plants: A Review
Wang, X., Cheng, Z.-M., Zhi, S., & Xu, F.
Czech Journal of Genetics and Plant Breeding, 52(No. 2), 41–54. 2016
doi:10.17221/151/2015-cjgpb
Triploid plants have larger organs, greater biomass, and strong stress resistance by preserving relatively larger amounts of photosynthetic energy. The undesirable spread of non-native invasive crop and horticultural plants into natural areas can also be reduced or eliminated by the use of triploids, because they tend to be sterile and seedless. Triploid plants have great economic value and have been useful for developing new agronomic, horticultural, and forestry plant varieties. Because of rapid advances in DNA sequencing technology, triploids may become a focus of genomic research in the future, and will create unprecedented opportunities for discovering and monitoring genomic and transcriptomic changes in unbalanced genomes. One of the new trends in genomics research is to create synthetic triploid plants as materials for the study of first genomic responses that occur immediately after triploid formation. Here, we summarize recent progress in the use of triploid plants, approaches for obtaining triploid plants, including natural selection, artificial hybridization, and endosperm regeneration, the obstacles to obtain triploids, and possible ways to overcome these difficulties. This summary of the scientific progress on triploid plants will promote understanding of how they can be generated and assist plant breeders to design new strategies for triploid breeding.

BreedIT
https://ibreedit.com/solutions/medic...abis-breeders/
https://ibreedit.com/contact-us-2-2/
By using our proven iBreedIT® IDSS (Intelligent Decision Support System) technology to improve Cannabis strains and to breed new varieties of Cannabis, we believe that BreedIT is perfectly aligned to address the world’s increasing need for better and more efficient commercial medical varieties of the plant.
Our technology enables more effective planning, management, and analysis of cannabis breeding data so that ideally breeders can produce crops in a more exacting manner, with measureable ingredients that are more consistent from one plant to the next. In this manner, the quantity of inactive or even undesired ingredients in the plant may be reduced. This consistency should allow us to create finely tuned cannabis crops for medicinal purposes. It is for this reason we believe that our technology may assist us to become a world leader in cannabis breeding.
The full medical benefits and potential uses of medical Cannabis are only starting to be scientifically examined in recent years. The full potential uses of different varieties and traits are an exciting new field for seed breeders around the world. By utilizing proven methodologies available in Cannabinoids Level and aroma,
Trichome Type,
Resin Quantity, Quality and adaptability.
They have a Medical Cannabis Brochure “The art of Breeding for Cannabis”

Cannabinoid Inheritance Relies on Complex Genetic Architecture.
Campbell, L. G., Dufresne, J., & Sabatinos, S. A.
Cannabis and Cannabinoid Research.(2019).
doi:10.1089/can.2018.0015
Introduction: Understanding the inheritance of cannabinoid compounds in Cannabis sativa will facilitate effective crop breeding and careful regulation of controlled substances. The production of two key cannabinoids, D-9- tetrahydrocannabinol (THC) and cannabidiol (CBD), is partially controlled by two additive loci. Here, we present the first study to search for evidence of alternate genetic models describing the inheritance and expression of cannabinoids. Materials and Methods: Using an information-theoretic approach, we estimated composite genetic effects (CGEs) of four cultivars with pure CBD or pure THC chemotypes, their F1 and F2 hybrid progeny, to identify genetic models that explain cannabinoid inheritance patterns. We also estimated the effective number of genetic factors that control differences in cannabinoid concentration (THC, CBD, and cannabichromene [CBC]). Results: Unlike previous research, we note nonadditive components of cannabinoid inheritance. Concentration of THC is a polygenic trait (three to four genetic factors). Both additive and dominance CGEs best explained THC expression patterns. In contrast, cytoplasmic genomes and additive genes may influence CBD concentration. Maternal additive effects and additive genetic effects apparently influence CBC expression. Conclusions: Cannabinoid inheritance is more complex than previously appreciated; among other genetic effects, cytogenetic and maternal contributions may be undervalued influences on cannabinoid ratios and concentrations. Further research on the environmental sensitivity of cannabinoid production is advised.

Cannabinoids vs. whole metabolome: Relevance of cannabinomics in analyzing Cannabis varieties
Pedro G. Vasquez-Ocmín, Guillaume Marti, Maxime Bonhomme, Fabienne Mathis, Sylvie Fournier, Stephane Bertani, Alexandre Maciuk Analytica Chimica Acta 1184 (2021) 339020 DOI: 10.1016/j.aca.2021.339020
https://www.researchgate.net/publica...abis_varieties
Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with D9 -THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.

Cannabis Breeder’s Rights ver 2.0.1
https://www.academia.edu/38353720/Ca..._ver_2.0.1.pdf
A licensing framework for cannabis and hemp breeders similar to Creative Commons for software and media. Twenty-two license designations describe propagation, sales, and attribute conditions. This schema was suggested by DJ Short as a means of conveying a breeder’s intentions for the genetics they release.

*Cannabis Domestication, Breeding History, Present-day Genetic Diversity, and Future Prospects
Robert C. Clarke & Mark D. Merlin
Critical Reviews in Plant Sciences Pages 1-35
Doi: 10.1080/07352689.2016.1267498
Humans and the Cannabis plant share an intimate history spanning millennia. Humans spread Cannabis from its Eurasian homelands throughout much of the world, and, in concert with local climatic and human cultural parameters, created traditional landrace varieties (cultivars resulting from a combination of natural and farmer selection) with few apparent signs of domestication. Cannabis breeders combined populations from widely divergent geographical regions and gene pools to develop economically valuable fiber, seed, and drug cultivars, and several approaches were used with varying results. The widespread use of single plant selections in cultivar breeding, inbreeding, and the adoption of asexual reproduction for commercial drug production, reduced genetic diversity and made many present-day cultivars susceptible to pathogens and pests. The great majority of drug Cannabis cultivars are now completely domesticated, and thus are entirely dependent on humans for their survival. Future ramifications remain to be realized.

Cannabis Endophytes and Their Application in Breeding and Physiological Fitness
Parijat Kusari, Souvik Kusari, Michael Spiteller, Oliver Kayser
Chapter 20 Pg 419 in Book “Cannabis sativa L. - Botany and Biotechnology”
DOI: 10.1007/978-3-319-54564-6_20
Plant-associated endophytes live in mutualistic association with their hosts wherein a plethora of physiological, chemical, and molecular interactions are responsible maintaining their association. In this chapter, we explore the multifaceted potential of endophytes harbored in Cannabis sativa L. plants in interacting not only with the host plants, but also with invading pathogens and associated endophytic microflora, resulting in diverse functional traits. These traits range from production of bioactive natural products, attenuation of virulence factors of invading phytopathogens, to providing host plant fitness and maintaining ecological interactions. We further elaborate the ecological relevance of endophytes harbored in the liverwort, Radula marginata that produces secondary metabolites structurally similar to those found in Cannabis plants. Until now, research on endophytic microflora of C. sativa prospected from different ecosystems has yielded interesting fundamental insights into plant-microbe and microbe-microbe interactions, which have direct or indirect biotechnological implications. Therefore, we discuss the possible benefits of using Cannabis and Radula endophytes in the pharmaceutical and agricultural sectors, and the biotechnological approaches required to commercialize selected endophytes

Editorial: Cannabis Genomics, Breeding and Production
Rachel G. Backer, Giuseppe Mandolino, Olivia Wilkins, Mahmoud A. ElSohly and Donald L. Smith
Front. Plant Sci. (2020)
doi: 10.3389/fpls.2020.591445
Cannabis sativa was illegal during most of the 20 th century, but has recently been decriminalized or even legalized in some jurisdictions. During the same period, scientific tools were developed, giving us unprecedented insights into how plants grow, evolve, interact with their environment, and synthesize metabolites. However, because cannabis was largely illegal as these advances were made, this plant has been woefully understudied, and continues to hold many mysteries. To move forward, and bring the benefits of cannabis to the forefront, the legal landscape must be streamlined to allow for efficient scientific investigation.The legal classification of cannabis and hemp in the United States and around the world is rapidly evolving which means there are ever-changing obstacles for producers and researchers alike. For example, there is confusion of whether cannabis state laws are superseded by federal law, a variety of factors that determine the extent of enforcement related to state-authorized cannabis activities, and questions surrounding the legality and approval process for CBD-based products. Also in Europe the relations between EU regulations and controls, and the attitude of national legislations toward cannabis is not without contradictions.

Cannabis plants having modified expression of thca synthase
https://patents.google.com/patent/WO2016189384A1/en
The invention relates to novel genetically modified plants and methods or materials, such as polynucleotides, expression cassettes, or vectors for producing the same. Moreover, the invention relates to altering the content of cannabinoids in plants and to medical compositions derived from such plants. In particular embodiments, the present invention relates to cannabis plants having modified expression of tetrahydrocannabinolic acid (THCA) synthase and methods of modifying the amount of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in cannabis by modifying expression of THCA synthase.

Cannabis sativa L. - Botany and Biotechnology
Suman Chandra, Hemant Lata, Mahmoud A. ElSohly
book 978-3-319-54563-9
ebook 978-3-319-54564-6 10.1007/978-3-319-54564-6
This book highlights current Cannabis research: its botany, authentication, biotechnology, in vitro propagation, chemistry, cannabinoids biosynthesis, metabolomics, genomics, biomass production, quality control, and pharmacology.
Cannabis sativa L. (Family: Cannabaceae) is one of the oldest sources of fiber, food and medicine. This plant has been of interest to researchers, general public and media not only due to its medicinal properties but also the controversy surrounding its illicit use. Cannabis has a long history of medicinal use in the Middle East and Asia, being first introduced as a medicine in Western Europe in the early 19th century. Due to its numerous natural constituents, Cannabis is considered a chemically complex species. It contains a unique class of terpeno-phenolic compounds (cannabinoids or phytocannabinoids), which have been extensively studied since the discovery of the chemical structure of tetrahydrocannabinol (?9-THC), commonly known as THC, the main constituent responsible for the plant’s psychoactive effects. An additionally important cannabinoid of current interest is Cannabidiol (CBD). There has been a significant interest in CBD and CBD oil (extract of CBD rich Cannabis) over the last few years because of its reported activity as an antiepileptic agent, particularly its potential use in the treatment of intractable epilepsy in children.

*Characteristics of the Diploid, Triploid, and Tetraploid Versions of a Cannabigerol-Dominant F1 Hybrid Industrial Hemp Cultivar, Cannabis sativa ‘Stem Cell CBG’
Seth Crawford, Brendan M. Rojas, Eric Crawford, Matthew Otten, Thecla A. Schoenenberger,
Andrea R. Garfinkel and Hsuan Chen
Genes 2021, 12, 923.
doi: 10.3390/genes12060923
https://www.mdpi.com/2073-4425/12/6/923/htm
Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa
crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, ‘TS1-3’ and ‘P163’, to produce the tetraploids ‘TS1-3 (4x)’ and ‘P163 (4x)’. The diploid, triploid, and tetraploid F1 hybrids from ‘TS1-3’ _ ‘P163’, ‘TS1-3 (4x)’ _ ‘P163’, and ‘TS1-3 (4x)’ _ ‘P163 (4x)’ were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture.

Not Cannabis specific
Chromosome engineering: power tools for plant genetics.
Chan, S. W. L.
Trends in Biotechnology, 28(12), 605–610. (2010).
doi:10.1016/j.tibtech.2010.09.002
The term ‘‘chromosome engineering’’ describes technologies in which chromosomes are manipulated to change their mode of genetic inheritance. This review examines recent innovations in chromosome engineering that promise to greatly increase the efficiency of plant breeding. Haploid Arabidopsis thaliana have been produced by altering the kinetochore protein CENH3, yielding instant homozygous lines. Haploid production will facilitate reverse breeding, a method that downregulates recombination to ensure progeny contain intact parental chromosomes. Another chromosome engineering success is the conversion of meiosis into mitosis, which produces diploid gametes that are clones of the parent plant. This is a key step in apomixis (asexual reproduction through seeds) and could help to preserve hybrid vigor in the future. New homologous recombination methods in plants will potentiate many chromosome engineering applications.

Cold acclimation induces distinctive changes in the chromatin state and transcript levels of COR genes in Cannabis sativa varieties with contrasting cold acclimation capacities
Boris F. Mayera, Mohamed Ali Ali-Benalia, Jordan Demonea, Annick Bertrandb and Jean-Benoit Charrona
Published in Physiologia plantarum 2015
DOI:10.1111/ppl.12318
Little is known about the capacity of Cannabis sativa to cold acclimate and develop freezing tolerance. This study investigates the cold acclimation capacity of nine C. sativa varieties and the underlying genetic and epigenetic responses. The varieties were divided into three groups based on their contrasting cold acclimation capacities by comparing the survival of non-acclimated and coldacclimated plants in whole-plant freeze tests. In response to the cold acclimation treatment, all varieties accumulated soluble sugars but only the varieties with superior capacity for cold acclimation could maintain higher levels throughout the treatment. In addition, the varieties that acclimated the most efficiently accumulated higher transcript levels of COR genes and genes involved in de novo DNA methylation while displaying locus- and variety-specific changes in the levels of H3K9ac, H3K27me3 and methylcytosine during cold acclimation. Furthermore, these hardy C. sativa varieties displayed significant increases in methylcytosine levels at COR gene loci when deacclimated, suggesting a role for locus-specific DNA methylation in deacclimation. This study uncovers the molecular mechanisms underlying cold acclimation in Cannabis sativa and reveals higher levels of complexity regarding how genetic, epigenetic, and environmental factors intertwine.

Complex Patterns of Cannabinoid Alkyl Side-Chain Inheritance in Cannabis
Welling, M. T., Liu, L., Raymond, C. A., Kretzschmar, T., Ansari, O., & King, G. J.
Scientific Reports, 9(1). (2019)
doi:10.1038/s41598-019-47812-2
The cannabinoid alkyl side-chain represents an important pharmacophore, where genetic targeting of alkyl homologs has the potential to provide enhanced forms of Cannabis for biopharmaceutical manufacture. Delta(9)-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase genes govern dicyclic (CBDA) and tricyclic (THCA) cannabinoid composition. However, the inheritance of alkyl side-chain length has not been resolved, and few studies have investigated the contributions and interactions between cannabinoid synthesis pathway loci. To examine the inheritance of chemical phenotype (chemotype), THCAS and CBDAS genotypes were scored and alkyl cannabinoid segregation analysed in 210 F2 progeny derived from a cross between two Cannabis chemotypes divergent for alkyl and cyclic cannabinoids. Inheritance patterns of F2 progeny were non-Gaussian and deviated from Mendelian expectations. However, discrete alkyl cannabinoid segregation patterns consistent with digenic as well as epistatic modes of inheritance were observed among F2 THCAS and CBDAS genotypes. These results suggest linkage between cannabinoid pathway loci and highlight the need for further detailed characterisation of cannabinoid inheritance to facilitate metabolic engineering of chemically elite germplasm.

*CROSS-HYBRIDIZATION OF DISTINCT HOMOZYGOUS CANNABIS PLANTS TO PRODUCE CONSISTENT EARLY FLOWERING SEEDS
Patent Application (Application #20190297821)
Seth S. Crawford, Eric B. Crawford
https://patents.justia.com/patent/20190297821
A method for developing seeds for consistent early flowering short-day Cannabis plants, the method including crossbreeding a short-day Cannabis plant with a day-neutral Cannabis plant, and collecting the seeds produced through crossbreeding.

Cultivars breeding and production of non-psychoactive medicinal cannabis with high CBD content.
[Article in Chinese]
Zhang JQ1, Chen SL1, Wei GF1, Ning K1, Wang CQ1, Wang L2, Chen H2, Dong LL1.
Zhongguo Zhong Yao Za Zhi. 2019 Nov;44(21):4772-4780.
doi: 10.19540/j.cnki.cjcmm.20191009.101.
The shortage of new cannabis varieties with low THC and high CBD content and irregular planting techniques have become the bottleneck for he development of non-psychoactive medicinal cannabis industry. Based on the cannabis germplasm resources, this paper proposes strategies for breeding high CBD content,seed-type and high-efficiency, dwarf non-psychoactive medicinal cannabis varieties through molecular marker development, assisted breeding, genetic engineering breeding and traditional breeding. According to the suitable ecological factors of non-psychoactive medicinal cannabis, the CBD content and grain yield of non-psychoactive medicinal cannabis can be improved by regulating the nutritional structure and illumination properties of non-psychoactive medicinal cannabis, scientific harvesting and storage. At the same time, in order to further accelerate the application of non-psychoactive medicinal cannabis, we can accelerate the selection of new varieties of non-psychoactive medicinal cannabis by mining genetic information of cannabis, and strengthen the application of information technology and automation of modern agriculture in the production of non-psychoactive medicinal cannabis. Provide basis for the cultivation and wide application of new non-psychoactive medicinal cannabis varieties with high quality and high yield.

Current status and future scenarios of hemp breeding
Paolo Ranalli
Euphytica 140: 121–131, 2004.
DOI: 10.1007/s10681-004-4760-0
Hemp is a multi-use crop, able to provide fiber, cellulose, seeds and seed oil, cannabinoid, and biomass. Integrating many agroindustrial chains, Cannabis is considered a crop model in which insights into specific metabolic pathways and biosynthetic processes are valuable for improvement of the plant for all sets of industrial derivatives. In this review the hemp breeding status is elucidated and many aspects are focused: (i) recovering, maintenance and characterization of genetic resources; (ii) widening of germplasm and genetic variability; (iii) marker-assisted selection and development of breeding programs; (iv) sexual differentiation; (v) monitoring of THC content. Modern hemp varieties for fiber and other specific end uses have been developed and new varieties are entering production. The scenario for the breeding advances in hemp relies on basic and applied research which provides insights to identify a strategy for the design of modified plants with enhanced performance. This is accounted by the dissection
of traits into components and the modification of single steps of the related metabolic pathways. These advances are provided by genomic techniques and are able: (i) to identify key genes encoding enzymes and regulatory factors
participating in cannabinoid, fiber and oil biosynthesis; (ii) to identify the mode of regulation of these genes; (iii) to characterize the function of the selected genes through higher, lower or specific expression incited by specific promoters. The identification of molecular markers for specific traits, gathered in a saturated linkage map, will have a remarkable impact on hemp breeding. The advances in basic and applied research make it possible to design methods for the identification of superior parents and cross combinations and the development of selection schemes that rely on less labour-intensive and time-consuming methods.

DEVELOPMENT OF CANNABIS CHEMOVARS FOR WHOLE PLANT MEDICINES BASED ON DATA FROM LABORATORY ASSAYS TO IMPROVE EFFICACY
Dr. Mark A. Lewis, Gary Hiller, Dr. Tamas Biro
NAPRO Poster find DOI or Link
Whole plant medicine is often touted as more effcacious than pure, single-compound, silver bullet preparations. This concept is based on the assumption that synergistic effects arise from the co-administration of certain compounds that improve effcacy, but do not present themselves when each compound is administered separately. Whole-plant products should not be viewed equally nor be considered better than single-compound options merely because they are whole-plant products. To be credible, “synergistic effects,” and related concepts of “entourage effects” and outputs, namely: (i) speciffed compounds, (ii) speciffed concentrations, and (ii) measureable effects stemming from co-administration of the speci_ed compounds at various ratios. Whether in the medical-use or adult-use context, this approach will allow healthcare practitioners and consumers alike to bene_t from an objective, evidence-based
approach to comparing and selecting whole-plant cannabis products. This approach was used to proactively guide the breeding programs and research and development initiatives presented herein. Traditionally, naturally occurring plants and plant extracts are screened in laboratory assays to identify prospects for therapeutic use after such plants are grown. Herein, we
employ data from laboratory assays that identify objectively measurable synergistic effects stemming from the combination of single-molecule compounds and how such data can be used to guide plant breeding to create plants that contain synergistic combinations of compounds in speci_ed concentrations designed to improve efficacy and user experience.

Development of ornamental Cannabis sativa L. cultivars: phytochemical, morphological, genetic characterization and propagation aspects.
Casano, S.
Acta Horticulturae, (1263), 283–290.(2019).*
doi:10.17660/actahortic.2019.1263.37*
Due to its attractive variegated foliage, ornamental Cannabis cultivars offer new marketing opportunities. During the last years various chimera have been detected and, while most of them are still in process of selection, Phytoplant Research (PR) has recently obtained from the Community Plant Variety Office (CPVO) the provisional protection of plant breeder’s rights for the dioecious female cultivar ‘Divina’ (CPVO file number: 2017/0149; granted with decision N° EU 50045 of 16 July 2018) characterized by a stable variegated foliage of vegetatively propagated plants. ‘Divina’ has a distinctive “mottled yellow and green leaf” phenotype from the cultivar ‘Pilar’ where the mutated type chimera was detected and isolated from a mutated donor greenhouse growing plant. Both cultivars show the same genotype when nuclear DNA fingerprinting is performed by using 15 SSR molecular markers. Additionally, by using a molecular marker for ?9-THCA/CBDA synthases it was confirmed that only the alleles for CBDA synthase can be detected in both cultivars; gas chromatography analysis also confirmed that both cultivars had a CBD chemotype. The content of the psychotropic compound ?9-THC in inflorescences of ‘Divina’ plants was lower than 0.4% in all the samples analysed at our laboratories. Microscope observations of “yellow” portions of stalks, leaves and inflorescence bracts confirm the presence of bulbous, sessile- and capitate-stalked trichomes. ‘Divina’ cuttings are more prone to fungal disease during in vivo rooting stage than ‘Pilar’ cuttings, however, satisfactory percentages of true to type rooted cuttings can be achieved by adopting good propagation practices. A protocol for all the stages of in vitro micropropagation has been established at our laboratories. When considering the sexual propagation of the cultivar, all the F1-hybrid offspring obtained by crossing a dioecious “green leaf” male plant with a ‘Divina’ plant showed a “green leaf” phenotype.


*Diversity In Cannabis
Etienne P.M. de Meijer
1994 book of his thesis
https://edepot.wur.nl/207065
In an effort to reduce the overproduction of a too limited number of arable food crops, several research programs focusing on industrial crops have been initiated recently in several countries in Northwest Europe (e.g., Hennink et al., 1994). The 'Dutch hemp research program' investigated from 1990 to 1994 the feasibility of hemp as an arable crop for the Netherlands and as a raw material for paper pulp production. In this context, breeding, agronomy, plant pathology, mechanization, processing and economics were subject of a comprehensive study, the overall results of which have been summarized by van Berlo (1993). Fibre hemp cultivation has a long history in the Netherlands (Hoogendoorn, 1993) although it has never been of great importance. The crop disappeared at the beginning of the 20th century (de Jonge, 1944). Two previous attempts to re-introduce fibre hemp have been reported (de Jonge, 1944; Friederich, 1960). Breeding of fibre hemp in the Netherlands seems unprecedented. In the framework of the present research program, it was considered necessary to investigate the prospects of breeding directed at increased stem yield potential, improved stem quality for pulp production, disease resistance and low psychoactive potency. A collection of Cannabis germplasm, which was presumed to cover the variation within the genus, was therefore established and evaluated at the DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO, Wageningen). This thesis reports on the composition of the collection and its evaluation for traits relevant to the introduction of hemp in crop rotations in the Netherlands and its utilization as a raw material for paper pulp. The present study shows strong analogy with recent evaluations of kenaf germplasm (Hibiscus cannabinus L), a bark fibre crop traditionally used for rope and textile production, and since the 1950s under investigation as a source of pulp fibre in the USA and Australia (Adamson & Bagby, 1975; Wood, 1990).

Dwarf germplasm: the key to giant Cannabis hempseed and cannabinoid crops
Small, E.
Genetic Resources and Crop Evolution,(2017). 65(4), 1071–1107.
doi:10.1007/s10722-017-0597-y
After a century of banishment, both euphoric (‘‘marijuana’’) and non-euphoric (‘‘industrial hemp’’) classes of Cannabis sativa are attracting billions of dollars of investment as new legitimate crops. Mostdomesticated C. sativa is very tall, a phenotype that is desirable only for hemp fibre obtained from the stems. However, because the principal demands today are for chemicals from the inflorescence and oilseeds from the infructescence, an architecture maximizing reproductive tissues while minimizing stems is appropriate. Such a design was the basis of the greatest short-term increases in crop productivity in the history of agriculture: the creation of short-stature (‘‘semi-dwarf’’), high-harvest-index grain cultivars, especially by ideotype breeding, as demonstrated during the ‘‘Green Revolution.’’ This paradigm has considerable promise for C. sativa. The most critical dwarfing character for breeding such productivity into C. sativa is contraction of internodes. This reduces stem tissues (essentially a waste product except for fibre hemp) and results in compact inflo- rescences (which, on an area basis, maximize cannabi- noid chemicals) and infructescences (which maximize oilseed production), as well as contributing to ease of harvesting and efficiency of production on an area basis. Four sources of germplasm useful for breeding semi-dwarf biotypes deserve special attention: (1) Naturally short northern Eurasian wild plants (often photoperiodically day-neutral, unlike like most bio- types) adapted to the stress of very short seasons by maximizing relative development of reproductive tissues. (2) Short, high-harvest-index, oilseed plants selected in northern regions of Eurasia. (3) ‘‘Indica type’’ marijuana, an ancient semi-dwarf cultigen tracing to the Afghanistan-Pakistan area. (4) Semi- dwarf strains of marijuana bred illegally in recent decades to avoid detection when grown clandestinely indoors for the black market. Although the high THC content in marijuana strains limits their usage as germplasm for low-THC cultivars, modern breeding techniques can control this variable. The current elimination of all marijuana germplasm from breeding of hemp cultivars is short-sighted because marijuana biotypes possess a particularly wide range of genes. There is an urgent need to develop public gene bank collections of Cannabis.

I liked this talk very much, helps people understand terpene chemotypes
Enabling the Next Generation Medicinal Breeding: Understanding Terpene Chemotypes in Cannabis
Kieth Allen from Steep Hill
https://www.labtube.tv/video/enablin...es-in-cannabis
Terpenes give cannabis its distinctive odors and flavors, and so variation in terpene content is a very important factor differentiating strains. As we look forward to a new industry wave of craft and medicinal breeding, it is clear that a full understanding of the genetic basis of oil profile variation will be an essential starting point. We have combined extensive terpene profile data with modern genomic approaches to identify the full set of genes (or close to it) responsible for producing the large range of terpenes found in this plant. With this knowledge in hand, we are mapping genetic variants to oil profile variation, which we hope will provide a roadmap for breeding to specific terpene profile end-points. Dr. Allen will discuss our work so far, discussing patterns we’ve found in the oil profile data, in terms of specific enzymology across different strains. This puzzle is further complicated by the multi-product nature of most terpene synthases and tissue-specific expression of different enzymes that make the same product, so Dr. Allen will additionally provide attendees with a primer on the fascinating peculiarities of the synthases that make these molecules.

Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization
Ernest Small
The Botanical Review
DOI:10.1007/s12229-015-9157-3
Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticatedplants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.

Foliar Sprays of Silver Thiosulfate Produce Male Flowers on Female Hemp Plants
Jessica D. Lubell, and Mark H. Brand
American Society for Horticultural Science 2018 Volume/Issue: Volume 28: Issue 6 743–747
DOI: https://doi.org/10.21273/HORTTECH04188-18
https://journals.ashs.org/horttech/v...ticle-p743.xml
Interest in hemp (Cannabis sativa) for its medicinal compounds, cannabidiol (CBD), and ?-9-tetrahydrocannabidiol (THC), continues to increase. Maximizing yield of CBD and/or THC requires female plants because female inflorescences accumulate significantly greater concentrations of these compounds than male inflorescences. Production of all female seed requires induction of female plants to develop male flowers that produce genetically female pollen. Growers would like access to feminized seed to produce all-female crops. We evaluated the efficacy of 0, 0.3, and 3 mm silver thiosulfate (STS) applied as a foliar spray (on three occasions 7 days apart) to produce male flowers on four strains of female hemp (having a THC concentration of ?0.3%), designated CBD hemp A, CBD hemp B, CBD hemp C, and industrial hemp. Silver thiosulfate at 3 mm was the most efficacious treatment for all strains. The majority of inflorescences had 100% male flowers at 3 mm STS, and terminal inflorescences had ?95% conversion to male flowers. Silver thiosulfate at 0.3 mm produced partial conversion to male flowers, whereas most inflorescences had around 50% male flowers, except for CBD hemp A, which demonstrated greater levels of masculinization. At 0.3 mm STS, terminal inflorescences of CBD hemp A had 91% conversion to male flowers. This study demonstrates that male flowers can be produced easily and consistently on female plants through application of foliar sprays of STS under short-day conditions.

Not Cannabis specific
*Gene discovered crucial to making crop plants produce clonal seeds
PhysOrg Jan 6, 2022
https://phys.org/pdf560694578.pdf
Researchers from KeyGene and Wageningen University & Research (WUR), in collaboration with colleagues from Japan and New Zealand, have discovered a gene that will make it possible to produce seeds from crops that are genetically identical to the mother plant and that do not need pollination. This phenomenon, called apomixis, enables plants with a desirable combination of traits to produce many offspring with the same desirable combination of genes as the mother plant. Together with researchers from the Japanese breeding company Takii and New Zealand's Plant & Food Research and Lincoln University, the KeyGene and WUR researchers explain in Nature Genetics how the gene works and the way it influenced the work of the 'father of genetics' Gregor Mendel. The discovery is expected to lead to major innovations in plant breeding over the coming years. The gene found has been given the name PAR, shortened from parthenogenesis, the process whereby egg cells grow into plant embryos without fertilization of the egg cells. The discovery marks a definitive breakthrough and crowns the research team's work that started at KeyGene over 15 years ago.

Genetic Architecture of Flowering Time and Sex Determination in Hemp (Cannabis sativa L.): A Genome-Wide Association Study.
Petit, J., Salentijn, E. M. J., Paulo, M.-J., Denneboom, C., & Trindade, L. M.
Frontiers in Plant Science, 11.(2020).
doi:10.3389/fpls.2020.569958
Flowering time and sex determination in hemp (Cannabis sativa L.) strongly influence fiber quality and seed production of this crop. The control of these traits is paramount for the breeding of new cultivars. Yet, little is known about the genetics underlying such complex traits and a better understanding requires in depth knowledge of the molecular mechanisms responsible for these traits. In this report, the genetic architecture of flowering time and sex determination in hemp was studied using a Genome-Wide Association Studies (GWAS) approach. Association studies were performed on a panel of 123 hemp accessions, tested in three contrasting environments, using a set of 600 K SNP markers. Altogether, eight QTLs were identified across environments; six for flowering time traits and two for sex determination. These QTLs covered genomic regions with 33 transcripts predicted to be involved in flowering and sex determination as well as a microRNA, miR156. Genes related to perception and transduction of light and transcription factors well-known to regulate flowering were identified in QTLs for flowering time traits. Transcription factors and genes involved in regulating the balance of phytohormones, specially auxins and gibberellic acid, were identified in QTLs for sex determination. Sex determination QTLs were associated with the development of male flowers in female plants and thus with the stability of sex determination in monecious plants. The present study elucidates relevant knowledge on the genetic mechanisms of flowering and sex determination traits in hemp, and provides new tools for hemp breeding

Not Cannabis Specific
Genome Elimination: Translating Basic Research into a Future Tool for Plant Breeding
Comai, L.
PLoS Biology, 12(6), e1001876. (2014).
doi:10.1371/journal.pbio.1001876
: During the course of our history, humankind has been through different periods of agricultural improvement aimed at enhancing our food supply and the performance of food crops. In recent years, it has become apparent that future crop improvement efforts will require new approaches to address the local challenges of farmers while empowering discovery across industry and academia. New plant breeding approaches are needed to meet this challenge to help feed a growing world population. Here I discuss how a basic research discovery is being translated into a potential future tool for plant breeding, and share the story of researcher Simon Chan, who recognized the potential application of this new approach— genome elimination—for the breeding of staple food crops in Africa and South America.

Hashish revival in Morocco
Pierre-Arnaud Chouvya, Kenza Afsahi
International Journal of Drug Policy
https://www.geopium.org/wp-content/u...al_Morocco.pdf
DOI: 10.1016/j.drugpo.2014.01.001
Background: In less than a decade, Morocco reportedly saw cannabis cultivation decrease by 65%, and hashish production is widely believed to have followed the same trend. Yetlarge anomalies exist between the alleged fall of hashish production in Morocco and international seizure data. While no explanation for such a discrepancy existed, the main hypothesis was that cannabis cultivation and hashish production had not declined to the extent suggested by the available information. Methods: Based on existing data, on interviews with various actors, from European police sources to Moroccan cannabis cultivators, and on field research in Morocco, this article reviews contradictory available data and confronts it with observations made in the field. Results: In the past decade cannabis cultivation underwent radical changes that could explain the discrepancy between official Moroccan cultivation and production data on the one hand, and international seizures on the other hand. The “traditional” kif cannabis variety is being rapidly replaced by hybrids with much larger resin yields and much higher potency. This unnoticed phenomenon, which slowly started in the early 2000s, explains how a two-third decline in cannabis cultivation was at least partially compensated for by three to five-fold yield increases. Conclusion: The fact that the massive ongoing switch to hybrid cultivation is largely unknown or unaccounted for is actually a serious issue, for it directly questions the economic strategies that are being implemented in part to reduce and suppress cannabis cultivation in the Rif.

How ancient viruses got cannabis high
(2018, November 26)
https://phys.org/news/2018-11-ancien...abis-high.html
Modern day hemp and marijuana evolved distinct chemistry thanks to ancient viruses that colonized the ancestral cannabis genome millions of years ago.
World's first cannabis chromosome map reveals the plant's evolutionary past and points to its future as potential medicine. THC and CBD, bioactive substances produced by cannabis and sought by medical patients and recreational users, sprung to life thanks to ancient colonization of the plant's genome by viruses, U of T researchers have found. The finding is only one of the insights revealed by the long-awaited cannabis genome map detailing gene arrangement on the chromosomes, published recently in the journal Genome Research. Among other revelations are discovery of a gene
responsible for the production of cannabichromene, or CBC, a lesser known cannabinoid, as the active substances in cannabis are known, and new insights into how strain potency is determined. "The chromosome map is an important foundational resource for further research which, despite cannabis' widespread use, has lagged behind other crops due to restrictive legislation," says Tim Hughes, a professor in the Donnelly Centre for Cellular and Biomolecular Research and co-leader of the study. Hughes is also a professor in the Department of Molecular Genetics and Senior Fellow at the Canadian Institute for Advancement of Research. The researchers expect the map will speed up breeding efforts to create new strains with desired medical properties as well as varieties that can be grown more sustainably, or with increased resistance to diseases and pests. The study was a three-part collaboration between Tim Hughes' team and those of Jonathan Page, of Aurora Cannabis and the University of British Columbia , and Harm van Bakel, of the Icahn School of Medicine at Mt Sinai in New York.

Not Cannabis specific
Hybrid recreation by reverse breeding in Arabidopsis thaliana
Erik Wijnker, Laurens Deurhof, Jose van de Belt, C Bastiaan de Snoo, Hetty Blankestijn, Frank Becker, Maruthachalam Ravi, Simon W L Chan, Kees van Dun, Cilia L C Lelivelt, Hans de Jong, Rob Dirks & Joost J B Keurentjes
NATURE PROTOCOLS | VOL.9 NO.4 | 2014 | 761
doi:10.1038/nprot.2014.049
Hybrid crop varieties are traditionally produced by selecting and crossing parental lines to evaluate hybrid performance. Reverse breeding allows doing the opposite: selecting uncharacterized heterozygotes and generating parental lines from them. With these, the selected heterozygotes can be recreated as F1 hybrids, greatly increasing the number of hybrids that can be screened in breeding programs. Key to reverse breeding is the suppression of meiotic crossovers in a hybrid plant to ensure the transmission of nonrecombinant chromosomes to haploid gametes. These gametes are subsequently regenerated as doubled-haploid (DH) offspring. Each DH carries combinations of its parental chromosomes, and complementing pairs can be
crossed to reconstitute the initial hybrid. Achiasmatic meiosis and haploid generation result in uncommon phenotypes among offspring owing to chromosome number variation. We describe how these features can be dealt with during a reverse-breeding experiment, which can be completed in six generations (~1 year).

Identification of QTLs for sex expression in dioecious and monoecious hemp (Cannabis sativa L.)
A.-M. Faux . X. Draye . M.-C. Flamand, A. Occre . P. Bertin
Euphytica
DOI: 10.1007/s10681-016-1641-2
Abstract Hemp (Cannabis sativa L.) is a diploid species including both dioecious and monoecious cultivars with hetero- and homomorphic sex chromosomes, respectively. It displays a high plasticity of sex expression, i.e., the ratio of female and male flowers. In this study, we investigated the role of sex chromosomes in the genetic determinism of sex expression in dioecious and monoecious hemp. The experimental
materials were three F1 segregating populations, two dioecious (C1 and C2: ‘Carmagnola’ $ 9 ‘Carmagnola’#), and one monoecious (UF: ‘Uso 31’ 9 ‘Fedora17’). A ‘sex’ phenotypic marker was mapped in C1 and C2. In total, 23, 42, and 26 AFLP markers (71 markers in total) were mapped to three, nine, and three co-segregation groups putatively located on sex chromosomes in C1, C2, and UF, respectively. Recombination rates with sex ranged from 0 to 0.5. Five sexlinked markers were detected in UF, revealing homologies between the X chromosomes of monoecious
hemp and the X and Y chromosomes of dioecious hemp. Five QTLs associated with quantitative variations in sex expression were identified in each map. Four markers associated with variations in sex expression in UF segregated with sex or accounted for a putative QTL in C1 or C2. Two QTLs and three of these markers were mapped in UF in a region homologous to the sex-locus region of the dioecious maps. Given these results, conducting further research on the genetic determinism of sex expression in hemp using a quantitative approach appears relevant.

*Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex.
Mohan Ram, H. Y., & Sett, R. (1982).
Theoretical and Applied Genetics, 62(4), 369–375.
doi:10.1007/bf00275107
Apical application of silver nitrate (AgNO~ ;50 and 100 ~tg per plant) and silver thiosulphate anionic complex (Ag(S203)~-; STS; 25, 50 and 100 ~tg per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 ~tg per plant of AgNO3 was ineffective and 150 ~tg treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 ~tg STS. The induced male flowers produced pollen grains that germinated on stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO3 in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.

Introduction to the Special Issue on Cannabis
Dennis J. Gray, Robert C. Clarke & Robert N. Trigiano
Critical Reviews in Plant Sciences, 35(5-6), 289–292.
DOI: 10.1080/07352689.2016.1267492
Decades of archaeological research and discovery around the world have concluded that the rise of modern civilization is strictly linked to when humans shifted from a hunter-gatherer/subsistence lifestyle to one that was civilized–urbanized, with people settled into permanent towns and cities (Cochran and Harpending, 2009; Scarre and Fagan, 2016). Civilization depended on the development
of agriculture, which occurred when certain key plants found to provide nutrition and protection were taken from nature and genetically modified to be of increasing use by humankind, a process termed as “domestication” (Leach, 2003; Zohary et al., 2012). Domestication is defined as a process by which wild plants are artificially evolved into crop plants by selection of traits that are advantageous or desirable to humans (Bennici, 2005; Acquaah, 2012). Domestication involves genetic modification by selection and/or breeding, creating crop plants that are increasingly improved in quality and yield, becoming abundant and dependable. The initial stages of domestication were critical because humans acted as (1) dispersal agents (managing the reproduction of cultivated plants and controlling the mobility, range, and density of domestic livestock); (2) agents of selection (favoring the reproductive success of particular phenotypic variants); and (3) ecosystem modifiers, which, along with natural environmental changes, alter the developmental conditions and hence the characteristics of associated organisms (Larson et al., 2013). The newfound ability to grow food allowed populations to coalesce and become stable, unleashing an explosion of cultural and technical development that continues through to the present (Cochran and Harpending, 2009). Plant domestication is considered to be the very cornerstone of civilization (Gray and Trigiano, 2010) because, without it, civilization as we know it today could not have developed.

Not Cannabis specific Machine learning in plant science and plant breeding Aalt Dirk Jan van Dijk, Gert Kootstra, Willem Kruijer, and Dick de Ridder
iScience 24, 101890, January 22, 2021 https://www.cell.com/iscience/pdf/S2...20)31087-7.pdf Technological developments have revolutionized measurements on plant genotypes and phenotypes, leading to routine production of large, complex data sets. This has led to increased efforts to extract meaning from these measurements and to integrate various data sets. Concurrently, machine learning has rapidly evolved and is now widely applied in science in general and in plant genotyping and phenotyping in particular. Here, we review the application of machine learning in the context of plant science and plant breeding.We focus on analyses at different phenotype levels, from biochemical to yield, and in connecting genotypes to these. In this way, we illustrate how machine learning offers a suite of methods that enable researchers to find meaningful patterns in relevant plant data.

Not Cannabis specific
Maternal control of seed size in plants
Na Li and Yunhai Li
Journal of Experimental Botany
doi: 10.1093/jxb/eru549
Seed size is a key determinant of evolutionary fitness, and is also one of the most important components of seed yield. In angiosperms, seed development begins with double fertilization, which leads to the formation of a diploid embryo and a triploid endosperm. The outermost layer of the seed is the seed coat, which differentiates from maternal integuments. Therefore, the size of a seed is determined by the co-ordinated growth of the embryo, endosperm, and maternal tissue. Recent studies have identified several factors that act maternally or zygotically to regulate seed size, and revealed possible mechanisms that underlie seed size control in Arabidopsis and rice. In this review, we summarize current research progress in maternal control of seed size and discuss the roles of several newly identified regulators in maternal regulation of seed growth.

*Microgametophyte Development in Cannabis sativa L. and First Androgenesis Induction Through Microspore Embryogenesis
Alberto Galán-Ávila, Edgar García-Fortea, Jaime Prohens and Francisco Javier Herraiz
Front. Plant Sci. 12:669424. May 2021
doi: 10.3389/fpls.2021.669424
https://www.ncbi.nlm.nih.gov/pmc/art...-12-669424.pdf
Development of double haploids is an elusive current breeding objective in Cannabis sativa L. We have studied the whole process of anther and pollen grain formation during meiosis, microsporogenesis, and microgametogenesis and correlated the different microgametophyte developmental stages with bud length in plants from varieties USO31 and Finola. We also studied microspore and pollen amyloplast content and studied the effect of a cold pretreatment to excised buds prior to microspore in vitro culture. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability of microspores and pollen grains contained in anthers was observed, and this allowed the identification of bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains. The starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis. Although at a low frequency, cold-shock pretreatment applied on buds can deviate the naturally occurring gametophytic pathway toward an embryogenic development. This represents the first report concerning androgenesis induction in C. sativa , which lays the foundations for double haploid research in this species.

Not Cannabis Specific
Molecular approaches for the fixation of plant hybrid vigor.
Siddiqi, I., Marimuthu, M. P. A., & Ravi, M.
Biotechnology Journal, 4(3), 342–347.(2009).
doi:10.1002/biot.200900018
Research in the plant sciences is inextricably linked to our dependence on plants as a source of food. With the growing population comes the challenge of increasing food supply and hence the yield of food crops. This issue is of particular relevance for developing countries. The green revolution beginning in the late 1960s, enabled countries such as India to achieve self-sufficiency in food production; however, the increases in yield have plateaued and the world is faced with having to meet the increasing demand for food with far greater constraints in the availability of the two main resources: land and water.The use of hybrids in agriculture is well known to lead to large yield increases of upto 20% for important crops such as maize and rice, a phenomenon known as hybrid vigor. However, hybrid vigor is not stable and declines in each subsequent generation due to segregation of alleles at the time of sexual reproduction. The instability of hybrids requires that seeds of any hybrid have to be synthesized anew at each generation by crossing of the two parental lines. From the perspective of agriculture, the interest in apomixis derives from the possibility of fixing hybrid vigor by the formation of asexual seeds through apomixis, bypassing the normal processes of meiosis and fertilization. The application of apomixis to plant breeding and seed production would have a major impact on crop production by allowing the fixation and propagation of any elite genotype however complex it may be. Apomixis does not occur naturally in the major food crops and would have to be introduced into food crops using biotechnological approaches

Not Cannabis specific
Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement
Stephen P. Moose, Rita H. Mumm
Plant Physiology, July 2008, Vol. 147, pp. 969–977
doi/10.1104/pp.108.118232
The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices has created the foundation for molecular plant breeding, an interdisciplinary science that is revolutionizing 21st century crop improvement. Though the methods of molecular plant breeding continue to evolve and are a topic of intense interest among plant breeders and crop scientists (for review, see Cooper et al., 2004; Nelson et al., 2004; Lo¨rz and Wenzel, 2005; Varshney et al., 2006; Eathington et al., 2007; Mumm, 2007), they have received relatively little attention from the majority of plant biologists engaged in basic scientific research. The objective of this article for an Editor’s Choice series on future advances in crop biotechnology is to briefly review important historical developments in molecular plant breeding, key principles influencing the current practice of molecular plant breeding, and factors that influence the adoption of molecular plant breeding in crop improvement programs. Furthermore, we emphasize how the application of molecular plant breeding is now contributing to discoveries of genes and their functions that open new avenues for basic plant biology research

Not Cannabis specific
Navigating complexity to breed disease-resistant crops.
Nelson, R., Wiesner-Hanks, T., Wisser, R., & Balint-Kurti, P.
Nature Reviews Genetics, 19(1), 21–33. (2017).
doi:10.1038/nrg.2017.82
| Plant diseases are responsible for substantial crop losses each year and pose a threat to global food security and agricultural sustainability. Improving crop resistance to pathogens through breeding is an environmentally sound method for managing disease and minimizing these losses. However, it is challenging to breed varieties with resistance that is effective, stable and broad-spectrum. Recent advances in genetic and genomic technologies have contributed to a better understanding of the complexity of host–pathogen interactions and have identified some of the genes and mechanisms that underlie resistance. This new knowledge is benefiting crop improvement through better-informed breeding strategies that utilize diverse forms of resistance at different scales, from the genome of a single plant to the plant varieties deployed across a region.

New developments in fiber hemp (Cannabis sativa L.) breeding
Elma M.J. Salentijna, Qingying Zhangb, Stefano Amaduccic, Ming Yangb, Luisa M. Trindadea,
Industrial Crops and Products Vol. 68, June 2015, pages 32-41
doi: 10.1016/j.indcrop.2014.08.011
Fiber hemp (Cannabis sativa L.) is a sustainable and high yielding industrial crop that can help to meet the high global demand for fibers. Hemp can be grown for fiber, seeds, and/or for dual purpose in a wide range of geographic zones and climates. Currently the main hemp producing regions in the world are China, Europe, and Canada. The number of new cultivars developed for each of these regions has gradually increased, with each region producing its own typical hemp cultivars for different purposes. In this article, the state of the art of fiber hemp breeding programs in Europe, China, and Canada are reviewed. The breeding strategies and tools used in the breeding of hemp cultivars are discussed. We also provide an overview of genetic diversity in hemp for different traits. In addition, the current knowledge of the main breeding goals for fiber hemp, which are an improvement of fiber quality and fiber yield, breeding for specific cannabinoid profiles, control of flowering behavior, male flowering control, and breeding of cultivars for specific environments are evaluated. Lastly, we discuss the inestimable value of next generation technologies to breed new hemp cultivars that are suitable for a biobased economy.
PHYLOS density report (2022)
https://8vjgattmsg1.typeform.com/to/...rce=phylos.bio

Potential of marker-assisted selection in hemp genetic improvement
G. Mandolino & A. Carboni
Euphytica 140: 107–120, 2004.
DOI: 10.1007/s10681-004-4759-6
The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars.
Barcaccia, G., Palumbo, F., Scariolo, F., Vannozzi, A., Borin, M., & Bona, S.
Frontiers in Plant Science, 11. (2020).
doi:10.3389/fpls.2020.573299
Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges—along with potential solutions—behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F1 hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs

PRESERVING HUMBOLDT COUNTY’S LEGACY GENETICS LELEHNIA DUBOIS & DANIEL HENDRICKS https://cannmedevents.com/2022/02/16...ource=hs_email

Protein changes in response to lead stress of lead-tolerant and lead-sensitive industrial hemp using SWATH technology
Cheng Xia, Li Hong, Su Yuan, Yang Yang, Du Guang Hui, Liu Fei Hu, Xu Yan Ping, Huang Xing, Deng Gang
PeerJ Preprints (2019)
DOI: 10.7287/peerj.preprints.27527v1
Hemp is a Pb-tolerant and Pb-accumulating plant, and the study of its tolerance mechanisms could facilitate the breeding of hemp with enhanced Pb tolerance and accumulation. In the present study, we took advantage of SWATH (sequential window acquisition of all theoretical mass spectra) technology to study the difference in proteomics between the Pb-tolerant seed-type hemp variety Bamahuoma (BM) and the Pbsensitive fiber-type hemp variety Yunma 1(Y1) under Pb stress (3g/kg soil). A total of 63 and 372 proteins differentially expressed under Pb stress relative to control conditions were identified with liquid chromatography electro spray ionization tandem mass spectrometry in BM and Y1, respectively; with each of these proteins being classified into 14 categories. Hemp adapted to Pb stress through accelerating ATP metabolism; enhancing respiration, light absorption and light energy transfer; promoting assimilation of intercellular nitrogen (N) and carbon (C); eliminating reactive oxygen species; regulating stomatal development and closure; improving exchange of water and CO2 in leaves; promoting intercellular transport; preventing aggregation of unfolded proteins; degrading misfolded proteins; and increasing the transmembrane transport of ATP in chloroplasts. Our results provide important reference protein and gene information for future molecular studies into the resistance and accumulation of Pb in hemp.

Not Cannabis Specific
Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping.
Seymour, D. K., Filiault, D. L., Henry, I. M., Monson-Miller, J., Ravi, M., Pang, A., … Maloof, J. N.
Proceedings of the National Academy of Sciences, 109(11), 4227–4232.(2012).
doi:10.1073/pnas.1117277109
Quantitative trait loci (QTL) mapping is a powerful tool for investigating the genetic basis of natural variation. QTL can be mapped using a number of different population designs, but recombinant inbred lines (RILs) are among the most effective. Unfortunately, homozygous RIL populations are time consuming to construct, typically requiring at least six generations of selfing starting from a heterozygous F1. Haploid plants produced from an F1 combine the two parental genomes and have only one allele at every locus. Converting these sterile haploids into fertile diploids (termed “doubled haploids,” DHs) produces immortal homozygous lines in only two steps. Here we describe a unique technique for rapidly creating recombinant doubled haploid populations in Arabidopsis thaliana: centromere-mediated genome elimination. We generated a population of 238 doubled haploid lines that combine two parental genomes and genotyped them by reduced representation Illumina sequencing. The recombination rate and parental allele frequencies in our population are similar to those found in existing RIL sets. We phenotyped this population for traits related to flowering time and for petiole length and successfully mapped QTL controlling each trait. Our work demonstrates that doubled haploid populations offer a rapid, easy alternative to RILs for Arabidopsis genetic analysis.

Results of Efforts by French and Ukranian Breeders to Reduce Cannabinoid Levels in Industrial Hemp (Cannabis sativa L.)
P. Holoborodko, V. Virovets, I. Laiko, S. Bertucelli, O. Beherec, G. Fournier https://www.votehemp.com/wp-content/...trial_Hemp.pdf.
Results are given of independent, multi-year efforts by French and Ukrainian breeders to create new, highly productive, monoecious hemp varieties without drug properties, while retaining an innate resistance to pests and diseases

NOT CANNABIS SPECIFIC
*Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant.
Wijnker, E., van Dun, K., de Snoo, C. B., Lelivelt, C. L. C., Keurentjes, J. J. B., Naharudin, N. S., Dirks, R.
Nature Genetics, 44(4), 467–470.(2012).
doi:10.1038/ng.2203
Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.

Reversal of Ethephon-Induced Feminization in Male Plants of Cannabis sativa by Ethylene Antagonists
H.Y.Mohan Ram, Rina Sett Zeitschrift fuer Pflanzenphysiologie 107(1): 85-89
https://www.sciencedirect.com/scienc...44328X11800127 Interaction between ethephon and the two ethylene antagonists — silver nitrate and cobalt chloride — applied exogenously to the male Cannabis sativa plants was studied. The feminization effect caused by two sprays of 960 mg· l-1 of ethephon could be overcome by 100 μg of AgNO3 or CoCl2. However, neither of the antagonists could suppress the feminization effect of 1920 mg · l-1 of ethephon sprayed twice.
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Seeded Unseeded Cannabis
Cannabis Business Times - September 2019 –
Mojave Richmond & Robert C. Clarke
https://magazine.cannabisbusinesstim.. .cbd-needs.aspx
Morocco, where crops destined for hashish production are seeded, is on a steady path toward all-female seedless crop production. The popularity of feminized seeds, which produce only female plants, has grown exponentially, yet nearly all modern Moroccan drug crops still do not start from these because many growers continue to sow seeds that they harvest (rather than purchasing new seeds), and crops grown from these seeds are half male and half female. Pollen fills the air in mid-summer, and by autumn every female plant is full of seeds. (When females are pollinated, their flower growth is reduced, and the seeds produced are undesirable for end consumers.) Morocco’s future of not harvesting seeds is clearly on the horizon as awareness amongst farmers and widespread availability of less expensive female seed are slowly becoming reality.

Selection for low ? 9-tetrahydrocannabinol content in Thai hemp cultivars
Weerapun Kunkaew, S. Julsrigival, S. Pinmanee, Prapatsorn Tipparat
January 2011 SABRAO journal of breeding and genetics 43(1):1-14
https://www.votehemp.com/PDF/sabrao_2011_43-1_1-14.pdf
Selection for reduced ? 9-tetrahydrocannabinol (THC) content in four Thai hemp cultivars (including V50, Mae Sa Mai, Huay Hoi and Pang Ung) was carried out in highland areas in the northern Thailand. Research work was conducted for two consecutive growing seasons during 2008 to 2009 at Pangda Royal Agricultural Station, Samoeng district, Chiang Mai province, Thailand. Results of selection indicated that after selecting for two successive generations, the average THC content of four Thai hemp cultivars reduced to 18.0-55.0% and cannabidiol (CBD) content increased to 20.0-127.0%. The results of selection also indicated that chemotype classification could be grouped by using the ratio of CBD/THC content as follows: non-drug type (CBD/THC>10.0), intermediate type (1.0?CBD/THC?10.0) and drug type (CBD/THC<1.0). Thus, selection for reduced THC content, high ratio of CBD/THC content could be used as alternative criteria for improving low THC content in hemp cultivars. As well, mass selection method was considered as an effective and suitable method for improving these THC and CBD traits.

Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype
Chiara Onofri, Etienne P.M. de Meijer, Giuseppe Mandolino
Phytochemistry. 2015 Aug;116:57-68.
doi: 10.1016/j.phytochem.2015.03.006.
Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.
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Does this mean THC synthase evolved or mutated from CBD synthase? Maybe man helped select the few that were THC synthase however they came to be, and spread them worldwide? I would not be surprised......
Man selects for THC and against CBD, in the case of Ganja, he selects for high resin contents regardless of Cannabinoid for Hashish, for hemp until recently there was little selections for Cannabinoids until after Cannabis became illegal less then 100 years ago, and very recently for CBD for mediacal varieties.
-SamS

Not Cannabis Specific
Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.
Sung, Y.-C., Lin, C.-P., Hsu, H.-J., Chen, Y.-L., & Chen, J.-C.
Frontiers in Plant Science, 10. (2019).
doi:10.3389/fpls.2019.01183
Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

Stability of cannabinoids in dried samples of cannabis dating from around 1896–1905.
Harvey, D. J.
Journal of Ethnopharmacology, 28(1), 117–128. (1990).
doi:10.1016/0378-8741(90)90068-5
Cannabinoids from three samples of cannabis obtained from the Pitt-Rivers Museum, Oxford, and dating from the turn of the century were examined by gas chromatography and mass spectometry for the presence of cannabinoids. Although the samples were from different geographical locations, the profiles of constituent cannabinoids were similar. In common with other aged material, most of the cannabinoid content was present as cannabinol (CBN), the main chemical degradation product of the major psychoactive constituent, delta-9-tetrahydrocannabinol (delta-9-THC). However, a
substantial concentration of CBN acid-A was also present; this compound is unstable to heat and readily undergoes decarboxylation to CBN. Methyl and propyl homologues of CBN, together with delta-9-THC and its naturally occurring acid-A were also found at low concentrations in all samples. Intermediates in the formation of CBN from delta-9-THC, previously identified in aged solutions of the drug, were absent or present in only trace concentrations. However, oxidation products involving hydroxylation at the benzylic positions, C-11 and C-l’, not seen in solution, were identified in substantial abundance. The results suggest that decomposition of cannabis samples may proceed more slowly than originally thought.

Not Cannabis specific
*Statistical genetic considerations for maintaining germ plasm collections
J. Crossa , C. M. Hernandez , P. Bretting , S. A. Eberhart , S. Taba
Theor Appl Genet (1993) 86:673-678
DOI: 10.1007/BF00222655
One objective of the regeneration of genetic populations is to maintain at least one copy of each allele present in the original population. Genetic diversity within populations depends on the number and frequency of alleles across all loci. The objectives of this study on outbreeding crops are: (1) to use probability models to determine optimal sample sizes for the regeneration for a number of alleles at independent loci; and (2) to examine theoretical considerations in choosing core subsets of a collection. If we assume that k-1 alleles occur at an identical low frequency of p0 and that the k(th) allele occurs at a frequency of 1-[(k-1)p0], for loci with two, three, or four alleles, each with a p0 of 0.05, 89-110 additional individuals are required if at least one allele at each of 10 loci is to be retained with a 90% probability; if 100 loci are involved, 134-155 individuals are required. For two, three, or four alleles, when p0 is 0.03 at each of 10 loci, the sample size required to include at least one of the alleles from each class in each locus is 150-186 individuals; if 100 loci are involved, 75 additional individuals are required. Sample sizes of 160-210 plants are required to capture alleles at frequencies of 0.05 or higher in each of 150 loci, with a 90-95% probability. For rare alleles widespread throughout the collection, most alleles with frequencies of 0.03 and 0.05 per locus will be included in a core subset of 25-100 accessions.

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We develop genetic markers that will tell you information about your plant, such as flowering time, terpene makeup, yield patterns, etc. With that information, you can supercharge “pheno-hunting” and select your backcrosses much more rapidly and confidently.
What are genetic markers?
Think of markers as tiny probes. These probes bind to DNA on the chromosome to visually map the plant’s genetic makeup for genes like sex, cannabinoid and terpene profile, yield, flowering time, and more. When you consider the chromosome as the backbone of DNA, there are limitless possibilities to what we can do to maximize your success!
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Targeted mutation of ?12 and ?15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil
Monika Bielecka, Filip Kaminski, Ian Adams, Helen Poulson, Raymond Sloan, Yi Li, Tony R. Larson, Thilo Winzer, Ian A. Graham
February 2014 Plant Biotechnology Journal 12(5)
DOI: 10.1111/pbi.12167
We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven ?12 desaturases and one of three ?15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have ?x+3 activity, while CSFAD3A activity was exclusively at the ?15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major ?12 and ?15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1?9) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2?6,9 and 18:2?9,15, were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2?9,15 from endogenous 18:1?9 when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop.

The Botany of Cannabis sativa L.
In, The Analytical Chemistry of Cannabis, 2016
Brian F. Thomas, Mahmoud A. ElSohly
10.1016/B978-0-12-804646-3.00001-1
CHEMICAL CONSTITUENTS AND PHENOTYPES OF C. SATIVA L.
CBN was the first cannabinoid to be isolated and identified from C. sativa. The elucidation of CBN led to speculation that the psychotropically active constituents of cannabis could be THCs. The nonpsychotropic compound CBD was subsequently isolated from Mexican marijuana and the structure was determined. Gaoni and Mechoulam, two pioneers of cannabis research, determined the structureof ?9-THC after finally succeeding in isolating and purifying this elusive compound (see Mechoulam Close-up: How to Pamper an Idea). Since then, the number of cannabinoids and other compounds isolated from cannabis has increased continually, with 545 now reported. Of these, 104 are phytocannabinoids (Table 1.3). From the isolation and structural elucidation of ?9-THC in 1964 until 1980, 61 phytocannabinoids were isolated and reported. Only nine new ones were characterized between 1981 and 2005, but 31 were reported between 2006 and 2010. The 13 chemical constituent type groups shown in Table 1.3 suggests the chemical complexity of the cannabis plant.
Table 1.3 Constituents of Cannabis sativa L.
No. Groups Number of Known Compounds
1 CBG type 17
2 CBC type 8
3 CBD type 8
4 ?9-THC type 18
5 ?8-THC type 2
6 CBL type 3
7 CBE type 5
8 CBN type 10
9 CBND type 2
10 CBT type 9
11 Miscellaneous 22
12 Total cannabinoids 104

The characterization of key physiological traits of medicinal cannabis (Cannabis sativa L.) as a tool for precision breeding
Erez Naim Feil, Luke W. Pembleton, Laura E. Spooner, Alix L. Malthouse, Amy Miner, Melinda Quinn, Renata M. Polotnianka, Rebecca C. Baillie, German C. Spangenberg and Noel O. I. Cogan
BMC Plant Biol (2021) 21:294
DOI: 10.1186/s12870-021-03079-2
https://bmcplantbiol.biomedcentral.c.. .21-03079-2.pdf
Background: For millennia, drug-type cannabis strains were extensively used for various medicinal, ritual, and inebriant applications. However, cannabis prohibition during the last century led to cultivation and breeding activi ties being conducted under clandestine conditions, while scientifc development of the crop ceased. Recently, the potential of medicinal cannabis has been reacknowledged and the now expanding industry requires optimal and scientifcally characterized varieties. However, scientifc knowledge that can propel this advancement is sorely lacking. To address this issue, the current study aims to provide a better understanding of key physiological and phenological traits that can facilitate the breeding of advanced cultivars.
Results: A diverse population of 121 genotypes of high-THC or balanced THC-CBD ratio was cultivated under a con trolled environment facility and 13 plant parameters were measured. No physiological association across genotypes attributed to the same vernacular classifcation was observed. Floral bud dry weight was found to be positively associ ated with plant height and stem diameter but not with days to maturation. Furthermore, the heritability of both plant height and days to maturation was relatively high, but for plant height it decreased during the vegetative growth phase. To advance breeding efcacy, a prediction equation for forecasting foral bud dry weight was generated, driven by parameters that can be detected during the vegetative growth phase solely.
Conclusions: Our fndings suggest that selection for taller and fast-growing genotypes is likely to lead to an increase in foral bud productivity. It was also found that the fnal plant height and stem diameter are determined by 5 inde pendent factors that can be used to maximize productivity through cultivation adjustments. The proposed prediction equation can facilitate the selection of prolifc genotypes without the completion of a full cultivation cycle. Future studies that will associate genome-wide variation with plants morphological traits and cannabinoid profle will enable precise and accelerated breeding through genomic selection approaches.

The Cream of the Crop: Biology, Breeding and Applications of Cannabis sativa
Susanne Schilling, Caroline A. Dowling, Jiaqi Shi. Louise Ryan, David Hunt, Eve OReilly, Antoinette S. Perry, Oliver Kinnane, Paul F. McCabe, and Rainer Melzer
DOI: 10.22541/au.160139712.25104053
Cannabis sativa is an extraordinarily versatile species. Hemp and its cousin marijuana, both C. sativa, have been used for millennia as a source of fibre, oil and for medicinal, spiritual and recreational purposes. Because the consumption of Cannabis can have psychoactive effects, the plant has been widely banned throughout the last century. In the past decade, evidence of its medicinal properties did lead to the relaxation of legislation in many countries around the world. Consequently, the genetics and development of Cannabis as well as Cannabis-derived products are the subject of renewed attention.Here, we review the biology of C. sativa, including recent insights from taxonomy, morphology and genomics, with an emphasis on the genetics of cannabinoid synthesis. Because the female Cannabis flower is of special interest as the site of cannabinoid synthesis, we explore flower development, flowering time well as the species’ unique sex determination system in detail. Furthermore, we outline the tremendous medicinal, engineering, and environmental opportunities that Cannabis bears. Together, the picture emerges that our understanding of Cannabis biology currently progresses at an unusual speed. A future challenge will be to preserve the multi-purpose nature of Cannabis, and to harness its medicinal properties and sustainability advantages simultaneously.

The Derivation of Modern Cannabis Varieties
Bailey Rahn, Brian J. Pearson, Robert N. Trigiano & Dennis J. Gray
Critical Reviews in Plant Sciences,
DOI:10.1080/07352689.2016.1273626
Considering the ancient importance of cannabis and the current trend toward its deregulation worldwide, it is imperative to develop best management practices to legitimize the crop. The genetic backgrounds of commercially-sold varieties must be determined in order to standardize the products produced from them and to efficiently improve them for future needs. Currently the
genetic backgrounds of most cannabis varieties are unknown or suspect. It is possible that some are merely clones of other varieties. By utilizing modern tools of genetic analyses, the identities of all or most existing varieties, as well as their parentages, will be determined. As with all major crops, understanding varietal identity is a crucial step required to modernize the cannabis industry. This
article describes and explores the derivation of 601 modern varieties in order to provide a fundamental point of reference for scientists to study cannabis genetics in the future.

The genetics of Cannabis – genomic variations of key synthases and their effect on cannabinoids content
Aparna Singh, Andriy Bilichak and Igor Kovalchuk
Genome (2020)
10.1139/gen-2020-0087
Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalisation in Canada, it is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allows for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single nucleotide polymorphisms in the coding regions of cannabinoid synthases play important role in determining plant chemotype. Deep understanding of how these variants affect enzymes activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profile. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.

*The Inheritance of Chemical Phenotype in Cannabis sativa L.
Etienne P. M. de Meijer, Manuela Bagatta, Andrea Carboni, Paola Crucitti,
V. M. Cristiana Moliterni, Paolo Ranalli and Giuseppe Mandolino
Genetics. 2003 Jan; 163(1): 335–346.
https://www.researchgate.net/publica...nabis_sativa_L
PMC1462421
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure Delta-9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F(1)'s were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F(1) plants were self-fertilized, and 10 inbred F(2) offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F(1) to the F(2)'s derived from it. A model involving one locus, B, with two alleles, B(D) and B(T), is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype B(D)/B(T) at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either B(D)/B(D) or B(T)/B(T)). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F(2) segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype-associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.

*The inheritance of chemical phenotype in Cannabis sativa L. (II): Cannabigerol predominant plants
E. P. M. de Meijer & K. M. Hammond
Euphytica (2005) 145: 189–198
DOI: 10.1007/s10681-005-1164-8
This paper aims to clarify the genetic mechanism that is responsible for the accumulation of cannabigerol (CBG) in certain phenotypes of Cannabis sativa L. CBG is the direct precursor of the cannabinoids CBD, THC and CBC. Plants strongly predominant in CBG have been found in different fibre hemp accessions. Inbred offspring derived from one such individual were crossed with true breeding THC predominant- and CBD predominant plants, respectively. The segregations in the cross progenies indicate that CBG accumulation is due to the homozygous presence of a minimally functional allele, tentatively called B0, at the single locus B that normally controls the conversion of CBG into THC (allele BT) and/or CBD (allele BD). The fact that CBG accumulating plants have so far been found in European fibre hemp populations that are generally composed of BD/BD plants, and the observation that the here investigated B0 allele possesses a residual ability to convert small amounts of CBG into CBD, make it plausible that this B0 is a mutation of normally functional BD. Therefore, B0 is considered as a member of the BD allelic series encoding a CBD synthase isoform with greatly weakened substrate affinity and/or catalytic capacity.

*The inheritance of chemical phenotype in Cannabis sativa L. (III): variation in cannabichromene proportion
E. P. M. de Meijer · K. M. Hammond · M. Micheler
Euphytica (2009) 165:293–311
DOI 10.1007/s10681-008-9787-1
The mechanism that controls the proportion of cannabichromene (CBC), a potential pharmaceutical, in the cannabinoid fraction of Cannabis sativa L. is explored. As with tetrahydrocannabinol (THC) and cannabidiol (CBD), CBC is an enzymatic conversion product of the precursor cannabigerol (CBG). CBC is reported to dominate the cannabinoid fraction of juveniles and to decline with maturation. This ontogeny was confirmed in inbred lines with different mature chemotypes. A consistent CBC presence was found in early leaves from a diverse clone collection, suggesting that CBC synthase is encoded by a fixed locus. Morphological variants possessing a ‘prolonged juvenile chemotype’ (PJC), a substantial proportion of CBC persisting up to maturity, are presented. PJC is associated with a reduced presence of floral bracts, bracteoles, and capitate-stalked trichomes. Genetic factors causing these features were independent of the allelic chemotype locus B that was previously postulated and regulates THC and CBD synthesis and CBG accumulation. In contrast to previously described Cannabis chemotypes, the cannabinoid composition of PJCs showed plasticity in that reduced light levels increased the CBC proportion. The ability of PJC plants to enable the production of pharmaceutical raw material with high CBC purity is demonstrated.

*The inheritance of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free plants
E. P. M. de Meijer, E K. M. Hammond, A Sutton
Euphytica (2009) 168:95–112
DOI 10.1007/s10681-009-9894-7
A genetic factor that blocks the cannabinoid biosynthesis in Cannabis sativa has been investigated. Crosses between cannabinoid-free material and high content, pharmaceutical clones were performed. F1s were uniform and had cannabinoid contents much lower than the mean parental value. Inbred F2 progenies segregated into discrete groups: a cannabinoid-free chemotype, a chemotype with relatively low cannabinoid content and one with relatively high content, in a monogenic 1:2:1 ratio. In our model the cannabinoid knockout factor is indicated as a recessive allele o, situated at locus O, which segregates independently from previously presented chemotype loci. The genotype o/o underlies the cannabinoid-free chemotype, O/o is expressed as an intermediate, low content chemotype, and O/O is the genotype of the high content chemotype. The data suggests that locus O governs a reaction in the pathway towards the phenolic cannabinoid precursors. The composition of terpenoids and various other compound classes of cannabinoid-free segregants remains unaffected. Backcrossing produced cannabinoid-free homologues of pharmaceutical production clones with potential applications in pharmacological research. A new variant of the previously presented allele ‘B 0’, that almost completely obstructs the conversion of CBG into CBD, was also selected from the source population of the cannabinoid knockout factor.

*The inheritance of chemical phenotype in Cannabis sativa L. (V): regulation of the propyl-/pentyl cannabinoid ratio, completion of a genetic model
E. P. M. de Meijer . K. M. Hammond
May 2016 Euphytica 210(2)
DOI 10.1007/s10681-016-1721-3
In order to complete a genetic model for the inheritance of chemotype in Cannabis, this paper explores the regulation of the propyl-/pentyl cannabinoid ratio. Plants almost pure in compounds with a C5 side chain are by far the most common, and such a chemotype can be considered a wild-type condition. Mutant progenitors with higher levels of the rarer cannabinoid THC-C3 (tetrahydrocannabivarin) were identified. Their propyl cannabinoid proportion in the total cannabinoid fraction (PC3) ranged from 14 to 69 %, which, through selective inbreeding, could be increased to highly specific lineage maxima. Inbred plants with maximised PC3 derived from the different progenitors, were then crossed with a pure C5 wild type and the PC3 distribution patterns of the F2s examined. Distinct patterns, compatible with oligogenic and polygenic segregation appeared. It was hypothesised that the PC3 regulating loci of the six source progenitors would be at least partially different, complementary, and additive in their phenotypical effect. So, high PC3 offspring from the different lineages were mutually crossed. Inbred lines derived from multi-cross hybrid combinations reached unprecedented PC3 levels of up to 96 % which supports the hypothesis. For the regulation of C3/C5 ratios, a model of a multiple locus A 1–A 2–…A n is proposed, with the pentyl- and propyl cannabinoid pathway being enhanced by alleles A pe1?n and A pr1?n, respectively.

Not Cannabis specific
Cannabis is an Annual, Dioecious, Heterozygous and an obligate out-crosser, not Monoecious, Homozygous like tomatoes which are not obligate out crossers, tomatoes are poor examples to compare to Cannabis.
Unraveling the genetic basis of hybrid vigor.
Birchler, J. A., Yao, H., & Chudalayandi, S.
Proceedings of the National Academy of Sciences, 103(35),(2006).
doi:10.1073/pnas.0605627103
he study of hybrid vigor and inbreeding depression traces back to Charles Darwin, who was the first scientist to examine the phenomenon in a systematic manner (1). Hybrid vigor, or heterosis, is the increase in stature, biomass, and fertility that characterizes the progeny of crosses between diverse parents such that the F1 is superior to the better of the two parents. In plants, this is basically achieved by a greater proliferation of cells in some but not all tissues (2). Inbreeding depression refers to the decline in the quantitative measure of these characters upon selffertilization or other forms of homozygosis of alleles (inbreeding). The genetic basis of heterosis has been debated for nearly a hundred years without an emerging consensus (3–5) (Fig. 1). An early view was that the combination of different alleles in an organism resulted in a superior state for growth and vigor compared with the presence of identical alleles (3). As genetic knowledge increased, the concept that inferior alleles of different genes in the two parents were complemented in the hybrid (6), thus leading to the superior characteristics, gained favor. Although the latter explanation is simple and easily envisioned, results that seemed to favor interactions of diverse alleles have been repeatedly found. In this issue of PNAS, the work of Semel et al. (7) examined an extensive set of quantitative traits in partial hybrids of domesticated tomato and a wild relative. They conclude that most traits that exhibit heterosis do so as a result of heterozygosity of the controlling genomic regions to produce traits superior to the better parent. They also suggest that heterosis was selected over evolutionary time for characteristics that impact reproductive success.

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa
Jonathan P. Wenger, Clemon J. DabneyIII, Mahmoud A. ElSohly, Suman Chandra, Mohamed M. Radwan, Chandrani G. Majumdar, and George D. Weiblen
American Journal of Botany 107(10): 1423–10. (2020)
doi:10.1002/ajb2.1550
PREMISE: How genetic variation within a species affects phytochemical composition is a fundamental question in botany. The ratio of two specialized metabolites in Cannabis sativa, tetrahydrocannabinol (THC) and cannabidiol (CBD), can be grouped into three main classes (THC-type, CBD-type, and intermediate type). We tested a genetic model associating these three groups with functional and nonfunctional alleles of the cannabidiolic acid synthase gene (CBDAS).
METHODS: We characterized cannabinoid content and assayed CBDAS genotypes of >300 feral C. sativa plants in Minnesota, United States. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and 12 Canadian-certified grain cultivars were also examined.
RESULTS: Frequencies of CBD-type, intermediate-type, and THC-type feral plants were 0.88, 0.11, and 0.01, respectively. Although total cannabinoid content varied substantially, the three groupings were perfectly correlated with CBDAS genotypes. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. Despite significant mean differences in total cannabinoid content, CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, and the same was true for industrial grain cultivars when plants exhibited >0.5% total cannabinoid content.
CONCLUSIONS: Our results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. Cannabinoid profiles and associated CBDAS segregation patterns suggest that feral C. sativa populations are potentially valuable experimental systems and sources of germplasm

Variations of 9-THC content in single plants of hemp varieties
Klemens Mechtler, Josef Bailer, Karl de Hueber
Industrial Crops and Products. 19(1), 19-24
DOI: 10.1016/S0926-6690(03)00077-3
Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. To test the distribution of 9-tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of five well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30–61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribution of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single-plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC values far outside this bandwidth.

Not Cannabis specific
When the going gets tough, the tough turn female: injury and sex expression in a sex-changing tree
Jennifer Blake-Mahmud, and Lena Struwe
American Journal of Botany 107(2): 339–349. May 2019
doi: 10.1002/ajb2.1427
https://www.ncib.nlm.nih.gov/pmc/articles/PMC7155049/
PREMISE: Plant sex is usually fixed, but in rare cases, sex expression is flexible and may be influenced by environmental factors. Theory links female sex expression to better health, but manipulative work involving the experimental change of health via injury is limited, particularly in sexually plastic species. A better understanding of mechanisms influencing shifts in sex is essential to our understanding of life history theory regarding trade-offs in sex allocation and differential mortality.
METHODS: We investigated the relationship between physiological stress and sex expression in sexually plastic striped maple trees (Acer pensylvanicum) by inflicting damage of various intensities (crown pruning, defoliation, and hydraulic restriction). We then monitored the sex expression of injured and control individuals for 2 years to assess the extent to which injury may cue changes in sex expression.
RESULTS: We found that severe damage such as full defoliation or severe pruning increased odds of changing sex to female and decreased odds of changing to male. In fact, no pruned male trees flowered male 2 years later, while all males in the control group flowered partially or fully male. After full defoliation, trees had 4.5 times higher odds of flowering female. Not all injury is equal; less-severe physical trauma did not affect the frequency of sex change to femaleness.
CONCLUSIONS: This work demonstrates that physical trauma in striped maple appears to exhibit a threshold effect in which only the most stressful of physiological cues instigate changes in sex expression, a phenomenon previously unknown, and that damage stress is strongly correlated with switching to femaleness. These findings have implications for population sex ratios and sustainability within an increasing stressful climate regime.

Not Cannabis specific
*WO2020239984A1 Gene for Parthenogensis Patent
https://patentimages.storage.googleapi s.com/d1/99/59/c922ddef2fd1b8/WO2020239984A1.pdf
https://patents.google.com/patent/WO2020239984A1/en
The invention provides the nucleotide sequence and amino acid sequences of the parthenogenesis gene of Taraxacum as well as (functional) homologues, fragments and variants thereof, which provides parthenogenesis as a part of apomixis. Also parthenogenetic plants and methods for making these are provided, as are molecular markers and methods of using these.
Title: Gene for Parthenogenesis
Field of the invention
The present invention relates to the field of biotechnology and in particular to plant biotechnology including plant breeding. The invention relates in particular to the identification and uses of genes relating to and useful e.g. in apomixis and haploid induction. The invention in particular relates to the gene that is associated with parthenogenesis, as well as the encoded protein, and fragments of both. The invention further relates to methods for suppressing and/or inducing parthenogenesis in plants and crops, to the use of the gene and/or the protein or their fragments for apomixis in particular in combination with apomeiotic gene(s), or for the production of haploid plants of which the chromosomes can be doubled to produce doubled haploids.
 
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BabTaHerbless

New member
May you Sam pardon my vibrancy but... I believe this could be a great, great project. something like a participated weed's Encyclopedia that recall me Diderot's one.
i guess you'll need a press office, LOL.

the kind of ambition i do really love.
Once more: great, great idea!
Keep it up!


respect
 

bsgospel

Bat Macumba
Query: and I think I already know the answer but here goes...

This McPartland paper from last year- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225593/

I'm thinking it goes under classification/taxonomy but there is quite a bit of history in here as well. How would you like to address papers which may fall into multiple categories?

Suggest any paper that needs to be in more than one SUBJECT and I will post it under more than one SUBJECT-SamS
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Cannabinoids


926 PubMed Cannabidiol Studies

[/COLOR]https://medicalmarijuana.procon.org/files/926 PubMed Cannabidiol S tudies.pdf

PubChem 7-Hydroxycannabidiol (Compound)
Chemical Co-Occurrences in Literature
https://pubchem.ncbi.nlm.nih.gov/com...ion=Literature

*A historical overview of chemical research on cannabinoids
Raphael Mechoulam , Lum?´r Hanus?
Chemistry and Physics of Lipids 108 (2000) 1–13
DOI: 10.1016/S0009-3084(00)00184-5
The chemical research on the plant cannabinoids and their derivatives over two centuries is concisely reviewed. The tortuous path leading to the discovery of the endogenous cannabinoids is described. Future directions, which will probably be followed are delineated.

A Network Pharmacology Approach to Identify Potential Molecular Targets for Cannabidiol’s Anti-Inflammatory Activity
Hang Ma,1,* Feng Xu,1,2 Chang Liu,1 and Navindra P. Seeram
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0025
Introduction: Published preclinical and clinical studies support the anti-inflammatory activity of CBD, but the molecular targets (e.g., genes and proteins) that are involved in its mechanisms of action remain unclear. Herein, a network-based pharmacology analysis was performed to aid in the identification of potential molecular targets for CBD’s anti-inflammatory activity. Materials and Methods: Target genes and proteins were obtained from several online databases, including Swiss target prediction, Online Mendelian Inheritance in Man, and the DrugBank database. A compound-targetdisease network was constructed with Cytoscape tool, and a network of protein–protein interactions was established with the Search Tool for the Retrieval of Interacting Genes/Proteins database. Lead proteins identified from the compound-target-disease network were further studied for their interactions with CBD by computational docking. In addition, biological pathways involved in CBD’s anti-inflammatory activity were identified with the Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes analysis. Results: A panel of proteins, including cellular tumor antigen p53, NF-kappa-B essential modulator, tumor necrosis factor (TNF) receptor, transcription factor p65, NF-kappa-B p105, NF-kappa-B inhibitor alpha, inhibitor of nuclear factor kappa-B kinase subunit alpha, and epidermal growth factor receptor, were identified as lead targets involved in CBD’s anti-inflammatory activity. This finding was further supported by molecular docking, which showed interactions between the lead proteins and CBD. In addition, several signaling pathways, including TNF, toll-like receptor, mitogen-activated protein kinases, nuclear factor kappa-light-chain-enhancer of activated B cells, and nucleotide-binding oligomerization domain-like receptors, were identified as key regulators in the mediation of CBD’s anti-inflammatory activity. Conclusion: A network-based pharmacology analysis identified potential molecular targets and signaling pathways for CBD’s anti-inflammatory activity. Findings from this study add to the growing body of data supporting the utilization of CBD as a promising anti-inflammatory natural product.

A New Cannabinoid, ¢6-Tetrahydrocannabinol 2¢-O-â-D-Glucopyranoside, Biotransformed by Plant Tissue
Hiroyuki Tanaka, Ryuji Takahashi, Satoshi Morimoto, and Yukihiro Shoyama
J. Nat. Prod. 1997, 60, 168-170
DOI: 10.1021/np9604846
?6-Tetrahydrocannabinol ( ¢6-THC, 1) was converted mainly to 1 2¢-O-â-D-glucopyranoside (2) using tissue segments of Pinellia ternata tubers. In time-course experiments, 1 was absorbed rapidly by the tissues and glucosylated.

A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis.
Berman, P., Futoran, K., Lewitus, G. M., Mukha, D., Benami, M., Shlomi, T., & Meiri, D.
Scientific Reports, 8(1).(2018).
doi:10.1038/s41598-018-32651-4
Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-?9- trans-tetrahydrocannabinol (?9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic efects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufcient to comprehend the medicinal and pharmacological efects of the whole plant. In this study we suggest a new ESI-LC/ MS/MS approach to identify phytocannabinoids from 10 diferent subclasses, and comprehensively profle the identifed compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identifed and used for profling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant efects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same efects. This study’s approach for phytocannabinoid profling can enable researchers and physicians to analyze the efects of specifc Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis

A new tetrahydrocannabinolic acid.
Mechoulam, R., Ben-Zvi, Z., Yagnitinsky, B., & Shani, A.
Tetrahedron Letters, 10(28), 2339–2341. (1969).
doi:10.1016/s0040-4039(01)88158-2
A major component of hashish is the acid I (1) which, though inactive per se, is converted on smoking into the active A'-tetrabydrocannabinol (al-TIE) (II) (2). We wish to report now the presence in some hashish samples of a second&'-TIE acid, III, which on heating, and presumably on smoking, is likewise converted into A'-TRC. In order to differentiate between the two acids we suggest that acid I be named 'I o'-THC acid A" and III ti $-TIE acid B". Careful chromatography of the cannabinoid acidic material of a hashish sole on silicic acid (Merck, 70-325 mesh) yielded cannabidiolic acid (3), (eluted with lO$ ether in pet. ether), A'-THC acid B (eluted with 50$ ether in pet. ether) end cannabigerolic acid (3)(eluted with ether). In this hashish sample III was present in 0.556 over-all concentration.

A new type of cannabinoid. Synthesis of cannabielsoic acid A by a novel photo-oxidative cyclisation.
Shani, A., & Mechoulam, R.
Journal of the Chemical Society D: Chemical Communications, (5), 273.(1970).
doi:10.1039/c29700000273
Two tricyclic dihydrobenzofuran cannabinoids
have been isolated and one of them has been synthesized
by an intramolecular photo-oxidative cyclisation which
involves attack by a phenoxy-group and molecular
oxygen on a double bond.

A novel phytocannabinoid isolated from Cannabis sativa L. with an in vivo cannabimimetic activity higher than ?9-tetrahydrocannabinol: ?9-Tetrahydrocannabiphorol
CinziaCitti, Pasquale Linciano, Fabiana Russo, Livio Luongo, Monica Iannotta, Sabatino Maione, Aldo Laganà, Anna Laura Capriotti, Flavio Forni , Maria Angela Vandelli, Giuseppe Gigli & Giuseppe Cannazza
Scientific Reports volume 9, Article number: 20335 (2019)
doi.org/10.1038/s41598-019-56785-1
-)-Trans-?9-tetrahydrocannabinol (?9-THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain infuences the biological activity of this cannabinoid. In particular, synthetic analogues of ?9-THC with a longer side chain have shown cannabimimetic properties far higher than ?9-THC itself. In the attempt to defne the phytocannabinoids profle that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of ?9-THC but with a seven-term alkyl side chain was identifed. The natural compound was isolated and fully characterized and its stereochemical confguration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-?9-tetrahydrocannabiphorol (?9-THCP). Along with ?9-THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identifed by match with its synthetic counterpart. The binding activity of ?9-THCP against human CB1 receptor in vitro (Ki=1.2nM) resulted similar to that of CP55940 (Ki=0.9nM), a potent full CB1 agonist. In the cannabinoid tetrad pharmacological test, ?9-THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity. The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difcult to explain by the presence of the sole ?9-THC.

A PCR marker Linked to a THCA synthase Polymorphism is a Reliable Tool to Discriminate Potentially THC-Rich Plants of Cannabis sativa L.
Christina Staginnus Ph.D., Siegfried Zörntlein Ph.D., Etienne de Meijer Ph.D.
Journal of Forensic Sciences
DOI: 10.1111/1556-4029.12448
Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant’s life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (BT and BD). The homozygous BT/BT genotype underlies the THC-predominant phenotype, BD/BD is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (BT/BD). Using PCR-based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC-predominant or the THC-intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species-specific marker system
was applied to a large number of casework samples and fiber hemp cultivars.

A Phase I, Randomized, Double?Blind, Placebo?Controlled, Single Ascending Dose, Multiple Dose, and*Food Efect Trial of*the*Safety, Tolerability and*Pharmacokinetics of*Highly Purifed Cannabidiol in*Healthy Subjects
Lesley*Taylor, Barry*Gidal, Graham*Blakey, Bola*Tayo, Gilmour*Morrison
CNS Drugs (2018) 32:1053–1067
doi: 10.1007/s40263-018-0578-5
Background A formal single ascending and multiple dose pharmacokinetic (PK) trial of cannabidiol (CBD) oral solution was required to determine the safety and tolerability of CBD, the maximum tolerated dose, and to examine the efect of food on CBD PK parameters.
Objective This trial assessed the safety, tolerability and PK of CBD oral solution in healthy adult volunteers, as well as the efect of food on CBD PK parameters.
Methods The study consisted of three*arms: single ascending dose (1500, 3000, 4500 or 6000*mg CBD [n=6 per group]/ placebo [n=8; 2 per CBD dose group]), multiple dose (750*or 1500*mg CBD [n=9 per group]/placebo [n=6; 3 per CBD dose group] twice daily), and food efect (1500*mg CBD single dose [n=12]). All subjects completed all trial arms and were analyzed as planned.
Results CBD was generally well tolerated. Diarrhea, nausea, headache, and somnolence were the most common adverse events (AEs) across all trial arms, with an increased incidence of some gastrointestinal and nervous system disorder AEs (most notably diarrhea and headache) apparent in subjects taking CBD compared with placebo. All AEs were of mild or moderate severity; none were severe or serious. There were no deaths or discontinuations in the trial. After single oral doses, CBD appeared rapidly in plasma; time to maximum plasma concentration (tmax) was approximately 4–5*h. The major circulating metabolite was 7-carboxy-CBD, then parent CBD, 7-hydroxy-CBD (active metabolite), and 6-hydroxy-CBD (a relatively minor metabolite). Plasma exposure to CBD [maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from time zero to time t (AUCt)] increased in a less than dose-proportional manner (Cmax slope 0.73; AUCt slope 0.64). Oral clearance of CBD was high (1111–1909*L/h) and apparent volume of distribution was large (20,963–42,849*L). CBD reached steady state after approximately 2*days, with moderate accumulation (1.8- to 2.6-fold) after 750*and 1500*mg CBD twice daily. After 7*days, a twofold increase in CBD dose resulted in 1.6- and 1.9-fold increases
in geometric mean Cmax and area under the plasma concentration-time curve over a dosing interval (AUC?), respectively. CBD elimination was multiphasic; the terminal elimination half-life was approximately 60*h after 750 and 1500*mg CBD twice daily; and efective half-life estimates ranged from 10 to 17*h. Cmax was 541.2*ng/mL and AUC? was 3236*ng·h/mL after 1500*mg CBD twice daily. A high-fat meal increased CBD plasma exposure (Cmax and AUCt) by 4.85- and 4.2-fold, respectively; there was no efect of food on tmax or terminal half-life.
Conclusion CBD was generally well tolerated. Most AEs were mild in severity; none were severe or serious. The safety and PK profle support twice-daily administration of CBD.

A PHYSIOLOGICALLY ACTIVE PRINCIPLE FROM CANNABIS
HAAGEN-SMIT, A. J., WAWRA, C. Z., KOEPFLI, J. B., ALLES, G. A., FEIGEN, G. A., & PRATER, A. N.
SATIVA (MARIHUANA).
Science, 91(2373), 602–603. (1940).
doi:10.1126/science.91.2373.602
While it has long been known that the physiological activity of Cannabis sativa (marihuana or hashish) is associated with its contained resins, no physiologically active crystalline material has heretofore been isolated.
We report in this note the isolation of such a substance. The hydrocarbon nonacosane and an oily product termed canabinol were first isolated by Wood, Spivey and Easterfie1d In 1938 Bergel, Todd and Work reported the preparation of a crystallilie p-nitro benzoate of cannabinol which could be used to separate the cannabinol from the oil by chromatographic adsorption methods. Recently an oily product which was named cannabidiol was isolated by Adams, Hunt and Clark. None of these well-defined products has exhibited the characteristic physiological activities that are shown by the crude drug though canabinol was found to be quite toxic. Reviews of the earlier work on the chemistry of Cannabis have been published by Walton and by Blatt.
Work on the separation of physiologically active fractions from alcoholic extracts of Cannabis sativa has been in progress for the past year in our laboratories. The extracts of Minnesota wild hemp used for the work were generously supplied by the Narcotics Laboratory, United States Treasury Department, and we are indebted to Nessrs. H. J. Anslinger and H. J. Wollner for their collaboration which made this work possible. The alcohol extract of the crude drug was diluted with water to yield a seventy per cent, alcohol solution, and this vas partitioned into petroleum ether. Salt forming compounds were extracted and then colored substances were largely removed by adsorption on zinc carbonate. The resultant resinous material was fractionally precipitated from methanol with water and there was obtained a physiologically active fraction of about one twentieth the weight of the crude resin material. This purified product 1~as fractionally distilled under 0.005 mm pressure, with the most active fraction distilling at 128O-135O C. This fraction is a red-colored oil which shows typical activity in dogs following an oral dose of 1.0 mg per kg. By cooling a solution of this oil in a methanol-acetic acid mixture, some crystalline material was obtained. This was then recrystallized several times from methanol to yield colorless needles melting at 128O-129O C.

A polar cannabinoid from Cannabis sativa var. Carma
Giovanni Appendino, Giana A, Simon Gibbons, Massimo E Maffei, Giorgio Gnavi, Gianpaolo Grassi ??
Natural product communications (Impact Factor: 0.92). 01/2008; 3(12):1977-1980.
DOI:10.1177/1934578x0800301207
ABSTRACT The aerial parts of Cannabis sativa var. Carma afforded a novel polar cannabinoid whose structure was established as rac-6?,7?-dihydro, 6?,7?-dihydroxycannabigerol (carmagerol, 1) on the basis of spectroscopic data and semisynthesis from cannabigerol (2a). The dihydroxylation of the ?-double bond was detrimental to the anti-bacterial activity.

A Random Walk Through a Cannabis Field
R. MECHOULAM, W. A. DEVANE, A. BREUER AND J. ZAHALKA
Pharmacology Biochemistry & Behavior, Vol. 40, pp. 461--464.
doi: 10.1016/0091-3057(91)90347-5.
The present overview covers various aspects of research going on in the Cannabis field in the Department of Natural Products at the Hebrew University. In the first part we discuss, and try to explain, the reason for the absence of the term Cannabis (and possibly also opium) in the Old Testament. In the second part we bring evidence that, contrary to widely held views, stereospecificity of cannabinoid action is extremely high, and in certain cases almost absolute. Previous results seem to have been due to impurities in the samples tested. (+)-Delta-I-THC, (+)-delta-6-THC and (+)-7-hyroxy-delta-6-THC, when purified sufficiently, exhibit activity of about 1% of that of the natural ( - ) enantiomers. A new labeled carmabinoid ligand has been prepared by catalytic reduction of (-)-7-hydroxy-delta-6-THC dimethylheptyl. The equatorial C-1 epimer obtained binds to the cannabinoid receptor with a K I of 40 pM. This compound is one of the most active cannabinoids tested so far for binding to the canabinoid receptor, and may become an important tool in cannabinoid research


A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples
Fidelia Cascini, Simona Martello
Forensic Science International 217 (2012) 134–138
DOI: 10.1016/j.forsciint.2011.10.041
In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drugtype from the fibre-type varieties.
A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency
plants, we chose the DDCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished.
As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested.

A Systematic Review on the Pharmacokinetics of Cannabidiol in Humans
Sophie A. Millar, Nicole L. Stone, Andrew S. Yates, Saoirse E. O'Sullivan
Frontiers in Pharmacology, 9.
doi:10.3389/fphar.2018.01365*
Background: Cannabidiol is being pursued as a therapeutic treatment for multiple conditions, usually by oral delivery. Animal studies suggest oral bioavailability is low, but literature in humans is not sufficient lacking. The aim of this review was to collate published data in this area.
Methods: A systematic search of PubMed and EMBASE (including MEDLINE) was conducted to retrieve all articles reporting pharmacokinetic data of CBD in humans.
Results: Of 792 articles retireved, 24 included pharmacokinetic parameters in humans. The half-life of cannabidiol was reported between 1.4-10.9 hours after oromucosal spray, 2-5 days after chronic oral administration, 24 hours after i.v., and 31 hours after smoking. Bioavailability following smoking was 31% however no other studies attempted to report the absolute bioavailability of CBD following other routes in humans, despite i.v formulations being available. The area-under-the-curve and Cmax increase in dose-dependent manners and are reached quicker following smoking/inhalation compared to oral/oromucosal routes. Cmax is increased during fed states and in lipid formulations. Tmax is reached between 0-4 hours.
Conclusions: This review highlights the paucity in data and some discrepancy in the pharmacokinetics of cannabidiol, is area despite its widespread use of cannabidiol in humans. Analysis and understanding of these properties such as bioavailability and half-life is critical to future therapeutic success, and robust data from a variety of formulations is required.

A Total Synthesis of dl-?1-Tetrahydrocannabinol, the Active Constituent of Hashish.
Mechoulam, R., & Gaoni, Y.
Journal of the American Chemical Society, 87(14), 3273–3275.(1965).
doi:10.1021/ja01092a065
We wish to report that we have completed the first total synthesis2 of (//-cannabidiol3 (la) and (//-A4-3,4-trans-tetrahydrocannabinol4·5 (II), the psychotomimetically active constituent of hashish (marihuana)

Accumulation of bioactive metabolites in cultivated medical Cannabis
Richard D. Richins, Laura Rodriguez-Uribe, Kiah Lowe, Rebekah Ferral, Mary A. O’Connell
DOI: 10.1371/journal.pone.0201119
There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

Accumulation of Cannabinoids in Glandular Trichomes of Cannabis (Cannabaceae)
Paul G. Mahlberg, Eun Soo Kim
June 2004 Journal of Industrial Hemp 9(1):15-36
DOI: 10.1300/J237v09n01_04
Sessile- and capitate-stalked secretory glands are sites of cannabinoid accumulation in Cannabis (Cannabaceae). Analyses show cannabinoids to be abundant in glands isolated from bracts or leaves of pistillate plants. Cannabinoids are concentrated in the secretory cavity formed as an intrawall cavity in the outer wall of the disc cells. Specialized plastids, lipoplasts, in the disc cells synthesize lipophilic substances, such as terpenes, that migrate through the plasma membrane and into the cell wall adjacent to the secretory cavity. These substances enter the cavity as secretory vesicles. An antibody probe for THC shows it to be most abundant along the surface of vesicles, associated with fibrillar material in the cavity, in the cell wall and in the cuticle; little THC was detected in the cytoplasm of disc or other cells. The phenol, phloroglucinol, is abundant in both gland types. A working hypothesis for the site of cannabinoid synthesis is proposed, and must be examined further. Knowledge of the mechanism of cannabinoid synthesis and localization can contribute to efforts to further reduce the THC content in hemp strains for potential agricultural use in the United States and elsewhere.

Acidic Cannabinoid Decarboxylation
Crist N. Filer
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0072
https://www.liebertpub.com/doi/pdf/1.../can.2021.0072
Introduction: Cannabis is a valuable plant, cultivated by humans for millennia. However, it has only been in the past several decades that biologists have begun to clarify the interesting Cannabis biosynthesis details, especially the production of its fascinating natural products termed acidic cannabinoids. Discussion: Acidic cannabinoids can experience a common organic chemistry reaction known as decarboxylation, transforming them into structural analogues referred to as neutral cannabinoids with far different pharmacology. This review addresses acidic and neutral cannabinoid structural pairs, when and where acidic cannabinoid decarboxylation occurs, the kinetics and mechanism of the decarboxylation reaction as well as possible future directions for this topic. Conclusions: Acidic cannabinoid decarboxylation is a unique transformation that has been increasingly investigated over the past several decades. Understanding how acidic cannabinoid decarboxylation occurs naturally as well as how it can be promoted or prevented during harvesting or storage is important for the various stakeholders in Cannabis cultivation.

Alkaloids of Cannabis sativa leaves.
El-Feraly, F. S., & Turner, C. E.
Phytochemistry, 14(10), 2304. (1975)
doi:10.1016/s0031-9422(00)91127-0
Plant. Cannabis sativa L. Cannabinaceae (leaves) Source. Research Institute of Pharmaceutical Sciences, University of Mississippi, Lot CQUF-72, QU-A(l)/C-70, S-62, grown in 1972. Previous work. On leaves: choline and trigonelline [ 11, muscarine [2], an unidentified betain [3], and cannabamines A-D, all as yet of unknown structure [4]. Present work. Dried ground leaves were extracted by percolation with 95’;/0 EtOH at room temp. After removal of solvent irl uacuo at 40”, the residue (14%) was partitioned between 2% citric acid and CHCI,. The aqueous phase was rendered alkaline with cont. NH, and extracted with CHCI,. The alkaloidal fraction thus obtained was
further purified by dissolving in 1N HCl and extraction with CHCl,, then alkalinized with cont. NH, and reextracted with CHCI,. This latter fraction was chromatographed on Si gel G.

Allergenic Properties of Naturally Occurring Cannabinoids.
Watson, E. S., Murphy, J. C., & Turner, C. E.
Journal of Pharmaceutical Sciences, 72(8), 954–955. (1983).
doi:10.1002/jps.2600720831
The guinea pig maximization test was used to determine the potential of seven cannabinoids to produce allergic contact dermatitis. A9-Tetrahydrocannabinol and cannahinol were found to be extreme (Grade V) sensitizers. Cannabidiol, A8-tetrahydrocannabinol, and cannabichromene were moderate (Grade 111) sensitizers. Cannabigerol and cannabinol methyl ether were not sensitizers. Most of the cannabinoids were found to be allergenically cross-reactive. Additionally, it was shown that the presence of a free 1’-hydroxyl group was required for sensitization, but not to elicit a response in sensitive animals.

AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor
Daniele Bolognini, Maria Grazia Cascio1, Daniela Parolaro and Roger G Pertwee
British Journal of Pharmacology (2012) 165 2561–2574
DOI:10.1111/j.1476-5381.2011.01503.x
BACKGROUND AND PURPOSE We have investigated how pre-incubating hCB2 CHO cells with the CB2 receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB2 receptors in these cells or their membranes.
EXPERIMENTAL APPROACH We tested the ability of AM630, SR144528 and of the CB1/CB2 receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB2 CHO cells or [35S]-GTPgS binding to membranes prepared from these cells, or to displace [3 H]-CP55940 from whole cells and membranes. Assays were also performed with the CB2 receptor partial agonist, D9 -tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively.
KEY RESULTS AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of D9 -tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [3 H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [35S]-GTPgS membrane assay.
CONCLUSIONS AND IMPLICATIONS These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB2 receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528.

An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol.
Morales, P., Reggio, P. H., & Jagerovic, N.
Frontiers in Pharmacology, 8. (2017).
doi:10.3389/fphar.2017.00422
Cannabidiol (CBD) has been traditionally used in Cannabis-based preparation, however historically, it has received far less interest as a single drug than the other components of Cannabis. Currently, CBD generates considerable interest due to its beneficial neuroprotective, antiepileptic, anxiolytic, antipsychotic, and anti-inflammatory properties. Therefore, the CBD scaffold becomes of increasing interest for medicinal chemists. This review provides an overview of the chemical structure of natural and synthetic CBD derivatives including the molecular targets associated with these compounds. A clear identification of their biological targets has been shown to be still very challenging.

Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog.
Citti, C., Linciano, P., Forni, F., Vandelli, M. A., Gigli, G., Laganà, A., & Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis. (2019). doi:10.1016/j.jpba.2019.06.049
Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use.

Analytical Classes of Cannabinol Compounds in Marihuana Resin
CHARLES C. FULTON
I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 14, No 5 1942
DOI:10.1021/i560105a010
In 1896 Wood, Spivey, and Easterfield isolated from can-nabis resin a toxic “red oil”, seemingly a definite chemical compound, which they named “cannabinol”. In 1899 they isolated a pure compound from the red oil (as the crystalline acetate) and transferred to it this name cannabinol. Their own work, and that of Cahn in 1931 showed that the “crude cannabinol” was not just an impure form of pure cannabinol, but rather a mixture of closely related compounds. Other authors, however, especially before 1931, continued to refer to the complex of related compounds as cannabinol. Blatt, in reviewing the literature in 1938, used “crude cannabinol” intentionally as a collective name for these compounds. In this paper all the compounds of the resin, whether they go into the distilled red oil or not, which are closely related to cannabinol, are referred to collectively as “cannabinol compounds”. In 1940 Adams, Hunt, and Clark reported the isolation of another of these related compounds, naming it “cannabidiol” Jacob and Todd have isolated still another, calling it “cannabol”. Pure cannabinol and cannabidiol are stated not to have the specific narcotic effect of marihuana, but Adams and his co-workers have shown that cannabidiol isomerizes to tetrahydrocannabinol (which dehydrogenation converts to cannabinol), and that this compound, and also hexahydrocannabinol, do have “marihuana activity”

Anhydrocannabisativine, a New Alkaloid from Cannabis sativa
Elsohly, M. A., Turner, C. E., Phoebe, C. H., Knapp, J. E., Schiff, P. L., & Slatkin, D. J.
L. Journal of Pharmaceutical Sciences, 67(1),124 (1978)
doi:10.1002/jps.2600670135
Ethanol extracts of the leaves and roots of a Mexican variant of Cannabis satiua L. (marijuana) afforded, after partitioning and chromatography, the new spermidine alkaloid, anhydrocannabisativine. The structure was determined by spectral analysis and semisynthesis.

Antibacterial Cannabinoids from Cannabis sativa: A Structure?Activity Study.
Appendino, G., Gibbons, S., Giana, A., Pagani, A., Grassi, G., Stavri, M., Rahman M. M.
Journal of Natural Product, 71(8), 1427–1430. (2008)
doi:10.1021/np8002673
Marijuana (Cannabis satiVa) has long been known to contain antibacterial cannabinoids, whose potential to address antibiotic resistance has not yet been investigated. All five major cannabinoids (cannabidiol (1b), cannabichromene (2), cannabigerol (3b), ?9-tetrahydrocannabinol (4b), and cannabinol (5)) showed potent activity against a variety of methicillin-resistant Staphylococcus aureus (MRSA) strains of current clinical relevance. Activity was remarkably tolerant to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids), and to carboxylation of the resorcinyl moiety (pre-cannabinoids). Conversely, methylation and acetylation of the phenolic hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were all detrimental for antibacterial activity. Taken together, these observations suggest that the prenyl moiety of cannabinoids serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore, while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.

Anti-Inflammatory Activity in Colon Models Is Derived from D9-Tetrahydrocannabinolic Acid That Interacts with Additional Compounds in Cannabis Extracts
Rameshprabu Nallathambi, Moran Mazuz, Aurel Ion, Gopinath Selvaraj, Smadar Weininger, Marcelo Fridlender, Ahmad Nasser, Oded Sagee, Puja Kumari, Diana Nemichenizer, Maayan Mendelovitz, Nave Firstein, Orly Hanin, Fred Konikoff, Yoram Kapulnik, Timna Naftali, and Hinanit Koltai
Cannabis and Cannabinoid Research, 2(1), 167–182.
DOI: 10.1089/can.2017.0027
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner. Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell cultureand colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

Anti-inflammatory cannabinoids in diet Towards a better understanding of CB2 receptor action?
Jürg Gertsch
[Communicative & Integrative Biology 1:1, 26-28; July/August/September 2008
https://www.ncbi.nlm.nih.gov/pmc/art...b0101_0026.pdf
DOI: 10.4161/cib.1.1.6568
The endocannabinoid system is an ancient lipid signaling network which in mammals modulates neuronal functions, inflammatory processes, and is involved in the aetiology of certain human lifestyle diseases, such as Crohn’s disease, atherosclerosis and osteoarthritis. The system is able to downregulate stress-related signals that lead to chronic inflammation and certain types of pain, but it is also involved in causing inflammation-associated symptoms, depending on the physiological context. The cannabinoid type-2 (CB2) receptor, which unlike the CB1 receptor does not induce central side effects, has been shown to be a promising therapeutic target. While CB1 receptor antagonists/inverse agonists are of therapeutic value, also CB2 receptor ligands including agonists are of pharmacological interest. Although the endocannabinoid system is known to be involved in the regulation of energy homoeostasis and metabolism (mainly via CB1 receptors) there was hitherto no direct link between food intake and cannabinoid receptor activation. Our recent finding that beta-caryophyllene, a ubiquitous lipohilic plant natural product, selectively binds to the CB2 receptor and acts as a full agonist is unexpected. Maybe even more unexpected is that oral administration of this dietary compound exerts potent anti-inflammatory effects in wild type mice but not in CB2 receptor (Cnr2-/-) knockout mice. Like other CB2 ligands also beta-caryophyllene inhibits the pathways triggered by activation of the toll-like receptor complex CD14/TLR4/ MD2, which typically lead to the expression of proinflammatory cytokines (IL-1?, IL-6; IL-8 and TNFalpha) and promotes a TH1 immune response. In this addendum, the CB2 receptor-dependent effect of beta-caryophyllene on LPS-triggered activation of the kinases Erk1/2 and JNK1/2 are further discussed with respect to the possibility that both CB2 inverse agonists and agonists, independent of their G-protein signaling, may block LPS-triggered activation of MAPKs, leading to inhibition of proinflammatory cytokine expression and attenuation of inflammation.

Anti-inflammatory properties of cannabichromene.
Wirth, P. W., Sue Watson, E., ElSohly, M., Turner, C. E., & Murphy, J.
Life Sciences, 26(23), 1991–1995.(1980)
doi:10.1016/0024-3205(80)90631-1
It was not known if Cannabichromene (CBC), which is a major constituent of drug types of Cannabis, has anti-inflammatory properties as do other cannabinoids. CBC was tested in vivo using the rat paw edema test and in vitro using the erythrocyte membrane stabilization assay. CBC was as effective as phenylbutazone (PBZ) at equivalent doses. Since CBC is less toxic than PBZ, larger doses may be given to produce a greater therapeutic effect. Cannabichromene (CBC) was first isolated from Cannabis sativa L.and its structure determined by Gaoni and Mechoulam in 1966 (I). For a long time CBC was mis-identified as cannabidiol (CBD) in most crude preparations from Cannabis, since CBC and CBD have the same relative retention time on most liquid phases used in gas chromatography analysis (2,3). An analytical procedure for the separation of CBC and CBD was reported in 1973 (3) and it is now known that CBC is more abundant than CBD in the more potent drug variants of Cannabis (4,5). Little pharmacological research has been done using CBC. Cannabis preparations have historically been used to relieve pain in both inflammatory and noninflammatory conditions (5). Investigations have shown A 9- THC to have anti-inflammatory effects (6,7,8,9,10), although some tests have failed to demonstrate this (11,12). The anti-inflammatory effects of CBD, cannabinol (CBN) and crude marihauna extract have also been tested (7,8) and all have been shown to have anti-inflammatory activity. The most potent anti-inflammatory activity among the cannabinoids is produced by Ag-THC followed, in decreasing order, by CBD, CBN, and crude marihauna. Recently a new synthesis of CBC was developed in this laboratory (13). This improved synthesis allowed the production of sufficient amounts of pure CBC to conduct anti-inflammatory testing. CBC was tested in vivo and in vitro to determine if CBC shares the anti-inflammatory effects of the other cannabinoids.

Application of a liquisolid technique to cannabis sativa extract compacts: Effect of liquid vehicles on the dissolution enhancement and stability of cannabinoids
Napaphak Jaipakdee, PeeraTabboon, Ekapol Limpongsa
International Journal of Pharmaceutics 10 November 2021, 121277
DOI: 10.1016/j.ijpharm.2021.121277
https://www.sciencedirect.com/scienc...838?via%3Dihub
This work describes the application of liquisolid technique to enhance cannabinoid dissolution from Cannabis sativa L. (CS) compacts. Effects of five vehicles, namely, volatile (ethanol) and nonvolatile (caprylocaproyl macrogolglycerides, polyethylene glycol 400, oleoyl macrogolglycerides and polysorbate 20) liquids, on tablet properties, dissolution and stability were investigated. The viscid oleoresin CS extract was mixed with vehicles before being transformed into free-flowing powder by the use of microcrystalline cellulose and colloidal silica as carrier and coating materials. Liquid vehicles had a nonsignificant effect on liquid load factor of CS extract. CS liquisolid compacts had acceptable tableting properties in terms of weight variation, friability, hardness, content uniformity and disintegration time. Different vehicles affected the hardness, disintegration, and wettability of CS compacts and thus the dissolution behaviors of cannabinoids to different extents. Dissolutions of cannabinoids from CS compacts were rate-limited by the disintegration process. Liquisolid formulations using nonvolatile liquids with low polarity or high hydrophilic-lipophilic balance yielded more than 90% cannabinoid dissolution. Stability studies revealed nonsignificant changes in tablet characteristics, cannabinoid content and dissolutions of CS compacts when stored at 5 ± 3 °C for 3 months. This work presents a general concept of how to successfully formulate CS extract with cannabinoid dissolution enhancement characteristics.
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Are cannabidiol and ?9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review
John M McPartland, Marnie Duncan, Vincenzo Di Marzo and Roger G Pertwee
British Journal of Pharmacology Volume 172, Issue 3, pages 737–753, February 2015
DOI: 10.1111/bph.12944
Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of ?9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and ?9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target.

Aromatic Prenyltransferase from Cannabis
Inventor Jonathan E. Page, Zakia Boubakir
US20120144523A1 Patent
Nucleic acid molecules from Cannabis sativa (cannabis, hemp, marijuana) have been isolated and characterized, and encode polypeptides having aromatic prenyltransferase activity. Specifically, the enzyme, CsPT1, is a geranylpyrophosphate olivetolate geranyltransferase, active in the cannabinoid biosynthesis step of prenylation of olivetolic acid to form cannabigerolic acid (CBGA). Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds. The polypeptides may be used in vivo or in vitro to produce cannabinoid compounds.

Association of Naturalistic Administration of Cannabis Flower and Concentrates With Intoxication and Impairment.
Bidwell, L. C., Ellingson, J. M., Karoly, H. C., YorkWilliams, S. L., Hitchcock, L. N., Tracy, B. L., … Hutchison, K. E.
JAMA Psychiatry.(2020).
doi:10.1001/jamapsychiatry.2020.0927
IMPORTANCE The rapidly growing legal cannabis market includes new and highly potent products, the effects of which, to our knowledge, have not previously been examined in biobehavioral research studies because of federal restrictions on cannabis research. OBJECTIVE To use federally compatible, observational methods to study high-Δ9- tetrahydrocannabinol (THC) legal market forms of cannabis.
DESIGN, SETTING, AND PARTICIPANTS In this cohort study with a between-groups design that was conducted in a community and university setting, cannabis flower users and concentrate users were randomly assigned to higher- vs lower-THC products within user groups. Participants completed a baseline and an experimental mobile laboratory assessment that included 3 points: before, immediately after, and 1 hour after ad libitum legal market flower and concentrate use. Of the 133 individuals enrolled and assessed, 55 regular flower cannabis users (41.4%) and 66 regular concentrate cannabis users (49.6%) complied with the study’s cannabis use instructions and had complete data across primary outcomes.
EXPOSURES Flower users were randomly assigned to use either 16% or 24% THC flower and concentrate users were randomly assigned to use either 70% or 90% THC concentrate that they purchased from a dispensary.
MAIN OUTCOMES AND MEASURES Primary outcome measures included plasma cannabinoids, subjective drug intoxication, and neurobehavioral tasks testing attention, memory, inhibitory control, and balance.
RESULTS A total of 121 participants completed the study for analysis: 55 flower users (mean [SD] age, 28.8 [8.1] years; 25 women [46%]) and 66 concentrate users (mean [SD] age, 28.3 [10.4] years; 30 women [45%]). Concentrate users compared with flower users exhibited higher plasma THC levels and 11-hydroxyΔ9 -THC (THC’s active metabolite) across all points. After ad libitum cannabis administration, mean plasma THC levels were 1016 (SE = 1380) μg/mL in concentrate users (to convert to millimoles per liter, multiply by 3.18) and 455 (SE = 503) μg/mL in flower users. Most neurobehavioral measures were not altered by short-term cannabis consumption. However, delayed verbal memory (F1,203 = 32.31; P < .001) and balance function (F1,203 = 18.88; P < .001) were impaired after use. Differing outcomes for the type of product (flower vs concentrate) or potency within products were not observed. CONCLUSIONS AND RELEVANCE This study provides information about the association of pharmacological and neurobehavioral outcomes with legal market cannabis. Short-term use of concentrates was associated with higher levels of THC exposure. Across forms of cannabis and potencies, users’ domains of verbal memory and proprioception-focused postural stability were primarily associated with THC administration

Associations between cannabinoids and growth stages of twelve industrial hemp cultivars grown outdoors in Atlantic Canada
NadaHammami, a Jean-PierrePrivé, b David L.Joly,a GaétanMoreau
Industrial Crops and Products Vol 172. 15 nov 2921
doi.org/10.1016/j.indcrop.2021.113997
Industrial hemp is increasingly grown and harvested for its cannabinoids of pharmaceutical interest. These compounds are generally obtained from plants harvested at maturity but not all cannabinoids are present or abundant during the last stage of hemp development. This study examined intraspecific cannabinoid variability during ontogenic development of hemp to identify growth stages and cultivars that optimize production of specific compounds. The cannabinoid content of twelve commercial industrial hemp (Cannabis sativa L. subsp. sativa) cultivars at three growth stages (i.e., flowering, grain filling and maturity) was determined by high performance liquid chromatography in an experimental field in Cocagne, New Brunswick, Canada. Most cannabinoids in acidic or neutral form were more abundant at maturity. However, cannabigerolic acid, a precursor to all acidic and neutral forms of cannabinoids mentioned in our study, was more abundant during the grain-filling stage. In contrast, cannabichromene was associated with the flowering stage and found in greater abundances in grain cultivars than in dual-purpose cultivars. The cultivar Katani exhibited higher concentrations of most cannabinoids while the cultivars Ferimon, Altair and Anka exhibited higher concentrations of cannabinoid acidic precursors. The current study could help optimize the targeted production of cannabinoids at specific growth stages and to identify the chemical phenotype of different hemp cultivars.
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Base-catalysed Double-bond lsomerizations of Cannabinoids: Structural and StereochemicaI Aspects
Morris Srebnik, Naphtali Lander, Aviva Breuer, and Raphael Mechoulam
J. CHEM. SOC. PERKIN TRANS. I 1984
https://pdfs.semanticscholar.org/fae...66db9943f6.pdf
DOI: 10.1039/P19840002881
Base-catalysed double- bond isomerization offers a convenient, high- yield route to a variety of new cannabinoids, as well as to compounds of this group which are accessible with some difficulty. By this route we have obtained optically active A2-tetrahydrocannabinols and A3-tetrahydrocannabinols, the A6-isomer of cannabidiol, and other novel cannabinoids.

BETA CARYOPHYLLENE - A TERPENE OR A CANNABINOID?
Noel Palmer
https://www.cbxsciences.com/blog/201...-a-cannabinoid
Most people understand that cannabis is responsible for producing cannabinoids; most notably delta-9 THC and CBD. What many people don’t fully understand is that cannabis produces other phytochemicals (chemicals produced by plants) that are therapeutically active. Terpenes are a class of phytochemicals produced by cannabis (and other plants), and in fact scientists believe that terpenes serve as the building blocks for cannabinoids in the cannabis plant. That being said, terpenes in and of themselves are considered to be therapeutically relevant in many ways.

Beta-caryophyllene is a dietary cannabinoid.
Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, Altmann KH, Karsak M, Zimmer A.
PNAS July 1, 2008 105 (26) 9099-9104
doi: 10.1073/pnas.0803601105
The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Bioactive Prenylogous Cannabinoid from Fiber Hemp (Cannabis sativa)
Federica Pollastro, Orazio Taglialatela-Scafati, Marco Allarà, Giovanni Appendino
Journal of Natural Products 74(9):2019-22 September 2011
DOI: 10.1021/np200500p
https://www.researchgate.net/publica...annabis_sativa
The waxy fraction from the variety Carma of fiber hemp (Cannabis sativa) afforded the unusual cannabinoid 4, identified as the farnesyl prenylogue of cannabigerol (CBG, 1) on the basis of its spectroscopic properties. A comparative study of the profile of 4 and 1 toward metabotropic (CB1, CB2) and ionotropic (TRPV1, TRPV2, TRPM8, TRPA1) targets of phytocannabinoids showed that prenylogation increased potency toward CB2 by ca. 5-fold, with no substantial difference toward the other end-points, except for a decreased affinity for TRPM8. The isolation of 4 suggests that C. sativa could contain yet-to-be-discovered prenylogous versions of medicinally relevant cannabinoids, for which their biological profiles could offer interesting opportunities for biomedical exploitation.

Biological Activity of Cannabichromene, its Homologs and Isomers
CARLTON E. TURNER, MAHMOUD A. ELSOHLY
August 1981 The Journal of Clinical Pharmacology 21(8-9 Suppl):283S-291S
DOI: 10.1002/j.1552-4604.1981.tb02606.x
Cannabichromene (GBC) is one of four major cannabinoids in Cannabis sativa L. and is the second most abundant cannabi noid in drug-type cannabis. Cannabichromene and some of its homologs, analogs, and isomers were evaluated for antiinflammatory, antibacterial, and antifungal activity. Antiinflammatory activity was evaluated by the carrageenan-induced rat paw edema and the erythrocyte membrane stabilization method. In both tests, CBC was superior to phenylbutazone. Antibacterial activity of CBC and its isomers and homologs was evaluated using gram-positive,
gram-negative, and acid-fast bacteria. Antifungal activity was evaluated using yeastlike and filamentous fungi and a dermatophyte. Antibacterial activity was strong, and the antifungal activity was mild to moderate.

Not quite true some were discovered earlier
BioMedican Discovers New Class of Cannabinoids; Sesqui CBG
Breakthrough discovery of a new class of Cannabinoids - Sesqui CBG
BioMedican
https://www.prnewswire.com/news-rele...0431-365750754
BioMedican has discovered a patent-pending process to biosynthesize a new class of rare Cannabinoids called Sesqui-Cannabinoids. In this class of rare Cannabinoids, there is only one paper that describes the properties of Sesqui-CBG in 2011, but there are several articles that note the presence of Sesqui-CBGA or even Sesqui-CBDA in plants, but only in the list of detected compounds (no properties described). The company filed a patent last month, and BioMedican can now produce Sesqui-CBG. In the next six months, the company expects to be able to produce Sesqui-CBD, Sesqui-CBN, Sesqui-THCV, Sesqui-THCA

Biosynthesis and Chemical Modifications of Minor Cannabinoids
Gia-Nam Nguyen, Oliver Kayser,
In book: eLS
DOI: 10.1002/9780470015902.a0028875
https://www.researchgate.net/publica...r_Cannabinoids
Minor cannabinoids are cannabinoids at very low abundances in Cannabis sativa L. essential oils (<0.5% of dried weight). The pharmacological properties of minor cannabinoids have never been determined or validated in clinical assays or in rational in vitro and in vivo assays. Due to this lack of biological and pharmacological information,
minor cannabinoids have great potential as future drug candidates. The biosynthesis of minor cannabinoids is not clear in C. sativa L., but, likely, chemical conversion in the trichomes influenced by light, temperature and ultraviolet (UV) radiation is the main catalytic mode. This chemical conversion may explain the high chemodiversity and low structural stability.

Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents.
Thomas, B. F., & ElSohly, M. A.
The Analytical Chemistry of Cannabis, 27–41. (2016).
doi:10.1016/b978-0-12-804646-3.00002-3
In addition to nucleic acids, proteins, lipids, and carbohydrates, cannabis produces a large number of additional constituents, or secondary metabolites, including phytocannabinoids, terpenoids, and phenylpropanoids.1,2 While phytocannabinoids are often referred to as the “active” ingredients in cannabis, these other chemical constituents have a broad spectrum of pharmacological properties and can contribute to the effects seen upon cannabis ingestion or combustion and inhalation, and may also be contained within and contribute to the activity of extracts, tinctures, and other cannabis formulations.3 This overview of cannabis constituents will focus on the phytocannabinoids, terpenoids, and flavonoids that make up a large percentage of the pharmacologically active ingredients of current or emerging interest.

Biosynthesis of cannflavins A and B from Cannabis sativa
L Kevin A Reaa , José A. Casarettoa , M. Sameer Al-Abdul-Wahidb , Arjun Sukumarana , Jennifer Geddes-McAlistera , Steven J. Rothsteina , Tariq A. Akhtara,
DOI: 10.1016/j.phytochem.2019.05.009
Cannflavin A and B are prenylated flavonoids that are unique to Cannabis sativa.
Cannflavins exhibit anti-inflammatory activity that is thirty times that of aspirin.
Cannflavins are synthesized via a branch point from the phenylpropanoid pathway.
A single prenyltransferase converts chrysoeriol to cannflavin A and B.
In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for anO-methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ? chrysoeriol ? cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

Biosynthesis of Nature-Inspired Unnatural Cannabinoids
Kevin J. H. Lim, Yan Ping Lim, Yossa D. Hartono, Maybelle K. Go, Hao Fan, Wen Shan Yew
Molecules 2021, 26(10), 2914;
DOI: 10.3390/molecules26102914
Natural products make up a large proportion of medicine available today. Cannabinoids from the plant Cannabis sativais one unique class of meroterpenoids that have shown a wide range of bioactivities and recently seen significant developments in their status as therapeutic agents for various indications. Their complex chemical structures make it difficult to chemically synthesize them in efficient yields. Synthetic biology has presented a solution to this through metabolic engineering in heterologous hosts. Through genetic manipulation, rare phytocannabinoids that are produced in low yields in the plant can now be synthesized in larger quantities for therapeutic and commercial use. Additionally, an exciting avenue of exploring new chemical spaces is made available as novel derivatized compounds can be produced and investigated for their bioactivities. In this review, we summarized the biosynthetic pathways of phytocannabinoids and synthetic biology efforts in producing them in heterologous hosts. Detailed mechanistic insights are discussed in each part of the pathway in order to explore strategies for creating novel cannabinoids. Lastly, we discussed studies conducted on biological targets such as CB1, CB2 and orphan receptors along with their affinities to these cannabinoid ligands with a view to inform upstream diversification efforts.

BIOSYNTHESIS OF PROPYL CANNABINOID ACID AND ITS BIOSYNTHETIC RELATIONSHIP WITH PENTYL AND METHYL CANNABINOID ACIDS*
Y. SHOYAMA, H. HIRANO and I. NISHIOKA
Phyrochemtirry, Vol. 23, No. 9, pp. 1909-1912, 1984
Doi: 10.1016/S0031-9422(00)84939-0
Biosynthesis of propyl cannabinoid acid has been determined by in vitro incubation with a crude enzyme solution from three strains of Cannabis sativa using “C-labelled cannabinoid acid. Biosynthetic relationships between methyl, propyl and pentyl cannabinoid acids have been demonstrated

Breeding Distinct Industrial Hemp Varieties for the 21st Century
Seth Crawford, Ph.D.
https://www.youtube.com/watch?v=lNDP...z0DPbFWwlMJDqk
Industrial scale production of non-psychoactive cannabinoids began in earnest in the US in 2015 under Section 7606 of the 2014 Farm Bill. Seed for planting was initially scarce due to DEA prohibitions on importation and a lack of natively developed cultivars. Our company began identifying specific breeding strategies to produce agronomically advanced cultivars of hybrid cannabis that still remain federally compliant as “industrial hemp”. Since 2016, these breeding programs have led to the creation of the first (a) high content type III (CBD) day neutral varieties (“autoflowering”), (b) predictably “early” flowering type III photoperiod varieties, (c) high content type IV (CBG) dominant day neutral varieties, and (d) predictably “early” flowering type IV photoperiod varieties. In each circumstance, the varieties developed are 100% female (verified using Medicinal Genomics’ youPCR colorimetric screen), with only 1 in 4000 plants sporting a male phenotype in the field. In 2018, our seed was used to plant over 4000 acres across the US. Our most recent experimental results demonstrate that pure type IV (CBG) varieties can produce CBG to THC ratios of over 300:1, which enables raw oil extractions below the federal 0.3% THC requirement.

Cal Trainers Warned Off CBD Use in Horses
https://www.thoroughbreddailynews.co.. .0431-365750754
This can be evinced in a California Horse Racing Board (CHRB) notification sent to trainers Friday, warning that use of these CBD products can lead to a positive “for CBD and/or CBD metabolites in blood and urine,” and that, because CBD is currently unclassified in California, a positive would by default result in a class 1, category A drug violation

Can You Pass the Acid Test? Critical Review and Novel Therapeutic Perspectives of D9-Tetrahydrocannabinolic Acid A
Guillermo Moreno-Sanz
DOI: 10.1089/can.2016.0008
D9-tetrahydrocannabinolic acid A (THCA-A) is the acidic precursor of D9-tetrahydrocannabinol (THC), the main psychoactive compound found in Cannabis sativa. THCA-A is biosynthesized and accumulated in glandular trichomes present on flowers and leaves, where it serves protective functions and can represent up to 90% of the total THC contained in the plant. THCA-A slowly decarboxylates to form THC during storage and fermentation and can further degrade to cannabinol. Decarboxylation also occurs rapidly during baking of edibles, smoking, or vaporizing, the most common ways in which the general population consumes Cannabis. Contrary to THC, THCA-A does not elicit psychoactive effects in humans and, perhaps for this reason, its pharmacological value is often neglected. In fact, many studies use the term ‘‘THCA’’ to refer indistinctly to several acid derivatives of THC. Despite this perception, many in vitro studies seem to indicate that THCA-A interacts with a number of molecular targets and displays a robust pharmacological profile that includes potential anti-inflammatory, immunomodulatory, neuroprotective, and antineoplastic properties. Moreover, the few in vivo studies performed with THCA-A indicate that this compound exerts pharmacological actions in rodents, likely by engaging type-1 cannabinoid (CB1) receptors. Although these findings may seem counterintuitive due to the lack of cannabinoid-related psychoactivity, a careful perusal of the available literature yields a plausible explanation to this conundrum and points toward novel therapeutic perspectives for raw, unheated Cannabis preparations in humans.


Cannabichromene
Federica Pollastro, Diego Caprioglio, Danilo Del Prete, F. Rogati, Alberto Minassi, Orazio Taglialatela-Scafati, Eduardo Muñoz, Giovanni Appendino
January 2018 Natural product communications 13(9):1189-1194
https://www.researchgate.net/publica...annabichromene
DOI: 10.1177/1934578X1801300922
Cannabinochromene (CBC, 1a) is the archetypal member of a class of more than twenty isoprenylated 5-hydroxy-7-alkyl(aralky)benzo[2H]pyranes first reported from Cannabis sativa L. but also occurring in unrelated plants (Rhododendron species) as well as liverworts and fungi. The chemistry, synthesis, and bioactivity of CBC (1a) is reviewed, highlighting its underexploited pharmacological potential and rich chemistry.

Cannabichromene, a new active principle in hashish.
Gaoni, Y., & Mechoulam, R.
Chemical Communications (London), (1), 20.(1966).
doi:10.1039/c19660000020
IT is generally assumed that the active principles of hashish are double bond or stereochemical
isomers of tetrahydrocannabinol.2 Recently we were indeed able to isolate, elucidate the structure
of, and synthesize the active A1-tetrahydrocannabinol (I).3 However we have been unable to detect the presence of any additional tetrahydrocannabinols in hashish. We now report the isolation of a second active constituent which is of a different structural type. For this new component we suggest the name cannabichromene and assign structure (11).

Cannabichromene is a cannabinoid CB2 receptor agonist.
Udoh, M., Santiago, M., Devenish, S., McGregor, I. S., & Connor, M.
British Journal of Pharmacology. (2019).
doi:10.1111/bph.14815
What is already known:
• The phytocannabinoid cannabichromene (CBC) has anti-nociceptive and antiinflammatory effects in vitro and in vivo
• How CBC exerts these effects is largely unknown
What this study adds:
• This study shows that CBC is a selective CB2 receptor agonist
• CBC has a higher in vitro efficacy than tetrahydrocannabinol, and activates CB2 receptor regulatory pathways Clinical Significance:
• Cannabis contains a CB2-selective compound that could reduce inflammation without producing intoxication
KEY RESULTS CBC activated CB2 but not CB1 receptors to produce a hyperpolarization of AtT20 cells. This activation wasinhibited by aCB2 antagonist AM630, and sensitive to pertussistoxin. Application of CBC reduced activation of CB2 receptors (but not CB1 receptors) by subsequent co-application of CP55,940, an efficacious CB1 and CB2 agonist. Continuous CBC application induced loss of cell surface CB2 receptors and desensitisation of the CB2-induced hyperpolarization.
CONCLUSIONS AND IMPLICATIONS CBC is a selective CB2 receptor agonist displaying higher efficacy than THC in hyperpolarising AtT20 cells. CBC can also recruit CB2 receptor regulatory mechanisms. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB2-mediated modulation of inflammation.

Cannabichromene, Related Phytocannabinoids, and 5-Fluoro-cannabichromene Have Anticonvulsant Properties in a Mouse Model of Dravet Syndrome
Lyndsey L. Anderson, Adam Ametovski, Jia Lin Luo, Declan Everett-Morgan, Iain S. McGregor, Samuel D. Banister, Jonathon C. Arnold
ACS Chem. Neurosci. 2021, XXXX, XXX, XXX-XXX
Cannabis-based products are increasingly being used to treat refractory childhood epilepsies such as Dravet syndrome. Cannabis contains at least 140 terpenophenolic compounds known as phytocannabinoids. These include the known anticonvulsant compound cannabidiol (CBD) and several molecules showing emergent anticonvulsant properties in animal models. Cannabichromene (CBC) is a phytocannabinoid frequently detected in artisanal cannabis oils used in the community by childhood epilepsy patients. Here we examined the brain and plasma pharmacokinetic profiles of CBC, cannabichromenic acid (CBCA), cannabichromevarin (CBCV), and cannabichromevarinic acid (CBCVA) following intraperitoneal administration in mice. The anticonvulsant potential of each was then tested against hyperthermia-induced seizures in the Scn1a+/– mouse model of Dravet syndrome. All phytocannabinoids within the CBC series were readily absorbed and showed substantial brain penetration (brain–plasma ratios ranging from 0.2 to 5.8). Anticonvulsant efficacy was evident with CBC, CBCA, and CBCVA, each significantly increasing the temperature threshold at which Scn1a+/– mice had a generalized tonic–clonic seizure. We synthesized a fluorinated derivative of CBC (5-fluoro-CBC), which showed improved brain penetration relative to the parent CBC molecule but not any greater anticonvulsant effect. Since CBC and derivatives are anticonvulsant in a model of intractable pediatric epilepsy, they may constitute part of the mechanism through which artisanal cannabis oils are anticonvulsant in patients.

Cannabichromevarin and Cannabigerovarin, Two New Propyl Homologues of Cannabichromene and Cannabigerol
YUKIHIRO SHOYAMA, HITOTOSHI HIRANO, MIYUKI ODA, TAKAO SOMEHARA, ITSUO NISHIOKA
Chemical and Pharmaceutical Bull 1975 Volume 23 Issue 8 Pages 1894-1895
doi: 10.1248/cpb.23.1894
Two new neutral cannabinoids, cannabichromevarin and cannabigerovarin, were isolated from the "Meao variant, "Thailand Cannabis and their structures were determined to be the homologues of cannabichromene and cannabigerol which have a propyl sidechain, respectively, on the basis of spectral and chemical evidences.

Cannabidiol, a nonpsychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT1A somatodendritic autoreceptors in the dorsal raphe nucleus
EM Rock, D Bolognini, CL Limebeer, MG Cascio, S Anavi-Goffer, PJ Fletcher, R Mechoulam, RG Pertwee and LA Parker
British Journal of Pharmacology (2012) 165 2620–2634
DOI:10.1111/j.1476-5381.2011.01621.x
BACKGROUND AND PURPOSE To evaluate the hypothesis that activation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis.
EXPERIMENTAL APPROACH The potential of systemic and intra-DRN administration of 5-HT1A receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT1A receptors and to modify the ability of the 5-HT1A agonist, 8-OH-DPAT, to stimulate [35S]GTPgS binding in rat brainstem membranes.
KEY RESULTS CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg-1 , but not 40 mg·kg-1 )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose–response curve) at enhancing the ability of 8-OH-DPAT to stimulate [ 35S]GTPgS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping.
CONCLUSIONS AND IMPLICATIONS These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT1A autoreceptors in the DRN

Cannabidiol, a novel inverse agonist for GPR12.
Brown KJ, Laun AS, Song ZH.
Biochem Biophys Res Commun. 2017 Nov 4;493(1):451-454. doi: 10.1016/j.bbrc.2017.09.001.
GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

Cannabidiol – A phytocannabinoid that widely affects sphingolipid metabolism under conditions of brain insulin resistance
Tomasz Charytoniuk , Klaudia Sztolsztener , Ewa Harasim-Symbor, Klaudia Berk , Adrian Chabowski , Karolina Konstantynowicz-Nowicka
Biomedicine & Pharmacotherapy 142 (2021)
DOI: 10.1016/j.biopha.2021.112057
https://www.sciencedirect.com/scienc...53332221008404
Obesity-related insulin resistance (IR) and attenuated brain insulin signaling are significant risk factors for neurodegenerative disorders, e.g., Alzheimer’s disease. IR and type 2 diabetes correlate with an increased concentration of sphingolipids, a class of lipids that play an essential structural role in cellular membranes and cell signaling pathways. Cannabidiol (CBD) is a nonpsychoactive constituent of Cannabis sativa plant that interacts with the endocannabinoidome. Despite known positive effects of CBD on improvement in diabetes and its aftermath, e.g., anti-inflammatory and anti-oxidant effects, there are no studies evaluating the effect of phytocannabinoids on the brain insulin resistance and sphingolipid metabolism. Our experiment was carried out on Wistar rats that received a high-fat diet and/or intraperitoneal CBD injections. In our study, we indicated inhibition of de novo synthesis and salvage pathways, which resulted in significant changes in the concentration of sphingolipids, e.g., ceramide and sphingomyelin. Furthermore, we observed reduced brain IR and decreased tau protein phosphorylation what might be protective against neuropathologies development. We believe that our research will concern a new possible therapeutic approach with Cannabis -plant derived compounds and within a few years, cannabinoids would be considered as prominent substances for targeting both metabolic and neurodegenerative pathologies.

Cannabidiol (CBD) as an Adjunctive Therapy in Schizophrenia: A Multicenter Randomized Controlled Trial.
McGuire, P., Robson, P., Cubala, W. J., Vasile, D., Morrison, P. D., Barron, R., … Wright, S.
American Journal of Psychiatry, 175(3), 225–231. (2018).
doi:10.1176/appi.ajp.2017.17030325
Objective: Research in both animals and humans indicates that cannabidiol (CBD) has antipsychotic properties. The authors assessed the safety and effectiveness of CBD in patients with schizophrenia. Method: In an exploratory double-blind parallel-group trial, patients with schizophrenia were randomized in a 1:1 ratio to receive CBD (1000 mg/day; N=43) or placebo (N=45) alongside their existing antipsychotic medication. Participants were assessed before and after treatment using the Positive and Negative Syndrome Scale (PANSS), the Brief Assessment of Cognition in Schizophrenia (BACS), the Global Assessment of Functioning scale (GAF), and the improvement and severity scales of the Clinical Global Impressions Scale (CGI-I and CGI-S). Results: After 6 weeks of treatment, compared with the placebo group, the CBD group had lower levels of positive psychotic symptoms (PANSS: treatment difference=21.4, 95% CI=22.5, 20.2) and were more likely to have been rated as improved (CGI-I: treatment difference=20.5, 95% CI= 20.8, 20.1) and as not severely unwell (CGI-S: treatment difference=20.3, 95% CI=20.5, 0.0) by the treating clinician. Patients who received CBD also showed greater improvements that fell short of statistical significance in cognitive performance (BACS: treatment difference=1.31, 95% CI=20.10, 2.72) and in overall functioning (GAF: treatment difference= 3.0, 95% CI=20.4, 6.4). CBD was well tolerated, and rates of adverse events were similar between the CBD and placebo groups. Conclusions: These findings suggest that CBD has beneficial effects in patients with schizophrenia. As CBD’s effects do not appear to depend on dopamine receptor antagonism, this agentmay represent a new class of treatment for the disorder.

Cannabidiol as the Substrate in Acid-Catalyzed Intramolecular Cyclization.
Marzullo, P., Foschi, F., Coppini, D. A., Fanchini, F., Magnani, L., Rusconi, S., … Passarella, D.
Journal of Natural Products.(2020).
doi:10.1021/acs.jnatprod.0c00436
The chemical reactivity of cannabidiol is based on its ability to undergo intramolecular cyclization driven by the addition of a phenolic group to one of its two double bonds. The main products of this cyclization are ?9 -THC (trans-?-9-tetrahydrocannabinol) and ?8 -THC (trans-?-8- tetrahydrocannabinol). These two cannabinoids are isomers, and the first one is a frequently investigated psychoactive compound and pharmaceutical agent. The isomers ?8 -iso-THC (trans-?-8-iso-tetrahydrocannabinol) and ?4(8)-iso-THC (trans-?-4,8-iso-tetrahydrocannabinol) have been identified as additional products of intramolecular cyclization. The use of Lewis and protic acids in different solvents has been studied to investigate the possible modulation of the reactivity of CBD (cannabidiol). The complete NMR spectroscopic characterizations of the four isomers are reported. High-performance liquid chromatography analysis and 1 H NMR spectra of the reaction mixture were used to assess the percentage ratio of the compounds formed.

Cannabidiol Claims and Misconceptions
Trends in Pharmacological Sciences
Ethan B. Russo
Doi: 10.1016/j.tips.2016.12.004
Once a widely ignored phytocannabinoid, cannabidiol now attracts great therapeutic interest, especially in epilepsy and cancer. As with many rising trends, various myths and misconceptions have accompanied this heightened public interest and intrigue. This forum article examines and attempts to clarify some areas of contention.

Cannabidiol Does Not Cause Significant Changes to Working Memory Performance in the N-Back Task
Éamon Jones and Styliani Vlachou
Pharmaceuticals 2021, 14, 1165.
DOI: 10.3390/ph14111165
Cannabis use can be traced back to several centuries before the Common Era, when it was used for industrial, medicinal and recreational purposes. More recently, over 100 different cannabinoid compounds have been identified, one of which is cannabidiol (CBD), a compound widely used for anti-inflammatory and anxiolytic treatment. The literature surrounding the cognitive effects of CBD is limited, with most studies focusing on the effects of other cannabinoids on cognition. To expand this literature, this study investigated whether CBD causes significant differences to working memory (WM) functioning, as measured by the N-back task. It was hypothesised that CBD does not cause statistically significant differences to WM. In all, 54 participants, 33 females and 21 males, were recruited, with a mean age of 32.63 years. Of these 54 participants, 26 reported using CBD and no other cannabinoids, while 28 reported not using any cannabinoid. The participants were instructed to answer a short online survey to gather basic demographic data and to complete an online N-back task to measure WM. For the computerised N-back task, the participants completed a practice and three test blocks, where they were instructed to respond to whether a series of letter stimuli were presented one trial back (1-back), two trials back (2-back) or three trials back (3-back). Multivariate analysis of covariance yielded no statistically significant difference on either response time or response accuracy data between groups after controlling for how long the participants use CBD and for what reason they use CBD. These results support our hypothesis that CBD does not cause significant changes to WM functioning. Further research is greatly needed to investigate the long-term effects of CBD use on WM and on general cognitive functioning

*Cannabidiol Does Not Convert to D9-Tetrahydrocannabinol
in an In Vivo Animal Model
Louise Wray,Colin Stott, Nicholas Jones, and Stephen Wright
Cannabis and Cannabinoid Research Volume 2.1, 2018
DOI: 10.1089/can.2017.0032
Cannabidiol (CBD) can convert to ??-tetrahydrocannabinol (THC) in vitro with prolonged exposure to simulated gastric fluid; however, in vitro conditions may not be representative of the in vivo gut environment. Using the minipig, we investigated whether enteral CBD converts to THC in vivo. Materials and Methods: Synthetic CBD (100 mg/mL) was administered orally in a sesame oil formulation twice daily to minipigs (N=3) in 15 mg/kg doses for 5 consecutive days. Blood samples were taken before and 1, 2, 4, and 6 h after morning doses on Days 1 and 5. Six hours after the final dose on Day 5, the animals were euthanized, and samples of gastrointestinal (GI) tract contents were obtained. Liquid chromatography with tandem mass spectrometry analysis determined CBD, THC, and 11-hydroxy-THC (11-OH-THC) concentrations. Lower limits of quantification: plasma CBD=1 ng/mL, plasma THC and 11-OH-THC=0.5 ng/mL, GI tract CBD=2 ng/mL, and GI tract THC and 11-OH-THC=1 ng/mL. Results: THC and 11-OH-THC were undetectable in all plasma samples. Maximum plasma concentrations (Cmax) of CBD were observed between 1 and 4 h on Days 1 and 5. CBD was present in plasma 6 h after administration on Days 1 (mean 33.6 ng/mL) and 5 (mean 98.8 ng/mL). Mean Cmax CBD values, 328 ng/mL (Day 1) and 259 ng/mL (Day 5), were within range of those achieved in clinical studies. Mean CBD exposure over 6 h was similar on Days 1 (921 h·ng/mL) and 5 (881 h·ng/mL). THC and 11-OH-THC were not detected in all GI tract samples. Mean CBD concentrations reached 84,500 ng/mL in the stomach and 43,900 ng/mL in the small intestine. Conclusions: Findings of the present study show that orally dosed CBD, yielding clinically relevant plasma exposures, does not convert to THC in the minipig, a species predictive of human GI tract function.

Cannabidiol: from an inactive cannabinoid to a drug with wide spectrum of action
Antonio Zuardi
Revista Brasileira de Psiquiatria 30(3):271-80 October 2008
DOI: 10.1590/S1516-44462008000300015
https://www.scielo.br/pdf/rbp/v30n3/a15v30n3.pdf
Objective: The aim of this review is to describe the historical development of research on cannabidiol.
Method: This review was carried out on reports drawn from Medline, Web of Science and SciELO.
Discussion: After the elucidation of the chemical structure of cannabidiol in 1963, the initial studies showed that cannabidiol was unable to mimic the effects of Cannabis. In the 1970’s the number of publications on cannabidiol reached a first peak, having the research focused mainly on the interaction with delta9-THC and its antiepileptic and sedative effects. The following two decades showed lower degree of interest, and the potential therapeutic properties of cannabidiol investigated were mainly the anxiolytic, antipsychotic and on motor diseases effects. The last five years have shown a remarkable increase in publications on cannabidiol mainly stimulated by the discovery of its anti-inflammatory, anti-oxidative and neuroprotective effects. These studies have suggested a wide range of possible therapeutic effects of cannabidiol on several conditions, including Parkinson’s disease, Alzheimer’s disease, cerebral ischemia, diabetes, rheumatoid arthritis, other inflammatory diseases, nausea and cancer.
Conclusion: In the last 45 years it has been possible to demonstrate that CBD has a wide range of pharmacological effects, many of which being of great therapeutic interest, but still waiting to be confirmed by clinical trials.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response
Long Chi Nguyen, Dongbo Yang, Vlad Nicolaescu, Thomas J. Best, Takashi Ohtsuki, Shao-Nong Chen, J. Brent Friesen, Nir Drayman, Adil Mohamed, Christopher Dann, Diane Silva, Haley Gula, Krysten A. Jones, J. Michael Millis, Bryan C. Dickinson, Savaş Tay, Scott A. Oakes, Guido F. Pauli, David O. Meltzer, Glenn Randall and Marsha Rich Rosner
Version 1. bioRxiv. Preprint. 2021 Mar 10.
doi: 10.1101/2021.03.10.432967
https://www.ncbi.nlm.nih.gov/pmc/art....10.432967.pdf
The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV- 2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARSCoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.



Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis?like disease in C57BL/6 mice
Ewa Kozela, Nirit Lev, Nathali Kaushansky, Raya Eilam. Neta Rimmerman, Rivka Levy, Avraham Ben?Nun, Ana Juknat, Zvi Vogel
British Journal of Pharmacology (2011) 163 1507–1519
doi: 10.1111/j.1476-5381.2011.01379.x
BACKGROUND AND PURPOSE Cannabis extracts and several cannabinoids have been shown to exert broad anti?inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.
EXPERIMENTAL APPROACH We used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation.
KEY RESULTS Treatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors.
CONCLUSIONS AND IMPLICATIONS CBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.

Cannabidiol Interferes with Establishment of ?9-Tetrahydrocannabinol-Induced Nausea Through a 5-HT1A Mechanism
Marieka V. DeVuono, Olivia La Caprara, Gavin N. Petrie, Cheryl L. Limebeer, Erin M. Rock, Matthew N. Hill, Linda A. Parker
Cannabis and Cannabinoid Research Dec 2020
DOI: 10.1089/can.2020.0083
Introduction: Cannabinoid hyperemesis syndrome (CHS) is characterized by intense nausea and vomiting brought on by the use of high-dose ?9-tetrahydrocannabinol (THC), the main psychotropic compound in cannabis. Cannabidiol (CBD), a nonpsychotropic compound found in cannabis, has been shown to interfere with some acute aversive effects of THC. In this study, we evaluated if CBD would interfere with THC-induced nausea through a 5-HT1A receptor mechanism as it has been shown to interfere with nausea produced by lithium chloride (LiCl). Since CHS has been attributed to a dysregulated stress response, we also evaluated if CBD would interfere with THC-induced increase in corticosterone (CORT).
Materials and Methods: The potential of CBD (5 mg/kg, ip) to suppress THC-induced conditioned gaping (a measure of nausea) was evaluated in rats, as well as the potential of the 5-HT1A receptor antagonist, WAY-100635 (WAY; 0.1 mg/kg, ip), to reverse the suppression of THC-induced conditioned gaping by CBD. Last, the effect of CBD (5 mg/kg, ip) on THC-induced increase in serum CORT concentration was evaluated.
Results: Pretreatment with CBD (5 mg/kg, ip) interfered with the establishment of THC-induced conditioned gaping (p=0.007, relative to vehicle [VEH] pretreatment), and this was reversed by pretreatment with 0.1 mg/kg WAY. This dose of WAY had no effect on gaping on its own. THC (10 mg/kg, ip) significantly increased serum CORT compared with VEH-treated rats (p=0.04). CBD (5 mg/kg, ip) pretreatment reversed the THC-induced increase in CORT.
Conclusions: CBD attenuated THC-induced nausea as well as THC-induced elevation in CORT. The attenuation of THC-induced conditioned gaping by CBD was mediated by its action on 5-HT1A receptors, similar to that of LiCl-induced nausea.

Cannabidiol is a partial agonist at dopamine D2High receptors, predicting its antipsychotic clinical dose.
Seeman P.
Transl Psychiatry. 2016 Oct 18;6(10):e920.
doi: 10.1038/tp.2016.195.
Although all current antipsychotics act by interfering with the action of dopamine at dopamine D2 receptors, two recent reports showed that 800 to 1000?mg of cannabidiol per day alleviated the signs and symptoms of schizophrenia, although cannabidiol is not known to act on dopamine receptors. Because these recent clinical findings may indicate an important exception to the general rule that all antipsychotics interfere with dopamine at dopamine D2 receptors, the present study examined whether cannabidiol acted directly on D2 receptors, using tritiated domperidone to label rat brain striatal D2 receptors. It was found that cannabidiol inhibited the binding of radio-domperidone with dissociation constants of 11?nm at dopamine D2High receptors and 2800?nm at dopamine D2Low receptors, in the same biphasic manner as a dopamine partial agonist antipsychotic drug such as aripiprazole. The clinical doses of cannabidiol are sufficient to occupy the functional D2High sites. it is concluded that the dopamine partial agonist action of cannabidiol may account for its clinical antipsychotic effects

Cannabidiol Monomethyl Ether. A New Neutral Cannabinoid
YUKIHIRO SHOYAMA, KEIKO KUBOE, ITSUO NISHIOKA, TATSUO YAMAUCHI
Chemical & Pharmaceutical Bull 1972 Volume 20 Issue 9 Pages 2072
doi: 10.1248/cpb.20.2072
No Abstract available

Cannabidiol or ?-Caryophylline which is doing the work?
FIND DOI or LINK
When examining the CB2 receptor and it’s natural agonists cannabidiol and ?-caryophylline and their binding affinity at the site, it becomes obvious quickly that there is a large disparity of binding affinity between the two substances. When looking at the data we find both with affinity in the nM range, however in analysis we find that ?-caryophylline has an almost 15x greater affinity at the site than CBD ?-caryophylline has a Ki of 155 nM (Å) And we find that CBD has a much higher Ki of 2283 nM (Ç)

Cannabidiol monotherapy for treatment-resistant schizophrenia.
Zuardi, A. W., Hallak, J. E. C., Dursun, S. M., Morais, S. L., Sanches, R. F., Musty, R. E., & Crippa, J. A. S.
Journal of Psychopharmacology, 20(5), 683–686. (2006).
doi:10.1177/0269881106060967
Cannabidiol (CBD), one of the major products of the marijuana plant, is devoid of marijuana’s typical psychological effects. In contrast, potential antipsychotic efficacy has been suggested based on preclinical and clinical data (Zuardi et al., 2002). In this report, we further investigated the efficacy and safety of CBD monotherapy in three patients with treatmentresistant schizophrenia (TRS). This was an in-patient study. All patients were given placebo for the initial 5 days, and from the 6th to 35th day (inclusive) they received CBD (initial oral dose of 40mg reaching 1280mg/day). On the 36th day, CBD treatment was discontinued and replaced by placebo for 5 days, which was subsequently switched to olanzapine for over 15 days. Efficacy, tolerability and side effects were assessed. One patient showed mild improvement, but two patients didn’t show any improvement during CBD monotherapy. All patients tolerated CBD very well and no side effects were reported. These preliminary data suggest that CBD monotherapy may not be effective for TRS.

Cannabidiol reduces ethanol consumption, motivation and relapse in mice.
Viudez-Martínez A, García-Gutiérrez MS, Navarrón CM, Morales-Calero MI, Navarrete F, Torres-Suárez AI, Manzanares J.
Addict Biol. 2018 Jan;23(1):154-164.
doi: 10.1111/adb.12495.
This study evaluated the effects of cannabidiol (CBD) on ethanol reinforcement, motivation and relapse in C57BL/6 J mice. The effects of CBD (60 mg/kg, i.p.) on blood ethanol concentration, hypothermia and handling-induced convulsions associated to acute ethanol administration were evaluated. The two-bottle choice paradigm was performed to assess the effects of CBD (30, 60 and 120 mg/kg/day, i.p.) on ethanol intake and preference. In addition, an oral ethanol self-administration experiment was carried out to evaluate the effects of CBD [a single s.c. administration of a microparticle formulation providing CBD continuous controlled release (30 mg/kg/day)] on the reinforcement and motivation for ethanol. The effects of CBD (60 and 120 mg/kg/day, i.p.) on ethanol-induced relapse were also evaluated. Gene expression analyses of tyrosine hydroxylase in ventral tegmental area and ?-opioid (Oprm1), cannabinoid (CB1 r and CB2 r) and GPR55 receptors in nucleus accumbens (NAcc) were carried out by real-time polymerase chain reaction. Cannabidiol reduced the ethanol-induced hypothermia and handling-induced convulsion but failed to modify blood ethanol concentration. CBD reduced ethanol consumption and preference in the two-bottle choice, significantly decreased ethanol intake and the number of effective responses in the oral ethanol self-administration, and reduced ethanol-induced relapse. Furthermore, the administration of CBD significantly reduced relative gene expression of tyrosine hydroxylase in the ventral tegmental area, Oprm1, CB1 r and GPR55 in the NAcc and significantly increased CB2 r in the NAcc. Taken together, these results reveal that the administration of CBD reduced the reinforcing properties, motivation and relapse for ethanol. These findings strongly suggest that CBD may result useful for the treatment of alcohol use disorders.

Cannabidiolic acid synthase, the chemotype?determining enzyme in the fiber?type Cannabis sativa
Futoshi Taura, Supaart Sirikantaramas, Yoshinari Shoyama, Kazuyoshi Yoshikai,
Yukihiro Shoyama, Satoshi Morimoto
FEBS Letters Volume 581, Issue 16 June 26, 2007 2929-2934
10.1016/j.febslet.2007.05.043
Cannabidiolic?acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic?acid into CBDA, the dominant cannabinoid constituent of the fiber?typeCannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene?specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic?acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug?typeCannabis plants.

Cannabigerol Monomethyl Ether, a New Component of Hemp.
YAMAUCHI, T., SHOYAMA, Y., MATSUO, Y., & NISHIOKA, I. (1968).
CHEMICAL & PHARMACEUTICAL BULLETIN, 16(6), 1164–1165.
doi:10.1248/cpb.16.1164
No abstract available

CANNABINEROLIC ACID, A CANNABINOID FROM CANNABIS SATIVA*
FUTOSHI TAURA, SATOSHI MORIMOTOt and YUKIHIRO SHOYAMA
Phytochemistry, Vol. 39, No. 2, pp. 457-458, 1995
doi: 10.1016/0031-9422(94)00887-Y
Investigation of the leaves of Cannabis sativa resulted in the isolation of a new cannabinoid, cannabinerolic acid. The structure of the new cannabinoid was established on the basis of spectroscopic and chemical evidence

Cannabinoid Biosynthesis using Noncanonical Cannabinoid Synthases
DOI: 10.1101/2020.01.29.926089
BioRxiv (2020)
Maybelle Go, Kevin Jie Han Lim. Wen Shan Yew
We have found enzymes from the berberine-bridge enzyme (BBE) superfamily (IPR012951) that catalyze the oxidative cyclization of the monoterpene moiety in cannabigerolic acid (CBGA) to form cannabielsoic acid B (CBSA). The enzymes are from a variety of organisms and are previously uncharacterized. This is the first report that describes enzymes that did not originate from the Cannabis plant that catalyze the production of cannabinoids. Out of 72 homologues chosen from the enzyme superfamily, six orthologues were shown to accept CBGA as a substrate and catalyze the biosynthesis of CBSA. The six enzymes discovered in this study are the first report of heterologous expression of BBEs that did not originate from the Cannabis plant that catalyze the production of cannabinoids using CBGA as substrate. This study details a new avenue for discovering and producing natural and unnatural cannabinoids.

Cannabinoid chemistry: an overview
Lumir Hanus, Raphael Mechoulam
https://www.researchgate.net/publica...ry_an_overview
Cannabis sativa probably originates from neolithic China [1]. However the exact period of its domestication is unknown. The first known record of the use of cannabis as a medicine was published in China 5000 years ago in the reign of the Emperor Chen Nung. It was recommended for malaria, constipation, rheumatic pains, absent-mindedness and female disorders. Later its use spread into India and other Asian countries, the Middle East, Asia, South Africa and South America. It was highly valued in medieval Europe. In Western Europe, particularly in England, cannabis was extensively used as a medicine during the 19th century, while in France it was mostly known as a “recreational” drug [2].

Cannabinoid Combination Induces Cytoplasmic Vacuolation in MCF-7 Breast Cancer Cells.
Schoeman, R., Beukes, N., & Frost, C.
Molecules, 25(20), 4682.(2020).
doi:10.3390/molecules25204682
Abstract: This study evaluated the synergistic anti-cancer potential of cannabinoid combinations across the MDA-MB-231 and MCF-7 human breast cancer cell lines. Cannabinoids were combined and their synergistic interactions were evaluated using median effect analysis. The most promising cannabinoid combination (C6) consisted of tetrahydrocannabinol, cannabigerol (CBG), cannabinol (CBN), and cannabidiol (CBD), and displayed favorable dose reduction indices and limited cytotoxicity against the non-cancerous breast cell line, MCF-10A. C6 exerted its effects in the MCF-7 cell line by inducing cell cycle arrest in the G2 phase, followed by the induction of apoptosis. Morphological observations indicated the induction of cytoplasmic vacuolation, with further investigation suggesting that the vacuole membrane was derived from the endoplasmic reticulum. In addition, lipid accumulation, increased lysosome size, and significant increases in the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) expression were also observed. The selectivity and ability of cannabinoids to halt cancer cell proliferation via pathways resembling apoptosis, autophagy, and paraptosis shows promise for cannabinoid use in standardized breast cancer treatment.

Cannabinoid Content in Cannabis Flowers and Homemade Cannabis-Based Products Used for Therapeutic Purposes in Argentina
Daniela Sedan, Cristian Vaccarini, Pablo Demetrio, Marcelo Morante, Romina Montiel, Alvaro Saurí, and Dario Andrinolo
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0117
https://www.liebertpub.com/doi/epub/.../can.2020.0117
Introduction: A recent law (DCTO-2020-883-APN-PTE—Law No. 27,350. Regulation) passed in Argentina put an end to the ban imposed for the last 60 years on cannabis cultivation within the country. The law permits restricted access to cannabis derivatives for medicinal, therapeutic, and palliative use by individuals and communities, allowing self- and community based cannabis production. This is cause for concern in view of the lack of quality controls for cannabis derivatives. The several varieties of cannabis grown in Argentina have different chemical pro􀂦les and are processed in a variety of ways— mostly by alcohol extraction or maceration at different temperatures and for different amounts of times—making the cannabinoid content of these preparations highly variable. Determining the characteristics of home- and community-grown cannabis products will facilitate the implementation of public policies conducive to their safety and improvement.
Objective: The aim of this study was to determine the cannabinoid chemotypes used for therapeutic purposes in Argentina and evaluate whether the cannabinoids present in homemade derivatives are comparable to those in commercially available products.
Materials and Methods: High performance liquid chromatography with ultraviolet and diode array detector (HPLC/UV-DAD) analysis of 436 samples (oils, resins, and inflorescences) was carried out to determine the identity and concentration of five cannabinoids: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), and cannabinol (CBN). From three different sources, the samples represent the type of medical cannabis preparations to which patients have access.
Results: The results indicate that the medium-to-low cannabinoid concentration in a significant number of homemade oil samples is similar to that found in commercial products. Most of the samples have a THC/CBD ratio >1 or only contain THC. Acidic cannabinoids were detected in homemade preparations, but were not reported in package inserts of commercial products.
Conclusions: Our results indicate that despite their considerable variability, homemade preparations as a whole show
cannabinoid levels and profiles equivalent to the commercially available products commonly used for medicinal, therapeutic, and palliative purposes in Argentina.
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Cannabinoid Content of Individual Plant Organs From Different Geographical Strains of Cannabis sativa L.
Hemphill, J. K., Turner, J. C., & Mahlberg, P. G.
Journal of Natural Products, 43(1), 112–122. (1980).
doi:10.1021/np50007a009
Individual plant organs from different geographical strains of Cannabis sativa L. were analyzed for their cannabinoid content by gas-liquid chromatography. Analyses showed that different plant parts from each strain varied quantitatively in their cannabinoid content. However, each plant part possessed a cannabinoid profile which characterized the chemical phenotype of that strain. Accumulation of a specific cannabinoid in high quantities that was uncharacteristic of that strain was found. Factors such as maturity of plant organ, sex of the plant, location of the plant organ on the plant and sampling procedures influenced the accumulation of cannabinoids. Pollen grains and seeds (intact or crushed) were found to lack detectable levels of cannabinoids. Based on these results, precautions that should be taken then accumulating data on the chemical phenotype of a Cannabzs plant are discussed.

Cannabinoid Decarboxylation: A Comparative Kinetic Study
Teresa Moreno, Peter Dyer, and Stephen Tallon
Ind. Eng. Chem. Res. (2020)
DOI: 10.1021/acs.iecr.0c03791
Cannabinoids like ?9 -tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG) are considered the main active components in Cannabis sativa L. and are obtained through the decarboxylation of their acid analogues (THCA, CBDA, and CBGA), which are the forms naturally present in the plant. The kinetics of this reaction were studied for hemp plant material in an oven at different temperatures (80?160 °C) and reaction times (5?120 min). The effect of oxygen and the amount of plant material on the reaction rate was also studied. The reactions follow first-order kinetics, with THCA showing the fastest decarboxylation rate. In all cases, a significant loss of neutral cannabinoids was observed at elevated temperatures and reaction times, although this can be minimized in the absence of oxygen. Two different kinetic models were used to fit the experimental data and to predict the optimum decarboxylation conditions to maximize THC or CBD concentration.

Cannabinoid-deficient Benin republic hemp (Cannabis sativa L.) improves semen parameters by reducing prolactin and enhancing anti-oxidant status
Abdullateef Isiaka Alagbonsi, Luqman Aribidesi Olayaki, Halimat Amin Abdulrahim, Thomson Sijuade Adetona and Gbemileke Tobiloba Akinyemi
Alagbonsi et al.
BMC Complementary and Alternative Medicine (2019) 19:132
DOI: 10.1186/s12906-019-2541-5
Background: Nigerian Cannabis sativa (hemp) causes male gonadotoxicity by inducing hyperprolactinemia, down-regulation of hypothalamic-pituitary-testicular axis, and oxidative stress. Benin republic hemp has been preferred by illicit users in Nigeria but its effect on male fertility is not understood. This study determined and compared the compositions of Benin republic hemp ethanol extract (BHE) and Nigerian hemp. The effects of BHE on semen parameters, reproductive hormones, and anti-oxidant status, and the possibility of bromocriptine (prolactin inhibitor) to abolish hemp-induced toxicities in rats were also investigated.
Methods: Thirty-six male Wistar rats were blindly randomized into 6 oral treatment groups (n = 6 each). Groups I (control) and II received normal saline and bromocriptine (3 mg/kg) respectively. Groups III and IV received 2 mg/kg of BHE alone and in combination with bromocriptine respectively, while groups V and VI received 10 mg/kg BHE alone and in combination with bromocriptine respectively. Comparisons among the groups were done by one-way analysis of variance, followed by post-hoc Tukey multiple comparison test. Statistical significance was considered at p < 0.05.
Results: The BHE has no cannabichromene and tetrahydrocannabinol but a very small quantity of cannabinol and higher quantity of fatty acids when compared to Nigerian hemp. Both doses of BHE increased sperm count, morphology and viability but not motility. Co-administration of BHE with bromocriptine lowered sperm count but increased sperm morphology and viability. Bromocriptine and/or BHE caused reduction in the plasma prolactin level, increase in the plasma superoxide dismutase activity, but no significant change in the plasma gonadotropin releasing hormone, follicle stimulating hormone (except for the increase in rats that received bromocriptine+ 10
mg/kg BHE), luteinizing hormone, estradiol, malondialdehyde and glutathione peroxidase. The 10 mg/kg BHE or bromocriptine+BHE (both doses) increased total anti-oxidant capacity and catalase.
Conclusions: The BHE improves semen parameters by reducing plasma prolactin and enhancing plasma anti-oxidant status. Its pro-fertility potential might be associated with its deficiency in the widely known gonadotoxic phytocannabinoids.

Cannabinoid Delivery Systems for Pain and Inflammation Treatment.
Bruni, N., Della Pepa, C., Oliaro-Bosso, S., Pessione, E., Gastaldi, D., & Dosio, F.
Molecules, 23(10), 2478. (2018).
doi:10.3390/molecules23102478*
There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

Cannabinoid Ester Constituents from High-Potency Cannabis sativa
Safwat A Ahmed, Samir A Ross, Desmond Slade, Mahmoud A Elsohly
July 2008 Journal of Natural Products 71(6):1119
DOI: 10.1021/np070454a
DOI: 10.1021/np800261x (correction for article)
Eleven new cannabinoid esters, together with three known cannabinoid acids and ? 9-tetrahydrocannabinol (? 9-THC), were isolated from a high-potency variety of Cannabis satiVa. The structures were determined by extensive spectroscopic analyses to be-fenchyl ? 9-tetrahydrocannabinolate (1), epi-bornyl ? 9-tetrahydrocannabinolate (2), R-terpenyl ? 9-tetrahydrocannabinolate (3), 4-terpenyl ? 9-tetrahydrocannabinolate (4), R-cadinyl ? 9-tetrahydrocannabinolate (5), ?-eudesmyl ? 9-tetrahydrocannabinolate (6), ?-eudesmyl cannabigerolate (7), 4-terpenyl cannabinolate (8), bornyl ? 9-tetrahydrocannabinolate (9), R-fenchyl ? 9-tetrahydrocannabinolate (10), R-cadinyl cannabigerolate (11), ? 9-tetrahydro-cannabinol (? 9-THC), ? 9-tetrahydrocannabinolic acid A (? 9-THCA), cannabinolic acid A (CBNA), and cannabigerolic acid (CBGA). Compound 8 showed moderate antimicrobial activity against Candida albicans ATCC 90028 with an IC 50 value of 8.5 µg/mL. CB-1 receptor assay indicated that the esters, as well as the parent acids, are not active.

Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after ?9-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716.
Higgs, S., Williams, C. M., & Kirkham, T. C.
Psychopharmacology, 165(4), 370–377. (2003).
doi:10.1007/s00213-002-1263-3
Rationale: Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability. Objectives: The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution. Methods: Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats. Results: D9 -tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/ kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a nonsignificant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent. Conclusions: These data are consistent with endocannabinoid involvement in the mediation of food palatability

Cannabinoid Inheritance Relies on Complex Genetic Architecture.
Campbell, L. G., Dufresne, J., & Sabatinos, S. A.
Cannabis and Cannabinoid Research.(2019).
doi:10.1089/can.2018.0015
Introduction: Understanding the inheritance of cannabinoid compounds in Cannabis sativa will facilitate effective crop breeding and careful regulation of controlled substances. The production of two key cannabinoids, D-9- tetrahydrocannabinol (THC) and cannabidiol (CBD), is partially controlled by two additive loci. Here, we present the first study to search for evidence of alternate genetic models describing the inheritance and expression of cannabinoids. Materials and Methods: Using an information-theoretic approach, we estimated composite genetic effects (CGEs) of four cultivars with pure CBD or pure THC chemotypes, their F1 and F2 hybrid progeny, to identify genetic models that explain cannabinoid inheritance patterns. We also estimated the effective number of genetic factors that control differences in cannabinoid concentration (THC, CBD, and cannabichromene [CBC]). Results: Unlike previous research, we note nonadditive components of cannabinoid inheritance. Concentration of THC is a polygenic trait (three to four genetic factors). Both additive and dominance CGEs best explained THC expression patterns. In contrast, cytoplasmic genomes and additive genes may influence CBD concentration. Maternal additive effects and additive genetic effects apparently influence CBC expression. Conclusions: Cannabinoid inheritance is more complex than previously appreciated; among other genetic effects, cytogenetic and maternal contributions may be undervalued influences on cannabinoid ratios and concentrations. Further research on the environmental sensitivity of cannabinoid production is advised.

Cannabinoid modulation of mother-infant interaction: is it just about milk?
Antonia Manduca, Patrizia Campolongo and Viviana Trezza
Rev. Neurosci. 2012; 23(5-6): 707–722
DOI 10.1515/revneuro-2012-0074
Mother-infant interactions are essential for proper neurobehavioral development of the offspring, and disruptions in those relationships may result in neuroendocrine, neurochemical and behavioral alterations at adulthood. The neural circuitries involved in mother-infant interactions have not been completely elucidated yet. The brain endocannabinoid system plays an essential role in prenatal and postnatal neurobehavioral development. Here, we will summarize and discuss the available findings about the role of endocannabinoids in three key aspects of mother-infant interactions in rodents: suckling, maternal behavior and separationinduced ultrasonic vocalizations (USVs). The studies reviewed here show that endocannabinoids are not only involved in suckling initiation and, therefore, in the feeding and growth of the offspring, but also regulate the emotional reactivity of rodent pups, as measured by the rate of isolation-induced USVs. Conversely, less information is available about endocannabinoid modulation of maternal behavior, and therefore more research in this direction is warranted. Indeed, since Cannabis sativa preparations are widely used by young people, including pregnant and lactating women, it is important to understand whether developmental exposure to cannabinoids interferes wit

Cannabinoid Modulation of Neuroinflammatory Disorders
Viviane M. Saito, Rafael M. Rezende and Antonio L. Teixeira
Current Neuropharmacology, 2012, 10, 159-166
https://www.ncbi.nlm.nih.gov/pmc/art.../CN-10-159.pdf
In recent years, a growing interest has been dedicated to the study of the endocannabinoid system. The isolation of Cannabis sativa main psychotropic compound, Δ9-tetrahydrocannabinol (THC), has led to the discovery of an atypical neurotransmission system that modulates the release of other neurotransmitters and participates in many biological processes, including the cascade of inflammatory responses. In this context, cannabinoids have been studied for their possible therapeutic properties in neuroinflammatory diseases. In this review, historic and biochemical aspects of cannabinoids are discussed, as well as their function as modulators of inflammatory processes and therapeutic perspectives for neurodegenerative disorders, particularly, multiple sclerosis.

Cannabinoid physiology and pharmacology: 30 years of progress
Allyn C.Howlett, Christopher S.Breivogel, Steven R.Childers, Samuel A.Deadwyler. Robert E.Hampson, Linda J.Porrino
Neuropharmacology Volume 47, Supplement 1, 2004, Pages 345-358
DOI: 10.1016/j.neuropharm.2004.07.030
Δ9-Tetrahydrocannabinol from Cannabis sativa is mimicked by cannabimimetic analogs such as CP55940 and WIN55212-2, and antagonized by rimonabant and SR144528, through G-protein-coupled receptors, CB1 in the brain, and CB2 in the immune system. Eicosanoids anandamide and 2-arachidonoylglycerol are the “endocannabinoid” agonists for these receptors. CB1 receptors are abundant in basal ganglia, hippocampus and cerebellum, and their functional activity can be mapped during behaviors using cerebral metabolism as the neuroimaging tool. CB1receptors couple to Gi/o to inhibit cAMP production, decrease Ca2+ conductance, increase K+ conductance, and increase mitogen-activated protein kinase activity. Functional activation of G-proteins can be imaged by [35S]GTPγS autoradiography. Post-synaptically generated endocannabinoids form the basis of a retrograde signaling mechanism referred to as depolarization-induced suppression of inhibition (DSI) or excitation (DSE). Under circumstances of sufficient intracellular Ca2+ (e.g., burst activity in seizures), synthesis of endocannabinoids releases a diffusible retrograde messenger to stimulate presynaptic CB1 receptors. This results in suppression of γ-aminobutyric acid (GABA) release, thereby relieving the post-synaptic inhibition. Tolerance develops as neurons adjust both receptor number and cellular signal transduction to the chronic administration of cannabinoid drugs. Future therapeutic drug design can progress based upon our current understanding of the physiology and pharmacology of CB1, CB2 and related receptors. One very important role for CB1 antagonists will be in the treatment of craving in the disease of substance abuse.

Cannabinoid Profiling of Hemp Seed Oil by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.
Citti, C., Linciano, P., Panseri, S., Vezzalini, F., Forni, F., Vandelli, M. A., & Cannazza, G.
Frontiers in Plant Science, 10.(2019).*
doi:10.3389/fpls.2019.00120
Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.

Cannabinoid research in the 2010s
Mauro Maccarrone and Steve PH Alexander
bph_1930 2409..2410
DOI:10.1111/j.1476-5381.2012.01930.x
Cannabis sativa is possibly the plant with the longest history of cultivation by man (Russo, 2007). It has long been exploited for its fibre; as a biomass converter, it has exceptional utility. For most people, however, there is the association of cannabis with ‘recreational drugs’, which has lead to the profusion of names associated with the plant and extracts thereof (marijuana,hashish, bhang, weed, grass, etc.). The ‘modern’ scientific era of cannabis research was prompted by the discovery of the major psychoactive ingredient in cannabis extracts (Gaoni and Mechoulam, 1964). This was, of course, D9-tetrahydrocannabinol or THC. Raphael Mechoulam has numerous publications, filled with seminal observations, including the identification of the two ‘best’ candidates for endogenous cannabinoid molecules: anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (Mechoulam et al., 1995). He has become something of an icon in the cannabis field, with this issue of BJP containing a series of original articles prompted by a symposium held in Jerusalem in November 2010 to celebrate his 80th birthday. The first issue, entitled ‘Cannabinoids in Biology and Medicine’, containing primarily reviews, was published in August 2011 (https://onlinelibrary.wiley.com/doi/...sue-7/issuetoc). Current research incannabinoid-related areas is vibrant, with the added focus of TRPV1 ion channels, PPAR nuclear receptors and the ‘orphan’ G-protein coupled receptors, GPR18, GPR55 and GPR119, as molecular targets of cannabinoids and cannabinoid-like molecules. Furthermore, the identification of endogenous agonists at cannabinoid receptors which lead to the demonstration of multiple routes for synthesis and transformation of these endocannabinoids has added to the molecular targets available for potential exploitation.

Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes.
Pawe? Rodziewicz, Stefan Loroch, Lukasz Marczak, Oliver Kayser
April 2019 Plant Science 284
doi: 10.1016/j.plantsci.2019.04.008
Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.

Cannabinoid system in the skin - a possible target for future therapies in dermatology
Piotr Kupczyk , Adam Reich, Jacek C Szepietowski
Exp Dermatol. 2009 Aug.
DOI: 10.1111/j.1600-0625.2009.00923.x
https://www.researchgate.net/publica..._P_Reich_A_Sze pietowski_JCCannabinoid_system _in_the_skin_-_a_possible_target_for_future_ therapies_in_dermatology_Exp_D ermatol_18669-679
Cannabinoids and their derivatives are group of more than 60 biologically active chemical agents, which have been used in natural medicine for centuries. The major agent of exogenous cannabinoids is Delta(9)-tetrahydrocannabinol (Delta(9)-THC), natural psychoactive ingredient of marijuana. However, psychoactive properties of these substances limited their use as approved medicines. Recent discoveries of endogenous cannabinoids (e.g. arachidonoylethanolamide, 2-arachidonoylglycerol or palmithyloethanolamide) and their receptors initiated discussion on the role of cannabinoid system in physiological conditions as well as in various diseases. Based on the current knowledge, it could be stated that cannabinoids are important mediators in the skin, however their role have not been well elucidated yet. In our review, we summarized the current knowledge about the significant role of the cannabinoid system in the cutaneous physiology and pathology, pointing out possible future therapeutic targets.

Cannabinoids
Nafiz K. Sheikh; Anterpreet Dua.
https://www.ncbi.nlm.nih.gov/books/NBK556062/
Cannabinoids, broadly speaking, are a class of biological compounds that bind to cannabinoid receptors. They are most frequently sourced from and associated with the plants of the Cannabis genus, including Cannabis sativa,Cannabis indica, and Cannabis ruderalis. The earliest known use of cannabinoids dates back 5,000 years ago in modern Romania, while the documentation of the earliest medical dates back to around 400 AD. However, formal extraction, isolation, and structural elucidation of cannabinoids have taken place rather recently in the late 19th and early 20th centuries . Since then, numerous advancements have been made in further isolating naturally occurring cannabinoids, synthesizing artificial equivalents, and discovering the endogenous the endocannabinoid system in mammals, reptiles, fish, and birds. Cannabinoids, in the context of medicine, come in three forms:
• Phytocannabinoids - derived naturally from flora
• Endocannabinoids - produced endogenously
• Synthetic Cannabinoids – created artificially

Cannabinoids Accumulation in Hemp (Cannabis sativa L.) Plants under LED Light Spectra and Their Discrete Role as a Stress Marker
Islam, M.J. Ryu, B.R. Azad, M.O.K. Rahman, M.H. Cheong, E.J. Lim, J.-D. Lim, Y.-S.
Biology 2021, 10, 710.
DOI: 10.3390 /biology10080710
https://www.ncbi.nlm.nih.gov/pmc/art...y-10-00710.pdf
Hemp adaptability through physiological and biochemical changes was studied under 10 LED light spectra and natural light in a controlled aeroponic system. Light treatments were imposed on 25 days aged seedlings for 16 h daily (300 µmol m−2 s −1 ) for 20 days. Plant accumulated highest Cannabidiol (CBD) in R7 :B2 :G1 light treatment, with relatively higher photosynthetic rate and lower reactive oxygen species, total phenol content, total flavonoid content, DPPH radical scavenging capacity, and antioxidant enzymatic activities. Tetrahydrocannabinol (THC) also accumulated at a higher level in white, R8 :B2 , and R7 :B2 :G1 light with less evidence of stress-modulated substances. These results indicated that CBD and THC have no or little relation with light-mediated abiotic stress in hemp plants. On the contrary, Tetrahydrocannabinolic acid (THCA) was accumulated higher in R6 :B2 :G1 :FR1 and R5 :B2 :W2 :FR1 light treatment along with lower photosynthetic rate and higher reactive oxygen species, total phenol content, total flavonoid content, DPPH radical scavenging capacity, and antioxidant enzymatic activities. However, Cannabidiolic acid (CBDA) was accumulated higher in R6 :B2 :G1 :FR1 light treatment with higher stress-modulated substances and lower physiological traits. CBDA was also accumulated higher in R8 :B2 and R7 :B2 :G1 light treatments with less evidence of stress-modulated substances. Besides, Greenlight influenced CBD and CBDA synthesis where FR and UV-A (along with green) play a positive and negative role in this process. Overall, the results indicated that the treatment R7 :B2 :G1 enhanced the medicinal cannabinoids most, and the role of THCA as a stress marker is more decisive in the hemp plant than in other cannabinoids under attributed light-mediated stress

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro
LS Whyte, L Ford, SA Ridge, GA Cameron, MJ Rogers and RA Ross
British Journal of Pharmacology (2012) 165 2584–2597
DOI:10.1111/j.1476-5381.2011.01519.x
BACKGROUND AND PURPOSE Both CB1 and CB2 cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects ofcannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro.
EXPERIMENTAL APPROACH Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing avb3 or with F-actin rings, or measurement of resorption area.
KEY RESULTS Human osteoclasts express both CB1 and CB2 receptors. CB2 expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, thedifferentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB1 and/or CB2 antagonists.
CONCLUSIONS AND IMPLICATIONS Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB2 receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease.

Cannabinoids and Cytochrome P450 Interactions.
Zendulka, O., Dovrt?lová, G., Nosková, K., Turjap, M., Šulcová, A., Hanuš, L., & Ju?ica, J.
Current Drug Metabolism, 17(3), 206–226.(2016).
doi:10.2174/1389200217666151210142051
Objective: This review consists of three parts, representing three different possibilities of interactions between cannabinoid receptor ligands of both exogenous and endogenous origin and cytochrome P450 enzymes
(CYPs). The first part deals with cannabinoids as CYP substrates, the second summarizes current knowledge on the influence of various cannabinoids on the metabolic activity of CYP, and the third outline a possible involvement of
the endocannabinoid system and cannabinoid ligands in the regulation of CYP liver activity.
Methods: We performed a structured search of bibliographic and drug databases for peer-reviewed literature using
focused review questions.
Results: Biotransformation via a hydrolytic pathway is the major route of endocannabinoid metabolism and the deactivation of substrates is characteristic, in contrast to the minor oxidative pathway via CYP involved in the bioactivation reactions. Phytocannabinoids are extensively metabolized by CYPs. The enzymes CYP2C9, CYP2C19, and CYP3A4 catalyze most of their hydroxylations. Similarly, CYP represents a major metabolic pathway for both synthetic cannabinoids used therapeutically and drugs that are abused. In vitro experiments document the mostly CYP inhibitory activity of the major phytocannabinoids, with cannabidiol as the most potent inhibitor of many CYPs. The drug-drug interactions between cannabinoids and various drugs at the CYP level are reported, but their clinical relevance remains unclear. The direct activation/inhibition of nuclear receptors in the liver cells by cannabinoids may result in a change of
CYP expression and activity. Finally, we hypothesize the interplay of central cannabinoid receptors with numerous nervous systems, resulting in a hormone-mediated signal towards nuclear receptors in hepatocytes.

Cannabinoids and Neuropathic Pain
P. Goya, N. Jagerovica, L. Hernandez-Folgadoa and M.I. Martin
Mini Reviews in Medicinal Chemistry, 2003, 3, 159-165
doi: 10.2174/1389557033487818
After a brief overview of the endocannabinoid system (CB receptors, and endocannabinoids) and of the cannabinergic ligands, some general issues related to cannabinoids and pain are commented. Finally, the most important findings regarding cannabinoids and neuropathic pain are discussed in detail.

Cannabinoids and neuroprotection in CNS inflammatory disease
Samuel J. Jackson, Lara T. Diemel, Gareth Pryce, David Baker
Journal of the Neurological Sciences 233 (2005) 21 – 25
DOI: 10.1016/j.jns.2005.03.002
The current failure of potent immunosuppressive agents to control progressive disease in multiple sclerosis has moved a focus from immunotherapy towards the need for neuroprotection. There is increasing evidence for cannabinoid-mediated control of symptoms, which is being more supported by the underlying biology. However there is accumulating evidence in vitro and in vivo to support the hypothesis that the cannabinoid system can limit the neurodegenerative possesses that drive progressive disease, and may provide a new avenue for disease control.

Cannabinoids and Reproduction: A Lasting and Intriguing History
Giovanna Cacciola, Rosanna Chianese, Teresa Chioccarelli, Vincenza Ciaramella,Silvia Fasano, Riccardo Pierantoni, Rosaria Meccariello, and Gilda Cobellis
Pharmaceuticals 2010, 3, 3275-3323;
doi:10.3390/ph3103275
https://pdfs.semanticscholar.org/f78...879.1582090724
Starting from an historical overview of lasting Cannabis use over the centuries, we will focus on a description of the cannabinergic system, with a comprehensive analysis of chemical and pharmacological properties of endogenous and synthetic cannabimimetic analogues. The metabolic pathways and the signal transduction mechanisms, activated by cannabinoid receptors stimulation, will also be discussed. In particular, we will point out the action of cannabinoids and endocannabinoids on the different neuronal networks involved in reproductive axis, and locally, on male and female reproductive tracts, by emphasizing the pivotal role played by this system in the control of fertility.

Cannabinoids and the immune system: Potential for the treatment of inflammatory diseases?
J. Ludovic Croxford , Takashi Yamamura
Journal of Neuroimmunology 166 (2005) 3 – 18
DOI: 10.1016/j.jneuroim.2005.04.023
Since the discovery of the cannabinoid receptors and their endogenous ligands, significant advances have been made in studying the physiological function of the endocannabinoid system. The presence of cannabinoid receptors on cells of the immune system and anecdotal and historical evidence suggesting that cannabis use has potent immuno-modulatory effects, has led to research directed at understanding the function and role of these receptors within the context of immunological cellular function. Studies from chronic cannabis smokers have provided much of the evidence for immunomodulatory effects of cannabis in humans, and animal and in vitro studies of immune cells such as T cells and macrophages have also provided important evidence. Cannabinoids can modulate both the function and secretion of cytokines from immune cells. Therefore, cannabinoids may be considered for treatment of inflammatory disease. This review article will highlight recent research on cannabinoids and how they interact with the immune system and also their potential use as therapeutic agents for a number of inflammatory disorders


Cannabinoids and Terpenes as Chemotaxonomic Markers in Cannabis
Elzinga S, Fischedick J, Podkolinski R, and Raber J
Natural Products Chemistry & Research
doi: 10.4172/2329-6836.1000181
In this paper, we present principal component analysis (PCA) results from a dataset containing 494 cannabis flower samples and 170 concentrate samples analyzed for 31 compounds. A continuum of chemical composition amongst cannabis strains was found instead of distinct chemotypes. Our data shows that some strains are much more reproducible in chemical composition than others. Strains labeled as indica were compared with those labeled as sativa and no evidence was found that these two cultivars are distinctly different chemotypes. PCA of “OG” and “Kush” type strains found that “OG” strains have relatively higher levels of ?-terpineol, fenchol, limonene, camphene, terpinolene and linalool where “Kush” samples are characterized mainly by the compounds trans-ocimene, guaiol, ?-eudesmol, myrcene and ?-pinene. The composition of concentrates and flowers were compared as well. Although the absolute concentration of compounds in concentrates is much higher, the relative composition of compounds between flowers and concentrates is similar.

Cannabinoids and the immune system: potential for the treatment of inflammatory diseases?
J Ludovic Croxford , Takashi Yamamura
J Neuroimmunol . 2005 Sep;166(1-2):3-18.
DOI:10.1016/j.jneuroim.2005.04.023
Since the discovery of the cannabinoid receptors and their endogenous ligands, significant advances have been made in studying the physiological function of the endocannabinoid system. The presence of cannabinoid receptors on cells of the immune system and anecdotal and historical evidence suggesting that cannabis use has potent immuno-modulatory effects, has led to research directed at understanding the function and role of these receptors within the context of immunological cellular function. Studies from chronic cannabis smokers have provided much of the evidence for immunomodulatory effects of cannabis in humans, and animal and in vitro studies of immune cells such as T cells and macrophages have also provided important evidence. Cannabinoids can modulate both the function and secretion of cytokines from immune cells. Therefore, cannabinoids may be considered for treatment of inflammatory disease. This review article will highlight recent research on cannabinoids and how they interact with the immune system and also their potential use as therapeutic agents for a number of inflammatory disorders.

Cannabinoids as novel anti-inflammatory drugs
Prakash Nagarkatti, Rupal Pandey, Sadiye Amcaoglu Rieder, Venkatesh L Hegde, and Mitzi Nagarkatti
Future Med Chem. 2009 October ; 1(7): 1333–1349.
doi:10.4155/fmc.09.93
https://europepmc.org/backend/ptpmcr...?accid=PMC2828 614&blobtype=pdf
Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of Δ9 -tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to a significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found on the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids, led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivo can constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components

Cannabinoids as pharmacotherapies for neuropathic pain: From the bench to the bedside.
Rahn, E. J., & Hohmann, A. G.
Neurotherapeutics, 6(4), 713–737. (2009).
doi:10.1016/j.nurt.2009.08.002
Neuropathic pain is a debilitating form of chronic pain resulting from nerve injury, disease states, or toxic insults. Neuropathic pain is often refractory to conventional pharmacotherapies, necessitating validation of novel analgesics. Cannabinoids, drugs that share the same target as
9 -tetrahydrocannabinol (9 -THC), the psychoactive ingredient in cannabis, have the potential to address this unmet need. Here, we review studies evaluating cannabinoids for neuropathic pain management in the clinical and preclinical literature. Neuropathic pain associated with nerve injury, diabetes, chemotherapeutic treatment, human immunodeficiency virus, multiple sclerosis, and herpes zoster infection is considered. In animals, cannabinoids attenuate neuropathic nociception produced by traumatic nerve injury, disease, and toxic insults. Effects of mixed cannabinoid CB1/CB2 agonists, CB2 selective agonists, and modulators of the endocannabinoid system (i.e., inhibitors of transport or degradation) are compared. Effects of genetic disruption of cannabinoid receptors or enzymes controlling endocannabinoid degradation on neuropathic nociception are described. Specific forms of allodynia and hyperalgesia modulated by cannabinoids are also considered. In humans, effects of smoked marijuana, synthetic 9 -THC analogs (e.g., Marinol, Cesamet) and medicinal cannabis preparations containing both 9 -THC and cannabidiol (e.g., Sativex, Cannador) in neuropathic pain states are reviewed. Clinical studies largely affirm that neuropathic pain patients derive benefits from cannabinoid treatment. Subjective (i.e., rating scales) and objective (i.e., stimulusevoked) measures of pain and quality of life are considered. Finally, limitations of cannabinoid pharmacotherapies are discussed together with directions for future research. Key Words: Endocannabinoid, marijuana, neuropathy, multiple sclerosis, chemotherapy, diabetes.

Cannabinoids Biology: The Search for New Therapeutic Targets
Christian C Felder, Amy K Chesterfield, Steve Moore
Molecular interventions 6(3):149-61
DOI: 10.1124/mi.6.3.6
https://www.researchgate.net/publica...peutic_Targets
Cannabinoids, in the form of marijuana plant extracts, have been used for thousands of years for a wide variety of medical conditions, ranging from general malaise and mood disorders to more specific ailments, such as pain, nausea, and muscle spasms. The discovery of tetrahydrocannabinol, the active principal in marijuana, and the identification and cloning of two cannabinoid receptors (i.e., CB 1 and CB 2) has subsequently led to biomedical appreciation for a family of endocannabinoid lipid transmitters. The biosynthesis and catabolism of the endocannabinoids and growing knowledge of their broad physiological roles are providing insight into potentially novel therapeutic targets. Compounds directed at one or more of these targets may allow for cannabinoid-based therapeutics with limited side effects and abuse liability.

Cannabinoids, Endocannabinoids, and Related Analogs in Inflammation
Sumner H. Burstein1,2,3 and Robert B. Zurier
The AAPS Journal, Vol. 11, No. 1, March 2009 (# 2009)
DOI: 10.1208/s12248-009-9084-5.
This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation. A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process.

Cannabinoids for Neuropathic Pain
Perry G. Fine & Mark J. Rosenfeld
Curr Pain Headache Rep (2014) 18:451
DOI 10.1007/s11916-014-0451-2
https://rsds.insctest1.com/wp-conten...athic-pain.pdf
Treatment options for neuropathic pain have limited efficacy and use is fraught with dose-limiting adverse effects. The endocannabinoid system has been elucidated over the last several years, demonstrating a significant interface with pain homeostasis. Exogenous cannabinoids have been demonstrated to be effective in a range of experimental neuropathic pain models, and there is mounting evidence for therapeutic use in human neuropathic pain conditions. This article reviews the history, pharmacologic development, clinical trials results, and the future potential of nonsmoked, orally bioavailable, nonpsychoactive cannabinoids in the management of neuropathic pain

Cannabinoids for the pharmaceutical industry
Colin G. Stott & Geoffrey W. Guy
Euphytica 140: 83–93, 2004
Doi: 10.1007/s10681-004-4757-8
Cannabis sativa, is a rich source of a variety of compounds, including cannabinoids, terpenoids and flavonoids. Their content depends upon the plant genetics, growth conditions, time of harvest and drying conditions. To date, more than 60 different cannabinoids have been identified in the plant. Cannabis has been used medicinally for 4000 years and remained in the British pharmacopaeia until 1932, and in the British Pharmaceutical Codex until 1949. Medical use has been prohibited in the UK since 1973. The principal cannabinoid, delta-9-tetrahydrocannabinol (THC) was first isolated in 1964; the first cannabinoid pharmaceutical product Marinol R _ (a synthetic THC product) was approved in the USA in 1985. The discovery of specific cannabinoid receptors in the early 1990s and subsequent identification of the endocannabinoids anandamide and 2-arachadonoylglycerol, led to a resurgence of interest in the field of cannabinoid medicine, especially within the pharmaceutical industry. Cannabidiol (CBD), as a non-psychoactive,cannabinoid is currently a cannabinoid of significant interest, showing a wide range of pharmacological activity. The other classes of compounds present in cannabis also have their own pharmacology (e.g. terpenoids, flavonoids). The potential for interaction and synergy between compounds within the plant, may play a role in the therapeutic potential of cannabis. This may explain why a cannabis-based medicine using extracts containing multiple cannabinoids, in defined ratios, and other non-cannabinoid fractions, may provide better therapeutic success and be better tolerated than the single synthetic cannabinoid medicines currently available. The development and employment of one of these medicines, Sativex R _ , is described.

Cannabinoids for Treatment of MS Symptoms: State of the Evidence.
Rice J, Cameron M.
Curr Neurol Neurosci Rep. 2018 Jun 19;18(8):50.
doi: 10.1007/s11910-018-0859-x.
PURPOSE OF REVIEW:
Cannabis and cannabinoids have been used medically and recreationally for thousands of years and recently there has been a growing body of research in this area. With increased access now that medical marijuana is available in many jurisdictions, patients and providers want to know more about the evidence for benefits and risks of cannabinoid use. This paper provides an overview of the available cannabinoid-based formulations, a summary of the highest quality evidence for the use of cannabinoids for treating spasticity and pain associated with multiple sclerosis (MS), and a discussion of possible dosing regimens based on information from these studies.
RECENT FINDINGS:
Two recent high-quality systematic reviews concluded that the only strong evidence for medical marijuana in neurological disorders was for reducing the symptoms of patient-reported spasticity and central pain in MS and that the only complementary and alternative medicine (CAM) intervention in MS with strong supportive evidence was cannabinoids. Based on this review, they concluded that nabiximols (Sativex oral spray), oral cannabis extract (OCE), and synthetic tetrahydrocannabinol (THC) are probably effective at reducing patient-reported symptoms of spasticity in people with MS, but OCE and synthetic THC were not found to be effective for reducing physician-administered measures of spasticity. In addition, nabiximols, OCE, and synthetic THC are probably effective at reducing MS-related pain. Cannabinoids were generally well-tolerated. However, cannabis use has been associated with an increased risk of psychosis and schizophrenia in at-risk individuals, there is growing evidence that cannabis can increase the risk for cardiovascular diseases, including myocardial infarction (MI), hypertension, heart failure, and stroke, and a recently recognized adverse effect of cannabis is cannabinoid hyperemesis syndrome. The medical use of cannabinoids remains controversial. While cannabinoids have been studied for a variety of neurologic disorders, there is strongest evidence to indicate benefits in treatment of spasticity and neuropathic pain in multiple sclerosis. Although the best dose for an individual remains uncertain, most participants in the studies discussed in this paper used between 20 and 40 mg of THC a day in divided doses. Adverse events in studies were generally more common in the groups using cannabinoid products but serious adverse events were rare and cannabis products were generally well-tolerated. Cannabis use does appear to be associated with increased risk of certain adverse events, including psychosis, cardiovascular diseases, and cannabinoid hyperemesis syndrome.

Cannabinoids for the treatment of refractory neuropathic pruritus in amyotrophic lateral sclerosis: A case report
Kelvin Lou , Shane Murphy , Clair Talbot
Palliat Med. 2021 Sep 11;
DOI: 10.1177/02692163211045314
https://journals.sagepub.com/doi/pdf...92163211045314
Background: Neuropathic symptoms have a wide variety of manifestations, ranging from pain to pruritus. Neuropathic pruritus is a type of chronic pruritus related to damaged small fibers. Cannabinoids have evidence to manage neuropathic symptoms. We present a case of refractory neuropathic pruritus that was successfully managed with the use of oral cannabinoids.
Case presentation: A 60-year-old male with amyotrophic lateral sclerosis with ongoing pruritus despite the use of standard neuropathic therapies.
Possible course of action: Sodium channel and N-methyl-D-aspartate receptor antagonists have evidence for neuropathic symptoms but can cause significant gastrointestinal side effects. Prescription cannabinoids such as nabiximol can be cost prohibitive to use in practice. Synthetic tetrahydrocannabinol products are dose limited by psychoactive side effects.
Formulation of a plan: A balanced oral cannabinoid from a licensed producer was preferred as it has evidence for neuropathic symptoms and is generally well tolerated.
Outcome: The patient showed improvement to his pruritus score from 7/10 to 3/10. There was initial increased sedation but tolerance developed quickly.
Lessons learned from case: Cannabinoids are possibly safe and effective in management of neuropathic pruritus. View on research problems: Additional research is needed to establish efficacy and safety.

Cannabinoids - from plant to patient.
Holdcroft A
5 April 2001, London, UK.
IDrugs. 2001 Jul;4(7):773-5.
The active constituents of cannabis (predominantly cannabinoids and possibly flavonoids) are more effective than a single cannabinoid in reducing muscle spasticity in a multiple sclerosis animal model. Possible interactions include both pharmacokinetic and pharmacodynamic effects. Synthetic cannabinoids with agonist or antagonist/inverse agonist effects are available with high affinity for cannabinoids receptors. Pharmaceutical products that block their uptake and metabolism may enhance the endocannabinoid system. Government- and charity-funded clinical trials of cannabis are proceeding in pain, both acute (MRC multicenter trial) and chronic, and multiple sclerosis (MRC and Multiple Sclerosis Society multicenter trials). The design of these trials should enable evidence to be presented to regulatory bodies documenting the medicinal uses of standardized cannabis plant material.

Special Issue: Cannabinoids in Biology and Medicine, Part I.
Guest Editors: Itai Bab and Steve Alexander
Volume 163, Issue 7
GET DOI’s
https://bpspubs.onlinelibrary.wiley... .14765381/163/7
Free downloads of 19 papers

Cannabinoids in Clinical Practice.
Williamson, E. M., & Evans, F. J.
Drugs, 60(6), 1303–1314. (2000).
doi:10.2165/00003495-200060060-00005
Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (?9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties of THC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapyinduced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.

Cannabinoids in models of chronic inflammatory conditions
Raphael Mechoulam, Percy F. Sumariwalla, Marc Feldmann & Ruth Gallily
Phytochemistry Reviews (2005) 4: 11–18
DOI 10.1007/s11101-004-1534-1
https://link.springer.com/content/pd...004-1534-1.pdf
Cannabis sativa has been used as an anti-inflammatory plant for millennia. However until the elucidation of the chemistry of its constituents and the discovery of the endogenous cannabinoid system only a limited amount of research had been done on the effects of the plant or its constituents on inflammation. In the present overview we summarize our work on the effects of the non-psychotropic cannabidiol (CBD) and of a synthetic cannabidiol-derived acid (HU-320) in animal models of arthritis. Both compounds block progression of the disease, when administered after its onset. Cannabidiol was equally effective was administered i.p. or orally. Significant protection of the joints against severe damage was noted. In vitro cannabidiol reduced lymphocyte proliferation, and TNF-a formation and blocked zymosan-triggered production of reactive oxygen intermediates (ROI). Ex vivo lymph node cells from CBD-treated mice showed a decrease of collagen II-specific proliferation and IFN-c production. A decreased release of TNF by knee synovial cells was also noted. A synthetic cannabidiol derivative, HU-320 also inhibited production of TNF and ROI by mouse macrophages in vitro and suppressed in vivo rise in serum TNF following endotoxin challenge. HU-320 showed no activity in a standard assay for THC-type psychotropic effects. These results suggest that CBD and HU-320 hold promise as potential novel anti-inflammatory agents.

Cannabinoids in neuroinflammation oxidative stress and neuro Excitotoxicity
Rosales-Corral S, Hernández L and Gallegos M
Pharm Anal Acta 2015, 6:3
DOI: 10.4172/2153-2435.1000346
https://www.researchgate.net/publica...inoids_in_Neur oinflammation_Oxidative_Stress _and_Neuro_Excitotoxicity
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Research on cannabinoids has been growing significantly in the last five years. More than fifty percent of this research corresponds to “cannabinoids and brain”, particularly about neurodegeneration. In this sense, there is evidence reporting that specific phyto cannabinoids show some specific action on each one of main pathogenic mechanisms involved in neurodegeneration such as oxidative stress, neuroinflammation and excitotoxicity. However, by using the same targets, cannabinoids may also induce the opposite effects, this is, excitotoxicity and inflammation. In fact, both tetrahydro cannabinol and cannabidiol activate cannabinoid receptors, but they also may act as antagonists of those receptors. It seems to be a dose-dependent issue; nonetheless, as reviewed in this paper, many other factors such as timing, type of cell and its state of activity even the activation of different, noncannabinoid receptorsseem to have a role related to those unexpected antagonic effects.[/FONT]

Cannabinoids in Parkinson’s Disease
Mario Stampanoni Bassi, Andrea Sancesario, Roberta Morace, Diego Centonze, and Ennio Iezzi
Cannabis and Cannabinoid Research Volume 2.1, 2017 DOI: 10.1089/can.2017.0002
The endocannabinoid system plays a regulatory role in a number of physiological processes and has been found altered in different pathological conditions, including movement disorders. The interactions between cannabinoids and dopamine in the basal ganglia are remarkably complex and involve both the modulation of other neurotransmitters (c-aminobutyric acid, glutamate, opioids, peptides) and the activation of different receptors subtypes (cannabinoid receptor type 1 and 2). In the last years, experimental studies contributed to enrich this scenario reporting interactions between cannabinoids and other receptor systems (transient receptor potential vanilloid type 1 cation channel, adenosine receptors, 5-hydroxytryptamine receptors). The improved knowledge, adding new interpretation on the biochemical interaction between cannabinoids and other signaling pathways, may contribute to develop new pharmacological strategies. A number of preclinical studies in different experimental Parkinson’s disease (PD) models demonstrated that modulating the cannabinoid system may be useful to treat some motor symptoms. Despite new cannabinoid-based medicines have been proposed for motor and nonmotor symptoms of PD, so far, results from clinical studies are controversial and inconclusive. Further clinical studies involving larger samples of patients, appropriate molecular targets, and specific clinical outcome measures are needed to clarify the effectiveness of cannabinoid based therapies

Cannabinoids in the Management of Acute Pain: A Systematic Review and Meta-analysis.
Gazendam, A., Nucci, N., Gouveia, K., Abdel Khalik, H., Rubinger, L., & Johal, H.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2019.0079
Objective: To synthesize the best evidence surrounding the efficacy of cannabinoids for acute pain in the clinical setting based on subjective pain scores and observed adverse effects. Design: Systematic review with meta-analysis. Data Sources: PubMed, Embase, Cochrane Databases, and Google Scholar. Eligibility Criteria: English-language randomized-controlled clinical trials comparing cannabinoids with placebo in patients with acute pain. Data Extraction and Synthesis: Study quality was assessed using the Cochrane risk of bias tool. All stages were conducted independently by a team of three reviewers. Data were pooled through meta-analysis and stratified by route of administration. Primary Outcomes and Measures: Patient-reported pain and adverse events (AEs). Results: Six trials (678 participants) were included examining oral (5 trials) and intramuscular (1 trial) cannabinoids. Overall, there was a small but statistically significant treatment effect favoring the use of cannabinoids over placebo (0.90, 95% confidence interval [CI]1.69 to0.1, i 2 = 65%, p = 0.03). When stratified by route of administration, intramuscular cannabinoids were found to have a significant reduction in pain relative to placebo (2.98, 95% CI4.09 to1.87, i 2 = 0%, p < 0.0001). No difference in effect was observed between oral cannabinoids and placebo (0.21, 95% CI0.64 to 0.22, i 2 = 3%, p = 0.34). Serious AEs were rare, and similar across the cannabinoid (14/374, 3.7%) and placebo groups (8/304, 2.6%). Conclusions: There is low-quality evidence indicating that cannabinoids may be a safe alternative for a small but significant reduction in subjective pain score when treating acute pain, with intramuscular administration resulting in a greater reduction relative to oral. Higher quality, long-term randomized-controlled trials examining whether there may be a role for cannabinoids in treating acute pain are required.

Cannabinoids in the treatment of pain and spasticity in multiple sclerosis.
Smith PF.
Curr Opin Investig Drugs. 2002 Jun;3(6):859-64.
There is a large amount of evidence to support the view that the psychoactive ingredient in cannabis, delta9-tetrahydrocannabinol (delta9-THC), and cannabinoids in general, can reduce muscle spasticity and pain under some circumstances. Cannabinoid (CB1) receptors in the CNS appear to mediate both of these effects and endogenous cannabinoids may fulfil these functions to some extent under normal circumstances. However, in the context of multiple sclerosis (MS), it is still questionable whether cannabinoids are superior to existing, conventional medicationsfor the treatment of spasticity and pain. In the case of spasticity, there are too few controlled clinical trials to draw any reliable conclusion at this stage. In the case of pain, most of the available trials suggest that cannabinoids are not superior to existing treatments; however, few trials have examined chronic pain syndromes that are relevant to MS. Whether or not cannabinoids do have therapeutic potential in the treatment of MS, a further issue will be whether synthetic cannabinoids should be used in preference to cannabis itself. Smoking cannabis is associated with significant risks of lung cancer and other respiratory dysfunction. Furthermore, delta9-THC, as a broad-spectrum cannabinoid receptor agonist, will activate both CB1 and CB2 receptors. Synthetic cannabinoids, which target specific cannabinoid receptor subtypes in specific parts of the CNS, are likely to be of more therapeutic use than delta9-THC itself. If rapid absorption is necessary, such synthetic drugs could be delivered via aerosol formulations.

Cannabinoids in treatment-resistant epilepsy: A review
Brooke K. O'Connell, David Gloss, Orrin Devinsky
Epilepsy & Behavior 70(Pt B) February 2017
https://www.researchgate.net/publica...lepsy_A_review
DOI: 10.1016/j.yebeh.2016.11.012
Treatment-resistant epilepsy (TRE) affects 30% of epilepsy patients and is associated with severe morbidity and increased mortality. Cannabis-based therapies have been used to treat epilepsy for millennia, but only in the last few years have we begun to collect data from adequately powered placebo-controlled, randomized trials (RCTs) with cannabidiol (CBD), a cannabis derivative. Previously, information was limited to case reports, small series, and surveys reporting on the use of CBD and diverse medical marijuana (MMJ) preparations containing: tetrahydrocannabinol (THC), CBD, and many other cannabinoids in differing combinations. These RCTs have studied the safety and explored the potential efficacy of CBD use in children with Dravet Syndrome (DS) and Lennox-Gastaut Syndrome (LGS). The role of the placebo response is of paramount importance in studying medical cannabis products given the intense social and traditional media attention, as well as the strong beliefs held by many parents and patients that a natural product is safer and more effective than FDA-approved pharmaceutical agents. We lack valid data on the safety, efficacy, and dosing of artisanal preparations available from dispensaries in the 25 states and District of Columbia with MMJ programs and online sources of CBD and other cannabinoids. On the other hand, open-label studies with 100 mg/ml CBD (Epidiolex®, GW Pharmaceuticals) have provided additional evidence of its efficacy along with an adequate safety profile (including certain drug interactions) in children and young adults with a spectrum of TREs. Further, Phase 3 RCTs with Epidiolex support efficacy and adequate safety profiles for children with DS and LGS at doses of 10- and 20-mg/kg/day. This article is part of a Special Issue titled "Cannabinoids and Epilepsy".
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Cannabinoids, interoception, and anxiety.
Andrade, A. K., Renda, B., & Murray, J. E.
Pharmacology Biochemistry and Behavior.(2019).
doi:10.1016/j.pbb.2019.03.006
The use of cannabis is rapidly gaining legal status across North America. Such dramatic legislative shifts have prompted an urgency in elucidating the stimulus effects of cannabis consumption. Cannabis use, though relatively safe compared to other drugs of abuse, has been associated with greater risk of mental health disorders, possibly via its primary psychoactive constituent, ?-9-tetrahydrocannabinol (THC). In this review, we discuss endocannabinoid activation and cannabis constituents from the perspective of subjective interoceptive (internally-perceived) states and how that relates to anxiety. Human studies have examined these subjective effects through use of self-report questionnaires. However, non-human studies use proxy methods of assessing anxiety states, such as elevated plus maze and fear conditioning paradigms. So far, this body of research has demonstrated that both endogenous and exogenous cannabinoid activation generally elicits biphasic effects on expression of the subjective state, with lower doses appearing to have anxiolytic properties and higher doses perceived as anxiogenic. Unfortunately, research with these compounds has been historically limited due to excessively tight regulatory control. Therefore, much work remains regarding the investigation of interactions between cannabinoid receptor activity and cannabis constituents on anxiety. Ongoing changes in legal status will hopefully mitigate the challenges faced by researchers attempting to access cannabis and THC that is inherently built in by federal and international classifications.

Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability
A Alhamoruni1 , KL Wright2 , M Larvin1 and SE O’Sullivan
British Journal of Pharmacology (2012) 165 2598–2610
DOI:10.1111/j.1476-5381.2011.01589.x
BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro.
EXPERIMENTAL APPROACH Confluent Caco-2 cell monolayers were treated for 24 h with IFNg and TNFa (10 ng·mL-1 ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB1, CB2, TRPV1, PPARg and PPARa. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids.
KEY RESULTS D9 -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB1 receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB1 antagonism. No role for the CB2 receptor was identified in these studies. Co-application of THC, cannabidiol or a CB1 antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation.
CONCLUSIONS AND IMPLICATIONS These findings suggest that locally produced endocannabinoids, acting via CB1 receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation.

Cannabinoids pharmacological effects are beyond the palliative effects: CB2 cannabinoid receptor agonist induced cytotoxicity and apoptosis in human colorectal cancer cells (HT-29)
Aylar Alenabi , Hassan Malekinejad
Mol Cell Biochem. 2021 Sep;476(9):3285-3301.
doi: 10.1007/s11010-021-04158-6
Colorectal cancer (CRC) is between the top three occurring cancers worldwide. The anticancer effects of Cannabinoid receptor 2 (CB2) agonist (GW833972A) in the presence and absence of its inverse agonist (SR144528) on Human colorectal adenocarcinoma cells (HT-29) was investigated. Following cell viability assays on HT-29 and HFF cells, the molecular mechanism(s) of cytotoxicity and apoptotic pathways of cell death were analyzed. The anticancer effects of CB2 agonist were measured with tumor cell migration and colony-forming assays. Real-time PCR and Western blotting techniques were used to examine any alterations in the expression of apoptotic genes. A concentration and time-dependent cytotoxicity of CB2 agonist with IC50 value of 24.92 ± 6.99 μM was obtained. The rate of lipid peroxidation was elevated, while the TNF-α concentration was declined, significantly (p < 0.05). CB2 agonist (50 μM) reduced the colony-forming capability by 83% and tumor cell migration by 50%. Apoptotic effects of CB2 agonist were revealed with the increase of apoptotic cells in Acridine orange/Ethidium bromide staining, clear DNA fragmentation, pro-apoptotic genes and proteins upregulation (Caspase-3 and p53), and significant downregulation of anti-apoptotic Bcl-2. All assessments demonstrated that CB2 agonist-induced effects were reversed by CB2 inverse agonist. These data suggest that CB2 agonists at micro-molar concentrations might be considered in the CRC treatment, and their effectiveness attributes to the apoptosis induction via upregulation of caspase-3 and p53 and downregulation of Bcl-2.

Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis
Mohamed M. Radwan, Suman Chandra, Shahbaz Gul and Mahmoud A. ElSohly
Molecules 2021, 26, 2774.
DOI: 10.3390/molecules26092774
https://www.researchgate.net/publica...ds_of_Cannabis
Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

CANNABINOIDS: POTENTIAL ANTICANCER AGENTS
Manuel Guzmán
Nature reviews VOLUME 3 | OCTOBER 2003 | 745
https://www.cannabis-med.org/data/pdf/en_2006_02_1.pdf
DOI: 10.1038/nrc1188
the active components of Cannabis sativa and their derivatives — exert palliative effects in cancer patients by preventing nausea, vomiting and pain and by stimulating appetite. In addition, these compounds have been shown to inhibit the growth of tumour cells in culture and animal models by modulating key cell-signalling pathways. Cannabinoids are usually well tolerated, and do not produce the generalized toxic effects of conventional chemotherapies. So, could cannabinoids be used to develop new anticancer therapies

Cannabinoids Production by Hairy Root Cultures of Cannabis sativa L.
Sayed Farag, Oliver Kayser
American Journal of Plant Sciences
DOI: 10.4236/ajps.2015.611188
Tetrahydrocannabinol (THC) derivatives are used clinically as analgesic, anti-inflammatory, appetite stimulant, anti-emetic and anti-tumor cannabinoids. THC and its related compounds are at present obtained by extraction from intact Cannabis plants or chemical synthesis, but plant cell cultures may be an alternative source of production. In the present study, hairy root cultures of C. sativa (Cannabaceae) were induced by incubation of aseptically grown callus culture with solid B5 medium supplemented with 4 mg/l naphthaleneacetic acid in darkness at 25 ?C. Hairy root growth profiles in shake flask, increased periodically during 35 days of growth cycle. The cannabinoid contents produced in minor levels and remained below 2.0 ?g/g dry weight. The contents of can-
nabinoid were analyzed by liquid chromatography and confirmed by mass spectrometry.

Cannabinoids Primer: The Natural synthesis and pre-cursors to cannabinoids
https://www.scribd.com/document/2493...-cannabinoids#


III.—Cannabinol. Part I
Thomas Barlow Wood, M.A., W. T. Newton Spivey, M.A. and Thomas Hill Easterfield, M.A., Ph.D.
J. Chem. Soc., Trans., 1899,75, 20-36
DOI: 10.1039/CT8997500020
IN a paper communicated to the Society in 1896 (Trans., 1896, 88, 539) the authors, under the name of “cannabinol,” described a physiologically active substance which they had isolated from ‘ charas,” the exuded resin of Indian hemp. From the constancy ofcomposition of a number of preparations of this substance obtained from different samples of “charas,” it was believed to be a definite chemical compound of the formula C18H,,02 ; this conclusion seemed
to be justified by the determination of the molecular weight, and by the examination of several derivatives. Since then, the authors have further examined cannabinol, and have found that it is a mixture of at least two compounds having similar physical characters. One of these, of the formula C21H2602, has been isolated, and it is proposed to retain the name cannabinol for this compound.

Cannabinol and Sleep: Separating Fact from Fiction
Jamie Corroon
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0006
https://www.liebertpub.com/doi/pdf/1.../can.2021.0006
In recent years, marketers of cannabis (i.e., marijuana) products have claimed that cannabinol (CBN) has unique sleep-promoting effects. Despite a plausible mechanism, it is possible that such claims are merely rooted in cannabis lore. The aim of this narrative review was to answer the question: ‘‘Is there sufficient clinical evidence to support claims that CBN has sleep-promoting effects?’’ A systematic search of PubMed/- MEDLINE was performed to evaluate the published evidence. The abstracts of 99 human studies were screened for relevance by the author and reviewed for compliance with the inclusion criteria. The characteristics and principal findings were extracted from eight full-text articles that met inclusion criteria for detailed review. Pre-clinical and clinical research investigating the effects of CBN is dated and limited, with the preponderance of human studies occurring in the 1970–1980s with small sample sizes lacking diversity in sociodemographic characteristics. Studies specifically assessing subjective effects associated with sleep, such as sedation or fatigue, are rare. Most importantly, published clinical trials investigating associations between CBN and validated sleep questionnaires and/or formal polysomnography were not identified in this review. In addition, evidence demonstrating that CBN itself elicits cannabis-like effects in humans is mixed, with the majority of available evidence demonstrating a lack of such an effect. Consequently, there is insuf- ficient published evidence to support sleep-related claims. Randomized controlled trials are needed to substantiate claims made by manufacturers of cannabis products containing CBN. These studies should specifically evaluate its effects on sleep through polysomnography, or at minimum, through validated sleep questionnaires, and use dosages significantly higher than those found in currently available cannabis products marketed for sleep (typically £ 5 mg). Individuals seeking cannabis-derived sleep aids should be skeptical of manufacturers’ claims of sleep-promoting effects.


Cannabis, a complex plant: different compounds and different effects on individuals.
Atakan, Z.
Therapeutic Advances in Psychopharmacology, 2(6), 241–254. (2012).
doi:10.1177/2045125312457586
Cannabis is a complex plant, with major compounds such as delta-9- tetrahydrocannabinol and cannabidiol, which have opposing effects. The discovery of its compounds has led to the further discovery of an important neurotransmitter system called the endocannabinoid system. This system is widely distributed in the brain and in the body, and is considered to be responsible for numerous significant functions. There has been a recent and consistent worldwide increase in cannabis potency, with increasing associated health concerns. A number of epidemiological research projects have shown links between dose-related cannabis use and an increased risk of development of an enduring psychotic illness. However, it is also known that not everyone who uses cannabis is affected adversely in the same way. What makes someone more susceptible to its negative effects is not yet known, however there are some emerging vulnerability factors, ranging from certain genes to personality characteristics. In this article we first provide an overview of the biochemical basis of cannabis research by examining the different effects of the two main compounds of the plant and the endocannabinoid system, and then go on to review available information on the possible factors explaining variation of its effects upon different individuals.

Cannabis and Bioactive Cannabinoids
Federica Messina, Ornelio Rosati, Massimo Curini, M. Carla Marcotullio
Studies in Natural Products Chemistry Volume 45, 2015, Pages 17-57
Chapter 2 - Cannabis and Bioactive Cannabinoids
DOI: 10.1016/B978-0-444-63473-3.00002-2
The therapeutic use of Cannabis dates back to ancient times and this plant has been used for centuries as remedy for a large number of diseases. Today it is well known that biological activity of Cannabis is related to the endocannabinoidsystem (ECS), a complex signaling network that comprises classical cannabinoidreceptors (CB1 and CB2), arachidonic acid-derived ligands, and enzymes degrading the endocannabinoids anandamide and 2-arachidonoyl glycerol, namely fatty acid amide hydrolase and monoacylglycerol lipase. The modulation of the ECS activity turned out to be a therapeutic promise in a wide range of diseases. A problem to the development of Cannabis and cannabinoid medications is the psychoactive property of natural or synthetic agonists, mediated by CB1 receptor. This review deals with the literature analysis of the important biological activities of Cannabis and the efforts aimed to the discovery of natural and nonnatural selective cannabinoids.
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Cannabis and its cannabinoids analysis by gas chromatography–mass spectrometry with Cold EI.
Amirav, A., Neumark, B., Margolin Eren, K. J., Fialkov, A. B., & Tal, N.
Journal of Mass Spectrometry, 56(6).(2021).
doi:10.1002/jms.4726
Cannabis extracts and products were analyzed by gas chromatography–mass spectrometry (GC–MS) with Cold EI for their full content including terpenes, sesquiterpenes, sesquiterpinols, fatty acids, delta 9-tetrahydrocannabinol (THC), cannabidiol (CBD), other cannabinoids, hydrocarbons, sterols, diglycerides, triglycerides, and impurities. GC–MS with Cold EI is based on interfacing GC and MS with supersonic molecular beams (SMB) along with electron ionization of vibrationally cold sample compounds in the SMB in a fly-through ion source (hence the name Cold EI). GC–MS with Cold EI improves all the performance aspects of GC–MS, enables the analysis of Cannabinoids with OH groups without derivatization, while providing enhanced molecular ions for improved identification, and enables internal quantitation without calibration. We found over 50 cannabinoid compounds including a new one with a Cold EI mass spectrum very similar to delta 9-THC as well as relatively large cannabinoids with molecular weight above m/z = 400. Because the analysis was universal in full scan and not targeted, we found impurities such as bromo CBD and fluticasone propionate and could monitor the formation of oxidized CBD during decarboxylation. In addition, GC–MS with Cold EI enabled nontargeted full analysis of terpenes, sesquiterpenes, and sesquiterpinols in cannabis extracts with good internal quantitation. GC–MS with Cold EI further served with very good sensitivity for the concentration determination of delta 9-THC in CBD-related products. Finally, cannabis drugs such as EP-1 used in Israel for treatment of epilepsy and for children with autism spectrum disorder (ASD) were analyzed for their full cannabinoids content for learning on the entourage effect and for drug activity optimization

Cannabis and the brain.
Iversen, L.
Brain, 126(6), 1252–1270. (2003).
doi:10.1093/brain/awg143
The active compound in herbal cannabis, D9-tetrahydrocannabinol, exerts all of its known central effects through the CB1 cannabinoid receptor. Research on cannabinoid mechanisms has been facilitated by the availability of selective antagonists acting at CB1 receptors and the generation of CB1 receptor knockout mice. Particularly important classes of neurons that express high levels of CB1 receptors are GABAergic interneurons in hippocampus, amygdala and cerebral cortex, which also contain the neuropeptides cholecystokinin. Activation of CB1 receptors leads to inhibition of the release of amino acid and monoamine neurotransmitters. The lipid derivatives anandamide and 2-arachidonylglycerol act as endogenous ligands for CB1 receptors (endocannabinoids). They may act as retrograde synaptic mediators of the phenomena of depolarizationinduced suppression of inhibition or excitation in hippocampus and cerebellum. Central effects of cannabinoids include disruption of psychomotor behaviour, shortterm memory impairment, intoxication, stimulation of appetite, antinociceptive actions (particularly against pain of neuropathic origin) and anti-emetic effects. Although there are signs of mild cognitive impairment in chronic cannabis users there is little evidence that such impairments are irreversible, or that they are accompanied by drug-induced neuropathology. A proportion of regular users of cannabis develop tolerance and dependence on the drug. Some studies have linked chronic use of cannabis with an increased risk of psychiatric illness, but there is little evidence for any causal link. The potential medical applications of cannabis in the treatment of painful muscle spasms and other symptoms of multiple sclerosis are currently being tested in clinical trials. Medicines based on drugs that enhance the function of endocannabinoids may offer novel therapeutic approaches in the future.

Cannabis compounds exhibit anti infammatory activity in vitro in COVID 19 related infammation in lung epithelial cells and pro infammatory activity in macrophages
Seegehalli M.Anil, Nurit Shalev, Ajjampura C.Vinayaka, Stalin Nadarajan, Dvora Namdar, Eduard Belausov, Irit Shoval, KarthikAnanth Mani, Guy Mechrez & Hinanit Koltai
Nature Scientifc Reports | (2021) 11:1462
DOI: 10.1038/s41598-021-81049-2
https://www.nature.com/articles/s41598-021-81049-2.pdf
Cannabis sativa is widely used for medical purposes and has anti-infammatory activity. This study intended to examine the anti-infammatory activity of cannabis on immune response markers associated with coronavirus disease 2019 (COVID-19) infammation. An extract fraction from C. sativa Arbel strain (FCBD) substantially reduced (dose dependently) interleukin (IL)-6 and -8 levels in an alveolar epithelial (A549) cell line. FCBD contained cannabidiol (CBD), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with FCBD and a FCBD formulation using phytocannabinoid standards (FCBD:std) reduced IL-6, IL-8, C–C Motif Chemokine Ligands (CCLs) 2 and 7, and angiotensin I converting enzyme 2 (ACE2) expression in the A549 cell line. Treatment with FCBD induced macrophage (diferentiated KG1 cell line) polarization and phagocytosis in vitro, and increased CD36 and type II receptor for the Fc region of IgG (FcγRII) expression. FCBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. FCBD:std, while maintaining antiinfammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-infammatory IL secretion in macrophages in comparison to FCBD. The phytocannabinoid formulation may show superior activity versus the cannabis-derived fraction for reduction of lung infammation, yet there is a need of caution proposing cannabis as treatment for COVID-19.

Cannabis for COVID-19: can cannabinoids quell the cytokine storm?
Emmanuel Shan Onaivi & Venkatanarayanan Sharma
Future Sci. OA (2020) 6(8), FSO625
DOI: 10.2144/fsoa-2020-0124
https://www.ncbi.nlm.nih.gov/pmc/art...soa-06-625.pdf
The recent emergence of SARS coronavirus 2 (SARS-CoV-2) has resulted in the alarming spread of the highly infectious and contagious COVID-19 pandemic that is causing catastrophic damage and affecting health, life and death around the world [1,2]. SARS-CoV-2 has also created a COVID-19 ‘infodemic’ crisis with an overabundance of information and misinformation about the origin of the virus, potential therapies, and whether it was engineered in the laboratory. However, the global pace of SARS-CoV-2 and COVID-19 research is providing rapid and critical advances in comparison to that of the previous SARS-CoV, Middle East respiratory syndrome (MERS) and HIV [1,3,4]. COVID-19 has a protean manifestation, and the cryptic transmission of SARS-CoV-2 is characterized by multiple chains of transmission, unlike the SARS-CoV and MERS-CoV viruses that have been reported to occur mainly through nosocomial transmission [3,4]. There are still many unknowns regarding COVID-19, but there are also important lessons to be gleaned from AIDS that are applicable to the COVID-19 pandemic. They are both zoonotic diseases with different mode of transmission, with no vaccine or cure yet; however, there is an effective antiretroviral therapy for AIDS [5,6]. Furthermore, cannabis and cannabinoids have been proposed and used as adjunctive treatment for AIDS-associated cachexia, and in reduction of disease symptoms [7,8]. The processes of inflammation are important in both the pathogenesis of AIDS and COVID-19 [6,8]. Cannabinoids are effective at suppressing immune and inflammatory functions [7–9], and their potential as an anti-inflammatory treatment in COVID-19 has been suggested

*Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers.
McKernan, K., Spangler, J., Zhang, L., Tadigotla, V., Helbert, Y., Foss, T., & Smith, D. R. (2016) F1000Research, 4, 1422. doi:10.12688/f1000research.7507.2
The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis
products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.

Cannabis Pharmacology:The Usual Suspects and a Few Promising Leads
Ethan Russo
Poster
https://ethanrusso.org/download/cann...omising-leads/
Outline of an Ideal Cannabis Classification Scheme
Combines shape, content and purpose
Basic class based on primary cannabinoid (e.g. Type I for THC)
Plant morphology (e.g., broad-leaf, compact vs. tall, spindly)
Specific cannabinoid content
Specific terpenoid content
Scent
Taste (when vaporized)
Uses/Effects (patient-oriented)

Cannabis Phenolics and their Bioactivities
Federica Pollastroa, Alberto Minassia and Luigia Grazia Fresu
Current Medicinal Chemistry, 2018, 25, 1160-1185
DOI: 10.2174/0929867324666170810164636
Background: Although Cannabis sativa L. is one of the most versatile plant species with multipurpose use both as medical, alimentary source and as psychoactive abuse, its biomedical relevance focused the attention on major cannabinoids. Phytochemical characterization of cannabis highlights the presence of various non-cannabinoids constituents including flavonoids, spiroindans, dihyrostilbenes, dihydrophenanthrenes, lignanamides, steroids and alkaloids. This review aims to identify polyphenols present in this plant, their biosynthesis, their bioactivities and their synthesis, when this occurred.
Methods: We undertook a systematic research focused on bibliographic databases including all noncannabinoids phenolics in various C. sativa strains from their isolation, structural elucidation, their biological activity to their synthesis.
Result: Nevertheless, attention has so far been focused only on cannabinoids (more than one hundred isolated), cannabis is a complex plant able to produce more than 480 chemical entities that represent almost all of the different biogenetic classes. Regarding phenolic compounds, the plant biosynthesises a
plethora of unique non-cannabinoids second metabolites, such as prenylated flavonoids, stilbenoids derivatives and lignanammides.
Conclusion: Cannabis is a plant with high pharmacological and nutrition values, its potentialities and applications are not only circumscribed to cannabinoids biological activities, but also defined by noncannabinoid compounds. The combination of other cannabinoids together with noncannabinoid components could enhance the beneficial effects of THC and could reduce undesirable side effects

Cannabisol, a novel D9-THC dimer possessing a unique methylene bridge, isolated from Cannabis sativa
Fazila Zulfiqar , Samir A. Ross, Desmond Slade, Safwat A. Ahmed, Mohamed M. Radwan, Zulfiqar Ali, Ikhlas A. Khan, Mahmoud A. ElSohly
Tetrahedron Letters 53 (2012) 3560–3562
https://dx.doi.org/10.1016/j.tetlet.2012.04.139
Cannabisol (1), a unique dimer of D9-tetrahydrocannabinol (D9-THC) with a methylene bridge, was isolated from Cannabis sativa. This is the first example of a C-bridged dimeric cannabinoid. The structure of 1 was unambiguously deduced by HRESIMS, GCMS, and NMR spectroscopy. A plausible biogenesis of 1 is described.

Cannabis sativa L.
Isvett Josefina Flores-Sanchez and Robert Verpoorte
Plant Cell Physiol (2008) 49 (12): 1767-1782. Plants
doi: 10.1093/pcp/pcn150
Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.

Cannabis Sativa L.: a comprehensive review on the analytical methodologies for cannabinoids and terpenes characterization.
Micalizzi G, Vento F, Alibrando F, Donnarumma D, Dugo P, Mondello L.
J Chromatogr A. 2020 Dec 30;1637:461864.
doi: 10.1016/j.chroma.2020.461864.
Undoubtedly, the enormous interest about cannabis cultivation mainly derives from the well-known pharmacological properties of cannabinoids and terpenes biosynthesized by the plants. ...Lastly, GC GC techniques are also reported for accurate identification and quantificatification of terpenes in complex cannabis matrices.

Cannabis sativa L. and Nonpsychoactive Cannabinoids: Their Chemistry and Role against Oxidative Stress, Inflammation, and Cancer
Federica Pellati, Vittoria Borgonetti, Virginia Brighenti, Marco Biagi, Stefania Benvenuti, and Lorenzo Corsi
Hindawi BioMed Research International Volume 2018, doi: 10.1155/2018/1691428
In the last decades, a lot of attention has been paid to the compounds present in medicinal Cannabis sativa L., such as ?9 - tetrahydrocannabinol (?9 -THC) and cannabidiol (CBD), and their efects on infammation and cancer-related pain. Te National Cancer Institute (NCI) currently recognizes medicinal C. sativa as an efective treatment for providing relief in a number of symptoms associated with cancer, including pain, loss of appetite, nausea and vomiting, and anxiety. Several studies have described CBD as a multitarget molecule, acting as an adaptogen, and as a modulator, in diferent ways, depending on the type and location of disequilibrium both in the brain and in the body, mainly interacting with specifc receptor proteins CB1 and CB2. CBD is present in both medicinal and fbre-type C. sativa plants, but, unlike ?9 -THC, it is completely nonpsychoactive. Fibre-type C. sativa (hemp) difers from medicinal C. sativa, since it contains only few levels of ?9 -THC and high levels of CBD and related nonpsychoactive compounds. In recent years, a number of preclinical researches have been focused on the role of CBD as an anticancer molecule, suggesting CBD (and CBD-like molecules present in the hemp extract) as a possible candidate for future clinical trials. CBD has been found to possess antioxidant activity in many studies, thus suggesting a possible role in the prevention of both neurodegenerative and cardiovascular diseases. In animal models, CBD has been shown to inhibit the progression of several cancer types. Moreover, it has been found that coadministration of CBD and ?9 -THC, followed by radiation therapy, causes an increase of autophagy and apoptosis in cancer cells. In addition, CBD is able to inhibit cell proliferation and to increase apoptosis in diferent types of cancer models. Tese activities seem to involve also alternative pathways, such as the interactions with TRPV and GRP55 receptor complexes. Moreover, the fnding that the acidic precursor of CBD (cannabidiolic acid, CBDA) is able to inhibit the migration of breast cancer cells and to downregulate the proto-oncogene c-fos and the cyclooxygenase-2 (COX-2) highlights the possibility that CBDA might act on a common pathway of infammation and cancer mechanisms, which might be responsible for its anticancer activity. In the light of all these fndings, in this review we explore the efects and the molecular mechanisms of CBD on infammation and cancer processes, highlighting also the role of minor cannabinoids and noncannabinoids constituents of ?9 -THC deprived hemp.


Cannabis sativa: The Plant of the Thousand and One Molecules
Christelle M. Andre, Jean-Francois Hausman and Gea Guerriero
Front. Plant Sci.
DOI: 10.3389/fpls.2016.00019
Cannabis sativa L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fiber since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibers. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenes and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the -omics technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered Cannabis cell culture, are presented.

Cannabisativine, a new alkaloid from cannabis sativa l. root.
Latter, H. L., Abraham, D. J., Turner, C. E., Knapp, J. E., Schiff, P. L., & Slatkin, D. J.
Tetrahedron Letters, 16(33), 2815–2818. (1975)
doi:10.1016/s0040-4039(00)75003-9
An ethanol extract' of the root of Cannabzs sativa L. afforded, after partltloning and repeated chromatography, a new spermldlne (pyrido[2,1-d] [1,5,9]triazacyclotridecine) slkalold, cannablsatlvine (I) with the chemical name of 13-(1,2-dihydroxyheptsnyl)-1,4,5,6,7,8,9,10,11,13,16,16a-dodecahydropyrldo[2,1-~][1,5,9]trlazacyclotrldec~n-2(3H)-one. Crystalllsatlon of I from acetone gave white plates nth mp 167-168O and [alg5 + 55.1° (c 0.53, CHC13)

Cannabis Species.
Bharat Singh and Ram Avtar Sharma
Secondary Metabolites of Medicinal Plants, 226–236. (2020).
doi:10.1002/9783527825578.c02-19
Cannabis sativa L. (Fam. – Cannabaceae) is an annual herbaceous plant and indigenous to Central Asia and India subcontinent. The phytochemicals in this plant species are secreted by the glandular trichomes found on the calyx and bracts of flowers. The Cannabis plant is used in the treatment of nausea and vomiting during chemotherapy; it also recommended to treat chronic pain and muscle spasms and central nervous system (CNS) and as anti-inflammatory (Li 1973; Gubellini et al. 2002; Brotchie 2003; Johnson et al. 2009; Radwan et al. 2009; Styrczewska et al. 2012; Tabrez et al. 2012; Borgelt et al. 2013; Whiting et al. 2015), as well as Parkinson’s disease (Pisani et al. 2005, 2010, 2011). Based on the 2500-year-old ancient reports unearthed in the Turpan district in Xinjiang, China, it was concluded that Cannabis plant was used for ancient customs/rituals and in medicines (Jiang et al. 2006; Manduca et al. 2012; Radwan et al. 2015). Cannabigerol and olivetol are bound into tetrahydrocannabinol and other neutral cannabinoids (Kajima and Piraux 1982; Elzinga et al. 2015)

Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performancesupercritical fluid chromatography
Mazzoccanti, O. H. Ismail, I. D’Acquarica, C. Villani, a C. Manzo,
M. Wilcox, A. Cavazzini and F. Gasparrini
Chem. Commun., 2017, 53, 12262
DOI:10.1039/c7cc06999e
By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (_)-D9-THC in medicinal marijuana (Bedrocans). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity.

Cannabis. X. The Isolation and Structures of Four New Propyl Cannabinoid Acids, Tetrahydrocannabivarinic Acid, Cannabidivarinic Acid, Cannabichromevarinic Acid and Cannabigerovarinic Acid, from Thai Cannabis, 'Meao Variant'
Yukihiro Shoyama, Hitotoshi Hirano, Itsuo Nishioka
Chemical and Pharmaceutical Bull. Volume 25 (1977) Issue 9
DOI:10.1248/cpb.25.2306
Four new cannabinoid acids, tetrahydrocannabivarinic acid, cannabidivarinic acid, cannabichromevarinic acid and cannabigerovarinic acid were isolated from Thai Cannabis, 'Meao variant'and these structures were elucidated on the basis of chemical and spectral data. This is the first example of the isolation of propyl cannabinoid acid from Cannabis.

CANNABIS XI. synthesis OF CANNABIGERORCINIC-CARBOXYL-14C ACID, CANNABIGERO-VARINIC-CARBOXYL-14C ACID, CANNABIDIVARINIC-CARBOXYL-14C ACID AND 1-CANNABICHROMEVARINIC-.CARBOXYL-l 4C ACID
SYNTHESIS

Yukihiro Shoyama, Hitotoshi Hirano and Itsuo Nishioka
Journal of Labelled Compounds and Radiopharmaceuticals Volume 14, Issue 6 1978 Pages 835-842
doi: 10.1002/jlcr.2580140607
The preparation of cannabigerorcinic?carboxyl?14C acid, cannabigerovarinic?carboxyl?14 C acid, cannabidivarinic?carboxyl?14C acid and dl?cannabichromevarinic?carbox yl?14C acid is described. The 14C?label was introduced into the carboxyl carbon via methyl magnesium carbonate?14C.

Cannabis. XII. Variations of Cannabinoid Contents in Several Strains of Cannabis sativa L. with Leaf-age, Season and Sex
HIROFUMI KUSHIMA, YUKIHIRO SHOYAMA, ITSUO NISHIOKA
Chemical and Pharmaceutical Bull. Volume 28 (1980) Issue 2
doi: 10.1248/cpb.28.594
A quantitative analysis procedure for tetrahydrocannabinolic acid, cannabichromenic acid, cannabidiolic acid and cannabigerolic acid monomethyl ether was established by gas chromatography and by a combined gas chromatography-preparative thin-layer chromatography method using cholestane as an internal standard. The variations of cannabinoid contents with leaf-age, season and sex were investigated in three kinds of physiological varieties" of Cannabis sativa L., the Mixican, the Minamioshihara No. 1 and the CBDA strain.

Cannabis. XIII. Two New Spiro-compounds, Cannabispirol and Acetyl Cannabispirol
YUKIHIRO SHOYAMA, ITSUO NISHIOKA
Chemical and Pharmaceutical Bull. 1978 Volume 26 Issue 12 Pages 3641-3646
doi: 10.1248/cpb.26.3641
Two new spiro-compounds, cannabispirol and acetyl cannabispirol, were isolated along with cannabispirone and cannabispirenone from the Japanese domestic cannabis and these structures were elucidated. The biogenetic relationship of spiro-compounds and cannabinoids was also discussed

Cannabis. XIV. Two new propyl cannabinoids, cannabicyclovarin and .DELTA.7-cis-iso-tetrahydrocannabivarin, from thai cannabis.
Yukihiro Shoyama, Satoshi Morimoto,Itsuo Nishioka
January 1981CHEMICAL & PHARMACEUTICAL BULLETIN 29(12):3720-3723
DOI: 10.1248/cpb.29.3720
Two new neutral propyl cannabinoids, cannabicyclovarin and ?7-cis-iso-tetrahydrocannabivarin have been isolated from Thai cannabis, "Meao strain."This is the first report of isolation of the latter, which has a novel skeleton, from natural sources.

Cannabis. XV. Preparation and Stability of ? 9 -Tetrahydrocannabinol-?-Cyclodextrin Inclusion Complex
Yukihiro Shoyama, Satoshi Morimoto, I. Nishioka
Journal of Natural Products 46(5):633-637 September 1983
DOI: 10.1021/np50029a007
Investigation is made of the preparation of the inclusion complex of ?9-tetrahydrocannabinol (1) with ?-cyclodextrin (?-CD) in solid form by the precipitation method. In order to estimate the inclusion complex formation, various spectral data (uv, cd, and emission spectra) have been studied. It has been estimated that the inclusion complex is highly stable under various conditions [light of 10,000 lux, oxygen existence, and high temperature (120°)] compared to 1 alone. The extensive application for a drug delivery system is also discussed.

CANNABIS, 21.' BIOTRANSFORMATION OF CANNABINOL TO ITS GLYCOSIDES BY IN VITRO PLANT TISSUE
HIROYUKI TANAKA, SATOSHI MIORIMOTO, YUKIHIRO SHOYAMA
Journal of Natural Products Vol. 56, No. 12,pp. 2068-2072, December 1993
DOI:10.1021/np50102a006
A marijuana compound, cannabinol , was converted to two metabolites using in vitro tissue of Pinellia ternata. The structures of the metabolites were determined to be cannabinol-0-P-D-glucopyranoside[ 27 and 9'-hydroxycannabinol-O-P-D-glucopyranosid13e1 by 'H nmr and "C nmr. From the time course experiments, 1 was absorbed rapidly in the tissues and glycosylated. Hydroxylation at the pentyl group occurred, and its metabolite was secreted in the medium.

Cannabis XXII. Synthesis of spiro-compounds
J Novák
Tetrahedron Letters 22(11):1063-1064 December 1981
DOI: 10.1016/S0040-4039(01)82866-5
O-methyl-cannabispirenone has been synthesized by two new methods applying spiroannelation on 5,7-dimethoxyindan-1-aldehyde. An alternative high yield synthesis of the latter has been elaborated.

Cannabitwinol, a Dimeric Phytocannabinoid from Hemp, Cannabis sativa L., Is a Selective Thermo-TRP Modulator.
Chianese, G., Lopatriello, A., Schiano-Moriello, A., Caprioglio, D., Mattoteia, D., Benetti, E., … Taglialatela-Scafati, O.
Journal of Natural Products. (2020).
doi:10.1021/acs.jnatprod.0c00668
https://pubs.acs.org/doi/suppl/10.10...668_si_001.pdf (supplemental materials)
Cannabitwinol (CBDD, 3), the second member of a new class of dimeric phytocannabinoids in which two units are connected by a methylene bridge, was isolated from a hemp (Cannabis sativa L.) industrial extract. The structural characterization of cannabitwinol, complicated by broadening of 1 H NMR signals and lack of expected 2D NMR correlations at room temperature, was fully carried out in methanol-d4 at ?30 °C. All the attempts to prepare CBDD by reaction of CBD with formaldehyde or its iminium analogue (Eschenmoser salt) failed, suggesting that this sterically congested dimer is the result of enzymatic reactions on the corresponding monomeric acids. Analysis of the cannabitwinol profile of transient receptor potential (TRP) modulation evidenced the impact of dimerization, revealing a selectivity for channels activated by a decrease of temperature (TRPM8 and TRPA1) and the lack of significant affinity for those activated by an increase of temperature (e.g., TRPV1). The putative binding modes of cannabitwinol with TRPA1 and TRPM8 were investigated in detail by a molecular docking study using the homology models of both channels.

Carboxylation of resorcinols with methylmagnesium carbonate. Synthesis of cannabinoid acids.
Mechoulam, R., & Ben-Zvi, Z.
Journal of the Chemical Society D: Chemical Communications, (7), 343.(1969).
doi:10.1039/c29690000343

CBN and D9-THC concentration ratio as an indicator of the age of stored marijuana samples
S. A. ROSS, M. A. EL Sohly
January 1997 Bulletin on narcotics 49(1):139-147
https://www.unodc.org/unodc/en/data-...1_page008.html
The concentration of D9-tetrahydrocannabinol (THC) and cannabinol (CBN) in cannabis plant material (marijuana) of different varieties stored at room temperature (20-22o Celsius (C)) over a four-year period was determined. The percentage loss of THC was proportional to the storage time. On average, the concentration of THC in the plant material decreased by 16.6% ±7.4 of its original value after one year and 26.8% ±7.3, 34.5% ±7.6 and 41.4% ±6.5 after two, three and four years, respectively. A relationship between the concentration ratio of CBN to THC and the storage time was developed and could serve as a guide in determining the approximate age of a given marijuana sample stored at room temperature.
Introduction
The stability of (-)-D9-tetrahydrocannabinol (D9-THC) has been the subject of several investigations (1-13). In 1970, Liskow (1) reported that marijuana deteriorates during storage at room temperature because of the loss of D9-THC at a rate of 3 to 5 per cent a month. Shoyama and others (3) were able to isolate cannabinolic acid (CBNA) from stored hemp but not from fresh hemp, and concluded that conversion of tetrahydrocannabinolic acid (THCA) to CBNA was effected by ultraviolet light and by storage and heat. The same conclusion was reached by Turner and others (4, 5), who reported that THC disappeared at a rate of 3.83, 5.38 and 6.92 per cent per year over two years when stored at -18o, 4o, and 22o C, respectively. The loss of THC was essentially complete at 37o C and 50o C. Fairbairn and others (6) reported that carefully prepared herbal or resin cannabis products are reasonably stable for one to two years if stored in the dark at room temperature.
Razdan and others (2) found thatD9-THC is much less stable than D8-THC and is converted mainly to CBN. The degradation of D9-THC to CBN in the plant material on storage was also proposed by Waller and others (7), Razdan and others (8), El-Kheir and others (9), Hanus and others (10) and Yotoriyana and others (11). Although CBN is the major observed decomposition product of THC, it could not account for the decrease in the concentration of THC over a period of time when the latter is kept under conditions suitable for decomposition (12). Turner and ElSohly (13) addressed this problem and proposed a possible pathway for the decomposition of THC to CBN which involvesformation of epoxy and hydroxylated intermediates. These include 9,10-dihydroxy-D6a(10a)-THC (racemic mixture) and 8,9-dihydroxy- D6a(10a)-THC(racemic mixture). They found that these intermediates could be detected only by gas chromatography as their trimethyl silyl (TMS) derivatives. They also indicated that these compounds were susceptible to heat and acid and that the final product was CBN.
In the present report, the change in the level of THC and CBN in stored marijuana was studied over a four-year period. THC and CBN were analysed annually in marijuana stored at room temperature and a correlation was developed between the ratio of CBN to THC and the age of the plant material. The empirical correlation could be used to estimate the age of a given marijuana sample.

Chapter 2 – Cannabis and Bioactive Cannabinoids
Federica Messina, Ornelio Rosati, Massimo Curini, M. Carla Marcotulli
Studies in Natural Products Chemistry
Volume 45, 2015, Pages 17–57
DOI: 10.1016/B978-0-444-63473-3.00002-2
The therapeutic use of Cannabis dates back to ancient times and this plant has been used for centuries as remedy for a large number of diseases. Today it is well known that biological activity of Cannabis is related to the endocannabinoid system (ECS), a complex signaling network that comprises classical cannabinoid receptors (CB1 and CB2), arachidonic acid-derived ligands, and enzymes degrading the endocannabinoids anandamide and 2-arachidonoyl glycerol, namely fatty acid amide hydrolase and monoacylglycerol lipase. The modulation of the ECS activity turned out to be a therapeutic promise in a wide range of diseases. A problem to the development of Cannabis and cannabinoid medications is the psychoactive property of natural or synthetic agonists, mediated by CB1 receptor. This review deals with the literature analysis of the important biological activities of Cannabis and the efforts aimed to the discovery of natural and nonnatural selective cannabinoids.

CANNAMIMETICS UNLOCKS HEMP’S CANNABINOID FAMILY TREE
PPM WHITE PAPER
TABLE OF CONTENTS
Understanding Phytosynthesis .............................. .............................. ...... 3
Cannamimetics?, Minor Cannabinoids and Bespoke Plant Medicine Profiles .... 7
Phytocannabinoid Series: Olivetol, Orcinol and Varinol .............................. 8
Cannabis sativa and Hemp .............................. .............................. ......... 12
Members of the CBD Family .............................. .............................. ....... 13
Genetic Engineering and Pharma Approaches .............................. ............... 16
PPM’s Scientific Advancements and Application of Cannamimetics ................. 21
Conclusion .............................. .............................. .............................. .... 22
Appendix: Why Plant Medicine and CBD Doesn’t Quite Fit the FDA’s SOP ...... 24
Meet the Authors .............................. .............................. ....................... 26
For More Information .............................. .............................. ................. 30
Bibliography .............................. .............................. ........................... 31


Chapter Thirteen – Effects of Cannabis and Cannabinoids in the Human Nervous System
Harold Kalant
The Effects of Drug Abuse on the Human Nervous System
Doi: 10.1016/B978-0-12-418679-8.00013-7
The endocannabinoid (EC) system, consisting of ECs, their synthesizing and degrading enzymes, specific transmembrane EC transporters and receptors, is located in both excitatory and inhibitory synapses of all the classical neurotransmitter types throughout the central and peripheral nervous systems, where it acts as a retrograde signaling mechanism to inhibit further release of transmitter. This form of synaptic plasticity is a major component of both rapid short-term and sustained long-term adaptive responses that underlie such processes as homeostasis, learning, memory, and extinction. The functional effects on any given pathway can be either inhibitory or excitatory, depending on whether excitatory (e.g., glutamatergic) or inhibitory (e.g., GABAergic) modulation normally predominates in that pathway. However, the dose-effect curves of EC activity are in many instances biphasic, because sustained strong activity leads to EC receptor desensitization and down-regulation, resulting in progressive loss or even reversal of the effect. Therefore the effects of cannabis and exogenous cannabinoids, of both plant and synthetic origin, are in many cases different from, or even opposite to, those of the EC system.
The functional effects of the EC system and of exogenous cannabinoids are compared with respect to neuronal growth and maturation, neuroprotection against toxic and traumatic damage, sensory pathways, nausea and vomiting, appetite and food intake, the sleep/wake cycle, affective responses and mood states, motor control, seizure activity and cognitive functions. Effects in laboratory animals are compared to those in humans, including both actual and potential therapeutic effects and adverse effects. The therapeutic effects in most instances correspond to the low-dose actions of the EC system, whereas the adverse effects generally correspond to the high-dose range. The exogenous cannabinoids are less selective in their actions than the EC system because they act on a much wider range of EC receptors throughout the nervous system. It is concluded that for most potential therapeutic applications the future will lie with the development of highly selective site-specific agents that act on individual components of the EC system, rather than on the whole system.

Characterisation of cannabinoid composition in a diverse Cannabis sativa L. germplasm collection
Matthew T. Welling, Lei Liu, Tim Shapter, Carolyn A. Raymond, Graham J. King
Euphytica (2016) 208:463–475
DOI 10.1007/s10681-015-1585-y
The ability to characterise cannabinoid chemical phenotype (chemotype) accurately is important for the development of Cannabis sativa L. cultivars specific for pharmacological, hemp fibre, or seed end use. Although a number of chemotyping and genotyping methods have previously been developed to predict and characterise cannabinoid composition, only a subset of the gene pool has been examined. A representative survey from a wide range of geographically and genetically diverse C. sativa accessions using liquid chromatography–mass spectrometry (LC–MS) cannabinoid profiling together with dominant and co-dominant DNA marker assays was performed. Overall variability of chemotype across the gene pool was found to be three-fold greater within heterozygote genotypes than previously reported. Interestingly, an individual plant of East Asian origin was found to exhibit a rare propyl alkyl cannabinoid homologue and a chemotype inconsistent with the predicted genotype. We propose that in order to carry out comprehensive screening of genetic resource collections and to identify chemotypic variants specific for end-use pharmacological applications, a strategy which adopts both cannabinoid profiling and the co-dominant DNA marker assay is required. Further research with consideration of propyl-alkyl-cannabinoid homologues should explore the relationship between chemotype and genotype in greater detail.

Characteristics of cannabinoids composition of Cannabis plants grown in Northern Thailand and its forensic application
Prapatsorn Tipparat, Surapol Natakankitkul, Pipop Chamnivikaipong, Sirot Chutiwat
Forensic science international 215(1-3):164-70 May 2011
DOI: 10.1016/j.forsciint.2011.05.006
The Thai government has recognized the possibility for legitimate cultivation of hemp. Further study of certain cannabinoid characteristics is necessary in establishing criteria for regulation of cannabis cultivation in Thailand. For this purpose, factors affecting characteristics of cannabinoids composition of Thai-grown cannabis were investigated. Plants were cultivated from seeds derived from the previous studies under the same conditions. 372 cannabis samples from landraces, three different trial fields and seized marijuana were collected. 100g of each sample was dried, ground and quantitatively analyzed for THC, CBD and CBN contents by GC-FID. The results showed that cannabis grown during March-June which had longer vegetative stages and longer photoperiod exposure, had higher cannabinoids contents than those grown in August. The male plants grown in trial fields had the range of THC contents from 0.722% to 0.848% d.w. and average THC/CBD ratio of 1.9. Cannabis in landraces at traditional harvest time of 75 days had a range of THC contents from 0.874% to 1.480% d.w. and an average THC/CBD ratio of 2.6. The THC contents and THC/CBD ratios of cannabis in second generation crops grown in the same growing season were found to be lower than those grown in the first generation, unless fairly high temperatures and a lesser amount of rainfall were present. The average THC content in seized fresh marijuana was 2.068% d.w. while THC/CBD ratios were between 12.6 and 84.09, which is 10-45 times greater than those of similar studied cannabis samples from the previous study. However, most Thai cannabis in landraces and in trial fields giving a low log(10) value of THC/CBD ratio at below 1 may be classified as intermediate type, whereas seized marijuana giving a higher log(10) value at above 1 could be classified as drug type. Therefore, the expanded information provided by the current study will assist in the development of criteria for regulation of hemp cultivation in Thailand.


Characterization of Cannabinoid Synthase Family members
Anthony Torres, Keith Allen, Christian Cizek, Kim Neubauer, Daniela Vergara, Robert M Givens, Kymron DeCesare, Donald P Land, Reginald J Gaudino
Cannabis sativa, a unique plant has a long history of domestication by humans for thousands of years it’s medicinal properties have been utilized.
https://www.researchgate.net/public...zation_of_Cannabinoid_Synthase_Family_Members

Characterization of a new type of mead fermented with Cannabis sativa L. (hemp)
Raffaele Romano, Alessandra Aiello, Lucia De Luca, Rosario Sica, Emilio Caprio, Fabiana Pizzolongo, Giuseppe Blaiotta,
Journal of Food Science Feb 2021
DOI: 10.1111/1750-3841.15614
Mead, one of the oldest existing drinks, is a fermented product based on honey, water, and the possible addition of spices and selected yeasts. In this work, various parts (inflorescences, leaves, and steams) of Cannabis sativa L. at different concentrations andSaccharomyces cerevisiae biotype M3/5 were added during mead fermentation. The physicochemical parameters (pH, alcoholic content, sugar content, titratable acidity, and organic acids) of the mead were assessed at the beginning and end of fermentation. Moreover, polyphenols, cannabidiol and volatile organic compounds were identified at the end of fermentation and compared with the control sample prepared without hemp and with only indigenous yeasts. The mead fermented with hemp showed the highest quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg. The volatile organic compounds found were mainly alcohols, esters and terpenes, which were present at higher concentrations in the mead prepared with C. sativa L. than in the control mead and conferred freshness and “hemp aroma” characteristics.
Practical Application
Inflorescences, leaves, and steams of Cannabis sativa L. were added at different concentrations during mead fermentation. This type of mead showed high quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg which have anxiolytic and neuro?protective properties. Moreover the volatile organic compounds found (mainly alcohols, esters, and terpenes) conferred freshness and “hemp aroma” characteristics.

Characterisation of Cannabis accessions with regard to cannabinoid content in relation to other plant characters
E.P.M. de Meijer, H.J. van der Kamp & F.A. van Eeuwijk
Euphytica 62: 187-200, 1992.
DOI:10.1007/BF00041753
Ninety seven Cannabis accessions were evaluated for cannabinoid content and non-chemical plant characters. Variation within populations for cannabinoid content, and consistency of chemical characters at the population level were investigated. The relationship between chemical and other plant characters was very limited. Leaflet width and phenological data can be used for a rough prediction of the chemical phenotype on a population level. Various combinations of cannabinoid content and other economic plant characters were observed, thus a breeding programme will not be hampered by strict linkage. For a selection programme a direct analysis of cannabinoids will be inevitable

Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway
Futoshi Taura, Shinji Tanaka, Chiho Taguchi, Tomohide Fukamizu, Hiroyuki Tanaka, Yukihiro Shoyama, Satoshi Morimoto
FEBS Letters 583 (2009) 2061–2066
doi:10.1016/j.febslet.2009.05.024
Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from
Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of
C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

Chemical and Physical Elicitation for Enhanced Cannabinoid Production in Cannabis.
Gorelick, J., & Bernstein, N. (2017).
Cannabis Sativa L. - Botany and Biotechnology, 439–456.
doi:10.1007/978-3-319-54564-6_21
Of the many medicinal plants with therapeutic potential, Cannabis sativa is, by far, the most promising in the near future for large scale utilization. However, the inherent chemical variability of plant based medicines must be addressed, before cannabis can be incorporated into modern medical practices. This chemical variability can only be controlled and potentially optimized if the underlying causes of the production of therapeutic compounds in cannabis is adequately understood. Many of the medically useful compounds produced by plants are the result of the plant stress response. Although not completely clear, there is a significant body of evidence suggesting a similar role for cannabinoids. Cannabinoids are implicating in both, biotic and abiotic stresses, including thermal, nutrient, and water stress, photoradiation, as well as bacterial and fungal pathogens. This chapter will explore the possible ecological roles of cannabinoids in cannabis and the potential utilization of these roles via biotic or abiotic elicitors.

Chemical and physical variations of cannabis smoke from a variety of cannabis samples in New Zealand.
Sheehan, T. J., Hamnett, H. J., Beasley, R., & Fitzmaurice, P. S.
Forensic Sciences Research, 1–11. (2018)
doi:10.1080/20961790.2018.1445937
Studies have compared the chemical properties of tobacco smoke to those of cannabis smoke, with the objective of identifying the chemical attributes responsible for the mutagenicity and
carcinogenicity of cannabis smoke. Comparative studies have included small sample sizes andproduced conflicting results. The aim of this study was to assess the major chemical and physical variations of cannabis smoke across a range of cannabis samples of different potencies and origins, sourced from the illegal market in New Zealand. Twelve cannabis samples were studied ranging from 1.0% to 13.4% delta-9-tetrahydrocannabinol (D9 THC) content. A smoking machine was used to smoke “joints” (cannabis cigarettes) and the chemical/physical properties of the smoke assessed. The chemical constituents of the smoke extracts were analysed by gas chromatography/mass spectrometry. A range of different chemical constituents (in addition to D9 THC) were identified and their concentrations estimated. Terpenoids were identified as the major variable in cannabis smoke, showing a 40-fold range in total terpenoid content. Analysis of the total particulate matter showed that significantly different levels of particulate matter were produced between the different cannabis samples, ranging from 14.6 to 66.3 mg/g of cannabis smoked. The D9 THC delivery efficiency during smoking was also investigated and produced consistent results showing a mean and median of 12.6% and 10.8%, respectively, of the theoretically available D9 THC (ranging from 7.2% to 28.0%).

Chemical and spectroscopic characterization data of ‘cannabidibutol’, a novel cannabidiol butyl analog
Cinzia Citti, Pasquale Linciano , Flavio Forni, Maria Angela Vandelli, Giuseppe Gigli, Aldo Lagana, Giuseppe Cannazza
Data in Brief 26, (2019)
DOI: 10.1016/j.dib.2019.104463
Cannabidibutol (CBDB), a novel butyl analog of cannabidiol, was identified as impurity of commercial cannabidiol (CBD) extracted from hemp (for full data and results interpretation see “Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog” Citti et al, 2019). The compound was isolated from a CBD sample and subject to a full characterization. First, a complete spectroscopic characterization was performed by Nuclear Magnetic Resonance (NMR): in particular, 1 H-NMR, 13C-NMR, COSY, HSQC and HMBC, which were followed by UV absorption and circular dichroism (CD) spectra. In order to confirm the structural identity and stereochemistry of the compound, a stereoselective synthesis of the trans isomer (1R,6R) was carried out and all the chemical and spectroscopic properties were analyzed. The synthesized compound was characterized by NMR (1 H-NMR, 13CNMR, COSY, HSQC and HMBC), Infra-Red spectroscopy (IR), UV and CD absorption, matching the results obtained for the natural isolated compound. With the analytical standard in hand, a simple high-performance liquid chromatography method coupled to UV detection (HPLC-UV) was developed and validated in house in terms of linearity, accuracy, precision, dilution integrity and stability. The present data might be useful to any researcher or industry that may run into a very common impurity of CBD extracted from hemp, so it can be easily compared with their own experimental data

Chemical Basis of Hashish Activity.
Mechoulam, R., Shani, A., Edery, H., & Grunfeld, Y.
Science, 169(3945), 611–612.(1970).
doi:10.1126/science.169.3945.611
A sample of hashish was extracted consecutively with petroleum ether, benzene, and methanol. When tested intravenously in monkeys only the petroleum-ether fraction was active. This material was further fractionated. The only active compound isolated was A-1-tetrahydrocannabinol. Cannabinol, cannabidiol, cannabichromene, cannabigerol, and cannabicyclol when administered together with Al-tetrahydrocannabinol do not cause a change in the activity of the latter, under the experimental conditions used. These results provide evidence that, except for Al-tetrahydrocannabinol, no other major, psychotomimetically active compounds are present in hashish.

Chemical ecology of Cannabis
David W. Pate
Journal of the International Hemp Association 2: 29, 32-37.
https://www.druglibrary.org/olsen/he.../iha01201.html
The production of cannabinoids and their associated terpenes in Cannabis is subject to environmental influences as well as hereditary determinants. Their biosynthesis occurs in specialized glands populating the surface of all aerial structures of the plant. These compounds apparently serve as defensive agents in a variety of antidessication, antimicrobial, antifeedant and UV-B pigmentation roles. In addition, the more intense ambient UV-B of the tropics, in combination with the UV-B lability of cannabidiol, may have influenced the evolution of an alternative biogenetic route from cannabigerol to tetrahydrocannabinol in some varieties.
Link but no PDF

Chemical stabilization of a ?9-tetrahydrocannabinol prodrug in polymeric matrix systems produced by a hot-melt method: Role of microenvironment pH.
Munjal, M., ElSohly, M. A., & Repka, M. A.
AAPS PharmSciTech, 7(3), E114–E125. (2006).
doi:10.1208/pt070371
This research was conducted in order to fabricate stable polyethylene oxide (PEO)-based transmucosal systems of a ?9-tetrahydrocannabinol (THC) prodrug, a hemisuccinate ester, using a hot-melt method. Since ?9-tetrahydrocannabinolhemisuccin ate (THC-HS) was heat labile, a series of processing aids were evaluated in order to facilitate hot-melt production at lower temperatures, thereby reducing THC-HS degradation. The stability of THC-HS was influenced both by the processing conditions such as heating time and temperature, and the postprocessing storage conditions. The type
of formulation additive also affected the extent of degradation. In the presence of polyethylene glycol (PEG)-400, the percentage of relative degradation of THC-HS to THC was 13.5% and 49.4% at 80°C and 120°C, respectively. In contrast, incorporation of vitamin E succinate (VES) reduced processing degradation to 2.1% and 9.2%, respectively, under the same conditions. Severe degradation of THC-HS was observed during storage, even under freezing conditions (?18°C). A VES-Noveon AA-1 combination was observed to best stabilize the prodrug systems both during processing and postprocessing. Stabilization of THC-HS was achieved in these polyethylene oxide matrices at 4°C, with almost 90% of theoretical drug remaining for up to 8 months. Investigation of the pH effect revealed that the pH of the microenvironment in these polymeric systems could be modulated to significantly improve the stability of THC-HS, degradation being the least in a relatively acidic medium.

ChemInform Abstract: Cannabimovone, a Cannabinoid with a Rearranged Terpenoid Skeleton from Hemp
Orazio Taglialatela-Scafati??, ??Alberto Pagani, Fernando Scala, Luciano De Petrocellis, Vincenzo Di Marzo, Gianpaolo Grassi, Giovanni Appendin ??
ChemInform 08/2010; 41(33).
DOI: 10.1002/chin.201033214
The absolute configuration of title compound (I) is tentatively assigned in analogy to that of cannabidiol

Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents
Rudolf Brenneisen
DOI: 10.1007/978-1-59259-947-9_2 https://www.researchgate.net/publica...s_Constituents
In book: Marijuana and the Cannabinoids November 2007
The Cannabis plant and its products consist of an enormous variety of chemicals. Some of the 483 compounds identified are unique to Cannabis, for example, the more than 60 cannabinoids, whereas the terpenes, with about 140 members forming the most abundant class, are widespread in the plant kingdom. The term “cannabinoids” [note: “ ” represents a group of C21 terpenophenolic compounds found until now uniquely in Cannabis sativa L. (1). As a consequence of the development of synthetic cannabinoids (e.g., nabilone [2], HU-211 [dexanabinol; ref. (3), or ajulemic acid [CT-3; ref. 4]) and the discovery of the chemically different endogenous cannabinoid receptor ligands (“endocannabinoids,” e.g., anandamide, 2-arachidonoylglycerol) (5,6), the term ’“phytocannabinoids’” was proposed for these particular Cannabis constituents

Chemometric Analysis of Cannabinoids: Chemotaxonomy and Domestication Syndrome
E. M. Mudge, S. J. Murch & P. N. Brown
Nature Scientific Reports | (2018) 8:13090
DOI:10.1038/s41598-018-31120-2
Cannabis is an interesting domesticated crop with a long history of cultivation and use. Strains have been selected through informal breeding programs with undisclosed parentage and criteria. The term “strain” refers to minor morphological diferences and grower branding rather than distinct cultivated varieties. We hypothesized that strains sold by diferent licensed producers are chemotaxonomically indistinguishable and that the commercial practice of identifying strains by the ratio of total THC and CBD is insufcient to account for the reported human health outcomes. We used targeted metabolomics to analyze 11 known cannabinoids and an untargeted metabolomics approach to identify 21 unknown cannabinoids. Five clusters of chemotaxonomically indistinguishable strains were identifed from the 33 commercial products. Only 3 of the clusters produce CBDA in signifcant quantities while the other 2 clusters redirect metabolic resources toward the THCA production pathways. Six unknown metabolites were unique to CBD-rich strains and/or correlated to CBDA and 3 unknowns were found only in THCrich strains. Together, these data indicate the domestication of the cannabis germplasm has resulted in a loss of the CBDA pathway in some strains and reallocation of resources between CBDA and THCA
pathways in others. The impact of domestication is a lack of chemical diversity and loss of biodiversity in modern cannabis strains.

Chemotaxonomic features associated with flavonoids of cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) in relation to hops (Humulus lupulus L.).
Vanhoenacker G1, Van Rompaey P, De Keukeleire D, Sandra P.
Nat Prod Lett. 2002 Feb;16(1):57-63.
DOI: 10.1080/1057563029001/4863
The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-beta-D-glucuronide (3), and apigenin-7-O-beta-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.

CHEMOTAXONOMY OF CANNABIS I. CROSSBREEDING BETWEEN CANNABIS SATIVA AND C. RUDERALIS, WITH ANALYSIS OF CANNABINOID CONTENT
JOHN A. BEUTLER, ARA H. DER MARDEROSIAN
DOI: 10.1007/BF02907934
A controlled cross between Cannabis sativa L. and C. ruderalis Janisch. gave progeny intermediate in both cannabinoid content and morphology. The progeny fell into two distinct populations, those whose tetrahydrocannabinol (THC) content was closer to the C. sativa parent (greater than 60% of total cannabinoids) and those whose THC content was closer to the C. ruderalis parent (less than 40% of total cannabinoids). The lower THC group was twice as frequent as the other group. Earliness of flowering, number of flowers, and height characteristics were intermediate between the parents.

Cold Plasma Up-Regulated Expressions of WRKY1 Transcription Factor and Genes Involved in Biosynthesis of Cannabinoids in Hemp (Cannabis sativa L.).
Iranbakhsh, A., Oraghi Ardebili, Z., Molaei, H., Oraghi Ardebili, N., & Amini, M.
Plasma Chemistry and Plasma Processing.(2020).
doi:10.1007/s11090-020-10058-2
Plasma technology as an eco-friendly efcient strategy has gained much attention in various industries, especially in food, medicine, and agriculture. This study aimed to explore the cold plasma-mediated changes in growth, anatomy, expression of a WRKY1 transcription factor, and transcription rates of four key genes involved in the biosynthesis of cannabinoids (pharmaceutically valuable secondary metabolites) in hemp (Cannabis sativa L.). The seeds were treated with cold plasma (dielectric barrier discharge; 0.84 W cm?2; exposure times of 0, 40, and 80 s). The plasma treatment of 40 s increased biomass in both shoot and roots by an average of 57%, whereas the treatment at 80 s delayed growth and reduced it by 48%. Seed priming with plasma up-regulated the WRKY1 transcription factor (mean=11.55 folds). Besides, the plasma treatments induced the expression of olivetolic acid cyclase by 42 folds. Furthermore, the plasma-primed seedlings also exhibited higher expression rates of olivetol synthase by 19 folds. With a similar trend, exposure to plasma stimulated transcription of cannabidiolic acid synthase by 12.4 folds. Up-regulations in ?9-tetrahydrocannabinolic acid synthase also occurred following seed priming with plasma by 25.6 folds. Seed priming with plasma exhibits high potency to up-regulate expressions of genes involved in the productions of secondary metabolites, like cannabinoids. These results imply that the plasma reception and signal transduction can alter expressions of genes at the transcriptional level through which plasma priming may improve plant protection and secondary metabolism.

Combined effects of THC and caffeine on working memory in rats
Leigh V Panlilio1 , Sergi Ferré1 , Sevil Yasar2 , Eric B Thorndike1 , Charles W Schindler1 and Steven R Goldberg1
British Journal of Pharmacology (2012) 165 2529–2538
DOI:10.1111/j.1476-5381.2011.01554.x
BACKGROUND AND PURPOSE Cannabis and caffeine are two of the most widely used psychoactive substances. D9 -Tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, induces deficits in short-term memory. Caffeine, a non-selective adenosine receptor antagonist, attenuates some memory deficits, but there have been few studies addressing the effects of caffeine and THC in combination. Here, we evaluate the effects of these drugs using a rodent model of working memory.
EXPERIMENTAL APPROACH Rats were given THC (0, 1 and 3 mg·kg-1 , i.p.) along with caffeine (0, 1, 3 and 10 mg·kg-1 , i.p.), the selective adenosine A1-receptor antagonist CPT (0, 3 and 10 mg·kg-1 ) or the selective adenosine A2A-receptor antagonist SCH58261 (0 and 5 mg·kg-1 ) and were tested with a delayed non-matching-to-position procedure in which behaviour during the delay was automatically recorded as a model of memory rehearsal.
KEY RESULTS THC alone produced memory deficits at 3 mg·kg-1 . The initial exposure to caffeine (10 mg·kg-1 ) disrupted the established pattern of rehearsal-like behaviour, but tolerance developed rapidly to this effect. CPT and SCH58261 alone had no significant effects on rehearsal or memory. When a subthreshold dose of THC (1 mg·kg-1 ) was combined with caffeine (10 mg·kg-1 ) or CPT (10 mg·kg-1 ), memory performance was significantly impaired, even though performance of the rehearsal-like pattern was not significantly altered.
CONCLUSION AND IMPLICATIONS Caffeine did not counteract memory deficits induced by THC but actually exacerbated them. These results are consistent with recent findings that adenosine A1 receptors modulate cannabinoid signalling in the hippocampus.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast
Xiaozhou Luo, Michael A. Reiter, Leo d’Espaux, Jeff Wong, Charles M. Denby, Anna Lechner, Yunfeng Zhang, Adrian T. Grzybowski, Simon Harth, Weiyin Lin, Hyunsu Lee, Changhua Yu, John Shin, Kai Deng, Veronica T. Benites, George Wang, Edward E. K. Baidoo, Yan Chen, Ishaan Dev, Christopher J. Petzold & Jay D. Keasling1
Nature Letter
doi: 10.1038/s41586-019-0978-9
Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1 . Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3 . However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4 , and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, ?9 -tetrahydrocannabinolic acid, cannabidiolic acid, ?9 - tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5 . We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6 , as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:eek:livetola te geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8 . Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9 . We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Computational characterization and epitope prediction for Bet-v1 like protein of Cannabis sativa
Zarrin Basharat
PeerJ Preprints (2016)
DOI: 10.7287/peerj.preprints.2305v1
Cannabis sativa encodes a Bet-v1 like protein is an allergen and a causuative agent of pollen allergy. Multiple sequence alignment of this protein revealed conserved residues in Betv1 domain. Identification of linear epitopes of this protein was done after preliminary bioinformatics characterization and structure prediction. Structure prediction was done using Modeller software and minimized using Swiss PDBViewer. Six linear epitopes were then, predicted using EMBOSS antigenic program. Phylogenetic analysis of Bet-v1 with other sequences demonstrated divergence patterns with allergens of other species but revealed conserved residues in allergenic epitopes. This study can serve as an informational aid in the development of hypoallergenic vaccine
for Cannabis sativa allergy.

Concerning the Isomerization of ?1-to ?1(6)-Tetrahydrocannabinol
Gaoni, Y., & Mechoulam, R.
Journal of the American Chemical Society, 88(23), 5673–5675. (1966).
doi:10.1021/ja00975a071
The acid-catalyzed isomerization of A l-3,4-trunstetrahydrocannabinol (A1-3,4-truns-THC) (Ia),* the major psychotomimetic principle in hashish, to Al(6)-3,Ctruns-THC (lIa)a is well do~umented.~~’~ In a recent communication Taylor, et a1.,3d have reported the synthesis of &A l-3,4-cis-THC (IIIa) and have claimed that this compound is similarly isomerized to dZ-A1(6)-3,4-cis-THC (IVa). We wish to point out that the compound described as IVa possesses in fact structure V, for which we suggest the name Ad(*)-iso-THC. * The nmr spectrum published3d and confirmed by us5 is more compatible with structure V than with IVa.6 The following revised assignments can be made: 6 (CDCL)' 1.94 (s) and 1.69 (s) (two olefinic methyl groups), 1.36 (s) (one methyl group a! to an oxygen atom), and 4.19 (br) ((2-3 proton). The allylic benzylic C-3 proton in V is nearly in the plane of the aromatic ring and is therefore strongly deshielded.8 Hydrogenation of IIIa and V gave in each case a mixture of two isomers which were separated on Florid. lo The dihydro-V isomers were different from the dihydro-IIIa isomers and from the known dihydro-Ia isomer^.^^'^^ The chromatographically more polar dihydro-V (isomer B) was, however, identical," except for optical activity, with the reduction product of ( -)-As-iso-THC (VI). l2 The nmr spectrum of isomer B contains the isopropyl splitting as two doublets (6 0.92 and 1.07; J = 6.5 cps), while in the spectrum of the chromatographically less polar isomer A it appears as one broader doublet (6 0.75; J = 5 CPS).

Constituents of cannabis sativa L. An improved method for the synthesis of dl-cannabichromene.
Eisohly, M. A., Boeren, E. G., & Turner, C. E.
Journal of Heterocyclic Chemistry, 15(4), 699–700.(1978).
doi:10.1002/jhet.5570150441
A new procedure was developed for the synthesis of cannabichromene (III) which involves reflux of equimolar amounts of olivetol (I), citral (II) and t-butylamine in toluene for 9 hours. The purification of III was best achieved by sodium borohydride reduction of unreacted II followed by column chromatography on 1% sodium hydroxide impregnated silica gel 60-PF. The yield of III (62.0%) was much higher than that reported in the literature.

Constituents of Cannabis sativa L. I: Propyl Homologs of Cannabinoids from an Indian Variant.
Fetterman, P. S., & Turner, C. E.
Journal of Pharmaceutical Sciences, 61(9), 1476–1477.(1972)
doi:10.1002/jps.2600610930
Cannabidivarin and tetrahydrocannabivarin are shown to be present in an Indian variant of Cannabis sariua L. (marijuana) in Mississippi. GC and mass spectrometry were used for identification. Indications are that these compounds are present as acids in fresh material.

Constituents of Cannabis Sativa L. II: Absence of Cannabidiol in an African Variant.
Turner, C. E., & Hadley, K.
Journal of Pharmaceutical Sciences, 62(2), 251–255.(1973).
doi:10.1002/jps.2600620214
Cannabidiol is shown to be absent in an African variant of Cannabis sariua L. (marijuana) grown in Mississippi. TLC, GC, and GC-mass spectrometry were used for identification. The absence of cannabidiol in a variant of African Cannabis questions the validity of published biosyntheses of the cannabinoids.

Constituents of Cannabis Sativa L. III: Clear and Discrete Separation of Cannabidiol and Cannabichromene.
Turner, C. E., & Hadley, K. W.
Journal of Pharmaceutical Sciences, 62(7),1083–1086. (1973).
doi:10.1002/jps.2600620706
Synthetic cannabidiol and cannabichromene were discretely separated by GLC using their trimethylsilyl ether derivatives. The mono and disilylated derivatives of cannabidiol were identified. This procedure was utilized in the analysis of Cannabis sativa L.

Constituents of Cannabis sativa L. IV: Stability of Cannabinoids in Stored Plant Material.
Turner, C. E., Hadley, K. W., Fetterman, P. S., Doorenbos, N. J., Quimby, M. W., & Waller, C.
Journal of Pharmaceutical Sciences, 62(10),1601–1605.(1973).
doi:10.1002/jps.2600621005
The ( -)-Ao-rrans-tetrahydrocannabinol content of Cannabis sarioa L. stored at - 18, 4, and 22 f 1 ' decomposed at a rate of 3.83. 5.38, and 6.92%, respectively, per year, whereas the material stored at 37 and 50" showed considerable decomposition. C. mica L. stored in the absence of direct light at - 18,4, and 22 f 1" was more stable than cannabis stored under nitrogen. These data indicate that for normal research use, storage under nitrogen at 0" is not mandatory. Cannabinol is not the only decomposition product of ( - )-AO-rrurrs-tetrahydrocannabinol. Tentative evidence supports the possible formation of hexahydrocannabinol as a decomposition product in stored C. sarica L.

Constituents of Cannabis sativa L. VI: Propyl Homologs in Samples of Known Geographical Origin.
Turner, C. E., Hadley, K., & Fetterman, P. S.
Journal of Pharmaceutical Sciences, 62(10), 1739–1741.(1973)
doi:10.1002/jps.2600621045
Recent developments in identifying propyl homologs of cannabinoids prompted this communication. We wish to report the presence of cannabidivarin’ and (-)- A9-truns-tetrahydrocannabivarin in freshly grown cannabis from known geographical locations. We routinely employed GLC and TLC? to identify the Cs-homologs. Some samples werc identified by combined GLC-mass spectrometry 3. Vollner rr ul. (1) identified cannabidivarin (I) from a sample of hashish in 1969. Gill et ul. (2) later identified ( -))-~g-tra~rs-tetrahydrocannabivarin (11) from a sample of tincture of cannabis. Merkus (3) reported the presence of cannabivarin (111) in samples of Nepal hashish. This research group reported the presence of I
and 11 in a sample of freshly grown Cannubis satiua L. from Indian seed stock (IN-B) grown in Mississippi (4). De Zeeuw era/. (5) reported that propyl cannabinoids seem to depend on sample origin: samples from countries
like India, Nepal, and Pakistan contained significant amounts of propyl cannabinoids, whereas samples from Middle Eastern and Mediterranean countries contained much lower amounts. Our own findings using only those variants from exact geographical locations confirm and extend the previously thought abundance of propyl cannabinoids in freshly grown C. satiuu L. (Table I). The percentages of I and 11 given in Table I are normalized reports. Each cannabinoid is reported as its percentage in regard to total cannabinoid content. These data were obtained by a GLC-computer4 analysis based on relative retention times of routine cannabis analysis5. Figure 1 of an African variant (seed code SA-E) contained 1.70x of I; 53.69% of 11; 2.75% of 111. cannabichromene (IV), and cannabidiol (VIII); 23.41 % of ( -)-A9--rruns-tetrahydrocannabinol (V); and 4.38 of cannabinol (VI). Peak number VII was tentatively identified as (- )-A9-~runs-tetrahydrocannabiorco16, first reported by Vree et al. (6)

Constituents of Cannabis sativa L. VII: Use of Silyl Derivatives in Routine Analysis.
Turner, C. E., Hadley, K. W., Henry, J., & Leonard Mole, M.
Journal of Pharmaceutical Sciences, 63(12),1872–1876.(1974)
doi:10.1002/jps.2600631209
Naturally occurring cannabinoids previously impossible to separate and analyze were quantitated on a routine basis using silylation. Relative retention times of many silylated cannabinoids are reported for the first time.

Constituents of Cannabis sativa L. IX: Stability of Synthetic and Naturally Occurring Cannabinoids in Chloroform.
Turner, C. E., & Henry, J. T.
Journal of Pharmaceutical Sciences, 64(2), 357–359.(1975)
doi:10.1002/jps.2600640244
that chloroform was a more efficient solvent for extracting
cannabinoids from Cannabis satiua L. than benzene, pentane, hexane, petroleum ether, ethanol, acetone, and ether. Moreover, cannabinoids’ extracted with chloroform were stable at ambient temperature
for 144 hr (1). Recently, Parker et al. (2) reported that synthetic
cannabidiol was unstable in spectrograde chloroform over an 8-day period. The authors stated that: “caution should be exercised in the use of chloroform as a solvent for prolonged extraction and storage of cannabidiol.” Since members of this research group (3- 6) and others (7) have employed chloroform as an extracting solvent and since a working group, sponsored by the United Nations on the chemistry of Cannabis and its components recently recommended that the procedure developed in these laboratories be used worldwide, it seemed imperative that additional data be presented in support of chloroform as the solvent of choice for extracting cannabinoids from Cannabis.
The basic procedure utilized in these laboratories is as follows. Samples of Cannabis are extracted at ambient temperature with nanograde chloroform3 for 1 hr4. After that time, the chloroform is removed in vacuo and an ethanolic solution containing a known
amount of the internal standard, androst-4-ene-3,17-dione, is added. Therefore, by allowing 15 min for workup, each sample is exposed to chloroform for a maximum of 75 min. Thus, if synthetic and naturally
occurring cannabinoids are stable in chloroform for 75 min, chloroform, as previously recommended (1, 7), would be the solvent of choice for extraction of naturally occurring cannabinoids found in crude drug
preparations from C. sativa L.

Constituents of Cannabis sativa L. XI: Cannabidiol and Cannabichromene in Samples of Known Geographical Origin.
Holley, J. H., Hadley, K. W., & Turner, C. E.
Journal of Pharmaceutical Sciences, 64(5), 892–895.(1975)
doi:10.1002/jps.2600640546
Recent development of simplified methods for the separation of cannabidiol (I) and cannabichromene (11) as well as reports supporting the interactions between cannabinoids prompted this communication. We wish to report the presence of I and I1 in freshly
grown Cannabis from known geographical origin. The separation of pure I and I1 by conventional GLC techniques was reported (l), but this separation was insufficient for quantitation. Turner and Hadley (2) obtained a clear and discrete separation of I and I1 by the use of trimethylsilyl ether derivatives. Turner et al. (3) then described the use of silyl derivatives in routine analysis, which provided a method
for quantitating I and 11. As early as 1970, Carlini et al. (4) observed that I could possibly block some effects of (-)-Ag transtetrahydrocannabinol (111). More recently, I was reported to block several effects of I11 in animal models and to potentiate the analgesic effects of cannabinol (5). Cannabidiol induced reduction of the hypothermic response to I11 in rats and rabbits (6). De Zeeuw et al. (7) reported that propyl cannabinoids seem to be more abundant in Cannabis grown in India, Nepal, and Pakistan. Turner and Hadley
(8), using only samples of known geographical locations, confirmed and extended the previously thought abundance of propyl cannabinoids. Following this example, we investigated many variants
of Cannabis from known geographical origins as to their abundance of I and I1 in freshly grown material.

Constituents of cannabis sativaL. XII-mass spectral fragmentation patterns for some. Cannabinoid acid as their TMS derivatives.
Billets, S., El-Feraly, F., Fetterman, P. S., & Turner, C. E.
Organic Mass Spectrometry, 11(7), 741–751.(1976)
doi:10.1002/oms.1210110708
Fragmentation schemes for a number of TMS derivatized cannabinoid acids are described. Specific fragmentation pathways and rearrangement mechanisms were determined based on the ion composition of deuterium labeled derivatives. Significant differences in the mode of fragmentation of the two isomeric A@-tetrahydrocannabinolic acids are described. These results are based on information obtained from deuterium labeling and high resolution data or by the extension of previously reported mechanisms for neutral cannabinoids. Comparisons of the fragmentation schemes between
the neutral and acidic cannabinoids are used to confirm many of the fragmentation mechanisms

Constituents of Cannabis sativa L. XIII: Stability of Dosage Form Prepared by Impregnating Synthetic (–)-?9-trans-Tetrahydrocannabinol on Placebo Cannabis Plant Material.
Lewis, G. S., & Turner, C. E.
Journal of Pharmaceutical Sciences, 67(6), 876–878.(1978).
doi:10.1002/jps.2600670645
Synthetic (- )-lg-trans- tetrahydrocannabinol impregnated on placebo Cannabis decomposed only 6.3% after being stored for 1 year at -18". Storage at 5" and room temperature under various conditions led to severe decomposition. The amount of cannabinol observed when ( -)-A9-trans- tetrahydrocannabinol decomposed indicates that cannabinol is not the only decomposition product.

Constituents of Cannabis sativa L., XIV: Intrinsic Problems in Classifying Cannabis Based on a Single Cannabinoid Analysis.
Turner, C. E., Elsohly, M. A., Cheng, P. C., & Lewis, G.
Journal of Natural Products, 42(3), 317–319.(1979).
doi:10.1021/np50003a017
It is generally accepted that Cartnabis consists of only one species, Cannabis satiza L., but because of a long history of cultivation in a wide range of diverse geographical locations, Cannabis has evolved into many different variants with pronounced morphological and chemical differences. Because of these differences, several classification procedures have been developed. Grli6 (1) first classified samples of Caiiitabis into various “ripening types” according to predominant cannabinoids : “unripe,” predominantly cannabidiolic acid (CBD.1) ; “intermediate.” cannabidiol (CBD) ; “ripe,” ( -)-ftram-

Constituents of cannabis sativa L. XVI. A possible decomposition pathway of ?9-tetrahydrocannabinol to cannabinol.
Turner, C. E., & Elsohly, M. A.
Journal of Heterocyclic Chemistry, 16(8), 1667–1668.(1979).
doi:10.1002/jhet.5570160834
A pathway is proposed for the decomposition of ?9-tetrahydroeannabinol (I) and its ?8-isomer (IX) with the eventual formation of cannabinol (II) through epoxy and hydroxylated intermediates.

Constituents of Cannabis sativa L. XVII. A Review of the Natural Constituents
Turner, C. E., Elsohly, M. A., & Boeren, E. G.
Journal of Natural Products, 43(2), 169–234.(1980).
doi:10.1021/np50008a001
Caiitiabis preparations have been used by man for over 5000 years (1). Early we was associated with medical applications. However, as newer and better medicinal agent-were discovered. the use of crude drugs from Cannabis lost favor in clinics throughout the world. This trend continued. and today there are no crude Cannabis drugs used in modern medical clinics. The same Cannabis preparations once accepted as therapeutically useful drugs have gained acceptance as innocuous drug of leisure in most countries of the
world. The rapid and broad increase in the use and abuse of these drugs resulted in Cannabis being controlled by international treaties. Local, national, and international agencies are charged with the responsibility of enforcing these treaties. Crude Cannabis preparations once of medical use are non- illegal. Thus, marihuana, hashish. dagga, bhang, ganja, hash oil, sinsemilla, etc., comprise the world’s most common and widely used group of illicit drugs. Worldwide, approximately 300 million people use one or more of these crude drugs. It is estimated that in the United States of America 36 million people have used some form of Cannabis.
Current scientific opinions about Cannabis are not aln ays based on rational and reproducible scientific facts, but on emotion. =although the Cannabis and health issue is of interest to all health-related scientific disciplines and agencies of the world, due to the emotional issue. it is possible that scientific experiments may never clarify the pharmacology of Cannabis. The general opinion held by much of the broadly diversified scientific community is that Cannabis preparations can be evaluated solely on ( -)-A9-tetrahydrocannabinol (A9-THC) content, thereby. neglecting other cannabinoids and chemicals in the crude drug. It is easy to understand how this concept came to be accepted, since lg-THC is always referenced as the “active compound” in Cannabis, and data from ‘*synthetic marihuana” (A9-THC) is taken as being synonymous with data from marihuana. This has fostered the impression that marihuana and other crude drugs from Cannabis are singular in composition and uniform in potency with hashish being “X” times as strong as marihuana. etc. Therefore, it is critical to the scientific continunity that a document fully elucidating the current state of the knowledge of naturally occurring constituent< in Cannabis be published. Although over 7000 scientific papers (4, 5) have been published on Cannabis and its constituents and many reviews have been written on Cannabis constituents and cannabinoid chemistry (6-17), all are of limited value in the natural profile of Cannabis and are of even less value for elucidating chemically T1-h)- confusion exist- and may continue to exist in the biological evaluation of the crude drugs obtained from Cannabis.

Constituents of Cannabis sativa L. XVIII—Electron voltage selected ion monitoring study of cannabinoids.
Turner, C. E., Bouwsma, O. J., Billets, S., & Elsohly, M. A
Biological Mass Spectrometry, 7(6), 247–256.(1980).
doi:10.1002/bms.1200070605
Cannabis suriua L. contains more than 420 different constituents. Sixty-one of these constituents are cannabinoids. The electron voltage selected ion monitoring technique was used in the study of this unique group of natural compounds. Mass spectra of each compound were recorded at various eV settings (5.5-21 eV). The electron voltage selected ion recordings were obtained by plotting the relative intensities of characteristic fragment ions against electron voltage. The method was proven useful in the structure analysis of cannabinoids since it requires only minute quantities of sample. It was possible to recognize homologs and to differentiate positional and double bond isomers as well as cannabinoids with the same molecular weight.

CONSTITUENTS OF CANNABIS SATIVA L. XXIIII: CANNABITETROL, A NEW POLYHYDROXYLATED CANNABINOID.
ElSohly, H. N., Boeren, E. G., Turner, C. E., & ElSohly, M. A.
The Cannabinoids: Chemical, Pharmacologic, and Therapeutic Aspects, 89–96.(1984).
doi:10.1016/b978-0-12-044620-9.50011-7
Cannabis sativa L. contains many classes of chemical constituents, the most important of which are the cannabinoids. To date, 61 cannabinoids have been isolated from Cannabis (1). Cannabinoids were defined by Mechoulam and Gaoni (2) "as the group of C ^ compounds typical of and present in Cannabis sativa, their carboxylic acids, analogs, and transformation products." The 61 cannabinoids can be broken down as follows: a - Cannabigerol (CBG) type: 6 known b - Cannabichromene (CBCtype: 4 known c - Cannabidiol (CBD) type: 7 known d - delta-9-Tetrahydrocannabinoltype: 9 known e - delta-8-Tetrahydrocannabinoltype: 2 known f - Cannabicyclol (CBL) type: 3 known g - Cannabielsoin (CBE) type: 3 know
h - Cannabinol (CBN) type: 6 known:
i - Cannabinodiol (CBND) type: 2 known
j - Cannabitriol (CBT) type: 6 known
k - Miscellaneous types: 9 known
1 - Other cannabinoids: 4 known
This work will report the details for the isolation and tentative structure of a new polyhydroxylated cannabinoids which was named cannabitetrol (I), and will review the type of polyhydroxylated cannabinoids isolated from Cannabis, e.g., the cannabitriols (V-X), cannabiripsol (XI) and 8-hydroxy-isohexahydrocannabivarin (XII). (-)-Cannabirtiol or (-)-CBO (V), (+)-cannabitriol or (+)-CBO (VI), (+/-)-9fl0dihydroxy-delta-

Constituents of Cannabis sativa, XXV. Isolation of Two New Dihydrostilbenes from a Panamanian Variant.
ElSohly, H. N., Ma, G.-E., Turner, C. E., & ElSohly, M. A.
Journal of Natural Products, 47(3), 445–452.(1984).
doi:10.1021/np50033a008
TWO new dihydrostilbene compounds (named cannabistilbenes I and 11) were isolated from a polar acidic fraction of a Panamanian variant of Cannabis sativa grown in Mississippi. The structure of cannabistilbene I was shown to be 3,4'-dihydroxy-5-methoxy-3'-(3-rnethylbut-2-enyl)-dihydrostilbene (1) from spectral data which was confirmed by synthesis. There is spectral evidence to indicate that cannabistilbene I1 could be represented by either structure 3 or 4. 6a(10a)-tetrahydrocannabinol (VII), (-)-10-ethoxy-9 hydroxydelta-6a(10a)-tetrahydrocannabinol (IX) and cannabidiolic acid tetrahydrocannabitriol ester (ester at the 9-hydroxy group with cannabidiolic acid, CBDA-CBO) (X) make up the subclass of
cannabinoids called cannabitriols.

Consumer perceptions of strain differences in Cannabis aroma.
Gilbert AN, DiVerdi JA (2018)
PLoS ONE 13(2): e0192247.
Doi: 10.1371/journal.pone.0192247
The smell of marijuana (Cannabis sativa L.) is of interest to users, growers, plant breeders, law enforcement and, increasingly, to state-licensed retail businesses. The numerous varieties and strains of Cannabis produce strikingly different scents but to date there have been few, if any, attempts to quantify these olfactory profiles directly. Using standard sensory evaluation techniques with untrained consumers we have validated a preliminary olfactory lexicon for dried cannabis flower, and characterized the aroma profile of eleven strains sold in the legal recreational market in Colorado. We show that consumers perceive differences among strains, that the strains form distinct clusters based on odor similarity, and that strain aroma profiles are linked to perceptions of potency, price, and smoking interest

Contents of Cannabinoids in Hemp Varieties Grown in Maryland
Xiaoyan Chen, Hua Deng, Janai A. Heise, David P. Puthoff, Nabeel Bou-Abboud, Hongtao Yu, and Jiangnan Peng
ACS Omega (2021)
DOI: 10.1021/acsomega.1c04992
https://pubs.acs.org/doi/pdf/10.1021/acsomega.1c04992
Coincident with the cannabis legalization and the increased interest in the medicinal use of the plant, the cannabis
marketplace and farming have seen tremendous growth. It is reported that there are more than 2000 cannabis varieties available to
customers. However, the data that is available to the growers and breeders regarding the cannabinoid contents of various varieties
remains low. Here, a high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous
separation and determination of 11 cannabinoids. A total of 104 hemp bud materials belonging to 20 varieties were collected from
farms in the state of Maryland and analyzed with the HPLC method. The contents of the 11 cannabinoids in various varieties were
compared and discussed, highlighting the varieties that showed a high yield of cannabinoids and good consistency that are more
appropriate for cannabinoid production.

Conversion of Cannabidiol (CBD) into Psychotropic Cannabinoids Including Tetrahydrocannabinol (THC): A Controversy in the Scientific Literature
Patricia Golombek, Marco Müller, Ines Barthlott, Constanze Sproll and Dirk W. Lachenmeier
Toxics 2020, 8, 41
DOI: 10.3390/toxics8020041
Cannabidiol (CBD) is a naturally occurring, non-psychotropic cannabinoid of the hemp plant Cannabis sativa L. and has been known to induce several physiological and pharmacological effects. While CBD is approved as a medicinal product subject to prescription, it is also widely sold over the counter (OTC) in the form of food supplements, cosmetics and electronic cigarette liquids. However, regulatory difficulties arise from its origin being a narcotic plant or its status as an unapproved novel food ingredient. Regarding the consumer safety of these OTC products, the question whether or not CBD might be degraded into psychotropic cannabinoids, most prominently tetrahydrocannabinol (THC), under in vivo conditions initiated an ongoing scientific debate. This feature review aims to summarize the current knowledge of CBD degradation processes, specifically the results of in vitro and in vivo studies. Additionally, the literature on psychotropic effects of cannabinoids was carefully studied with a focus on the degradants and metabolites of CBD, but data were found to be sparse. While the literature is contradictory, most studies suggest that CBD is not converted to psychotropic THC under in vivo conditions. Nevertheless, it is certain that CBD degrades to psychotropic products in acidic environments. Hence, the storage stability of commercial formulations requires more attention in the future.

Conversion of cannabidiol to ?9-tetrahydrocannabinol and related cannabinoids in artificial gastric juice, and their pharmacological effects in mice
Kazuhito Watanabe · Yuka Itokawa · Satoshi Yamaori · Tatsuya Funahashi · Toshiyuki Kimura · Toshiyuki Kaji · Noriyuki Usami · Ikuo Yamamoto
Forensic Toxicol (2007) 25:16–21
DOI 10.1007/s11419-007-0021-y
Cannabidiol (CBD), a nonpsychoactive cannabinoid, was found to be converted to 9? –hydroxyhexahydrocannabinol (9? -OH-HHC) and 8-hydroxyiso - hexahydrocannabinol (8-OH-iso -HHC) together with ?9 -tetrahydrocannabinol (?9 -THC), a psychoactive cannabinoid, and cannabinol in artificial gastric juice. These cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS) by comparison with the spectral data of the authentic compounds. Pharmacological effects of 9? -OH-HHC and 8-OH-iso -HHC in mice were examined using catalepsy, hypothermia, pentobarbital- induced sleep prolongation, and antinociception against acetic acid-induced writhing as indices. The ED50 values (effective dose producing a 50% reduction of control; mg/kg, i.v.) of 9? -OH-HHC and 8-OH-iso -HHC for the cataleptogenic effect were 8.0 and 30.4, respectively. 8-OH-iso -HHC (10 mg/kg, i.v.) produced a significant hypothermia from 15 to 90 min after administration, although 9? -OH-HHC failed to induce such an effect at the same dose. However, both HHCs (10 mg/ kg, i.v.) significantly prolonged pentobarbital-induced sleeping time by 1.8 to 8.0 times as compared with the control solution with 1% Tween 80-saline. The ED50 values (mg/kg, i.v.) of 9? -OH-HHC and 8-OH-iso –HHC for the antinociceptive effect were 14.1 and 39.4, respectively. The present study demonstrated that CBD can be converted to ?9 -THC and its related cannabinoids, 9? - OH-HHC and 8-OH-iso -HHC, in artifi cial gastric juice, and that these HHCs show ?9 -THC-like effects in mice, although their pharmacological effects were less potent than those of ?9 -THC

Cross-Reactivity of Various Tetrahydrocannabinol Metabolites with a Monoclonal Antibody against Tetrahydrocannabinolic Acid
Kazuhito Watanabe, Midori Matsuda, Yuji Tateoka, Toshiyuki Kimura, Tamihide Matsunaga, Hiroyuki Tanaka, Yukihiro Shoyama, Ikuo Yamamoto
Journal of Health Science 2000 Volume 46 Issue 4 Pages 310-313
doi: 10.1248/jhs.46.310
A monoclonal antibody (MAb-4A4) against ?9-tetrahydrocannabinolic acid (THCA) was prepared and its cross-reactivity analyzed with various cannabinoids including tetrahydrocannabinol metabolites by competitive enzyme-linked immunoassay (ELISA). ?9-THC (1-100?g/ml) could be determined by the ELISA. Many metabolites of ?8-THC and ?9-THC reacted with the antibody, and their cross-reactivities were 44-157% of ?8-THC. However, the antibody did not recognize the lipophilic compounds cholesterol, testosterone, ?-carotene, androstene-3, 17-dione or an endogenous cannabinoid, anandamide.

CULTIVATION, EXTRACTION, AND ANALYSIS OF CANNABIS SATIVA L.
Doorenbos, N. J., Fetterman, P. S., Quimby, M. W., & Turner, C. E.
Annals of the New York Academy of Sciences, 191(1 Marijuana), 3–14. (1971)
doi:10.1111/j.1749-6632.1971.tb13982.x
Cannabis sativa L. (marijuana) has been cultivated at the University of Mississippi since 1968 under contract support from the National Institute of Mental Health. The primary objective is to provide fully defined standardized marijuana for research. Confiscated marijuana of unknown history is often adulterated. C. sutiva was grown on three sites in Mississippi in 1968 and on two sites in 1969 and 1970. The soil types were loess (clay) on campus (site A), sandy loam some 300 miles south of campus (site B), and clay loam in the delta region of the state (site C). Site C was used only in 1968. Land was prepared by conventional agricultural methods and treated with slag at the rate of two tonslacre. Seeds were planted mechanically or by hand at depths of about one-half inch in 40-inch-wide rows with a side dressing of 13-13-13 fertilizer at the rate of 300 pounds/acre. Plantings were made in May, June, and July. Seeds germinated in less than one week if the soil was moist. Weeds were controlled by cultivators and hand hoes. No herbicides or insecticides were used. Weed control was especially important to the young plants, since their growth was greatly inhibited by competition for light, water, and soil nutrients. Plants were watered at least once a week when it did not rain. Plants were usually thinned to 12-18 inches apart when 12-24 inches high. This was best accomplished by clipping an inch above the ground, since uprooting frequently set back or killed neighboring plants. This method allowed greater branching and leaf and flower development. Plants were also side-dressed one or two additional times during the growing season. Many seed types have been planted in this study. One cannot help being amazed at the many morphological variations observed among plants produced by these seed types, and it must be concluded that Cannabis is either a genus composed of more than 100 species or a single species that has not stabilized and that has many variations. We prefer to look upon these plants as a single species, Cannabis sutiva, as first named by Linnaeus. Plants within a given seed type tended to resemble each other and grow at similar rates, although

Current and Future Needs and Applications for Cannabis
Dennis J. Gray, Hailie Baker, Kayla Clancy, Robert C. Clarke, Kymron
deCesare, John Fike, Matthew J. Gibbs, Franjo Grotenhermen, Nolan C. Kane, Kyle G. Keepers, Donald P. Land, Ryan C. Lynch, J. Paul Mendieta, Mark Merlin, Kirsten Mu?ller-Vahl, Christopher S. Pauli, Brian J. Pearson, Bailey Rhan, Travis C. Ruthenberg, C. J. Schwartz,
Critical Reviews in Plant Sciences
DOI: 10.1080/07352689.2017.1284529
With the resurgence of interest in cannabis, for fiber, oil, and drug uses, we now can realistically look forward to consider future needs required to establish it as a modern crop. With the anticipation of future funding opportunities, it is likely that many avenues for its further domestication will become available. Here, we have asked the experts featured in this Special Issue to provide their thoughts and recommendations. There remains some confusion in the taxonomy of the genus Cannabis. At present, we consider drug and hemp varieties to be a single species, Cannabis sativa L. with three subspecies - indica, sativa, and ruderalis. However, a different diagnosis of species contained in the genus is
discussed in some chapters, as mentioned in the definitions section of this Special Issue. This emphasizes the need to further study the relatedness among members of the genus Cannabis. Efforts outlined in the works of Lynch and colleagues (this issue) and Vergara and colleagues (this issue), with future investigations to understand the genome(s) of cannabis, will certainly elucidate these relationships.

Decarboxylation of ?9-tetrahydrocannabinol: Kinetics and molecular modeling.
Perrotin-Brunel, H., Buijs, W., Spronsen, J. van, Roosmalen, M. J. E. van, Peters, C. J., Verpoorte, R., & Witkamp, G.-J.
Journal of Molecular Structure, 987(1-3), 67–73.(2011).
doi:10.1016/j.molstruc.2010.11.061
Efficient tetrahydrocannabinol (D9 -THC) production from cannabis is important for its medical application and as basis for the development of production routes of other drugs from plants. This work presents one of the steps of D9 -THC production from cannabis plant material, the decarboxylation reaction, transforming the D9 -THC-acid naturally present in the plant into the psychoactive D9 -THC. Results of experiments showed pseudo-first order reaction kinetics, with an activation barrier of 85 kJ mol1 and a pre-exponential factor of 3.7 108 s1 . Using molecular modeling, two options were identified for an acid catalyzed b-keto acid type mechanism for the decarboxylation of D9 -THC-acid. Each of these mechanisms might play a role, depending on the actual process conditions. Formic acid proved to be a good model for a catalyst of such a reaction. Also, the computational idea of catalysis by water to catalysis by an acid, put forward by Li and Brill, and Churchev and Belbruno was extended, and a new direct keto-enol route was found. A direct keto-enol mechanism catalyzed by formic acid seems to be the best explanation for the observed activation barrier and the pre-exponential factor of the decarboxylation of D9 -THC-acid. Evidence for this was found by performing an extraction experiment with Cannabis Flos. It revealed the presence of short chain carboxylic acids supporting this hypothesis. The presented approach is important for the development of a sustainable production of D9 -THC from the plant.

Not Cannabis specific
Decarboxylation Reaction Mechanism
ww.youtube.com/watch?v=833x8dRlpb8

Decarboxylation Study of Acidic Cannabinoids: A Novel Approach Using Ultra-High-Performance Supercritical Fluid Chromatography/Photodiode Array-Mass Spectrometry
Mei Wang, Yan-Hong Wang, Bharathi Avula, Mohamed M. Radwan, Amira S. Wanas, John van Antwerp, Jon F. Parcher, Mahmoud A. ElSohly, and Ikhlas A. Khan
Cannabis and Cannabinoid Research
Volume 1.1, 2016 Pg 262-271
DOI: 10.1089/can.2016.0020
Introduction:
Decarboxylation is an important step for efficient production of the major active components in cannabis, for example, D9 -tetrahydrocannabinol (D9-THC), cannabidiol (CBD), and cannabigerol (CBG). These cannabinoids do not occur in significant concentrations in cannabis but can be formed by decarboxylation of their corresponding acids, the predominant cannabinoids in the plant. Study of the kinetics ofdecarboxylation is of importance for phytocannabinoid isolation and dosage formulation for medical use. Efficient analytical methods are essential for simultaneous detection of both neutral and acidic cannabinoids.
Methods: C. sativa extracts were used for the studies. Decarboxylation conditions were examined at 80C, 95C, 110C, 130C, and 145C for different times up to 60 min in a vacuum oven. An ultra-high performance supercritical fluid chromatography/photodiode array-mass spectrometry (UHPSFC/PDA-MS) method was used for the analysis of acidic and neutral cannabinoids before and after decarboxylation.
Results: Decarboxylation at different temperatures displayed an exponential relationship between concentration and time indicating a first-order or pseudo-first-order reaction. The rate constants for D9-tetrahydrocannabinolicacid-A (THCA-A) were twice those of the cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA). Decarboxylation
of THCA-A was forthright with no side reactions or by-products. Decarboxylation of CBDA and CBGA was not as straightforward due to the unexplained loss of reactants or products.
Conclusion: The reported UHPSFC/PDA-MS method provided consistent and sensitive analysis of phytocannabinoids and their decarboxylation products and degradants. The rate of change of acidic cannabinoid concentrations over time allowed for determination of rate constants. Variations of rate constants with temperature yielded values for reaction energy.

?8?Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors
Sándor Bátkai, Partha Mukhopadhyay, B?la Horváth, Mohanraj Rajesh, Rachel Y Gao, Anu Mahadevan, Mukkanti Amere, Natalia Battista, Aron H Lichtman, Lisa A Gauson, Mauro Maccarrone, Roger G Pertwee, Pál Pacher
British Journal of Pharmacology (2012) 165 2450–2461
https://doi.org/10.1111/j.1476-5381.2011.01410.x
BACKGROUND AND PURPOSE Activation of cannabinoid CB2 receptors protects against various forms of ischaemia-reperfusion (I/R) injury. D8 -Tetrahydrocannabivarin (D8 -THCV) is a synthetic analogue of the plant cannabinoid D9 -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB2 receptors. Here, we assessed effects of D8 -THCV and its metabolite 11-OH-D8 -THCVon CB2 receptors and against hepatic I/R injury.
EXPERIMENTAL APPROACH Effects in vitro were measured with human CB2 receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.
KEY RESULTS Displacement of [3 H]CP55940 by D8 -THCV or 11-OH-D8 -THCV from specific binding sites in CHO cell membranes transfected with human CB2 receptors (hCB2) yielded Ki values of 68.4 and 59.95 nM respectively. D8 -THCV or 11-OH-D8 -THCV inhibited forskolin-stimulated cAMP production by hCB2 CHO cells (EC50 = 12.95 and 14.3 nM respectively). D8 -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-a, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of D8 -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB2 receptor antagonist attenuated the protective effects of D8 -THCV, while a CB1 antagonist tended to enhance it.
CONCLUSIONS AND IMPLICATIONS D8 -THCV activated CB2 receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB2 receptor activation.

Delta-8-THC: Delta-9-THC's nicer younger sibling?
Jessica S Kruger , Daniel J Kruge r
J Cannabis Res 2022 Jan 4;4(1):4.
doi: 10.1186/s42238-021-00115-8.
https://www.ncbi.nlm.nih.gov/labs/pm...rticle_115.pdf
Background: Products containing delta-8-THC became widely available in most of the USA following the 2018 Farm Bill and by late 2020 were core products of hemp processing companies, especially where delta-9-THC use remained illegal or required medical authorization. Research on experiences with delta-8-THC is scarce, some state governments have prohibited it because of this lack of knowledge.
Objective: We conducted an exploratory study addressing a broad range of issues regarding delta-8-THC to inform policy discussions and provide directions for future systematic research.
Methods: We developed an online survey for delta-8-THC consumers, including qualities of delta-8-THC experiences, comparisons with delta-9-THC, and open-ended feedback. The survey included quantitative and qualitative aspects to provide a rich description and content for future hypothesis testing. Invitations to participate were distributed by a manufacturer of delta-8-THC products via social media accounts, email contact list, and the Delta8 Reddit.com discussion board. Participants (N = 521) mostly identified as White/European American (90%) and male (57%). Pairwise t tests compared delta-8-THC effect rating items; one-sample t tests examined responses to delta-9-THC comparison items.
Results: Most delta-8-THC users experienced a lot or a great deal of relaxation (71%); euphoria (68%) and pain relief (55%); a moderate amount or a lot of cognitive distortions such as difficulty concentrating (81%), difficulties with short-term memory (80%), and alerted sense of time (74%); and did not experience anxiety (74%) or paranoia (83%). Participants generally compared delta-8-THC favorably with both delta-9-THC and pharmaceutical drugs, with most participants reporting substitution for delta-9-THC (57%) and pharmaceutical drugs (59%). Participant concerns regarding delta-8-THC were generally focused on continued legal access.
Conclusions: Delta-8-THC may provide much of the experiential benefits of delta-9-THC with lesser adverse effects. Future systematic research is needed to confirm participant reports, although these studies are hindered by the legal statuses of both delta-8-THC and delta-9-THC. Cross-sector collaborations among academics, government officials, and representatives from the cannabis industry may accelerate the generation of knowledge regarding delta-8-THC and other cannabinoids. A strength of this study is that it is the first large survey of delta-8 users, limitations include self-report data from a self-selected convenience sample.

Δ 9 - cis -Tetrahydrocannabinol: Natural Occurrence, Chirality, and Pharmacology
Michael A. Schafroth, Giulia Mazzoccanti, Ines Reynoso-Moreno, Reto Erni, Federica Pollastro, Diego Caprioglio, Bruno Botta, Gianna Allegrone, Giulio Grassi, Andrea Chicca, Francesco Gasparrini, Jürg Gertsch, Erick M. Carreira, and Giovanni Appendino
Journal of Natural Products XXXXXX)July 2021
DOI: 10.1021/acs.jnatprod.1c00513
https://www.researchgate.net/publica...d_Pharmacology
The cis-stereoisomers of Δ⁹-THC [(−)-3 and (+)-3] were identified and quantified in a series of low-THC-containing varieties of Cannabis sativa registered in Europe as fiber hemp and in research accessions of cannabis. While Δ⁹-cis-THC (3) occurs in cannabis fiber hemp in the concentration range of (−)-Δ⁹-trans-THC [(−)-1], it was undetectable in a sample of high-THC-containing medicinal cannabis. Natural Δ⁹-cis-THC (3) is scalemic (ca. 80–90% enantiomeric purity), and the absolute configuration of the major enantiomer was established as 6aS,10aR [(−)-3] by chiral chromatographic comparison with a sample available by asymmetric synthesis. The major enantiomer, (−)-Δ⁹-cis-THC [(−)-3], was characterized as a partial cannabinoid agonist in vitro and elicited a full tetrad response in mice at 50 mg/kg doses. The current legal discrimination between narcotic and non-narcotic cannabis varieties centers on the contents of “Δ⁹-THC and isomers” and needs therefore revision, or at least a more specific wording, to account for the presence of Δ⁹-cis-THCs [(+)-3 and (−)-3] in cannabis fiber hemp varieties.


D9 -Tetrahydrocannabinol (D9 -THC) attenuates mouse sperm motility and male fecundity
Daniel J Morgan, Charles H Muller, Natalia A Murataeva, Brian J Davis and Ken Mackie
British Journal of Pharmacology (2012) 165 2575–2583
DOI:10.1111/j.1476-5381.2011.01506.x
BACKGROUND AND PURPOSE Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1 cannabinoid receptor agonist, and D9 -tetrahydracannabinol (D9 -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of D9 -THC on male fecundity were determined.
EXPERIMENTAL APPROACH Effects of D9 -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of D9 -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by D9 -THC-treated males.
KEY RESULTS Pretreatment of sperm for 15 min with 1 mM D9 -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 mM WIN 55,212-2 or 10 mM D9 -THC for 30 min reduced sperm ATP levels. In sperm lacking CB1 receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of D9 -THC persisted. Administration of 50 mg·kg-1 D9 -THC to male mice just before mating caused a 20% decrease in embryonic litter size.
CONCLUSIONS AND IMPLICATIONS D9 -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or D9 -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB1 receptordependent whereas that of D9 -THC is not.

Delta-9-Tetrahydrocannabinolic Acid B: A Mechanism for its Formation in Cannabis
Crist N. Filer Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0216
There appears to be consensus among Cannabis biologists that delta-9-tetrahydrocannabinolic acid A (THCA-A) is the exclusive product of the enzyme THCA synthase. This then leaves an open question for formation of the THCA-A structural isomer, delta-9-tetrahydrocannabinolic acid B (THCA-B), discovered as a minor product in Cannabis in 1969. With no reasonable biochemical pathway to explain the presence of THCA-B in Cannabis, a synthetic route was next considered. Using established literature precedent, a photochemical mechanism has been proposed for the conversion of THCA-A to cannabidiolic acid (CBDA), followed by conversion of CBDA to THCA-B employing bond-breaking and bond-forming reactions. Find Pdf

Delta-9-THC based monotherapy in fibromyalgia patients on experimentally induced pain, axon reflex flare, and pain relief.
Schley M1, Legler A, Skopp G, Schmelz M, Konrad C, Rukwied R.
Curr Med Res Opin. 2006 Jul;22(7):1269-76.
DOI:10.1185/030079906X112651
OBJECTIVE: Fibromyalgia (FM) is a chronic pain syndrome characterized by a distinct mechanical hyperalgesia and chronic pain. Recently, cannabinoids have been demonstrated as providing anti-nociceptive and anti-hyperalgesic effects in animal and human studies. Here, we explored in nine FM patients the efficacy of orally administered delta-9-tetrahydrocannabinol (THC) on electrically induced pain, axon reflex flare, and psychometric variables.
RESEARCH DESIGN AND METHODS: Patients received a daily dose of 2.5-15 mg of delta-9-THC, with a weekly increase of 2.5 mg, as long as no side effects were reported. Psychometric variables were assessed each week by means of the West Haven-Yale Multidimensional Pain Inventory (MPI), Pittsburgh Sleep Quality Index (PSQI), Medical outcome survey-short form (MOS SF-36), the Pain Disability Index (PDI), and the Fibromyalgia Impact Questionnaire (FIQ). In addition, patients recorded daily, in a diary, their overall pain intensity on a numeric scale. Each week, pain and axon reflex flare was evoked experimentally by administration of high intensity constant current pulses (1 Hz, pulse width 0.2 ms, current increase stepwise from 2.5-12.5 mA every 3 minutes) delivered via small surface electrodes, attached to the volar forearm skin.
MAIN OUTCOME MEASURES: Daily pain recordings by the patient, experimentally induced pain, and axon reflex flare recorded by a laser Doppler scanner.
RESULTS: Five of nine FM patients withdrew during the study due to adverse side effects. Delta-9-THC had no effect on the axon reflex flare, whereas electrically induced pain was significantly attenuated after doses of 10-15 mg delta-9-THC (p < 0.05). Daily-recorded pain of the FM patients was significantly reduced (p < 0.01).
CONCLUSIONS: This pilot study demonstrated that a generalized statement that delta-9-THC is an analgetic drug cannot be made. However, a sub-population of FM patients reported significant benefit from the delta-9-THC monotherapy. The unaffected electrically induced axon reflex flare, but decreased pain perception, suggests a central mode of action of the cannabinoid.

?9-Tetrahydrocannabinol content in cannabis samples seized in Novi Sad during 2008
MAJA DJURENDI?-BRENESEL, NIKŠA AJDUKOVI?, KATARINA ŠTAJNIC-RISTI?, VLADIMIR PILIJA and IGOR VESELINOVI?
J. Serb. Chem. Soc. 75 (7) 893–902 (2010)
doi: 10.2298/JSC091019070D
The three main cannabinoids ?9-tetrahydrocannabinol (?9-THC), cannabidiol (CBD) and cannabinol (CBN) were identified and determined quantitatively using a GCD (GC-EI) instrument in 280 samples of illicit herbal cannabis, seized by the Police authorities in Novi Sad, during 2008. The samples were sent to the Institute of Forensic Medicine, Clinical Center Vojvodina, for forensic chemical analysis. The cannabinoid content of the samples enabled the classification of the cannabis into three chemical phenotypes and the differentiation into drug and textile-cannabis, using the Waller classification index. This differentiation has great forensic significance in the classification of certain cases as a criminal action. The experimental results showed that the ?9-THC content in illicitly circulated cannabis slightly decreased from January to December 2008, as did the quality of the drug-cannabis. The reasons for the quality variations could lie in the geographical origin of the cannabis plants, the conditions of plants storage, various parts of the plants in samples and the time elapsed between harvesting and chemical analysis.

Delta-9-Tetrahydrocannabivarin as a Marker for the Ingestion of Marijuana versus Mannol : Results of a Clinical Study
ElSohly, M. A., deWit, H., Wachtel, S. R., Feng, S., & Murphy, T. P.
Journal of Analytical Toxicology, 25(7), 565–571. (2001)
doi:10.1093/jat/25.7.565
Delta-9-Tetrahydrocannabinol (THC), the main psychologically active ingredient of the cannabis plant (marijuana), has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metaholite of THC, 11-nor-A% tetrahydrocannabinol.9.carboxy lic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana positive drug test is the result of the ingestion of marijuana (or a related product) or whether it results from the sole use of Marinol. We have previously proposed the use of Delta-9—tetrahydrocannabivarin (THCV, the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV is a natural component of most cannabis products along with THC and does not exist in Marinol. We have also reported that THCV is metabolized by human hepatocytes to 11-nor-tetrahydrocannabivarln-9- carboxylic acid (THCV.COOH); therefore, the presence of the latter in a urine specimen would indicate that the donor must have used marijuana or a related product (with or without Marino]). in this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Four subjects (male and female) participated in the study in a three session, within-subject, crossover design. The sessions were conducted at one week intervals. Each subject received, in separate sessions and in randomized order, an oral dose of Marinol (15 rag), a smoked dose of THC (16.88 mg) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV). Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration. Subjects were then transported home, were given sample collection containers and logbooks, and were instructed to record at home the volume and time of every urine collection for 24 h, and once a day for the remainder of a week (6 days). Subjects were also instructed to freeze the urine samples until the next session. All urine samples were analyzed by GC-MS for THC.COOH and THCV-COOH using solid-phase extraction and derivatization procedure on RapidTrace | and TBDMS as the derivative. The method had a limit of detection of 1.0 ng/mL and 1.0 ng/mL for THCV-COOH and THC-COOH, respectively.

Delta 9-Tetrahydrocannabivarin as a Marker for the Ingestion of Marijuana versus Marinol(R): Results of a Clinical Study.
ElSohly, M. A., deWit, H., Wachtel, S. R., Feng, S., & Murphy, T. P.
Journal of Analytical Toxicology, 25(7), 565–571.(2001).
doi:10.1093/jat/25.7.565
Ag-Tetrahydrocannabinol (THC), the main psychologically active ingredient of the cannabis plant (marijuana), has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metaholite of THC, 11-nor-A% tetrahydrocannabinol.9.carboxy lic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana positive drug test is the result of the ingestion of marijuana (or a related product) or whether it results from the sole use of Marinol. We have previously proposed the use of Ag-tetrahydrocannabivarin (THCV, the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV is a natural component of most cannabis products along with THC and does not exist in Marinol. We have also reported that THCV is metabolized by human hepatocytes to 11-nor-Ag-tetrahydrocannabivarln-9-carboxylic acid (THCV.COOH); therefore, the presence of the latter in a urine specimen would indicate that the donor must have used marijuana or a related product (with or without Marino]). in this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Four subjects (male and female) participated in the study in a threesession, within-subject, crossover design. The sessions were conducted at one.week intervals. Each subject received, in separate sessions and in randomized order, an oral dose of Marinol (15 rag), a smoked dose of THC (16.88 mg) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV). Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration. Subjects were then transported home, were given sample collection containers and logbooks, and were instructed to record at home the volume and time of every urine collection for 24 h, and once a day for the remainder of a week (6 days). Subjects were also instructed to freeze the urine samples until the next session. All urine samples were analyzed by GC-MS for THC.COOH and THCV-COOH using solid-phase extraction and derivatization procedure on RapidTrace | and TBDMS as the derivative. The method had a limit of detectionof 1.0 ng/mL and 1.0 ng/mL for THCV-COOH and THC-COOH, respectively.

D9 -Tetrahydrocannabinol (D9 -THC) attenuates mouse sperm motility and male fecundity
Daniel J Morgan, Charles H Muller, Natalia A Murataeva, Brian J Davis and Ken Mackie
British Journal of Pharmacology (2012) 165 2575–2583
DOI:10.1111/j.1476-5381.2011.01506.x
BACKGROUND AND PURPOSE Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1 cannabinoid receptor agonist, and D9 -tetrahydracannabinol (D9 -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of D9 -THC on male fecundity were determined.
EXPERIMENTAL APPROACH Effects of D9 -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of D9 -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by D9 -THC-treated males.
KEY RESULTS Pretreatment of sperm for 15 min with 1 mM D9 -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 mM WIN 55,212-2 or 10 mM D9 -THC for 30 min reduced sperm ATP levels. In sperm lacking CB1 receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of D9 -THC persisted. Administration of 50 mg·kg-1 D9 -THC to male mice just before mating caused a 20% decrease in embryonic litter size.
CONCLUSIONS AND IMPLICATIONS D9 -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or D9 -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB1 receptordependent whereas that of D9 -THC is not.

9-Tetrahydrocannabinol (THC), 11-Hydroxy-THC, and 11-Nor-9-carboxy-THC Plasma Pharmacokinetics during and after Continuous High-Dose Oral THC.
Schwilke, E. W., Schwope, D. M., Karschner, E. L., Lowe, R. H., Darwin, W. D., Kelly, D. L., … Huestis, M. A.
Clinical Chemistry, 55(12), 2180–2189.(2009).
doi:10.1373/clinchem.2008.122119
BACKGROUND: 9 -Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis and an active cannabinoid pharmacotherapy component. No plasma pharmacokinetic data after repeated oral THC administration are available. METHODS: Six adult male daily cannabis smokers resided on a closed clinical research unit. Oral THC capsules (20 mg) were administered every 4 –8 h in escalating total daily doses (40 –120 mg) for 7 days. Free and glucuronidated plasma THC, 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THC COOH) were quantified by 2-dimensional GC-MS during and after dosing. RESULTS: Free plasma THC, 11-OH-THC, and THCCOOH concentrations 19.5 h after admission (before controlled oral THC dosing) were mean 4.3 (SE 1.1), 1.3 (0.5), and 34.0 (8.4) g/L, respectively. During oral dosing, free 11-OH-THC and THCCOOH increased steadily, whereas THC did not. Mean peak plasma free THC, 11-OH-THC, and THCCOOH concentrations were 3.8 (0.5), 3.0 (0.7), and 196.9 (39.9) g/L, respectively, 22.5 h after the last dose. Escherichia coli -glucuronidase hydrolysis of 264 cannabinoid specimens yielded statistically significant increases in THC, 11-OH-THC, and THCCOOH concentrations (P 0.001), but conjugated concentrations were underestimated owing to incomplete enzymatic hydrolysis. CONCLUSIONS: Plasma THC concentrations remained 1 g/L for at least 1 day after daily cannabis smoking and also after cessation of multiple oral THC doses. We report for the first time free plasma THC concentrations after multiple high-dose oral THC throughout the day and night, and after Escherichia coli -glucuronidase hydrolysis. These data will aid in the interpretation of plasma THC concentrations after multiple oral doses.

Not Cannabis specific
Demystifying the liverwort Radula marginata, a critical review on its taxonomy, genetics, cannabinoid phytochemistry and pharmacology.
Hussain, T., Espley, R. V., Gertsch, J., Whare, T., Stehle, F., & Kayser, O.
Phytochemistry Reviews.(2019).
doi:10.1007/s11101-019-09638-8
Radula marginata Taylor is a subtropical liverwort, endemic to the Northern island of New Zealand in the Pacific region. Despite its significant potential as an alternative source of a THC-like phytocannabinoid, the biosynthesis of this compound remains elusive. Following the discovery of a bibenzyl cis-tetrahydrocannabinol (perrottetinene) in R. marginata, research has focused on genetic fingerprinting and transcriptomic analysis for the genes involved in its biosynthesis. More recently, its pharmacological activity with the endocannabinoid receptors (CB1 and CB2) has been demonstrated, paving the way to more extensive research on this liverwort. Here, we provide an in-depth review of the current research. Firstly, we describe the taxonomy and phytogeography of R. marginata, followed by the symbiosis and evolutionary aspects of bryophytes in general, with a specific focus on liverworts. We then review the physiology and anatomy from scanning electron microscopy data. The presence of the oil bodies and their functional insights for the storage of secondary metabolites, such as perrottetinene, are illustrated. We review the available genetic and transcriptomic resources for R. marginata. We discuss pharmacological aspects of the natural bi-benzyl cis-perrottetinene as compared to THC. The reported partial agonistic activity of this compound at CB1 and CB2 receptors, and the need for further study for the treatment of inflammatory conditions, is considered. Moreover, given that THC has beneficial effects topically, we discuss the potential use of R. marginata for dermatological conditions. Finally, we elaborate on the ethnobotanical aspects and the legal and ethical position of plant collection in New Zealand. Overall, this review gives the first detailed overview of R. marginata. We include the morphology, taxonomy, anatomy, physiology, evolution as well as the genetic basis linking it with electron microscopic studies and its biological effects.

Designing microorganisms for heterologous biosynthesis of cannabinoids.
Carvalho, Â., Hansen, E. H., Kayser, O., Carlsen, S., & Stehle, F.
FEMS Yeast Research, 17(4).(2017).
doi:10.1093/femsyr/fox037
During the last decade the use of medical Cannabis has expanded globally and legislation is getting more liberal in many countries, facilitating the research on cannabinoids. The unique interaction of cannabinoids with the human endocannabinoid system make these compounds an interesting target to be studied as a therapeutic agent for the treatment of several medical conditions. However, currently there are important limitations in the study, production and use of cannabinoids as pharmaceutical drugs. Besides the main constituent tetrahydrocannabinolic acid, the structurally related compound cannabidiol is of high interest as drug candidate. From the more than 100 known cannabinoids most of them are mostly unknown with regard to their pharmacological profile and in very low amounts extractable. Today, cannabinoids are isolated from the strictly regulated Cannabis plant and the supply of compounds with sufficient quality is a major problem. Biotechnological production could be an attractive alternative mode of production. Herein, we explore the potential use of synthetic biology as an alternative strategy for synthesis of cannabinoids 2 in heterologous hosts. We summarize the current knowledge surrounding cannabinoids biosynthesis and present a comprehensive description of the key steps of the genuine and artificial pathway, systems biotechnology needs and platform optimization.

Really great paper
*Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis.
Giese MW, Lewis MA, Giese L, Smith KM.
J AOAC Int.
DOI: 10.5740/jaoacint.15-116
The requirements for an acceptable cannabis assay have changed dramatically over the years resulting in a large number of laboratories using a diverse array of analytical methodologies that have not been properly validated. Due to the lack of sufficiently validated methods, we conducted a single- laboratory validation study for the determination of cannabinoids and terpenes in a variety of commonly occurring cultivars. The procedure involves high- throughput homogenization to prepare sample extract, which is then profiled for cannabinoids and terpenes by HPLC-diode array detector and GC-flame ionization detector, respectively. Spike recovery studies for terpenes in the range of 0.03-1.5% were carried out with analytical standards, while recovery studies for ?9-tetrahydrocannabinolic acid, cannabidiolic acid, ?9-tetrahydrocannabivarinic acid, and cannabigerolic acid and their neutral counterparts in the range of 0.3-35% were carried out using cannabis extracts. In general, accuracy at all levels was within 5%, and RSDs were less than 3%. The interday and intraday repeatabilities of the procedure were evaluated with five different cultivars of varying chemotype, again resulting in acceptable RSDs. As an example of the application of this assay, it was used to illustrate the variability seen in cannabis coming from very advanced indoor cultivation operations.

Development of a Novel Microwave Distillation Technique for the Isolation of Cannabis sativa L. Essential Oil and Gas Chromatography Analyses for the Comprehensive Characterization of Terpenes and Terpenoids, Including Their Enantio-Distribution
Giuseppe Micalizzi, Filippo Alibrando, Federica Vento, Emanuela Trovato, Mariosimone Zoccali, Paolo Guarnaccia, Paola Dugo, Luigi Mondello
Molecules 2021, 26(6), 1588;
DOI: 10.3390/molecules26061588
https://www.mdpi.com/1420-3049/26/6/1588
A microwave distillation method was optimized for the extraction and isolation of cannabis essential oil from fresh and dried hemp inflorescences. The developed method enabled us to obtain a distilled product rich in terpenes and terpenoid compounds, responsible of the typical and unique smell of the cannabis plant. The distillate from different hemp cultivars, including Kompolti, Futura 75, Carmagnola, Felina 32 and Finola were characterized by using a gas chromatograph equipped with both mass spectrometer and flame ionization detectors. In a single chromatographic run, the identity and absolute amounts of distilled compounds were determined. Peak assignment was established using a reliable approach based on the usage of two identification parameters, named reverse match, and linear retention index filter. Absolute quantification (mg g−1) of the analytes was performed using an internal standard method applying the flame ionization detector (FID) response factors according to each chemical family. An enantio-GC-MS method was also developed in order to evaluate the enantiomeric distribution of chiral compounds, an analytical approach commonly utilized for establishing the authenticity of suspicious samples.

Detection and Identification of Compounds in Cannabis
Coy W. Waller, Kathy W. Hadley, Carlton E. Turner
In book: Marihuana 1976
DOI: 10.1007/978-3-642-51624-5_2
The discussion in this chapter is restricted to three categories:1. basic analytical developments applicable to synthetic can-nabinoids and extracts from cannabis preparations; 2. application of electron voltage-mass fragment graphs as a potential tool in identifying cannabinoids and their metabolites in body fluids and fermentation processes; 3. new alkaloids that have been isolated and identified from C. sativa L.

Detection of Cannabinoids by LC–MS-MS and ELISA in Breast Milk
Cristina Sempio, Erica Wymore, Claire Palmer, Maya Bunik, Thomas K Henthorn,Uwe Christians, Jost Klawitter
Journal of Analytical Toxicology, bkaa142,
DOI: 10.1093/jat/bkaa142
Cannabis is the most commonly used drug of abuse in pregnancy and after delivery. However, little is known regarding the disposition of cannabinoids in breast milk, although delta-9-tetrahydrocannabinol (THC), the main psychoactive component, is highly lipophilic. Quantification of cannabinoids in breastmilk is essential for clinical monitoring and research studies and breastmilk banks mainly rely on ELISA in terms of screening for cannabinoids. To support clinical studies on disposition of cannabinoids in breastmilk, we validated a high-performance liquid chromatography–tandem mass spectrometry (LC–MS-MS) assay for the simultaneous quantification of 12 cannabinoids and their metabolites in human breast milk. Said assay was based upon a simple one-step protein precipitation, online column extraction and detection in the positive multiple reaction monitoring mode. After successful validation, the assay was used to analyze 30 samples from a clinical research study that had tested negative using an ELISA kit that is commonly used by breastmilk banks. In human breast milk, depending on the analyte, the lower limits of quantification of the LC–MS-MS assay ranged from 0.39 to 7.81 ng/mL. Acceptance criteria for intra- and inter-batch accuracy (85–115%) and imprecision (<15%) were met for all compounds. Mean extraction efficiencies were above 60% for all analytes. Mean matrix effect ranged from –12.5% to 44.5% except of THC- glucuronide for which significant matrix effects were noted. No carry-over was detected. Although cannabinoid-negative based on the ELISA, all 30 samples tested positive for THC using LC–MS-MS (0.8-130 ng/mL) and several also for 11-OH-THC, THCCOOH, CBD and CBG. We validated a sensitive and specific assay for the quantification of 12 cannabinoids in human breastmilk that outperformed an ELISA commonly used by breastmilk banks.

Determination of cannabidiol in plasma by electron-capture gas chromatography.
Jones, A. B., Elsohly, M. A., Bedford, J. A., & Turner, C. E.
Journal of Chromatography B: Biomedical Sciences and Applications, 226(1), 99–105.(1981).
doi:10.1016/s0378-4347(00)84210-3
A procedure was developed for the analysis of cannabidiol (CBD) in blood plasma. Tetrahydrocannabidiol was used as an internal standard and was added prior to extraction. The plasm& extracts were derivatized with pentafluorobenzyl bromide and the product purified on a mini-column of Florisil. The pentafluorobenzyl derivatives were then analyzed by gas
&bromatograpby ok a 5% OV-225 column using an electroncapture detector_ A detection limit of 50 ng CBD per ml of plasma was observed. The procedure was used

Determination of Tetrahydrocannabinolic Acid-Carrier Protein Conjugate by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry and Antibody Formation
Yuri Goto, Yumi Shima, Satoshi Morimoto, Yukihiro Shoyama, Hiroki Murakami, Akihiko Kusai and Kazutetsu Nojima
ORGANIC MASS SPECTROMETRY, VOL. 29, 668-671 (1994)
DOI:10.1002/oms.1210291115
In order to form the monoclonal antibody against tetrahydrocannabinolic acid (THCA), THCA was coupled with bovine serum albumin (BSA) or human serum alhumin (HSA) using fl-alanine as a linker or without fl-alanine to
produce individual antigen conjugates. The number of haptens contained in the antigen conjugates was determined by matrix-assisted laser desorption ionization mass spectrometry using BSA or HSA as an internal standard. The
formation of monoclonal antibody is also discussed

Development of Cannabinoids in Flowers of Industrial Hemp (Cannabis sativa L.): A Pilot Study
Rui Yang, Erin Berthold, Christopher R Mccurdy, Joshua H. Freeman
Journal of Agricultural and Food Chemistry XXXXXX) May 2020
DOI: 10.1021/acs.jafc.0c01211
field study was performed to investigate the development of cannabinoids in flowers of industrial hemp using three day-length-sensitive and two day-length-neutral varieties. Flower samples were analyzed for cannabinoids on a weekly basis from 2 to 4 weeks postanthesis to plant senescence. Results indicate that total THC, CBD, and CBG significantly increased as flowers matured, reaching the greatest concentration during 6 to 7 weeks postanthesis. After a plateau stage of varied length for different varieties, the peak concentrations declined as plants senesced. Total THC was above the 0.3% threshold from 4 weeks postanthesis to the end of the growing season for day-length-sensitive varieties, but this only occurred during 6 to 7 weeks postanthesis for day-length-neutral varieties. The CBD/THC ratio in flowers dynamically changed during the entire reproductive stage for all of the evaluated varieties. The current study provides vital information for successful cultivation of industrial hemp.

Developmental Plasticity of the Major Alkyl Cannabinoid
Chemotypes in a Diverse Cannabis Genetic Resource Collection

Matthew T. Welling, Lei Liu1, Carolyn A. Raymond, Omid Ansari, and
Graham J. King
Frontiers in Plant Science | 1 October 2018 | Volume 9 | Article 1510
doi: 10.3389/fpls.2018.01510

Development of the Industrial Chain for the Production of Cannabigerol (CBG): from Plant to Pure Compound
Xavier Nadal Roura, PhD
Manager Extraction R&D Department, Phytoplant Research S.L.
Presented at CANN 10 Tel Aviv, Israel June 5 2017
https://www.phytoplantresearch.com/u...e-Compound.pdf

Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and D9 -tetrahydrocannabinol in BV-2 microglial cells
Ana Juknat, Maciej Pietr, Ewa Kozela1, Neta Rimmerman, Rivka Levy, Giovanni Coppola, Daniel Geschwind and Zvi Vogel
British Journal of Pharmacology (2012) 165 2512–2528
DOI:10.1111/j.1476-5381.2011.01461.x
BACKGROUND AND PURPOSE Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoidD9 -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways.
EXPERIMENTAL APPROACH To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC.
KEY RESULTS Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 byTHC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2a/p8/ATF4/CHOP-TRIB3 pathway. Furthermore,CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. CONCLUSIONS AND IMPLICATIONS These observations indicated thatCBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity.

Discovery of KLS-13019, a Cannabidiol-Derived Neuroprotective Agent, with Improved Potency, Safety, and Permeability
William A. Kinney, Mark E. McDonnell, Hua Marlon Zhong, Chaomin Liu, Lanyi Yang, Wei Ling, Tao Qian, Yu Chen, Zhijie Cai, Dean Petkanas,†and Douglas E. Brenneman
ACS Med. Chem. Lett. 2016, 7, 424?428
DOI: 10.1021/acsmedchemlett.6b00009
Cannabidiol is the nonpsychoactive natural component of C. sativa that has been shown to be neuroprotective in multiple animal models. Our interest is to
advance a therapeutic candidate for the orphan indication hepatic encephalopathy (HE). HE is a serious neurological disorder that occurs in patients with cirrhosis or liver failure. Although cannabidiol is effective in models of HE, it has limitations in terms of safety and oral bioavailability. Herein, we describe a series of side chain modifi ed resorcinols that were designed for greater hydrophilicity and “ drug likeness” , while varying hydrogen bond donors, acceptors, architecture, basicity, neutrality, acidity, and polar surface area within the pendent group. Our primary screen evaluated the ability of the test agents to prevent damage to hippocampal neurons induced by ammonium acetate and ethanol at clinically relevant concentrations. Notably, KLS-13019 was 50-fold more potent and >400-fold safer than cannabidiol and exhibited an in vitro profile consistent with improved oral bioavailability.

Distribution of cannabinoid synthase genes in non-Cannabis organisms
Niranjan Aryal* , Debbie Figueroa Orellana and Jamal Bouie
Journal of Cannabis Research 2019, 1(2019):8
Doi: .1186/s42238-019-0008-7
The discovery of phytocannabinoid synthesizing enzymes, tetrahydrocannabinolic acid synthase (THCAs) and cannabidiolic acid synthase (CBDAs) was a breakthrough in Cannabis research. However, their evolutionary aspects and distribution across organisms has not been adequately studied. We searched for THCAs and CBDAs genes in organisms other than Cannabis plants using the database available in NCBI. Both cannabinoid synthases seem to be widely distributed in the plant kingdom. Of several complete or partial sequences of cannabinoid synthases-likes, CBDAs-like from Morus notabilis matched closely to CsCBDAs and CsTHCAs. When amino acid sequences of CsTHCAs, CsCBDAs and MnCBDAs-like were compared to each other, and to the motif database stored in Expasy, all three proteins contained the FAD_PCMH (PCMH-type FAD-binding) domain indicating the conservation of this domain in cannabinoid synthases. Apart from FAD binding, Berberine Bridge Enzyme (BBE-likes), which catalyzes the synthesis of isoquinoline alkaloids in many plants such as mulberry, poplas and citrus, were the other most closely related enzymes to CsTHCAs and CsCBDAs. We also searched for THCAs and CBDAs in fungal and bacterial kingdom but could not find any notably similar sequence. However, partial mRNA from FAD binding enzyme from Trametes versicolor and 6-hydroxy D nicotine oxidase from Aspergillus saccharolyticus matched the CsTHCA sequence and a partial mRNA from a hypothetical protein in Pneumocystis carinii was the most closely matched fungal enzyme to the CsCBDA. Our database search showed that Morus notabilis from mulberry family could be the candidate plant for further studies. Comparative transcriptomic and metabolomic studies for mulberry and Cannabis plants could provide a much clear concepts on the co-evolution of these syanthases. Moreover, the understanding of cannabinoid synthesis pathway is still evolving, in-depth bioinformatics and functional analysis of the enzymes involved are required for pharmaceutical research and industrial advancement.

Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars
Eduviges Borroto Fernandez, Ph.D.; Verena Peterseil, 1 M.Sc. Gerald Hackl, 1 Dipl.Ing.; Sonja Menges, 2 Ph.D.; Etienne de Meijer, 3 Ph.D.; and Christina Staginnus, 4 Ph.D.
J Forensic Sci, 2019
doi: 10.1111/1556-4029.14242
In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% BT allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one BT allele, reflects that plant breeding efforts have resulted in a fixation of the BT allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals

Do cannabinoids have a therapeutic role in transplantation?
Nagarkatti, M., Rieder, S. A., Hegde, V. L., Kanada, S., & Nagarkatti, P.
Trends in Pharmacological Sciences, 31(8), 345–350.(2010).
doi:10.1016/j.tips.2010.05.006
Cannabinoids have emerged as powerful drug candidates for the treatment of inflammatory and autoimmune diseases due to their immunosuppressive properties. Significant clinical and experimental data on the use of cannabinoids as anti-inflammatory agents exist in many autoimmune disease settings, but virtually no studies have been undertaken on their potential role in transplant rejection. Here we suggest a theoretical role for the use of cannabinoids in preventing allograft rejection. The psychotropic properties of CB1 agonists limit their clinical use, but CB2 agonists may offer a new avenue to selectively target immune cells involved in allograft rejection. Moreover, development of mixed CB1/CB2 agonists that cannot cross the blood–brain barrier may help prevent their undesired psychotropic properties. In addition, manipulation of endocannabinoids in vivo by activating their biosynthesis and inhibiting cellular uptake and metabolism may offer another pathway to regulate immune response during allograft rejection.

Dose-Related Inhibition of Capsaicin Responses by Cannabinoids CBG, CBD, THC and their Combination in Cultured Sensory Neurons
Uma Anand Christian Oldfield, Barbara Pacchetti, Praveen Anand, Mikael H Sodergren
DOI: 10.2147/JPR.S336773
Journal of Pain Research 2021:14
https://www.dovepress.com/getfile.php?fileID=76128
Background: The analgesic effects of Cannabis sativa are mediated by ∆9 tetrahydrocannabinol (THC), but the contributions of other bioactive complex components, including cannabigerol (CBG) and cannabidiol (CBD), are unclear. We describe the individual and combined effects of CBG, CBD and THC, on blocking capsaicin responses in dorsal root ganglion (DRG) neurons, in an in vitro model of nociceptor hypersensitivity.
Materials and Methods: Adult rat DRG were dissected and enzyme digested to obtain a neuronal suspension in BSF2 medium containing 2% fetal calf serum, and the neurotrophic factors NGF and GDNF. After 48 h, cultured neurons were loaded with Fura-2 AM, to determine the effects of cannabinoids on capsaicin responses using calcium imaging. In control experiments, neurons were treated with vehicle, followed by 1 µM capsaicin. In cannabinoid treated cultures, CBG, CBD or THC were applied individually, or combined (1:1:1 ratio), followed by 1 µM capsaicin. Data from n = 6 experiments were analysed with Student’s t-test and Pearson’s correlation coefficient.
Results: CBG, CBD and THC, applied individually, elicited dose-related calcium influx in a subset of DRG neurons, and a corresponding dose-related reduction of subsequent responses to capsaicin. Maximum inhibition of capsaicin responses was observed at 30 µM CBG, 100 µM CBD, and 100 µM THC individually, and with combined CBD+CBG +THC (1:1:1) at 90 µM. THC+CBD+CBG combined in a 1:1:1 proportion has the potential to enhance the potency of these compounds applied individually. There was a high correlation between cannabinoid-mediated calcium influx and reduction of capsaicin responses: CBG = −0.88, THC = −0.97, CBD = −0.99 and combined CBG + THC + CBD = −1.00.
Conclusion: CBG, CBD and THC demonstrated potent dose-related inhibition of capsaicin responses in DRG neurons when applied individually in vitro, and enhanced when applied in combination, being most effective at 90 μM. Thus, efficacy and tolerability of THC could be improved in combination with CBG and CBD at optimal concentrations, which deserve further studies in vivo

Not Cannabis specific
Double Duty for a Conserved Glutamate in Pyruvate Decarboxylase: Evidence of the Participation in Stereoelectronically Controlled Decarboxylation and in Protonation of the Nascent Carbanion/Enamine Intermediate
Danilo Meyer, Piotr Neumann, Christoph Parthier, Rudolf Friedemann, Natalia Nemeria, Frank Jordan, and Kai Tittmann
Biochemistry. 2010 September 21; 49(37): 8197–8212. doi:10.1021/bi100828r
https://www.ncbi.nlm.nih.gov/labs/pm...ihms232199.pdf
Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2- lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate. Variants carrying either an isofunctional (Glu473Asp) or isosteric (Glu473Gln) substitution exhibit a residual catalytic activity of less than 0.1% but accumulate different intermediates at the steady state. Whereas the predecarboxylation intermediate 2-lactyl-ThDP is accumulated in Glu473Asp because of a 3000-fold slower decarboxylation compared to that of the wild-type enzyme, Glu473Gln is not impaired in decarboxylation but generates a long-lived 2-hydroxyethylThDP carbanion/enamine postdecarboxylation intermediate. CD spectroscopic analysis of the protonic and tautomeric equilibria of the cocatalytic aminopyrimidine part of ThDP indicates that an acidic residue is required at position 473 for proper substrate binding. Wild-type PDC and the Glu473Asp variant bind the substrate analogue acetylphosphinate with the same affinity, implying a similar stabilization of the predecarboxylation intermediate analogue on the enzyme, whereas Glu473Gln fails to bind the analogue. The X-ray crystallographic structure of 2-lactyl-ThDP trapped in the decarboxylation-deficient variant Glu473Asp reveals a common stereochemistry of the intermediate C2α stereocenter; however, the scissile C2α–C(carboxylate) bond deviates by ~25–30° from the perpendicular “maximum overlap” orientation relative to the thiazolium ring plane as commonly observed in ThDP enzymes. Because a reactant-state stabilization of the predecarboxylation intermediate can be excluded to account for the slower decarboxylation, the data suggest a strong stereoelectronic effect for the transition state of decarboxylation as supported by additional DFT studies on models. To the best of our knowledge, this is a very rare example in which the magnitude of a stereoelectronic effect could be experimentally estimated for an enzymatic system. Given that variant Glu473Gln is not decarboxylation-deficient, electrostatic stress can be excluded as a driving force for decarboxylation. The apparent dual function of Glu473 further suggests that decarboxylation and protonation of the incipient carbanion are committed and presumably proceed in the same transition state.

Early phytocannabinoid chemistry to endocannabinoids and beyond
Raphael Mechoulam, Lumír O. Hanuš, Roger Pertwee and Allyn C. Howlett
Nature Reviews / Neuroscience
DOI: 10.1038/nrn3811
Isolation and structure elucidation of most of the major cannabinoid constituents — including ?9-tetrahydrocannabinol (?9-THC), which is the principal psychoactive molecule in Cannabis sativa was achieved in the 1960s and 1970s. It was followed by the identification of two cannabinoid receptors in the 1980s and the early 1990s and by the identification of the endocannabinoids shortly thereafter. There have since been considerable advances in our understanding of the endocannabinoid system and its function in the brain, which reveal potential therapeutic targets for a wide range of brain disorders.

Effect of Cannabidiol and ?9 -Tetrahydrocannabinol on Driving Performance A Randomized Clinical Trial
Thomas R. Arkell, Frederick Vinckenbosch, Richard C. Kevin, Eef L. Theunissen, Iain S. McGregor, Johannes G. Ramaekers
JAMA. 2020;324(21):2177-2186.
doi:10.1001/jama.2020.21218
IMPORTANCE Cannabis use has been associated with increased crash risk, but the effect of cannabidiol (CBD) on driving is unclear.
OBJECTIVE To determine the driving impairment caused by vaporized cannabis containing ?9 -tetrahydrocannabinol (THC) and CBD.
DESIGN, SETTING, AND PARTICIPANTS A double-blind, within-participants, randomized clinical trial was conducted at the Faculty of Psychology and Neuroscience at Maastricht University in the Netherlands between May 20, 2019, and March 27, 2020. Participants (N = 26) were healthy occasional users of cannabis.
INTERVENTIONS Participants vaporized THC-dominant, CBD-dominant, THC/CBD-equivalent, and placebo cannabis. THC and CBD doses were 13.75 mg. Order of conditions was randomized and balanced.
MAIN OUTCOMES AND MEASURES The primary end point was standard deviation of lateral position (SDLP; a measure of lane weaving) during 100 km, on-road driving tests that commenced at 40 minutes and 240 minutes after cannabis consumption. At a calibrated blood alcohol concentration (BAC) of 0.02%, SDLP was increased relative to placebo by 1.12 cm, and at a calibrated BAC of 0.05%, SDLP was increased relative to placebo by 2.4 cm. RESULTS Among 26 randomized participants (mean [SD] age, 23.2 [2.6] years; 16 women), 22 (85%) completed all 8 driving tests. At 40 to 100 minutes following consumption, the SDLP was 18.21 cm with CBD-dominant cannabis, 20.59 cm with THC-dominant cannabis, 21.09 cm with THC/CBD-equivalent cannabis, and 18.28 cm with placebo cannabis. SDLP was significantly increased by THC-dominant cannabis (+2.33 cm [95% CI, 0.80 to 3.86]; P < .001) and THC/CBD-equivalent cannabis (+2.83 cm [95% CI, 1.28 to 4.39]; P < .001) but not CBD-dominant cannabis (?0.05 cm [95% CI, ?1.49 to 1.39]; P > .99), relative to placebo. At 240 to 300 minutes following consumption, the SDLP was 19.03 cm with CBD-dominant cannabis, 19.88 cm with THC-dominant cannabis, 20.59 cm with THC/CBD-equivalent cannabis, and 19.37 cm with placebo cannabis. The SDLP did not differ significantly in the CBD (?0.34 cm [95% CI, ?1.77 to 1.10]; P > .99), THC (0.51 cm [95% CI, ?1.01 to 2.02]; P > .99) or THC/CBD (1.22 cm [95% CI, ?0.29 to 2.72]; P = .20) conditions, relative to placebo. Out of 188 test drives, 16 (8.5%) were terminated due to safety concerns.
CONCLUSIONS AND RELEVANCE In a crossover clinical trial that assessed driving performance during on-road driving tests, the SDLP following vaporized THC-dominant and THC/CBD-equivalent cannabis compared with placebo was significantly greater at 40 to 100 minutes but not 240 to 300 minutes after vaporization; there were no significant differences between CBD-dominant cannabis and placebo. However, the effect size for CBD-dominant cannabis may not have excluded clinically important impairment, and the doses tested may not represent common usage

Effect of Cannabidiol on the Long-Term Toxicity and Lifespan in the Preclinical Model Caenorhabditis elegans
M. Hunter Land, Marton L. Toth, Laura MacNair, Siva A. Vanapalli, Timothy W. Lefever, Erica N. Peters, and Marcel O. Bonn-Miller
Cannabis and Cannabinoid Research, 2020
DOI: 10.1089/can.2020.0103
Introduction: Despite widespread use of cannabidiol (CBD), no lifelong toxicity study has been published to date. Caenorhabditis elegans is often used in preclinical lifelong toxicity studies, due to an estimated 60–80% of their genes having a human ortholog, and their short lifespan of *2–3 weeks. In this study, we examined both acute and long-term exposure studies of CBD at physiologically relevant concentrations.
Materials and Methods: Acute toxicity was determined by treating day 1 adults with a wide range of CBD concentrations (0.4 lM to 4 mM) and assessing mortality and motility compared to control animals. Thermotolerance was examined by treating adult animals with CBD (0.4 lM to 4 mM) and exposing them to 37C for 4 h, and then scoring for the number of alive animals treated with CBD compared to controls. Long-term toxicity was assessed by exposing day 1 adults to 10, 40, and 100 lM CBD until all animals perished. Control animals had no active drug exposure.
Results: We report both acute and long-term exposure studies of CBD to adult C. elegans at physiologically relevant concentrations. Acute toxicity results showed that no animal died when exposed to 0.4–4000 lM CBD. The thermotolerance study showed that 40 lM CBD, but not other treatment levels, significantly increased resistance to heat stress by 141% compared to the untreated controls. Notably, whole-life exposure of C. elegans to 10–100 lM CBD revealed a maximum life extension of 18% observed at 40 lM CBD. In addition, motility analysis of the same groups revealed an increase in late-stage life activity by up to 206% compared to controls.
Conclusion: These results serve as the only CBD lifelong exposure data in an in vivo model to date. While further research into the lifelong use of CBD should be carried out in mammalian models, the C. elegans model indicates a lack of long-term toxicity at physiologically relevant concentrations.

Effect of Colonization of Trichoderma harzianum on Growth Development and CBD Content of Hemp (Cannabis sativa L.)
Ioanna Kakabouki, Alexandros Tataridas, Antonios Mavroeidis, Angeliki Kousta, Stella Karydogianni, Charikleia Zisi, Varvara Kouneli, Antigolena Folina, Aristidis Konstantas, Panayiota Papastylianou,
Microorganisms 9(518) March 2021
DOI: 10.3390/microorganisms9030518
https://www.researchgate.net/publica...nabis_sativa_L
Trichoderma harzianum, as a natural endophytic biocontrol agent, can ameliorate plant development, nutrient uptake, and resistance to biotic and abiotic stresses. This study aimed to investigate the effect of Trichoderma harzianum inoculation on agronomical and quality characteristics of two monoecious hemp (Cannabis sativa L.) varieties, Fedora 17 and Felina. A greenhouse pot experiment was conducted in a completely randomized design of two treatments of Trichoderma harzianum with a low and high dose of the fungus (T1 and T2). The significance of differences between treatments was estimated by using a Fisher’s test with a significance level p = 0.05. The root density of both varieties was significantly affected by treatments, and higher values were recorded in Fedora 17 (2.32 mm cm−3). The Arbuscular Mycorrhizal Fungi (AMF) colonization of the root system and the soil emission of CO2 were higher after the inoculation of Trichoderma harzianum. The highest values of plant height and dry weight were noticed for T2, especially in variety Felina. Trichoderma harzianum positively influenced characteristics of inflorescences such as their number, fresh weight moisture, and compactness in both varieties, while the dry weight, length, and dry yield of inflorescences were not improved. Finally, the fertigation of Trichoderma harzianum in hemp plants was beneficial by increasing the cannabidiol (CBD) content, especially in T2 treatment (4 × 1012 CFU kg−1).

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on nocturnal sleep and early-morning behavior in young adults.
Nicholson AN1, Turner C, Stone BM, Robson PJ.
J Clin Psychopharmacol. 2004 Jun;24(3):305-13.
DOI: 10.1097/01.jcp.0000125688.05091.8f
The effects of cannabis extracts on nocturnal sleep, early-morning performance, memory, and sleepiness were studied in 8 healthy volunteers (4 males, 4 females; 21 to 34 years). The study was double-blind and placebo-controlled with a 4-way crossover design. The 4 treatments were placebo, 15 mg Delta-9-tetrahydrocannabinol (THC), 5 mg THC combined with 5 mg cannabidiol (CBD), and 15 mg THC combined with 15 mg CBD. These were formulated in 50:50 ethanol to propylene glycol and administered using an oromucosal spray during a 30-minute period from 10 pm. The electroencephalogram was recorded during the sleep period (11 pm to 7 am). Performance, sleep latency, and subjective assessments of sleepiness and mood were measured from 8:30 am (10 hours after drug administration). There were no effects of 15 mg THC on nocturnal sleep. With the concomitant administration of the drugs (5 mg THC and 5 mg CBD to 15 mg THC and 15 mg CBD), there was a decrease in stage 3 sleep, and with the higher dose combination, wakefulness was increased. The next day, with 15 mg THC, memory was impaired, sleep latency was reduced, and the subjects reported increased sleepiness and changes in mood. With the lower dose combination, reaction time was faster on the digit recall task, and with the higher dose combination, subjects reported increased sleepiness and changes in mood. Fifteen milligrams THC would appear to be sedative, while 15 mg CBD appears to have alerting properties as it increased awake activity during sleep and counteracted the residual sedative activity of 15 mg THC.

Effect of Genotype and Growing Year on the Nutritional, Phytochemical, and Antioxidant Properties of Industrial Hemp (Cannabis sativa L.) Seeds
Maria Irakli, Eleni Tsaliki, Apostolos Kalivas, Fotios Kleisiaris, Eirini Sarrou and Catherine M Cook
Antioxidants 2019, 8, 491;
doi:10.3390/antiox8100491
Cannabis sativa L. seeds have been an important source of protein, oil, and dietary fiber for human and animals. Currently, there is a growing interest in the commercial products of these seeds, which are recognized as a legitimate source of medicaments, cosmeceuticals, and nutraceuticals. The objective of this study was to investigate the nutritional, phytochemical composition, and antioxidant properties of seeds from seven hemp cultivars grown in Greece for three consecutive years. All the measured parameters strongly varied under the influence of growing year and genotype. In particular, protein, oil, and carbohydrates’ content of hemp seeds as well as fatty acids’ composition were mainly affected by genotype, whereas the growing year had a major effect on phytochemical components and antioxidant activity, which was determined by the 2,20 -azino-bis (3-ethylbenzthiazoline sulfonate) (ABTS) and ferric-reducing antioxidant power (FRAP) assays. Moreover, a predominant effect of the year was observed for phenolic profiles as determined by high-performance liquid chromatography and total carotenoids’ content. This study suggests that hemp seeds could be a promising food crop as a result of their high nutritive traits and antioxidant potential. A comparison of the studied cultivars, showed that Finola seeds had the highest oil and protein contents and, thus, appeared to be the most promising cultivar for cultivation in Greece.

EFFECT OF LIGHT QUALITY ON CANNABINOID CONTENT OF CANNABIS SATIVA L. (CANNABACEAE)
PAUL G. MAHLBERG AND JOHN K. HEMPHILL
BOT. GAZ. 144(1):43-48. 1983.
DOI:10.1086/337342
ditions, filtered green, blue, and red light, and darkness-were analyzed by gas-liquid chromatography for their cannabinoid content. The highest content of cannabinoids, predominantly A'-tetrahydrocannabinol (A'-THC) in this strain, occurred in the youngest leaves of daylight-grown plants. Leaves at successively lower nodes of this control condition and all treated plants subsequently grown in daylight contained progressively lower levels of cannabinoids. Leaves from plants grown under filtered green light and darkness contained significantly lower levels of A'-THC than those from plants grown in daylight. However, the A'-THC content of leaves from plants grown under shaded daylight and filtered red and blue light did not differ significantly from the A'-THC content in daylight controls, indicating that these conditions did not alter the synthetic rate of this cannabinoid. The cannabichromene (CBC) content of plants grown under filtered red and green light and darkness differed from the CBC content in plants grown in daylight, indicating that the formation of this cannabinoid was independent of A'-THC. Leaves from plants grown under filtered red and green light and darkness recovered the capacity to synthesize typical levels of A'-THC and CBC when placed under daylight conditions. Plants from all light and dark treatments, when subsequently placed under daylight conditions for 66 days, attained levels of cannabinoid synthesis comparable to the daylight controls

Effects of Cannabidiol and Delta-9-Tetrahydrocannabinol on Emotion, Cognition, and Attention: A Double-Blind, Placebo-Controlled, Randomized Experimental Trial in Healthy Volunteers.
Woelfl, T., Rohleder, C., Mueller, J. K., Lange, B., Reuter, A., Schmidt, A. M., … Leweke, F. M.
Frontiers in Psychiatry, 11.(2020).
doi:10.3389/fpsyt.2020.576877
The two main phytocannabinoids—delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD)—have been extensively studied, and it has been shown that THC can induce transient psychosis. At the same time, CBD appears to have no psychotomimetic potential. On the contrary, emerging evidence for CBD’s antipsychotic properties suggests that it may attenuate effects induced by THC. Thus, we investigated and compared the effects of THC and CBD administration on emotion, cognition, and attention as well as the impact of CBD pre-treatment on THC effects in healthy volunteers. We performed a placebo-controlled, double-blind, experimental trial (GEI-TCP II; ClinicalTrials.gov identifier: NCT02487381) with 60 healthy volunteers randomly allocated to four parallel intervention groups, receiving either placebo, 800 mg CBD, 20 mg THC, or both cannabinoids. Subjects underwent neuropsychological tests assessing working memory (Letter Number Sequencing test), cognitive processing speed (Digit Symbol Coding task), attention (d2 Test of Attention), and emotional state (adjective mood rating scale [EWL]). Administration of CBD alone did not influence the emotional state, cognitive performance, and attention. At the same time, THC affected two of six emotional categories—more precisely, the performance-related activity and extraversion—, reduced the cognitive processing speed and impaired the performance on the d2 Test of Attention. Interestingly, pre-treatment with CBD did not attenuate the effects induced by THC. These findings show that the acute intake of CBD itself has no effect per se in healthy volunteers and that a single dose of CBD prior to THC administration was insufficient to mitigate the detrimental impact of THC in the given setting. This is in support of a complex interaction between CBD and THC whose effects are not counterbalanced by CBD under all circumstances.

Effects of cannabinoids and cannabinoid?enriched Cannabisextracts on TRP channels and endocannabinoid metabolic enzymes
Luciano De Petrocellis, Alessia Ligresti, Aniello Schiano Moriello, Marco Allarà, Tiziana Bisogno, Stefania Petrosino, Colin G Stott, Vincenzo Di Marzo
British Journal of Pharmacology (2011) 163 1479–1494
doi: 10.1111/j.1476-5381.2010.01166.x
BACKGROUND AND PURPOSE Cannabidiol (CBD) and ?9?tetrahydrocannabinol (THC) interact with transient receptor potential (TRP) channels and enzymes of the endocannabinoid system.
EXPERIMENTAL APPROACH The effects of 11 pure cannabinoids and botanical extracts [botanical drug substance (BDS)] from Cannabis varieties selected to contain a more abundant cannabinoid, on TRPV1, TRPV2, TRPM8, TRPA1, human recombinant diacylglycerol lipase ? (DAGL?), rat brain fatty acid amide hydrolase (FAAH), COS cell monoacylglycerol lipase (MAGL), human recombinant N?acylethanolamine acid amide hydrolase (NAAA) and anandamide cellular uptake (ACU) by RBL?2H3 cells, were studied using fluorescence?based calcium assays in transfected cells and radiolabelled substrate?based enzymatic assays. Cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THCVA) were also tested.
KEY RESULTS CBD, CBG, CBGV and THCV stimulated and desensitized human TRPV1. CBC, CBD and CBN were potent rat TRPA1 agonists and desensitizers, but THCV-BDS was the most potent compound at this target. CBG-BDS and THCV-BDS were the most potent rat TRPM8 antagonists. All non-acid cannabinoids, except CBC and CBN, potently activated and desensitized rat TRPV2. CBDV and all the acids inhibited DAGLa. Some BDS, but not the pure compounds, inhibited MAGL. CBD was the only compound to inhibit FAAH, whereas the BDS of CBC > CBG > CBGV inhibited NAAA. CBC = CBG > CBD inhibited ACU, as did the BDS of THCVA, CBGV, CBDA and THCA, but the latter extracts were more potent inhibitors.
CONCLUSIONS AND IMPLICATIONS These results are relevant to the analgesic, anti-inflammatory and anti-cancer effects of cannabinoids and Cannabis extracts

Effects of long term storage on secondary metabolite profiles of cannabis resin.
Grafström K, Andersson K, Pettersson N, Dalgaard J, Dunne S
Forensic Sci Int. 2019 Aug;301:331-340.
doi: 10.1016/j.forsciint.2019.05.035
The structural identification and the monitoring of the relative concentrations of a wide range of major (3) and minor secondary (16) metabolites used as marker substances for profiling of cannabis resin using GC-FID at the Swedish National Forensic Centre (NFC) has facilitated the mapping of their chemical and physical behaviors over a period of 48months whilst stored under different conditions (exposure to light, exposure to air, temperature). In all cases the behavior of this group of sesquiterpenes, sesquiterpenoids, cannabinoids and waxes could be directly related to their chemical lability/functionality. In particular, the identification of homologue triads for both ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD) together with a group of seemingly chemically inert substances (for example, cannabicyclol(CBL) and the waxes (n-alkanes)) has created new tools for the establishment of common origins between samples of cannabisresins aged under different conditions. Since sampling of the resin blocks in NFC's method for profiling of cannabis resin is made below the surface, the effects of light incursion were found to be negligible. The effects of exposure to air (and indirectly temperature) were found to be more significant, not unexpectedly as many of the observed transformations were based on oxidation or rearrangement processes.

Effects of Smoked Cannabis and Oral ?9-Tetrahydrocannabinol on Nausea and Emesis After Cancer Chemotherapy: A Review of State Clinical Trials
Richard E. Musty, Rita Rossi
Journal of Cannabis Therapeutics, Vol. 1(1) 2001
https://www.cannabis-med.org/data/pdf/2001-01-2.pdf
10.1300/J175v01n01_03
Background. In 1999 the Institute of Medicine (IOM) issued a report entitled Marijuana and Medicine (Joy, Watson and Benson, 1999). It recommended the development of cannabinoid drug delivery systems which might be effective for nausea, vomiting and AIDS wasting syndrome, among other chronic disorders. The report went on to recognize that patients should be allowed to smoke marijuana if they failed to achieve relief from approved symptoms that could be relieved by cannabinoid drugs with rapid onset. Recommended criteria of the report included: access to marijuana within 24 hours of submission by a physician, supervision that allows for assessment of treatment effectiveness, and an oversight strategy comparable to an institutional review board. In this context a review of previously unpublished state-run clinical trials with Cannabis sativa (marijuana and/ or ?9-tetrahydrocannabinol capsules) to test efficacy in reducing nausea and vomiting following cancer chemotherapy is warranted. The impetus for these studies came from individual state legislatures responding to constituents’ claims that smoking marijuana reduced or blocked nausea and vomiting. Methods. Technical reports were obtained from 6 states which had conducted clinical trials. Each protocol was examined for the procedure used, the experimental design of the clinical trial and the results obtained. Data were available on 748 patients who smoked marijuana prior to and/or after cancer chemotherapy and 345 patients who used the oral THC capsule.
Results. Patients who smoked marijuana experienced 70-100% relief
from nausea and vomiting, while those who used the THC capsule
experienced 76-88% relief.
Conclusions. On the basis of these studies, it appears that smoked
marijuana can be a very successful treatment for nausea and vomiting
following cancer chemotherapy. The development of smokeless inhalation devices could certainly reduce yhe potential harm from smoking marijuana.

Effects of ultraviolet-B radiation on the growth, physiology and cannabinoid production of Cannabis sativa L
J. Lydon
January 1986 Thesis (Ph. D.)--University of Maryland, College Park, 1985. Vita. Includes bibliographical references (leaves 78-95).
GET DOI or link
The concentration of cannabinoids in Cannabis sativa L. is correlated with high ultraviolet-B (UV-B) radiation environments. ..delta../sup 9/-Tetrahydrocannabinolic acid and cannabidiolic acid, both major secondary products of C. sativa, absorb UV-B radiation and may function as solar screens. The object of this study was to test the effects of UV-B radiation on the physiology and cannabinoid production of C. sativa. Drug and fiber-type C. sativa were irradiated with three levels of UV-B radiation for 40 days in greenhouse experiments. Physiological measurements on leaf tissues were made by infra-red gas analysis. Drug and fiber-type control plants had similar CO/sub 2/ assimilation rates from 26 to 32/sup 0/C. Drug-type control plant had higher dark respiration rates and stomatal conductances than fiber-type control plants. The concentration of ..delta../sup 9/-THC, but not of other cannabinoids) in both vegetative and reproductive tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation. The increased level of ..delta../sup 9/-THC found in leaves after irradiation may account for the physiological and morphological insensitivity to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidoil (CBD). Resin stripped form fresh fiber-type floral tissue by sonication was spotted on filter paper and irradiated continuously for 7 days. Cannabidiol (CBD) gradually decreased when irradiated but ..delta../sup 9/-THC and cannabichromene did not.

Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L .
Zirpel, B., Kayser, O., & Stehle, F.
Journal of Biotechnology, 284, 17–26. (2018).
doi:10.1016/j.jbiotec.2018.07.031
Cannabinoids are secondary natural products from the plant Cannabis sativa L. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the ? 9 - tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - ? 9 -tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the Cterminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52 °C to 57 °C. We generated two CBDAS variants, C_S116A and C_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production

Endogenous cannabinoids and appetite
Tim C. Kirkham* and Claire M. Williams
Nutrition Research Reviews (2001), 14, 65–86
DOI: 10.1079/NRR200118
Since pre-history, Cannabis sativa has been exploited for its potent and manifold pharmacological actions. Amongst the most renowned of these actions is a tendency to provoke ravenous eating. The characterization of the psychoactive principals in cannabis (exogenous cannabinoids) and, more recently, the discovery of specific brain cannabinoid receptors and their endogenous ligands (endocannabinoids) has stimulated research into the physiological roles of endocannabinoid systems. In this review, we critically discuss evidence from the literature that describe studies on animals and human subjects to support endocannabinoid involvement in the control of appetite. We describe the hyperphagic actions of the exogenous cannabinoid, 9-tetrahydrocannabinol, and the endogenous CB1 ligands, anandamide and 2-arachidonylglycerol, and present evidence to support a specific role of endocannabinoid systems in appetitive processes related to the incentive and reward properties of food. A case is made for more comprehensive and systematic analyses of cannabinoid actions on eating, in the anticipation of improved therapies for disorders of appetite and body weight, and a better understanding of the biopsychological processes underlying hunger.

Engineering yeasts as platform organisms for cannabinoid biosynthesis.
Zirpel, B., Degenhardt, F., Martin, C., Kayser, O., & Stehle, F.
Journal of Biotechnology, 259, 204–212 (2017).
doi:10.1016/j.jbiotec.2017.07.008
? 9 -tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa L. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme’s coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and 3 geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.
Husain, R., Weeden, H., Bogush, D., Deguchi, M., Soliman, M., Potlakayala, S., … Rudrabhatla, S.
PLOS ONE, 14(8), (2019).
doi:10.1371/journal.pone.0221570
Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.

Enzymatic Activation of Cannabinoid Pro-Drugs,
THESIS, Anna Theresa Margiotta
https://digscholarship.unco.edu/cgi/...context=theses


Enzymological Evidence for Cannabichromenic Acid Biosynthesis
Satoshi Morimoto, Kengo Komatsu, Futoshi Taura, and Yukihiro Shoyama
J. Nat. Prod. 1997, 60, 854-857
DOI: 10.1021/np970210y
An enzyme involved in the biosynthesis of cannabichromenic acid was identified in young leaves of Cannabis sativa . The enzyme, named cannabichromenic acid synthase, catalyzed the oxidocyclization of cannabigerolic acid to cannabichromenic acid. The biosynthetic mechanism for the formation of cannabichromenic acid was similar to those of 1 –tetrahydrocannabinolic acid and cannabidiolic acid

Evaluation of prevalent phytocannabinoids in the acetic acid model of visceral nociception.
Booker, L., Naidu, P. S., Razdan, R. K., Mahadevan, A., & Lichtman, A. H.
Drug and Alcohol Dependence, 105(1-2), 42–47(2009).
doi:10.1016/j.drugalcdep.2009.06.00
Considerable preclinical research has demonstrated the efficacy of 9-tetrahydrocannabinol (9-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of 9-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, 9-THC and CBN bound to the CB1 receptor and produced antinociceptive effects. The CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB1 receptor with similar affinity as 9-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of 9-THC when both drugs were given in combination. Importantly, the antinociceptive effects of 9-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of 9-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of 9-THC.

Evaluation of thermo-chemical conversion temperatures of cannabinoid acids in hemp (Cannabis sativa L.) biomass by pressurized liquid extraction
Kenneth J. Olejar, and Chad A. Kinney
Journal of Cannabis Research (2021) 3:40
DOI: 10.1186/s42238-021-00098-6
https://www.ncbi.nlm.nih.gov/pmc/art...Article_98.pdf
Background: Cannabinoids are increasingly becoming compounds of medical interest. However, cannabis plants only produce carboxylated cannabinoids. In order to access the purported medical benefits of these compounds, the carboxylic acid moiety must be removed. This process is typically performed by heating the plant material or extract; however, cannabinoids being thermolabile can readily degrade, evaporate, or convert to undesired metabolites. Pressurized liquid extraction (PLE) operates using a pseudo-closed system under pressure and temperature. While pressure is maintained at 11 MPa, temperature can be varied from ambient to 200 °C.
Methods: Temperatures were evaluated (80 to 160 °C) using PLE for the thermo-chemical conversion of cannabinoid acids utilizing water as the solvent in the first step of extraction with subsequent extraction with ethanol. Optimum temperatures were established for the conversion of 6 cannabinoid acids to their neutral cannabinoid forms. Cannabinoid acid conversion was monitored by HPLC.
Results: The use of PLE for thermo-chemical decarboxylation has resulted in a rapid decarboxylation process taking merely 6 min. The temperatures established here demonstrate statistically significant maxima and minima of cannabinoids and their parent cannabinoid acids. One-way ANOVA analysis shows where individual cannabinoids are statistically different, but the combination of the maxima and minima provides temperatures for optimum thermo-chemical conversion. CBC, CBD, CBDV, and CBG have an optimum temperature of conversion of 140 °C, while THC was 120 °C for 6 min.
Discussion: Decarboxylation of cannabinoid acids is necessary for conversion to the bioactive neutral form. The pseudo-closed chamber of the PLE makes this an ideal system to rapidly decarboxylate the cannabinoid acids due to pressure and temperature, while minimizing loss typically associated with conventional thermal-decarboxylation. This study established the optimum temperatures for thermo-chemical conversion of the cannabinoid acids in water and provides the groundwork for further development of the technology for industrial scale application.

Evidence that the plant cannabinoid ?9-tetrahydrocannabivarin is a cannabinoid CB1and CB2 receptor antagonist.
Thomas, A., Stevenson, L. A., Wease, K. N., Price, M. R., Baillie, G., Ross, R. A., & Pertwee, R. G.
British Journal of Pharmacology, 146(7), 917–926. (2005).
doi:10.1038/sj.bjp.0706414
1 D9-tetrahydrocannabivarin (THCV) displaced [3H]CP55940 from specific binding sites on mouse brain and CHO-hCB2 cell membranes (Ki ¼ 75.4 and 62.8 nM, respectively).
2 THCV (1 mM) also antagonized CP55940-induced stimulation of [35S]GTPgS binding to these membranes (apparent KB ¼ 93.1 and 10.1 nM, respectively).
3 In the mouse vas deferens, the ability of D9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent KB-value (96.7 nM) approximating the
apparent KB-values for its antagonism of CP55940- and R-( þ )-WIN55212-induced stimulation of [35S]GTPgS binding to mouse brain membranes.
4 THCV also antagonized R-( þ )-WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent KB-values (1.5, 1.2, 4.6 and 10.3 nM, respectively).
5 THCV (100 nM) did not oppose clonidine, capsaicin or ()-7-hydroxy-cannabidiol-dimethylheptyl-induced inhibition of electrically evoked contractions of the vas deferens.
6 Contractile responses of the vas deferens to phenylephrine hydrochloride or b,g-methylene-ATP were not reduced by 1 mM THCV or R-( þ )-WIN55212, suggesting that THCV interacts with R-( þ )-
WIN55212 at prejunctional sites.
7 At 32 mM, THCV did reduce contractile responses to phenylephrine hydrochloride and b,gmethylene-ATP, and above 3 mM it inhibited electrically evoked contractions of the vas deferens in an
SR141716A-independent manner. 8 In conclusion, THCV behaves as a competitive CB1 and CB2 receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R-( þ )-WIN55212 in this tissue than in brain membranes. The bases of these agonist-and tissue-dependent effects remain to be established

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes
Oier Aizpurua-Olaizola, Umut Soydaner, Ekin O?ztu?rk, Daniele Schibano, Yilmaz Simsir, Patricia Navarro, Nestor Etxebarria, and Aresatz Usobiaga
DOI: 10.1021/acs.jnatprod.5b00949
https://www.ncbi.nlm.nih.gov/pubmed/26836472
https://www.cannabisreports.com/news...is-chemotypes/

Exploring the Synthesis of Cannabinoids in Medicinal Cannabis – A Qualitative and Quantitative Proteomic Analysis of Laser-Microdissected Trichomes in an LC-MS-based Bottom Up Approach
Nizar Happyana, Stefan Loroch, Albert Sickmann, Oliver Kayser
Introduction: The Cannabis sativa trichomes are of high interest in biomedical research since they are the main site of cannabinoid production. More than 100 cannabinoids have been identified and structurally elucidated, like the prominent psychoactive tetrahydrocannbinolic acid (THCA) and cannabidiolic acid (CBDA). Although the distribution of the cannabinoid metabolites is well studied [1], the very complex biosynthesic pathways is only sparsely understood, not at least, because of the
limited available information on the Cannabis proteome.
Aim of this work: In a shotgun proteomics approach we want to quantify the known key players of the cannabinoid synthesis pathway in Cannabis trichomes and identify new proteins via sequence similarity searches.
Methods: Extracted proteins of laser-microdissected Cannabis trichomes were digested with trypsin and separated in a 2d approach, using high-pH reversed phase and low-pH reversed phase HPLC coupled to an Orbitrap benchtop instrument (U3000 RSLCnano, QExactive; Thermo Scientific). Mascot database search was performed against Uniprot - Cannabaceae (~500 Proteins) in a Pyrococcus furiosus backround for false discovery rate estimation [2].
Results: Using 2d-HPLC separation, we were able to identify 39 Cannabis related proteins including the major key players of cannabinoid synthesis like the tetrahydrocannabinolic acid (THCA) synthase, the olivetolic acid cyclase and the cannbidiolic acid (CBDA) synthase. In a second experiment we could show significant qualitative and quantitative differences in the trichome proteome of four Cannabis varieties. Combining these data with metabolic profiling will lead to a deeper understanding of the cannabinoid synthesis and therefore be of increased interest in research of bioactive compounds.

Extracting Fresh Cannabis with Hydrodynamic Methods
Riley Kirk
Extraction Magazine
https://extractionmagazine.com/2021/...namic-methods/
Cannabis extraction techniques have evolved with the industry. Extractors are searching for the most productive, efficient, green, full-spectrum methods to keep up with the demand. One of the newest techniques brought into the cannabis world by the company iaso™ is a process that involves producing nanoemulsions with fresh plant product. Founded in 2015, iaso introduced the concept of hydrodynamic extraction with PhytoX™ at the Terpenes and Testing World Conference in 2018. The process involves taking fresh Cannabis plants to produce droplets of oil suspended in water, aka nanoemulsions. The goal of developing this new extraction technique was to overcome common obstacles in Cannabis extraction such as consistency, efficiency, bioavailability, and safety.

Extraction of naturally occurring cannabinoids: an update
Lutfun Nahar | Shaikh Jamal Uddin | Ashraful Alam | Satyajit D. Sarker
Phytochemical Analysis. 2020;1–14
DOI: 10.1002/pca.2987
Introduction: Organic molecules that interact with the cannabinoid receptors are called cannabinoids, which can be endogenous, natural or synthetic compounds. They possess similar pharmacological properties as produced by the plant, Cannabis sativa L. Before cannabinoids can be analysed, they need to be extracted from the matrices.
Objective: To review literature on the methods and protocols for the extraction of naturally occurring cannabinoids.
Methodology: An extensive literature search was performed incorporating several databases, notably, Web of Knowledge, PubMed and Google Scholar, and other relevant published materials. The keywords used in the search, in various combinations, with cannabinoids and extraction being present in all combinations, were Cannabis, hemp, cannabinoids, Cannabis sativa, marijuana, and extraction.
Results: In addition to classical maceration with organic solvents, e.g. ethanol, pressurised solvent extraction, solvent heat reflux, Soxhlet extraction, supercritical fluid extraction, ultrasound-assisted extraction and microwave-assisted extraction, are routinely used nowadays for the extraction of cannabinoids from plant materials and cannabis consumer products. For the extraction of cannabinoids from biological samples, e.g. human blood, and also from food and beverages, and wastewater, solidphase extraction and its variants, as well as liquid–liquid extraction are commonly used. Parameters for extraction can be optimised by response surface methodology or other mathematical modelling tools. There are at least six US patents on extraction of cannabinoids available to date.
Conclusions: Irrespective of the extraction method, extraction temperature, extraction time and extraction pressure play a vital role in overall yield of extraction. Solvent polarity can also be an important factor in some extraction methods.

Falling Film Evaporators
Asia Mayfield
Extraction Magazine
https://extractionmagazine.com/2021/...m-evaporators/
Most cannabis extraction methods require a solvent like ethanol. To create the final product, technicians need a reliable, cost-effective way to separate the solvent from the biomass. One way to do this is to use falling film evaporation.
Falling film evaporators have been used for years in industries as varied as agriculture and seawater desalination. These evaporators are now common in the cannabis space as well, largely because their use can increase an extract maker’s energy efficiency saving time and money. Falling film evaporators use the physics of evaporation and gravity combined with heat in a vacuum to separate the solvent from cannabinoids and terpenes.

“Finola” Cannabis Cultivation for Cannabinoids Production in Thessaloniki-Greece
Dani Fadel, Najoie Assaad, Gabriel Alghazal, Zeinab Hamouche & Diamanto Lazari
Journal of Agricultural Science; Vol. 12, No. 7; 2020
https://www.researchgate.net/publica...aloniki-Greece
Cannabis has garnered a great deal of new attention in the past couple of years due to the increasing hopes of its legalization for recreational use and indications for medicinal benefit. The increasing consumption and cultivation has led to a multiplication of scientific studies. Focus was placed in this study foremost on yielding morphological data (length of the plant, inflorescence fresh and dry weight) for appropriate mechanical harvest and biochemical cannabinoids analysis of the industrial cannabis “Finola” that is newly grown in Greece. The average, standard error and the coefficient of variation were estimated in case of necessity and the correlation among all results was done using Microsoft Excel 2010 and Minitab 19 Software. Furthermore, three chemical analyses for TLC and NMR techniques were applied for analysis. The Cannabinoid quality or chemotype analysis was also calculated. After extraction and isolation of cannabinoids using ethanol and other separation
compounds, cannabinoid acids, tetrahydrocannabinol (THC), cannabidiol (CBD) and some other cannabinoids were extracted, isolated, identified and isolated with no delays or limitations. Finola cannabis provided a scientific background that may be considered by the Lebanese growers to accelerate and improve the relative mentality and to provide a collection of relevant scientific information, upon which the field of cannabis analysis can continue to grow.

First direct evidence for the mechanism of .DELTA.1-tetrahydrocannabinolic acid biosynthesis.
Taura, F., Morimoto, S., Shoyama, Y., & Mechoulam, R.
Journal of the American Chemical Society, 117(38), 9766–9767 (1995).
doi:10.1021/ja00143a024
Numerous plausible hypotheses have been advanced regarding the biogenesis of A'-tetrahydrocannabinol (AI-THC, la), the psychoactive constituent of marihuana (Cannabis satiua L.);I however, they all lack experimental support. Thus all hypothetical biogenetic schemes assume that Al tetrahydrocannabinolic acid (AI-THCA, lb), the precursor of AI-THC, is formed by cyclization from cannabidiolic acid (2).2 We now present
experimental evidence that establishes that lb is actually formed from cannabigerolic acid (3b) through oxidocyclization by a new enzyme, AI-THCA synthase, and that 2, a plausible intermediate, is not a substrate for this enzymatic reaction.

Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L.
Ross, S. A., ElSohly, M. A., Sultana, G. N. N., Mehmedic, Z., Hossain, C. F., & Chandra, S.
Phytochemical Analysis, 16(1), 45–48. (2005).
doi:10.1002/pca.809
Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavonol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-O-sophoroside by spectroscopic methods including high-field two-dimensional NMR experiments. The characterisation of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannabinoids and reported data. The identified cannabinoids were ?9 - tetrahydrocannabiorcol, cannabidivarin, cannabicitran, ?9 -tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, ?9 -tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol , 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-Oethylcannabitriol.

Frequent cannabis users demonstrate low knowledge of cannabinoid content and dosages.
Kruger, D. J., Kruger, J. S., & Collins, R. L.
Drugs: Education, Prevention and Policy, 1–7. (2020). doi:10.1080/09687637.2020.1752150
Background: The legal status of cannabis is rapidly evolving, outpacing systematic research and educational efforts. Aims: We investigated knowledge of cannabinoid content and dosages among frequent cannabis users. Methods: A brief survey was administered in April 2019 (N ¼ 472) at a cannabis advocacy event in a state with legal medical and recreational adult cannabis use. Findings: Most participants (67%) used cannabis every day, 85% used cannabis for health or medical purposes. Knowledge of cannabis was mostly from participants’ own experiences (78%), with some receiving information from a medical cannabis caregiver or dispensary (23%) or their primary care provider (18%). The majority reported not knowing the effective dosages of THC (53% of participants) or CBD (68% of participants), other participants gave average estimates of 91 mg and 177 mg, respectively. Participants’ average estimates for high-THC (52%) and high-CBD (53%) as well as low-THC (28%), and low-CBD (30%) strains of cannabis were considerably higher than currently accepted definitions. Men, European Americans, and participants who had medical cannabis cards were more accurate in cannabinoid concentration estimates. Conclusions: Frequent cannabis users reported low knowledge of and substantially overestimated cannabinoid content. The importance of education and research on cannabinoid dosages grows with increasing cannabis accessibility.

Gene duplication and divergence affecting drug content in Cannabis sativa
George D. Weiblen, Jonathan P. Wenger, Kathleen J. Craft, Mahmoud A. ElSohly, Zlatko Mehmedic, Erin L. Treiber, M. David Marks
New Phytologist 17 July 2015
DOI: 10.1111/nph.13562
Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically.
Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified.
Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content.
Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

Genomic and Chemical Diversity of Commercially Available High-CBD Industrial Hemp Accessions
Matthew S. Johnson and Jason G. Wallace
Front. Genet. 12:682475.
doi: 10.3389/fgene.2021.682475
https://www.ncbi.nlm.nih.gov/pmc/art...-12-682475.pdf
High consumer demand for cannabidiol (CBD) has made high-CBD hemp (Cannabis sativa) an extremely high-value crop. However, high demand has resulted in the industry developing faster than the research, resulting in the sale of many hemp accessions with inconsistent performance and chemical profiles. These inconsistencies cause significant economic and legal problems for growers interested in producing high-CBD hemp. To determine the genetic and phenotypic consistency in available high-CBD hemp varieties, we obtained seed or clones from 22 different named accessions meant for commercial production. Genotypes (∼48,000 SNPs) and chemical profiles (% CBD and THC by dry weight) were determined for up to 8 plants per accession. Many accessions–including several with the same name–showed little consistency either genetically or chemically. Most seed-grown accessions also deviated significantly from their purported levels of CBD and THC based on the supplied certificates of analysis. Several also showed evidence of an active tetrahydrocannabinolic acid (THCa) synthase gene, leading to unacceptably high levels of THC in female flowers. We conclude that the current market for high-CBD hemp varieties is highly unreliable, making many purchases risky for growers. We suggest options for addressing these issues, such using unique names and developing seed and plant certification programs to ensure the availability of high-quality, verified planting materials

GC-MS Metabolite Profile and Identification of Unusual Homologous Cannabinoids in High Potency Cannabis sativa
Josep Basas-Jaumandreu, F. Xavier C. de las Heras
Planta Med
DOI: 10.1055/a-1110-1045
Phytochemical investigation of the lipids extracted from seeds of Cannabis sativaby GC-MS showed 43 cannabinoids, 16 of which are new. The extract is dominated by ?9-tetrahydrocannabinolic acid (A) and its neutral derivative trans-?9-tetrahydrocannabinol-C5 (THC) Cis and trans-?9-tetrahydrocannabinol-C7 isomers with an ethyl-pentyl branched chain together with minor amounts of trans-?9-tetrahydrocannabinol with a methyl-pentyl C6 branched side chain were identified as new natural compounds. Four cannabichromene isomers with a C5 side chain are postulated to be derived from the double bond migration at the terminal isoprenyl unit. C7 cannabichromene together with the neutral and acidic forms of cannabinol-C7 were also detected. The mass spectrum of these homologues as trimethylsilyl (TMS) derivatives are presented, and the fragmentation patterns are discussed.


Hashish components. Photochemical production of cannabicyclol from cannabichromene.
Crombie, L., Ponsford, R., Shani, A., Yagnitinsky, B., & Mechoulam, R.
Tetrahedron Letters, 9(55), 5771–5772.(1968).
doi:10.1016/s0040-4039(00)76346-5
Revision of the proposed structure for cannabicyclol has been suggested. We now report that cannabichromene (II) 3,495 can be converted into cannabicyclol by photochemical means: this supports the revised structure (I, R = n-C5Hll> and provides a convenient preparative method.
Cannabichromene (750 mg.) in tert. butanol-acetone (l:l, 180 ml.) was irradiated for 41 hr. (450 Watt Hanovia lamp, Pyrex deep well, nitrogen). After chromatography on Florisil (90 g.) cannabicyclol (340 mg.), and recovered chromene (150 mg.), were isolated by elution with light
petroleum containing 1% and 2.5% ether. Crystallised from pentane, the former, m.p. 145-146', was found to be identical (I.R., N.M.R., T.L.C., V.P.C. and mixed m.p.) with cannabicyclol of natural origin.

Hashish—IV.THE ISOLATION AND STRUCTURE OF CANNABINOLIC CANNABIDIOLIC AND CANNABIGEROLIC ACIDS
Mechoulam, R., & Gaoni, Y.
Tetrahedron, 21(5), 1223–1229. (1965).
doi:10.1016/0040-4020(65)80064-3
methyl esters of cannabinolic, cannabidiolic and cannabigerolic acids are shown possess structures VIb, IVb and Ib respectively.

HASHISH-VII’ THE ISOMERIZATION OF CANNABIDIOL TO TETRAHYDROCANNABINOLS
Gaoni, Y., & Mechoulam, R.
Tetrahedron, 22(4), 1481–1488. (1966)
doi:10.1016/s0040-4020(01)99446-3
Depending on the reaction conditions used, the physiologically active products obtained by Adams on isomerization of the inactive cannabidiol (Ia) with acids are shown to be either Al(@) tetrahydrocannabiiol (II) or a mixture of II, At tetrahydmcanna binol (IIIa) and the two isomers of 1 cthoxy hexahydrocanna binol (VIIIa, VIIIb).

Hashish. XII. Stereoelectronic factor in the chloranil dehydrogenation of cannabinoids. Total synthesis of dl-cannabichromene.
Mechoulam, R., Yagnitinsky, B., & Gaoni, Y.
Journal of the American Chemical Society, 90(9), 2418–2420. (1968).
doi:10.1021/ja01011a037
We wish to present evidence suggesting that the dehydrogenation of hydroaromatic compounds by chloranil can be added to the list of reactions in which stereoelectronic factors have been found to influence the course, stereochemistry, and rate. Among these are enolization, protonation and bromination of enols,‘ bimolecular elimination^,^ oxidation of allylic alcohols by manganese dioxide,6 dichlorodicyanoquinone,7 and chromic acid,s dichlorodicyanoquinone dehydrogenation of ketones,g etc. A 1-3,4-trans-Tetrahydrocannabinol (A I-THC) (I) lo is converted into cannabinol (11) in 90% yield on boiling with chloranil in benzene for 2 hr while A1(6)-THC (111) and A '-3,4-cis-THC (VI)lza remain almost unchanged under identical experimental conditions for up to 20 hr. The same applies also to cannabidiol (IV),13 which does not yield the corresponding biphenyl derivative, and to the chroman V, l4 which is not converted into VIII. l6

Hashish—XIII. ON THE NATURE OF THE BEAM TEST
Mechoulam, R., Ben-Zvi, Z., & Gaoni, Y.
Tetrahedron, 24(16), 5615–5624 (1968).
.doi:10.1016/0040-4020(68)88159-1
The purple colour produced by treatment of cannabidiol (I) with 5% etbanolic potassium hydroxide (Beam teat) is due to the anions of the hydroxy-quinont II and its dimcr III. Compounds II and III are formed from I by air oxidation during the reaction. The diquioone III is reduced in the mass spectrometer to a M+ + 4 species (probably the dihydroquinone).

Heterogeneity in the Composition of Marijuana Seized in California
James Richard Burgdorf, Beau Kilmer, Rosalie Liccardo Pacula
Drug Alcohol Depend . 2011 August 1; 117(1): 59–61. doi:10.1016/j.drugalcdep.2010.11.031.
Background—Marijuana contains multiple cannabinoids. Most attention is given to delta-9-tetrahydrocannabinol (THC) which produces euphoria and in some cases anxiety and panic reactions. Research suggests that another cannabinoid, cannabidiol (CBD), may offset some of these effects. Thus, there is growing interest in the health consequences of the THC to CBD ratio
for marijuana.
Methods— Using data from over 5,000 marijuana samples in California from 1996-2008, we examine changes in the median THC-level, median CBD-level, and median THC:CBD-ratio.
Results— The median THC-level and median THC:CBD-ratio has dramatically increased for seizures in California, particularly north of the Mexican border.
Conclusion— Research on the consequences of the THC:CBD ratio should continue, especially as more attention is devoted to thinking about how to regulate marijuana for medical and recreational use. Researchers should also consider the lack of uniformity in the chemical composition of marijuana when evaluating its health effects

HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp.
Tolomeo, F., Russo, F., Vandelli, M. A., Biagini, G., Capriotti, A. L., Laganà, A., … Citti, C.
Journal of Pharmaceutical and Biomedical Analysis, 203, 114215.(2021).
doi:10.1016/j.jpba.2021.114215
A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in “pure” commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01–1.00 g/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.

Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe.
Tsochatzis, E.D.; Alberto Lopes, J.; Holland, M.V.; Reniero, F.; Palmieri, G.; Guillou, C.
Molecules 2021, 26, 793.
doi: 10.3390/molecules26040793
The rapid diffusion of new psychoactive substances (NPS) presents unprecedented challenges to both customs authorities and analytical laboratories involved in their detection and characterization. In this study an analytical approach to the identification and structural elucidation of a novel synthetic cannabimimetic, quinolin-8-yl-3-[(4,4-difluoropiperidin-1-yl) sulfonyl]-4-methylbenzoate (2F-QMPSB), detected in seized herbal material, is detailed. An acid precursor 4-methyl-3-(4,4-difluoro-1-piperidinylsulfonyl) benzoic acid (2F-MPSBA), has also been identified in the same seized material. After extraction from the herbal material the synthetic cannabimimetic, also referred to as synthetic cannabinoid receptor agonists or “synthetic cannabinoids”, was characterized using gas chromatography-mass spectrometry (GC-MS), 1H, 13C, 19F and 15N nuclear magnetic resonance (NMR) and high-resolution tandem mass spectrometry (HR-MS/MS) combined with chromatographic separation. A cheminformatics platform was used to manage and interpret the analytical data from these technique

Identification and Characterization of Cannabimovone, a Cannabinoid from* * Cannabis sativa, as a Novel PPAR?*Agonist via a Combined Computational and Functional Study
Fabio Arturo Iannotti, Fabrizia De Maio, Elisabetta Panza, Giovanni Appendino,* * Orazio Taglialatela?Scafati, Luciano De Petrocellis and Pietro Amodeo and Rosa Maria Vitale
Molecules 2020, 25, 1119;
doi:10.3390/molecules25051119
Phytocannabinoids (pCBs) are a large family of meroterpenoids isolated from the plant Cannabis sativa.*?9?Tetrahydrocannabino l (THC) and cannabidiol (CBD) are the best investigated phytocannabinoids due to their relative abundance and interesting bioactivity profiles. In addition to various targets, THC and CBD are also well?known agonists of peroxisome proliferator?activated receptor gamma (PPAR?), a nuclear receptor involved in energy homeostasis and lipid metabolism. In the search of new pCBs potentially acting as PPAR?* agonists, we identified cannabimovone (CBM), a structurally unique abeo?menthane pCB, as a novel PPAR?*modulator via a combined computational and experimental approach. The ability of CBM to act as dual PPAR?/?*agonist was also evaluated. Computational studies suggested a different binding mode toward the two isoforms, with the compound able to recapitulate the pattern of H?bonds of a canonical agonist only in the case of PPAR?. Luciferase assays confirmed the computational results, showing a selective activation of PPAR?*by CBM in the low micromolar range. CBM promoted the expression of PPAR?* target genes regulating the adipocyte differentiation and prevented palmitate?induced insulin signaling impairment. Altogether, these results candidate CBM as a novel bioactive compound potentially useful for the treatment of insulin resistance?related disorders.

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells
Satoshi Morimoto 1 , Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama and Futoshi Taura
Journal of Biological Chemistry 282, 20739-20751
DOI: 10.1074/jbc.M700133200
Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and ?1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

Identification and Determination of Cannabinoids in both Commercially Available and Cannabis Oils Stored Long Term
Mamoru Yotoriyama, Eiji Ishiharajima, Yoko Kato, Ikuo Yamamoto
Journal of health science 51(4):483-487 August 2005
DOI: 10.1248/jhs.51.483
Among cannabinoids (CNs) in two commercially available and long-term stored cannabis oils obtained from the seeds of Cannabis sativa L. in Japan, tetrahydrocannabinol (THC), canabidiol (CBD), cannabinol (CBN), and cannabichromene were identified and determined using high-performance thin-layer chromatography and capillary gas chromatography-mass speectrometry after partitioning extraction with n-hexane/acetonitrile. CNs were determined in two commercially available cannabis oils, designated "Hemp oil" (THC, 16.3 mu g/g and CBN, 5.9 mu g/g) and "Taima-yu" (CBD, 26.1 mu g/g). The concentration of CBD in a cannabis oil that had been stored for 20 years was 107 4 mu g/g. However, no other CNs including THC were detected in the oil. The CBD content in the original oil was estimated to be about 130 mu g/g from the determination values of the two periods. These results suggest that cannabis oils generally contain CNs to some extent.

Identification and Quantitation of 11 .Nor-AgTetrahydrocannabivarin-9-Carboxylic Acid, a Major Metabolite of Ag-Tetrahydrocannabivarin
Mahmoud A. EISohly, Shixia Feng, Timothy P. Murphy, Anthony W. Warrington, Samir Ross, Allison Nimrod,
Zlatko Mehmedic, and Neil Fortner
DOI: 10.1093/jat/25.6.476
Journal of Analytical Toxicology, Vol. 25, September 2001 After incubation of &9-tetrahydrocannabivarin with human hepatocytes, a major metabolic product was detected by gas chromatography-mass spectrometry that showed identical
retention time and mass spectrum to the synthetic 11-nor-Agtetrahydrocannabivarin-9-carboxylic acid (11-nor-A%THCV-9-
COOH). Analysis of human urine specimens from marijuana users and plasma samples from Marinol | users showed that 11-nor-AgTHCV-9-COOH was only present in urine specimens of marijuana users. These results supported the conclusion that identification of 11-nor-Ag-THCV-9-COOH in a donor's urine specimen indicates the use or ingestion of cannabis-related product(s) and would not explain the sole use of Marinol.

Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol.
Linciano, P., Citti, C., Russo, F., Tolomeo, F., Laganà, A., Capriotti, A. L., … Cannazza, G.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-79042-2
The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and ?9 -tetrahydrocannabinol (?9 -THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and ?9 -THCB) and a heptyl-(in CBDP and ?9 -THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that flls the gap between the pentyl and heptyl homologs of CBD and ?9 -THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and ?9 -tetrahydrocannabihexol (?9 -THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and ?9 -THCH have been already identifed and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and ?9 -tetrahydrocannabinol monomethyl ether (?9-THCM). The unambiguously identifcation in cannabis extract of the n-hexyl homologues of CBD and ?9 -THC diferent from the corresponding methylated isomers (CBDM, CBGM and ?9 -THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantifcation of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of ?9 -THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confrmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and ?9 -THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

Identification of candidate genes affecting ?9-tetrahydrocannabinol biosynthesis in Cannabis sativa.
Marks MD, Tian L, Wenger JP, Omburo SN, Soto-Fuentes W, He J, Gang DR, Weiblen GD, Dixon RA (2009)
J Exp Bot 60: 3715–3726
doi: 10.1093/jxb/erp210
https://www.ncbi.nlm.nih.gov/pmc/art...pdf/erp210.pdf
https://www.ncbi.nlm.nih.gov/pmc/art...pdf/erp210.pdf
RNA isolated from the glands of a D9-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.


Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.
Gagne SJ, Stout JM, Liu E, Boubakir Z, Clark SM, Page JE
Proc Natl Acad Sci USA 109: 12811–12816. (2012)
doi: 10.1073/pnas.1200330109.
!9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The !rst intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from
cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis "owers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase- like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes
requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric "+# barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Identification of Phenotypic Characteristics in Three Chemotype Categories in the Genus Cannabis
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
HortScience 2021
DOI: 10.21273/HORTSCI15607-20
Modern Cannabis cultivars are morphologically distinguished by their leaflet shapes (wide for ‘‘Indica’’ and narrow for ‘‘Sativa’’) by users and breeders. However, there are no scientific bases or references for determining the shape of these leaflets. In addition, these two categories contained mostly THC dominant (high THC) cultivars while excluded CBD dominant (high CBD) and intermediate (intermediate level of both THC and CBD) cultivars. This study investigated the phenotypic variation in 21 Cannabis cultivars covering three chemical phenotypes, referred to as chemotypes, grown in a commercial greenhouse. Thirty morphological traits were measured in the vegetative, flowering, and harvest stages on live plants and harvested inflorescences. The collected data were subjected to correlation analysis, hierarchical clustering, principal component analysis, and canonical correlation analysis with preassigned chemotypes. Canonical correlation analysis assigned individual plants to their chemotypes with 92.9% accuracy. Significant morphological differences were identified. Traits usable as phenotype markers for CBD dominant cultivars included light-green and narrow leaflets, a greater number of primary and secondary serrations, loose inflorescences, dense and resinous trichomes, and Botrytis cinerea resistance. Traits for intermediate cultivars included deep-green and medium-wide leaflets, more primary and secondary serrations, medium compact inflorescences, trichomes that are less dense and less resinous, and Botrytis cinerea resistance. Traits for THC dominant cultivars included deep-green and wide leaflets, large and compact inflorescences, dense and resinous trichomes, and Botrytis cinerea susceptibility. The results of this study provide a comprehensive profile of morphological traits of modern Cannabis cultivars and provides the first such profile for CBD dominant and intermediate cultivars. Additionally, this study included the traits of inflorescences, which have not been compared between three chemotypes in the literature. Phenotype markers identified in this study can facilitate preliminary cultivar identification and selection on live plants before or as a supplement to chemical and genetic analysis.

Identification of terpenoid chemotypes among high (?)-trans-?9-tetrahydrocannabinol-producing Cannabis sativa L. cultivars.
Fischedick JT (2017)
Cannabis Cannabinoid Res 2: 34–47
Doi: 10.1089/can.2016.0040
https://www.researchgate.net/publica...va_L_Cultivars
Introduction: With laws changing around the world regarding the legal status of Cannabis sativa (cannabis) it is important to develop objective classification systems that help explain the chemical variation found among various cultivars. Currently cannabis cultivars are named using obscure and inconsistent nomenclature. Terpenoids, responsible for the aroma of cannabis, are a useful group of compounds for distinguishing cannabis cultivars with similar cannabinoid content.
Methods: In this study we analyzed terpenoid content of cannabis samples obtained from a single medical cannabis dispensary in California over the course of a year. Terpenoids were quantified by gas chromatography with flame ionization detection and peak identification was confirmed with gas chromatography mass spectrometry. Quantitative data from 16 major terpenoids were analyzed using hierarchical clustering analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).
Results: A total of 233 samples representing 30 cultivars were used to develop a classification scheme based on quantitative data, HCA, PCA, and OPLS-DA. Initially cultivars were divided into five major groups, which were subdivided into 13 classes based on differences in terpenoid profile. Different classification models were compared with PLS-DA and found to perform best when many representative samples of a particular class were included.
Conclusion: A hierarchy of terpenoid chemotypes was observed in the data set. Some cultivars fit into distinct chemotypes, whereas others seemed to represent a continuum of chemotypes. This study has demonstrated an approach to classifying cannabis cultivars based on terpenoid profile.

Identification through synthesis of an active .DELTA.1(6)-tetrahydrocannabinol metabolite.
Ben-Zvi, Z., Mechoulam, R., & Burstein, S.
Journal of the American Chemical Society, 92(11), 3468–3469.(1970).
doi:10.1021/ja00714a043
Sir : We recently reported' the isolation and partial elucidation of the structure of a A1(6)-tetrahydrocannabinol (A 1(6j-THC) metabolite obtained from the urine of rabbits injected with A l@)-THC (Ia) tritiated at C-2. On the basis of mass spectral analysis and certain chemical transformations, we deduced that the metabolite is a hydroxylated derivative of Ia. The new hydroxyl group was shown to be allylic, and we tentatively suggested structure IIa. The metabolite was only obtained in minute amounts which were not Ia,R=H b, R = COCHj CHjOR I Ha, R = H c, R = COCH, 5 IIIa, R = H Va,R=H b, R =COCH; b, R = COCH, CH2R I b, R = COCH, VIIa, R = H b,R=OH (1) S. H. Burstein, F. Menezes, E. Williamson, and R. Mechoulam, Nature, 225,87 (1970). sufficient for a nuclear magnetic resonance determination; hence the problem was approached through synthesis. We report now that we have completed the preparation of IIa and have shown that it is identical with the metabolite

Impact of N, P, K, and Humic Acid Supplementation on the Chemical Profile of Medical Cannabis (Cannabis sativa L)
Bernstein Nirit, Jonathan Gorelick, Roei Zerahia, Sraya Koch
June 2019 Frontiers in Plant Science 10
DOI: 10.3389/fpls.2019.00736
Mineral nutrition is a major factor affecting plant growth and function. Increasing evidence supports the involvement of macro and micronutrients in secondary metabolism. The use of the appropriate nutritional measures including organic fertilizers, supplements, and biostimulants is therefore a vital aspect of medicinal plant production including medical cannabis. Due to legal restriction on cannabis research, very little information is available concerning the effects of nutritional supplements on physiological and chemical properties of medical cannabis, and their potential role in standardization of the active compounds in the plant material supplied to patients. This study therefore evaluated the potential of nutritional supplementations, including humic acids (HAs) and inorganic N, P, and K to affect the cannabinoid profile throughout the plant. The plants were exposed to three enhanced nutrition treatments, compared to a commercial control treatment. The nutrition treatments were supplemented with HA, enhanced P fertilization, or enhanced NPK. The results demonstrate sensitivity of cannabinoids metabolism to mineral nutrition. The nutritional supplements affected cannabinoid content in the plants differently. These effects were location and organ specific, and varied between cannabinoids. While the P enhancement treatment did not affect THC, CBD, CBN, and CBG concentrations in the flowers from the top of the plants, a 16% reduction of THC concentration was observed in the inflorescence leaves. Enhanced NPK and HA treatments also produced organ-specific and spatially specific responses in the plant. NPK supplementation increased CBG levels in flowers by 71%, and lowered CBN levels in both flowers and inflorescence leaves by 38 and 36%, respectively. HA was found to reduce the natural spatial variability of all of the cannabinoids studied. However, the increased uniformity came at the expense of the higher levels of cannabinoids at the top of the plants, THC and CBD were reduced by 37 and 39%, respectively. Changes in mineral composition were observed in specific areas of the plants. The results demonstrate that nutritional supplements influence cannabinoid content in cannabis in an organ- and spatial-dependent manner. Most importantly, the results confirm the potential of environmental factors to regulate concentrations of individual cannabinoids in medical cannabis. The identified effects of nutrient supplementation can be further developed for chemical control and standardization in cannabis.

Industrial Hemp: Renewed Opportunities for an Ancient Crop,
John Fike
Critical Reviews in Plant Sciences,
DOI: 10.1080/07352689.2016.1257842
Hemp (Cannabis sativa L.) has been a species of value to humans for much of our history given its broad adaptation and multiple uses. The plant is thought to have originated in Eurasia but has been carried to much of the rest of the world, largely for use as a fiber crop. Declining needs for fiber and competition from other plant fiber sources began to reduce demands for hemp. In turn, concern over psychotropically potent forms of hemp (i.e., marijuana) would lead to the crop’s effective prohibition during much of the 20th century. Growing recognition of the many uses for hemp beyond the traditional rope, cordage, and canvas has helped revive interest in the crop, and a majority of US states have reduced restrictions to allow research with the plant. Although hemp now appears on the verge of returning to favor in the United States, there will be much to learn to make it a viable crop competitive with other commodities. Variety and photoperiodicity, site suitability, end use (grain, fiber, or dual purposes) and management, and the interactions of these factors will have a strong impact on crop productivity and suitability for post-harvest use. In addition, the harvest and processing technologies (particularly for fibers and essential oils) that are needed to optimize the plant’s value are limited or lacking in the United States. Disease and pest issues are often considered of little concern for hemp, but these likely will grow as the plant’s range expands. Opportunities for hemp have increased with the recognition that the crop offers growing and diverse uses for not only its fibers, but for its seed grain and essential oils as well. Several studies indicate that hemp grains are nutritious as feed and food additives and its essential oils are of interest given a number of pharmacologically beneficial properties. Although full of promise given its numerous potential benefits and uses, building markets for these products will be a critical (and likely slow) part of hemp’s development into a useful agronomic species for US growers.

INFLUENCE OF AGROCLIMATIC CONDITIONS ON CONTENT OF MAIN CANNABINOIDS IN INDUSTRIAL HEMP (Cannabis sativa L.)
Vladimir Sikora, Janoš Berenji, Dragana Latkovi? (2011)
DOI: 10.2298/GENSR1103449S
Influence of agroclimatic conditions on content of main cannabinoids in industrial hemp (Cannabis sativa L.)-Genetika, Vol 43, No. 3,449 -456. In a six-year field experiment eight industrial hempvarieties were examined for ? 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) contents. The study analyzed the influence of growing degree days (GDD), soil temperature at 5 cm, air humidity, and growing season precipitation on the levels of the main cannabinoids in this crop.Agroclimatic conditions do not influence THC and CBD contents in industrial hemp in the same way. THC synthesis and accumulation are under the significant positive influence of GDD and air humidity and under the negative influence of precipitation, while soil temperature at 5 cm has no significant effect on it. Soil temperature at 5 cm has a significant positive effect on the CBD content, as do GDD. Precipitation has a negative influence on the CBD content of industrial hemp, while air humidity has no influence on it.

Influence of Altitude on Phytochemical Composition of Hemp Inflorescence: A Metabolomic Approach
Luca Giupponi, Valeria Leoni, Radmila Pavlovic, Anna Giorgi
Molecules 25(6):1381 March 2020
https://www.researchgate.net/publica...lomic_Approach
The phytochemical profiling of hemp inflorescences of clonal plants growing in different conditions related to altitude was investigated. Four strains of industrial hemp (Cannabis sativa L., family Cannabaceae) of Kompolti variety were selected and cloned to provide genetically uniform material for analyses of secondary metabolites (terpenes, cannabinoids, and flavonoids) at two different elevations: mountain (Alagna Valsesia 1200 m ASL) and plains (Vercelli Province 130 m ASL). Environmental conditions influenced by elevation have proven to be important factors inducing variations in hemp inflorescences' secondary metabolite composition. In fact, all plants grown at altitude exhibited a higher total amount of terpenes when compared with plains counterparts, with β-Myrcene, trans-Caryophyllene and α-Humulene as the main contributors. A metabolomic, un-targeted approach performed by HPLC-Q-Exactive-Orbitrap ® -MS platform with subsequent data processing performed by Compound Discoverer™ software, was crucial for the appropriate recognition of many metabolites, clearly distinguishing mountain from plains specimens. Cannabidiolic acid CBDA was the most abundant phytocannabinoid, with significantly higher concentrations in the mountain samples. The metabolic pathway of CBGA (considered as the progenitor/precursor of all cannabinoids) was also activated towards the production of CBCA, which occurs in considerably 3 times higher quantities than in the clones grown at high altitude. Isoprenoid flavones (Cannaflavins A, B, and C) were correspondingly upregulated in mountain samples, while apigenin turned out to be more abundant in plains samples. The possibility to use hemp inflorescences in pharmaceutical/nutraceutical applications opens new challenges to understand how hemp crops respond in terms of secondary metabolite production in various environments. In this regard, our results with the applied analytical strategy may constitute an effective way of phytochemical profiling hemp inflorescences.

Influence of Light Spectra on the Production of Cannabinoids.
Amrein, P., Rinner, S., Pittorino, T., Espel, J., & Schmidmayr, D.
Medical Cannabis and Cannabinoids, 1–8.(2020).
doi:10.1159/000510146
In recent years, more attention has been paid to cannabis from both medical and political points of view. This study investigates the influence of 5 different light spectra on the active substance content in THC-poor hemp of the Alessia chemotype II variety. The focus is on comparing conventional growing under metal halide lamps with growing under high-pressure sodium (HPS) vapor lamps with regard to different spectra of LED lighting modules. Growing was carried out in 10 growing boxes under controlled and mostly identical conditions for all boxes. The photoperiod during the vegetative phase was 18 h light and photosynthetic photon flux density ?520 ?mol?m?2 s?1. The flowering phase was 12 h light and ?540 ?mol?m?2 s?1. During the experiment, CO2, temperature, and humidity were measured and logged. Additionally, weekly measurements of chlorophyll, electric conductivity of the fertilizer, activity measurement (salt content) of the soil, and pH value of the soil were checked. The content of cannabinoids was measured by high-performance liquid chromatography (HPLC). Plant height and growth were monitored during the whole experiment by cameras taking pictures every 30 min and loading them onto a cloud storage platform. Cannabinoid content was measured using HPLC. Plant wet weight was determined at the end of the experiment and showed that plants under the high pressure lamp treatment had less flower weight than those under the LED treatment. In conclusion, it could be shown that certain LED spectra can considerably increase the amount of cannabinoids with respect to conventional illumination (HPS).

Influence of photoperiodism on cannabinoid content of Cannabis sativa L.
Valle JR, Vieira JE, Aucélio JG, Valio IF.
Bull Narc. 1978 Jan-Mar;30(1):67-8.
https://www.unodc.org/unodc/en/data-...1_page008.html
Cannabis sativa plants submitted to 10 and 12 hours of natural light showed different content in cannabinoids. An increase of exposure to natural light of only 2 hours a day, at least, doubled the average amoung of THC, but decreased that of cannabichromene

inhalation of cannabidiol (CBD) in rats.
Javadi-Paydar, M., Creehan, K. M., Kerr, T. M., & Taffe, M. A.
Pharmacology Biochemistry and Behavior, 172741. (2019).
doi:10.1016/j.pbb.2019.172741
Rationale: Cannabidiol (CBD), a compound found in many strains of the Cannabis genus, is increasingly available in e-cigarette liquids as well as other products. CBD use has been promoted for numerous purported benefits which have not been rigorously assessed in preclinical studies. Objective: To further validate an inhalation model to assess CBD effects in the rat. The primary goal was to determine plasma CBD levels after vapor inhalation and compare that with the levels observed after injection. Secondary goals were to determine if hypothermia is produced in male Sprague-Dawley rats and if CBD affects nociception measured by the warm water tail-withdrawal assay. Methods: Blood samples were collected from rats exposed for 30 minutes to vapor generated by an ecigarette device using CBD (100, 400 mg/mL in the propylene glycol vehicle). Separate experiments assessed the body temperature response to CBD in combination with nicotine (30 mg/mL) and the anti-nociceptive response to CBD. Results: Vapor inhalation of CBD produced concentration-related plasma CBD levels in male and female Wistar rats that were within the range of levels produced by 10 or 30 mg/kg, CBD, i.p.. Dose-related hypothermia was produced by CBD in male Sprague-Dawley rats, and nicotine (30 mg/mL) inhalation enhanced this effect. CBD inhalation had no effect on anti-nociception alone or in combination with ? 9 - tetrahydrocannabinol inhalation. Conclusions: The vapor-inhalation approach is a suitable pre-clinical model for the investigation of the effects of inhaled CBD. This route of administration produces hypothermia in rats, while i.p. injection does not, at comparable plasma CBD levels

In planta imaging of ?9-tetrahydrocannabinolic acid in Cannabis sativa L. with hyperspectral coherent anti-Stokes Raman scattering microscopy
Erik T. Garbacik, Roza P. Korai, Eric H. Frater, Jeroen P. Korterik,
Cees Otto, Herman L. Offerhaus
Journal of Biomedical Optics 18(4), 046009 (April 2013)
DOI: 10.1117/1.JBO.18.4.046009
Nature has developed many pathways to produce medicinal products of extraordinary potency and specificity with significantly higher efficiencies than current synthetic methods can achieve. Identification of these mechanisms and their precise locations within plants could substantially increase the yield of a number of natural pharmaceutics. We report label-free imaging of ?9-tetrahydrocannabinolic acid (THCa) in Cannabis sativa L. using coherent anti-Stokes Raman scattering microscopy. In line with previous observations we find high concentrations of THCa in pistillate flowering bodies and relatively low amounts within flowering bracts.
Surprisingly, we find differences in the local morphologies of the THCa-containing bodies: organelles within bracts are large, diffuse, and spheroidal, whereas in pistillate flowers they are generally compact, dense, and have
heterogeneous structures. We have also identified two distinct vibrational signatures associated with THCa, both in pure crystalline form and within Cannabis plants; at present the exact natures of these spectra remain an open question.

In Situ Decarboxylation-Pressurized Hot Water Extraction for Selective Extraction of Cannabinoids from Cannabis sativa. Chemometric Approach
Yannick Nuapia, Kgomotso Maraba, Hlanganani Tutu, Luke Chimuka and Ewa Cukrowska
Molecules 2021, 26, 3343.
DOI: 10.3390/ molecules26113343
https://www.researchgate.net/publica...etric_Approach
Isolation of the therapeutic cannabinoid compounds from Cannabis Sativa L. (C. Sativa) is important for the development of cannabis-based pharmaceuticals for cancer treatment, among other ailments. The main pharmacological cannabinoids are THC and CBD. However, THC also induces undesirable psychoactive effects. The decarboxylation process converts the naturally occurring acidic forms of cannabinoids, such as cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA), to their more active neutral forms, known as cannabidiol (CBD) and tetrahydrocannabinol (THC). The purpose of this study was to selectively extract cannabinoids using a novel in situ decarboxylation pressurized hot water extraction (PHWE) system. The decarboxylation step was evaluated at different temperature (80–150 ◦C) and time (5–60 min) settings to obtain the optimal conditions for the decarboxylation-PHWE system using response surface methodology (RSM). The system was optimized to produce cannabis extracts with high CBD content, while suppressing the THC and CBN content. The identification and quantification of cannabinoid compounds were determined using UHPLC-MS/MS with external calibration. As a result, the RSM has shown good predictive capability with a p-value < 0.05, and the chosen parameters revealed to have a significant effect on the CBD, CBN and THC content. The optimal decarboxylation conditions for an extract richer in CBD than THC were set at 149.9 ◦C and 42 min as decarboxylation temperature and decarboxylation time, respectively. The extraction recoveries ranged between 96.56 and 103.42%, 95.22 and 99.95%, 99.62 and 99.81% for CBD, CBN and THC, respectively.

Introduction to Recent Advances in Cannabinoid Research Robert B Laprairie and Will Costain in Book, Recent Advances in Cannabinoid Research (2019) https://www.intechopen.com/chapters/66581
https://www.intechopen.com/books/7040
DOI: 10.5772/interchopen.85814 https://www.researchgate.net/publica...inoid_Research
On October 17, 2018, Canada became the first G20 nation to legalize the use of Cannabis sativa for both medicinal and recreational purposes. This change in legislation and end of prohibition are indicative of a larger global movement to understand Cannabis—and the bioactive chemicals present within Cannabis known as the cannabinoids—for its potential
biomedical uses, harms, and economic values. Currently, interest in Cannabis and canna binoid research is surging as the many knowledge gaps in basic biology, pharmacology, epidemiology, and clinical efficacy are identified. The purpose of this book is to summarize some leading areas of research in the cannabinoid field where knowledge gaps have
been or are being actively addressed. The research described herein spans between basic biological and clinical research. As the editors of this text, we are grateful to the work.

Investigation of mechanism of drug-induced cardiac injury and torsades de pointes in cynomolgus monkeys
DL Misner, C Frantz, L Guo, MR Gralinski, PB Senese, J Ly,
M Albassam and KL Kolaja
British Journal of Pharmacology (2012) 165 2771–2786 2771
DOI:10.1111/j.1476-5381.2011.01756.x
BACKGROUND AND PURPOSE
Drug candidates must be thoroughly investigated for their potential cardiac side effects. During the course of routine toxicological assessment, the compound RO5657, a CCR5 antagonist, was discovered to have the rare liability of inducing torsades de pointes (polymorphic ventricular arrhythmia) in normal, healthy animals. Studies were conducted to determine the molecular mechanism of this arrhythmia.
EXPERIMENTAL APPROACH
Toxicological effects of repeat dosing were assessed in naïve monkeys. Cardiovascular effects were determined in conscious telemetry-implanted monkeys (repeat dosing) and anaesthetized instrumented dogs (single doses). Mechanistic studies were performed in guinea-pig isolated hearts and in cells recombinantly expressing human cardiac channels.
KEY RESULTS
In cynomolgus monkeys, RO5657 caused a low incidence of myocardial degeneration and a greater incidence of ECG abnormalities including prolonged QT/QTc intervals, QRS complex widening and supraventricular tachycardia. In telemetry-implanted monkeys, RO5657 induced arrhythmias, including torsades de pointes and in one instance, degeneration to fatal ventricular fibrillation. RO5657 also depressed both heart rate (HR) and blood pressure (BP), with no histological evidence of myocardial degeneration. In the anaesthetized dog and guinea-pig isolated heart studies, RO5657 induced similar cardiovascular effects. RO5657 also inhibited Kv11.1 and sodium channel currents.
CONCLUSIONS AND IMPLICATIONS
The molecular mechanism of RO5657 is hypothesized to be due to inhibition of cardiac sodium and Kv11.1 potassium channels. These results indicate that RO5657 is arrhythymogenic due to decreased haemodynamic function (HR/BP), decreased conduction and inhibition of multiple cardiac channels, which precede and are probably the causative factors in the observed myocardial degeneration.

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa.
Zagzoog, A., Mohamed, K. A., Kim, H. J. J., Kim, E. D., Frank, C. S., Black, T., … Laprairie, R. B.
Scientific Reports, 10(1).(2020).
doi:10.1038/s41598-020-77175-y
The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of ?9 -tetrahydrocannabinol (?9 -THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent efects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by ?9 -THC is known to bring about the ‘high’ associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating efects that are potentially therapeutic. CB2R activation by certain cannabinoids has been associated with anti-infammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifcally, ?9 -THC, ?9 -tetrahydrocannabinolic acid (?9 - THCa), ?9 -tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their afnity to receptors, ability to inhibit cAMP accumulation, ?arrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic efects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis-derived molecules.

Iodine-Promoted Aromatization of p?Menthane-Type Phytocannabinoids
Federica Pollastro, Diego Caprioglio, Patrizia Marotta, Aniello Schiano Moriello, Luciano De Petrocellis, Orazio Taglialatela-Scafati, and Giovanni Appendino
J. Nat. Prod. 2018 Mar 23;81(3):630-633.
DOI: 10.1021/acs.jnatprod.7b00946
Treatment with iodine cleanly converts various p menthane-type phytocannabinoids and their carboxylated precursors into cannabinol (CBN, 1a ). The reaction is superior to previously reported protocols in terms of simplicity and substrate range, which includes not only tricyclic tetrahydrocannabinols such as ?9 -THC (2a ) but also bicyclic phytocannabinoids such as cannabidiol (CBD, 3a ). Lower homologues from the viridin series (2c and 3c , respectively) afforded cannabivarin (CBV), a non-narcotic compound that, when investigated against a series of ionotropic (thermo-TRPs) biological endpoints of phytocannabinoids, retained the submicromolar TRPA1- activating and TRPM8-inhibiting properties of CBN, while also potently activating TRPV2. Treatment with iodine provides an easy access to CBN (1a ) from crude extracts and side-cuts of the purification of ?9 -THC and CBD from respectively narcotic Cannabis sativa (marijuana) and
fiber hemp, substantially expanding the availability of this compound and, in the case of fiber hemp, dissecting it from narcotic phytocannabinoids.

Isolation and Pharmacological Evaluation of Minor Cannabinoids from High-Potency Cannabis sativa.
Radwan MM, ElSohly MA, El-Alfy AT, Ahmed SA, Slade D, Husni AS, Manly SP, Wilson L, Seale S, Cutler SJ, Ross SA
J Nat Prod.
doi: 10.1021/acs.jnatprod.5b00065
Seven new naturally occurring hydroxylated cannabinoids (1-7), along with the known cannabiripsol (8), have been isolated from the aerial parts of high-potency Cannabis sativa. The structures of the new compounds were determined by 1D and 2D NMR spectroscopic analysis, GC-MS, and HRESIMS as 8?-hydroxy-?9-tetrahydrocannabinol (1), 8?-hydroxy-?9-tetrahydrocannabinol (2), 10?-hydroxy-?8-tetrahydrocannabinol (3), 10?-hydroxy-?8-tetrahydrocannabinol (4), 10?-hydroxy-?9,11-hexahydrocannabinol (5), 9?,10?-epoxyhexahydrocannabinol (6), and 11-acetoxy-?9-tetrahydrocannabinolic acid A (7). The binding affinity of isolated compounds 1-8, ?9-tetrahydrocannabinol, and ?8-tetrahydrocannabinol toward CB1 and CB2 receptors as well as their behavioral effects in a mouse tetrad assay were studied. The results indicated that compound 3, with the highest affinity to the CB1 receptors, exerted the most potent cannabimimetic-like actions in the tetrad assay, while compound 4 showed partial cannabimimetic actions. Compound 2, on the other hand, displayed a dose-dependent hypolocomotive effect only.

Isolation of a high affinity cannabinoid for human CB1 receptor from a medicinal cannabis variety: D9-Tetrahydrocannabutol, the butyl homologue of D9-tetrahydrocannabinol.
Linciano, P., Citti, C., Luongo, L., Belardo, C., Maione, S., Vandelli, M. A., … Cannazza, G.
Journal of Natural Products. (2019).
doi:10.1021/acs.jnatprod.9b00876
The butyl homologues of ?9 -tetrahydrocannabinol, ?9 -tetrahydrocannabutol (?9 -THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of ?9 -THC, ?9 -THCB, showed an affinity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (?)-trans-?9 -THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on ?9 -THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of ?9 -THCB toward the CB1 receptor.


Isolation of Cannabisativine, an Alkaloid, from Cannabis sativa L. Root.
Turner, C. E., Hsu, M. H., Knapp, J. E., Schiff, P. L., & Slatkin, D. J.
Journal of Pharmaceutical Sciences, 65(7), 1084–1085.(1976)
doi:10.1002/jps.2600650736
An ethanol extract of the root of a Mexican variant of Cannabis satiua L. (marijuana) afforded, after partitioning and chromatography, the new spermidine alkaloid cannabisativine.

Isolation, Structure, and Partial Synthesis of an Active Constituent of Hashish.
Gaoni, Y., & Mechoulam, R.
Journal of the American Chemical Society, 86(8), 1646–1647.(1964).
doi:10.1021/ja01062a046
Hashish (marihuana), the psychotomimetically active resin of the female flowering tops of Cannabis sativa L. is one of the most widely used illicit narcotic drugs. A number of groups have reported the isolation of active constituents.2 Most of these substances are not fully characterized, and comparisons with or between them are difficult. We now wish to report the isolation of an active constituent of hashish to which we assign structure I ( 1- 3,4-iraws-tetrahydrocannabinol).3 This is the first active component whose constitution is fully elucidated.

Isomers of THC
WHO Expert Committee on Drug Dependence Critical Review
https://www.who.int/medicines/access...rsTHC.pdf?ua=1
This is an advance copy distributed to the participants of the 41st Expert Committee on Drug Dependence, before it has been formally published by the World Health Organization. The document may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in part or in whole, in any form or by any means without the permission of the World Health Organization. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. The World Health Organization does not warrant that the information contained in this publication is complete and correct and shall not be liable for any damages incurred as a result of its use.

It Is Our Turn to Get Cannabis High: Put Cannabinoids in Food and Health Baskets.
Salami, S. A., Martinelli, F., Giovino, A., Bachari, A., Arad, N., & Mantri, N.
Molecules, 25(18), 4036. (2020).
doi:10.3390/molecules25184036
Cannabis is an annual plant with a long history of use as food, feed, fiber, oil, medicine, and narcotics. Despite realizing its true value, it has not yet found its true place. Cannabis has had a long history with many ups and downs, and now it is our turn to promote it. Cannabis contains approximately 600 identified and many yet unidentified potentially useful compounds. Cannabinoids, phenolic compounds, terpenoids, and alkaloids are some of the secondary metabolites present in cannabis. However, among a plethora of unique chemical compounds found in this plant, the most important ones are phytocannabinoids (PCs). Over hundreds of 21-22-carbon compounds exclusively produce in cannabis glandular hairs through either polyketide and or deoxyxylulose phosphate/methylerythritol phosphate (DOXP/MEP) pathways. Trans-?9-tetrahydrocannabinol (?9-THC) and cannabidiol (CBD) are those that first come to mind while talking about cannabis. Nevertheless, despite the low concentration, cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabinodiol (CBND), and cannabinidiol (CBDL) may have potentially some medical effects. PCs and endocannabinoids (ECs) mediate their effects mainly through CB1 and CB2 receptors. Despite all concerns regarding cannabis, nobody can ignore the use of cannabinoids as promising tonic, analgesic, antipyretic, antiemetic, anti-inflammatory, anti-epileptic, anticancer agents, which are effective for pain relief, depression, anxiety, sleep disorders, nausea and vomiting, multiple sclerosis, cardiovascular disorders, and appetite stimulation. The scientific community and public society have now increasingly accepted cannabis specifically hemp as much more than a recreational drug. There are growing demands for cannabinoids, mainly CBD, with many diverse therapeutic and nutritional properties in veterinary or human medicine. The main objective of this review article is to historically summarize findings concerning cannabinoids, mainly THC and CBD, towards putting these valuable compounds into food, feed and health baskets and current and future trends in the consumption of products derived from cannabis.

Itudinal Adaptation precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.
Stout JM, Boubakir Z, Ambrose SJ, Purves RW, Page JE (2012
Plant J 71: 353–365
doi: 10.1111/j.1365-313X.2012.04949.x.
The psychoactive and analgesic cannabinoids (e.g. D9 -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g)1 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

Keep Off the Grass: Artisanal versus Pharmaceutical Cannabidiol in Pediatric Refractory Epilepsy Patients (710)
Nathan Cohen, Joan Conry, John Schreiber
American Academy of Neurology, April 14, 2020; 94 (15 Supplement)
https://n.neurology.org/content/94/1...t/710.abstract
Objective:*We hypothesize that cannabidiol (CBD) levels will be higher in patients taking pharmaceutical versus artisanal CBD and higher CBD levels will be associated with increased side effects and decreased seizure frequency.
Background:*CBD is a neuroactive Cannabis-derivative with antiseizure properties. Pharmaceutical CBD was FDA-approved for the management of seizures associated with Lennox-Gastaut syndrome (LGS) and Dravet syndrome in June 2018 but artisanal CBD has utilized in various preparations since the late 1970s. These CBD preparations have varying manufacturing techniques and contain variable amounts of CBD/THC. It is our practice, when appropriate, to encourage transition from artisanal to pharmaceutical CBD.
Design/Methods:*This is an IRB-approved retrospective chart review. We included patients with epilepsy treated with artisanal and/or pharmaceutical CBD who had serum CBD levels. Patients were identified by treating neurologists.
We recorded name, medical record number, date of birth, gender, epilepsy diagnosis, artisanal CBD dosage/concentration, pharmaceutical CBD dose, serum CBD level, seizure history, response to medication (percent reduction in motor seizures), and reported side effects.
Results:*31 patients met inclusion criteria, 48% female, mean age 10.1-years-old (range 3-20-years-old). Epilepsy syndromes were LGS 32%, Dravet 6%, and other epilepsy syndromes 62%. This includes 22 patients participating in a pharmaceutical CBD expanded access program from 9/2017-3/2019. Mean serum CBD level was 30.1 ng/mL in artisanal group and 124 ng/mL in the pharmaceutical group. At last follow-up (median follow-up 11.8 months (IQR 3.6-17.5 months), patients on artisanal CBD had 70% increase in overall seizures, while prescription CBD group had 39% reduction. 11 patients reported adverse effects (somnolence, emesis, diarrhea, diminished appetite), 6 of whom discontinued CBD due to side effects (all were in the prescription CBD group).
Conclusions:*Pharmaceutical CBD achieves higher serum CBD levels and achieves better seizure control than artisanal CBD in refractory pediatric epilepsy patients. This group also reported increased adverse side effects.
The use of medical cannabis to treat various medical conditions has grown in recent years. While not always legal, artisanal CBD has been available longer, so some people have been using it to treat epilepsy for years," study author*Nathan T. Cohen, M.D.,*says. "They may want to reconsider because our research indicates that pharmaceutical CBD may indeed be more effective than artisanal CBD."
The use of medical cannabis to treat various medical conditions has grown in recent years. While not always legal, artisanal CBD has been available longer, so some people have been using it to treat epilepsy for years," study author*Nathan T. Cohen, M.D.,*says. "They may want to reconsider because our research indicates that pharmaceutical CBD may indeed be more effective than artisanal CBD."

Kinetics of CBD, Δ 9-THC Degradation and Cannabinol Formation in Cannabis Resin at Various Temperature and pH Conditions
Wuttichai Jaidee , Ittipon Siridechakorn , Siwames Nessopa , Vanuchawan Wisuitiprot , Nathareen Chaiwangrach , Kornkanok Ingkaninan , Neti Waranuch
Cannabis Cannabinoid Res 2021 Jun 4.
doi: 10.1089/can.2021.0004
https://www.liebertpub.com/doi/pdf/1.../can.2021.0004
Cannabidiol (CBD), cannabinol (CBN), and Δ9-tetrahydrocannabinol (Δ9-THC) are major cannabinoids in cannabis resin and products. The kinetic of the chemical reaction of resin cannabis is important for product development and storage. A few reports are available in the literature on the rate of CBD and Δ9-THC degradation, and CBN formation in dried resin and solutions of various pH. Materials and Methods: Thermal degradation of CBD, Δ9-THC, and formation of CBN was studied at 50°C, 60°C, 70°C, and 80°C for dried cannabis resin. The effect of pH and temperature on cannabinoids transformation in cannabis solution was also examined at pH 2, 4, 6, 8, 10, and 12 and at 40°C, 50°C, 60°C, and 70°C. High-performance chromatography coupled with diode-array detection (HPLC-DAD) was used for the analysis of CBD, CBN, and Δ9-THC transformation. The values of activation energies (Ea), shelf-life (t90% - t110%), and rate constant (k) were calculated for the CBD, Δ9-THC, and CBN. The effect of temperature and pH on the dried cannabis resin was adequately modeled with the Arrhenius equation. Results: The results indicated that the chemical kinetics in the thermal degradation of CBD, Δ9-THC, and formation of CBN were the zero-order, pseudo-zero-order, and first-order reactions, respectively, in cannabis resin. The first-order and pseudo-first-order degradation kinetics were evidenced for CBD and Δ9-THC, respectively, in cannabis solutions, whereas the zero-order formation kinetic was detected for the CBN. The transformation rate of the CBD, CBN, and Δ9-THC increased with increasing temperature, especially as temperature increased to 70°C at pH 2.0. The optimum pH for CBD stability was between pH 4 and 6, whereas the optimum pH for Δ9-THC stability was between pH 4 and 12. Conclusion: The major cannabinoids (CBD, CBN, and Δ9-THC) reacted more quickly at high temperature and in an acidic solution. Especially, the minimum transformation of CBD, CBN, and Δ9-THC was achieved by using on a low temperature, slightly to moderately acidic pH values, and short-time processing. These results may help to improve the storage condition of CBD, CBN, and Δ9-THC products and in the manufacturing process.
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Letter to the Editor: On the Reluctant Decarboxylation of THCA-B

Crist N. Filer
Cannabis and Cannabinoid Research 2021
DOI: 10.1089/can.2021.0003
https://www.liebertpub.com/doi/pdf/1...?download=true
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Letter to the Editor: The Controversial Cannabidiol Cyclization: Its Conceptual Origin
Crist N. Filer
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0099
The acid catalyzed cyclization of cannabidiol (through its isopropenyl group) to both delta-9-tetrahydrocannabinol and its more thermodynamically stable olefin isomer delta-8-tetrahydrocannabinol has been known for 80 years. This reaction has also prompted recent controversy with the suggestion and detailed rebuttal1 that this same chemistry might occur during human oral ingestion of cannabidiol. Although its synthetic utility is diminished because of its nature as a product mixture, this cyclization is still unique and fascinating in that nonintoxicating cannabidiol is directly converted to several intoxicating cannabinoids. Only rarely in medicinal chemistry does a single step bond-forming reaction accomplish such a dramatic pharmacological transformation. It is widely acknowledged2 that the first experimental report of the cannabidiol cyclization can be attributed to natural products chemist Roger Adams at the University of Illinois in 1940 during a prolific program of Cannabis chemistry exploration.3 Remarkably, Adams also described this transformation with only an incomplete structural understanding of the substances involved.

Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice
Sabine Steffens, Niels R. Veillard, Claire Arnaud, Graziano Pelli, Fabienne Burger, Christian Staub, Andreas Zimmer, Jean-Louis Frossard & François Mach
Nature volume 434, pages782–786 (2005)
DOI: 10.1038/nature03389
Atherosclerosis is a chronic inflammatory disease, and is the primary cause of heart disease and stroke in Western countries1. Derivatives of cannabinoids such as delta-9-tetrahydrocannabinol (THC) modulate immune functions2 and therefore have potential for the treatment of inflammatory diseases. We investigated the effects of THC in a murine model of established atherosclerosis. Oral administration of THC (1?mg?kg-1 per day) resulted in significant inhibition of disease progression. This effective dose is lower than the dose usually associated with psychotropic effects of THC. Furthermore, we detected the CB2 receptor (the main cannabinoid receptor expressed on immune cells2,3) in both human and mouse atherosclerotic plaques. Lymphoid cells isolated from THC-treated mice showed diminished proliferation capacity and decreased interferon-? secretion. Macrophage chemotaxis, which is a crucial step for the development of atherosclerosis1, was also inhibited in vitro by THC. All these effects were completely blocked by a specific CB2 receptor antagonist4. Our data demonstrate that oral treatment with a low dose of THC inhibits atherosclerosis progression in the apolipoprotein E knockout mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, THC or cannabinoids with activity at the CB2 receptor may be valuable targets for treating atherosclerosis.

Managing Plant Nutrition to Produce Elevated Cannabinoids
Ozadia Plant Science Sap & Leaf Analysis

In this webinar, Ozadia Plant Science Chief Science Officer, John Kempf, discusses the important factors surrounding cannabinoid levels, how to increase them with proper plant nutrition, and their relationships with microbial organisms and their environments. John and the listeners have a Q&A session at the end of the video that provides additional insights to compliment the webinar. Please watch this video to learn more about these topics and how the Ozadia system for holistic plant nutrition can help you!

Marijuana and Paraquat.
Turner, C. E.
JAMA: The Journal of the American Medical Association, 240(17), 1857.(1978)
doi:10.1001/jama.1978.03290170039017
Becauseof the news coverage of paraquat and marijuana, we wish to (1) report some data on Cannabis sativa L contaminated with the herbicide paraquat (1,1'dimethyl-4,4 ' -dipyridilium dichloride) and (2) present some facts regarding marijuana with or without paraquat that will be useful to the medical science community. In late 1975 Mexico initiated a program to eradicate cannabis by spraying illicit fields with aqueous paraquat.' In theory this was subsequently to reduce the flow of crude drugs from cannabis. This program was intensified in 1976. It is estimated that 36 million Americans have tried marijuana2; thus, any agent used chemically to control cannabis is important to a large segment of our society and medical scientists. In September 1977 we were requested by the National Institute of Drug Abuse (NIDA) to screen confiscated Mexican cannabis for paraquat. To this end, 100 samples of kilogram bricks of cannabis confiscated after Jan 1, 1976, were analyzed quantita¬ tively for paraquat using the proce¬ dure recommended by Chevron Chem¬ ical Co, Ortho Division. With this method paraquat can be determined at concentration levels down to 1 ppm. Thirteen of the 100 samples con¬ tained paraquat, the identity of which was carried out by isolation, spectral analyses, and comparison with au¬ thentic paraquat. Fifty-four samples were confiscated in 1976, and four (7.4%) were positive. Forty-six sam¬ ples were confiscated in 1977, with nine (19.56%) testing positive. All positive samples were confiscated after Oct 20, 1976. (The total number of samples confiscated after this date was 61.) Therefore, it can be esti¬ mated that after Oct 20, 1976, the possibility of a Mexican-grown mari¬ juana sample containing 1 ppm or more of paraquat is 13 out of 61 (21.31%). It can not be said, however, that current street marijuana will be positive for paraquat 21.31% of the time. Samples analyzed by our group were from seized amounts of more than 90 kg, and none of this material ever reached the street. All the para¬ quat-contaminated samples were seized in California, Arizona, and Texas, and the paraquat concentra¬ tion within these samples ranged from 3.3 to 2,264 ppm. If all material from the 100 confis¬ cated samples (4,122.635 kg) had reached the street, 4,122,635 cigarettes containing approximately 1 g of marijuana could have been prepared. The normalized paraquat con¬ tent would be 0.347 mg per gram cigarette. Exact data are not available on the absolute amount of paraquat that will survive the smoking process. However, based on preliminary data from within NIDA research groups, we can say 96% of the paraquat decomposes to bipyridine, and some detectable paraquat is in the remaining 4%.3 Exactly how much paraquat would be available to the lung tissue is not known, and it is therefore impossible to state categorically the level of paraquat in marijuana that would cause lung problems

Marijuana Use by Breastfeeding Mothers and Cannabinoid Concentrations in Breast Milk.
Bertrand, K. A., Hanan, N. J., Honerkamp-Smith, G., Best, B. M., & Chambers, C. D.
Pediatrics, e20181076. (2018).
doi:10.1542/peds.2018-1076
https://www.researchgate.net/publica...in_Breast_Milk
BACKGROUND AND OBJECTIVE: Marijuana is the most commonly used recreational drug among breastfeeding women. With legalization of marijuana in several US states and a 1990 study in which authors documented psychomotor deficits in infants breastfed by mothers using marijuana, there is a need for information on potential exposure to the breastfed infant. Our objective with this study was to quantify cannabinoids in human milk after maternal marijuana use. Methods: Between 2014 and 2017, 50 breastfeeding women who reported marijuana use provided 54 breast milk samples to a research repository, Mommy's Milk. Concentrations of ?-9-tetrahydrocannabinol (?9-THC), 11-hydroxy-?-9-tetrahydrocannabinol, cannabidiol, and cannabinol were measured by using liquid chromatography mass spectrometry electrospray ionization. Results: ?9-THC was detectable in 34 (63%) of the 54 samples up to ?6 days after last reported use; the median concentration of ?9-THC was 9.47 ng/mL (range: 1.01-323.00). Five samples had detectable levels of 11-hydroxy-?-9-tetrahydrocannabinol (range: 1.33-12.80 ng/mL) or cannabidiol (range: 1.32-8.56 ng/mL). The sample with the highest concentration of cannabidiol (8.56 ng/mL) did not have measurable ?9-THC. Cannabinol was not detected in any samples. The number of hours since last use was a significant predictor of log ?9-THC concentrations (-0.03; 95% confidence interval [CI] -0.04 to -0.01; P = .005). Adjusted for time since last use, the number of daily uses and time from sample collection to analysis were also significant predictors of log ?9-THC concentrations (0.51; 95% CI 0.03 to 0.99; P = .039; 0.08; 95% CI 0.00 to 0.15; P = .038, respectively). Conclusions: ?9-THC was measurable in a majority of breast milk samples up to ?6 days after maternal marijuana use.

Medicinal cannabis: Principal cannabinoids concentration and their stability evaluated by a high performance liquid chromatography coupled to diode array and quadrupole time of flight mass spectrometry method.
Citti, C., Ciccarella, G., Braghiroli, D., Parenti, C., Vandelli, M. A., & Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis, 128, 201–209. 2016).*
doi:10.1016/j.jpba.2016.05.033*
In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UVdetection (HPLC-UV)for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabisbased medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs


Medicinal chemistry endeavors around the phytocannabinoids.
Stern E, Lambert DM.
Chem Biodivers. 2007 Aug;4(8):1707-28.
DOI:10.1002/cbdv.200790149
Over the past 50 years, a considerable research in medicinal chemistry has been carried out around the natural constituents of Cannabis sativa L. Following the identification of Delta9-tetrahydrocannabinol (Delta9-THC) in 1964, critical chemical modifications, e.g., variation of the side chain at C3 and the opening of the tricyclic scaffold, have led to the characterization of potent and cannabinoid receptor subtype-selective ligands. Those ligands that demonstrate high affinity for the cannabinoid receptors and good biological efficacy are still used as powerful pharmacological tools. This review summarizes past as well as recent developments in the structure-activity relationships of phytocannabinoids.

Measuring the bioactivity of phytocannabinoid cannabidiol from cannabis sources, and a novel non-cannabis source.
D. Cushing, S. Kristipati, R. Shastri, & B. Joseph
Journal of Medical Phyto Research Vol. 1, Article 2, (8-23)
Doi: 10.31013/1002b
Phytocannabinoid Cannabidiol (CBD) has been shown to elicit a great many immunological benefits. It acts on the endocannabinoid system, namely through interactions with cannabinoid receptor 2 (CB2). CBD-CB2 affinity, which we refer to as bioactivity, is rarely tested for clinical samples. We believe that uncontrolled variation in bioactivity levels have been silently confounding many CBD experiments. In our four-part study, we validate an efficient bioactivity test that can enable greater scientific control over CBD studies. We use it to compare the bioactivity of CBD obtained from different plant organs, and we also studied whether processing methods play a role in determining bioactivity. We also examine the bioactivity and processing factors of a novel non-cannabis plant capable of producing CBD in commercial quantities, named Humulus Kriya (H. Kriya, U.S. Patent No. 15/932,529, 2018). We also test the bioactivity of some CBD isolates/extracts currently sold in the market, and compare them with a CBD product called ImmunAG, which was extracted from the inflorescence of H. Kriya. We find that the CBD from the inflorescence of the plant produces the highest bioactivity, followed by the apical buds/leaves, the petioles, and finally the stalk. We find that H. Kriya has a bioactivity profile similar to Cannabis Sativa. We find that the bioactivity levels among cannabis-based commercial CBD products are quite low, and variable. We find significantly higher bioactivity levels in ImmunAG.

Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates
Joseph Molnar, D Szabó, Rozalia Pusztai Yukihiro Shoyama
Anticancer research 20(2A):861-7 March 2000
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in the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Membrane Filtration for Cannabis Extracts
Colby McCoy
Extraction Magazine
https://extractionmagazine.com/2021/...abis-extracts/
Most extracts undergo winterization, which strips away lipids from the final product. Extracts then undergo a color removal process to provide a desirable amber hue by removing chlorophyll. Extractors may use distillation or chromatography to further refine concentrates. This process can be both long and complicated.
That said, there is another solution for post-processing of extracts. Enter membrane filtration, also known as organic solvent nanofiltration (OSN), which streamlines the filtration process. According to Buonomenna and Bae [1], OSN “allows separations of organic mixtures down to a molecular level by simply applying a pressure gradient over a membrane and recovery with possibility of reuse of the organic solvents.” Membrane filters are physical barriers with pores sized so small that pressure must be applied to push liquid through for filtration.

Metabolic fingerprinting of Cannabis sativa L. cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes.
Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R (2010)
Phytochemistry 71: 2058–2073
doi: 10.1016/j.phytochem.2010.10.001
Cannabis sativa L. is an important medicinal plant. In order to develop cannabis plant material as a medicinal product quality control and clear chemotaxonomic discrimination between varieties is a necessity. Therefore in this study 11 cannabis varieties were grown under the same environmental conditions. Chemical analysis of cannabis plant material used a gas chromatography flame ionization detection method that was validated for quantitative analysis of cannabis monoterpenoids, sesquiterpenoids, and cannabinoids. Quantitative data was analyzed using principal component analysis to determine which compounds are most important in discriminating cannabis varieties. In total 36 compounds were identified and quantified in the 11 varieties. Using principal component analysis each cannabis variety could be chemically discriminated. This methodology is useful for both chemotaxonomic discrimination of cannabis varieties and quality control of plant material.

Metabolism of ?1(6)-Tetrahydro-cannabinol, an Active Marihuana Constituent.
BURSTEIN, S. H., MENEZES, F., WILLIAMSON, E., & MECHOULAM, R.
Nature, 225(5227), 87–88.(1970).
doi:10.1038/225087a0
A METHOD for preparing specifically tritiated (—)-?1(6)-tetrahydrocannabinol (I) was recently developed in our laboratories1. We would now like to report some of the results we have obtained in studying the metabolic fate of this material in the rabbit. Several publications dealing with the metabolism of cannabinoids have appeared2,3 in which other species were used, but none of the metabolites was identified. Moreover, in some cases the methods used to obtain the labelled cannabinoids would not yield radiochemically pure substances.

Metabolites of cannabidiol identified in human urine.
Harvey DJ1, Mechoulam R.
Xenobiotica. 1990 Mar;20(3):303-20.
DOI: 10.3109/00498259009046849
1. Urine from a dystonic patient treated with cannabidiol (CBD) was examined by g.l.c.-mass spectrometry for CBD metabolites. Metabolites were identified as their trimethylsilyl (TMS), [2H9]TMS, and methyl ester/TMS derivatives and as the TMS derivatives of the product of lithium aluminium deuteride reduction. 2. Thirty-three metabolites were identified in addition to unmetabolized CBD, and a further four metabolites were partially characterized. 3. The major metabolic route was hydroxylation and oxidation at C-7 followed by further hydroxylation in the pentyl and propenyl groups to give 1"-, 2"-, 3"-, 4"- and 10-hydroxy derivatives of CBD-7-oic acid. Other metabolites, mainly acids, were formed by beta-oxidation and related biotransformations from the pentyl side-chain and these were also hydroxylated at C-6 or C-7. The major oxidized metabolite was CBD-7-oic acid containing a hydroxyethyl side-chain. 4. Two 8,9-dihydroxy compounds, presumably derived from the corresponding epoxide were identified. 5. Also present were several cyclized cannabinoids including delta-6- and delta-1-tetrahydrocannabinol and cannabinol. 6. This is the first metabolic study of CBD in humans; most observed metabolic routes were typical of those found for CBD and related cannabinoids in other species.

Microbial metabolism of cannflavin A and B isolated from Cannabis sativa.
Ibrahim, A. K., Radwan, M. M., Ahmed, S. A., Slade, D., Ross, S. A., ElSohly, M. A., & Khan, I. A.
Phytochemistry, 71(8-9), 1014-1019. (2010).
doi:10.1016/j.phytochem.2010.02.011
Microbial metabolism of cannflavin A (1) and B (2), two biologically active flavonoids isolated from Cannabis sativa L., produced five metabolites (3–7). Incubation of 1 and 2 with Mucor ramannianus (ATCC 9628) and Beauveria bassiana (ATCC 13144), respectively, yielded 600S,700-dihydroxycannflavin A (3), 600S,700-dihydroxycannflavin A 7-sulfate (4) and 600S,700-dihydroxycannflavin A 40 -O-a-L-rhamnopyranoside (5), and cannflavin B 7-O-b-D-4000-O-methylglucopyranoside (6) and cannflavin B 7-sulfate (7), respectively. All compounds were evaluated for antimicrobial and antiprotozoal activity.

Microwave-Assisted Extraction of Cannabinoids inHemp Nut Using Response Surface Methodology:Optimization and Comparative Study
Chih-Wei Chang, Ching-Chi Yen, Ming-Tsang Wu, Mei-Chich Hsu and Yu-Tse Wu
Molecules 2017, 22, 1894;
doi:10.3390/molecules22111894
Hemp nut is commonly incorporated into several food preparations; however, most countries set regulations for hemp products according to their cannabinoid content. In this study, we have developed an efficient microwave-assisted extraction (MAE) method for cannabinoids (i.e., ? 9-tetrahydrocannabinol, cannabidiol, and cannabinol) in hemp nut. Optimization of the MAE procedure was conducted through single factor experiments and response surface methodology (RSM). A comparative study was also conducted to determine the differences in the extraction yields and morphology of hemp nut between MAE and reference extraction methods, namely heat reflux extraction (HRE), Soxhlet extraction (SE), supercritical fluid extraction (SFE), and ultrasound assisted extraction (UAE). Among the independent variables in RSM, the temperature was the most significant parameter. The optimal conditions of MAE were as follows: extraction solvent of methanol, microwave power of 375 W, temperature of 109 °C, and extraction time of 30 min. Compared with reference extraction methods, MAE achieved the highest extraction yields of total cannabinoids in hemp nut (6.09 ? g/g for MAE; 4.15 ? g/g for HRE; 5.81 ? g/g for SE; 3.61 ? g/g for SFE; 3.73 ? g/g for UAE) with the least solvent consumption and shortest time. Morphological observations showed that substantial cell rupturing occurred in the microstructure of hemp nut after MAE, indicating enhanced dissolution of the target compounds during the extraction process. The MAE method is thus a rapid, economic, and environmentally friendly extraction method that is both effective and practical for industrial applications

Minor Cannabinoids: Biosynthesis, Molecular Pharmacology and Potential Therapeutic Uses
Kenneth B. Walsh, Amanda E. McKinney, and Andrea E. Holme
Front. Pharmacol., 29 November 2021 DOI:10.3389/fphar.2021.777804 https://www.frontiersin.org/articles...21.777804/full
The medicinal use of Cannabis sativa L. can be traced back thousands of years to ancient China and Egypt. While marijuana has recently shown promise in managing chronic pain and nausea, scientific investigation of cannabis has been restricted due its classification as a schedule 1 controlled substance. A major breakthrough in understanding the pharmacology of cannabis came with the isolation and characterization of the phytocannabinoids trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This was followed by the cloning of the cannabinoid CB1 and CB2 receptors in the 1990s and the subsequent discovery of the endocannabinoid system. In addition to the major phytocannabinoids, Δ9-THC and CBD, cannabis produces over 120 other cannabinoids that are referred to as minor and/or rare cannabinoids. These cannabinoids are produced in smaller amounts in the plant and are derived along with Δ9-THC and CBD from the parent cannabinoid cannabigerolic acid (CBGA). While our current knowledge of minor cannabinoid pharmacology is incomplete, studies demonstrate that they act as agonists and antagonists at multiple targets including CB1 and CB2 receptors, transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPARs), serotonin 5-HT1a receptors and others. The resulting activation of multiple cell signaling pathways, combined with their putative synergistic activity, provides a mechanistic basis for their therapeutic actions. Initial clinical reports suggest that these cannabinoids may have potential benefits in the treatment of neuropathic pain, neurodegenerative diseases, epilepsy, cancer and skin disorders. This review focuses on the molecular pharmacology of the minor cannabinoids and highlights some important therapeutic uses of the compounds.

Minor oxygenated cannabinoids from high potency Cannabis sativa L.
Safwat A. Ahmed, Samir A. Ross, Desmond Slade, Mohamed M. Radwan, Ikhlas A. Khan, Mahmoud A. ElSohly
Phytochemistry 117 (2015) 194–199
DOI: 10.1016/j.phytochem.2015.04.007
Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC–MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9a-hydroxyhexahydrocannabinol, 7-oxo-9a-hydroxyhexa-hydrocannabinol,10a-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, D9-THC aldehyde A, 8-oxo-D9-THC, 10aa-hydroxy-10-oxo-D8-THC, 9a-hydroxy-10-oxo-D6a,10a-THC, and 10S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0 lg/mL), moderate antileishmanial (IC50 14.0 lg/mL) and mild antimalarial activity against Plasmodium falciparum (D6clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3 lg/mL, respectively.


Minnesota wild hemp: a crucial botanical source in early cannabinoid discovery
Crist N. Filer
Journal of Cannabis Research (2020) 2:25
doi 10.1186/s42238-020-00031-3
Renewed and sustained Cannabis chemistry exploration was initiated by Roger Adams at the University of Illinois Chemistry Department with cooperation from the Treasury Department Narcotics Laboratory in the early 1940’s. This partnership and time investment by both parties made practical sense. Adams was able to explore natural products chemistry and the Narcotics Laboratory began to clarify the chemistry mysteries of Cannabis. Minnesota wild hemp, often viewed as just a roadside weed, was employed as the critical botanical source. Based on its widespread cultivation during World War II, this was also a very pragmatic decision. Although the unique Illinois – Washington D. C. collaboration lasted only a few short years (1939–1942), the stunning results included the isolation and extensive characterization of cannabidiol, the structure elucidation and total synthesis of cannabinol as well as the identification of the tetrahydrocannabinol structure as an intoxicating pharmacophore. Furthermore, this research well prepared many junior chemists for prolific careers in both academia as well as industry, inspired the discoveries of later Cannabis investigators and also provided a successful model of a productive academic-government partnership.

Mississippi-grown marihuana —Cannabis sativa cultivation and observed morphological variations.
Quimby, M. W., Doorenbos, N. J., Turner, C. E., & Masoud, A.
Economic Botany, 27(1), 117–127. (1973).
doi:10.1007/bf02862224
For more than a generation Cannabis sativa Linnaeus has perhaps received more attention from the news media than any other species of plant. During the days of the Great Depression at least some people foresaw difficulties that lay ahead in connection with this species. One has only to read the fine address presented by the Honorable Harry J. Anslinger (1) before the New York Herald Tribune Forum in New York City on October 25, 1938. Much of what was said about marihuana then applies today. To quote a brief statement made in reference to the fight against drugs,"...each victory leads to a new field of battle. Our most recent enemy is Marihuana, the use of which as a narcotic was virtually unknown in the United States a decade ago .... It is a peril--in some ways the worst we have met -- and it concerns us all." The increasing importance of the plant as a menace has made a closer study urgent. In Mississippi we are cultivating cannabis for use in research, and some of this work will be described in this report. First, however, let us examine briefly the history of this plant. Over a period of many centuries this annual, and usually dioecious, species has spread from its original home in Central Asia to most tropical and temperate areas of the world. Some writers believe that the spread to China began some 4,500 years ago (2). Ancient Chinese works mention the two forms of the plant, male and female (3). The migratory Scythians are said by some (4) to have introduced the plant into the Danube estuary and Asia Minor nearly 35 centuries ago. From there it gradually made its way into Europe and Africa. Information on the spread and use of Cannabis is readily available. The plant reached the New World in post Columbian times and here, as in many other parts of the world, it has become a weed in some areas and it persists year after year. Dewey (6) reports that cannabis was brought to New England in 1632 and to Virginia in 1649 from England. The same source includes much interesting information concerning the early cultivation in the colonies and, later, elsewhere in what is now the continental United States. Its spread to other parts of the world is also considered.

Modeling cannabinoids from a large-scale sample of Cannabis sativa chemotypes.
Vergara, D., Gaudino, R., Blank, T., & Keegan, B.
PLOS ONE, 15(9), e0236878. (2020).
doi:10.1371/journal.pone.0236878
The widespread legalization of Cannabis has opened the industry to using contemporary analytical techniques for chemotype analysis. Chemotypic data has been collected on a large variety of oil profiles inherent to the cultivars that are commercially available. The unknown gene regulation and pharmacokinetics of dozens of cannabinoids offer opportunities of high interest in pharmacology research. Retailers in many medical and recreational jurisdictions are typically required to report chemical concentrations of at least some cannabinoids. Commercial cannabis laboratories have collected large chemotype datasets of diverse Cannabis cultivars. In this work a data set of 17,600 cultivars tested by Steep Hill Inc., is examined using machine learning techniques to interpolate missing chemotype observations and cluster cultivars into groups based on chemotype similarity. The results indicate cultivars cluster based on their chemotypes, and that some imputation methods work better than others at grouping these cultivars based on chemotypic identity. Due to the missing data and to the low signal to noise ratio for some less common cannabinoids, their behavior could not be accurately predicted. These findings have implications for characterizing complex interactions in cannabinoid biosynthesis and improving phenotypical classification of Cannabis cultivars.

Modification of??9-THC-actions by cannabinol and cannabidiol in the rat.
Fernandes, M., Schabarek, A., Coper, H., & Hill, R.
Psychopharmacologia, 38(4), 329–338. (1974).
doi:10.1007/bf00429130
Cannabinol (CBN) and Cannabidiol (CBD) were tested in several
test procedures known to be altered by Ag-tetrahydroeannabinol (THC) or crude cannabis preparations. They were inactive in doses up to 80 mg/kg in tests on animal motility, food and water intake, body temperature and catalepsy. In contrast, CBD enhanced the hexobarbitone "sleeping time" more pronounced than Ag-THC whereas CBN increased the "sleeping time" only slightly. When administered in combination CBD prolonged all actions of THC, whereas CBN selectively blocked the effect of THC on hexobarbitone "sleeping time". The enhancement by CBD is best explained by an inhibition of THC-metabolism.

Molecular, biochemical and genetic aspects of cannabinoids
Eric Murillo-Rodriguez
June 2015
DOI: 10.13140/RG.2.1.1863.9523
The full book can be read at:
https://www.researchgate.net/publica...2d29e/download
Cannabinoids are a group of terpenophenolic compounds present in Cannabis (Cannabis sativa L). The broader definition of cannabinoids refers to a group of substances that are structurally related to delta-9-tetrahydrocannabinol (?9-THC) or that bind to cannabinoid receptors. Before the 1980’s, it was often speculated that cannabinoids produced their physiological and behavioral effects via nonspecific interaction with cell membranes, instead of interacting with specific membrane-bound receptors. The discovery of the first cannabinoid receptors in the 1980s helped to resolve this debate. These receptors are common in animals, and have been found in mammals, birds, fish, and reptiles. There are currently two known types of cannabinoid receptors, termed CB1 and CB2. The cannabinoid system has been around for over 600 million years…even before the dinosaurs!! The cannabinoid system is present in species such as hydra, mollusks, and insects, leading to speculation on the physiological importance of such a system preserved throughout evolution. To date, the presence in the central nervous system of specific lipids that bind naturally to the CB1/CB2 cannabinoid receptors has been documented. Pharmacological experiments have shown that injection of those compounds induces cannabimimetic effects. The family of endogenous cannabinoids or endocannabinoids comprises Arachidonoylethanolamine, 2-araquidonylglycerol, Virodhamine, noladinether and N-arachidonyldopamine. The endocannabinoids have an active role in modulating diverse neurobiological functions, such as learning and memory, feeding, pain perception and sleep generation. The system of endogenous cannabinoids is present in several species, including humans, leading to speculation regarding the neurobiological role of the endocannabinoid system in diverse functions. Hence, I thought it was time to bring out an editorial book on the subject containing advanced and up-to-date scientific information on this special and exclusive topic. I expect that such a book is likely to attain global circulation among students, teachers and researchers alike. Fortunately, in response to our appeal, a number of leading scientists in the field across the globe agreed to contribute to the book. Thus, this book deals with various aspects of the cannabinoid and endocannabinoid system, from phenomena to molecular processes. I am sincerely grateful to all the contributors for keeping. The purpose of the book is to provide a comprehensive understanding of the endocannabinoid system with adequate emphasis on the pharmacological and molecular foundation and is directed at all who are interested in cannabinoid research.

Molecular docking analysis of phyto-constituents from Cannabis sativa with pfDHFR
Temitope . David1, Niyi S. Adelakun, Olaposi . Omotuyi, Damilohun S. Metibemu,Oluwafemi E. Ekun Gabriel O. eniafe, Olumide K. Inyang, Bamidele Adewumi, Ojochenemi A. Enejoh, Raymond T. Owolabi, Eunice I. Oribamise
Bioinformation 14(9): 574-579 (2018)
doi: 10.6026/97320630014574.
Available antimalarial drugs have been associated with numerous side effects, which include skin rashes and myelo-suppression. Therefore, it is of interest to explore compounds from natural source having drug-like properties without side effect. This study focuses on the screening of compounds from Cannabis sativa against malaria Plasmodium falciparum dihydrofolate reductase for antimalarial properties using Glide (Schrodinger maestro 2018-1). The result showed that phytochemicals from Cannabis sativa binds with a higher affinity and lower free energy than the standard ligand with isovitexin and vitexin having a glide score of -11.485 and -10.601 respectively, sophoroside has a glide score of -9.711 which is lower than the cycloguanil (co-crystallized ligand) having a glide score of -6.908. This result gives new perception to the use of Cannabis sativa as antimicrobial agent.

Monoclonal antibody against tetrahydrocannabinolic acid distinguishes Cannabis sativa samples from different plant species
Hiroyuki Tanaka, Yukihiro Shoyama
Forensic Science International 106 (1999) 135–146
DOI: 10.1016/S0379-0738(99)00193-0
The cross-reaction of anti-D -THCA MAb against other cannabinoids was very wide. However, other naturally occurring and synthetic phenolics including opium alkaloids did not react to the MAb. Using this ELISA, this paper reports application of the competitive ELISA for detection of marijuana samples. The ELISA described here was very sensitive to the ether extracts of marijuana samples when compared to those of other plants. The assay provided a sensitive method useful for the judge of marijuana samples.

*Multiplex qPCR and Cannabis Microbiome sequencing reveals several Bacteria and Fungi Native to Cannabis flowers
Kevin McKernan, Jessica Spangler, Lei Zhang, Vasisht Tadigotla, Yvonne Helbert, Douglas Smith
Color Poster:
https://system.na3.netsuite.com/core...7c1f3&_xt=.pdf
PDF:
https://www.medicinalgenomics.com/wp...owers_sbmt.pdf

Natural cannabinoids: Templates for drug discovery
Ganesh Thakur, Rick Duclos, Alexandros Makriyannis
January 2006 Life Sciences 78(5):454-66
DOI: 10.1016/j.lfs.2005.09.014
Recent studies have elucidated the biosynthetic pathway of cannabinoids and have highlighted the preference for a C-3 n-pentyl side chain in the most prominently represented cannabinoids from Cannabis sativa and their medicinally important decarboxylation products. The corresponding C-3 n-propyl side chain containing cannabinoids are also found, although in lesser quantities. Structure-activity relationship (SAR) studies performed on Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the key psychoactive ingredient of Cannabis, and its synthetic analogues have identified the C-3 side chain as the key pharmacophore for ligand affinity and selectivity for the known cannabinoid receptors and for pharmacological potency. Interestingly, the terminal n-pentyl saturated hydrocarbon side chain of endocannabinoids also plays a corresponding crucial role in conferring similar properties. This review briefly summarizes the biosynthesis of cannabinoids and endocannabinoids and focuses on their side chain SAR

New Frontier Data Report on Delta-8-THC:Emerging Trends in North America & Europe
New Frontier Data
http://info.newfrontierdata.com/2021...tory-landscape
With the expansion of global legal cannabis markets over the past decade, regulatory frameworks remain fragmented across national and state borders. In our latest report, 2021 Cannabis Regulatory Landscape: Emerging Trends in North America & Europe, we examine how and why regulations are evolving and explore the impact of cannabis consumer sentiment and behavior as a driving force behind regulatory evolution and standardization.
Key Report Findings:
*Consumer sentiment will influence regulations with 66% of cannabis consumers agreeing that legal cannabis is safer than illegal cannabis, and 77% of consumers citing potency as a “very” or “extremely” important driver in purchasing decisions.
*Potency caps in future legislation will likely include limits on THC to protect younger and inexperienced users, and in the absence of a unified regulatory structure, states are evaluating potency caps individually.
*Inconsistent CBD regulations in Europe have created a patchwork landscape for CBD products where average per capita annual spend on CBD was projected to reach €21Billion in 2020.

New potential therapeutic applications for certain phytocannabinoids revealed by pharmacological discoveries
Roger Pertwee
https://cannabis-med.org/members/wp-...11/Pertwee.pdf

NMR Assignments of the Major Cannabinoids and Cannabiflavonoids Isolated from Flowers of Cannabis sativa
Young Hae Choi, Arno Hazekamp, Anja M. G. Peltenburg-Looman, Michel Frédérich, Cornelis Erkelens, Alfons W. M. Lefeber and Robert Verpoorte
Phytochem. Anal. 15, 345–354 (2004)
DOI: 10.1002.pca.787
The complete 1H- and 13C-NMR assignments of the major Cannabis constituents, ?9-tetrahydrocannabinol, tetrahydrocannabinolic acid, ?8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid,
cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical shift of H-1? of the C5 side chain and 2?-OH. It was also observed that carboxylic acid substitution reduces intermolecular hydrogen bonding resulting in a sharpening of the H-5? signal in cannabinolic acid in deuterated chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3? and C-4? of cannflavin A and B were determined by HMBC spectra.

Non-cannabinoid constituents from a high potency Cannabis sativa variety.
Radwan, M. M., ElSohly, M. A., Slade, D., Ahmed, S. A., Wilson, L., El-Alfy, A. T., Ross, S. A.
Phytochemistry, 69(14), 2627–2633. (2008) doi:10.1016/j.phytochem.2008.07.010
Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10- dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and b-sitosteryl-3-O-b-D-glucopyranoside-20 - O-palmitate (6). In addition, five known compounds, a-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and b-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2–4 were inactive as analgesics.

Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb
Angelo A. Izzol, Francesca Borrelli1, Raffaele Capasso1, Vincenzo Di Marzo, and Raphael Mechoulam
TIPS-730; No of Pages 13
doi:10.1016/j.tips.2009.07.006 A
D9 -tetrahydrocannabinol binds cannabinoid (CB1 and CB2) receptors, which are activated by endogenous compounds (endocannabinoids) and are involved in a wide range of physiopathological processes (e.g. modulation of neurotransmitter release, regulation of pain perception, and of cardiovascular, gastrointestinal and liver functions). The well-known psychotropic effects of D9 - tetrahydrocannabinol, which are mediated by activation of brain CB1 receptors, have greatly limited its clinical use. However, the plant Cannabis contains many cannabinoids with weak or no psychoactivity that, therapeutically, might be more promising than D9 - tetrahydrocannabinol. Here, we provide an overview of the recent pharmacological advances, novel mechanisms of action, and potential therapeutic applications of such non-psychotropic plant-derived cannabinoids. Special emphasis is given to cannabidiol, the possible applications of which have recently emerged in inflammation, diabetes, cancer, affective and neurodegenerative diseases, and to D9 -tetrahydrocannabivarin, a novel CB1 antagonist which exerts potentially useful actions in the treatment of epilepsy and obesity.

Novel D9-tetrahydrocannabinol formulation Namisol® has beneficial pharmacokinetics and promising pharmacodynamic effects
Linda E. Klumpers, Tim L. Beumer, Johan G. C. van Hasselt, Astrid Lipplaa, Lennard B. Karger, H. Daniël Kleinloog, Jan I. Freijer, Marieke L. de Kam & Joop M. A. van Gerven
Br J Clin Pharmacol / 74:1 / 42–53
DOI:10.1111/j.1365-2125.2012.04164.x
AIMS Among the main disadvantages of currently available D9-tetrahydrocannabinol (THC) formulations are dosing difficulties due to poor pharmacokinetic characteristics. Namisol® is a novel THC formulation, designed to improve THC absorption. The study objectives were to investigate the optimal administration route, pharmacokinetics (PK), pharmacodynamics (PD) and tolerability of Namisol®.
METHODS This first in human study consisted of two parts. Panel I included healthy males and females (n = 6/6) in a double-blind, double-dummy, randomized, crossover study with sublingual (crushed tablet) and oral administration of Namisol® (5 mg THC). Based on these results, male and female (n = 4/5) participants from panel I received oral THC 6.5 and 8.0 mg or matching placebo in a randomized, crossover, rising dose study during panel II. PD measurements were body sway; visual analogue scales (VAS) mood, psychedelic and heart rate.THC and 11-OH-THC population PK analysis was performed.
RESULTS Sublingual administration showed a flat concentration profile compared with oral administration. Oral THC apparent t1/2 was 72–80 min, tmax was 39–56 min and Cmax 2.92–4.69 ng ml-1.THC affected body sway (60.8%, 95% CI 29.5, 99.8), external perception (0.078 log mm, 95% CI 0.019, 0.137), alertness (-2.7 mm, 95% CI -4.5, -0.9) feeling high (0.256 log mm, 95% CI 0.093, 0.418) and heart rate (5.6 beats min–1, 95% CI 2.7, 6.5). Namisol® was well tolerated.
CONCLUSIONS Oral Namisol® showed promising PK and PD characteristics. Variability and tmax of THC plasma concentrations were smaller for Namisol® than reported for studies using oral dronabinol and nabilone. This study was performed in a limited number of healthy volunteers. Therefore, future research on Namisol® should study clinical effects in patient populations.

Novel Solventless Extraction Technique to Preserve Cannabinoid and Terpenoid Profiles of Fresh Cannabis Inflorescence
Ethan B. Russo, Jeremy Plumb and Venetia L. Whiteley
Molecules Volume 26 Issue 18
DOI: 10.3390/molecules26185496
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-05496.pdf
Despite its use by humans for thousands of years, the technology of cannabis usage and extraction is still evolving. Given that the primary pharmacological compounds of interest are cannabinoid and terpenoids found in greatest abundance in capitate glandular trichomes of unfertilized female inflorescences, it is surprising that older techniques of hashish making have received less technological advancement. The purpose of this study was to employ organically grown cannabis and to isolate pure trichomes from freshly picked flowers via exposure to vapor from solid CO2, commonly known as “dry ice”, followed by their isolation via sifting through a 150 µ screens while maintaining the cold chain. Biochemical analysis was undertaken on fresh flower, frozen-sifted flower by-products, treated trichomes (Kryo-Kief™), dried flower, dried sifted flower by-product and dried kief. The dry ice process successfully concentrated cannabinoid content as high as 60.7%, with corresponding concentration and preservation of monoterpenoids encountered in fresh flower that are usually lost during the conventional cannabis drying and curing process. The resulting dried sifted flower by-product after dry ice processing remains a usable commodity. This approach may be of interest to pharmaceutical companies and supplement producers pursuing cannabis-based medicine development with an eye toward full synergy of ingredients harnessing the entourage effect

Nutrient concentrations, digestibility, and cannabinoid concentrations of industrial hemp plant components.
Kleinhenz, M. D., Magnin, G., Ensley, S. M., Griffin, J. J., Goeser, J., Lynch, E., & Coetzee, J. F.
Applied Animal Science, 36(4), 489–494.(2020).
doi:10.15232/aas.2020-02018
Objective: The cultivation and production of industrial hemp [Cannabis sativa containing <0.3% tetrahydrocannabinol (THC)] has increased. Information regarding the nutrient composition and cannabinoid concentration of different plant parts are deficient. Materials and Methods: Single lots of industrial hemp plants and by-products of cannabinoid production were obtained from a licensed research station located in south-central Kansas. Samples obtained were (1) whole industrial hemp plants (no roots), (2) stalks remaining after seed harvesting, (3) unprocessed female flowers intended for cannabinoid extraction, (4) whole seed heads for seed production, (5) leaves obtained from the drying process, (6) chaff obtained after seed harvesting and cleaning, and (7) processed female flowers after cannabinoid extraction. Plant materials were submitted for nutrient concentration and digestibility analysis, and for cannabinoid concentration determination, to a commercial nutrition laboratory. Results and Discussion: Dry matter of the plant material ranged from 65 to 96.6%. Crude protein ranged from 5.3 to 24.5%. Calcium concentration was from 1.0 to 5.7% DM. The plants tested had high fiber concentrations, with NDF ranging from 28 to 80% and ADF ranging from 18 to 65% DM. Total digestible nutrients was 19.8 to 61.5. Six of the 10 cannabinoids tested were detected in all samples. Cannabidiolic acid, cannabidiol, and ?9-tetrahydrocannabinolic acid A were detected in all samples at the highest concentrations. Implications and Applications: These findings will assist livestock producers in using industrial hemp in animal feeds through consideration of both the nutritional and cannabinoid concentrations in the ration.

O-Methyl Phytocannabinoids: Semi-synthesis, Analysis in Cannabis Flowerheads, and Biological Activity.
Caprioglio, D., Allegrone, G., Pollastro, F., Valera, S., Lopatriello, A., Collado, J., … Taglialatela-Scafati, O.
Planta Medica.*(2019).*
doi:10.1055/a-0883-5383*
A general protocol for the selective mono-O-methylation of resorcinyl phytocannabinoids was developed. The availability of semisynthetic monomethyl analogues of cannabigerol, cannabidiol, and cannabidivarin (1a–3a, respectively) made it possible to quantify these minor phytocannabinoids in about 40 different chemotypes of fiber hemp. No chemotype significantly accumulated mono-O-methyl cannabidiol (2b) or its lower homologue (3b), while at least three chemotypes containing consistent amounts (? 400 mg/kg) of O-methylcannabigerol (1b) were identified. O-Methylation of alkyl phytocannabinoids (1b–3b) does not significantly change the activity on peroxisome proliferator-activated receptors in contrast to what was reported for phenethyl analogues.


On a New Cannabinoid Classification System: A Sight on the Illegal Market of Novel Psychoactive Substances
Vadim Shevyrin, Vladimir Melkozerov, Gregory W. Endres, Yuri Shafran, and Yuri Morzherin
Cannabis and Cannabinoid Research Volume 1.1, 2016
DOI: 10.1089/can.2016.0004
Synthetic cannabinoids are one of the most rapidly expanding classes of novel psychoactive substances found in illegal markets. These substances have evolved to the point that many examples no longer fit with the traditional
cannabinoid classification system, where assignment of these compounds is difficult and ambiguous, leading to inconsistencies in regard to their chemical structures. This and other drawbacks can result in misunderstandings between forensic scientists and legal disciplines, complicating efforts toward improving the inadequacies of current antidrug laws. After a critical review, we offer an updated yet simplified cannabinoid classification system with the intention to facilitate interdisciplinary communication.

One-flow synthesis of tetrahydrocannabinol and cannabidiol using homo- and heterogeneous Lewis acids
Victor R. L. J. Bloemendal, Bram Spierenburg, Thomas J. Boltje, Jan C. M. van Hest & Floris P. J. T. Rutjes
Journal of Flow Chemistry (2021)
DOI: 10.1007/s41981-020-00133-2
Continuous flow chemistry holds great potential for the production of biologically relevant molecules. Herein, we present an approach for the continuous synthesis of cannabidiol and tetrahydrocannabinol in a one-flow system. The designed route consists of a reaction cascade involving Friedel-Crafts alkylation, subsequent ring opening and cyclisation in up to 45% yield. The reactions were successfully performed using both hetero- and homogeneous Lewis acids in continuous flow and provide yields that are similar to comparable batch processes.

One-Pot Total Synthesis of Cannabinol via Iodine-Mediated Deconstructive Annulation.
Caprioglio, D., Mattoteia, D., Minassi, A., Pollastro, F., Lopatriello, A., Mu?oz, E., … Appendino, G.
Organic Letters.(2019).*
doi:10.1021/acs.orglett.9b02258*
The thermal degradation of cannabichromene (CBC, 3) is dominated by cationic reactions and not by the pericyclic rearrangements observed in model compounds. The rationalization of these differences inspired the development of a process that coupled, in an aromatization-driven single operational step, the condensation of citral and alkylresorciniols to homoprenylchromenes and their in situ deconstructive annulation to benzo[c]chromenes. This process was applied to a total synthesis of cannabinol (CBN, 5) and to its molecular editing.

Not Cannabis specific
Organic Solvent Nanofiltration in Pharmaceutical Industry.
Buonomenna, M. G., & Bae, J.
Separation & Purification Reviews, 44(2), 157–182.( 2014).
doi:10.1080/15422119.2014.918884
Organic solvent nanofiltration (OSN) is a promising energy and waste efficient unit process to separate mixtures down to a molecular level, which gained attention in the pharmaceutical industry, in particular in the process development of Active Pharmaceutical Ingredients (APIs). This paper focuses on all the aspects related to OSN (i.e. membrane materials, commercial membranes, transport theories, applications) to understand the role of this technology in pharmaceutical industry. The most important results in last five years on OSN applications in the process development of APIs are reviewed extensively. Keywords: Organic Solvent Nanofiltration; Solvent resistant nanofiltration; Active Pharmaceutical Ingredients (APIs) ; Solvent exchange; Product purification; Genotoxin Impurities (GTIs); Membrane enhanced peptide synthesis (MEPS); Hybrid processes

Origin and evolution of the cannabinoid oxidocyclase gene family
Robin van Velzen, & M. Eric Schranz
bioRxiv Dec 20 (2020)
Doi: 10.1101/2020.12.18.423406
https://www.biorxiv.org/content/10.1...406v1.full.pdf
Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic meta-analyses, we propose a comprehensive classification of three main clades and seven subclades that is intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid oxidocyclase gene copy number variation may have less functional relevance than previously thought. Instead, we propose that cannabinoid phenotype is primarily determined by presence/absence of single-copy genes. Increased sampling across Cannabis’ native geographic range is likely to uncover additional cannabinoid oxidocyclase gene sequence variation.

Pharmacogenetics of Cannabinoids
Szymon Hryhorowicz, Michal Walczak, Oliwia Zakerska-Banaszak, Ryszard S?omski, andMarzena Skrzypczak-Zieli?ska
Eur J Drug Metab Pharmacokinet. 2018; 43(1): 1–12.
doi: 10.1007/s13318-017-0416-z
Although the application of medical marijuana and cannabinoid drugs is controversial, it is a part of modern-day medicine. The list of diseases in which cannabinoids are promoted as a treatment is constantly expanding. Cases of significant improvement in patients with a very poor prognosis of glioma or epilepsy have already been described. However, the occurrence of side effects is still difficult to estimate, and the current knowledge of the therapeutic effects of cannabinoids is still insufficient. In our opinion, the answers to many questions and concerns regarding the medical use of cannabis can be provided by pharmacogenetics. Knowledge based on proteins and molecules involved in the transport, action, and metabolism of cannabinoids in the human organism leads us to predict candidate genes which variations are responsible for the presence of the therapeutic and side effects of medical marijuana and cannabinoid-based drugs. We can divide them into: receptor genes—CNR1, CNR2, TRPV1, and GPR55, transporters—ABCB1, ABCG2, SLC6A, biotransformation, biosynthesis, and bioactivation proteins encoded by CYP3A4, CYP2C19, CYP2C9, CYP2A6, CYP1A1, COMT, FAAH, COX2, ABHD6, ABHD12 genes, and also MAPK14. This review organizes the current knowledge in the context of cannabinoids pharmacogenetics according to individualized medicine and cannabinoid drugs therapy

Pharmacokinetics and metabolism of the plant cannabinoids, delta9-tetrahydrocannabinol, cannabidiol and cannabinol.
Huestis MA
Handb Exp Pharmacol. 2005;(168):657-90.
DOI: 10.1007/3-540-26573-2_23
Increasing interest in the biology, chemistry, pharmacology, and toxicology of cannabinoids and in the development of cannabinoid medications necessitates an understanding of cannabinoid pharmacokinetics and disposition into biological fluids and tissues. A drug's pharmacokinetics determines the onset, magnitude, and duration of its pharmacodynamic effects. This review of cannabinoid pharmacokinetics encompasses absorption following diverse routes of administration and from different drug formulations, distribution of analytes throughout the body, metabolism by different tissues and organs, elimination from the body in the feces, urine, sweat, oral fluid, and hair, and how these processes change over time. Cannabinoid pharmacokinetic research has been especially challenging due to low analyte concentrations, rapid and extensive metabolism, and physicochemical characteristics that hinder the separation of drugs of interest from biological matrices--and from each other--and lower drug recovery due to adsorption of compounds of interest to multiple surfaces. delta9-Tetrahydrocannabinol, the primary psychoactive component of Cannabis sativa, and its metabolites 11-hydroxy-delta9-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol are the focus of this chapter, although cannabidiol and cannabinol, two other cannabinoids with an interesting array of activities, will also be reviewed. Additional material will be presented on the interpretation of cannabinoid concentrations in human biological tissues and fluids following controlled drug administration.

Pharmacological evaluation of the natural constituent of Cannabis sativa, cannabichromene and its modulation by ?9-tetrahydrocannabinol.
DeLong, G. T., Wolf, C. E., Poklis, A., & Lichtman, A. H.
Drug and Alcohol Dependence, 112(1-2), 126–133.(2010).
doi:10.1016/j.drugalcdep.2010.05.019
In contrast to the numerous reports on the pharmacological effects of 9-tetrahydrocannabinol (THC), the pharmacological activity of another substituent of Cannabis sativa, cannabichromene (CBC) remains comparatively unknown. In the present study, we investigated whether CBC elicits cannabinoid activity in the tetrad assay, which consists of the following four endpoints: hypomotility, antinociception, catalepsy, and hypothermia. Because cannabinoids are well documented to possess anti-inflammatory properties, we examined CBC, THC, and combination of both phytocannabinoids in the lipopolysaccharide (LPS) paw edema assay. CBC elicited activity in the tetrad that was not blocked by the CB1 receptor antagonist, rimonabant. Moreover, a behaviorally inactive dose of THC augmented the effects of CBC in the tetrad that was associated with an increase in THC brain concentrations. Both CBC and THC elicited dose-dependent anti-inflammatory effects in the LPS-induced paw edema model. The CB2 receptor, SR144528 blocked the anti-edematous actions of THC, but not those produced by CBC. Isobolographic analysis revealed that the anti-edematous effects of these cannabinoids in combination were additive. Although CBC produced pharmacological effects, unlike THC, its underlying mechanism of action did not involve CB1 or CB2 receptors. In addition, there was evidence of a possible pharmacokinetic component in which CBC dosedependently increased THC brain levels following an i.v. injection of 0.3 mg/kg THC. In conclusion, CBC produced a subset of behavioral activity in the tetrad assay and reduced LPS-induced paw edema through a noncannabinoid receptor mechanism of action. These effects were augmented when CBC and THC were co-administered.

Pharmacological interaction between cannabidiol and??9-tetrahydrocannabinol.
Karniol, I. G., & Carlini, E. A.
Psychopharmacologia, 33(1), 53–70.(1973).
doi:10.1007/bf00428793
The pharmacological interaction between cannabidiol (CBD) and (--)Ag-trans-tetrahydrocannabinol (AS-TI-IC) has been studied in rabbits, mice and rats by administering mixtures containing varying amounts of both substances. CBD blocked the following effects of Ag-THC: catatonia in mice, corneal areflexia in rabbits, the increased defecation and decreased ambulation after chronic treatment and exposures of rats in an open field arena, and the aggressiveness of rats previously stressed by REM sleep deprivation. On the other hand, CBD potentiated the AS-TttC-induced analgesia in mice and the Ag-THC-impairing effect on climbing rope performance of rats. These interactions are tentatively explained by postulating that CBD directly antagonizes the excitatory effects and/or indirectly potentiates the depressant effects of Ag-THC.

Phenotypic Characterization of the Endocannabinoid-Degrading Enzyme Alpha/Beta-Hydrolase Domain 6 Knockout Rat
Katsuhiko Noguchi, i Katsumi Kadekawa, Saori Nishijima, Mayuko Sakanashi, Shiho Okitsu-Sakurayama, Sayomi Higa-Nakamine, Hideyuki Yamamoto, and Kimio Sugaya
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0011
https://www.liebertpub.com/doi/pdf/1.../can.2021.0011
Introduction: Alpha/beta-hydrolase domain 6 (ABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol, a high-efficiency endogenous cannabinoid. Although the endocannabinoid system has been suggested to be involved in regulation of bladder function, the roles of ABHD6 in the control of micturition remain unknown. To elucidate the physiological and pathological roles of ABHD6 in vivo, we examined phenotypes of ABHD6 knockout rats (Abhd6/) generated by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated proteins system.
Materials and Methods: Age-matched knockout and wild-type (WT) rats of both sexes were used.
Results: Expression of ABHD6, assessed by quantitative real-time polymerase chain reaction and Western blot analysis, was clearly diminished in Abhd6/ rats compared with WT rats. Mutant rats had a normal appearance, and the body weight and food consumption were similar to those of WT rats. The interval between bladder contractions assessed by continuous cystometry was significantly shorter in ABHD6 knockout rats than in WT rats when the bladder was stimulated with acetic acid. Mechanical paw withdrawal thresholds measured by von Frey testing were significantly lowered in the knockout rats than in WT rats. The plasma levels of prostaglandin E2 (PGE2) and the stable metabolite of PGE2 in Abhd6–/– rats were twice as high as that in WT rats.
Conclusions: Deletion of the ABHD6 gene in rats causes more frequent urination in the stimulated bladder and hyperalgesia to non-noxious mechanical stimuli along with increased plasma PGE2

Phytocannabinoids: a unified critical inventory
Lum´?r Ond?rej Hanu?s, Stefan Martin Meyer, Eduardo Mu˜noz, Orazio Taglialatela-Scafatid and Giovanni Appendino
Natural Product Reports 2016 The Royal Society of Chemistry
DOI: 10.1039/c6np00074f
Cannabis sativa L. is a prolific, but not exclusive, producer of a diverse group of isoprenylated resorcinyl polyketides collectively known as phytocannabinoids. The modular nature of the pathways that merge into the phytocannabinoid chemotype translates in differences in the nature of the resorcinyl side-chain and the degree of oligomerization of the isoprenyl residue, making the definition of phytocannabinoid elusive from a structural standpoint. A biogenetic definition is therefore proposed, splitting the phytocannabinoid chemotype into an alkyl- and a b-aralklyl version, and discussing the relationships between phytocannabinoids from different sources (higher plants, liverworts, fungi). The startling diversity of cannabis phytocannabinoids might be, at least in part, the result of non-enzymatic transformations induced by heat, light, and atmospheric oxygen on a limited set of major constituents (CBG, CBD, D9 -THC and CBC and their corresponding acidic versions), whose degradation is detailed to emphasize this possibility. The diversity of metabotropic (cannabinoid receptors), ionotropic (thermosTRPs), and transcription factors (PPARs) targeted by phytocannabinoids is discussed. The integrated inventory of these compounds and their biological macromolecular end-points highlights the opportunities that phytocannabinoids offer to access desirable drug-like space beyond the one associated to the narcotic target CB1.

Phytocannabinoids and Cannabimimetic Drugs: Recent Patents in Central Nervous System Disorders
Roberta Ranieri, Daniela Marasco, Maurizio Bifulco, Anna Maria Malfitano
Recent Patents on CNS Drug Discovery 10(999):1-1 May 2016
DOI: 10.2174/1574889810666160517123938
https://www.researchgate.net/publica...stem_Disorders
Background: Starting from the chemical structure of phytocannabinoids, isolated from Cannabis sativa plant, research groups designed numerous cannabimimetic drugs. These compounds according to their activities can be partial, full agonists and antagonists of cannabinoid receptors. Anecdotal reports and scientific studies described beneficial properties of cannabinoids and their derivatives in several pathological conditions like neurological and neuropsychiatric disorders, and in many other diseases ranging from cancer, atherosclerosis, stroke, hypertension, inflammatory related disorders, and autoimmune diseases. Methods: In this study, starting from the endocannabinoid mechanism of action in neuronal signaling, we highlight and discuss potential application and recent patents of cannabimimetic drugs in neurological disorders. Results: The cannabinoid CB1 receptor was considered particularly interesting for therapeutic approaches in neurological diseases, because primarily expressed by neurons of the central nervous system. In many experimental models, these drugs act via this receptor, however, CB1 receptor independent mechanisms have been also described. Furthermore, endogenous ligands of cannabinoid receptors, the endocannabinoids, are potent modulators of the synaptic function in the brain. In neurological diseases, numerous studies reported modulation of the levels of endocannabinoids according to the phase of the disease and its progression. Conclusions: Finally, although the study of the mechanisms of action of these compounds is still unsolved, many reports and patents strongly suggest therapeutic potential of these compounds in neurological diseases

Phytocannabinoids beyond the Cannabis plant - do they exist?
Gertsch, J., Pertwee, R. G., & Di Marzo, V.
British Journal of Pharmacology, 160(3), 523–529.(2010).
doi:10.1111/j.1476-5381.2010.00745.x
It is intriguing that during human cultural evolution man has detected plant natural products that appear to target key protein receptors of important physiological systems rather selectively. Plants containing such secondary metabolites usually belong to unique chemotaxa, induce potent pharmacological effects and have typically been used for recreational and medicinal purposes or as poisons. Cannabis sativa L. has a long history as a medicinal plant and was fundamental in the discovery of the endocannabinoid system. The major psychoactive Cannabis constituent D9 -tetrahydrocannabinol (D9 -THC) potently activates the G-protein-coupled cannabinoid receptor CB1 and also modulates the cannabinoid receptor CB2. In the last few years, several other non-cannabinoid plant constituents have been reported to bind to and functionally interact with CB receptors. Moreover, certain plant natural products, from both Cannabis and other plants, also target other proteins of the endocannabinoid system, such as hydrolytic enzymes that control endocannabinoid levels. In this commentary we summarize and critically discuss recent findings.

Phytocannabinoids in Cannabis sativa: Recent studies on biosynthetic enzymes.
Taura F, Sirikantaramas S, Shoyama Y, Shoyama Y, Morimoto S (2007)
Chem Biodiv 4: 1649–1663
DOI: 10.1002/cbdv.200790145
Introduction
Tetrahydrocannabinolic Acid Synthase
1. Purification and Characterization
2. Molecular Cloning and Heterologous Expression
3. Localization and Possible Physiological Function
Cannabidiolic Acid Synthase
Cannabichromenic Acid Synthase
Biosynthetic Mechanism Leading to Cannabigerolic Acid
1. Geranylpyrophosphate:Olivetola te Geranyltransferase
2. Origin of the Monoterpene Moiety
3. Origin of the Alkylresorcinol Moiety
Conclusions
”…To overcome these difficulties, from the middle of 1990s, several research groups, including our laboratory, started to investigate the enzymes involved in cannabinoid biosynthesis, and successfully established the biosynthetic pathway of major phytocannabinoids as illustrated in Scheme 1. In the pathway, THCA, CBDA, and CBCA are biosynthesized from the common precursor CBGA by the action of unique oxidoreductases, i.e., THCA synthase, CBDA synthase, and CBCA synthase, respectively [21 – 23]. On the other hand, CBGA is synthesized by alkylation of olivetolic acid (OLA) with geranylpyrophosphate (GPP) by a novel prenyltransferase called geranylpyrophosphate:eek:livatola te geranyltransferase (GOT) [24]. This article reviews individual biosynthetic enzymes and related topics.”

Phytocannabinoids: Origins and Biosynthesis.
Gülck, T., & Møller, B. L.
Trends in Plant Science.(2020).
doi:10.1016/j.tplants.2020.05.005
Phytocannabinoids are bioactive natural products found in some flowering plants, liverworts, and fungi that can be beneficial for the treatment of human ailments such as pain, anxiety, and cachexia. Targeted biosynthesis of cannabinoids with desirable properties requires identification of the underlying genes and their expression in a suitable heterologous host. We provide an overview of the structural classification of phytocannabinoids based on their decorated resorcinol core and the bioactivities of naturally occurring cannabinoids, and we review current knowledge of phytocannabinoid biosynthesis in Cannabis, Rhododendron, and Radula species. We also highlight the potential in planta roles of phytocannabinoids and the opportunity for synthetic biology approaches based on combinatorial biochemistry and protein engineering to produce cannabinoid derivatives with improved properties.

Phytocannabinoids Unraveling the Complex Chemistry and Pharmacology of Cannabis sativa
Kinghorn, A. D., Falk, H., Gibbons, S., & Kobayashi, J. (Eds.).
In Book
Phytocannabinoids. Progress in the Chemistry of Organic Natural Products.(2017).
doi:10.1007/978-3-319-45541-9
Phytochemistry of Cannabis sativa L. …....Pg1 Mahmoud A. ElSohly, Mohamed M. Radwan, Waseem Gul, Suman Chandra, and Ahmed Galal
Synthesis of Phytocannabinoids . . . . . . . . . .Pg37 Michael A. Schafroth and Erick M. Carreira
Molecular Pharmacology of Phytocannabinoids . . . . . . . Pg61 Sarah E. Turner, Claire M. Williams, Leslie Iversen, and Benjamin J. Whalley
Molecular Targets of the Phytocannabinoids: A Complex Picture . . . . . Pg103 Paula Morales, Dow P. Hurst, and Patricia H. Reggio

Phytocannabinomics: Untargeted metabolomics as a tool for cannabis chemovar differentiation
Andrea Cerratoa, Cinzia Cittibc, Giuseppe Cannazzabc, Anna Laura Capriottia, Chiara Cavalierea, Giampaolo Grassid, Federico Marinia, Carmela Maria Montonea, Roberta Parise, Aldo Laganà
Talanta Volume 230, 1 August 2021, 122313
DOI: 10.1016/j.talanta.2021.122313
Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.
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PHYTOCANNABINOIDS: WHAT IS ON THE HORIZON?
Cayman Currents Issue 34 Fall 2020
https://www.caymanchem.com/cms/cayma...CMS/800233.pdf
Cannabis: Our Key to the Endocannabinoid System Page 2
Phytocannabinoid Testing Standards Page 5
Degradants Formed During Phytocannabinoid Processing Page 7
Basic Research Cannabinoids Page 11
Phytocannabinoid Reference Table Page 12
International Regulation & Control of Hemp & Cannabinoids Page 13
As Δ9-THC remains a controlled substance in almost all countries, new avenues are opening for the other non-regulated Cannabis constituents. Much is still left to be uncovered in knowing how Cannabis stimulates the endocannabinoid system in the body and to substantiate its anecdotal and scientific merits as a potential therapeutic. Cayman continues to keep an eye on the horizon by supporting law enforcement agencies in the quick identification of illicit synthetic cannabinoids, legal Cannabis industry purveyors in the safe manufacture of their products, and basic science researchers in the pursuit of understanding the endocannabinoid system and how it is affected by Cannabis.

Phytochemical Screening of Cannabis Sativa L. I: Constituents of an Indian Variant.
Mole, M. L., & Turner, C. E.
Journal of Pharmaceutical Sciences, 63(1), 154–156.(1974)
doi:10.1002/jps.2600630144
AS-trans-Tetrahydrocannabivarin, a mixture of sterols (campesterol, stigmasterol, and p-sitosterol), and the amino acid L-proline were isolated from an Indian variant of Cannabis satiua L. Characterizations were accomplished by the usual spectral methods, except for the sterols which were subjected to GLCmass spectral analysis.

Phytochemistry of Cannabis sativa L
Mahmoud A. ElSohly, Mohamed M. Radwan, Waseem Gul,
Suman Chandra, and Ahmed Galal
DOI: 10.1007/978-3-319-45541-9_1
In book: Phytocannabinoids January 2017
Cannabis sativa L. (Cannabaceae) is found in a variety of habitats and altitudes, ranging from sea level to the alpine foothills of the Himalayas from which it possibly originated [1]. Cannabis cultivation and use are 5000 to 6000 years old, making it difficult to pinpoint the origin of this species [2]. Furthermore, it is one of the oldest plant sources of food and textile fiber [3]. Cultivation of C. sativa for textile fiber originated from Egypt and western Asia and was introduced subsequently to Europe between 1000 and 2000 B.C. and to South America (Chile) in 1545. Over 60 years later (1606), hemp cultivation was introduced to North America (Port Royal, Canada) [4]. Current federal laws in the United States prohibit the cultivation of C. sativa, including hemp. Cannabis sativa has a rich history of medicinal use dating back to ancient times. The first account of its medicinal use came from the Middle East and Asia during the sixth century B.C. Its introduction into western medicine occurred much later, during the early nineteenth century [5, 6]. This species has been indicated in the treatment of pain, glaucoma, nausea, depression, and neuralgia [7–11]. The therapeutic value of phytocannabinoids has also been employed for HIV/AIDS symptom management and multiple sclerosis treatment
Cannabis (Cannabis sativa, or hemp) and its constituents—in particular the cannabinoids—have been the focus of extensive chemical and biological research for almost half a century since the discovery of the chemical structure of its major active constituent, ?9-tetrahydrocannabinol (?9-THC). The plant’s behavioral and psychotropic effects are attributed to its content of this class of compounds, the cannabinoids, primarily ?9-THC, which is produced mainly in the leaves and flower buds of the plant. Besides ?9-THC, there are also non-psychoactive cannabinoids with several medicinal functions, such as cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG), along with other non-cannabinoid constituents belonging to diverse classes of natural products. Today, more than 560 constituents have been identified in cannabis. The recent discoveries of the medicinal properties of cannabis and the cannabinoids in addition to their potential applications in the treatment of a number of serious illnesses, such as glaucoma, depression, neuralgia, multiple sclerosis, Alzheimer’s, and alleviation of symptoms of HIV/AIDS and cancer, have given momentum to the quest for further understanding the chemistry, biology, and medicinal properties of this plant. This contribution presents an overview of the botany, cultivation aspects, and the phytochemistry of cannabis and its chemical constituents. Particular emphasis is placed on the newly-identified/isolated compounds. In addition, techniques for isolation of cannabis constituents and analytical methods used for qualitative and quantitative analysis of cannabis and its products are also reviewed


PKS Activities and Biosynthesis of Cannabinoids and Flavonoids in Cannabis sativa L. Plants
Isvett Josefina, Flores-Sanchez, Robert Verpoorte
Plant and Cell Physiology, Volume 49, Issue 12, December 2008, Pages 1767–1782,
doi: 10.1093/pcp/pcn150
Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.

Placebo Effects of Edible Cannabis: Reported Intoxication Effects at a 30-Minute Delay
Mallory J. E. Loflin, Ph.D., Mitch Earleywine, Ph.D., Stacey Farmer, M.A., Melissa Slavin M.A., Rachel Luba, B.S. and Marcel Bonn-Miller, Ph.D.
DOI: 10.1080/02791072.2017.1354409
Previous research has demonstrated the ability of non-active smoked cannabis cigarettes to induce subjective effects of intoxication (i.e., placebo effect). No studies have been conduced to test whether edible forms of cannabis, which are associated with a significant delay in onset of effect, are able to induce a placebo effect. In the present study, 20 participants were told that they would receive an edible cannabis lollipop containing a high dose of tetrahydrocannabinol (THC), but were instead given a placebo control. Measures of intoxication and mood were taken at baseline, 30 minutes, and 60 minutes post-ingestion of the placebo lollipop. Results of four repeated-measures ANOVAs found significant and quadratic changes across time in cannabis (ARCI m-scale) intoxication (F(2,18) = 4.90, p = .01, ?2 = .22) and negative mood (F(2,18) = 3.99, p = .05, ?2 = .19). Changes in positive mood and the overall measure of general intoxication (ARCI) failed to reach significance. The present study provides preliminary evidence that a placebo effect can be induced with inert edible agents when participants are told that they are receiving active THC. This is the first known study to demonstrate an edible cannabis intoxication placebo effect.
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner.
Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of
concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.


PLANT AND BRAIN CANNABINOIDS: THE CHEMISTRY OF MAJOR NEW PLAYERS IN PHYSIOLOGY.
Hanuš, L., & Mechoulam, R.
Selected Topics in the Chemistry of Natural Products, 49–75.(2007).
doi:10.1142/9789812790781_0003
Cannabis sativa L. (hemp) is a dioecious annual flowering plant. Marihuana is the Spanish name for the dried leaves and female flowering tops of the hemp plant. Hashish is the resin which originates from these female flowering tops. The hemp’s natural homeland is most likely in the regions north of Afghanistan and the Altai mountains of southern Siberia (Russia). It is not clear when and where cultivation of hemp (Cannabis sativa L.) started. It seems most likely that the cultivation of hemp may have originated in northeastern Asia (north and north-east China and southeastern Siberia). Recent discoveries from Southern Moravia in the Czech Republic indicate that the inhabitants who lived at two of the most famous eastern Gravettian settlements, the upper paleolithic sites of Pavlov and Dolní Vìstonice, some 29 000 to 22 000 years ago were expert weavers. Excavations by American and Czech scientists revealed the evidence of plant fibres used in the manufacture of textiles, basketry, cordage and perhaps netting.1 However the plant(s) used have not been identified and wild hemp is just one of the possibilities. Cultivated hemp had many uses in ancient neolitic China. One of the earliests records of hemp use by man comes from a 12 000 year-old neolitic site at Yuan-shan (Taiwan island). Excavated items included coarse, sandy pottery with hemp cord marks covering the surface, and an incised, rodshaped stone beater used to pound hemp.2,3 A fragment of hemp cloth was discovered in 1972 in a grave from the Chou dynasty (1122–249 B.C.) in Shansi province (China). It is the oldest preserved specimen of cannabis.4

Plant cannabinoids: a neglected pharmacological treasure trove
Raphael Mechoulam British Journal of Pharmacology (2005) 146, 913–915
doi: 10.1038/sj.bjp.0706415
Most of the cannabinoids in Cannabis sativa L. have not been fully evaluated for their pharmacological activity. A publication in this issue presents evidence that a plant cannabinoid, D9-tetrahydrocannabivarin is a potent antagonist of anandamide, a major endogenous cannabinoid. It seems possible that many of the non-psychoactive constituents of this plant will be of biological interest

Plant-Derived Cannabinoids Modulate the Activity of Transient Receptor Potential Channels of Ankyrin Type-1 and Melastatin Type-8.
De Petrocellis, L., Vellani, V., Schiano-Moriello, A., Marini, P., Magherini, P. C., Orlando, P., & Di Marzo, V.
Journal of Pharmacology and Experimental Therapeutics, 325(3), 1007–1015. doi:10.1124/jpet.107.134809
The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and 9 -tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2 in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2 elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC50 60 nM) and the least potent being CBG and CBD acid (EC50 3.4 –12.0 M). CBCalso activated MO-sensitive DRG neurons, although with lower potency (EC50 34.3 M). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2 elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC50 70 –160 nM), whereas CBD acid was the least potent compound (IC50 0.9 –1.6 M). CBG inhibited Ca2 elevation also in icilin-sensitive DRG neurons with potency (IC50 4.5 M) similar to that of anandamide (IC50 10 M). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

Potential impacts of soil microbiota manipulation on secondary metabolites production in Cannabis
Bulbul Ahmed, Mohamed Hijri
Journal of Cannabis Research 3(1):25 July 2021
DOI: 10.1186/s42238-021-00082-0
https://www.researchgate.net/publica...on_in_cannabis
Background Cannabis growing practices and particularly indoor cultivation conditions have a great influence on the production of cannabinoids. Plant-associated microbes may affect nutrient acquisition by the plant. However, beneficial microbes influencing cannabinoid biosynthesis remain largely unexplored and unexploited in cannabis production. Objective To summarize study outcomes on bacterial and fungal communities associated with cannabis using high-throughput sequencing technologies and to uncover microbial interactions, species diversity, and microbial network connections that potentially influence secondary metabolite production in cannabis. Materials and method A mini review was conducted including recent publications on cannabis and their associated microbiota and secondary metabolite production. Results In this review, we provide an overview of the potential role of the soil microbiome in production of cannabinoids, and discussed that manipulation of cannabis-associated microbiome obtained through soil amendment interventions of diversified microbial communities sourced from natural forest soil could potentially help producers of cannabis to improve yields of cannabinoids and enhance the balance of cannabidiol (CBD) and tetrahydrocannabinol (THC) proportions. Conclusion Cannabis is one of the oldest cultivated crops in history, grown for food, fiber, and drugs for thousands of years. Extension of genetic variation in cannabis has developed into wide-ranging varieties with various complementary phenotypes and secondary metabolites. For medical or pharmaceutical purposes, the ratio of CBD to THC is key. Therefore, studying soil microbiota associated with cannabis and its potential impact on secondary metabolites production could be useful when selecting microorganisms as bioinoculant agents for enhanced organic cannabinoid production.

Preformulation Studies of a Prodrug of ?9-Tetrahydrocannabinol.
Thumma, S., Majumdar, S., ElSohly, M. A., Gul, W., & Repka, M. A. AAPS PharmSciTech, 9(3), 982–990.(2008).
doi:10.1208/s12249-008-9136-7
Preformulation studies were performed on a hemiglutarate ester prodrug of ?9 - tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a Tg close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at ?18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO

Preliminary Experiments on the Chemistry and Pharmacology of Cannabis.
GILL, E. W., PATON, W. D. M., & PERTWEE, R. G.
Nature, 228(5267), 134–136.(1970).
doi:10.1038/228134a0
There are at least six pharmacologically effective components of cannabis. Their effects on mice include a lowering of body temperature, catalepsy, analgesia and an extension of barbiturate sleeping time, with doses from 25 to 200 mg/kg.

Preliminary Observations on Cardiac Activities of Cannabis sativa L. Root Extracts.
Mole, M. L., Buelke, J., & Turner, C. E.
Journal of Pharmaceutical Sciences, 63(7),1169–1170(1974)
doi:10.1002/jps.2600630738
Rodger (1) reported that whiskey extracts (about 45% alcohol) of Cannabis roots have been used by American Indians and others to treat “dropsy.” Additionally, the extracts were reported to have a digiVol. 63, No. 7, July 1974 1 1169 talis-like effect on the heart. Subsequently, Ten Ham et al. (2) reported that the cardiac action of an ethanol-water extract (50% alcohol) of Cannabis roots was due solely to the potassium content of an infused extract (140 mmoles). This action was exemplified by the following changes in the guinea pig ECG: bradycardia, T wave and QRS complex inversion, and recovery of the heart when infusion was discontinued. We wish to report our preliminary findings indicating the presence of substances other than potassium in Cannabis roots of Mexican origin1 that display cardiac activities. Dried, ground root material was percolated consecutively with hexane, chloroform, 96% ethanol, water, and 5% hydrochloric acid. The potassium content of the first four extracts prepared for infusion2 was determined by atomic absorption spectroscopy and found to be 0.109, 0.181, 16.9, and 36.0 mmoles, respectively”. Ten Ham et al. (2) reported the potassium content of their infusion-prepared ethanol-water extract by flame photometery to be 140 mmoles/liter. Cardiac activity, although different from that observed by Ten Ham et al. (2), was found in the hexane, chloroform, and ethanol extracts; howevet, no activity of a detrimental nature was observed in the aqueous or 5% hydrochloric acid extracts

Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol.
Fellermeier M, Zenk MH (1998)
FEBS Lett 427: 283–285
DOI: 10.1016/S0014-5793(98)00450-5
https://www.sciencedirect.com/scienc...14579398004505
A new enzyme, geranylpyrophosphate:eek:livetola te geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.

Preparative Isolation of Cannabinoids from Cannabis sativa by Centrifugal Partition Chromatography.
Hazekamp, A., Simons, R., Peltenburg?Looman, A., Sengers, M., van Zweden, R., & Verpoorte, R.
Journal of Liquid Chromatography & Related Technologies, 27(15), 2421–2439.(2004).
doi:10.1081/jlc-200028170
A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large-scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (2)-D9 -(trans)-tetrahydrocannabinol (D9 -THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (2)-D9 -(trans)-tetrahydrocannabinolic acid-A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug-type and a fiber-type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material

Production of ?1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.
Taura, F., Dono, E., Sirikantaramas, S., Yoshimura, K., Shoyama, Y., & Morimoto, S.
Biochemical and Biophysical Research Communications, 361(3), 675–680 (2007)
doi:10.1016/j.bbrc.2007.07.079
D1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of D1 -tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted 1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Production of D9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing D9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Bastian Zirpel . Felix Stehle . Oliver Kayser
Biotechnol Lett (2015) 37:1869–1875
DOI: 10.1007/s10529-015-1853-x
Objective The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of D9- tetrahydrocannabinolic acid (THCA) using whole cells.
Results Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Ko- magataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after 192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1) THCA could be produced by 10.5 gCDW l-1 before enzyme activity was lost.
Conclusion
Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production

Prospects for cannabinoid therapies in basal ganglia disorders
Javier Fernández?Ruiz , Miguel Moreno?Martet, Carmen Rodríguez?Cueto , Cristina Palomo?Garo , María Gómez?Cañas, Sara Valdeolivas, Manuel Guzmán, Carmen Guaza,, Julián Romero
doi: 10.1111/j.1476-5381.2011.01365.x
Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinson's disease (PD) and Huntington's disease (HD), two of the most important disorders affecting the basal ganglia. Two pharmacological profiles have been proposed for cannabinoids being effective in these disorders. On the one hand, cannabinoids like ?9?tetrahydrocannabinol or cannabidiol protect nigral or striatal neurons in experimental models of both disorders, in which oxidative injury is a prominent cytotoxic mechanism. This effect could be exerted, at least in part, through mechanisms independent of CB1 and CB2 receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB2receptors leads to a slower progression of neurodegeneration in both disorders. This effect would be exerted by limiting the toxicity of microglial cells for neurons and, in particular, by reducing the generation of proinflammatory factors. It is important to mention that CB2 receptors have been identified in the healthy brain, mainly in glial elements and, to a lesser extent, in certain subpopulations of neurons, and that they are dramatically up?regulated in response to damaging stimuli, which supports the idea that the cannabinoid system behaves as an endogenous neuroprotective system. This CB2receptor up?regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB2 receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB2 receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation

Psychopharmacology of the endocannabinoids: far beyond anandamide
F A Pamplona , R N Takahashi
J Psychopharmacol 2012 Jan;26(1):7-22.
DOI: 10.1177/0269881111405357
The study of endocannabinoid pharmacology has proceeded from the discovery of Δ9-tetrahydrocannabinol, the main psychoactive compound in Cannabis sativa, to the identification of an endogenous endocannabinoid system that is essential for physiological modulation of neuronal functions. We have not yet achieved a complete understanding of the various roles of the endocannabinoids, but this is one of the fastest-growing fields in psychopharmacology. This review starts with a brief historical description of the discovery of the endocannabinoids and then focuses on recent pharmacological advances and recently discovered endocannabinoid mechanisms of action (e.g. functional selectivity, allosterism, and receptor trafficking). Finally, we will discuss the contention that the existence of evidence-based therapeutic applications for cannabinoids and the wide range of physiological functions affected by endocannabinoids suggests that the careful study of the endocannabinoid system may lead to the development of novel therapeutic drugs with higher societal acceptability and lower side effects profiles.


Purification and Characterization of Cannabidiolic-acid Synthase
from Cannabis sativa L.
BIOCHEMICAL ANALYSIS OF A NOVEL ENZYME THAT CATALYZES THE OXIDOCYCLIZATION OF
CANNABIGEROLIC ACID TO CANNABIDIOLIC ACID*
Futoshi Taura, Satoshi Morimoto, and Yukihiro Shoyama
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 29, Issue of July 19, pp. 17411–17416, 1996
DOI:10.1074/jbc.271.29.17411
We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of D1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s21) is lower than that for the latter (0.19 s21). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid

Qualitative and Quantitative Aspects of the Inheritance of Chemical Phenotype in Cannabis
Giuseppe Mandolino, Manuela Bagatta, Andrea Carboni, Paolo Ranalli, Etienne de Meijer
March 2003 Journal of Industrial Hemp 8(2):51-72
DOI: 10.1300/J237v08n02_04
Four crosses were made between Cannabis plants with “pure” CBD and THC chemotypes. The F1 plants obtained were self-fertilised to produce F2s. Chemotypical distributions were analysed by gas-chromatography. A segregation analysis of the different F2 progenies obtained showed that chemotype, estimated as CBD/THC ratio, behaves as a qualitative character, and a model for a single locus B, with two co-dominant alleles, BD and BT is described. The CBD/THC ratios in the F1 offsprings were found to be significantly different in the heterozygous plants from the different pedigrees. The amount of CBD plus THC in the same pedigrees was also described. Heterosis was found to be a common feature, but not a general one, of cannabinoid accumulation in the F1s. Distribution of the values of cannabinoid content in classes was found to be normal. RAPD markers linked to the segregating chemotypes (“pure” CBD and “pure” THC) were identified by bulk segregant analysis, and the degree of linkage of these markers with the chemotype was described.

Quality Control of Traditional Cannabis Tinctures: Pattern, Markers, and Stability.
Peschel, W
Scientia Pharmaceutica, 84(3), 567–584.(2016).
doi:10.3390/scipharm84030567
Traditional tinctures of Cannabis sativa L. became obsolete before elucidation of the main cannabinoids and routine quality testing for medicines. In view of increasing medicinal use of cannabinoids and associated safety concerns, tinctures from a ?9-tetrahydrocannabinol (THC)-type chemovar were studied. High-performance liquid chromatography with diode-array detection (HPLC/DAD) was used to determine THC, ?9-tetrahydrocannabinolic acid A (THCA), cannabinol (CBN), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannflavin A/B, and total phenolics. Derived group and ratio markers describe absolute and relative profiles when varying plant part (flos, folium), extraction solvent (EtOH percentage), storage conditions (‘shelf’ or ‘fridge’ up to 15 months), and pasteurization (2 h 70 ?C, 20 min 80 ?C). Tinctures from female flowering tops contained ten-fold more cannabinoids than tinctures from leaves; tinctures (80%–90% EtOH) contained ten-fold more cannabinoids than tinctures (40% EtOH). The analysis of CBGA + CBG, the main co-cannabinoids aside from THCA + THC, appears more relevant than CBDA + CBD. The decarboxylation of THCA to THC—the main change during storage of freshly prepared tinctures—is after 15 months in the ‘fridge’ comparable to 3 months on the ‘shelf’. Minimally increased CBN totals did not correlate to diminished totals of THCA and THC (up to 15% after 3 months ‘shelf’, 45% after 15 months ‘fridge’). Instead, total cannabinoids or acidic/neutral cannabinoid ratios are better stability markers. Moderate changes after pasteurization and partial losses below 10% for total cannabinoids after 9 months ‘fridge’ indicate possibilities for a reasonable shelf life. Yet storage and use of non-stabilized tinctures remain critical without authorized specification and stability data because a consistent cannabinoid content is not guaranteed.

Quantitative Analysis of Cannabinoids from Cannabis sativa Using
1H-NMR

Arno HAZEKAMP, Young Hae CHOI, and Robert VERPOORTE
July 2004 CHEMICAL & PHARMACEUTICAL BULLETIN 52(6):718-21
DOI: 10.1248/cpb.52.718
A 1H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of d 4.0—7.0 in the 1H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.

Quantitative determination and validation of 17 cannabinoids in cannabis and hemp using liquid chromatography-tandem mass spectrometry.
McRae, G., & Melanson, J. E.
Analytical and Bioanalytical Chemistry. (2020).
doi:10.1007/s00216-020-02862-8
The increase in production of cannabis for medical and recreational purposes in recent years has led to a corresponding increase in laboratories performing cannabinoid analysis of cannabis and hemp. We have developed and validated a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that is simple, reliable, specific, and accurate for the analysis of 17 cannabinoids in cannabis and hemp. Liquid-solid sample extraction coupled with dilution into a calibration ranges from 10 to 10,000 ng/mL and LC-MS/MS analysis provides quantification of samples ranging from 0.002 to 200 mg/g (0.0002 to 20.0%) in matrix. Linearity of calibration curves in methanol was demonstrated with regression r 2 ? 0.99. Within-batch precision (0.5 to 6.5%) and accuracy (91.4 to 108.0%) and between-batch precision (0.9 to 5.1%) and accuracy (91.5 to 107.5%) were demonstrated for quality control (QC) samples in methanol. Within-batch precision (0.2 to 3.6%) and accuracy (1.4 to 6.1%) and between-batch precision (1.4 to 6.1 %) and accuracy (90.2 to 110.3%) were also evaluated with a candidate cannabis certified reference material (CRM). Repeatability (1.5 to 12.4% RSD) and intermediate precision (2.2 to 12.8% RSD) were demonstrated via analysis of seven cannabis samples with HorRat values ranging from 0.3 to 3.1. The method provides enhanced detection limits coupled with a large quantitative range for 17 cannabinoids in plant material. It is suitable for a wide range of applications including routine analysis for delta-9-tetrahydrocannabinol (?9 -THC), delta-9-tetrahydrocannabinolic acid (?9 -THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and can

Recent Advances in the Chemistry of Hashish.
Mechoulam, R., & Gaoni, Y.
Progress in the Chemistry of Organic Natural Products , 175–213.(1967).
doi:10.1007/978-3-7091-8164-5_6*
Cannabis sativa L. (family Moraceae) is a dioecious plant of which two varieties exist, var. indica and var. non indica or typica. The flowering top of the female plant is covered with glandular hairs which secrete a resin. The resin formation ceases when the seeds mature and its function is to protect them during the ripening period. The cannabis resin is known as "hashish" in the Middle East and Europe and as "charas" in India. Frequently not only the resin but the whole flowering top is collected and used. It is then known as "ganja" in India, "kif" in North Africa, "dagga" in South Africa, "marihuana" in North America, "maconha" in Brazil, etc.

Recent applications of mass spectrometry for the characterization of cannabis and hemp phytocannabinoids: From targeted to untargeted analysis.
Capriotti, A. L., Cannazza, G., Catani, M., Cavaliere, C., Cavazzini, A.,Cerrato, A. Laganà, A.
Journal of Chromatography A, 1655, 462492.(2021).
doi:10.1016/j.chroma.2021.4624
This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analyzers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.



Reexamining Cannabidiol (CBD): Conversion to Tetrahydrocannabinol (THC) Using Only Heat
Robert Daniels, Omer Yassin, John Miguel Toribio, José A Gascón, Gregory A. Sotzing
January 2023, Pre-print
Abstract: Cannabidiol (CBD) containing products have flooded consumer markets in the past few years as the United States, Canada, and several European countries have legalized the cultivation of hemp. Due to complex legal histories of marijuana and cannabinoids, the stability and safety of CBD is still largely uncertain as research has been restricted globally. Conversion of CBD to its psychoactive isomers, delta-9-Tetrahydrocannabinol (Δ9-THC) and delta-8-Tetrahydrocannabinol (Δ8-THC), presents a significant safety issue for consumers and producers of CBD products. For some users, unintentional conversion, leading to intoxication, could significantly impair routine activities while others may seek to intentionally convert CBD for recreational use. Previous studies investigating the stability of CBD have focused mainly on replicating conditions experienced during long-term storage at room temperature or lower. Here, we report the thermal stability of CBD at 175°C. After 30 minutes of heating, both Δ8-THC and Δ9-THC were produced in significant amounts (~4.5% and 2.0%) in aerobic and anaerobic conditions without an acid catalyst. Dynamic 1 H-NMR experiments and computational electronic structure calculations were used to characterize possible reaction paths from CBD to THC. Our findings support an energetically feasible reaction route that is favorable due to both an increase in phenol acidity at high temperatures and intramolecular OH-hydrogen bonding.
Full text: https://www.researchgate.net/public...n_to_Tetrahydrocannabinol_THC_Using_Only_Heat

Regulation of Cannabinoid CB1 Receptors in the Central Nervous System by Chronic Cannabinoids
Laura J. Sim-Selley
Critical Reviews™ in Neurobiology Volume 15, 2003 Issue 2 30 pages
DOI: 10.1615/CritRevNeurobiol.v15.i2.10
Marijuana produces a number of characteristic behaviors in humans and animals, including memory impairment, antinociception, and locomotor and psychoactive effects. However, tolerance and dependence to cannabinoids develops after chronic use, as demonstrated both clinically and in animal models. The potential therapeutic benefits of certain cannabinoid-mediated effects, as well as the use of marijuana for its psychoactive properties, has raised interest in understanding the cellular adaptations produced by chronic administration of this class of drugs. The primary active constituent of marijuana, delta9-tetrahydrohydrocannabinol (THC), binds to specific G-protein-coupled receptors. The central nervous system (CNS) effects of THC are mediated by CB1 receptors, which couple primarily to inhibitory G-proteins. High levels of CB1 receptors are found in the basal ganglia, hippocampus, cortex, and cerebellum, consistent with the profile of behavioral effects. Studies over the past decade have determined that CB1 receptors undergo downregulation and desensitization following chronic administration of THC or synthetic cannabinoid agonists. In general, these adaptations are regionally widespread and of considerable magnitude, and are thought to contribute to tolerance to cannabinoid- mediated behavioral effects. Adaptation at the effector level has been more difficult to characterize, although it appears that alterations in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity may be particularly important in cannabinoid dependence. A striking characteristic of CB1 receptor adaptation is the region dependence of the magnitude and rate of development of downregulation and desensitization. These regional differences may provide interesting insights into the mechanisms of CB1 receptors receptor signaling in different brain regions. Moreover, region-specific adaptations in CB1 receptors following chronic cannabinoid administration may produce differential adaptations at the in vivo level.

Regulation of nausea and vomiting by cannabinoids
Linda A Parker, Erin M Rock
doi: 10.1111/j.1476-5381.2010.01176.x
Considerable evidence demonstrates that manipulation of the endocannabinoid system regulates nausea and vomiting in humans and other animals. The anti?emetic effect of cannabinoids has been shown across a wide variety of animals that are capable of vomiting in response to a toxic challenge. CB1 agonism suppresses vomiting, which is reversed by CB1 antagonism, and CB1 inverse agonism promotes vomiting. Recently, evidence from animal experiments suggests that cannabinoids may be especially useful in treating the more difficult to control symptoms of nausea and anticipatory nausea in chemotherapy patients, which are less well controlled by the currently available conventional pharmaceutical agents. Although rats and mice are incapable of vomiting, they display a distinctive conditioned gaping response when re?exposed to cues (flavours or contexts) paired with a nauseating treatment. Cannabinoid agonists (?9?THC, HU?210) and the fatty acid amide hydrolase (FAAH) inhibitor, URB?597, suppress conditioned gaping reactions (nausea) in rats as they suppress vomiting in emetic species. Inverse agonists, but not neutral antagonists, of the CB1 receptor promote nausea, and at subthreshold doses potentiate nausea produced by other toxins (LiCl). The primary non?psychoactive compound in cannabis, cannabidiol (CBD), also suppresses nausea and vomiting within a limited dose range. The anti?nausea/anti?emetic effects of CBD may be mediated by indirect activation of somatodendritic 5?HT1A receptors in the dorsal raphe nucleus; activation of these autoreceptors reduces the release of 5?HT in terminal forebrain regions. Preclinical research indicates that cannabinioids, including CBD, may be effective clinically for treating both nausea and vomiting produced by chemotherapy or other therapeutic treatments.


Relationship between cannabinoids content and composition of fatty acids in hempseed oils.
Petrovi?, M., Debeljak, Ž., Kezi?, N., & Džidara, P.
Food Chemistry, 170, 218–225. (2015).
doi:10.1016/j.foodchem.2014.08.039
Hempseed oils acquired on the Croatian markets were characterised by cannabinoid content and fatty acid composition. The new method for determination of cannabinoid content was developed and validated in the range of 0.05–60 mg/kg, and the content of tetrahydrocannabinol varied between 3.23 and 69.5 mg/kg. Large differences among the samples were obtained for phenotype ratio suggesting that not all of analysed hempseed oils were produced from industrial hemp. Sample clustering based on cannabinoid content assigned samples to two groups closely related to the phenotype ratios obtained. The results of this study confirm that hempseed oil is a good source of polyunsaturated fatty acids, especially c-linolenic and stearidonic acid, but the content varies a lot more than the omega-6/omega-3 ratio. The grouping of samples on fatty acid content assigned samples to two groups which were consistent with the groups obtained based on cannabinoid content clustering

Relative pharmacological potency in mice of optical isomers of Δ1-tetrahydrocannabinol.
Jones, G., Pertwee, R. G., Gill, E. W., Paton, W. D. M., Nilsson, I. M., Widman, M., & Agurell, S.
Biochemical Pharmacology, 23(2), 439–446.(1974).
doi:10.1016/0006-2952(74)90435-3
The tritium-labelled unnatural enantiomorph of A’-tetrahydrocannabinol (A’-THC) was synthesized. The ‘H-( +)-A’-THC had a specific activity of I.3 Ci,‘mmole and an optical purity of ca. 97”,,. The equipotent molar ratio for (f ) and ( -)-At-tetrahydrocannabinols was determined in mice by an established behavioural bioassay. The (+)-A’-THC was found to be significantly less potent than the laevorotatory Isomer, the mean potency ratio being 13 (95 per cent confidence limits: 7 and 24). Brain levels of (+)-A’-THC and its metabolites were measured in mice 20 min after intravenous injection of 3H-( +)-At-THC (2 mg kg) and were compared with the corresponding levels of (-I-A’-THC and its metabolites. With the exception of the concentrations of one metabolite. no statistically significant differences were observed between the mean levels of enantiomorphs of the cannabinoidsin the brain. In the case of the single metabolite (which was tentatively assigned the structure of 7-hydroxy-A’-THC) the brain level of the dextrorotatoryisomer was l,8-times higher than that of the laevorotatory isomer, a difference which was statistically significant. On incubation in vitro vvith an enriched mouse liver homogenate. (+ )-A’-THC was partially metabolized to more polar compounds; the principal metabolite was shown to be (+)-7-hydroxy-A’-THC. It was concluded that the differences in the psychopharmacological potencies in uiuo of the optical isomers of A’-THC are determined within the central nervous system and are not due to gross differences in metabolism or body distribution.

Safety and pharmacokinetics of oral delta-9-tetrahydrocannabinol in healthy older subjects: A randomized controlled trial
Amir I.A.Ahmed, GekeA.H.van den Elsen, Angela Colbers, Marjolein A. van der Marck, David M.Burger, TonB.Feuthe, Marcel G.M.Olde Rikkert, Cornelis Kramersc
European Neuropsychopharmacology(2014) 24, 1475–1482
DOI: 10.1016/j.euroneuro.2014.06.007
There is a great concern about the safety of THC-based drugs in older people( >65 years),as most of THC-trials did not include such group. In this phase 1, randomized, double-blind, double-dummy, placebo-controlled, cross-overtrial, we evaluated the safety and pharmacokinetics of three oral doses of Namisols, a novel THC in tablet form, in older subjects. Twelve healthy older subjects (6male;meanage72+5 years) randomly received a single oral dose of 3 mg, 5mg, or 6.5mg of THC or matching placebo, in a crossover manner, on each intervention day. The data for 11 subjects were included in the analysis. The data of 1 subject were excluded due to non compliance to study medication THC was safe and well tolerated. The most frequently reported adverse events (AEs) were drowsiness (27%) and drymouth (11%). Subjects reported more AEs with THC 6.5 mg than with3mg (p=0.048), 5mg (p=0.034) and placebo (p=0.013). There was a wide inter-individual variability in plasma concentrations of THC. Subjects for whom the Cmax fell within the sampling period (over2h), Cmax was 1.42– _4.57 ng/mLand Tmax was 67–92 min.TheAUC0–2 h (n=11) was1.67–3.51 ng/mL .Overall, the pharmacodynamic effects of THC were smaller than effects previously reported in young adults.

Season-long characterization of high-cannabinoid hemp (Cannabis sativa L.) reveals variation in cannabinoid accumulation, flowering time, and disease resistance
George M. Stack, Jacob A.Toth, Craig H. Carlson, Ali R. Cala, Mariana I. Marrero-Gonzalez, Rebecca L. Wilk, Deanna Gentner, Jamie L. Crawford, Glenn Philippe, Jocelyn K. C. Rose, Donald R. Viands, Christine D, Smart, Lawrence B, Smart
GCB bioenergy. 2021;00:1-16
DOI: 10.1111/gcbb.12793
GCB Bioenergy. 2021;00:1–16.
https://onlinelibrary.wiley.com/doi/...111/gcbb.12793
Given the dramatic rise in high-cannabinoid hemp (Cannabis sativa L.) production in the last decade, there is an increasingly urgent need to characterize available germ-plasm and develop knowledge to accelerate the breeding of uniform and stable cultivars. Despite persistent cultivation of hemp cultivars for grain and or fiber around
the world, the diversity and genetic underpinning of key traits for breeding and cultivation are poorly understood. For 30 high-cannabidiol hemp cultivars replicated on two field sites, we sought to evaluate yield, agronomic performance, and disease resistance while also conducting a detailed study of cannabinoid accumulation over the course of floral maturation. We observed significant variation in both within and among cultivars. During the growing season, the plants clustered into five groups by growth rate and varied in flowering time from photoperiod insensitive to photoperiod sensitive with very short critical photoperiods. Based on the observed ratio of total potential cannabidiol (CBD) to total potential tetrahydrocannabinol (THC), there was segregation for cannabinoid chemotype in some seeded cultivar populations. Analysis of cannabichromene (CBC) production revealed that some cultivars had a discretely lower CBD:CBC ratio than the others. There was a continuous range of powdery mildew severity by cultivar, with one that had little to no observed powdery mildew suggesting it might have genetic resistance. Biomass production at harvest was strongly influenced by location and cultivar, and there was variation by cultivar in the relative cannabinoid production in shoot tip samples compared with whole plant samples.
While our results provide preliminary guidance regarding relative performance of current cultivars, our analyses indicate a need for additional hemp breeding to provide stable, uniform, and legally compliant cultivars with improved disease resistance and flowering times optimized for the latitudes of different growing locations.

Secondary metabolism in cannabis
Flores-Sanchez, I. J., & Verpoorte, R.
(2008). Phytochemistry Reviews, 7(3), 615–639
doi:10.1007/s11101-008-9094-4
Cannabis sativa L. is an annual dioecious plant from Central Asia. Cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans are
some of the secondary metabolites present in C. sativa. Earlier reviews were focused on isolation and identification of more than 480 chemical compounds;
this review deals with the biosynthesis of the secondary metabolites present in this plant. Cannabinoid biosynthesis and some closely related pathways that involve the same precursors are disscused.

Selective Extraction of Cannabinoid Compounds from Cannabis Seed Using Pressurized Hot Water Extraction
Yannick Nuapia, Hlanganani Tutu , Luke Chimuka and Ewa Cukrowska
Molecules 2020, 25, 1335;
doi:10.3390/molecules25061335
Phytochemicals of Cannabis sativa mainly for the use in the different industries are that of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Pressurized hot water extraction (PHWE) is seen as an efficient, fast, green extraction technique for the removal of polar and semi-polar compounds from plant materials. The PHWE technique was applied to extract cannabinoid compounds from Cannabis sativa seed. Response surface methodology was used to investigate the influence of extraction time (5–60 min), extraction temperature (50–200 ?C) and collector vessel temperature (25–200 ?C) on the recovery of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), cannabichromene (CBG) and cannabigerol (CBC) from Cannabis sativa seed by PHWE. The identification and semi quantification of cannabinoid compounds were determined using GCXGC-TOFMS. The results obtained from different extractions show that the amount of THC and CBN was drastically decreasing in the liquid extract when the temperature rose from 140 to 160 ?C in the extraction cell and the collector0 s vessel. The optimal conditions to extract more CBD, CBC, and CBG than THC and CBN were set at 150 ?C, 160 ?C and 45 min as extraction temperature, the temperature at collector vessel, and the extraction time, respectively. At this condition, the predicted and experimental ratio of THCt (THC + CBN)/CBDt (CBD + CBC+ CBG) was found to be 0.17 and 0.18, respectively. Therefore, PHWE can be seen as an alternative to the classic extraction approach as the efficiency is higher and it is environmentally friendly

Separation of the discriminative stimulus effects of stereoisomers of ?2- and ?3-tetrahydrocannabinols in pigeons.
Järbe, T. U. C., Hiltunen, A. J., Mechoulam, R., Srebnik, M., Breuer, A.
European Journal of Pharmacology, 156(3), 361–366.(1988).
doi:10.1016/0014-2999(88)90281-6
Pigeons, trained to discriminate between the presence or absence of tetrahydrocannabinol (THC) (I) (0.56mg/kg), were tested with (1S,4R)-A2-THC (II) (1-17.5 mg/kg), with the C-1 epimers of (4R)-A2-THC acetate, namely (1S,4R)-A2-THC acetate (IliA) (3-17.5 mg/kg) and (1R,4R)-A2-THC acetate (IIIB) (1-17.5 mg/kg) and with the enantiomers of A3-THC acetate, namely (1S)-A3-THC acetate (IVA) (1-10 mg/kg) and (1R)-A3-THC acetate (IVB) (3-30 mg/kg). The results indicated that (I) was considerably more potent than any of the other compounds evaluated (EDs0 of compound I = 0.18 and 0.25 mg/kg at the two post-injection intervals examined, 90 and 270 min, respectively). Furthermore, of the two A2-THC acetates, compound (IIIB) was active whereas compound (IliA) was not in comparable doses. The parent phenol of compound (IliA), namely (II), was also inactive. Comparison of the pair of enantiomers, (IVA) and (IVB), showed the former to be significantly more potent than the latter. We have thus shown that the A1-THC-like cue properties are separated in the stereoisomers of A 2- and A3-THC.


Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype.
Chiara Onofri, Etienne P M de Meijer, Giuseppe Mandolino
Phytochemistry
DOI: 10.1016/j.phytochem.2015.03.006
Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.

Single molecule sequencing of THCA synthase reveals copy number variation in modern drug type Cannabis sativa L.
Kevin J. McKernan, Yvonne Helbert, Vasisht Tadigotla, Stephen McLaughlin, Jessica Spangler, Lei Zhang, Douglas Smith
https://www.biorxiv.org/content/10.1101/028654v1
doi:*10.1101/028654
Cannabinoid expression is an important genetically determined feature of*cannabis*that presents clinical and legal implications for patients seeking cannabinoid specific therapies like Cannabidiol (CBD).
Cannabinoid, terpenoid, and flavonoid marker assisted selection can accelerate breeding efforts by offering genetic tools to select for desired traits at an early stage in growth. To this end, multiple models for chemotype inheritance have been described suggesting a complex picture for chemical phenotype determination.
Here we explore the potential role of copy number variation of THCA Synthase using phased single molecule sequencing and demonstrate that copy number and sequence variation of this gene is common and suggests a more nuanced view of chemotype prediction.

Source of cannabinoids: what is available, what is used, and where does it come from?
Specchio N1, Pietrafusa N1, Cross HJ2.
Epileptic Disord. 2020 Jan 13.
doi: 10.1684/epd.2019.1121
Cannabis sativa L. is an ancient medicinal plant wherefrom over 120 cannabinoids are extracted. In the past two decades, there has been increasing interest in the therapeutic potential of cannabis-based treatments for neurological disorders such as epilepsy, and there is now evidence for the medical use of cannabis and its effectiveness for a wide range of diseases. Cannabinoid treatments for pain and spasticity in patients with multiple sclerosis (Nabiximols) have been approved in several countries. Cannabidiol (CBD), in contrast to tetra-hydro-cannabidiol (THC), is not a controlled substance in the European Union, and over the years there has been increasing use of CBD-enriched extracts and pure CBD for seizure disorders, particularly in children. No analytical controls are mandatory for CBD-based products and a pronounced variability in CBD concentrations in commercialized CBD oil preparations has been identified. Randomized controlled trials of plant-derived CBD for treatment of Lennox-Gastaut syndrome (LGS) and Dravet syndrome (DS) have provided evidence of anti-seizure effects, and in June 2018, CBD was approved by the Food and Drug Administration as an add-on antiepileptic drug for patients two years of age and older with LGS or DS. Medical cannabis, with various ratios of CBD and THC and in different galenic preparations, is licensed in many European countries for several indications, and in July 2019, the European Medicines Agency also granted marketing authorisation for CBD in association with clobazam, for the treatment of seizures associated with LGS or DS. The purpose of this article is to review the availability of cannabis-based products and cannabinoid-based medicines, together with current regulations regarding indications in Europe (as of July 2019). The lack of approval by the central agencies, as well as social and political influences, have led to significant variation in usage between countries.

Stereochemical Assignments for the Two Enantiomeric Pairs of 9,10-Dihydroxy-?6a(10a)-Tetrahydrocannabinols. X-Ray Crystal Structure Analysis of (±) Trans-cannabitriol.
McPhail, A. T., ElSohly, H. N., Turner, C. E., & ElSohly, M. A.
Journal of Natural Products, 47(1), 138–142. (1984).*
doi:10.1021/np50031a020
gle-crystal X-ray analysis of (k )-cannabitriol (I), isolated from Panamanian Cannabis, confirmed the structure of previously isolated (+)- and (-)-cannabitriol and defined the relative stereochemistry at the asymmetric centers. Based on the results obtained from this study, all compounds previously named cannabitriol f(+), (-), or (*)I should henceforth be designated trans-cannabitriol with appropriate sign of optical rotation, the other enantiomeric pair of (* )-9, 10-dihydroxy-AG'"oa'-tetrahydrocannabinols being (2)-cis-cannabitriol (4).

Stereospecific cyclizations and isomerizations of cannabichromene and related cannabinoids.
Yagen, B., & Mechoulam, R.
Tetrahedron Letters, 10(60), 5353–5356.(1969).
doi:10.1016/s0040-4039(01)88962-0
Ve haVe reported the iaomerizaticm by p-tolneneulphonic aid of $d& -trfrrbgrdroeannabinol
( &i&C) (Is) and cannabichromene (IIa) to 4(8) A --&- THC (III) end related reaction8 (1,2).
Crombie (3)haa Pointed out thatmostof the compoundswhichare obtained from cannabichromeme cmbe formed through cation A. We report nom aome o~ervationa which throw additiolul light on the oubject. Cannabichromene (111) (2) in q etbylene chloride reacts with excess lO$ berm trifluorido etherate in the same l olyent at -2G" for 5 hr. to gire lG$ tetracyclic diether (IV) (4), 67% n8- 3,4-ois -iswTltC (V) (3) and 1% starting wbterial. None of them incmeric A --*-3+trana&o -TKC (1) was isolated or detected by vpc. The structure of V was errtrbliahed bybiydrogenation to the IcnoVn dihydrocompound (1). The cyclization apparently fakes place vith cennabichromene (IIa) in coaforution~cthrough cation A.

Stereospecific synthesis of (-)-.DELTA.1- and (-)-.DELTA.1(6)-tetrahydrocannabinols.
Mechoulam, R., Braun, P., & Gaoni, Y.
Journal of the American Chemical Society, 89(17), 4552–4554. (1967).
doi:10.1021/ja00993a072
A number of total syntheses of racemic A1-tetrahydrocannabinol (A1-THC) (I) and A1@)-THC (11) have been publishedrecently. * No resolution has been reported so far, and hence these syntheses are of limited value in psychobiological research for which compounds with the natural modification are required. We report now a facile, stereospecific synthesis leading to (-)-A'@)-THC and thence to (-)-A1-THC. A pinane derivative was chosen as a starting material for the following reasons: (i) its bulky dimethyl-methylene bridge was expected to provide stereochemical control of the reactions; (ii) as optically pure apinene is readily available in both the (+) and (-) modifications, an entry into the n

Steroids of Cannabis sativa root.
Slatkin, D. J., Knapp, J. E., Schiff, P. L., Turner, C. E., & Leonard Mole,
Phytochemistry, 14(2), 580–581.*(1975).*
doi:10.1016/0031-9422(75)85135-1
PIant. Cannabis .sativa L. Cannabinaceac (root). val of the solvent irr WOW at 10 . the residue (2,8”,,) Sozt~c~. Research Institute of Pharmaceutical was partitioned bctbveen H,O and C‘HC’l, to give Sciences. University of Mississippi; Lot MC-A(~)- fractions of 0.35 and I%“,,. rcspectivclh. The
C-69 grown in I97 I. PI.c~~~~‘oLIS hawk. On roots: fric- C’HC13 fraction was then partitioned between light dclin [ 11, epifricdejinol [ 1). X-(I’-hydroxy-/j- petrol. and 1 o”,, phenylethyl)-/,-hydroxy-r~.u/7~~-cinnamamide . aq. MeOH and the light petrol. fraction (0.1 I”,,) was chromatogrn(,hcd o\c‘r silicic choline [2]. and neurine 121. ac1 ‘d Pwsmt work. The dried, ground roots wcse Elution with light petrol CHCl, (1 :9) gnvc a extracted by percolation with MeOH. After remo- sterol mixture which crystalliFcd from McOH : m.p. 135-136”; ~5:: 3450, 2960, 1460, 1375 and 1061 cm- ‘. GC-MS analysis showed the mixture to be composed of sitosterol(52”/,) (M+ 414) campester01 (28%) (M+ 400) and stigmasterol (20%) (Mf 412). The spectra were consistent with those of authentic samples. Elution with CHCl, gave a fraction which, on preparative TLC over silica gel G using EtOAcCHCl,
(1:9), afforded a steroidal mixture which crystallized from MeOH; m.p. 90-91”; [IX];”+ 80.3” (c 0.94, CHCl,); n”,$)” 243 nm (log E 4.20); vky: 2940, 2870, 1665, 1616, 1440 and 1375 cm- ‘.GC-MS analysis showed the mixture to be composed of stigmast-4-en-3-one(sitost-4-en-3-one) (49%) (M+ 412) campest-4-en-3-one (28’j/,) (M+398) and stigmast-4,22-dien-3-one (23%) (M’ 410). Direct comparison (m.p., m.m.p., IR, GC-MS) with an authentic sample confirmed the identity. Further elution with CHCl, gave a fraction
which, on preparative TLC over silica gel G using EtOAc-CHCl, (1:9), gave a steroidal mixture which crystallized from light petrol. (45 mg); m.p. 127--l 29’; [Y]:,” - 74.1” (c 1 .O, CHCl,); Jb;‘j:” 238 nm (log E 4.02); ~fi,f; 3420, 2960, 2938, 2830, 1663, 1625, 1460, 1385 and 1062 cm- ‘. GC-MS analysis showed the mixture to be composed of stigmast-5- en-3/I-ol-7-one (7-keto-/I-sitosterol) (53%) (M’ 428) campest-5-en-3/I-ol-7-one (25%) (M+ 414) and stigmast-5,22-dien-3/?-ol-7-one (22%) (M’426). Direct comparison (m.p.. m.m.p., IR, GCMS) with an authentic sample [3] confirmed the identity.

Structure activity relationship of four tetrahydrocannabinols and the pharmacological activity of five semi-purified extracts of Cannabis sativa.
Carlini EA, Santos M, Claussen U, Bieniek D, Korte F.
Psychopharmacologia (1970) 18:82–93.
doi: 10.1007/BF00402387
The structure activity relationships of four tetrahydroeannabinols and
the pharmacological activity of five semi-purified extracts from Cannabis sativa were studied using four biological methods: corneal areflexia in rabbits (Gayer test) and eatatonia, decrease of motor activity and suppression of isolation-induced aggressiveness in mice. Modifications in the structure of pure, natural A'-THC rendered the resultant compounds inactive only when activity was measured by the Gayer test; by the other three methods the activity ranged from 1/sth to equal to the activity of pure A"-THC. It was concluded that of the methods employed, the Gayer test was the only useful procedure to measure AP-THC content in mixtures. This was confirmed by the relationship found between A 9-THC content and activity using five semi-purified extracts.


Structure determination and absolute configuration of cannabichromanone derivatives from high potency Cannabis sativa.
Ahmed SA, Ross SA, Slade D, Radwan MM, Khan IA, ElSohly MA
Tetrahedron Lett 49: 6050–6053 (2008a)
doi: 10.1016/j.tetlet.2008.07.178
Three new cannabichromanone derivatives were isolated from high potency cannabis, along with the known cannabichromanone. Full spectroscopic data, including the use of electronic circular dichroism and Mosher ester analysis to determine the absolute configuration of these compounds, are reported. All isolates were tested for antimicrobial, antimalarial, antileishmanial, and anti-oxidant activities.

Structure of Cannabidiol, a Product Isolated from the Marihuana Extract of Minnesota Wild Hemp. I
ROGER ADAMS, MADISON HUNT, AND J. H. CLARK
Journal of the American Chemical Society*volume 62, issue 1, P196-200 (1940)?DOI:10.1021/ja01858a058
Marihuana is the term commonly used in the United States to represent those portions of the Cannabis sativa or hemp plant which are capable of inducing somatic and psychic changes in humans. It is also familiarly known as hashish, bhang, charas, and ganja. The activity of an extract of the plant is found to vary widely, and to be dependent on the source of the hemp. Previous
investigators, for the most part, have studied the resin obtained by working up hashish of Indian origin from the variety of hemp known as Cannabis indica. In this investigation, Minnesota wild hemp, cut after flowering had begun and
before the seed had "set" in the female tops, was used as a raw material. It was extracted with ethanol and the so-called "red oil" containing the active principle or principles was obtained by distillation under diminished pressure.
Numerous investigators have studied the active red oil from Cannabis sativa and indica but only a single pure substance other than nonacosane has yet been isolated from the mixture of products present. This was called cannabinol by Wood, Spivey and Easterfield, and was purified through its crystalline acetate. They assigned the formula C21H2~02. This formula was confirmed and the constitution investigated by Cahn,3 who proposed structure I in which the positions of the hydroxyl and n-amyl groups are uncertain.
Cannabinol is very toxic but has no marihuana activity. A knowledge of the structure of this compound is significant, however, due to the fact that the active red oil, even though derived from various sources, gives an analysis not substantially different from that of pure cannabinol

Studies in Chemistry and Biology of Non-psychotropic Cannabinoids August 2015
Edition: 1st Book Publisher: Lambert Academic
Yehoshua Maor
https://www.researchgate.net/publica...c_Cannabinoids
Cannabinoids, both plant and endogenous compounds, exert a very large spectrum of physiological actions over almost every system tested to date. Most of these actions are associated with the neural and immune systems. However cardiovascular effects are also well established. Most of the effects are caused by the activation of two receptors, CB1 and CB2. Plant based cannabinoids are isolated from Cannabis sativa herbs or produced as synthetic cannabinoid analogues. The most important classes of natural phytocannabinoids are ?9-tetrahydrocannabinols (?9THC), cannabidiols(CBD). Much work has been done on ?9-THC, mainly due to the fact that other plant cannabinoids do not bind to the known cannabinoid receptors. In spite of their lack ofpsychotropic activity many of these neutral compounds exert biological activity systems in the body. However, the mechanism for their pharmacological action is unclear. This work represents a further step towards understanding the biological activities of non-psychotropic cannabinoids.

Studies on tetrahydrocannabinolic acid synthase that produces the acidic precursor of tetrahydrocannabinol, the pharmacologically active cannabinoid in marijuana
Futoshi Taura
Drug Discov Ther. 2009; 3(3):83-87.
https://pdfs.semanticscholar.org/c36...808.1560054937
Tetrahydrocannabinol (THC), the psychoactive component of marijuana, is now regarded as a promising medicine because this cannabinoid has been shown to exert a variety of therapeutic activities. It has been demonstrated that THC is generated from the acidic precursor, tetrahydrocannabinolic acid (THCA) by nonenzymatic decarboxylation, and that THCA is biosynthesized by THCA synthase, which catalyzes a unique biosynthetic reaction, the stereospecific oxidative cyclization of the geranyl group of the substrate cannabigerolic acid. Molecular characterization of THCA synthase has revealed its structural characteristics and reaction mechanism. THCA synthase is the first cannabinoid synthase to be studied and is potentially attractive target for various biotechnological applications as it produces the direct precursor of THC. This review describes the research history of this enzyme, i.e., purification, molecular cloning, biochemical characterization, and possible biotechnological application of THCA synthase.

Study of cannabinoids content in marihuana samples (Cannabis sativa L.) cultivated in several regions of Colombia
VITAE, REVISTA DE LA FACULTAD DE QUÍMICA FARMACÉUTICA ISSN 0121-4004 Volumen 16 número 2, año 2009. Universidad de Antioquia, Medellín, Colombia. págs. 237-244
https://www.scielo.org.co/scielo.php...42009000200008
SPANISH
ESTUDIO DEL CONTENIDO DE CANABINOIDES EN MUESTRAS DE MARIHUANA (Cannabis sativa L.) CULTIVADAS EN VARIAS REGIONES DE COLOMBIA
Néstor M. FLORIAN R. 1,2*, Fabián PARADA A. 2 y William F. GARZÓN M. 2,3 Recibido: Febrero 12 de 2009 Aceptado: Mayo 21 de 2009 RESUMEN El presente estudio analiza el contenido de cannabinoides en muestras de Cannabis sativa L. cultivadas ilícitamente en Colombia. En primer término se optimizan las condiciones para la extracción y cuantificación de Cannabidiol (CBD), ?9-tetrahidrocannabinol (THC) y Cannabinol (CBN) a partir de una muestra vegetal mediante cromatografía de gases con detector de ionización de llama (CG-FID), validando el respectivo método analítico. Se analizan muestras procedentes de cuatro regiones colombianas (región de los Llanos Orientales, LL; región del Cauca, CA; región de la Sierra Nevada de Santa Marta, SN; región del Eje Cafetero, EC) , determinando cuantitativamente la presencia de CBD, THC y CBN. El contenido promedio de THC en las muestras de la región LL es de 15.74 % ± 2.92, en la región CA de 10.98% ± 6.70, en la región SN de 2.81% ± 1.72 y en la región EC de 1.87% ± 1.25. El alto contenido de THC en las muestras vegetales de Cannabis de los Llanos Orientales y el Cauca podría ser indicativo del empleo de variedades mejoradas, lo cual genera una gran preocupación en torno a los mayores efectos potenciales de la droga entre los consumidores. Palabras clave: Cannabis sativa, marihuana, cannabidiol, cannabinol. ABSTRACT The present study analyzes the cannabinoids content in samples of Cannabis sativa L. cultivated illicitly in Colombia. The physicochemical conditions are optimized for the extraction and quantification of Cannabidiol (CBD), 9-Tetrahidrocannabinol (THC) and Cannabinol (CBN) starting from a vegetable sample using gas chromatography with flame ionization detector (GC-FID), validating the respective analytic method. Samples coming from four diferent Colombian regions are analyzed (Llanos Orientales, LL; Cauca, CA; Sierra Nevada de Santa Marta, SN; Eje Cafetero, EC). The study let determination of CBD, THC and CBN samples quantitatively. The average content of THC in the samples of the region LL is of 15.74% ± 2.92, in the region CA is of 10.98% ± 6.70, in the region SN is of 2.81% ± 1.72 and in the region EC of 1.87 % ± 1.25. The higher content of THC in vegetable samples of Cannabis from the Llanos Orientales (LL) and Cauca (CA) could be indicative of the employment of improved varieties. This fact generates a great concern about the potential effects produced by the drug in consumers. Keywords: Cannabis sativa, marihuana, cannabidiol, cannabinol. V

Supercritical carbon dioxide extraction of cannabinoids from Cannabis sativa L. plant material
Laura Rovetto, Niccolo Aieta
Journal of Supercritical Fluids The 129 March 2017
DOI: 10.1016/j.supflu.2017.03.014
Cannabis sativa L. extracts with high concentration of Δ⁹-tetrahydrocannabinolic acid (THCA) and Δ⁹-tetrahydrocannabinol (THC) were obtained by supercritical carbon dioxide (CO2) extraction. The objective of this work was to investigate the cannabinoid concentration of the extracts, the overall process yield under different extraction conditions, and the effect of ethanol as co-solvent. Extraction experiments were carried out with multistage pressure increments and at constant pressure of 17, 24 and 34 MPa and 328 K with flow rate of 200 g/min of CO2. At 34 MPa apparent solubilities of extracts were determined for four different Cannabis sativa L. strains with variable cannabinoids initial content. Extraction yield was highly dependent on pressure and plant material starting composition. The use of ethanol as a co-solvent was investigated with two different approaches, i.e. constant co-solvent flow, and by applying pulses of ethanol at different times though the extraction procedure. The obtained extracts were fractionated in 3 separators in a cascade configuration of decreasing temperature and pressure. The cannabinoid composition of the extracts was determined with HPLC analysis. Process extraction efficiency as high as 92% was achieved.

Synthesis and Antimicrobial Activities of Certain Cannabichromene and Cannabigerol Related Compounds.
Elsohly, H. N., Turner, C. E., Clark, A. M., & Elsohly, M. A.
Journal of Pharmaceutical Sciences, 71(12), 1319–1323.(1982).
doi:10.1002/jps.2600711204*
Cannabichromene homologs, analogs, and isomers as well as the CI-homolog and isomer of cannabigerol were prepared and tested for their antimicrobial and antifungal properties. Spectral data of all compounds synthesized are presented.

Synthetic cannabinoids: Analysis and metabolites.
ElSohly, M. A., Gul, W., Wanas, A. S., & Radwan, M. M.
Life Sciences, 97(1), 78–90.(2014).
doi:10.1016/j.lfs.2013.12.212
Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a 23 wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity 24 toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that 25 could possess high therapeutic activity without many side effects. However, underground laboratories have 26 used the information generated by the scientific community to develop these compounds for illicit use as 27 marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular 28 emphasis on the methods used for their identification in the herbal products with which they are mixed and 29 identification of their metabolites in biological specimens.


Taming THC: potential cannabis synergy and phytocannabinoid terpenoid entourage effects
Ethan B Russo
British Journal of Pharmacology (2011) 163 1344–1364
doi: 10.1111/j.1476-5381.2011.01238.x
Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis chemotypes expressing high titres of each component for future study. This review will explore another echelon of phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, ??pinene, linalool, ??caryophyllene, caryophyllene oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour when inhaled from ambient air at serum levels in the single digits ng·mL?1. They display unique therapeutic effects that may contribute meaningfully to the entourage effects of cannabis?based medicinal extracts. Particular focus will be placed on phytocannabinoid?terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation, depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin?resistant Staphylococcus aureus). Scientific evidence is presented for non?cannabinoid plant components as putative antidotes to intoxicating effects of THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be proposed. Phytocannabinoid?terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new therapeutic products is possible from this venerable plant.

Terpenes/Terpenoids in Cannabis: Are They Important?
Lumír Ond?ej, Hanuš, Yotam Hod
Med Cannabis Cannabinoids, 1-36 (2020)
DOI: 10.1159/000509733
Cannabis sativa plant has not only cannabinoids as crucial compounds but also the other compounds that play important role as synergistic and/or entourage compound. Cannabis/hemp plant materials and essential oils were analyzed with the help of gas chromatography/mass spectrometry detector for the content of terpenes and terpenoids. The main terpenes/terpenoids and their abundance in the samples were evaluated. Results of this study will be helpful in the next evaluation of these compound in mixture with cannabinoids and their importance in medical treatment.

Terpenoids and Phytocannabinoids Co-Produced in Cannabis Sativa Strains Show Specific Interaction for Cell Cytotoxic Activity.
Namdar, D., Voet, H., Ajjampura, V., Nadarajan, S., Mayzlish-Gati, E., Mazuz, M., … Koltai, H.
Molecules, 24(17), 3031. (2019).
doi:10.3390/molecules24173031
: Mixtures of different Cannabis sativa phytocannabinoids are more active biologically than single phytocannabinoids. However, cannabis terpenoids as potential instigators of phytocannabinoid activity have not yet been explored in detail. Terpenoid groups were statistically co-related to certain cannabis strains rich in ? 9 -tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), and their ability to enhance the activity of decarboxylase phytocannabinoids (i.e., THC or CBD) was determined. Analytical HPLC and GC/MS were used to identify and quantify the secondary metabolites in 17 strains of C. sativa, and correlations between cannabinoids and terpenoids in each strain were determined. Column separation was used to separate and collect the compounds, and cell viability assay was used to assess biological activity. We found that in “high THC” or “high CBD” strains, phytocannabinoids are produced alongside certain sets of terpenoids. Only co-related terpenoids enhanced the cytotoxic activity of phytocannabinoids on MDA-MB-231 and HCT-116 cell lines. This was found to be most effective in natural ratios found in extracts of cannabis inflorescence. The correlation in a particular strain between THCA or CBDA and a certain set of terpenoids, and the partial specificity in interaction may have influenced the cultivation of cannabis and may have implications for therapeutic treatments

Tetrahydrocannabinol Homologs with Marihuana Activity. IX1.
Adams, R., Loewe, S., Jelinek, C., & Wolff, H.
Journal of the American Chemical Society, 63(7), 1971–1973 (1941).
doi:10.1021/ja01852a052
In a previous paper it was reported that the
tetrahydrocannabinols (I and 11) produced by isomerizing cannabidiol were very potent in marihuana and one or the other or both probably represented at least part of the active
material in the red oil of hemp. A synthetic tetrahydrocannabinol (111) with the double bond conjugated to the benzene ring was prepared3 and was demonstrated to have marihuana activity. It was much less effective than the tetrahydrocannabinols from cannabidiol but nevertheless about as effective as “purified red oil” from This same optically inactive tetrahydrocannabinol (TIT) was described about the same time by Ghosh, Todd and Wilkinson in the report of an investigation on the synthesis of cannabinol, but no pharmacological tests on the product were reported. In a recent article by Ghosh, Todd and Wright these authors describe the testing of the product
I11 and have confirmed the pharmacological activity as published by us last year. By the Gayer test similar results were obtained as by the ataxia test used by us, the “Bioassay by Approximation” evaluating motor incoordination of dogs. They also report in this most recent article the condensation of pulegone and olivetol to give a product reported as a mixture of tetrahydrocannabinols, which showed about 40% of the physiological activity of the pure compound (111)

Tetrahydrocannabinolic acid, a genuine substance of tetrahydrocannabinol.
Toshio Yamauchi, Yukihiro Shoyama, Itsuo Nishioka
Published in Chemical & pharmaceutical bulletin Vol 15 (1967)Issue 7
DOI:10.1248/cpb.15.1075
Isolation of tetrahydrocannabinolic acid (abbreviated to THCA) was first described by F. Korte, et al in 1965, but in his latest lecture he mentioned that their sample was the molecular compound with dimethylformamide. We wish to report the isolation of pure delta-2-THCA, a main component in Mexican hemp cultivated in Japan, with the aid of chromatography on cellulose powder impregnated with dimethylformamide and n-hexane as an eluant, followed by preparative thin-layer chromatography with n-hexane-EtOAc.

Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes.
Supaart Sirikantaramas, Futoshi Taura, Yumi Tanaka, Yu Ishikawa, Satoshi Morimoto, Yukihiro Shoyama
Plant and Cell Physiology
DOI: 10.1093/pcp/pci166
Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription–PCR (RT–PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.

THC Accumulation in Glands of Cannabis
Paul G. Mahlberg and Eun Soo Kim,
The Hemp Report
Volume 3, Issue 17, Summer 2001 ISSN 1498-8135
https://www.hempreport.com/issues/17/malbody17.html

THC (TETRAHYDROCANNABINOL) ACCUMULATION IN GLANDS OF CANNABIS (CANNABACEAE)
Paul G. Mahlberg and Eun Soo Kim
The Hemp Report Volume 3, Issue 17, Summer 2001 ISSN 1498-8135
https://www.hempreport.com/issues/17/malbody17.html
DOI: 10.1300/J237v09n01_0
THC (delta 9-tetrahydrocannabinol) is known to be present in flowering plants of Cannabis. However, its location in the plant and particularly in the cell remains less generally known. Our studies have been directed to determining where these compounds are localized at the whole plant and cellular level, with a long term objective to determine the organelle or membrane in the cell in their synthesis. It also should be possible to identify the gene responsible for synthesis of these compounds, THC in particular, and modulate this gene so as to develop strains of Cannabis with no THC, or no cannabinoids. Such strains would be intended for hemp agriculture.
The first phase of this study is to determine localization of THC in the plant. As part of this program we initiated an effort to accumulate a germplasm collection of various strains of world-wide distribution that are utilized in hemp cultivation in the classical sense, as well as seed oil strains and those with various levels of THC. We have utilized these strains for analyses of cannabinoids and their distribution; they also will serve as a germplasm source for future studies.
The purposes of this study are: a) to determine where cannabinoids are localized in the plant, and in which specific tissue, and b) to determine where within the cell or tissue cannabinoids are localized. Preliminary studies show it to be in the gland. Historically, it has been reported that a glandless mutant was detected at one time, but is now lost. If THC is in the gland, and glandless mutants can be produced, it should then be possible to reduce significantly the THC content of the plant. Such a plant, with its low THC content, would be a potentially important strain for the industrial hemp industry.

The absolute configuration of ?1-tetrahydrocannabinol, the major active constituent of hashish.
Mechoulam, R., & Gaoni, Y.
Tetrahedron Letters, 8(12), 1109–1111.*(1967).
doi:10.1016/s0040-4039(00)90646-4*
We have recently reported 283 the isolation, structure elucidation and partial synthesis of (-)- A’-tetrahydrocannabinol (I), the major active principle in hashish, as well as a total synthesis of the racemate of I. We wish now to present data showing that the absolute configuration of the naturally occuring (-)-I is D. relative to D-(+)-glyceraldehyde. Adams has reported that tetrahydrocannabidiol (III) obtained by reduction of cannabidiol which has since been shown to possess structure 115, can be oxidized to menthane carboxylic acid (IVa). The anilide of IVa, thus obtained did not depress the melting point of the anilide of IVa prepared from menthol (V) through the menthyl chloride (VI) followed by carbonation of the Grignard derivative of VI. Unfortunately the rotation of the anilide of IVa prepared by the degradation of the natural product was not reported.

Not Cannabis specific
The biochemistry and medical significance of the flavonoids
Bent H. Havsteen
Pharmacology & Therapeutics 96 (2002) 67– 202
DOI:10.1016/S0163-7258(02)00298-X
Flavonoids are plant pigments that are synthesised from phenylalanine, generally display marvelous colors known from flower petals, mostly emit brilliant fluorescence when they are excited by UV light, and are ubiquitous to green plant cells. The flavonoids are used by botanists for taxonomical classification. They regulate plant growth by inhibition of the exocytosis of the auxin indolyl acetic acid, as well as by induction of gene expression, and they influence other biological cells in numerous ways. Flavonoids inhibit or kill many bacterial strains, inhibit important viral enzymes, such as reverse transcriptase and protease, and destroy some pathogenic protozoans. Yet, their toxicity to animal cells is low. Flavonoids are major functional components of many herbal and insect preparations for medical use, e.g., propolis (bee’s glue) and honey, which have been used since ancient times. The daily intake of flavonoids with normal food, especially fruit and vegetables, is 1–2 g. Modern authorised physicians are increasing their use of pure flavonoids to treat many important common diseases, due to their proven ability to inhibit specific enzymes, to simulate some hormones and neurotransmitters, and to scavenge free radicals

The biogenesis of cannabinoids in Cannabis sativa.
Kajima, M., & Piraux, M.
Phytochemistry, 21(1), 67–69.
doi:10.1016/0031-9422(82)80016-2 (1982).
annabigerol and olivetol are incorporated into tetrahydrocannabinol and other neutral cannabinoids.
Cannabidiol is not taken up in any of the neutral cannabinoids and thus it appears the latter are formed from neutral
precursors.

The Biosynthesis of Cannabinoids
Lydia Kariuki
Terpenes And Testing Magazine
https://terpenesandtesting.com/the-b...-cannabinoids/

The Biosynthesis of Cannabinoids
F. Degenhardt, F. Stehle, O. Kayser
Handbook of Cannabis and Related Pathologies. Chapter 2
DOI: 10.1016/B978-0-12-800756-3.00002-8
This chapter focuses on the pathway which leads to the biosynthesis of phytocannabinoids in C. sativa L.
• CBGA is the central precursor of phytocannabinoid biosynthesis in Cannabis.
• CBGAS, only three enzymes—THCAS, CBDAS, and CBCAS—are involved in the biosynthesis of phytocannabinoids in Cannabis plants.
• Sequences of CBDAS and THCAS are known.
• The carboxyl group in CBGA seems to be essential for the enzymatic reactions catalyzed by CBDAS, CBCAS, and THCAS.
• The diversity of more than 60 cannabinoids is the result of nonenzymatic modifications.
• Propyl cannabinoids occur by the prenylation of divarinic acid (DA) with geranyl diphosphate (GPP).

The biosynthesis of the cannabinoids
M. Nazir Tahir, Fred Shahbazi, Simon Rondeau-Gagné & John F. Trant
Journal of Cannabis Research volume 3, Article number: 7 (2021)
DOI: 10.1186/s42238-021-00062-4
https://jcannabisresearch.biomedcent.. .38-021-00062-4
Cannabis has been integral to Eurasian civilization for millennia, but a century of prohibition has limited investigation. With spreading legalization, science is pivoting to study the pharmacopeia of the cannabinoids, and a thorough understanding of their biosynthesis is required to engineer strains with specific cannabinoid profiles. This review surveys the biosynthesis and biochemistry of cannabinoids. The pathways and the enzymes’ mechanisms of action are discussed as is the non-enzymatic decarboxylation of the cannabinoic acids. There are still many gaps in our knowledge about the biosynthesis of the cannabinoids, especially for the minor components, and this review highlights the tools and approaches that will be applied to generate an improved understanding and consequent access to these potentially biomedically-relevant materials.

The Bivalent Rewarding and Aversive properties of ?9-tetrahydrocannabinol are Mediated Through Dissociable Opioid Receptor Substrates and Neuronal Modulation Mechanisms in Distinct Striatal Sub-Regions
Christopher Norris, Hanna J. Szkudlarek, Brian Pereira, Walter Rushlow & Steven R. Laviolette
https://www.nature.com/articles/s41598-019-46215-7.pdf
une 2019 Scientific Reports IN PRESS
DOI: 10.1038/s41598-019-46215-7
The primary psychoactive compound in cannabis, ?9-tetrahydrocannabinol (THC), is capable of producing bivalent rewarding and aversive affective states through interactions with the mesolimbic system. However, the precise mechanisms underlying the dissociable effects of THC are not currently understood. In the present study, we identify anatomically dissociable effects of THC within the rat nucleus accumbens (NAc), using an integrative combination of behavioral pharmacology and in vivo neuronal electrophysiology. We report that the rewarding vs. aversive stimulus properties of THC are both anatomically and pharmacologically dissociable within distinct anterior vs. posterior sub-regions of the NAc. While the rewarding effects of THC were dependent upon local ?-opioid receptor signaling, the aversive effects of THC were processed via a ?-opioid receptor substrate. Behaviorally, THC in the posterior NASh induced deficits in social reward and cognition whereas THC in the anterior NAc, potentiated opioid-related reward salience. In vivo neuronal recordings demonstrated that THC decreased medium spiny neuron (MSN) activity in the anterior NAc and increased the power of gamma (?) oscillations. In contrast, THC increased MSN activity states in the posterior NASh and decreased ?-oscillation power. These findings reveal critical new insights into the bi-directional neuronal and pharmacological mechanisms controlling the dissociable effects of THCin mesolimbic-mediated affective processing.

The Cannabinoid Content of Legal Cannabis in Washington State Varies Systematically Across Testing Facilities and Popular Consumer Products.
Jikomes, N., & Zoorob, M.
Scientific Reports, 8(1).(2018).
doi:10.1038/s41598-018-22755-2
The majority of adults in the U.S. now have state-legal access to medical or recreational cannabis products, despite their federal prohibition. Given the wide array of pharmacologically active compounds in these products, it is essential that their biochemical profle is measured and reported to consumers, which requires accurate laboratory testing. However, no universal standards for laboratory testing protocols currently exist, and there is controversy as to whether all reported results are legitimate. To investigate these concerns, we analyzed a publicly available seed-to-sale traceability dataset from Washington state containing measurements of the cannabinoid content of legal cannabis products from state-certifed laboratories. Consistent with previous work, we found that commercial Cannabis strains fall into three broad chemotypes defned by the THC:CBD ratio. Moreover, we documented systematic diferences in the cannabinoid content reported by diferent laboratories, relative stability in cannabinoid levels of commercial fower and concentrates over time, and diferences between popular commercial strains. Importantly, interlab diferences in cannabinoid reporting persisted even after controlling for plausible confounds. Our results underscore the need for standardized laboratory methodologies in the legal cannabis industry and provide a framework for quantitatively assessing laboratory quality

The Delta-8 THC Controversy, What’s Really At Stake?
Bill Weinberg
Project CBD (April 19 2021)
https://www.projectcbd.org/politics/...UauxAPj1h-fBM0
Even as the edifice of cannabis prohibition crumbles state by state, the federal illegality of the plant and its principal psychotropic componentTHC continues to drive a quest for loopholes in the relevant statutes. Hence the recent focus on Delta-8 tetrahydrocannabinol — a psychoactive but less potent cannabinoid than the more common and notorious Delta-9 THC. Is there any truth to the claim that the 2018 Farm Bill inadvertently legalized Delta-8 THC, as some entrepreneurs maintain? That certainly wasn’t the spirit or intent of the Farm Bill, which did not seek to legalize psychoactive cannabis compounds. The Farm Bill removed hemp – defined as cannabis with less than 0.3 percent THC – from the purview of the Controlled Substances Act. But the Farm Bill never actually mentions Delta-8 THC. And there’s considerable disagreement among hemp advocates regarding the legal status of this compound and what it means for the cannabis industry as a whole.

The Essential Medicinal Chemistry of Cannabidiol (CBD)
Kathryn M. Nelson, Jonathan Bisson, Gurpreet Singh, James G. Graham, Shao-Nong Chen, J. Brent Friesen, Jayme L. Dahlin, Matthias Niemitz, Michael A. Walters, and Guido F. Pauli
J. Med. Chem. 2020, 63, 12137−12155
Doi: 10.1021/acs.jmedchem.0c00724
This Perspective of the published essential medicinal chemistry of cannabidiol (CBD) provides evidence that the popularization of CBD-fortified or CBD-labeled health products and CBD-associated health claims lacks a rigorous scientific foundation. CBD’s reputation as a cure-all puts it in the same class as other “natural” panaceas, where valid ethnobotanicals are reduced to single, purportedly active ingredients. Such reductionist approaches oversimplify useful, chemically complex mixtures in an attempt to rationalize the commercial utility of natural compounds and exploit the “natural” label. Literature evidence associates CBD with certain semiubiquitous, broadly screened, primarily plantbased substances of undocumented purity that interfere with bioassays and have a low likelihood of becoming therapeutic agents. Widespread health challenges and pandemic crises such as SARS-CoV-2 create circumstances under which scientists must be particularly vigilant about healing claims that lack solid foundational data. Herein, we offer a critical review of the published medicinal chemistry properties of CBD, as well as precise definitions of CBD-containing substances and products, distilled to reveal the essential factors that impact its development as a therapeutic agent.

The dimerization of ?9-tetrahydrocannabinolic acid A (THCA-A).
Cuadari, A., Pollastro, F., Unciti-Broceta, J. D., Caprioglio, D., Minassi, A., Lopatriello, A., … Appendino, G
Aca Pharmaceutica Sinica B. . (2019).
doi:10.1016/j.apsb.2019.06.007*
The renewed interest in dimeric salicylates as broad-spectrum anti-inflammatory and antidiabetic agents provided a rationale to investigate the dimerization of the substituted salicylate D9 -tetrahydrocannabinolic acid (THCA-A, 3a) as a strategy to solve its instability to decarboxylation and to generate analogues and/or pro-drugs of this native pre-cannabinoid. Activation of the carboxylic group with the DCC-HOBt-DMAP protocol afforded a high yield of the OBt ester 4, that was next converted into the highly crystalline di-depsidic dimer 5 upon treatment with DMAP. The mono-depsidic dimer 6 was also formed when the reaction was carried out with partially decarboxylated THCA-A samples. The structure of the depsidic dimers was established by spectroscopic methods and by aminolysis of 5 into the pre-cannabinoid amide 7. Both dimers showed excellent shelf stability and did not generate significant amounts of D9 -THC upon heating. However, only the didepsidic dimer 5 activated PPAR-g, the major target of pre-cannabinoids, but strong binding to serum proteins abolished this activity, also shielding it from the action of esterases.

The dual neuroprotective–neurotoxic profile of cannabinoid drugs
Yosef Sarne, Fadi Asaf, Miriam Fishbein, Mikhal Gafni, Ora Keren
British Journal of Pharmacology (2011)163 1391–1401
doi: 10.1111/j.1476-5381.2011.01280.x
Extensive in vitro and in vivo studies have shown that cannabinoid drugs have neuroprotective properties and suggested that the endocannabinoid system may be involved in endogenous neuroprotective mechanisms. On the other hand, neurotoxic effects of cannabinoids in vitro and in vivo were also described. Several possible explanations for these dual, opposite effects of cannabinoids on cellular fate were suggested, and it is conceivable that various factors may determine the final outcome of the cannabinoid effect in vivo. In the current review, we focus on one of the possible reasons for the dual neuroprotective/neurotoxic effects of cannabinoids in vivo, namely, the opposite effects of low versus high doses of cannabinoids. While many studies reported neuroprotective effects of the conventional doses of cannabinoids in various experimental models for acute brain injuries, we have shown that a single administration of an extremely low dose of ?9?tetrahydrocannabinol (THC) (3–4 orders of magnitude lower than the conventional doses) to mice induced long?lasting mild cognitive deficits that affected various aspects of memory and learning. These findings led to the idea that this low dose of THC, which induces minor damage to the brain, may activate preconditioning and/or postconditioning mechanisms and thus will protect the brain from more severe insults. Indeed, our recent findings support this assumption and show that a pre? or a postconditioning treatment with extremely low doses of THC, several days before or after brain injury, provides effective long?term cognitive neuroprotection. The future therapeutical potential of these findings is discussed

The Effect of Electrical Lighting Power and Irradiance on Indoor-Grown Cannabis Potency and Yield
David J. Potter,
J Forensic Sci, May 2012, Vol. 57, No. 3
doi: 10.1111/j.1556-4029.2011.02024.x
The floral development and potencies [D9-tetrahydrocannabinol (THC) contents] of cannabis plants were compared when grown indoors under high-pressure sodium lamps consuming electrical power at three densities (270, 400, and 600 W?m2). After a 3-week vegetative phase, plants were grown for 8 weeks, with lamps maintaining an artificial day length of 12 h. Foliar and floral yields were measured. Gas chromatography was used to measure the content of the psychoactive cannabinoid THC. Mean yields per unit of electrical power in each lighting regime ranged from 0.9 to 1.6 g ? W, the highest being achieved in the lowest irradiance regime. The individual potencies of the separated leaf and flower materials were not affected by increasing irradiance. However, there was a corresponding increase in the overall potency of the aerial plant tissue. This was because of the plants in brighter conditions producing a higher proportion of floral material.

The effect of five day dosing with THCV on THC-induced cognitive, psychological and physiological effects in healthy male human volunteers: A placebo-controlled, double-blind, crossover pilot trial.
Englund A1, Atakan Z2, Kralj A2, Tunstall N2, Murray R2, Morrison P2.
J Psychopharmacol.
DOI: 10.1177/0269881115615104
Rationale: Cannabis is mostly grown under illegal and unregulated circumstances, which seems to favour a product increasingly high in its main cannabinoid ?-9-tetrahydrocannabinol (THC). ?-9-tetrahydrocannabivarin (THCV) is a relatively untested cannabinoid which is said to be a cannabinoid receptor neutral antagonist, and may inhibit the effects of THC. Objectives: To explore the safety and tolerability of repeated THCV administration and its effects on symptoms normally induced by THC in a sample of healthy volunteers. Methods: Ten male cannabis users (<25 use occasions) were recruited for this within-subjects, placebo-controlled, double-blind, cross-over pilot study. 10mg oral pure THCV or placebo were administered daily for five days, followed by 1mg intravenous THC on the fifth day. Results: THCV was well tolerated and subjectively indistinguishable from placebo. THC did not significantly increase psychotic symptoms, paranoia or impair short-term memory, while still producing significant intoxicating effects. Delayed verbal recall was impaired by THC and only occurred under placebo condition (Z=-2.201, p=0.028), suggesting a protective effect of THCV. THCV also inhibited THC-induced increased heart rate (Z=-2.193, p=0.028). Nine out of ten participants reported THC under THCV condition (compared to placebo) to be subjectively weaker or less intense (?2=6.4, p=0.011). THCV in combination with THC significantly increased memory intrusions (Z=-2.155, p=0.031). Conclusion: In this first study of THC and THCV, THCV inhibited some of the well-known effects of THC, while potentiating others. These findings need to be interpreted with caution due to a small sample size and lack of THC-induced psychotomimetic and memory-impairing effect, probably owing to the choice of dose.

The Effect of Light Spectrum on the Morphology and Cannabinoid Content of Cannabis sativa L.
Gianmaria Magagninia, Gianpaolo Grassia, Stiina Kotirantab
Med Cannabis Cannabinoids 2018;1:19–27
DOI: 10.1159/000489030
Cannabis sativa L. flowers are the main source of ?-9 tetrahydrocannabinol (THC) used in medicine. One of the most important growth factors in cannabis cultivation is light; light quality, light intensity, and photoperiod play a big role in a successful growth protocol. The aim of the present study was to examine the effect of 3 different light sources on morphology and cannabinoid production. Cannabis clones were grown under 3 different light spectra, namely
high-pressure sodium (HPS), AP673L (LED), and NS1 (LED). Light intensity was set to ?450 µmol/m2/s measured from the canopy top. AThe photoperiod was 18L:6D/21 days during the vegetative phase and 12L:12D/46 days during the generative phase, respectively. At the end of the experiment, plant dry weight partition, plant height, and cannabinoid content (THC, cannabidiol [CBD], tetrahydrocannabivarin
[THCV], cannabigerol [CBG]) were measured under different light treatments. The experiment was repeated twice. The 3 light treatments (HPS, NS1, AP673L) resulted in differences in cannabis plant morphology and in cannabinoid content, but not in total yield of cannabinoids. Plants under HPS treatment were taller and had more flower dry weight than those under treatments AP673L and NS1. Treatment NS1 had the highest CBG content. Treatments NS1 and AP673L had higher CBD and THC concentrations than the HPS treatment. Results were similar between experiments 1 and 2. Our results show that the plant morphology can be manipulated with the light spectrum. Furthermore, it is possible to affect the accumulation of different cannabinoids to increase the potential of medicinal grade cannabis. In conclusion, an optimized light spectrum improves the value and quality of cannabis. Current LED technology showed significant differences in growth habit and cannabinoid profile compared to the traditional HPS light source. Finally, no difference of flowering time was observed under different R:FR (i.e., the ratio between red and far-red light).

Not Cannabis Specific
The effect of ultraviolet radiation on the accumulation of medicinal compounds in plants
Wen Jing Zhang, Lars Olof Björn
Fitoterapia 80 (2009) 207–218
doi:10.1016/j.fitote.2009.02.006
A review is given of how the production by plants of compounds useful as medicines or raw materials for manufacture of medicines is influenced by ultraviolet radiation, particularly by UVB radiation (280–315 nmwavelength). The compounds considered in this review are flavonoids and other phenolics, alkaloids (especially indole terpenoid and purine alkaloids), essential oils
and other terpenoids, cannabinoids, glucosinolates and isothiocyanates, and compounds having human hormone activity. A short account is also given of ultraviolet signalling in plants. The review concludes with a discussion of the possible evolutionary mechanisms that have led to the evolution of UV-B regulation of secondary metabolite accumulation.

The Effects of Food on Cannabidiol Bioaccessibility
Khadijeh Mozaffari, Stephanie Willette, Ben F. Lucker, Sarah E. Kovar, Francisco Omar Holguin and Ivette Guzman
Molecules 2021, 26, 3573.
DOI: 10.3390/molecules26123573
https://www.mdpi.com/1420-3049/26/12/3573
Cannabidiol (CBD) is a hydrophobic non-psychoactive compound with therapeutic characteristics. Animal and human studies have shown its poor oral bioavailability in vivo, and the impact of consuming lipid-soluble CBD with and without food on gut bioaccessibility has not been explored. The purpose of this research was to study the bioaccessibility of CBD after a three-phase upper digestion experiment with and without food, and to test lipase activity with different substrate concentrations. Our results showed that lipase enzyme activity and fatty acid absorption increased in the presence of bile salts, which may also contribute to an increase in CBD bioaccessibility. The food matrix used was a mixture of olive oil and baby food. Overall, the fed-state digestion revealed significantly higher micellarization efficiency for CBD (14.15 _ 0.6% for 10 mg and 22.67 _ 2.1% for
100 mg CBD ingested) than the fasted state digestion of CBD (0.65 _ 0.7% for 10 mg and 0.14 _ 0.1% for 100 mg CBD ingested). The increase in bioaccessibility of CBD with food could be explained by the fact that micelle formation from hydrolyzed lipids aid in bioaccessibility of hydrophobic molecules. In conclusion, the bioaccessibility of CBD depends on the food matrix and the presence of lipase and bile salts.

The endocannabinoid nervous system: unique opportunities for therapeutic intervention.
A C Porter et al.
Pharmacol Ther. 2001 Apr.
DOI:10.1016/S0163-7258(01)00130-9
http://citeseerx.ist.psu.edu/viewdoc...=rep1&type=pdf
The active principle in marijuana, Delta(9)-tetrahydrocannabinol (THC), has been shown to have wide therapeutic application for a number of important medical conditions, including pain, anxiety, glaucoma, nausea, emesis, muscle spasms, and wasting diseases. Delta(9)-THC binds to and activates two known cannabinoid receptors found in mammalian tissue, CB1 and CB2. The development of cannabinoid-based therapeutics has focused predominantly on the CB1 receptor, based on its predominant and abundant localization in the CNS. Like most of the known cannabinoid agonists, Delta(9)-THC is lipophilic and relatively nonselective for both receptor subtypes. Clinical studies show that nonselective cannabinoid agonists are relatively safe and provide therapeutic efficacy, but that they also induce psychotropic side effects. Recent studies of the biosynthesis, release, transport, and disposition of anandamide are beginning to provide an understanding of the role of lipid transmitters in the CNS. This review attempts to link current understanding of the basic biology of the endocannabinoid nervous system to novel opportunities for therapeutic intervention. This new knowledge may facilitate the development of cannabinoid receptor-targeted therapeutics with improved safety and efficacy profiles.

The Endogenous Cannabinoid System Regulates Seizure Frequency and Duration in a Model of Temporal Lobe Epilepsy
Melisa J. Wallace, Robert E. Blair, Katherine W. Falenski, Billy R. Martin and Robert J. DeLorenzo
Journal of Pharmacology and Experimental Therapeutics October 2003, 307 (1) 129-137;
DOI: 10.1124/jpet.103.051920
Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract ?9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acylactivating enzyme in Cannabis sativa trichomes
Jake M Stout, Zakia Boubakir, Stephen J Ambrose, Jonathan E Page
February 2012 The Plant Journal 71(3):353-65
DOI: 10.1111/j.1365-313X.2012.04949.x
The psychoactive and analgesic cannabinoids (e.g. ?(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

The identification of tetrahydrocannabinolic acid synthase in the non-aqueous secretions of the storage cavities from Cannabis sativa glandular trichomes
P Rodziewicz, S Loroch, I Feldmann, C Schumbrutzki, O Kayser
Planta Medica International Open 4 2017
DOI: 10.1055/s-0037-1608363
http://www.tb.bci.tu-dortmund.de/Pos...Rodziewicz.pdf
Cannabis sativa is an important herbaceous species cultivated since ancient times due to its unique medical and recreational properties, but also as a source of valuable seed oil and high quality fibre. Among numerous phytochemicals synthesized in this herb, cannabinoids represent the unique class of secondary metabolites, which largely contribute to the pharmacological properties of this species. More than 100 cannabinoids have been described in the literature, but Δ 9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) are present in the largest quantities in this plant. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers and their main reservoirs are storage cavities of these hair-like structures.
Tetrahydrocannabinolic acid synthase catalyzes the unique oxidative cyclization of cannabigerolic acid (CBGA) into THCA, which is a direct precursor of the mind-affecting compound – Δ 9- tetrahydrocannabinol (THC). Thus, it is considered to be a key enzyme controlling the psychoactive properties of C. sativa. In previous research it was suggested that THCA synthase is secreted into the storage cavities of glandular trichomes, where it might also synthesize the final product – THCA [2]. However, no direct evidence on protein level was presented.
To examine the putative extracellular location of THCA synthase, the storage cavity secretions from C. sativa trichomes were obtained and submitted for proteomic analysis. By using mass spectrometry we were able to identify the THCA synthase in the non-aqueous content of the storage cavities from C. sativa glandular trichomes.

The ISO-Tetrahydrocannabinols.
Israel Journal of Chemistry, 6(5), 679–690.(1968).
Gaoni, Y., & Mechoulam, R.
doi:10.1002/ijch.196800086
Synthetic routes are described which lead to is 0 -tetrahydrocannablnols, a new series of structural isomers of the natural 6 i-tetrahydrocannabinol. In this group of compounds a dihydrobenzopyran ring system is formed by cyclization of one of the phenolic groups of a cannabinoid with the Ci carbon in the terpene moiety of the molecule.

The Nonpsychoactive Cannabinoid Cannabidiol Inhibits 5-Hydroxytryptamine3A Receptor-Mediated Currents in Xenopus laevis Oocytes
Keun-Hang Yang, Sehamuddin Galadari, Dmytro Isaev, Georg Petroianu, Toni S. Shippenberg, and Murat Oz
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 2
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC2872948/pdf/zpt547.pdf
The effect of the plant-derived nonpsychotropic cannabinoid, cannabidiol (CBD), on the function of hydroxytryptamine (5-HT)3A receptors expressed in Xenopus laevis oocytes was investigated using two-electrode voltage-clamp techniques. CBD reversibly inhibited 5-HT (1 M)-evoked currents in a concentration-dependent manner (IC50 0.6 M). CBD (1 M) did not alter specific binding of the 5-HT3A antagonist [3 H]3-(5-methyl-1H-imidazol-4-yl)-1-(1-methylindol-3-yl)propan-1-one (GR65630), in oocytes expressing 5-HT3A receptors. In the presence of 1 M CBD, the maximal 5-HT-induced currents were also inhibited. The EC50 values were 1.2 and 1.4 M, in the absence and presence of CBD, indicating that CBD acts as a noncompetitive antagonist of 5-HT3 receptors. Neither intracellular BAPTA injection nor pertussis toxin pretreatment (5 g/ml) altered the CBDevoked inhibition of 5-HT-induced currents. CBD inhibition was inversely correlated with 5-HT3A expression levels and mean 5-HT3 receptor current density. Pretreatment with actinomycin D, which inhibits protein transcription, decreased the mean 5-HT3 receptor current density and increased the magnitude of CBD inhibition. These data demonstrate that CBD is an allosteric inhibitor of 5-HT3 receptors expressed in X. laevis oocytes. They further suggest that allosteric inhibition of 5-HT3 receptors by CBD may contribute to its physiological roles in the modulation of nociception and emesis.


The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions
Pasquale LincianoabFabiana RussoaCinzia CittiacFrancesco TolomeocRoberta ParisdFlavia FulviodNicola PecchionieMaria Angela VandelliaAldo LaganàcfAnna Laura CapriottifGiuseppe BiaginigLuigi CarbonecGiuseppe GiglicGiuseppe Cannazza
Talanta 2021
DOI: 10.1016/j.talanta.2021.122704
The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).
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The phytocannabinoid, ?9-tetrahydrocannabivarin, can act through 5-HT1A receptors to produce anti-psychotic effects
November 2014 British Journal of Pharmacology 172(5)
DOI: 10.1111/bph.13000 Maria grazia Cascio, Erica Zamberletti, Pietro Marini, Roger G Pertwee
Background and purpose: This study aimed to address the questions of whether ?(9)-tetrahydrocannabivarin (THCV) can (i) enhance activation of 5-HT1 A receptors in vitro and (ii) induce any apparent 5-HT?A receptor-mediated antipsychotic effects in vivo. Experimental approach: In vitro studies investigated the effect of THCV on targeting by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) of 5-HT?A receptors in membranes obtained from rat brainstem or human 5-HT?A CHO cells, using [(35)S]-GTP?S and 8-[(3)H]-OH-DPAT binding assays. In vivo studies investigated whether THCV induces signs of 5-HT?A receptor-mediated antipsychotic effects in rats. Key results: THCV (i) potently, albeit partially, displaced 8-[(3) H]-OH-DPAT from specific binding sites in rat brainstem membranes; (ii) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of receptors in these membranes; (iii) produced concentration-related increases in 8-[(3)H]-OH-DPAT binding to specific sites in membranes of human 5-HT?A receptor-transfected CHO cells; and (iv) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of these human 5-HT?A receptors. In phencyclidine-treated rats, THCV, like clozapine (i) reduced stereotyped behaviour; (ii) decreased time spent immobile in the forced swim test; and (iii) normalized hyperlocomotor activity, social behaviour and cognitive performance. Some of these effects were counteracted by the 5-HT?A receptor antagonist, WAY100635, or could be reproduced by the CB? antagonist, AM251. Conclusions and implications: Our findings suggest that THCV can enhance 5-HT?A receptor activation, and that some of its apparent antipsychotic effects may depend on this enhancement. We conclude that THCV has therapeutic potential for ameliorating some of the negative, cognitive and positive symptoms of schizophrenia.

The phytocannabinoid ?9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum.
Ma, Y.-L., Weston, S. E., Whalley, B. J., & Stephens, G. J.
British Journal of Pharmacology, 154(1), 204–215.(2008).
doi:10.1038/bjp.2008.57
Background and purpose: The phytocannabinoid D9 -tetrahydrocannabivarin (D9 -THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of D9 -THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS.
Experimental approach: Effects of D9 -THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. Key results: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by D9 -THCV, and also by the CB1 receptor antagonist AM251; D9 -THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, D9 -THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with D9 -THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; D9 -THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of D9 -THCV and WIN55 were attenuated by the GABAA receptor antagonist bicuculline methiodide.
Conclusions and implications: We show for the first time that D9 -THCV acts as a functional CB1 receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. D9 -THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB1 receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst D9 -THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

The Phytochemical Diversity of Commercial Cannabis in the United States
Christiana J. Smith, Daniela Vergara, Brian Keegan, Nick Jikomes
BioRxiv 2021
DOI: 10.1101/2021.07.05.451212
https://www.biorxiv.org/content/10.1...212v1.full.pdf
The legal status of Cannabis is changing, fueling an increased diversity of Cannabis-derived products. Because Cannabis contains dozens of chemical compounds with potential psychoactive or medicinal effects, understanding its phytochemical diversity is crucial. The legal Cannabis industry heavily markets products to consumers based on widely used labelling systems purported to predict the effects of different Cannabis “strains.” We analyzed the cannabinoid and terpene content of tens of thousands of commercial Cannabis samples across six US states, finding distinct chemical phenotypes (chemotypes) which are reliably present. After careful descriptive analysis of the phytochemical diversity and comparison to the commercial labels commonly attached to Cannabis samples, we show that commercial labels do not consistently align with the observed chemical diversity. However, certain labels are statistically overrepresented for specific chemotypes. These results have important implications for the classification of commercial Cannabis, the design of animal and human research, and the regulation of legal Cannabis marketing.

The protective mechanism of cannabidiol in cardiac injury: A systematic review of non-clinical studies.
Shayesteh MR, Haghi-Aminjan, Mousavi MJ, Momtaz S, Abdollahi M.
Curr Pharm Des. 2019 Jul 9. [Epub ahead of print]
doi: 10.2174/2210327909666190710103103.
BACKGROUND: Cardiac disease is accounted as the leading cause of worldwide morbidity and mortality, mainly in association with induction of inflammation and oxidative stress. The disease is characterized by the overproduction of reactive oxygen and/or nitrogen species (ROS/RNS), and reduced antioxidant capacity. Cannabidiol (CBD) is a non-psychoactive ingredient of marijuana that reported to be safe and well tolerated in patients. Due to its pleiotropic effect, CBD has been shown to exert cytoprotective effects. This study intended to clarify the mechanisms and the potential role of CBD regarding cardiac injuries treatment.
METHODS: A systematic literature search was conducted, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, in the electronic databases including PubMed, Web of Science, Scopus, and Embase up to July 2018 using predefined search terms in the titles and abstracts by two independent investigators. Accordingly, a set of pre-specified inclusion and exclusion criteria were considered and 8 articles were ultimately included in this study.
RESULTS: Our findings obviously demonstrate that CBD has multi-functional protective assets to improve cardiac injuries; preliminary through scavenging of free radicals, and reduction of oxidative stress, apoptosis, and inflammation.
CONCLUSION: CBD can protect against cardiac injuries, mainly through its anti-oxidant, anti-inflammatory, and anti-apoptotic effects on the basis of non-clinical studies.

The psychoactive plant cannabinoid, ?9-tetrahydrocannabinol, is antagonized by ?8- and ?9-tetrahydrocannabivarin in mice in vivo.
Pertwee, R. G., Thomas, A., Stevenson, L. A., Ross, R. A., Varvel, S. A., Lichtman, A. H., … Razdan, R. K.
British Journal of Pharmacology, 150(5), 586–594. (2009).
doi:10.1038/sj.bjp.0707124
Background and purpose: To follow up in vitro evidence that D9-tetrahydrocannabivarin extracted from cannabis (D9-THCV) is a CB1 receptor antagonist by establishing whether synthetic D9
-tetrahydrocannabivarin (O-4394) and D8-tetrahydrocannabivarin (O-4395) behave as CB1 antagonists in vivo.
Experimental approach: O-4394 and O-4395 were compared with eD9 -THCV as displacers of [3H]-CP55940 from specific CB1 binding sites on mouse brain membranes and as antagonists of CP55940 in [35S]GTPgS binding assays performed with mouse brain membranes and of R-( þ )-WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg1 (i.v.) D9-tetrahydrocannabinol in mice was then investigated.
Key results: O-4394 and O-4395 exhibited similar potencies to eD9 -THCV as displacers of [3H]-CP55940 (Ki ¼ 46.6 and 64.4 nM, respectively) and as antagonists of CP55940 in the [35S]GTPgS binding assay (apparent KB ¼ 82.1 and 125.9 nM, respectively) and R-( þ )-WIN55212 in the vas deferens (apparent KB ¼ 4.8 and 3.9 nM respectively). At i.v. doses of 0.1, 0.3, 1.0 and/or 3 mg kg1 O-4394 and O-4395 attenuated D9 -tetrahydrocannabinol-induced anti-nociception (tail-flick test) and hypothermia (rectal temperature). O-4395 but not O-4394 also antagonized D9-tetrahydrocannabinol-induced ringimmobility. By themselves, O-4395 and O-4394 induced ring immobility at 3 or 10 mg kg1 (i.v.) and antinociception at doses above 10 mg kg1 (i.v.). O-4395 also induced hypothermia at 3 mg kg1 (i.v.) and above.
Conclusions and implications: O-4394 and O-4395 exhibit similar in vitro potencies to eD9 -THCV as CB1 receptor ligands and as antagonists of cannabinoid receptor agonists and can antagonize D9-tetrahydrocannabinol in vivo.

The role of derivatization techniques in the analysis of plant cannabinoids by gas chromatography mass spectrometry.
Fodor, B., & Molnár-Perl, I.
TrAC Trends in Analytical Chemistry, 95, 149–158.(2017).
doi:10.1016/j.trac.2017.07.022
Derivatization is the most powerful contribution to the identification and quantification of plant cannabinoids (p-CBDs) by gas chromatography-mass spectrometry (GC-MS): providing volatile derivatives with eminent properties (high selectivity, outstanding sensitivity and mass spectrometric peculiarities). These derivatives are excellent candidates to determine the main p-CBDs, like tetrahydrocannabivarin (THCV), tetrahydrocannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), cannabicyclol (CBCL), tetrahydrocannabinol (THC), cannabigerol (CBG), cannabinol (CBN), 11-hydroxy-∆ 9-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH). Identification and quantification of p-CBDs is required partly as trace constituents in complex biological matrices of drug users, partly as main
components in seizure samples: in both cases, in extremely different ratios. GC proposals published between 2000 and 2017, along with outstanding pioneer contributions, are reviewed. Procedures, without derivatization and applying various alkylsilyl, acylation and/or
esterification techniques were listed, compared and criticized. Further sorting was based on the reagent type, on examined matrices, on enrichment/detection related acquisition protocols and on analytical performance characteristics.

The therapeutic potential of cannabis.
Baker D, Pryce G, Giovannoni G, Thompson AJ.
Lancet Neurol. 2003 May;2(5):291-8.
DOI: 10.1016/S1474-4422(03)00381-8
Research of the cannabinoid system has many similarities with that of the opioid system. In both instances, studies into drug-producing plants led to the discovery of an endogenous control system with a central role in neurobiology. Few compounds have had as much positive press from patients as those of the cannabinoid system. While these claims are investigated in disorders such as multiple sclerosis spasticity and pain, basic research is discovering interesting members of this family of compounds that have previously unknown qualities, the most notable of which is the capacity for neuroprotection. Large randomised clinical trials of the better known compounds are in progress. Even if the results of these studies are not as positive as many expect them to be, that we are only just beginning to appreciate the huge therapeutic potential of this family of compounds is clear.

The Therapeutic Potential of Cannabis and Cannabinoids
Franjo Grotenhermen, and Kirsten Müller-Vahl,
Dtsch Arztebl Int. 2012 Jul; 109(29-30): 495–501.
doi: 10.3238/arztebl.2012.0495
Background Cannabis-based medications have been a topic of intense study since the endogenous cannabinoid system was discovered two decades ago. In 2011, for the first time, a cannabis extract was approved for clinical use in Germany.
Methods Selective literature review
Results
Cannabis-based medications exert their effects mainly through the activation of cannabinoid receptors (CB1 and CB2). More than 100 controlled clinical trials of cannabinoids or whole-plant preparations for various indications have been conducted since 1975. The findings of these trials have led to the approval of cannabis-based medicines (dronabinol, nabilone, and a cannabis extract [THC:CBD=1:1]) in several countries. In Germany, a cannabis extract was approved in 2011 for the treatment of moderate to severe refractory spasticity in multiple sclerosis. It is commonly used off label for the treatment of anorexia, nausea, and neuropathic pain. Patients can also apply for government permission to buy medicinal cannabis flowers for self-treatment under medical supervision. The most common side effects of cannabinoids are tiredness and dizziness (in more than 10% of patients), psychological effects, and dry mouth. Tolerance to these side effects nearly always develops within a short time. Withdrawal symptoms are hardly ever a problem in the therapeutic setting.
Conclusion
There is now clear evidence that cannabinoids are useful for the treatment of various medical conditions.
Knowledge about the therapeutic potential of cannabis products has been greatly improved by a large number of clinical trials in recent years (1– 5). In October 2008, the German Medical Association, the National Association of Statutory Health Insurance Physicians, and the Drug Commission of the German Medical Association issued the following statement at a hearing of the Health Committee of the German Federal Parliament (Bundestag): “The benefit of treatment with cannabinoids for a number of medical indications has been shown in controlled trials in which predominantly standardized and/or synthetic cannabinoid preparations were used. The use of such preparations may therefore be reasonable for patients in whom conventional treatment does not achieve adequate relief of symptoms such as spasticity, pain, nausea, vomiting, or loss of appetite” (6). The first cannabis-based medication was approved for use in Germany in 2011. In this article we present the current state of knowledge on the therapeutic application of cannabinoid medications.

Thermal stability of cannabinoids in dried cannabis: a kinetic study
Juris Meija, Garnet Mcrae, Christopher Owen Miles, Jeremy E Melanson
Analytical and Bioanalytical Chemistry January 2021
10.1007/s00216-020-03098-2
This study was undertaken to quantitatively explore the effect of temperature on the degradation of cannabinoids in dried cannabis flower. A total of 14 cannabinoids were monitored using liquid chromatography and tandem mass spectrometry in temperature environments from ? 20 to + 40 °C lasting up to 1 year. We find that a network of first-order degradation reactions is well-suited to model the observed changes for all cannabinoids. While most studies focus on high-temperature effects on the cannabinoids, this study provides high-precision quantitative assessment of room temperature kinetics with applications to shelf-life predictions and age estimates of cannabis products.

Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.
Yuanyuan Liu, Panpan Zhu, Sen Cai, George Haughn and Jonathan E. Page
Plant Mol Biol (2021).
DOI: 10.1007/s11103-021-01129-9
Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate theTHCAS promoter in planta. The O-Box element within the proximal region of the THCASpromoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.

Time course of cannabinoid accumulation and chemotype development during the growth of Cannabis sativa L
D. Pacifico · F. Miselli · A. Carboni · A. Moschella · G. Mandolino
Euphytica (2008) 160:231–240
DOI 10.1007/s10681-007-9543-y
The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gaschromatographic
analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R £ 15R) £ NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe
II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype
III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103 days after sowing, and consistent with the ratios determined on mature inflorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80 days from sowing, and were shown to be simultaneous during the growth period, irrespective of the. Callus cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.

(−)-trans-Δ9‑Tetrahydrocannabiphorol Content of Cannabis sativa Inflorescence from Various Chemotypes
Justin Bueno and Eric A. Greenbaum
J. Nat. Prod. 2021, 84, 531−536
DOI: .10.1021/acs.jnatprod.0c01034
The (− )-trans -Δ9 -tetrahydrocannabiphorol (Δ9 -THCP, 1 ) content of the inflorescence from six Cannabis sativa chemotypes, including 14 plants of distinct genotypes, and two extracts was determined quantitatively via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This represents the first comprehensive quantitative screening for 1 from various C. sativa chemotypes. Compound 1 was detected in all 13 inflorescence samples originating from “ (− )-trans -Δ9 –tetrahydrocannabinol (Δ9 -THC, 2 ) dominant” C. sativa chemotypes, but was not detected in a “ cannabidiol (CBD, 3 ) dominant” chemotype. The inflorescence content of 1 ranged approximately from 0.0023% to 0.0136% (w/w). Comprehensive inflorescence sampling was performed for each specimen investigated. A trend between inflorescence cannabinoid potency and the location of which the inflorescence was sampled on the C. sativa plant was observed for the three cannabinoids tested (1−3 ). The preliminary results obtained indicate Δ9 -THCP (1 ) may have a higher degree of prevalence in C. sativa inflorescence than previously estimated.
((−)-trans-Δ9-Tetrahydrocannabiphorol Content of Cannabis sativa Inflorescence from Various Chemotypes. (Supporting Info pdf Find and add)

Transformation potential of cannabinoids during their passage through engineered water treatment systems: A perspective.
Apul, O. G., Rowles, L. S., Khalid, A., Karanfil, T., Richardson, S. D., & Saleh, N. B.
Environment International, 137, 105586. (2020).*
doi:10.1016/j.envint.2020.105586*
Cannabinoids are incipient contaminants with limited literature in the context of water treatment. With increasing positive public opinion toward legalization and their increasing use as a pharmaceutical, cannabinoids are expected to become a critical class of pollutant that requires attention in the water treatment industry. The destructive removal of cannabinoids via chlorination and other oxidation processes used in drinking water and wastewater treatment requires careful investigation, because the oxidation and disinfection byproducts (DBPs) may pose significant risks for public health and the environment. Understanding transformation of cannabinoids is the first step toward the development of management strategies for this emerging class of contaminant in natural and engineered aquatic systems. This perspective reviews the current understanding of cannabinoid occurrence in water and its potential transformation pathways during the passage through drinking water and wastewater treatment systems with chlorination process. The article also aims to identify research gaps on this topic, which demand attention from the environmental science and engineering community.

Translational Investigation of the Therapeutic Potential of Cannabidiol (CBD): Toward a New Age
José A. Crippa, Francisco S. Guimarães , Alline C. Campos, Antonio W. Zuardi
Front. Immunol. 9:2009.
doi: 10.3389/fimmu.2018.02009
Background: Among the many cannabinoids in the cannabis plant, cannabidiol (CBD) is a compound that does not produce the typical subjective effects of marijuana.
Objectives: The aim of the present review is to describe the main advances in the development of the experimental and clinical use of cannabidiol CBD in neuropsychiatry.
Methods: A non-systematic search was performed for studies dealing with therapeutic applications of CBD, especially performed by Brazilian researchers.
Results: CBD was shown to have anxiolytic, antipsychotic and neuroprotective properties. In addition, basic and clinical investigations on the effects of CBD have been carried out in the context of many other health conditions, including its potential use in epilepsy, substance abuse and dependence, schizophrenia, social phobia, posttraumatic stress, depression, bipolar disorder, sleep disorders, and Parkinson.
Discussion: CBD is an useful and promising molecule that may help patients with a number of clinical conditions. Controlled clinical trials with different neuropsychiatric populations that are currently under investigation should bring important answers in the near future and support the translation of research findings to clinical settings

Treatment of Tourette Syndrome with Delta-9- Tetrahydrocannabinol (D9 -THC): No Influence on Neuropsychological Performance
Kirsten R Mu¨ller-Vahl, Heidrun Prevedel, Karen Theloe, Hans Kolbe, Hinderk M Emrich, and Udo Schneider
Neuropsychopharmacology (2003) 28, 384–388
•DOI: 10.1038/sj.npp.1300047
Previous studies provide evidence that marijuana (Cannabis sativa) and delta-9-tetrahydrocannabinol (D9 -THC), the major psychoactive ingredient of marijuana, respectively, are effective in the treatment of tics and behavioral problems in Tourette syndrome (TS). It, therefore, has been speculated that the central cannabinoid receptor system might be involved in TS pathology. However, in healthy marijuana users there is an ongoing debate as to whether the use of cannabis causes acute and/or long-term cognitive deficits. In this randomized double-blind placebo-controlled study, we investigated the effect of a treatment with up to 10 mg D9 -THC over a 6-week period on neuropsychological performance in 24 patients suffering from TS. During medication and immediately as well as 5–6 weeks after withdrawal of D9 -THC treatment, no detrimental effect was seen on learning curve, interference, recall and recognition of word lists, immediate visual memory span, and divided attention. Measuring immediate verbal memory span, we even found a trend towards a significant improvement during and after treatment. Results from this study corroborate previous data suggesting that in patients suffering from TS, treatment with D9 -THC causes neither acute nor long-term cognitive deficits. Larger and longer-duration controlled studies are recommended to provide more information on the adverse effect profile of THC in patients suffering from TS.

Ultrasound-Assisted Extraction of Cannabinoids from Cannabis Sativa L. Optimized by Response Surface Methodology.
Agarwal, C., Máthé, K., Hofmann, T., & Csóka, L.
Journal of Food Science, 83(3), 700–710. (2018).
doi:10.1111/1750-3841.14075
Ultrasonication was used to extract bioactive compounds from Cannabis sativa L. such as polyphenols, flavonoids, and cannabinoids. The influence of 3 independent factors (time, input power, and methanol concentration) was evaluated on the extraction of total phenols (TPC), flavonoids (TF), ferric reducing ability of plasma (FRAP) and the overall yield. A face-centered central composite design was used for statistical modelling of the response data, followed by regression and analysis of variance in order to determine the significance of the model and factors. Both the solvent composition and the time significantly affected the extraction while the sonication power had no significant impact on the responses. The response predictions obtained at optimum extraction conditions of 15 min time, 130 W power, and 80% methanol were 314.822 mg GAE/g DW of TPC, 28.173 mg QE/g DW of TF, 18.79 mM AAE/g DW of FRAP, and 10.86% of yield. A good correlation was observed between the predicted and experimental values of the responses, which validated the mathematical model. On comparing the ultrasonic process with the control extraction, noticeably higher values were obtained for each of the responses. Additionally, ultrasound considerably improved the extraction of cannabinoids present in Cannabis.

Uncovering the psychoactivity of a cannabinoid from liverworts associated with a legal high.
Chicca, A., Schafroth, M. A., Reynoso-Moreno, I., Erni, R., Petrucci, V., Carreira, E. M., & Gertsch, J.
Science Advances, 4(10), eaat2166.(2018).
doi:10.1126/sciadv.aat2166
Phytochemical studies on the liverwort Radula genus have previously identified the bibenzyl (?)-cis-perrottetinene (cis-PET), which structurally resembles (?)-?9 -trans-tetrahydrocannabinol (?9 -trans-THC) from Cannabis sativa L. Radula preparations are sold as cannabinoid-like legal high on the internet, even though pharmacological data are lacking. Herein, we describe a versatile total synthesis of (?)-cis-PET and its (?)-trans diastereoisomer and demonstrate that both molecules readily penetrate the brain and induce hypothermia, catalepsy, hypolocomotion, and analgesia in a CB1 receptor–dependent manner in mice. The natural product (?)-cis-PET was profiled on major brain receptors, showing a selective cannabinoid pharmacology. This study also uncovers pharmacological differences between ?9 -THC and PET diastereoisomers. Most notably, (?)-cis-PET and (?)-trans-PET significantly reduced basal brain prostaglandin levels associated with ?9 -trans-THC side effects in a CB1 receptor–dependent manner, thus mimicking the action of the endocannabinoid 2-arachidonoyl glycerol. Therefore, the natural product (?)-cis-PET is a psychoactive cannabinoid from bryophytes, illustrating the existence of convergent evolution of bioactive cannabinoids in the plant kingdom. Our findings may have implications for bioprospecting and drug discovery and provide a molecular rationale for the reported effects upon consumption of certain Radula preparations as moderately active legal highs.

Using Cannabidiol as a potential postharvest treatment to maintain quality and extend the shelf life of strawberries.
Inselberg, H., & do Nascimento Nunes, M. C.
Postharvest Biology and Technology, 173, 111416.(2021).
doi:10.1016/j.postharvbio.2020.111416
Cannabis has been used in ancient medicine to treat a wide array of medical issues. Specifically, Cannabidiol (CBD), a non-psychoactive component of cannabis, has been linked to containing antimicrobial properties. However, research surrounding the potential use of CBD as an antimicrobial agent is still preliminary. This study aims to examine the potential of CBD oil as a postharvest treatment used by consumers at home to reduce microbial growth and extend the shelf life of strawberries. CBD oil was applied to fresh fruit after harvest followed by storage at 1 ?C for 8 days and 10 ?C for to 8 days. Strawberries were evaluated for visual quality and microbial load before and during storage. Results from this study showed that CBD oil was effective at maintaining the visual appearance of strawberries, above the minimum threshold of a visual rating score of 3, compared to the fruit that was not treated. It was also found that CBD oil was effective at reducing the microbial load on treated strawberries compared to fruit that was not treated. This research shows that CBD oil has the potential to be used by consumers at home as an effective antimicrobial treatment and to extend strawberry shelf life. visual appearance of strawberries, above the minimum threshold of a visual rating score of 3, compared to the fruit that was not treated. It was also found that CBD oil was effective at reducing the microbial load on treated
strawberries compared to fruit that was not treated. This research shows that CBD oil has the potential to be used by consumers at home as an effective antimicrobial treatment and to extend strawberry shelf life.

Utilisation of Design of Experiments Approach to Optimise Supercritical Fluid Extraction of Medicinal Cannabis.
Rochfort, S., Isbel, A., Ezernieks, V., Elkins, A., Vincent, D., Deseo, M. A., & Spangenberg, G. C.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-66119-1
Carbon dioxide supercritical fuid extraction (CO2 SFE) is a clean and cost-efective method of extracting cannabinoids from cannabis. Using design of experiment methodologies an optimised protocol for extraction of medicinal cannabis bud material (population of mixed plants, combined THC:CBD approximately 1:1.5) was developed at a scale of one kg per extraction. Key variables investigated were CO2 fow rate, extraction time and extraction pressure. A total of 15 batches were analysed for process development using a two-level, full factorial design of experiments for three variable factors over eleven batches. The initial eleven batches demonstrated that CO2 fow rate has the most infuence on the overall yield and recovery of the key cannabinoids, particularly CBD. The additional four batches were conducted as replicated runs at high fow rates to determine reproducibility. The highest extraction weight of 71g (7.1%) was obtained under high fow rate (150g/min), with long extraction time (600min) at high pressure (320bar). This method also gave the best recoveries of THC and CBD. This is the frst study to report the repeated extraction of large amounts of cannabis (total 15kg) to optimise the CO2 SFE extraction process for a pharmaceutical product.

Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods.
Namdar, D., Mazuz, M., Ion, A., & Koltai, H.
Industrial Crops and Products, 113, 376–382.(2018).
doi:10.1016/j.indcrop.2018.01.060
In the last decade, recognition of the therapeutic abilities of Cannabis sativa has risen, along with the need to standardize its products. Standardization requires grading the methods for growing the plant and extracting the active compounds accumulated in its inflorescence. We explored the results of different methods used today and their effect on the levels of compounds extracted from inflorescences positioned along the C. sativa flowering stem. The polarity of the solvent used for the extraction, drying processes and separation methods influenced the chemical composition of the extract. However, regardless of extraction and analytical methods applied, the amounts of cannabinoids and terpenoids in the inflorescences decreased with the position of the sampled in- florescence from top to bottom of the flowering stem. These results have significant implications for the development of growth protocols for C. sativa cultivation and flower extraction methods to standardize cannabisbased products

Very low doses of ?8-THC increase food consumption and alter neurotransmitter levels following weight loss.
Avraham, Y., Ben-Shushan, D., Breuer, A., Zolotarev, O., Okon, A., Fink, N., … Berry, E. M. (2004)
Pharmacology Biochemistry and Behavior, 77(4), 675–684.
doi:10.1016/j.pbb.2004.01.015
We have investigated the effect of 0.001 mg/kg D8 -tetrahydrocannabinol (THC) on food consumption, cognitive function, and neurotransmitters in mice. Sabra mice were treated with vehicle, THC, or THC+CB1 antagonist (SR141716A). The mice were fed for 2.5 h a day for 9 or 50 days. In the 9-day schedule, THC-treated mice showed a 16% increase in food intake compared with controls ( P<001) was recorded. During the course of the 9- and 50-day experimental protocol, all mice lost about 20% and 10% of their original weight, respectively, to reach approximately the same weights, which were not significantly different between the different treatment groups. In addition, THC caused an increase in activity ( P<05) Cognitive function showed a tendency to improve (P<06) in the THC-treated mice, which was reversed by the antagonist for Days 4 and 5 of the maze (P<01 and P<05, ) , respectively). Significant decreases in dopamine and serotonin (5-HT) levels were found both in the hypothalamus (P<01) and the hippocampus (P<01, P<05) ), respectively, while norepinephrine (NE) levels showed tendency to increase in both the hypothalamus and hippocampus. D8 -THC increased food intake significantly more (P<05) than did D9 -THC, while performance and activity were similar. Thus, D8 -THC (0.001 mg/kg) caused increased food consumption and tendency to improve cognitive function, without cannabimimetic side effects. Hence, a low dose of THC might be a potential therapeutic agent in the treatment of weight disorders.

Why Is THCA Decarboxylation Faster than CBDA? an in Silico Perspective
Weiying He, Paul J. Foth, Markus Roggen, Glenn M. Sammis, Pierre Kennepohl
Doi: 10.26434/chemrxiv.12909887.v1
Tetrahydrocannabinol acid (THCA) and cannabidiol acid (CBDA), the two crucial organic components in cannabis and hemp, decarboxylate at different rates to their more active neutral forms. Theoretical calculations are used herein to analyze how the remote annulated ring or pendant substituent influences the rate determining steps of the decarboxylation processes. The uncatalyzed keto-enol tautomerization that precedes decarboxylation is found to be extremely slow in both cases albeit with a ten-fold preference for CBDA. A single molecule of methanol dramatically enhances the reaction rates by allowing for tautomerization through a more favorable six-membered ring transition state. Methanol-catalyzed tautomerization is found to be faster in THCA than in CBDA. This difference results from both the larger dipole moment of the THCA scaffold as well as its greater rigidity relative to CBDA. The greater dipole moment leads to a somewhat better binding of methanol. The lower entropic penalty in THCA towards tautomerization leads to faster decarboxylation


X-Ray structure of cannabispiran: a novel Cannabis constituent.
Ottersen, T., Aasen, A., El-Feraly, F. S., & Turner, C. E.
Journal of the Chemical Society, Chemical Communications, (15), 580 (1976)
doi:10.1039/c39760000580
Cannabispiran, a spiro-compound with a novel skeletal class was isolated from the leaves of Cannabis sativa L. and X-ray crystallographic studies indicated that the compound was 7'-hydroxy-5'-methoxyspiro- (cyclohexane-1, 1'-indan) -4-one (1). COMBINED gas chromatographic-mass spectrometric analysis of the alcoholic extract of an Indian Cannabis (marihuana) variant revealed the presence of a novel constituent which had the same relative retention time1 as ( -)-As-transtetrahydrocannabinol both before silylation (0.43) and after silylation (0.21). This compound was isolated by adsorption chromatography on silica gel and was obtained as colourless crystals, m.p. 178-179 "C, and was optically inactive. Analysis indicated the formula C,,H,,O, which was confirmed by high-resolution mass spectral data.

Yield and cannabinoids contents in different cannabis (Cannabis sativa L.) genotypes for medical use
Anežka Janatováa, Adéla Fra?kováb, Pavel Tlustošc, Karel Hamouza, Mat?j Božikb,
Pavel Klou?ekb,
Industrial Crops & Products 112 (2018) 363–367
Doi: 10.1016/j.indcrop.2017.12.006
In the last decades, there has been a significant increase in the number of lifestyle and auto-immune diseases, such as various cancers or multiple sclerosis. In countries where cannabis is decriminalized for medical purposes, it is most often prescribed for these diagnoses. Today, over 700 different cannabis genotypes are being bred, and it is very important to describe in detail their cultivation, potential yields, chemical profile and stability, to be recommended to a particular patient with a specific diagnosis. The aim of this study was to evaluate the in- florescence yields and the content of ?9 -tetrahydrocannabinol (?9 -THC) and cannabidiol (CBD) of seven traditional genotypes of cannabis – Conspiracy Kush, Nurse Jackie, Jilly Bean, Nordle, Jack Cleaner 2, Jack Skellington and National Health Services. The plants were grown under controlled climatic conditions during six growing cycles at a density of 9 plants/m2 . Dried inflorescences from each plant were homogenized and analyzed by gas chromatography with flame ionization detection. The average yield per plant was 21.02 ± 3.33 g and the highest yields showed genotype Nurse Jackie (24.74 ± 6.11 g). The lowest yields were shown by genotype Jack Skellington (15.41 ± 4.02 g). Average ?9 -THC levels for each variety in all 6 growing cycles ranged from 15.69 ± 2.6 % to 19.31 ± 2.47 % (w/w). The lowest contents of ?9 -THC were measured in the Nordle genotype and the highest values were found in the Jack Cleaner 2 and Jack Skellington genotypes. Average CBD levels in the plants ranged from 0.45 ± 0.1 % to 0.57 ± 0.08 % (w/w) over six individual cycles. This study shows that among genotypes studied, the best parameters – high yield and stable cannabinoids production – are shown by genotypes Nurse Jackie and Jilly Bean

Yielding Morphological Characteristics and Biochemical Analysis of "Karma Lemon" Cannabis Producing Cannabinoids in Thessaloniki-Greece
Dani Fadel, Najoie Assaad, Nour Wahab & Diamanto Lazari
Journal of Agricultural Science Vol. 12, No. 7; 2020
https://www.researchgate.net/publica...aloniki-Greece
Cannabis has been widely used by humans over many centuries as a source of fiber, oil and for medicinal purposes. Its use was illicit in numerous countries, including Greece and Lebanon. “Karma Lemon”, one of the newest cannabis strain originated from Italy, is selected in this study to analyze its components using various techniques starting from the extraction, isolation and identification of cannabinoids using separatory compounds and NMR techniques as well as the main important morphological traits of this strain to be harvested at an
appropriate time for medicinal uses in Greece and later on, in Lebanon. Thirty different samples were selected from the field of respected “Hemp Way Company” in Thessaloniki and studied for morphological traits. These were related to the length of the plant at harvest time (1.809 m) needed for the use of combines and the weight of inflorescence (213.5 g fresh and 40.8 g dry) for oil or seed production. Three samples of Karma Lemon cannabis strain inflorescence were analyzed at the Laboratory of Pharmacognosy, School of Pharmacy, in the Aristotle University of Thessaloniki in Greece, after proper extraction and isolation using ethanol and other separation compounds. TLC and NMR techniques were used to visualize and identify cannabinoids present after isolation. Cannabinoid acids, CBG, CBN, THC, CBD and other cannabinoids were identified and isolated.
 
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IC Cannabinoid Receptors

2-Linoleoylglycerol Is a Partial Agonist of the Human Cannabinoid Type 1 Receptor that Can Suppress 2-Arachidonolyglycerol and Anandamide Activity.
Lu, L., Williams, G., & Doherty, P.
Cannabis and Cannabinoid Research. (2019).
doi:10.1089/can.2019.0030
Introduction: The cannabinoid type 1 (CB1) receptor and cannabinoid type 2 (CB2) receptor are widely expressed in the body and anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are their best characterized endogenous ligands. The diacylglycerol lipases (diacylglycerol lipase alpha and diacylglycerol lipase beta) not only synthesize essentially all the 2-AG in the body but also generate other monoacylglycerols, including 2- linoleoylglycerol (2-LG). This lipid has been proposed to modulate endocannabinoid (eCB) signaling by protecting 2-AG from hydrolysis. However, more recently, 2-LG has been reported to be a CB1 antagonist. Methods: The effect of 2-LG on the human CB1 receptor activity was evaluated in vitro using a cell-based reporter assay that couples CB1 receptor activation to the expression of the b-lactamase enzyme. Receptor activity can then be measured by a b-lactamase enzymatic assay. Results: When benchmarked against 2-AG, AEA, and arachidonoyl-2¢-chloroethylamide (a synthetic CB1 agonist), 2-LG functions as a partial agonist at the CB1 receptor. The 2-LG response was potentiated by JZL195, a drug that inhibits the hydrolysis of monoacylglycerols. The 2-LG response was also fully inhibited by the synthetic CB1 antagonist AM251 and by the natural plant derived antagonist cannabidiol. 2-LG did not potentiate, and only blunted, the activity of 2-AG and AEA. Conclusions: These results support the hypothesis that 2-LG is a partial agonist at the human CB1 receptor and capable of modulating the activity of the established eCBs.

A CB2-Selective Cannabinoid Suppresses T-Cell Activities
and Increases Tregs and IL-10
Rebecca H. Robinson & Joseph J. Meissler & Xiaoxuan Fan & Daohai Yu & Martin W. Adler & Toby K. Eisenstein
J Neuroimmune Pharmacol
DOI 10.1007/s11481-015-9611-3
We have previously shown that agonists selective for the cannabinoid receptor 2 (CB2), including O-1966, inhibit the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, predominantly through effects
on T-cells. Current studies explored the mechanism of this immunosuppression by O-1966 using mouse spleen cells. Treatment with O-1966 dose-relatedly decreased levels of the active nuclear forms of the transcription factors NF-?B and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures generated using a PCR T-cell activation array showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. Further, an increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures. Pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-related decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. These data support the potential of CB2-selective agonists as useful therapeutic agents to prolong graft survival in transplant patients, and strengthens their potential as a new class of immunosuppressive agents with broader applicability.

A Putative ‘Pre-Nervous’ Endocannabinoid System in Early
Echinoderm Development
G.A. Buznikov, L.A. Nikitina, V.V. Bezuglov, M.E.Y. Francisco, G. Boysen, I.N. Obispo-Peak, R.E. Peterson, E.R. Weiss, H. Schuel, B.R.S. Temple, A.L. Morrow, J.M. Lauder. Dev Neurosci 2010;32:1–18
DOI: 10.1159/00023575
Embryos and larvae of sea urchins ( Lytechinus variegatus,
Strongylocentrotus droebachiensis , Strongylocentrotus purpuratus, Dendraster excentricus), and starfish ( Pisaster ochraceus ) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8–16 cell and mid-blastula 2 stage. ‘Perturb-andrescue’ experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from
the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [ 3 H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [ 3 H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs
fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D 1 , D2 or _1 -adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the
multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury
Bela Horváth, Lital Magid, Partha Mukhopadhyay, Sándor Bátkail, Mohanraj Rajesh, Ogyi Park, Galin Tanchian, Rachel Y Gao, Catherine E Goodfellow, Michelle Glass, Raphael Mechoulam and Pál Pacher
British Journal of Pharmacology (2012) 165 2462–2478
DOI:10.1111/j.1476-5381.2011.01381.x
BACKGROUND AND PURPOSE Cannabinoid CB2 receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. EXPERIMENTAL APPROACH We have investigated the effects of a novel CB2 receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7- dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. KEY RESULTS Displacement of [3 H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB2 or CB1 receptors (hCB1/2) yielded Ki values of 6 nM and 1.4 mM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB2 CHO cells (EC50 = 162 nM) and yielded EC50 of 26.4 nM in [35S]GTPgS binding assays using hCB2 expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic proinflammatory chemokines (CCL3 and CXCL2), TNF-a, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-a production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-a. Pretreatment with a CB2 receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB1 antagonist tended to enhance them.
CONCLUSION AND IMPLICATIONS HU-910 is a potent CB2 receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury.

A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ.
Ramot et al. (2013),
PeerJ 1:e40;
DOI 10.7717/peerj.40
Cannabinoid receptors (CB) are expressed throughout human skin epithelium. CB1 activation inhibits human hair growth and decreases proliferation of epidermal keratinocytes. Since psoriasis is a chronic hyperproliferative, inflammatory skin disease, it is conceivable that the therapeutic modulation of CB signaling, which can inhibit both proliferation and inflammation, could win a place in future psoriasis management. Given that psoriasis is characterized by up-regulation of keratins K6 and K16, we have investigated whether CB1 stimulation modulates their expression in human epidermis. Treatment of organ-cultured human skin with the CB1-specific agonist, arachidonoyl-chloro-ethanolamide (ACEA), decreased K6 and K16 staining intensity in situ. At the gene and protein levels, ACEA also decreased K6 expression of cultured HaCaT keratinocytes, which show some similarities to psoriatic keratinocytes. These effects were partly antagonized by the CB1-specific antagonist, AM251. While CB1- mediated signaling also significantly inhibited human epidermal keratinocyte proliferation in situ, as shown by K6/Ki-67-double immunofluorescence, the inhibitory effect of ACEA on K6 expression in situ was independent of its anti-proliferative effect. Given recent appreciation of the role of K6 as a functionally important protein that regulates epithelial wound healing in mice, it is conceivable that the novel CB1- mediated regulation of keratin 6/16 revealed here also is relevant to wound healing. Taken together, our results suggest that cannabinoids and their receptors constitute a novel, clinically relevant control element of human K6 and K16 expression.

A novel probe for the cannabinoid receptor.
Devane, W. A., Breuer, A., Sheskin, T., Jaerbe, T. U. C., Eisen, M. S., & Mechoulam, R. (1992).
Journal of Medicinal Chemistry, 35(11), 2065–2069 doi:10.1021/jm00089a018
The 1,1-dimethylheptyl (DMH) homologue of 7-hydroxy-delta 6-tetrahydrocannabinol (3) is the most potent cannabimimetic substance reported so far. Hydrogenation of 3 leads to a mixture of the epimers of 5'-(1,1-dimethylheptyl)-7-hydroxyhexahydrocannabinol or to either the equatorial (7) or to the axial epimer (8), depending on the catalysts and conditions used. Compound 7 discriminates for delta 1-THC (2) in pigeons (ED50 = 0.002 mg/kg, after 4.5 h), at the potency level of 3, and binds to the cannabinoid receptor with a KD of 45 pM, considerably lower than the Ki of 180 pM measured for compound 3 and the Ki of 2.0 nM measured for CP-55940 (1), a widely employed ligand. Tritiated 7 was used as a novel probe for the cannabinoid receptor.

A time dependent contribution of hippocampal CB1, CB2, and PPAR? receptors to cannabidiol induced disruption of fear memory consolidation.
Raymundi, A. M., Silva, T. R., Zampronio, A. R., Guimarães, F. da S., Bertoglio, L. J., & Stern, C. A. J.
British Journal of Pharmacology. (2019).
doi:10.1111/bph.14895
In preclinical studies, cannabidiol (CBD) mitigates fear memories by facilitating their extinction or interfering with their generalization and reconsolidation. The brain regions and mechanisms underlying these effects, and their temporal window, are still poorly understood. Here, we have investigated related questions in the dorsal hippocampus (DH) during contextual fear consolidation.

Absence of entourage: Terpenoids commonly found in Cannabis sativa do not modulate the functional activity of ?9-THC at human CB1 and CB2 receptors
Marina Santiago, Shivani Sachdev, Jonathon C Arnold, Iain S McGregor, and Mark Connor
Doi: 10.1101/569079
Introduction Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids, may act in concert to elicit therapeutic effects. Cannabinoids such as ?9-tetrahydrocannabinol (?9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), however, it is not known if terpenoids present inCannabis also affect cannabinoid receptor signalling. Therefore, we examined 6 common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signalling in vitro.
Materials and Methods Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real-time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 minutes. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels.
Results ?-Pinene, ?-pinene, ?-caryophyllene, linalool, limonene and ?-myrcene (up to 30-100 µM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by ?9-THC (10µM): (CB1: control, 59±7%; with terpenoids (10 µM each) 55±4%; CB2: ?9-THC 16±5%, with terpenoids (10 µM each) 17±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with ?9-THC and signalling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 minutes the control hyperpolarization recovered by 63±6%, in the presence of the terpenoids recovery was 61±5%.
Discussion None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signalling of the phytocannabinoid agonist ?9-THC. These results suggest that if a phytocannabinoid-terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signalling pathways that do not involve potassium channels, however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioural effect of Cannabis

Activation of Cannabinoid Receptor 2 Prevents Colitis-Associated Colon Cancer through Myeloid Cell De-activation Upstream of IL-22 Production.
Becker, W., Alrafas, H. R., Wilson, K., Miranda, K., Culpepper, C., Chatzistamou, I., … Nagarkatti, P. S.
iScience, 23(9), 101504.(2020).
doi:10.1016/j.isci.2020.101504
Intestinal disequilibrium leads to inflammatory bowel disease (IBD), and chronic inflammation predisposes to oncogenesis. Antigen-presenting dendritic cells (DCs) and macrophages can tip the equilibrium toward tolerance or pathology. Here we show that delta-9-tetrahydrocannabinol (THC) attenuates colitis-associated colon cancer and colitis induced by anti-CD40. Working through cannabinoid receptor 2 (CB2), THC increases CD103 expression on DCs and macrophages and upregulates TGF-b1 to increase T regulatory cells (Tregs). THC-induced Tregs are necessary to remedy systemic IFNg and TNFa caused by anti-CD40, but CB2- mediated suppression of APCs by THC quenches pathogenic release of IL-22 and IL-17A in the colon. By examining tissues from multiple sites, we confirmed that THC affects DCs, especially in mucosal barrier sites in the colon and lungs, to reduce DC CD86. Using models of colitis and systemic inflammation we show that THC, through CB2, is a potent suppressor of aberrant immune responses by provoking coordination between APCs and Tregs.

Activation of CB1 receptors on GABAergic interneurons in the ventrolateral orbital cortex induces analgesia.
Yuwei, W., Dongqin, F., Qiaofen, G., Li, Y., Qian, Z., Han, J., … Liu, Y.
Neuroscience Letters, 135286. (2020).
doi:10.1016/j.neulet.2020.135286
The prefrontal ventrolateral orbital cortex (VLO) is involved in antinociception. It has been found that dopamine receptors, adrenoceptors, serotonin receptors and ?-opioid receptors are involved in this effect through direct/indirect activation of the VLO output neurons. However, the effect of CB1 receptors on the VLO modulation of pain has not been studied. In this study, we investigated whether activation of CB1 receptors in the VLO modulates nociception. A common peroneal nerve (CPN) ligation model was used to induce neuropathic pain in male mice. On day 13 after CPN ligation, spontaneous firing of the VLO pyramidal neurons was recorded and CB1 receptor level in the VLO was detected. Mechanical allodynia was measured after HU210 was microinjected into the VLO. Relative contribution of CB1 receptors on GABAergic neurons and glutamatergic neurons was determined by CB1 receptor knockdown using a viral strategy. Our data indicated that on day 13 after nerve injury, spontaneous firing of the VLO pyramidal neurons reduced significantly but was enhanced by intraperitoneal injection of HU210 (20 ?g/kg), a potent CB1 receptor agonist. Expression of CB1 receptor in the VLO was up-regulated. Microinjection of HU210 into the VLO attenuated allodynia, and this effect was blocked by pre-microinjection of specific CB1 receptor antagonist AM281. Deletion of CB1 receptors on GABAergic neurons in the VLO can completely block HU210-induced analgesia. Thus, it can be concluded that activation of CB1 receptors on GABAergic interneurons in the VLO may be involved in analgesia effect of cannabinoids.

Advances Towards The Discovery of GPR55 Ligands.
Morales P, Jagerovic N
Curr Med Chem. 2016;23(20):2087-100.
DOI:10.2174/0929867323666160425113836
The G-protein-coupled receptor 55 (GPR55) was identified in 1999. It was proposed as a novel member of the endocannabinoid system due to the fact that some endogenous, plant-derived and synthetic cannabinoid ligands act on GPR55. However, the complexity of the cellular downstream signaling pathways related to GPR55 activation delayed the discovery of selective GPR55 ligands. It was only a few years ago that the high throughput screening of libraries of pharmaceutical companies and governmental organizations allowed to identify selective GPR55 agonists and antagonists. Since then, several GPR55 modulator scaffolds have been reported. The relevance of GPR55 has been explored in diverse physiological and pathological processes revealing its role in inflammation, neuropathic pain, bone physiology, diabetes and cancer. Considering GPR55 as a new promising therapeutic target, there is a clear need for new selective and potent GPR55 modulators. This review will address a current structural update of GPR55 ligands.

Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two
McPartland, J. M., MacDonald, C., Young, M., Grant, P. S., Furkert, D. P., & Glass, M. (2017).
Cannabis and Cannabinoid Research, 2(1), 87–95.
doi:10.1089/can.2016.0032
Introduction:Cannabis biosynthesizes ?9-tetrahydrocannabinolic acid (THCA-A), which decarboxylates into ?9-tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB1).
Results:The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB1*and hCB2, equating to approximate Ki*values of 3.1?M and 12.5?M, respectively. THC showed 62-fold greater affinity at hCB1*and 125-fold greater affinity at hCB2. In efficacy tests, THCA-A (10?M) slightly inhibited forskolin-stimulated cAMP at hCB1, suggestive of weak agonist activity, and no measurable efficacy at hCB2.
Discussion:The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10?M approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact—from its inevitable decarboxylation into THC—and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB1or CB2.

Agonists at GPR119 mediate secretion of GLP-1 from mouse enteroendocrine cells through glucose independent pathways
H Lan, HV Lin, CF Wang, MJ Wright, S Xu, L Kang, K Juhl, JA Hedrick and TJ Kowalski
doi: 10.1111/j.1476-5381.2011.01754.x.
BACKGROUND AND PURPOSE
The G protein-coupled receptor 119 (GPR119) mediates insulin secretion from pancreatic b cells and glucagon-like peptide 1 (GLP-1) release from intestinal L cells. While GPR119-mediated insulin secretion is glucose dependent, it is not clear whether or not GPR119-mediated GLP-1 secretion similarly requires glucose. This study was designed to address the glucose dependence of GPR119-mediated GLP-1 secretion, and to explore the cellular mechanisms of hormone secretion in L cells versus those in b cells.
EXPERIMENTAL APPROACH
GLP-1 secretion in response to GPR119 agonists and ion channel modulators, with and without glucose, was analysed in the intestinal L cell line GLUTag, in primary intestinal cell cultures and in vivo. Insulin secretion from Min6 cells, a pancreatic b cell line, was analysed for comparison.
KEY RESULTS
In GLUTag cells, GPR119 agonists stimulated GLP-1 secretion both in the presence and in the absence of glucose. In primary mouse colon cultures, GPR119 agonists stimulated GLP-1 secretion under glucose-free conditions. Moreover, a GPR119 agonist increased plasma GLP-1 in mice without a glucose load. However, in Min6 cells, GPR119-mediated insulin secretion
was glucose-dependent. Among the pharmacological agents tested in this study, nitrendipine, an L-type voltage-dependent calcium channel blocker, dose-dependently reduced GLP-1 secretion from GLUTag cells, but had no effect in Min6 cells in the absence of glucose.
CONCLUSIONS AND IMPLICATIONS
Unlike that in pancreatic b cells, GPR119-mediated GLP-1 secretion from intestinal L cells was glucose-independent in vitro and in vivo, probably because of a higher basal calcium tone in the L cells.

Allosteric Modulation: An Alternate Approach Targeting the Cannabinoid CB1 Receptor.
Nguyen T, Li JX, Thomas BF, Wiley JL, Kenakin TP, Zhang Y.
November 2016Medicinal Research Reviews 37(3)
DOI: 10.1002/med.21418
The cannabinoid CB1 receptor is a G protein coupled receptor and plays an important role in many biological processes and physiological functions. A variety of CB1 receptor agonists and antagonists, including endocannabinoids, phytocannabinoids, and synthetic cannabinoids, have been discovered or developed over the past 20 years. In 2005, it was discovered that the CB1 receptor contains allosteric site(s) that can be recognized by small molecules or allosteric modulators. A number of CB1 receptor allosteric modulators, both positive and negative, have since been reported and importantly, they display pharmacological characteristics that are distinct from those of orthosteric agonists and antagonists. Given the psychoactive effects commonly associated with CB1 receptor agonists and antagonists/inverse agonists, allosteric modulation may offer an alternate approach to attain potential therapeutic benefits while avoiding inherent side effects of orthosteric ligands. This review details the complex pharmacological profiles of these allosteric modulators, their structure-activity relationships, and efforts in elucidating binding modes and mechanisms of actions of reported CB1 allosteric modulators. The ultimate development of CB1 receptor allosteric ligands could potentially lead to improved therapies for CB1-mediated neurological disorders.

Allosteric Modulators of the CB1 Cannabinoid Receptor: A Structural Update Review Paula Morales, Pilar Goya, Nadine Jagerovic, and Laura Hernandez-Folgado
Cannabis and Cannabinoid Research
DOI: 10.1089/can.2015.0005
In 2005, the first evidence of an allosteric binding site at the CB1R was provided by the identification of three indoles of the company Organon that were allosteric enhancers of agonist binding affinity and, functionally, al- losteric inhibitors of agonist activity. Since then, structure–activity relationships of indoles as CB1R modulators have been reported. Targeting the allosteric site on CB1R, new families structurally based on urea and on 3- phenyltropane analogs of cocaine have been discovered as CB1R-negative allosteric modulators (NAMs), respec- tively, by Prosidion and by the Research Triangle Park. Endogenous allosteric ligands of different nature have been identified more recently. Thus, the therapeutic neuroprotection application of lipoxin A4, an arachidonic acid derivative, as an allosteric enhancer of CB1R activity has been confirmed in vivo. It was also the case of the steroid hormone, pregnenolone, whose negative allosteric effects on D9-tetrahydrocannabinol (D9-THC) were reproduced in vivo in a behavioral tetrad model and in food intake and memory impairment assays. Curi- ously, the peroxisome proliferator-activated receptor-c agonist fenofibrate or polypeptides such as pepcan-12 have been shown to act on the endocannabinoid system through CB1R allosteric modulation. The mechanistic bases of the effects of the phytocannabinoid cannabidiol (CBD) are still not fully explained. However, there is evidence that CBD behaves as an NAM of D9-THC- and 2-AG. Allosteric modulation at CB1R offers new oppor- tunities for therapeutic applications. Therefore, further understanding of the chemical features required for allo- steric modulation as well as their orthosteric probe dependence may broaden novel approaches for fine-tuning the signaling pathways of the CB1R.

An endocannabinoid catabolic enzyme FAAH and its paralogs in an early land plant reveal evolutionary and functional relationship with eukaryotic orthologs.
Haq, I., & Kilaru, A.
Scientific Reports, 10(1).(2020).
doi:10.1038/s41598-020-59948-7
Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identifed nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37°C and pH 8.0, with higher specifcity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identifed that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

An Examination of Anthocyanins’ and Anthocyanidins’ Affinity for Cannabinoid Receptors
Gabriele Korte, Andrea Dreiseitel, Peter Schreier, Anett Oehme, Sanja Locher, Goeran Hajak, and Philipp G. Sand
Journal of Medicinal Food, 12(6), 1407–1410.
doi:10.1089/jmf.2008.0243
A growing body of evidence suggests that anthocyanins and anthocyanidins may possess analgesic properties in addition to neuroprotective and anti-inflammatory activities. These functionalities suggest a role for the cannabinoid receptor (CB) in mediating biological effects. Competitive radioligand binding assays identified cyanidin (Ki¼ 16.2 mM) and delphinidin (Ki ¼ 21.3 mM) as ligands with moderate affinity to human CB1. For CB2, similar affinities were achieved by cyanidin (Ki ¼ 33.5 mM), delphinidin (Ki ¼ 34.3 mM), and peonidin (Ki ¼ 46.4 mM). Inhibition constants >50 mM were obtained for pelargonidin, malvidin, cyanidin-3,5-di-O-glucoside, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and cyanidin-3- O-rutinoside for both CB subtypes.


Anandamide, an Endogenous Cannabinoid Receptor Ligand, Also Interacts with 5-Hydroxytryptamine (5-HT) Receptor.
Toshiyuki KIMURA, Tomoko OHTA, Kazuhito Watanabe, Ikuo YAMAMOTO
Biological & Pharmaceutical Bulletin 21(3):224-6 April 1998
DOI: 10.1248/bpb.21.224
Interactions of anandamide (N-arachidonylethanolamide), an endogenous compound for cannabinoid receptors, with the receptors for 5-hydroxytryptamine (5-HT), benzodiazepine, and gamma-aminobutyric acid(A) (GABA[A]) receptors in bovine synaptic membrane were examined. Anandamide decreased the 5-HT receptor bindings at concentrations of 1-100 microM, although it did not cause any change in benzodiazepine or GABA(A) receptor bindings. A high concentration of anandamide, 100 microM, significantly decrease both [3H]5-HT and [3H]ketanserin bindings. The present study revealed that the pharmacological activity of anandamide might be partially mediated through the 5-HT receptor.

*Anandamide, an endogenous ligand of the cannabinoid receptor, induces hypomotility and hypothermia in vivo in rodents
Jacqueline N.Crawley, Rebecca L.Corwin, John K.Robinson, Christian C.Felder, William A.Devane, Julius Axelrod
Pharmacology Biochemistry and Behavior Volume 46, Issue 4, December 1993, Pages 967-972
DOI: 10.1016/0091-3057(93)90230-q
Anandamine (arachidonylethanolamide), an arachidonic acid derivative isolated from the porcine brain, displays binding characteristics indicative of an endogenous ligand for the cannabinoid receptor. The functional activity of anandamide was tested in vivo using behavioral and physiological paradigms in laboratory rodents. At IP doses from 2 to 20 mg/kg in mice, anandamide significantly decreased spontaneous motor activity in a Digiscan open field. Rectal body temperature significantly decreased at doses of 10 and 20 mg/kg in rats. At doses from 0.03 to 30 mg/kg, anandamide had no significant effect on chow consumption in ad lib fed rats. Over the dose range of 2–20 mg/kg, anandamide did not show anxiolytic properties in the mouse light ? dark exploration model of anxiety. Over the dose range of 0.3–3 mg/kg, anandamide had no effect on choice accuracy or session duration in the delayed nonmatching to sample memory task (DNMTS) in rats. These results demonstrate that anandamide has biological and behavioral effects in awake rodents, some of which are similar to the reported actions of THC.

Anandamide uptake explained?
Fowler, C. J.
Trends in Pharmacological Sciences, 33(4), 181–185.(2012).
doi:10.1016/j.tips.2012.01.001
The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol are removed from the extracellular space by a process of cellular uptake followed by metabolism. Although the enzymes responsible for endocannabinoid metabolism have been well characterised, the processes involved in uptake have been the subject of much controversy. Recent studies, however, have identified intracellular transport proteins (fatty acid binding proteins 5 and 7, heat shock protein 70, albumin, and fatty acid amide hydrolase-like AEA transporter protein) that shuttle AEA from the plasma membrane to its metabolic enzymes. Proteins such as the fatty acid amide hydrolase-like anandamide transporter protein may be useful targets for novel therapeutic strategies aimed at potentiating AEA signalling. In this article I review the current state of the art of endocannabinoid uptake.

Anticonvulsive and anti-epileptogenesis effects ofEchinacea purpurea root extract, an involvement of CB2 receptor
Masoumeh Gholami , Jamal Amri , Saeed Pazhoohan , Mehdi Sadegh
J Complement Integr Med. 2021 Aug 31.
doi: 10.1515/jcim-2020-0219
Objective: Phytocannabinoids beyond the Δ9-tetrahy-drocannabinol have shown anticonvulsive effects. Also, alkylamides from Echinacea purpurea have been proved as cannabinomimetics. We examined the effect of the hydroalcoholic root extract of E. purpurea on pentylenetetrazol (PTZ)-induced tonic-clonic seizures and kindling model of epileptogenesis and the involvement of CB2 receptors as the mediator of this effect.
Methods: Male Wistar rats (200 ± 20 g) were used. Single intraperitoneal (i.p.) injection of PTZ (80 mg/kg) was used to induce tonic-clonic seizures. The kindling model of epileptogenesis was induced by daily injections of PTZ (37 mg/kg; i.p. for 15 days). Latency and duration of the stages were monitored for analysis. The hydroalcoholic root extract ofE. purpurea was injected (i.p.) 20 min before seizure induction at the doses of 10, 50, 100 and 200 mg/kg. CB2 receptor antagonist SR144528 was injected (0.1 mg/kg; i.p.) 20 min before the Echinacea injection.
Results: In the tonic-clonic model, pretreatment with E. purpurea at the doses of 100 and 200 mg/kg significantly increased latencies to S2-S6, while it significantly decreased S6 duration and mortality rate. SR144528 injection before the injection of 100 mg/kg of E. purpurea significantly prevented the effects of the extract on S4-S6 latencies. In the kindling model, E. purpurea at the doses of 100 and 200 mg/kg significantly delayed epileptogenesis and decreased mortality rate, while SR144528 injection before the injection of 100 mg/kg of E. purpurea significantly blocked this effect of the extract.
Conclusion: These findings revealed the anticonvulsive and antiepileptogenesis effects of the E. purpurea root extract, which can be mediated by CB2 receptors.
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Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review
John McPartland, Marnie Duncan, Vincenzo Di Marzo, Roger G Pertwee
British Journal of Pharmacology 172(3) September 2014
DOI: 10.1111/bph.12944
https://www.researchgate.net/publica...tematic_review
Based on evidence that the therapeutic properties of Cannabis preparations are not solely dependent on the presence of Δ9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid type-1 (CB1) receptors, thus behaving like first generation CB1 inverse agonists, such as rimonabant. Here we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent preclinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action, and thus appear very unlikely to produce unwanted central nervous system effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low affinity CB1 ligand which can nevertheless affect CB1 activity in vivo in an indirect manner, whilst THCV is a high affinity CB1 ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 antagonism. THCV also has high affinity for CB2 and signals as a partial agonist, a departure from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology, and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target.

Beta-caryophyllene is a dietary cannabinoid.
Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, Altmann KH, Karsak M, Zimmer A.
PNAS July 1, 2008 105 (26) 9099-9104
doi: 10.1073/pnas.0803601105
The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Brain CB2 Receptors: Implications for Neuropsychiatric Disorders
Michelle Roche, and David P Finn
Pharmaceuticals 2010, 3, 2517-2553;
doi:10.3390/ph3082517

https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4033937/pdf/pharmaceuticals-03-02517.pdf
Although previously thought of as the peripheral cannabinoid receptor, it is now accepted that the CB2 receptor is expressed in the central nervous system on microglia, astrocytes and subpopulations of neurons. Expression of the CB2 receptor in the brain is significantly lower than that of the CB1 receptor. Conflicting findings have been reported on the neurological effects of pharmacological agents targeting the CB2 receptor under normal conditions. Under inflammatory conditions, CB2 receptor expression in the brain is enhanced and CB2 receptor agonists exhibit potent anti-inflammatory effects. These findings have prompted research into the CB2 receptor as a possible target for the treatment of neuroinflammatory and neurodegenerative disorders. Neuroinflammatory alterations are also associated with neuropsychiatric disorders and polymorphisms in the CB2 gene have been reported in depression, eating disorders and schizophrenia. This review will examine the evidence to date for a role of brain CB2 receptors in neuropsychiatric disorders.

Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells.
Sachdev S, Boyd R, Grimsey NL, Santiago M, Connor M. 2019.
PeerJ 7:e7733 (2019)
DOI: 10.7717/peerj.7733
Background. Synthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of coconsumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid 19 -tetrahydrocannabinol (19 -THC). Methods. The effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2. Results. BFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or 19 -THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or 19 -THC to AtT20 cells. Discussion. BFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the ‘‘contamination’’ was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

(+)-Cannabidiol analogues which bind cannabinoid receptors but exert peripheral activity only
Ester Fride , Cfir Feigin, Datta E Ponde, Aviva Breuer, Lumír Hanus, Nina Arshavsky, Raphael Mechoulam
Eur J Pharmacol . 2004 Dec 15;506(2):179-88.
doi: 10.1016/j.ejphar.2004.10.049.
https://www.researchgate.net/publica..._activity_only
Delta9-Tetrahydrocannabinol (Delta9-THC) and (-)-cannabidiol are major constituents of the Cannabis sativa plant with different pharmacological profiles: (-)-Delta9-tetrahydrocannabinol, but not (-)-cannabidiol, activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. We have tested a series of (+)-cannabidiol derivatives, namely, (+)-cannabidiol-DMH (DMH-1,1-dimethylhepty, (+)-7-OH-cannabidiol-DMH, (+)-7-OH- cannabidiol, (+)-7-COOH- cannabidiol and (+)-7-COOH-cannabidiol-DMH, for central and peripheral (intestinal, antiinflammatory and peripheral pain) effects in mice. Although all (+)-cannabidiols bind to cannabinoid CB1 and CB2 receptors, only (+)-7-OH-cannabidiol-DMH was centrally active, while all (+)-cannabidiol analogues completely arrested defecation. The effects of (+)-cannabidiol-DMH and (+)-7-OH-cannabidiol-DMH were partially antagonized by the cannabinoid CB1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716), but not by the cannabinoid CB2 receptor antagonist N-1S)-endo-1,3,3-trimethil bicyclo [2.2.1] heptan-2-yl-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), and had no effect on CB1(-/-) receptor knockout mice. (+)-Cannabidiol-DMH inhibited the peripheral pain response and arachidonic-acid-induced inflammation of the ear. We conclude that centrally inactive (+)-cannabidiol analogues should be further developed as antidiarrheal, antiinflammatory and analgesic drugs for gastrointestinal and other peripheral conditions.
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Cannabigerol Action at Cannabinoid CB1 and CB2 Receptors and at CB1–CB2 Heteroreceptor Complexes Gemma Navarro, Katia Varani, Irene Reyes-Resina, Verónica Sánchez de Medina, Rafael Rivas-Santisteban, Carolina Sánchez-Carnerero Callado, Fabrizio Vincenzi, Salvatore Casano, Carlos Ferreiro-Vera, Enric I. Canela, Pier Andrea Borea, Xavier Nadal and Rafael Franco Front. Pharmacol. 9:632. (2018)
doi: 10.3389/fphar.2018.00632
Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB1 (CB1 R) and CB2 (CB2 R) receptors and the effects of the compound on agonist activation of those receptors and of CB1 –CB2 heteroreceptor complexes. Using [3 H]-CP-55940, CBG competed with low micromolar Ki values the binding to CB1 R and CB2 R. Homogeneous binding in living cells, which is only technically possible for the CB2 R, provided a 152 nM Ki value. Also interesting, CBG competed the binding of [3 H]-WIN-55,212-2 to CB2 R but not to CB1 R (Ki : 2.7 versus > 30 m M). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1–1 m M. cAMP, pERK, b -arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB2 R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB2 R. The effect of CBG on CB1 R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling.

Cannabinoid CB Receptor Activation Attenuates Fentanyl-Induced Respiratory Depression
Carmen A. Zavala, Ana C. Thomaz, Vishakh Iyer, Ken Mackie, and Andrea G. Hohmann
Cannabis and Cannabinoid Research 2021
DOI: 10.1089/can.2020.0059
https://www.liebertpub.com/doi/epub/.../can.2020.0059
Introduction: Overdose fatalities associated with the opioid epidemic are predictably attributable to drug-induced respiratory depression. In terms of illicit opioid abuse, fentanyl is the synthetic opioid responsible for the largest number of overdose deaths. There is, therefore, an urgent need to identify safe and effective therapeutics that can attenuate fentanyl-induced respiratory depression. Identi cation of effective alternate analgesic strategies that lessen the respiratory depression associated with narcotics would also help improve current strategies for pain management. Our laboratory recently reported that the G protein-biased CB cannabinoid receptor agonist LY2828360 suppressed chemotherapy-induced neuropathic nociception and attenuated both morphine tolerance and physical dependence in paclitaxel-treated mice. However, the impact of LY2828360 on other undesirable side effects of opioids, such as opioid-induced respiratory depression, remains unknown.
Materials and Methods: We used whole-body plethysmography to assess the impact of the CB cannabinoid agonist
LY2828360 on fentanyl-induced respiratory depression using wild-type (WT) and CB knockout (CB KO) mice.
Results: Fentanyl reduced minute ventilation and respiratory frequency without altering tidal volume in both WT and CB KO mice. In WT mice, the high dose of fentanyl (0.2 mg/kg intraperitoneal [i.p.]) produced a greater suppression of respiratory parameters compared with the low dose of fentanyl (0.1 mg/kg i.p.). Coadministration of a behaviorally active dose of LY2828360 (3 mg/kg i.p.) with fentanyl (0.2 mg/kg i.p.) attenuated fentanyl-induced respiratory depression in WT mice. Notably, LY2828360 (3 mg/kg i.p.) did not attenuate fentanyl-induced respiratory depression in CB KO mice, consistent with mediation by CB receptors. Moreover, LY2828360 (3 mg/kg i.p.) alone lacked intrinsic effects on respiratory parameters in either WT or CB KO mice.
Conclusion: The combination of a CB agonist with fentanyl may represent a safer adjunctive therapeutic strategy compared with a narcotic analgesic alone by attenuating the development of opioid-induced respiratory depression. Moreover, the CB agonist, administered alone, did not alter respiration. Our  ndings suggest that the CB cannabinoid agonist LY2828360 may provide CB -mediated protection against fentanyl-induced respiratory depression, a detrimental and unwanted side effect of opioid use and abuse.

Cannabinoid (CB-1) receptor antagonists: a molecular approach to treating acute cannabinoid overdose.
Skolnick, P., & Crystal, R
Journal of Neural Transmission. (2019).
doi:10.1007/s00702-019-02132-7
The legalization of cannabis for both recreational and medical use in the USA has resulted in a dramatic increase in the number of emergency department visits and hospital admissions for acute cannabinoid overdose (also referred to as cannabis intoxication and cannabis poisoning). Both “edibles” (often sold as brownies, cookies, and candies) containing large amounts of ?9 -tetrahydrocannabinol and synthetic cannabinoids (many possessing higher potencies and efcacies than ?9 -tetrahydrocannabinol) are responsible for a disproportionate number of emergency department visits relative to smoked cannabis. Symptoms of acute cannabinoid overdose range from extreme lethargy, ataxia, and generalized psychomotor impairment to feelings of panic and anxiety, agitation, hallucinations, and psychosis. Treatment of acute cannabinoid overdose is currently supportive and symptom driven. Converging lines of evidence indicating many of the symptoms which can precipitate an emergency department visit are mediated through activation of cannabinoid1 receptors. Here, we review the evidence that cannabinoid1 receptor antagonists, originally developed for indications ranging from obesity to smoking cessation and schizophrenia, provide a molecular approach to treating acute cannabinoid overdose.

Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons drives overconsumption of palatable food and obesity
Inigo Ruiz de Azua, Elena Martin-Garcia, Laura Domingo-Rodriguez, Alejandro Aparisi Rey, Diego Pascual Cuadrado, Larglinda Islami, Petri Turunen, Floortje Remmers, Beat Lutz & Rafael Maldonado
Neuropsychopharmacology (2021)
Palatable food can promote overfeeding beyond homeostatic requirements, thereby constituting a major risk to obesity. Here, the lack of cannabinoid type 1 receptor (CB1) in dorsal telencephalic glutamatergic neurons (Glu-CB1-KO) abrogated the overconsumption of palatable food and the development of obesity. On low-fat diet, no genotype differences were observed. However, under palatable food conditions, Glu-CB1-KO mice showed decreased body weight and food intake. Notably, Glu-CB1-KO mice were protected from alterations in the reward system after high-fat diet feeding. Interestingly, obese wild-type mice showed a superior olfactory detection as compared to mutant mice, suggesting a link between overconsumption of palatable food and olfactory function. Reconstitution of CB1 expression in olfactory cortex in high-fat diet-fed Glu-CB1-KO mice using viral gene delivery partially reversed the lean phenotype concomitantly with improved odor perception. These findings indicate that CB1 in cortical glutamatergic neurons regulates hedonic feeding, whereby a critical role of the olfactory cortex was uncovered as an underlying mechanism.

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/ threonine kinases to activate ERK in neuronal cells
George D Dalton and Allyn C Howlett
British Journal of Pharmacology (2012) 165 2497–2511
DOI:10.1111/j.1476-5381.2011.01455.x
BACKGROUND AND PURPOSE Signalling networks that regulate the progression of cannabinoid CB1 receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB1 receptor-stimulated ERK phosphorylation in a neuronal cell model.
EXPERIMENTAL APPROACH Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB1 receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.
KEY RESULTS The time-course of CB1 receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB1 receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires Gi/o bg subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by theprotein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB1 receptor-stimulated, ligand-independent, transactivation of multiple RTKs.
CONCLUSIONS AND IMPLICATIONS The complex expression of CB1 receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB1-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways
O Gomez, A Sanchez?Rodriguez, MQU Le, C Sanchez?Caro, F Molina?Holgado, E Molina?Holgado
British Journal of Pharmacology (2011) 163 1520–1532
doi: 10.1111/j.1476-5381.2011.01414.x
BACKGROUND AND PURPOSE The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocytedifferentiation , we exposed differentiating cultures of oligodendrocytes with cannabinoid CB1, CB2 and CB1/CB2 receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity.
EXPERIMENTAL APPROACH Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB1, CB2 and CB1/CB2 receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB1 receptor) and AM630 (CB2 receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation.
KEY RESULTS ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation.
CONCLUSIONS AND IMPLICATIONS Activation of cannabinoid CB1 or CB2 receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

Cannabinoid Receptor 1 Binding Activity and Quantitative Analysis of Cannabis sativa L. Smoke and Vapor
Justin FISCHEDICK, Frank VAN DER KOOY,* and Robert VERPOORTE
Chem. Pharm. Bull. 58(2) 201—207 (2010)
DOI: 10.1248/cpb.58.201
Cannabis sativa L. (cannabis) extracts, vapor produced by the Volcano® vaporizer and smoke made from burning cannabis joints were analyzed by GC-flame ionization detecter (FID), GC-MS and HPLC. Three different medicinal cannabis varieties were investigated Bedrocan®, Bedrobinol® and Bediol®. Cannabinoids plus other components such as terpenoids and pyrolytic by-products were identified and quantified in all samples. Cannabis vapor and smoke was tested for cannabinoid receptor 1 (CB1) binding activity and compared to pure D9 -tetrahydrocannabinol (D9 -THC). The top five major compounds in Bedrocan® extracts were D9 -THC, cannabigerol (CBG), terpinolene, myrcene, and cis-ocimene in Bedrobinol® D9 -THC, myrcene, CBG, cannabichromene (CBC), and camphene in Bediol® cannabidiol (CBD), D9 -THC, myrcene, CBC, and CBG. The major components in Bedrocan® vapor (1.0 mg/g) were D9 -THC, terpinolene, myrcene, CBG, cis-ocimene and CBD in Bedrobinol® D9 -THC, myrcene and CBD in Bediol® CBD, D9 -THC, myrcene, CBC and terpinolene. The major components in Bedrocan® smoke (1.0 mg/g) were D9 -THC, cannabinol (CBN), terpinolene, CBG, myrcene and cis-ocimene in Bedrobinol® D9 -THC, CBN and myrcene in Bediol® CBD, D9 -THC, CBN, myrcene, CBC and terpinolene. There was no statistically significant difference between CB1 biinding of pure Delta(9)-THC compared to cannabis smoke and vapor at an equivalent concentration of Delta(9)-THC.

Cannabinoid receptor expression in non-small cell lung cancer. Effectiveness of tetrahydrocannabinol and cannabidiol inhibiting cell proliferation and epithelial-mesenchymal transition in vitro.
Milian, L., Mata, M., Alcacer, J., Oliver, M., Sancho-Tello, M., Martín de Llano, J. J., … Carda, C.
PLOS ONE, 15(2), (2020).
doi:10.1371/journal.pone.0228909
Background/Objective Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines. Methods Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10–100 ?M), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF). Results The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increased survival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used. Conclusions The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.

Cannabinoid Receptor Interacting Protein (CRIP) 1a competition with ?-arrestin for CB1 receptor binding sites
Lawrence C. Blume, Theresa Patten, Khalil Eldeeb, Sandra Leone-Kabler, Alexander A. Ilyasov, Bradley M. Keegan, Jeremy E. O’Neal, Caroline E. Bass, Roy R. Hantgan, W. Todd Lowther, Dana E. Selley, and Allyn C. Howlett
Molecular Pharmacology Fast Forward. Published on November 28, 2016
DOI: 10.1124/mol.116.104638
Cannabinoid Receptor Interacting Protein1a (CRIP1a) is a CB1 receptor (CB1R) distal Cterminus-associated protein that alters CB1R interactions with G-proteins (Blume et al.,2015; Smith et al.,2015). We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with ?-arrestin proteins that interact with the CB1R at the C-terminal. Co-immunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or ?-arrestin, but CRIP1a and ?-arrestin fail to co-immunoprecipitate with each other. This suggests a competition for CRIP1a and ?-arrestin binding to the CB1R, which we hypothesized could attenuate the action of ?-arrestin to mediate CB1R internalization. We determined that agonist mediated reduction of the density of cell surface endogenously-expressed CB1Rs was clathrinand dynamin-dependent, and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a over-expression attenuated CP55940-mediated GFP-CB1R as well as endogenous ?-arrestin redistribution to punctae, and conversely, CRIP1a knock-down augmented ?-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer
peptide reduced CB1R-CRIP1a association. Co-immunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with ?-arrestin1, and phosphorylated central or distal C-terminal peptides competed
for association with ?-arrestin2. Thus, CRIP1a can compete with ?-arrestins for interaction with C-terminal CB1R domains which could affect agonist-driven, ?-arrestin-mediated internalization of the CB1R.

Cannabinoid receptor signalling in neurodegenerative diseases: a potential role for membrane fluidity disturbance
M Maccarrone, G Bernardi, A Finazzi Agrò, D Centonze
doi: 10.1111/j.1476-5381.2011.01277.x
Type?1 cannabinoid receptor (CB1) is the most abundant G?protein?coupled receptor (GPCR) in the brain. CB1 and its endogenous agonists, the so?called ‘endocannabinoids (eCBs)’, belong to an ancient neurosignalling system that plays important functions in neurodegenerative and neuroinflammatory disorders like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. For this reason, research on the therapeutic potential of drugs modulating the endogenous tone of eCBs is very intense. Several GPCRs reside within subdomains of the plasma membranes that contain high concentrations of cholesterol: the lipid rafts. Here, the hypothesis that changes in membrane fluidity alter function of the endocannabinoid system, as well as progression of particular neurodegenerative diseases, is described. To this end, the impact of membrane cholesterol on membrane properties and hence on neurodegenerative diseases, as well as on CB1 signalling in vitro and on CB1?dependent neurotransmission within the striatum, is discussed. Overall, present evidence points to the membrane environment as a critical regulator of signal transduction triggered by CB1, and calls for further studies aimed at better clarifying the contribution of membrane lipids to eCBs signalling. The results of these investigations might be exploited also for the development of novel therapeutics able to combat disorders associated with abnormal activity of CB1.

Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation
Ismael Galve-Roperh, Valerio Chiurchiù, Javier Díaz-Alonso, Monica Bari, Manuel Guzmán, Mauro Maccarrone
Progress in Lipid Research 52 (2013) 633–650
DOI: 10.1016/j.plipres.2013.05.004
https://www.researchgate.net/publica...ifferentiation
Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB1) and type-2 (CB2) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB1 and CB2 receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease.

Cannabinoid receptor type-1: breaking the dogmas
Arnau Busquets Garcia, Edgar Soria-Gomez, Luigi Bellocchio, and Giovanni Marsicano
DOI: 10.12688/f1000research.8245.1
The endocannabinoid system (ECS) is abundantly expressed in the brain. This system regulates a plethora of physiological functions and is composed of cannabinoid receptors, their endogenous ligands (endocannabinoids), and the enzymes involved in the metabolism of endocannabinoids. In this review, we highlight the new advances in cannabinoid signaling, focusing on a key component of the ECS, the type-1 cannabinoid receptor (CB1). In recent years, the development of new imaging and molecular tools has demonstrated that this receptor can be distributed in many cell types (e.g., neuronal or glial cells) and intracellular compartments (e.g., mitochondria). Interestingly, cellular and molecular effects are differentially mediated by CB1 receptors according to their specific localization (e.g., glutamatergic or GABAergic neurons). Moreover, this receptor is expressed in the periphery, where it can modulate periphery-brain connections. Finally, the better understanding of the CB1 receptor structure led researchers to propose interesting and new allosteric modulators. Thus, the advances and the new directions of the CB1 receptor field will provide new insights and better approaches to profit from its interesting therapeutic profile.

Cannabinoid receptor 2 activation alleviates septic lung injury by promoting autophagy via inhibition of inflammatory mediator release.
Liu, A. P., Yuan, Q. H., Zhang, B., Yang, L., He, Q. W., Chen, K., … Zhan, J.
Cellular Signalling, 109556. (2020).
doi:10.1016/j.cellsig.2020.109556
Septic lung injury is one of main causes of high mortality in severe patients. Inhibition of excessive inflammatory response is considered as an effective strategy for septic lung injury. Previous studies have shown that cannabinoid receptor 2 (CB2), a G protein-coupled receptor, play an important role in immunosuppression. Whether CB2 can be used as a therapeutic target for septic lung injury is unclear. The aim of this study is to explore the role of CB2 in sepsis and its potential mechanism. In this study, treatment with HU308, a specific agonist of CB2, could reduce lung pathological injury, decrease the level of inflammatory cytokines and strengthen the expression of autophagy-related gene after cecal ligation puncture (CLP)-induced sepsis in mice. Similar results were obtained in RAW264.7 macrophages after LPS treatment. Furthermore, the effect of HU308 could be blocked by autophagy blocker 3-MA in vivo and in vitro. These results suggest that CB2 serves as a protective target for septic lung injury by decreasing inflammatory factors, which is associated with the enhancement of autophagy.

Cannabinoid Receptor 2 Functional Variant Contributes to the Risk for Pediatric Inflammatory Bowel Disease.
Strisciuglio C1, Bellini G, Miele E, Martinelli M, Cenni S, Tortora C, Tolone C, Miraglia Del Giudice E, Rossi F.
DOI: 10.1097/MCG.0000000000000755
GOALS:
We conducted a case-control association analysis to establish the role of a common CB2 functional variant, Q63R, in the susceptibility to inflammatory bowel disease (IBD).
BACKGROUND:
Endocannabinoids may limit intestinal inflammation through cannabinoid receptor 1 and/or 2 (CB1, CB2).
STUDY:
We genotyped 217 pediatric IBD patients [112 Crohn's disease (CD), 105 ulcerative colitis (UC)] and 600 controls for the CB2-Q63R variant by Taqman assay. Data were collected from clinical records on age at diagnosis, disease activity, duration and location, extraintestinal manifestations, therapy, clinical relapses, and need for surgery.
RESULTS:
We found a significant association of the CB2-R63 variant with IBD (allele frequencies, P=0.04; genotype distributions, P=0.0006), in particular with CD (allele frequencies, P=0.002; genotype distributions, P=0.00005) and with UC only for genotype distributions (P=0.03). RR carriers showed an increased risk for developing IBD [odds ratio (OR)=1.82; P=0.0002 for IBD; OR=2.02; P=10 for CD; OR=1.63; P=0.02 for UC at 95% confidence interval]. Upon genotype-phenotype evaluation, RR patients showed an increased frequency of moderate-to-severe disease activity at diagnosis in the case of both CD and UC (P=0.01 and P=0.02, respectively) and also an earlier clinical relapse in UC (P=0.04). In UC, all the clinical features related to the CB2 risk allele were still significantly associated with the variant when analyzed using a multivariate logistic regression model (P=0.001).
CONCLUSIONS:
The CB2-Q63R variant contributes to the risk for pediatric IBD, in particular CD. The R63 variant is associated with a more severe phenotype in both UC and CD. Taken together, our data point toward the involvement of the CB2 receptor in the pathogenesis and clinical features of pediatric IBD.

Cannabinoid receptor 2: Potential role in immunomodulation and neuroinflammation Review
Slava Rom, and Yuri Persidsky
J Neuroimmune Pharmacol. 2013 June ; 8(3): 608–620
doi:10.1007/s11481-013-9445-9
https://www.ncbi.nlm.nih.gov/pmc/art...ihms453729.pdf
An accumulating body of evidence suggests that endocannabinoids and cannabinoid receptors type 1 and 2 (CB1, CB2) play a significant role in physiologic and pathologic processes, including cognitive and immune functions. While the addictive properties of marijuana, an extract from the Cannabis plant, are well recognized, there is growing appreciation of the therapeutic potential of cannabinoids in multiple pathologic conditions involving chronic inflammation (inflammatory bowel disease, arthritis, autoimmune disorders, multiple sclerosis, HIV-1 infection, stroke, Alzheimer’s disease to name a few), mainly mediated by CB2 activation. Development of CB2 agonists as therapeutic agents has been hampered by the complexity of their intracellular signaling, relative

Cannabinoid Receptor Type 2 Activation in Atherosclerosis and Acute Cardiovascular Diseases
Federico Carbone, François Mach, Nicolas Vuilleumier and Fabrizio Montecucco
Current Medicinal Chemistry, 2014, 21,
DOI: 10.2174/0929867321666140915141332
https://www.researchgate.net/publica...inoid_Receptor _Type_2_Activation_in_Atherosc lerosis_and_Acute_Cardiovascul ar_Diseases
In the last decades, the cannabinoid system (comprising synthetic and endogenous cannabinoid agonists and antagonists, their receptors and degrading enzymes) has been shown to induce potent immunomodulatory activities in atherogenesis and acute ischemic complications. Different from the other cannabinoid receptors in which controversial results are reported, the selective activation of the cannabinoid receptor type 2 (CB2) has been shown to play anti-inflammatory and protective actions within atherosclerotic vessels and downstream ischemic peripheral organs. CB2 is a transmembrane receptor that triggers protective intracellular pathways in cardiac, immune and vascular cells in both human and animal models of atherosclerosis. Considering basic research data, medications activating CB2 function in the
circulation or peripheral target organs might be a promising approach against atherogenesis. This review updates evidence from preclinical studies on different CB2-triggered pathways in atherosclerosis and acute ischemic events.

Cannabinoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and functional involvement in nervous system structures — A short review
Ivana Svíženská, Petr Dubový, Alexandra Šulcová
Pharmacology, Biochemistry and Behavior 90 (2008) 501–511
doi:10.1016/j.pbb.2008.05.010
http://bonhamchemistry.com/wp-conten...oid_Review.pdf
In the last 25 years data has grown exponentially dealing with the discovery of the endocannabinoid system consisting of specific cannabinoid receptors, their endogenous ligands, and enzymatic systems of their biosynthesis and degradation. Progress is being made in the development of novel agonists and antagonists with receptor subtype selectivity which should help in providing a greater understanding of the physiological role of the endocannabinoid system and perhaps also in a broad number of pathologies. This could lead to advances with important therapeutic potential of drugs modulating activity of endocannabinoid system as hypnotics, analgesics, antiemetics, antiasthmatics, antihypertensives, immunomodulatory drugs, antiphlogistics, neuroprotective agents, antiepileptics, agents influencing glaucoma, spasticity and other “movement disorders“, eating disorders, alcohol withdrawal, hepatic fibrosis, bone growth, and atherosclerosis. The aim of this review is to highlight distribution of the CB1 and CB2 receptor subtypes in the nervous system and functional involvement of their specific ligands.

CANNABINOID RECEPTORS AND THEIR ENDOGENOUS AGONISTS
Christian C. Felder, Michelle Glass
Annu. Rev. Pharmacol. Toxicol. 1998. 38:179–200
DOI: 10.1146/annurev.pharmtox.38.1.179
https://www.researchgate.net/publica...enous_agonists
Marijuana has been in use for over 4000 years as a therapeutic and as a recreational drug. Within the past decade, two cannabinoid receptor types have been identified, their signal transduction characterized, and an endogenous lipid agonist
isolated from mammalian tissues. The CB1 cannabinoid receptor is widely distributed in mammalian tissues, with the highest concentrations found in brain neurons. CB1 receptors are coupled to modulation of adenylate cyclase and ion channels. The CB2 receptor is found in cells of the immune system and is coupled to inhibition of adenylate cyclase. Both receptor types selectively bind 19-THC, the active principle in marijuana, and anandamide (arachidonylethanolamide), an endogenous cannabimimetic eicosanoid. Progress is being made in the development of novel agonists and antagonists with receptor subtype selectivity, mice with genetic deletion of the cannabinoid receptors, and receptor-specific antibodies, which should help in providing a better understanding of the physiological role of the cannabinoid receptors.

Cannabinoid Receptors Are Absent in Insects
JOHN MCPARTLAND, VINCENZO DI MARZO, LUCIANO DE PETROCELLIS, LISON MERCER, AND MICHELLE GLASS
THE JOURNAL OF COMPARATIVE NEUROLOGY 436:423–429 (2001)
doi: 10.1002/cne.1078
The endocannabinoid system exerts an important neuromodulatory role in mammals. Knockout mice lacking cannabinoid (CB) receptors exhibit significant morbidity. The endocannabinoid system also appears to be phylogenetically ancient—it occurs in mammals, birds, amphibians, fish, sea urchins, leeches, mussels, and even the most primitive animal with a nerve network, the Hydra. The presence of CB receptors, however, has not been examined in terrestrial invertebrates (or any member of the Ecdysozoa). Surprisingly, we found no specific binding of the synthetic CB ligands [3
H]CP55,940 and [3 H]SR141716A in a panel of insects: Apis mellifera, Drosophila melanogaster, Gerris marginatus, Spodoptera frugiperda, and Zophobas atratus. A lack of functional CB receptors was confirmed by the inability of tetrahydrocannabinol (THC) and HU210 to activate G-proteins in insect tissues, utilizing a guanosine-59-O-(3-[35]thio)-triphosphate (GTPgS) assay. No orthologs of human CB receptors were located in the Drosophila genome, nor did we find orthologs of fatty acid amide hydrolase. This loss of CB receptors appears to be unique in the field of comparative neurobiology. No other known mammalian neuroreceptor is understood to be missing in insects. We hypothesized that CB receptors were lost in insects because of a dearth of ligands; endogenous CB ligands are metabolites of arachidonic acid, and insects produce little or no arachidonic acid or endocannabinoid ligands, such as anandamide

Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs
Erika Cottonea, Valentina Pomattoa, Fulvio Cerria, Ezio Campanticoa, Ken Mackieb, Massimiliano Delperoa, Alda Guastallaa, Claudio Datia, Patrizia Bovolina, and Maria Fosca Franzoni
Fish Physiol Biochem . 2013 October ; 39(5): 1287–1296.
doi:10.1007/s10695-013-9783-9.
Cannabinoids, the bioactive constituents of Cannabis sativa , and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does
exist, few data have been reported on bony fish. In the goldfish, Carassius auratus , the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A
and CB2B receptors, while the percentage of identity is lower with the pufferfish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis
shows the CB2 receptor as two major bands of about 53 kDa and 40 kDa, and other faint bands with apparent molecular masses around 70 kDa, 57 kDa and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of quantitative Real-Time PCR. Our
results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other non-mammalian vertebrates.

Cannabinoid Receptors as Therapeutic Targets
Spiro Pavlopoulos, Ganesh Thakur, Spyros P Nikas, Alexandros Makriyannis
Current Pharmaceutical Design 12 (14):1751.69· February 2006
DOI: 10.2174/138161206776873743
https://www.researchgate.net/publica...peutic_Targets
The cannabinoid receptors CB1 and CB2 are family A, G-protein Coupled Receptors that mediate the effects of cannabinoids, a class of compounds that are so named because the first members were isolates of the cannabis plant. In recent history, there has been much anecdotal evidence that the potent and diverse physiological responses produced by these compounds can be turned to therapeutic benefit for a wide variety of maladies. The remarkable abundance of cannabinoid receptors and the discovery of several endogenous ligands along with enzyme and transporter proteins for which they are substrates, suggests that an endogenous cannabinoid neuromodulatory system is an important mediator of biological function. For these reasons CB1 and CB2 receptors are attractive targets for the design of therapeutic ligands. The action of these receptors, however, may also be modulated by manipulating the enzymes and membrane transporters that regulate the endogenous ligands. Despite the range of physiological processes and activities that are mediated by cannabinoid receptors, it is clear that it is possible to produce ligands that result in differential responses. In this paper, we review the pharmacophoric elements that lead to these differential responses and in order to discuss them in context we present an overview of structural aspects governing cannabinoid receptor function, the cannabinergic system and its physiological functions.

Cannabinoid regulation of brain reward processing with an emphasis on the role of CB1 receptors: a step back into the future
George Panagis, Brian Mackey, Styliani Vlachou
Front. Psychiatry, 31 July 2014
DOI: 10.3389/fpsyt.2014.00092
https://www.frontiersin.org/articles...014.00092/full

Over the last decades, the endocannabinoid system has been implicated in a large variety of functions, including a crucial modulation of brain-reward circuits and the regulation of motivational processes. Importantly, behavioral studies have shown that cannabinoid compounds activate brain reward mechanisms and circuits in a similar manner to other drugs of abuse, such as nicotine, alcohol, cocaine, and heroin, although the conditions under which cannabinoids exert their rewarding effects may be more limited. Furthermore, there is evidence on the involvement of the endocannabinoid system in the regulation of cue- and drug-induced relapsing phenomena in animal models. The aim of this review is to briefly present the available data obtained using diverse behavioral experimental approaches in experimental animals, namely, the intracranial self-stimulation paradigm, the self-administration procedure, the conditioned place preference procedure, and the reinstatement of drug-seeking behavior procedure, to provide a comprehensive picture of the current status of what is known about the endocannabinoid system mechanisms that underlie modification of brain-reward processes. Emphasis is placed on the effects of cannabinoid 1 (CB[SUB]1[/SUB]) receptor agonists, antagonists, and endocannabinoid modulators. Further, the role of CB[SUB]1[/SUB]receptors in reward processes is investigated through presentation of respective genetic ablation studies in mice. The vast majority of studies in the existing literature suggest that the endocannabinoid system plays a major role in modulating motivation and reward processes. However, much remains to be done before we fully understand these interactions. Further research in the future will shed more light on these processes and, thus, could lead to the development of potential pharmacotherapies designed to treat reward-dysfunction-related disorders.

Cannabinoid Signaling Through Non-CB1R/Non-CB2R Targets in Microglia
Neta Rimmerman, Ewa Kozela, Rivka Levy, Zvi Vogel, Ana Juknat
endoCannabinoids, Actions at Non-CB1/CB2 Cannabinoid Receptors 143-171 (2012) book
DOI:10.1007/978-1-4614-4669-9_7
Several Cannabis constituents, including the psychoactive Δ9-tetrahydrocannabinol and the non-psychoactive cannabidiol, exert anti-inflammatory and anti-neurodegenerative effects in various experimental models. Here we review the interaction of cannabinoids (plant, endogenous or synthetic) with microglia, which are considered to be the immune cells of the brain. We describe the functional endocannabinoid system (ligands, receptors, and enzymes) in microglial cell models. In addition, we review the activity of cannabinoid ligands at non-CB1/non-CB2 GPCR targets, mainly GPR55 and GPR18, with focus on microglia. Finally, we discuss non-CB1/non-CB2-mediated effects of cannabinoid ligands on microglial migration, transcriptional regulation, and anti-inflammation in a multiple sclerosis-like model.

Cannabinoid Type 2 Receptor Activation Reduces the Progression of Kidney Fibrosis Using a Mouse Model of Unilateral Ureteral Obstruction
Mallory L. Swanson, Kevin R. Regner, Bob M. Moore II, and Frank Park Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0127
Background: Kidney fibrosis is a hallmark consequence of all forms of chronic kidney disease with few available treatment modalities.
Material and Methods: In this study, we performed the unilateral ureteral obstruction (UUO) procedure to investigate the effects of a selective cannabinoid type 2 (CB2) agonist receptor, SMM-295, as a nephroprotective therapy.
Results: SMM-295 was demonstrated to exhibit 50-fold selectivity over the cannabinoid type 1 (CB1) receptor with an EC50 *2 nM. Four other off-targets were identified in the safety panel, but only at the highest concentration (5 mM) tested in the assay demonstrating the relative selectivity and safety of our compound. Administration of SMM-295 (12 mg/kg IP daily) in UUO mice led to a significant decrease of 33% in tubular damage compared to the vehicle-treated UUO mice after 7 days. Consistent with these findings, there was a significant decrease in a-smooth muscle actin and fibronectin, which are markers of tubulointerstitial fibrosis, as determined by Western blot analysis. DNA damage as detected by a classic marker, c-H2AX, was significantly reduced by 50% in the SMM-295 treatment group compared to vehicle treatment. Genetic knockout of CB2 or administration of a CB2 inverse agonist did not exhibit any beneficial effect on tubulointerstitial fibrosis or kidney tubule injury.
Conclusions: In conclusion, our study provides new evidence that SMM-295 can therapeutically target the CB2 receptor with few, if any, physiological off-target sites to reduce kidney tissue damage and slow the fibrotic progression in a mouse model of kidney fibrosis.
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Cannabinoid Type 2 Receptors Mediate a Cell Type-Specific Plasticity in the Hippocampus.
Stempel AV, Stumpf A, Zhang HY, Özdo?an T, Pannasch U, Theis AK, Otte DM, Wojtalla A, Rácz I, Ponomarenko A, Xi ZX, Zimmer A, Schmitz D.
Neuron. 2016 May 18;90(4):795-809. doi: 10.1016/j.neuron.2016.03.034.
Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission

Cannabinoids and Cannabinoid Receptors: The Story so Far
Fred Shahbazi, Victoria Grandi, Abhinandan Banerjee, and John F. Trant
iScience 23, July 24, 2020
DOI: 10.1016/j.isci.2020.101301
Like most modern molecular biology and natural product chemistry, understanding cannabinoid pharmacology centers around molecular interactions, in this case, between the cannabinoids and their putative targets, the G-protein coupled receptors (GPCRs) cannabinoid receptor 1 (CB1 ) and cannabinoid receptor 2 (CB2 ). Understanding the complex structure and interplay between the partners in this molecular dance is required to understand the mechanism of action of synthetic, endogenous, and phytochemical cannabinoids. This review, with 91 references, surveys our understanding of the structural biology of the cannabinoids and their target receptors including both a critical comparison of the extant crystal structures and the computationally derived homologymodels, as well as an in depth
discussion about the binding modes of the major cannabinoids. The aim is to assist in situating structural biochemists, synthetic chemists, and molecular biologists who are new to the field of cannabis research.

Cannabinoids and Neuropathic Pain
P. Goya, N. Jagerovica, L. Hernandez-Folgadoa and M.I. Martin
Mini Reviews in Medicinal Chemistry, 2003, 3, 159-165

doi: 10.2174/1389557033487818

After a brief overview of the endocannabinoid system (CB receptors, and endocannabinoids) and of the cannabinergic ligands, some general issues related to cannabinoids and pain are commented. Finally, the most important findings regarding cannabinoids and neuropathic pain are discussed in detail.

Cannabinoids CB2 receptors, one new promising drug target for chronic and degenerative pain conditions in equine veterinary patients.
Sánchez-Aparicio, P., Florán, B., Rodríguez, V. D., Ibancovichi, J., Varela-Guerrero, J. A., & Recillas, S.
Journal of Equine Veterinary Science, (2019).
doi:10.1016/j.jevs.2019.102880
Osteoarticular equine disease is a common cause of malady, in general, its therapy is supported on steroids and non steroidal anti-inflammatories, nevertheless, many side effects may develop when these drugs are administered, nowadays the use of new alternatives for this pathology attention is demanded, in that sense, cannabinoid CB2 agonists may represent a novel alternative. Cannabinoids belongs to a group of molecules known by them psychoactive properties; they are synthetized by the Cannabis sativa plant, better known as marijuana. The aim of this study was to contribute to understand the pharmacology of cannabinoid CB2 receptors and its potential utilization on equine veterinary patients with a chronic degenerative painful condition.
In animals, two main receptors for cannabinoids are recognized, the cannabinoid receptor type-1 and the cannabinoid receptor type-2. Once they are activated, booth receptors exert a wide range of physiological responses, as nociception modulation. Recently it has been proposed the use of synthetic cannabinoids type-2 agonist, those receptors looks to confer antinociceptive properties but without the undesired psychoactive side effects, for that reason, veterinary patients, whit chronical degenerative diseases as osteoarthritis may alleviate one of the most common symptom, the pain, which in some cases for several reasons, as patient individualities, or side effects produced for more conventional treatments can´t be attended in the best way.

Cannabinoids for Neuropathic Pain
Perry G. Fine & Mark J. Rosenfeld

Curr Pain Headache Rep (2014) 18:451
DOI 10.1007/s11916-014-0451-2
https://rsds.insctest1.com/wp-conten...athic-pain.pdf

Treatment options for neuropathic pain have limited efficacy and use is fraught with dose-limiting adverse effects. The endocannabinoid system has been elucidated over the last several years, demonstrating a significant interface with pain homeostasis. Exogenous cannabinoids have been demonstrated to be effective in a range of experimental neuropathic pain models, and there is mounting evidence for therapeutic use in human neuropathic pain conditions. This article reviews the history, pharmacologic development, clinical trials results, and the future potential of nonsmoked, orally bioavailable, nonpsychoactive cannabinoids in the management of neuropathic pain.

Cannabinoids Inhibit T-cells via Cannabinoid Receptor 2 in an In Vitro Assay for Graft Rejection, the Mixed Lymphocyte Reaction
Rebecca Hartzell Robinson & Joseph J. Meissler & Jessica M. Breslow-Deckman & John Gaughan & Martin W. Adler & Toby K. Eisenstein
J Neuroimmune Pharmacol 2013 Dec;8(5):1239-50.
DOI 10.1007/s11481-013-9485-1
Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. This study tested the capacity of ?9-
tetrahydrocannabinol (?9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation. Both CB2-selective agonists and ?9-THC significantly suppressed the MLR in a dose dependent
fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2-selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR. Further, the CB2-selective agonists completely inhibited proliferation of purified T-cells activated by anti-CD3 and anti- CD28 antibodies. T-cell function was decreased by the CB2
agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly decreased in the cannabinoid treated cells. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients

Cannabis and endocannabinoid modulators: Therapeutic promises and challenges
Igor Grant, B. Rael Cahn
Clinical Neuroscience Research 5 (2005) 185–199
doi:10.1016/j.cnr.2005.08.015
The discovery that botanical cannabinoids such as delta-9 tetrahydrocannabinol exert some of their effect through binding specific
cannabinoid receptor sites has led to the discovery of an endocannabinoid signaling system, which in turn has spurred research into the mechanisms of action and addiction potential of cannabis on the one hand, while opening the possibility of developing novel therapeutic agents on the other. This paper reviews current understanding of CB1, CB2, and other possible cannabinoid receptors, their arachidonic acid derived ligands (e.g. anandamide; 2 arachidonoyl glycerol), and their possible physiological roles. CB1 is heavily represented in the central nervous system, but is found in other tissues as well; CB2 tends to be localized to immune cells. Activation of the endocannabinoid system can result in enhanced or dampened activity in various neural circuits depending on their own state of activation. This suggests that one function of the endocannabinoid system may be to maintain steady state. The therapeutic action of botanical cannabis or of synthetic molecules that are agonists, antagonists, or which may otherwise modify endocannabinoid metabolism and activity indicates they may have promise as neuroprotectants, and may be of value in the treatment of certain types of pain, epilepsy, spasticity, eating disorders, inflammation, and possibly blood pressure control.

CB receptor ligands from plants.
Woelkart K1, Salo-Ahen OM, Bauer R.
Curr Top Med Chem. 2008;8(3):173-86.
DOI: 10.2174/156802608783498023
Advances in understanding the physiology and pharmacology of the endogenous cannabinoid system have potentiated the interest of cannabinoid receptors as potential therapeutic targets. Cannabinoids have been shown to modulate a variety of immune cell functions and have therapeutic implications on central nervous system (CNS) inflammation, chronic inflammatory conditions such as arthritis, and may be therapeutically useful in treating autoimmune conditions such as multiple sclerosis. Many of these drug effects occur through cannabinoid receptor signalling mechanisms and the modulation of cytokines and other gene products. Further, endocannabinoids have been found to have many physiological and patho-physiological functions, including mood alteration and analgesia, control of energy balance, gut motility, motor and co-ordination activities, as well as alleviation of neurological, psychiatric and eating disorders. Plants offer a wide range of chemical diversity and have been a growing domain in the search for effective cannabinoid ligands. Cannabis sativa L. with the known plant cannabinoid, Delta(9-)tetrahydrocannabinol (THC) and Echinacea species with the cannabinoid (CB) receptor-binding lipophilic alkamides are the best known herbal cannabimimetics. This review focuses on the state of the art in CB ligands from plants, as well their possible therapeutic and immunomodulatory effects.

CB2 and TRPV1 receptors mediate cannabinoid actions on MDR1 expression in multidrug resistant cells
Jonathon C. Arnold, Phoebe Hone, Michelle L. Holland, John D. Allen
Pharmacological reports: PR 64(3):751-7 May 2012
DOI: 10.1016/S1734-1140(12)70871-X
Background: Cannabis is the most widely used illicit drug in the world that is often used by cancer patients in combination with conventional anticancer drugs. Multidrug resistance (MDR) is a major obstacle in the treatment of cancer. An extensively characterized mechanism of MDR involves overexpression of P-glycoprotein (P-gp), which reduces the cellular accumulation of cytotoxic drugs in tumor cells.
Methods: Here we examined the role of cannabinoid receptors and transient receptor potential vanilloid type 1 (TRPV1) receptors in the effects of plant-derived cannabinoids on MDR1 mRNA expression in MDR CEM/VLB100 cells which overexpress P-gp due to MDR1 gene amplification.
Results: We showed that both cannabidiol (CBD) and _9-tetrahydrocannabinol (_9-THC) (10 ?M) transiently induced the MDR1 transcript in P-gp overexpressing cells at 4 but not 8 or 48 h incubation durations. CBD and THC also concomitantly increased P-gp activity as measured by reduced accumulation of the P-gp substrate Rhodamine 123 in these cells with a maximal inhibitory effect observed at 4 h that slowly diminished by 48 h. CEM/VLB100 cell lines were shown to express CB2 and TRPV1 receptors. _9-THC effects on MDR1 expression were mediated by CB2 receptors. The effects of CBD were not mediated by either CB2 or TRPV1 receptors alone, however, required activation of both these receptors to modulate MDR1 mRNA expression.
Conclusion: This is the first evidence that CB2 and TRPV1 receptors cooperate to modulate MDR1 expression.

Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons drives overconsumption of palatable food and obesity
Inigo Ruiz de Azua, Elena Martin-Garcia, Laura Domingo-Rodriguez, Alejandro Aparisi Rey, Diego Pascual Cuadrado, Larglinda Islami, Petri Turunen, Floortje Remmers, Beat Lutz & Rafael Maldonado
Neuropsychopharmacology (2021)
Palatable food can promote overfeeding beyond homeostatic requirements, thereby constituting a major risk to obesity. Here, the lack of cannabinoid type 1 receptor (CB1) in dorsal telencephalic glutamatergic neurons (Glu-CB1-KO) abrogated the overconsumption of palatable food and the development of obesity. On low-fat diet, no genotype differences were observed. However, under palatable food conditions, Glu-CB1-KO mice showed decreased body weight and food intake. Notably, Glu-CB1-KO mice were protected from alterations in the reward system after high-fat diet feeding. Interestingly, obese wild-type mice showed a superior olfactory detection as compared to mutant mice, suggesting a link between overconsumption of palatable food and olfactory function. Reconstitution of CB1 expression in olfactory cortex in high-fat diet-fed Glu-CB1-KO mice using viral gene delivery partially reversed the lean phenotype concomitantly with improved odor perception. These findings indicate that CB1 in cortical glutamatergic neurons regulates hedonic feeding, whereby a critical role of the olfactory cortex was uncovered as an underlying mechanism.


CB2 Cannabinoid Receptor As Potential Target against Alzheimer's Disease
Ester Aso, Isidro Ferrer
Front. Neurosci.
https://www.nature.com/articles/npjamd201612
DOI: 10.3389/fnins.2016.00243
The CB2 receptor is one of the components of the endogenous cannabinoid system, a complex network of signaling molecules and receptors involved in the homeostatic control of several physiological functions. Accumulated evidence suggests a role for CB2 receptors in Alzheimer's disease (AD) and indicates their potential as a therapeutic target against this neurodegenerative disease. Levels of CB2 receptors are significantly increased in post-mortem AD brains, mainly in microglia surrounding senile plaques, and their expression levels correlate with the amounts of A?42 and ?-amyloid plaque deposition. Moreover, several studies on animal models of AD have demonstrated that specific CB2 receptor agonists, which are devoid of psychoactive effects, reduce AD-like pathology, resulting in attenuation of the inflammation associated with the disease but also modulating A? and tau aberrant processing, among other effects. CB2 receptor activation also improves cognitive impairment in animal models of AD. This review discusses available data regarding the role of CB2 receptors in AD and the potential usefulness of specific agonists of these receptors against AD.

Computational Prediction and Biochemical Analyses of New Inverse Agonists for the CB1 Receptor
Caitlin E. Scott, Kwang H. Ahn, Steven T. Graf, William A. Goddard III, Debra A. Kendall, Ravinder Abrol
J. Chem. Inf. Model
DOI: 10.1021/acs.jcim.5b00581
Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C.E. et al. Protein Sci. 2013, 22, 101-113; Ahn, K.H. et al. Proteins 2013, 81, 1304-1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.
Köfalvi, A., Moreno, E., Cordomí, A., Cai, N.-S., Fernández-Dueñas, V., Ferreira, S. G., … Ferré, S.
BMC Biology, 18(1). (2020). doi:10.1186/s12915-020-0739-0
Background: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gimediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level.


Crystal Structure of the Human Cannabinoid Receptor CB1
Tian Hua, Kiran Vemuri, Mengchen Pu, Lu Qu, Gye Won Han, Yiran Wu,1 Suwen Zhao, Wenqing Shui, Shanshan Li, Anisha Korde, Robert B. Laprairie, Edward L. Stahl, Jo-Hao Ho, Nikolai Zvonok, Han Zhou, Irina Kufareva, Beili Wu, Qiang Zhao, Michael A. Hanson, Laura M. Bohn, Alexandros Makriyannis, Raymond C. Stevens, and Zhi-Jie Liu
Doi: 10.1016/j.cell.2016.10.004
Cannabinoid receptor 1 (CB1) is the principal target of ?9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

D9 -Tetrahydrocannabinol and N-arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC-1B cells
Douglas McHugh, Jeremy Page, Emily Dunn and Heather B Bradshaw
DOI:10.1111/j.1476-5381.2011.01497.x
BACKGROUND AND PURPOSE Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells.
EXPERIMENTAL APPROACH Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis.
KEY RESULTS AEA, D9 -tetrahydrocannabinol (D9 -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB1 receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB1 and CB2 receptor ligands at GPR18 receptors, including D9 -THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and D9 -THC were antagonized by Pertussis toxin, AM251 and cannabidiol.
CONCLUSIONS AND IMPLICATIONS An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease

*Determination and Characterization of a Cannabinoid Receptor in Rat Brain
December 1988 Molecular Pharmacology 34(5):605-13
William Devane, F.A. III Dysarz, M.R. Johnson, Allyn C Howlett
https://citeseerx.ist.psu.edu/viewdo...=rep1&type=pdf
The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific
binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.

Diabetes and Cannabinoid CB1 receptor deficiency promote similar early onset aging-like changes in the skin
Ermelindo C Leal , Liane I F Moura , Roksana M Pirzgalska , Dorinda Marques-da-Silva , Catherine Ledent , Attila Köfalvi,, Eugenia Carvalho
Exp Gerontol. 2021 Aug 24;154:111528.
DOI: 10.1016/j.exger.2021.111528
Background: The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model.
Methods: We evaluated several signals of skin aging in wild-type control (WT), WT streptozotocin-induced type 1 diabetic mice (WT DM), CB1R knockout (CB1RKO) and CB1RKO DM mice. We quantified markers of inflammation, angiogenesis, antioxidant enzymes and collagen content. Moreover, we evaluate reactive oxygen species (ROS) levels and macrophage phenotype, M1 and M2.
Results: CB1R expression is decreased in the skin of WT DM mice and collagen levels are decreased in the skin of WT DM, CB1RKO and CB1RKO DM mice. Additionally, the absence of CB1R correlated with higher expression of pro-inflammatory markers, also evident in WT DM or CB1RKO DM mice. Moreover, the M1/M2 macrophage ratio and ROS levels were significantly elevated but in the diabetic WT and the CB1RKO mice, consistent with a significant decrease in the antioxidant capacity of the skin.
Conclusions: Our results indicate that CB1R deficiency in the skin may lead to accelerated skin aging due to the increased production of ROS, a decrease in the antioxidant defenses and a higher pro-inflammatory environment. A significant decrease in the CB1R expression may be a significant contributing factor to the early aging-like changes in diabetes.
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Different receptor mechanisms underlying phytocannabinoid? versus synthetic cannabinoid?induced tetrad effects: Opposite roles of CB1/CB2 versus GPR55 receptors.
Wang, X., Galaj, E., Bi, G., Zhang, C., He, Y., Zhan, J., … Xi, Z.
British Journal of Pharmacology. (2019).
doi:10.1111/bph.14958
Background and Purpose
Cannabis or cannabinoids produce characteristic tetrad effects – analgesia, hypothermia, catalepsy, and suppressed locomotion, which are generally believed to be mediated by the activation of cannabinoid CB1 receptors (CB1Rs). Given recent findings of CB2 and GPR55
receptors in the brain, we examined whether CB2 and GPR55 receptors are also involved in cannabinoid action.
Experimental Approach
We first compared ?-THC-, WIN55212-2-, or XLR11-induced tetrad effects between wildtype (WT) and each genotype of CB1-, CB2-, or GPR55-knockout mice, and then observed the effects of antagonists of these receptors on cannabinoid-induced tetrad effects in WT mice.
Key Results
Systemic administration of ?9-THC, WIN55212-2, or XLR11 produced dose-dependent tetrad effects in WT mice. Genetic deletion or pharmacological blockade of CB1Rs abolished the tetrad effects produced by all three cannabinoids. Unexpectedly, genetic deletion of CB2Rs also abolished analgesia and catalepsy produced by ?9-THC or WIN55212-2, but not by XLR11. Microinjections of ?9-THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1-KO, mice. CB2-KO mice displayed a reduction in intra-ventricle ?9-THC-induced analgesia and catalepsy. In contrast to CB1Rs and CB2Rs, genetic deletion of GPR55s caused enhanced responses to ?9-THC or WIN55212-2. Pharmacological blockade of CB1, CB2, or GPR55 receptors produced alterations similar to those observed in each genotype mouse line.
Conclusions and implications
These findings suggest that in addition to CB1Rs, both CB2Rs and GPR55s are also involved in some pharmacological effects produced by cannabinoids. CB1/CB2Rs, in contrast to GPR55 receptors, appear to play opposite roles in cannabinoid action.

Differential effect of opioid and cannabinoid receptor blockade on heroin?seeking reinstatement and cannabinoid substitution in heroin?abstinent rats
L Fattore, MS Spano , V Melis, P Fadda, W Fratta
British Journal of Pharmacology (2011) 163 1550–1562
doi: 10.1111/j.1476-5381.2011.01459.x
BACKGROUND AND PURPOSE Opioids and cannabinoids interact in drug addiction and relapse. We investigated the effect of the opioid receptor antagonist naloxone and/or the cannabinoidCB1 receptor antagonist rimonabant on cannabinoid?induced reinstatement of heroin seeking and on cannabinoid substitution in heroin?abstinent rats.
EXPERIMENTAL APPROACH Rats were trained to self-administer heroin (30 mg·kg-1 per infusion) under a fixed-ratio 1 reinforcement schedule. After extinction of self-administration (SA) behaviour, we confirmed the effect of naloxone (0.1–1 mg·kg-1 ) and rimonabant (0.3–3 mg·kg-1 ) on the reinstatement of heroin seeking induced by priming with the CB1 receptor agonist WIN55,212-2 (WIN, 0.15–0.3 mg·kg-1 ). Then, in a parallel set of heroin-trained rats, we evaluated whether WIN (12.5 mg·kg-1 per infusion) SA substituted for heroin SA after different periods of extinction. In groups of rats in which substitution occurred, we studied the effect of both antagonists on cannabinoid intake.
KEY RESULTS Cannabinoid-induced reinstatement of heroin seeking was significantly attenuated by naloxone (1 mg·kg-1 ) and rimonabant (3 mg·kg-1 ) and fully blocked by co-administration of sub-threshold doses of the two antagonists. Moreover, contrary to immediate (1 day) or delayed (90 days) drug substitution, rats readily self-administered WIN when access was given after 7, 14 or 21 days of extinction from heroin, and showed a response rate that was positively correlated with the extinction period. In these animals, cannabinoid intake was increased by naloxone (1 mg·kg-1) and decreased by rimonabant (3 mg·kg-1).
CONCLUSIONS AND IMPLICATIONS Our findings extend previous research on the crosstalk between cannabinoid and opioid receptors in relapse mechanisms, which suggests a differential role in heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats.

Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing
Karen Wright[SUP]1 [/SUP], Nicholas Rooney, Mark Feeney, Jeremy Tate, Duncan Robertson, Melanie Welham, Stephen Ward
doi: 10.1016/j.gastro.2005.05.026
https://www.researchgate.net/publica..._Wound_Healing Background & aims: Two G-protein-coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function.
Methods: The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments.
Results: CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1-lysophosphatidic acid 1 heteromeric receptor complex.
Conclusions: CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity.


Differential signalling in human cannabinoid CB1 receptors and their splice variants in autaptic hippocampal neurones
Alex Straiker, Jim Wager-Miller, Jacqueline Hutchens and Ken Mackie
British Journal of Pharmacology (2012) 165 2660–2671
DOI:10.1111/j.1476-5381.2011.01744.x
BACKGROUND AND PURPOSE Cannabinoids such as D9 - tetrahydrocannabinol, the major psychoactive component of marijuana and hashish, primarily act via cannabinoid CB1 and CB2 receptors to produce characteristic behavioural effects in humans. Due to the tractability of rodent models for electrophysiological and behavioural studies, most of the studies of cannabinoidreceptor action have used rodent cannabinoid receptors. While CB1 receptors are relatively well-conserved among mammals, human CB1 (hCB1) differs from rCB1 and mCB1 receptors at 13 residues, which may result in differential signalling. In addition, two hCB1 splice variants (hCB1a and hCB1b) have been reported, diverging in their amino-termini relative to hCB1 receptors. In this study, we have examined hCB1 signalling in neurones.
EXPERIMENTAL APPROACH hCB1, hCB1a hCB1b or rCB1 receptors were expressed in autaptic cultured hippocampal neurones from CB1 -/- mice. Such cells express a complete endogenouscannabinoid signalling system. Electrophysiological techniques were used to assess CB1 receptor-mediated signalling. KEY RESULTS Expressed in autaptic hippocampal neurones cultured from CB1 -/- mice, hCB1, hCB1a and hCB1b signal differentially from one another and from rodent CB1 receptors. Specifically, hCB1 receptors inhibit synaptic transmission less effectively than rCB1 receptors. CONCLUSIONS AND IMPLICATIONS Our results suggest that cannabinoid receptor signalling in humans is quantitatively very different from that in rodents. As the problems of marijuana and hashish abuse occur in humans, our results highlight the importance of studying hCB1 receptors. They also suggest further study of the distribution and function of hCB1 receptor splice variants, given their differential signalling and potential impact on human health.

Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse.
Ruehle S, Wager-Miller J, Straiker A, Farnsworth J, Murphy MN, Loch S, Monory S, Mackie K, Lutz B.
J Neurochem. 2017 Jun 13.
doi: 10.1111/jnc.14099
Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117 bp and 186 bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation because of the absence of two glycosylation sites in mCB1b. On-cell western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced MAPK phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.

Discovery and Isolation of Anandamide and Other Endocannabinoids
Lumír O. Hanuš
Chemistry & Biodiversity Volume 4, Issue 8 Pages 1828-1841 2007
doi: 10.1002/cbdv.200790154
1. Introduction. – Cannabis had many uses as a cultivated plant already in Neolithic China [1]. The first known record of the use of cannabis as a medicine was published in China 5000 years ago in the reign of the Emperor Chen Nung. However, it was not until the 19th century that cannabis became a common medicine in Europe, and its analgesic, anticonvulsant, anti-spasmodic, anti-emetic, and hypnotic uses were recognized. The Assyrians, who ruled large parts of the Middle East for nearly a millennium, ca. 3000 years ago, have left us a pharmaceutical legacy on hundreds of clay tablets. Cannabis was one of the major drugs of their pharmacopoeia. They named this plant according to its use. Campbell [2] identified the Sumerian term a-zal-la and the Akkadian term azulla as cannabis on the basis of their similarities to the Syrian azal, meaning >to spin?. He also took the Assyrian word gurgurangu as another reference to cannabis because of its similarity to garganinj, the Persian word for cannabis. Building on these similarities, Campbell then identified the Sumerian drug gan-zi-gun-nu as hashish (a robber (gan) who spins away (gun-nu) the soul (zi)), and this word has been translated as >the drug that takes away

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the ?2*subunit of GABAA*receptors?*
Baur R, Gertsch J, Sigel E.*
PeerJ*1:e149*(2013).*
DOI: 10.7717/peerj.149?NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant ?1?2?2*GABAA*receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

Dynamic regulation of the endocannabinoid system: implications for analgesia
Devi Rani Sagar, A Gemma Gaw, Bright N Okine, Stephen G Woodhams, Amy Wong, David A Kendall and Victoria Chapman
Molecular Pain 2009, 5:59
doi:10.1186/1744-8069-5-59
https://journals.sagepub.com/doi/pdf...1744-8069-5-59
The analgesic effects of cannabinoids are well documented, but these are often limited by psychoactive side-effects. Recent studies indicate that the endocannabinoid system is dynamic and altered under different pathological conditions, including pain states. Changes in this receptor system include altered expression of receptors, differential synthetic pathways for endocannabinoids are expressed by various cell types, multiple pathways of catabolism and the generation of biologically active metabolites, which may be engaged under different conditions. This review discusses the evidence that pain states alter the endocannabinoid receptor system at key sites involved in pain processing and how these changes may inform the development of cannabinoid-based analgesics.

Effect of Cannabinoid 2 Receptor Modulation on the Peripheral Immune Response in Central Nervous System Injury-Induced Immunodeficiency Syndrome
Saki Sultana, Ian Burkovskiy, Juan Zhou, Melanie M. Kelly, and Christian Lehmann
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0130
https://www.researchgate.net/publica...iency_Syndrome
Acute central nervous system (CNS) injury, such as stroke, spinal cord injury, or traumatic brain injury can result in dysregulated immune response, and the condition is known as CNS injury-induced immunodeficiency syndrome (CIDS). The endocannabinoid system is an important homeostatic regulator in the CNS and immune system. Activation of cannabinoid 2 receptors (CB2R) on immune cells has been reported to dampen inflammation, suggesting a potential role of CB2R in the peripheral immune response following CNS injury. In this study, we have investigated the effect of CB2R modulation on the peripheral immune response during CIDS. Materials and Methods: Experimental CNS injury was induced in C57BL/6 mice through intracerebral injection of the vasopressor peptide, endothelin-1. A selective CB2R agonist (HU308) was used as an early treatment before the onset of CIDS and AM630, a selective CB2R antagonist, was administered as a later-phase therapy to combat the systemic immunodeficiency following the CNS injury. The peripheral immune response to endotoxin was studied 24 h after the CNS injury using intravital microscopy to examine leukocyte activation within the intestinal microcirculation in mice. Brain infarct size, and plasma levels of cytokines and soluble adhesion molecules were measured as additional parameters for the assessment of treatment outcomes. Results: Our results showed that early CB2R activation with HU308 reduced brain injury size and restored leukocyte response to endotoxin in the peripheral microcirculation. Late CB2R inhibition with AM630 also improved the peripheral leukocyte response to endotoxin and did not exacerbate the extent of brain injury. Discussion: CB2R activation has the potential to mitigate CNS injury as an early treatment by limiting neuroinflammation and preventing the development of CIDS. At the later stage with already-established CIDS, treatment may require dampening CB2R activation to improve the patient's outcome.

Effects of cannabinoid receptor type 2 in respiratory syncytial virus infection in human subjects and mice.
Tahamtan, A., Samieipoor, Y., Nayeri, F. S., Rahbarimanesh, A. A., Izadi, A., Rashidi-Nezhad, A., … Salimi, V.
Virulence, 9(1), 217–230.(2017).
doi:10.1080/21505594.2017.1389369
An accumulating body of evidence suggests that the endocannabinoid system plays a significant role in pathophysiological processes and impacts disease severity. Here we investigate the possible role of a cannabinoid receptor type 2 (CB2) functional variant in determining disease severity and the potential pharmacological therapeutic effects of CB2 activation in viral respiratory infection. The common missense variant (CAA/CGG; Q63R) of the gene-encoding CB2 receptor (CNR2) was evaluated in 90 inpatient and 90 outpatient children with acute respiratory tract infection (ARTI). The frequency distribution of respiratory syncytial virus (RSV)-the main cause of severe cases of bronchiolitis and pneumonia in children-was studied in all collected samples. The mechanism through which CB2 affects clinical outcomes in case of RSV infection was studied in Balb/c mice model using AM630 as a CB2 antagonist. The potential therapeutic effect of CB2 activation during RSV infection was studied using a selective agonist, JWH133. The CB2 Q63R variation was associated with increased risk of hospitalization in children with ARTI. Children carrying the QQ genotype were more prone to developing severe ARTI (OR=3.275, 95% CI: 1.221-8.705; p=0.019). Of all the children enrolled in the study, 83 patients (46.1%) were found positive for RSV infection. The associated risk of developing severe ARTI following RSV infection increased more than two-fold in children carrying the Q allele (OR=2.148, 95% CI: 1.092-4.224; p=0.026). In mice, the blockade of CB2 by AM630 during RSV infection enhanced the influx of BAL cells and production of cytokines/chemokines while exaggerating lung pathology. CB2 activation by JWH133 reduces the influx of BAL cells and production of cytokines/chemokines while alleviating lung pathology. Collectively, CB2 is associated with RSV severity during infancy and may serve as a therapeutic target in RSV infection through the alleviation of virus-associated immunopathology.

Effects of palmitoylation of Cys415 in helix 8 of the CB1 cannabinoid receptor on membrane localization and signalling
Sergio Oddi, Enrico Dainese, Simone Sandiford, Filomena Fezza, Mirko Lanuti, Valerio Chiurchiù, Antonio Totaro, Giuseppina Catanzaro, Daniela Barcaroli, Vincenzo De Laurenzi, Diego Centonze, Somnath Mukhopadhyay, Jana Selent, Allyn C Howlett and Mauro Maccarrone
British Journal of Pharmacology (2012) 165 2635–2651
DOI:10.1111/j.1476-5381.2011.01658.x
BACKGROUND AND PURPOSE The CB1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB1 receptor by analysing the functional consequences of site-specific mutation of Cys415, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.
EXPERIMENTAL APPROACH The palmitoylation state of CB1 receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys415 was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [35S]GTPgS, cAMP and co-immunoprecipitation assays. KEY RESULTS Endogenous CB1 receptors in rat brain were palmitoylated.Mutation of Cys415 prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB1 receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins.
CONCLUSIONS AND IMPLICATIONS CB1 receptors were post-translationally modified by palmitoylation. Mutation of Cys415 provides a receptor that is functionally impaired in terms of membrane targeting and signalling.

Endocannabinoid genetic variation enhances vulnerability to THC reward in adolescent female mice.
Burgdorf, C. E., Jing, D., Yang, R., Huang, C., Hill, M. N., Mackie, K., … Rajadhyaksha, A. M.
Science Advances, 6(7), eaay1502. (2020).
doi:10.1126/sciadv.aay1502
Adolescence represents a developmental period with the highest risk for initiating cannabis use. Little is known about whether genetic variation in the endocannabinoid system alters mesolimbic reward circuitry to produce vulnerability to the rewarding properties of the exogenous cannabinoid ?9 -tetrahydrocannabinol (THC). Using a genetic knock-in mouse model (FAAHC/A) that biologically recapitulates the human polymorphism associated with problematic drug use, we find that in adolescent female mice, but not male mice, this FAAH polymorphism enhances the mesolimbic dopamine circuitry projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and alters cannabinoid receptor 1 (CB1R) levels at inhibitory and excitatory terminals in the VTA. These developmental changes collectively increase vulnerability of adolescent female FAAHC/A mice to THC preference that persists into adulthood. Together, these findings suggest that this endocannabinoid genetic variant is a contributing factor for increased susceptibility to cannabis dependence in adolescent females

Endocannabinoid Modulation Using Monoacylglycerol Lipase Inhibition in Tourette Syndrome: A Phase 1 Randomized, Placebo-Controlled Study
Kirsten R. Müller-Vahl, Carolin Fremer, Chan Beals, Jelena Ivkovic, Henrik Loft, Christoph Schindler
Pharmacopsychiatry Nov 2021
DOI 10.1055/a-1675-3494
https://www.thieme-connect.com/produ...-1675-3494.pdf
Introduction Tourette syndrome (TS) is a complex neurodevelopmental disorder characterized by chronic motor and vocaltics. While consistently effective treatment is lacking, evidence indicates that the modulation of endocannabinoid system is potentially beneficial. Lu AG06466 (previously ABX-1431) is a highly selective inhibitor of monoacylglycerol lipase, the primary enzyme responsible for the degradation of the endocannabinoid ligand 2-arachidonoylglycerol. This exploratory study aimed to determine the effect of Lu AG06466 versus placebo on tics and other symptoms in patients with TS.
Methods In this phase 1b cross-over study, 20 adult patients with TS on standard-of-care medications were randomized to a single fasted dose of Lu AG06466 (40 mg) or placebo in period 1, followed by the other treatment in period 2. The effects on tics, premonitory urges, and psychiatric comorbidities were evaluated using a variety of scaled approaches at different time points before and after treatment.
Results All scales showed an overall trend of tic reduction, with two out of three tic scales (including the Total Tic Score of
the Yale Global Tic Severity Score) showing a significant effect of a single dose of Lu AG06466 versus placebo at various timepoints. Treatment with Lu AG06466 resulted in a significant reduction in premonitory urges versus placebo. Single doses of Lu AG06466 were generally well-tolerated, and the most common adverse events were headache, somnolence, and fatigue.
Conclusion In this exploratory trial, a single dose of Lu AG06466 showed statistically significant positive effects on key
measures of TS symptoms

Endocannabinoid signaling at the periphery: 50 years after THC.
Mauro Maccarrone, Itai Bab, Tama´s B?ro, Guy A. Cabral, Sudhansu K. Dey, Vincenzo Di Marzo, Justin C. Konje, George Kunos, Raphael Mechoulam, Pal Pacher, Keith A. Sharkey, and Andreas Zimmer
Trends in Pharmacological Sciences, 36(5), 277–296.(2015).
doi:10.1016/j.tips.2015.02.008
In 1964, the psychoactive ingredient of Cannabis sativa, D9 -tetrahydrocannabinol (THC), was isolated. Nearly 30 years later the endogenous counterparts of THC, collectively termed endocannabinoids (eCBs), were discovered: N-arachidonoylethanolamine (anandamide) (AEA) in 1992 and 2-arachidonoylglycerol (2-AG) in 1995. Since then, considerable research has shed light on the impact of eCBs on human health and disease, identifying an ensemble of proteins that bind, synthesize, and degrade them and that together form the eCB system (ECS). eCBs control basic biological processes including cell choice between survival and death and progenitor/stem cell proliferation and differentiation. Unsurprisingly, in the past two decades eCBs have been recognized as key mediators of several aspects of human pathophysiology andthushave emergedtobe amongthemost widespread and versatile signaling molecules ever discovered. Here some ofthepioneersofthis research field review the state oftheartof critical eCBfunctions inperipheralorgans.Our community effort is aimed at establishing consensus views on the relevance of the peripheral ECS for human health and disease pathogenesis, as well as highlighting emerging challenges and therapeutic hopes

Endocannabinoid signalling: has it got rhythm?
Linda K Vaughn,Gerene Denning, Kara L Stuhr, Harriet de Wit, Matthew N Hill, and Cecilia J Hillard
Br J Pharmacol. 2010 Jun; 160(3): 530–543.
doi: 10.1111/j.1476-5381.2010.00790.x https://www.ncbi.nlm.nih.gov/labs/pm...es/PMC2931554/ Endogenous cannabinoid signalling is widespread throughout the body, and considerable evidence supports its modulatory role in many fundamental physiological processes. The daily and seasonal cycles of the relationship of the earth and sun profoundly affect the terrestrial environment. Terrestrial species have adapted to these cycles in many ways, most well studied are circadian rhythms and hibernation. The purpose of this review was to examine literature support for three hypotheses: (i) endocannabinoid signalling exhibits brain region-specific circadian rhythms; (ii) endocannabinoid signalling modulates the rhythm of circadian processes in mammals; and (iii) changes in endocannabinoid signalling contribute to the state of hibernation. The results of two novel studies are presented. First, we report the results of a study of healthy humans demonstrating that plasma concentrations of the endocannabinoid, N-arachidonylethanolamine (anandamide), exhibit a circadian rhythm. Concentrations of anandamide are threefold higher at wakening than immediately before sleep, a relationship that is dysregulated by sleep deprivation. Second, we investigated differences in endocannabinoids and congeners in plasma from Marmota monaxobtained in the summer and during the torpor state of hibernation. We report that 2-arachidonoylglycerol is below detection in M. monax plasma and that concentrations of anandamide are not different. However, plasma concentrations of the anorexigenic lipid oleoylethanolamide were significantly lower in hibernation, while the concentrations of palmitoylethanolamide and 2-oleoylglycerol were significantly greater in hibernation. We conclude that available data support a bidirectional relationship between endocannabinoid signalling and circadian processes, and investigation of the contribution of endocannabinoid signalling to the dramatic physiological changes that occur during hibernation is warranted.


Endocannabinoid tone versus constitutive activity of cannabinoid receptors
Allyn C. Howlett, Patricia H. Reggio , Steven R. Childers , Robert E. Hampson , Nadine M. Ulloa, Dale G. Deutsch
British Journal of Pharmacology (2011) 163 1329–1343
doi: 10.1111/j.1476-5381.2011.01364.x
This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2?arachidonoylglycerol (2?AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis?à?vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2?AG is well characterized, and 2?AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid?binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands

Endocannabinoids and neurodegenerative diseases
Vincenzo Micale, Carmen Mazzola, Filippo Drago
Pharmacological Research 56 (2007) 382–392
doi:10.1016/j.phrs.2007.09.008
The cannabinoid CB1 and CB2 receptors, the endogenous endocannabinoid (EC) ligands anandamide (AEA) and 2-arachidonylethanolamide, and the degradative enzymes fatty acid amide hydrolase (FAAH) and monoglyceride lipase (ML) are key elements of the EC system implicated in different physiological functions including cognition, motor activity and immune responses. Thus, both the possible neuroprotective role of ECs and their modulating action on neurotransmitter systems affected in several neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and multiple sclerosis (MS) are currently under investigation. Accumulating data show an unbalance in the EC system (i.e. decrease of neuronal cannabinoid CB1 receptors, increase of glial cannabinoid CB2 receptors and over-expression of FAAH in astrocytes) in experimental models of AD as well as in post-mortem brain tissue of AD patients, suggesting its possible role in inflammatory processes and in neuroprotection. However, the mechanisms of the EC modulation of immune response are not fully understood. By contrast, in HD a reduced EC signaling, given both by the loss of cannabinoid CB1 receptors and decrease of ECs in brain structures involved in movement control as basal ganglia, has been well documented in preclinical and clinical studies. Thus, in the present review we discuss recent data concerning the role of the EC system in the pathophysiology of AD and HD, two neurodegenerative diseases characterized by cognitive deficit and motor impairment, respectively. We focus on the effects of compounds modulating the EC system (agonists/antagonists of cannabin

Evaluation of phytocannabinoids from high-potency Cannabis sativa using in vitro bioassays to determine structure–activity relationships for cannabinoid receptor 1 and cannabinoid receptor 2
Afeef S. Husni, Christopher R. McCurdy, Mohamed M. Radwan, Safwat A. Ahmed, Desmond Slade, Samir A. Ross, Mahmoud A. ElSohly, Stephen J. Cutler
Med Chem Res (2014) 23:4295–4300
DOI 10.1007/s00044-014-0972-6
Cannabis has been around for thousands of years and has been used recreationally, medicinally, and for fiber. Over 500 compounds have been isolated from Cannabis sativa with approximately 105 being cannabinoids.
Of those 105 compounds, D9-tetrahydrocannabinol has been determined as the primary constituent, which is also responsible for the psychoactivity associated with Cannabis. Cannabinoid receptors belong to the large superfamily of G protein-coupled receptors. Targeting the cannabinoid receptors has the potential to treat a variety of conditions such as pain, neurodegeneration, appetite, immune function, anxiety, cancer, and others. Developing in vitro bioassays to determine binding and functional activity of compounds has the ability to lead researchers to develop a safe and effective drug that may target the cannabinoid receptors. Using radioligand binding and functional bioassays, a structure–activity relationship for major and minor cannabinoids was developed.

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction.
Michael C. J. Chang, Daniel J. Berkery, +4 authors Herbert Schuel
Published in Molecular reproduction and development 1993
DOI:10.1002/mrd.1080360416
Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation

Evidence for novel cannabinoid receptors
Malcolm Begg, Pal Pacher, Sandor Batkai, Douglas Osei-Hyiaman, Laszlo Offertaler, Fong Ming Mo, Jie Liu, George Kunos
Pharmacology & Therapeutics 106 (2005) 133 – 145
doi:10.1016/j.pharmthera.2004.11.005
https://citeseerx.ist.psu.edu/viewdo...1.1.1080.8989& rep=rep1&type=pdf
Cannabinoids, including the bioactive constituents of the marijuana plant, their synthetic analogs, and endogenous lipids with cannabinoid-like activity, produce their biological effects by interacting with specific receptors. To date, two G protein-coupled cannabinoid receptors have been identified by molecular cloning, CB1 receptors mainly expressed in the brain and mediating most of the neurobehavioral effects of cannabinoids and CB2 receptors expressed by immune and hematopoietic tissues. Recent findings indicate that some cannabinoid effects are not mediated by either CB1 or CB2 receptors, and in some cases there is compelling evidence to implicate additional receptors in these actions. These include transient receptor potential vanilloid 1 (TRPV1) receptors and as-yet-unidentified receptors implicated in the endothelium-dependent vasodilator effect of certain cannabinoids and in the presynaptic inhibition of glutamatergic neurotransmission in the hippocampus. The case for these additional receptors is being reviewed here

Evidence that (_)-7-hydroxy-4#-dimethylheptyl-cannabidiol activates a non-CB1, non-CB2, non-TRPV1 target in the mouse vas deferens
Roger G. Pertweea, Adele Thomasa, Lesley A. Stevensona,Yehoshua Maorb, Raphael Mechoulam
Neuropharmacology 48 (2005) 1139e1146
doi:10.1016/j.neuropharm.2005.01.010
Previous experiments showed that R-(C)-WIN55212-induced inhibition of electrically-evoked contractions of mouse vasa deferentia could be antagonized by cannabidiol in a manner that appeared to be competitive but not to involve direct competition for established cannabinoid receptors. We have now discovered that (_)-7-hydroxy-4#-dimethylheptyl-cannabidiol (7-OH-DMHCBD) inhibits electrically-evoked contractions of the vas deferens (EC50Z13.3 nM). This it appeared to do by acting on prejunctional neurones as 100 nM 7-OH-DMH-CBD did not attenuate contractile responses to phenylephrine or b,g-methylene-ATP. Although 7-OH-DMH-CBD was antagonized by SR141716A, it was less susceptible to antagonism by this CB1 receptor antagonist than R-(C)-WIN55212. 7-OH-DMH-CBD was also antagonized by cannabidiol (1 mM; apparent KBZ222.2 nM) but not by the CB2 receptor antagonist, SR144528 (32 nM), or by naloxone (300 nM), ruthenium red (1 mM) or capsazepine (10 mM). Yohimbine (100 nM) enhanced the ability of 7-OH-DMH-CBD to inhibit electrically-evoked contractions. R-(C)-WIN55212 was also potentiated by 100 nM yohimbine, possibly reflecting ongoing sequestration of Gi/o proteins from CB1 receptors by a2-adrenoceptors. Our results suggest that 7-OH-DMH-CBD may activate a neuronal target in the vas deferens that is not a CB1, CB2, TRPV1, opioid or a2-adrenergic receptor but do not exclude the possibility that it also activates CB1 receptors.

Experimental Methods to Study the Role of the Peripheral Cannabinoid Receptor in Immune Function
Nancy E. Buckley, Diep Burbridge, Manop Buranapramest, Tanya Ferguson, Renee Y. Paau
DOI:
10.1385/1-59259-999-0:19
https://link.springer.com/protocol/1...59259-999-0:19
Pg 19-40 In book:
Marijuana and Cannabinoid Research Methods and Protocols ed. By Emmanuel S. Onaivi

Marijuana components, such as Δ-9-tetrahydrocannabinol, and endogenous cannabinoids, such as anandamide and 2-arachydonoylglycerol, alter diverse immune functions. Two cannabinoid receptors have been discovered to date, the central cannabinoid receptor (CB[SUB]1[/SUB]R) and the peripheral cannabinoid receptor (CB[SUB]2[/SUB]R). The CB[SUB]1[/SUB]R is expressed predominantly in the central nervous system. The CB[SUB]2[/SUB]R is expressed mainly in cells of the immune system, suggesting that the CB[SUB]2[/SUB]R is involved in immunoregulatory events. Cannabinoids have been shown to modulate diverse immune functions including cytokine production, lymphocyte proliferation, and humoral and cell-mediated immune responses. In addition, cannabinoids have been shown to induce different signal transduction pathways. However, the role of cannabinoids and their receptors in the immune system remains unclear. The objective of the experimental methods described herein is to investigate the role of CB[SUB]2[/SUB]R activation in specific splenocyte and macrophage functions using a mouse lacking the CB[SUB]2[/SUB]R. Interestingly, our findings, thus far suggest that basal CB[SUB]2[/SUB]R activation modulates lymphocyte proliferation and cytokine secretion and macrophage phagocytic activity. Therefore, data obtained using the methodology described in this chapter will help us elucidate the role of cannabinoids and the CB[SUB]2[/SUB]R in the immune system.

Fatty Acid Modulation of the Endocannabinoid System and the Effect on Food Intake and Metabolism
Shaan S. Naughton, Michael L.Mathai, Deanne H. Hryciw, and Andrew J. McAinch
International Journal of Endocrinology Volume 2013, 11 pages
DOI: 10.1155/2013/361895
Endocannabinoids and their G-protein coupled receptors (GPCR) are a current research focus in the area of obesity due to the system’s role in food intake and glucose and lipid metabolism. Importantly, overweight and obese individuals often have higher circulating levels of the arachidonic acid-derived endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) and an altered pattern of receptor expression. Consequently, this leads to an increase in orexigenic stimuli, changes in fatty acid synthesis, insulin sensitivity, and glucose utilisation, with preferential energy storage in adipose tissue. As endocannabinoids are products of dietary fats, modification of dietary intake may modulate their levels, with eicosapentaenoic and docosahexaenoic acid based endocannabinoids being able to displace arachidonic acid from cell membranes, reducing AEA and 2-AG production. Similarly, oleoyl ethanolamide, a product of oleic acid, induces satiety, decreases circulating fatty acid concentrations, increases the capacityfor ??-oxidation, and is capable of inhibiting the action of AEA and 2-AG in adipose tissue.Thus, understanding how dietary fats alter endocannabinoid system activity is a pertinent area of research due to public healthmessages promoting a shift towards plant derived fats, which are rich sources of AEA and 2-AGprecursor fatty acids, possibly encouraging excessive energy intake and weight gain.

Flavonoids from Perovskia atriplicifolia and Their in Vitro Displacement of the Respective Radioligands for Human Opioid and Cannabinoid Receptors.
Tarawneh, A., León, F., Pettaway, S., Elokely, K. M., Klein, M. L., Lambert, J., … Cutler, S. J.
Journal of Natural Products, 78(6), 1461–1465 (2015).
doi:10.1021/acs.jnatprod.5b00218
Bioassay-guided fractionation of the leaves of Perovskia atriplicifolia (Russian sage) resulted in the isolation of four previously known flavonoid derivatives, 5-hydroxy- 6,7,3?,4?-tetramethoxyflavone (1), 5,7-dihydroxy-6,3?,4?-trimethoxyflavone (2), 5-hydroxy-6,7,4?-trimethoxyflavone (3), and 5,7-dihydroxy-6,4?-dimethoxyflavone (4). Compounds 1, 3, and 4 showed displacement of the radioligand for the cloned human ? opioid receptor with Ki values ranging from 3.1 to 26.0 ?M. In addition, the binding mode of the compounds in the active site of the ? opioid receptor was investigated through molecular modeling algorithms. This study may have implications in better understanding non-nitrogenous ? opioid receptor ligands.

FROM PHYTOCANNABINOIDS TO CANNABINOID RECEPTORS AND ENDOCANNABINOIDS: PLEIOTROPIC PHYSIOLOGICAL AND
PATHOLOGICAL ROLES THROUGH COMPLEX PHARMACOLOGY

Alessia Ligresti, Luciano De Petrocellis, and Vincenzo Di Marzo
Physiol Rev 96: 1593–1659, 2016
doi:10.1152/physrev.00002.2016
Apart from having been used and misused for at least four millennia for, among others, recreational and medicinal purposes, the cannabis plant and its most peculiar chemical components, the plant cannabinoids (phytocannabinoids), have the merit to have led humanity to discover one of the most intriguing and pleiotropic endogenous signaling systems, the endocannabinoid system (ECS). This review article aims to describe and critically discuss, in the most comprehensive possible manner, the multifaceted aspects of 1 ) the pharmacology and potential impact on mammalian physiology of all major phytocannabinoids, and not only of the most famous one _9 -tetrahydrocannabinol, and 2 ) the adaptive prohomeostatic physiological, or maladaptive pathological, roles of the ECS in mammalian cells, tissues, and organs. In doing so, we have respected the chronological order of the milestones of the millennial route from medicinal/recreational cannabis to the ECS and beyond, as it is now clear that some of the early steps in this long path, which were originally neglected, are becoming important again. The emerging picture is rather complex, but still supports the belief that more important discoveries on human physiology, and new therapies, might come in the future from new knowledge in this field.

Functional Crosstalk between CB and TRPV1 Receptors Protects Nigrostriatal Dopaminergic Neurons in the MPTP Model of Parkinson’s Disease
Rayul Wi, Young Cheul Chung, and Byung Kwan Jin
Hindawi Journal of Immunology Research Volume 2020, Article ID 5093493, doi: 10.1155/2020/5093493
The present study examined whether crosstalk between cannabinoid (CB) and transient potential receptor vanilloid type 1 (TRPV1) could contribute to the survival of nigrostriatal dopamine neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD). MPTP induced a significant loss of nigrostriatal dopamine neurons and glial activation in the substantia nigra (SN) and striatum (STR) as visualized by tyrosine hydroxylase (TH) or macrophage antigen complex-1 (MAC-1) or glial fibrillary acidic protein (GFAP) immunocytochemistry, respectively. RT-PCR analysis shows the upregulation of inducible nitric oxide synthase, interleukin-1?, and tumor necrosis factor-? in microglia in the SN in vivo, indicating the activation of the inflammatory system. By contrast, treatment with capsaicin (a specific TRPV1 agonist) increased the survival of dopamine neurons in the SN and their fibers and dopamine levels in the STR in MPTP mice. Capsaicin neuroprotection is accompanied by inhibiting MPTP-induced glial activation and production of inflammatory cytokines. Treatment with AM251 and AM630 (CB1/2 antagonists) abolished capsaicin-induced beneficial effects, indicating the existence of a functional crosstalk between CB and TRPV1. Moreover, treatment with anandamide (an endogenous agonist for both CB and TRVP1) rescued nigrostriatal dopamine neurons and reduced gliosis-derived neuroinflammatory responses in MPTP mice. These results suggest that the cannabinoid and vanilloid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with neuroinflammation.

GPR55 – a putative “type 3” cannabinoid receptor in inflammation
Hyewon Yang, Juan Zhou and Christian Lehmann
J Basic Clin Physiol Pharmacol 2016; 27(3): 297–302
DOI 10.1515/jbcpp-2015-0080
G protein-coupled receptor 55 (GPR55) shares numerous cannabinoid ligands with CB1 and CB2 receptors despite low homology with those classical cannabinoid receptors. The pharmacology of GPR55 is not yet fully elucidated; however, GPR55 utilizes a different signaling system and downstream cascade associated with the receptor. Therefore, GPR55 has emerged as a putative “type 3” cannabinoid receptor, establishing a novel class of cannabinoid receptor. Furthermore, the recent evidence of GPR55-CB1 and GPR55-CB2 heteromerization along with its broad distribution from central nervous system to peripheries suggests the importance of GPR55 in various cellular processes and pathologies and as a potential therapeutic target in inflammation.

GPR55 and its Interaction with Membrane Lipids: Comparison with Other Endocannabinoid-Binding Receptors Valeria Gasperi, Enrico Dainese, Sergio Oddi, Mauro Maccarrone Current Medicinal Chemistry 20(1) November 2012 DOI: 10.2174/0929867311302010064
https://www.researchgate.net/publica...ding_Receptors
A number of integral membrane G protein-coupled receptors (GPCRs) share common structural features (including palmytoilated aminoacid residues and consensus sequences specific for interaction with cholesterol) that allow them to interact with lipid rafts, membrane cholesterol-rich microdomains able to regulate GPCR signalling and functions. Among GPCRs, type-1 and type-2 cannabinoid receptors, the molecular targets of endocannabinoids (eCBs), control many physiological and pathological processes through the activation of several signal transduction pathways. Recently, the orphan GPR55 receptor has been proved to be activated by many eCBs, thus leading to the hypothesis that it might be the "type-3" cannabinoid receptor. While the biological activity of eCBs and the influence of membrane lipids on their functions are rather well established, information regarding GPR55 is still scarce and often controversial. Based on this background, here we shall review current data about GPR55 pharmacology and signalling, highlighting its involvement in several pathophysiological conditions. We shall also outline the structural features that allow GPR55 to interact with cholesterol and to associate with lipid rafts; how the latter lipid microdomains impact the biological activity of GPR55 is also addressed, as well as their potential for the discovery of new therapeutics useful for the treatment of those human diseases that might be associated with alterations of GPR55 activity.

GPR55: Current Knowledge and Future Perspectives of a Purported “Type-3” Cannabinoid Receptor
Moriconi#, I. Cerbara#, M. Maccarrone, and A. Topai
In the last decade, accumulated evidence highlighted that GPR55 might be activated by several classical cannabinoid ligands, making this orphan receptor the main candidate to be considered as the “third” cannabinoid receptor. The investigation of its pharmacology has often provided divergent and more intricate results, that have complicated the understanding of the physiological role of GPR55. Nevertheless, the patent analysis regarding GPR55 outlines the fair interest of big pharmaceutical companies, especially in the first years of this decade. This investigation provides a brief overview of the current “state of the art” of our knowledge of GPR55, giving particular emphasis to its functional selectivity. This property could account for controversial roles of GPR55, whose pharmacology and downstream signaling is known to vary significantly both in ligand- and system-dependent manners. In addition, we gain insights into the challenging aspect of finding out novel GPR55 modulators, by analyzing conserved structural and functional motifs that, together with future studies, could help to elucidate its mechanism of action and to design more selective and potent small-molecules directed towards GPR55. Preliminary data highlight remarkable differences, but also intriguing commonalities, between GPR55 and other members of class A G-protein-coupled receptors. It is anticipated that, in the next future, novel lead candidates targeting GPR55 could represent new tools to better understand GPR55-mediated human diseases and, hopefully, generate an innovative class of effective next-generation therapeutics.

Heterogeneity of the Endocannabinoid System Between Cerebral Cortex and Spinal Cord Oligodendrocytes
R. Moreno-Luna, P. F. Esteban, B. Paniagua-Torija, A. Arevalo-Martin, D. Garcia-Ovejero & E. Molina-Holgado
Molecular Neurobiology (2020)
doi: 10.1007/s12035-020-02148-1
In the last years, regional differences have been reported between the brain and spinal cord oligodendrocytes, which should be considered when designing therapeutic strategies for myelin repair. Promising targets to achieve myelin restoration are the different components of the endocannabinoid system (ECS) that modulate oligodendrocyte biology, but almost all studies have been focused on brain-derived cells. Therefore, we compared the ECS between the spinal cord and cerebral cortex-derived oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (OLs). Cells from both regions express synthesizing and degrading enzymes for the endocannabinoid 2-arachidonoylglycerol, and degrading enzymes increase with maturation, more notably in the spinal cord (monoglyceride lipase-MGLL, alpha/beta hydrolase domain-containing 6-ABHD6, and alpha/beta hydrolase domain-containing 12-ABHD12). In addition, spinal cord OPCs express higher levels of the synthesizing enzymes diacylglycerol lipases alpha (DAGLA) and beta (DAGLB) than cortical ones, DAGLA reaching statistical significance. Cells from both the cortex and spinal cord express low levels of NAEs synthesizing enzymes, except for the glycerophosphodiester phosphodiesterase 1 (GDE-1) but high levels of the degrading enzyme fatty acid amidohydrolase (FAAH) that increases with maturation. Finally, cells from both regions show similar levels of CB1 receptor and GPR55, but spinal cord-derived cells show significantly higher levels of transient receptor potential cation channel V1 (TRPV1) and CB2. Overall, our results show that the majority of the ECS components could be targeted in OPCs and OLs from both the spinal cord and brain, but regional heterogeneity has to be considered for DAGLA, MGLL, ABHD6, ABHD12, GDE1, CB2, or TRPV1.

Identification and cloning of three novel human G protein-coupled receptor genes GPR52, GPR53 and GPR55: GPR55 is extensively expressed in human brain.
Sawzdargo, M., Nguyen, T., Lee, D. K., Lynch, K. R., Cheng, R., Heng, H. H. Q, George, S.R., O’Dowd, B. F.
Molecular Bain Research, 64(2), 193–198. (1999).
doi:10.1016/s0169-328x(98)00277-0
The G protein-coupled receptor GPCR family share a structural motif of seven transmembrane segments with large numbers of Ž . conserved residues in those regions. Here, we report the identification and cloning of two novel human intronless GPCR genes, GPR52, GPR55 and a pseudogene CGPR53. GPR55 was identified from the expressed sequence tags EST database whereas GPR52 and Ž . pseudogene CGPR53 originated from the high throughput genome HTG database. A partial cDNA clone obtained from the IMAGE Ž . Consortium of GPR55 was used to screen a human genomic library to acquire the full length gene. GPR52 and CGPR53 were amplified from human genomic DNA using primers based on the HTG sequences. GPR55 and GPR52 encode receptors of 319 and 361 amino acids, respectively. GPR55 gene was mapped to chromosome 2q37, using fluorescence in situ hybridization FISH , and its mRNA Ž . transcripts have been detected in the caudate nucleus and putamen, but not in five other brain regions. Human receptors showing the highest amino acid identity to GPR55 include P2Y5 29% , GPR23 30% , GPR35 27% and CCR4 23% . GPR52 gene localized to Ž. Ž. Ž. Ž. chromosome 1q24 shares the highest identity with GPR21 71% , histamine H2 27% and 5-HT4 26% human receptors. Ž. Ž. Ž. CGPR53 is a pseudogene mapped to chromosome 6p21 that demonstrates the highest similarity to the MRG 35% , MAS 28% and C5a 24% human Ž. Ž. Ž. receptor genes

Impaired vessel relaxation response and increased infarct size in smooth muscle cannabinoid receptor 1 knockout mice
Lin-Na Wang, Meng-Dan Xing, Wan-Ting Qu, Cong-Bei Wang, Zhi-Qiang Liu, Jing Han, Wei Ren, Yan-Ning Qiao
Microvasc Res . 2022 Jan;139:104263.
doi: 10.1016/j.mvr.2021.104263.
Cannabinoids are reported to regulate cardiovascular functions. Cannabinoid receptors 1 (CB1Rs) are widely expressed in both the neuronal system and vascular system, but the contribution of CB1Rs in vascular smooth muscle (CB1RSM) to cardiovascular functions is not clear yet. In this research, we analyzed the effects of CB1RSM on blood pressure, vasoconstriction, and vasodilation abilities by using conditionally CB1R knockout mice (CB1RSMKO). The results show no significant difference in basal blood pressure between the conscious CB1RSMKO and control mice, indicating that CB1RSM is not essential for basal blood pressure maintenance. The constriction of the CB1RSMKO mesenteric artery in vitro was not significantly altered compared with that of the control mice. In contrast, the relaxation to CB1R agonist 2-AG or WIN55212-2 was decreased in CB1RSMKO vessels, suggesting that activation of CB1RSM mediates the vasodilation effect of cannabinoids. Ischemia stroke mouse model was used to further identify the potential function of CB1RSM in pathological conditions, and the results showed that the infarct volume in CB1RSMKO mice is significantly increased compared with the control littermates. These results suggest that vascular CB1R may not play a central role in basal vascular health maintenance but is protective in ischemia states, such as stroke. The protection function may be mediated, at least partly, by the relaxation effect of CB1RSM-dependent activities of endocannabinoids.
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In silico patent searching reveals a new cannabinoid receptor.
Baker D1, Pryce G, Davies WL, Hiley CR.
Trends Pharmacol Sci. 2006 Jan;27(1):1-4.
DOI: 10.1016/j.tips.2005.11.003
Increasing evidence suggests that some cannabinoids mediate their effects independently of the known cannabinoid CB(1) and CB(2) receptors. Two recently published patents indicate that several cannabinoid receptor ligands also bind to the orphan G-protein-coupled receptor GPR55. This receptor is reported to be expressed in several tissues and might function in lipid or vascular biology. Thus, GPR55 might represent a new cannabinoid receptor.

Not Cannabis specifically
Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain
Arieh Moussaieff, Neta Rimmerman, Tatiana Bregman, Alex Straiker,
Christian C. Felder,_ Shai Shoham, Yoel Kashman, Susan M. Huang,
Hyosang Lee, Esther Shohami, Ken Mackie, Michael J. Caterina,
J. Michael Walker, Ester Fride, and Raphael Mechoulam
FASEB J. 22, 3024–3034 (2008)
doi: 10.1096/fj.07-101865
Burning of Boswellia resin as incense has been part of religious and cultural ceremonies for millennia and is believed to contribute to the spiritual exaltation associated with such events. Transient receptor potential vanilloid (TRPV) 3 is an ion channel implicated in the perception of warmth in the skin. TRPV3 mRNA has also been found in neurons throughout the brain; however, the role of TRPV3 channels there remains unknown. Here we show that incensole acetate (IA), a Boswellia resin constituent, is a potent TRPV3 agonist that causes anxiolytic-like and antidepressive-like behavioral effects in wild-type (WT) mice
with concomitant changes in c-Fos activation in the brain. These behavioral effects were not noted in TRPV3_/_ mice, suggesting that they are mediated via
TRPV3 channels. IA activated TRPV3 channels stably expressed in HEK293 cells and in keratinocytes from TRPV3_/_ mice. It had no effect on keratinocytes from TRPV3_/_ mice and showed modest or no effect on TRPV1, TRPV2, and TRPV4, as well as on 24 other receptors, ion channels, and transport proteins. Our results imply that TRPV3 channels in the brain may play
a role in emotional regulation. Furthermore, the biochemical and pharmacological effects of IA may provide a biological basis for deeply rooted cultural and religious traditions.

Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos.
Zuccarini, G., D’Atri, I., Cottone, E., Mackie, K., Shainer, I., Gothilf, Y., … Merlo, G. R.
International Journal of Molecular Sciences, 21(1), (2019). 168.
doi:10.3390/ijms21010168
The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake

Involvement of 5-hydroxytryptamine1A receptors in ?9-tetrahydrocannabinol-induced catalepsy-like immobilization in mice
Nobuaki Egashira, Tomomi Matsuda, Emi Koushi, Kenichi Mishima, Katsunori Iwasaki, Yukihiro Shoyama, Michihiro Fujiwara
European Journal of Pharmacology 550 (2006) 117–122
doi:10.1016/j.ejphar.2006.08.051
The present study investigated the involvement of 5-hydroxytryptamine1A (5-HT1A) receptors in ?9-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization in mice. THC (10 mg/kg, i.p.) induced catalepsy-like immobilization but had no effect on motor coordination in the rota-rod test. The selective cannabinoid CB1 receptor antagonist rimonabant (3 mg/kg, i.p.) completely antagonized THC-induced catalepsy-like immobilization. The 5-HT1A/5-HT7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 0.3 and 1 mg/kg, i.p.) and 5-HT1A receptor partial agonist buspirone (0.06 and 0.1 mg/kg, i.p.) inhibited this THC-induced catalepsy-like immobilization. Moreover, the selective 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohezane carboxamide dihydrochloride (WAY100635; 0.3 or 1 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization by 8-OH-DPAT (1 mg/kg) or buspirone (0.06 mg/kg). In contrast, the selective 5-HT7 receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl] phenol hydrochloride (SB269970) had no effect on this inhibitory effect of 8-OH-DPAT. On the other hand, WAY100635 (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). These findings suggest that the 5-HT1A receptors are involved in THC-induced catalepsy-like immobilization.

Isolation of a high affinity cannabinoid for human CB1 receptor from a medicinal cannabis variety: D9-Tetrahydrocannabutol, the butyl homologue of D9-tetrahydrocannabinol.
Linciano, P. et al.
J. Nat. Prod. 2020 Jan 24;83(1):88-98.
DOI: 10.1021/acs.jnatprod.9b00876
The butyl homologues of ?9 -tetrahydrocannabinol, ?9 -tetrahydrocannabutol (?9 -THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) infl orescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute confi gurations of the isolated cannabinoids were confi rmed by comparison with the spectroscopic data of the respective
compounds obtained by stereoselective synthesis. The butyl homologue of ?9 -THC, ?9 -THCB, showed an affi nity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (? )-trans -?9 -THC. Docking studies suggested the key bonds responsible for THC-like binding affi nity for the CB1 receptor. The formalin test in vivo was performed on ?9 -THCB in order to reveal possible analgesic and anti-infl ammatory properties. The tetrad test in mice showed a partial agonistic activity of ?9 -THCB toward the CB1 receptor.

Major Phytocannabinoids and Their Related Compounds: Should We Only Search for Drugs That Act on Cannabinoid Receptors?
Leontina Elena Filipiuc, Daniela Carmen Ababei, Teodora Alexa-Stratulat, Cosmin Vasilica Pricope, Veronica Bild, Raluca Stefanescu, Gabriela Dumitrita Stanciu, and Bogdan-Ionel Tamba
Pharmaceutics 2021, 13, 1823.
DOI: 10.3390/ pharmaceutics13111823
https://www.ncbi.nlm.nih.gov/pmc/art...s-13-01823.pdf
The most important discoveries in pharmacology, such as certain classes of analgesics or chemotherapeutics, started from natural extracts which have been found to have effects in traditional medicine. Cannabis, traditionally used in Asia for the treatment of pain, nausea, spasms, sleep, depression, and low appetite, is still a good candidate for the development of new compounds. If initially all attention was directed to the endocannabinoid system, recent studies suggest that many of the clinically proven effects are based on an intrinsic chain of mechanisms that do not necessarily involve only cannabinoid receptors. Recent research has shown that major phytocannabinoids and their derivatives also interact with non-cannabinoid receptors such as vanilloid receptor 1, transient receptor ankyrin 1 potential, peroxisome proliferator-activated receptor-gamma or glitazone receptor, G55 protein-coupled receptor, and nuclear receptor, producing pharmacological effects in diseases such as Alzheimer’s, epilepsy, depression, neuropathic pain, cancer, and diabetes. Nonetheless, further studies are needed to elucidate the precise mechanisms of these compounds. Structure modulation of phytocannabinoids, in order to improve pharmacological effects, should not be limited to the exploration of cannabinoid receptors, and it should target other courses of action discovered through recent research.

Membrane-mediated action of the endocannabinoid anandamide on membrane proteins: implications for understanding the receptor-independent mechanism.
Medeiros D, Silva-Gonçalves LC, da Silva AM, Dos Santos Cabrera MP, Arcisio-Miranda M.
Sci Rep. 2017 Jan 27;7:41362.
doi: 10.1038/srep41362.
Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.

Mitochondria: a possible nexus for the regulation of energy homeostasis by the endocannabinoid system?
Christopher Lipina , Andrew J Irving , Harinder S Hundal
American journal of physiology. Endocrinology and metabolism (2014)
doi: 10.1152/ajpendo.00100.2014
The endocannabinoid system (ECS) regulates numerous cellular and physiological processes through the activation of receptors targeted by endogenously produced ligands called endocannabinoids. Importantly, this signaling system is known to play an important role in modulating energy balance and glucose homeostasis. For example, current evidence indicates that the ECS becomes overactive during obesity whereby its central and peripheral stimulation drives metabolic processes that mimic the metabolic syndrome. Herein, we examine the role of the ECS in modulating the function of mitochondria, which play a pivotal role in maintaining cellular and systemic energy homeostasis, in large part due to their ability to tightly coordinate glucose and lipid utilization. Because of this, mitochondrial dysfunction is often associated with peripheral insulin resistance and glucose intolerance as well as the manifestation of excess lipid accumulation in the obese state. This review aims to highlight the different ways through which the ECS may impact upon mitochondrial abundance and/or oxidative capacity and, where possible, relate these findings to obesity-induced perturbations in metabolic function. Furthermore, we explore the potential implications of these findings in terms of the pathogenesis of metabolic disorders and how these may be used to strategically develop therapies targeting the ECS.

Modulation of the endocannabinoid system: Neuroprotection or neurotoxicity?
Christopher J. Fowler, Maria Luisa Rojo, Antonio Rodriguez-Gaztelumendi
Experimental Neurology 224 (2010) 37–47
doi:10.1016/j.expneurol.2010.03.021
There is now a large volume of data indicating that compounds activating cannabinoid CB1 receptors, either directly or indirectly by preventing the breakdown of endogenous cannabinoids, can protect against neuronal damage produced by a variety of neuronal “insults”. Given that such neurodegenerative stimuli result in increased endocannabinoid levels and that animals with genetic deletions of CB1 receptors are more susceptible to the deleterious effects of such stimuli, a case can be made for an endogenous neuroprotective role of endocannabinoids. However, this is an oversimplification of the current literature, since (a) compounds released together with the endocannabinoids can contribute to the neuroprotective effect; (b) other proteins, such as TASK-1 and PPARα, are involved; (c) the CB1 receptor antagonist/inverse agonist rimonabant has also been reported to have neuroprotective properties in a number of animal models of neurodegenerative disorders. Furthermore, the CB2 receptor located on peripheral immune cells and activated microglia are potential targets for novel therapies. In terms of the clinical usefulness of targeting the endocannabinoid system for the treatment of neurodegenerative disorders, data are emerging, but important factors to be considered are windows of opportunity (for acute situations such as trauma and ischemia) and the functionality of the target receptors (for chronic neurodegenerative disorders such as Alzheimer's disease).

New ursane triterpenoids from Ficus pandurata and their binding affinity for human cannabinoid and opioid receptors
Amgad I. M. Khedr, Sabrin R. M. Ibrahim, Gamal A. Mohamed, Hany E. A. Ahmed, Amany S. Ahmad, Mahmoud A. Ramadan, Atef E. Abd El-Baky, Koji Yamada, Samir A. Ross
Arch. Pharm. Res.
DOI 10.1007/s12272-016-0784-y
Phytochemical investigation of Ficus pandurata Hance (Moraceae) fruits has led to the isolation of two new triterpenoids, ficupanduratin A [1b -hydroxy-3b -acetoxy-11a -methoxy-urs-12-ene] (11 ) and ficupanduratin B [21a hydroxy-3b -acetoxy-11a -methoxy-urs-12-ene] (17 ), along with 20 known compounds: a -amyrin acetate (1 ), a –amyrin (2 ), 3b -acetoxy-20-taraxasten-22-one (3 ), 3b -acetoxy-11a methoxy- olean-12-ene (4 ), 3b -acetoxy-11a -methoxy-12-ursene (5 ), 11-oxo-a -amyrin acetate (6 ), 11-oxo-b -amyrin acetate (7 ), palmitic acid (8 ), stigmast-4,22-diene-3,6-dione (9 ), stigmast-4-ene-3,6-dione (10 ), stigmasterol (12 ), b –sitosterol (13 ), stigmast-22-ene-3,6-dione (14 ), stigmastane- 3,6-dione (15 ), 3b ,21b -dihydroxy-11a -methoxy-olean-12-ene (16 ), 3b -hydroxy-11a -methoxyurs-12-ene (18 ), 6-hydroxystigmast-4,22-diene-3-one (19 ), 6-hydroxystigmast-4-ene-3-one (20 ), 11a ,21a -dihydroxy-3b -acetoxy-urs-12-ene(21 ), and b -sitosterol-3-O -b -D -glucopyranoside (22 ). Compound 21 is reported for the first time from a natural source. The structures of the 20 compounds were elucidated on the basis of IR, 1D (1 H and 13 C), 2D (1 H–1 H COSY, HSQC, HMBC and NOESY) NMR and MS spectroscopic data, in addition to comparison with literature data. The isolated compounds were evaluated for their anti-microbial, antimalarial, anti-leishmanial, and cytotoxic activities. In addition, their radioligand displacement affinity on opioid and cannabinoid receptors was assessed. Compounds 4 , 11 , and 15 exhibited good affinity towards the CB2 receptor, with displacement values of 69.7, 62.5 and 86.5 %, respectively. Furthermore, the binding mode of the active compounds in the active site of the CB2 cannabinoid receptors was investigated through molecular modeling

No more pain upon Gq-protein-coupled receptor activation: role of endocannabinoids
Sherry Shu-Jung Hu, Yu-Cheng Ho and Lih-Chu Chiou
European Journal of Neuroscience, Vol. 39, pp. 467–484, 2014
doi:10.1111/ejn.12475
https://onlinelibrary.wiley.com/doi/...1111/ejn.13231
Marijuana has been used to relieve pain for centuries. The analgesic mechanism of its constituents, the cannabinoids, was only revealed after the discovery of cannabinoid receptors (CB1 and CB2) two decades ago. The subsequent identification of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), and their biosynthetic and degradation enzymes discloses the therapeutic potential of compounds targeting the endocannabinoid system for pain control. Inhibitors of the anandamide and 2-AG degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, respectively, may be superior to direct cannabinoid receptor ligands as endocannabinoids are synthesized on demand and rapidly degraded, focusing action at generating sites. Recently, a promising strategy for pain relief was revealed in the periaqueductal gray (PAG). It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C–diacylglycerol lipase enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. Here, we introduce the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, particularly in the PAG. We also review recent studies disclosing the GqPCR–phospholipase C–diacylglycerol lipase–2-AG retrograde disinhibition mechanism in the PAG, induced by activating several GqPCRs, including metabotropic glutamatergic (type 5 metabotropic glutamate receptor), muscarinic acetylcholine (M1/M3), and orexin 1 receptors. Disinhibition mediated by type 5 metabotropic glutamate receptor can be initiated by glutamate transporter inhibitors or indirectly by substance P, neurotensin, cholecystokinin and capsaicin. Finally, the putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is discussed.

Novel Natural and Synthetic Ligands of the Endocannabinoid System
Lumir Hanus, Raphael Mechoulam
Current Medicinal Chemistry 17(14):1341-59 February 2010
DOI: 10.2174/092986710790980096
In this review we describe recent advances in the chemistry of novel CB(1)/CB(2) agonists, CB(1) antagonists, selective CB(2) agonists, fatty acid amide hydrolase inibitors, monoglyceride (MGL) and diglyceride (DAGL) inhibitors and cannabinoid-type agonists and antagonists of non CB(1)/CB(2) receptors. In view of recent interest in the activities of fatty acid amides of amino acids (N-acyl amino acids) a list of this type of compounds was compiled and is presented as a Table. We conclude that further synthetic work based on both the plant cannabinoids and on the endocannabinoids may lead to novel therapeutics and that the identification and the elucidation of the biological profile of the myriad of endogenous N-acyl amino acids and related compounds may enhance the already wide spectrum of lipidomics.

One for the Price of Two…Are Bivalent Ligands Targeting Cannabinoid Receptor Dimers Capable of Simultaneously Binding to both Receptors?
Glass, M., Govindpani, K., Furkert, D. P., Hurst, D. P., Reggio, P. H., & Flanagan, J. U.
Trends in Pharmacological Sciences, 37(5), 353–363.(2016).
doi:10.1016/j.tips.2016.01.010
Bivalent ligands bridging two G-protein-coupled receptors (GPCRs) provide valuable pharmacological tools to target oligomers. The success of therapeutically targeting the cannabinoid CB1 receptor has been limited, in part due to its widespread neuronal distribution. Therefore, CB1 ligands targeting oligomers that exhibit restricted distribution or altered pharmacology are highly desirable, and several bivalent ligands containing a CB1 pharmacophore have been reported. Bivalent ligand action presumes that the ligand simultaneously binds to both receptors within the dimeric complex. However, based on the current understanding of CB1 ligand binding, existing bivalent ligands are too short to bind both receptors simultaneously. However, ligands with longer linkers may not be the solution, because evidence suggests that ligands enter CB1 through the lipid bilayer and, thus, linkers are unlikely to exit the receptor through its external face. Thus, the entire premise of designing bivalent ligands targeting CB1 must be revisited.

Peripheral, but not central effects of cannabidiol derivatives: Mediation by CB1 and unidentified receptors
Ester Fridea, Datta Pondec, Aviva Breuerc, Lumir Hanusˇ
Neuropharmacology 48 (2005) 1117e1129
http://citeseerx.ist.psu.edu/viewdoc...=rep1&type=pdf
Delta-9 tetrahydrocannabinol (D9 -THC) and ()-cannabidiol (()-CBD) are major constituents of the Cannabis sativa plant with different pharmacological profiles: (D9 -THC activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. ()-CBD possesses no, or very weak affinity for these receptors. We tested a series of (C)- and ()-CBD derivatives for central and peripheral effects in mice. None of the ()-CBD derivatives were centrally active, yet most inhibited intestinal motility. Of the five (C)-CBD derivatives, all with CB1 receptor affinity, only (C)-7-OH-CBD-DMH (DMHZ1,1-dimethylheptyl), acted centrally, while all five arrested defecation. The effects of (C)-CBD-DMH and (C)-7-OH-CBD-DMH were inhibited by the CB1 receptor antagonist SR141716. The CB2 receptor antagonist SR144528, and the vanilloid TRPV1 receptor antagonist capsazepine, had no influence. Further, the ()-CBD derivatives ()-7-COOH-CBD and ()-7-COOH-CBD-DMH, displayed antiinflammatory activity. We suggest that (C)-CBD analogues have mixed agonist/antagonist activity in the brain. Second, ()-CBD analogues which are devoid of cannabinoid receptor affinity but which inhibit intestinal motility, suggest the existence of a non-CB1, non-CB2 receptor. Therefore, such analogues should be further developed as antidiarrheal and/or antiinflammatory drugs. We propose to study the therapeutic potential of ()- and (C)-CBD derivatives for complex conditions such as inflammatory bowel disease and cystic fibrosis

Pharmacology of Cannabinoid Receptors Review No.2/2-2008
Bela Szabo
Biotrend Reviews No.2 2008
https://www.academia.edu/6267916/Pha...ew_No.2_2-2008
https://www.scribd.com/document/2931...nnabinoids-pdf
Until 20 years ago, only vague explanations existed over the mechanism of action of products from Cannabis sativa. It was thought, for example, that the main psychotropic compound in the plant, Delta-9 tetrahydrocannabinol, acts by changing celll membrane properties due to its high lipophilicity. This scarcity of knowledge started to change in 1990 with thw identification of a G protein-coupled receptor for Delta-9 tetrahydrocannabinol, the CB1 cannabinoid receptor. Since then a tremendous amount of information accumulated: additional receptors were discovered and endogenous agonists (endocannabinoids) of the receptors were identified. Many new synthetic agonists and antagonists of the receptors have been developed. Intensive research is carried out to clarify the physiological and pathophysiological roles of endocannabinoids. And it is probed how diseases can be treated by exogenous cannabinoid receptor agonists or antagonists or by modulators of the biosynthesis or degradation of endocannabinoids.

Plant-Derived Cannabinoids Modulate the Activity of Transient Receptor Potential Channels of Ankyrin Type-1 and Melastatin Type-8.
De Petrocellis, L., Vellani, V., Schiano-Moriello, A., Marini, P., Magherini, P. C., Orlando, P., & Di Marzo, V.
Journal of Pharmacology and Experimental Therapeutics, 325(3), 1007–1015. doi:10.1124/jpet.107.134809
The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and 9 -tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2 in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2 elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC50 60 nM) and the least potent being CBG and CBD acid (EC50 3.4 –12.0 M). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC50 34.3 M). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2 elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC50 70 –160 nM), whereas CBD acid was the least potent compound (IC50 0.9 –1.6 M). CBG inhibited Ca2 elevation also in icilin-sensitive DRG neurons with potency (IC50 4.5 M) similar to that of anandamide (IC50 10 M). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

Prejunctional and peripheral effects of the cannabinoid CB1 receptor inverse agonist rimonabant (SR 141716)
Hester van Diepen & Eberhard Schlicker & Martin C. Michel
Naunyn-Schmiedeberg’s Arch Pharmacol (2008) 378:345–369
DOI: 10.1007/s00210-008-0327-2

https://link.springer.com/content/pdf/10.1007/s00210-008-0327-2.pdf
Rimonabant is an inverse agonist specific for cannabinoid receptors and selective for their cannabinoid-1 (CB1) subtype. Although CB1 receptors are more abundant in the central nervous system, rimonabant has many effects in the periphery, most of which are related to prejunctional modulation of transmitter release from autonomic nerves. However, CB1 receptors are also expressed in, e.g., adipocytes and endothelial cells. Rimonabant inhibits numerous cardiovascular cannabinoid effects, including the decrease of blood pressure by central and peripheral (cardiac and vascular) sites of action, with the latter often being endothelium dependent. Rimonabant may also antagonize cannabinoid effects in myocardial infarction and in hypotension associated with septic shock or liver cirrhosis. In the gastrointestinal tract, rimonabant counteracts the cannabinoid-induced inhibition of secretion and motility. Although not affecting most cannabinoid effects in the airways, rimonabant counteracts inhibition of smoothmuscle contraction by cannabinoids in urogenital tissues and may interfere with embryo attachment and outgrowth of blastocysts. It inhibits cannabinoid-induced decreases of intraocular pressure. Rimonabant can inhibit proliferation of, maturation of, and energy storage by adipocytes. Among the many cannabinoid effects on hormone secretion, only some are rimonabant sensitive. The effects of rimonabant on the immune system are not fully clear, and it may inhibit or stimulate proliferation in several types of cancer. We conclude that direct effects of rimonabant on adipocytes may contribute to its clinical role in treating obesity. Other peripheral effects, many of which occur prejunctionally, may also contribute to its overall clinical profile and lead to additional indications as well adverse events.



Sch35966 is a potent, selective agonist at the peripheral cannabinoid receptor (CB2) in rodents and primates
W Gonsiorek , CA Lunn , X Fan , G Deno , J Kozlowski and RW Hipkin
British Journal of Pharmacology (2007) 151, 1262–1271
doi:10.1038/sj.bjp.0707336
https://bpspubs.onlinelibrary.wiley... .sj.bjp.0707336
Background and purpose: The peripheral cannabinoid receptor (CB2) is expressed on peripheral immune cells and is thought to have a role in the immunosuppressive effects of cannabinoids. Historically, there have been few potent, CB2-selective agonists to assess the contribution of CB2 to this phenomenon. The studies presented here describe the synthesis of 8,10- bis[(2,2-dimethyl-1-oxopropyl)oxy]-11-methyl-1234-tetrahydro-6H-benzoquinolizin-6-one (Sch35966), which binds with low nanomolar potency to CB2 in both primates and rodents. Experimental approach: The affinity, potency and efficacy of Sch35966 and other cannabinoid ligands at CB2 was assessed using competition binding assays vs [3 H]CP55,940, [35S]GTPgS exchange, cAMP accumulation and cell chemotaxis assays. Key results: We showed that Sch35966 has 4450-fold selectivity for CB2 binding vs the central cannabinoid receptor (CB1) in primates (humans and cynomolgus monkeys) and rodents (rats and mice). Sch35966 is an agonist as it effectively inhibited forskolin-stimulated cAMP synthesis in CHO-hCB2 cells, stimulated [35S]GTPgS exchange and directed chemotaxis in cell membranes expressing CB2. In all species examined, Sch35966 was more potent, more efficacious and more selective than JWH-015 (a commonly used CB2-selective agonist). Conclusions and implications: Taken together, the data show that Sch35966 is a potent and efficacious CB2-selective agonist in rodents and primates.

Sex difference in brain CB1 receptor availability in man.
Laurikainen, H., Tuominen, L., Tikka, M., Merisaari, H., Armio, R.-L., Sormunen, E., … Hietala, J.
NeuroImage. (2018).
doi:10.1016/j.neuroimage.2018.10.013
The endocannabinoid system (ECS) has a widespread neuromodulatory function in the central nervous system and is involved in important aspects of brain function including brain development, cortical rhythms, plasticity, reward, and stress sensitivity. Many of these effects are mediated via the cannabinoid CB1 receptor (CB1R) subtype. Animal studies convincingly show an interaction between the ECS and sex hormones, as well as a sex difference of higher brain CB1R in males. Human in vivo studies of sex difference have yielded discrepant findings. Gender differences in CB1R availability were investigated in vivo in 11 male and 11 female healthy volunteers using a specific CB1R tracer FMPEP-d2 and positron emission tomography (PET). Regional FMPEP-d2 distribution volume was used as a proxy for CB1R availability. In addition, we explored whether CB1R availability is linked to neuropsychological functioning. Relative to females, CB1R availability was on average 41% higher in males (p=0.002) with a regionally specific effect larger in the posterior cingulate and retrosplenial cortices (p=0.001). Inter-subject variability in CB1R availability was similar in both groups. Voxel-based analyses revealed an inverse association between CB1R availability and visuospatial working memory task performance in both groups (p=0.001) Inter-subject variability in CB1R availability was 36 similar in both groups. Voxel-based analyses revealed an inverse association between CB1R availability and visuospatial working memory task performance in both groups (p<0.001). A CB1R sex difference with a large effect size was observed and should be considered in the design of CB1R-related studies on neuropsychiatric disorders. The behavioural correlates and clinical significance of this difference remain to be further elucidated, but our studies suggest an association between CB1R availability and working memory.

Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced ?-Endorphin Release.
Gao F, Zhang LH, Su TF, Li L, Zhou R, Peng M, Wu CH, Yuan XC, Sun N, Meng XF, Tian B, Shi J, Pan HL, Li M.
Mol Neurobiol. 2016 Aug;53(6):3616-3625.
doi: 10.1007/s12035-015-9291-2.
Activation of cannabinoid receptor-2 (CB2) results in ?-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the G?? subunits from Gi/o, but not G?s, were involved in CB2 receptor activation-induced ?-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced ?-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced ?-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced ?-endorphin release through Gi/o-G??-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists.

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors Iu Raïch, Rafael Rivas-Santisteban, Alejandro Lillo, Jaume Lillo, Irene Reyes-Resina, Xavier Nadal, Carlos Ferreiro-Vera, Veronica Sanchez de Medina, Maria Majellaro, Eddy Sotelo, Gemma Navarro, Rafael Franco
Pharmacological Research 174 (2021)
DOI: 10.1016/j.phrs.2021.105970
We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2′ _-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation

Skeletal lipidomics: regulation of bone metabolism by fatty acid amide family
Itai Bab, Reem Smoum, Heather Bradshaw, Raphael Mechoulam
British Journal of Pharmacology (2011) 163 1441–1446
DOI: 10.1111/j.1476-5381.2011.01474.x
There is increasing evidence demonstrating that fatty acid derivatives play a key regulatory role in a variety of tissues. However, the study of skeletal lipidomics is just emerging and global strategies, such as targeted lipidomics, have not been applied to bone tissue. Such strategies hold great promises as in the case of genomics and proteomics. A partial profile of endocannabinoids and endocannabinoid?like compounds has demonstrated the presence of several long?chain fatty acid amides (FAAs), some of which displaying potent effects on osteoblasts, the bone forming cells and osteoclasts, the bone resorbing cells. In the skeleton, the FAAs activate the CB1 cannabinoid receptor present in sympathetic nerve terminals as well as CB2 cannabinoid receptor, the Gi?protein coupled receptor GPR55, and the transient receptor potential vanilloid type ion channel expressed by osteoblasts and/or osteoclasts. This review on the skeletal FAA system focuses on the production of FAAs in the skeleton and their net bone anabolic and anti?catabolic activity resulting from the stimulation of bone formation and inhibition of bone resorption. As the FAA family holds great promise as a basis for the treatment of osteoporosis and other diseases involving bone, further studies should aim towards the complete profiling of these lipids and their receptors in bone tissue, followed by elucidation of their function and mechanism of action.

Small Molecules from Nature Targeting G-Protein Coupled Cannabinoid Receptors: Potential Leads for Drug Discovery and Development
Charu Sharma, Bassem Sadek, Sameer N. Goyal, Satyesh Sinha, Mohammad Amjad Kamal, and Shreesh Ojha
Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 238482, 26 pages
Doi: 10.1155/2015/238482
The cannabinoid molecules are derived from Cannabis sativa plant which acts on the cannabinoid receptors types 1 and 2 (CB1 and CB2) which have been explored as potential therapeutic targets for drug discovery and development. Currently, there are numerous cannabinoid based synthetic drugs used in clinical practice like the popular ones such as nabilone, dronabinol, and ?9 tetrahydrocannabinol mediates its action throughCB1/CB2 receptors.However, these synthetic based Cannabis derived compounds are known to exert adverse psychiatric effect and have also been exploited for drug abuse. This encourages us to find out an alternative and safe drug with the least psychiatric adverse effects. In recent years, many phytocannabinoids have been isolated from plants other than Cannabis. Several studies have shown that these phytocannabinoids show affinity, potency, selectivity, and efficacy towards cannabinoid receptors and inhibit endocannabinoid metabolizing enzymes, thus reducing hyperactivity of endocannabinoid systems. Also, these naturally derived molecules possess the least adverse effects opposed to the synthetically derived cannabinoids. Therefore, the plant based cannabinoid molecules proved to be promising and emerging therapeutic alternative. The present review provides an overview of therapeutic potential of ligands and plants modulating cannabinoid receptors that may be of interest to pharmaceutical industry in search of new and safer drug discovery and development for future therapeutics.

So what do we call GPR18 now?
SPH Alexander
DOI:10.1111/j.1476-5381.2011.01731.x
British Journal of Pharmacology (2012) 165 2411–2413 2411
In this issue of the British Journal of Pharmacology, the second associated with a celebration of the 80th birthday of Raphael Mechoulam, Doug McHugh, Heather Bradshaw and colleagues describe their investigations into the orphan GPCR, GPR18 (McHugh et al., 2012). They observed that N-arachidonoylglycine (NAGly, an endogenous fatty acid: amino acid conjugate; see Figure 1A), D9 - tetrahydrocannabinol (THC, the major psychoactive component of the cannabis plant; see Figure 1A) and anandamide (AEA, an endogenous cannabinoid agonist; see Figure 1A) function as ‘full’ agonists at GPR18 expressed heterologously in HEK293 cells, as measured by phosphorylation of ERK1/2, with that rank order of potency. This leads me to pose the question: what do we call GPR18 now? The short answer to that question, of course, is that we wait for a decision from the appropriate Nomenclature Committee of IUPHAR – the Union of Basic and Clinical Pharmacology (https://www.iuphar.org/nciuphar.html). So what options are open to them? Guidance from NC-IUPHAR suggests naming the receptor after the endogenous ligand or group of ligands. Of the ligands examined for activity at recombinant GPR18, NAGly was the most potent in the current study (McHugh et al., 2012). Is GPR18, therefore, a NAGly (or potentially a NAGly1 or NAG1) receptor?

Spinal cannabinoid CB1 or CB2 receptors activation attenuates mechanical allodynia in streptozotocin-induced diabetic rats
Maryna Rodrigues Gonçalves, Milena Santana da Conceição, Carlos Henrique Alves Jesus, Aléxia Thamara Gasparin, Evelize Stacoviaki Rosa, Joice Maria da Cunha
Behav Pharmacol. 2020 Aug 13.
doi: 10.1097/FBP.0000000000000580.
Diabetes is a chronic disease associated with a high number of complications such as peripheral neuropathy, which causes sensorial disturbances and may lead to the development of diabetic neuropathic pain (DNP). The current treatment for DNP is just palliative and the drugs may cause severe adverse effects, leading to discontinuation of treatment. Thus, new therapeutic targets need to be urgently investigated. Studies have shown that cannabinoids have promising effects in the treatment of several pathological conditions, including chronic pain. Thus, we aimed to investigate the acute effect of the intrathecal injection of CB1 or CB2 cannabinoid receptor agonists N-(2-chloroethyl)-5Z, 8Z, 11Z, 14Z-eicosatetraenamide (ACEA) or JWH 133, respectively (10, 30 or 100 ?g/rat) on the mechanical allodynia associated with experimental diabetes induced by streptozotocin (60 mg/kg; intraperitoneal) in rats. Cannabinoid receptor antagonists CB1 AM251 or CB2 AM630 (1 mg/kg) were given before treatment with respective agonists to confirm the involvement of cannabinoid CB1 or CB2 receptors. Rats with diabetes exhibited a significant reduction on the paw mechanical threshold 2 weeks after diabetes induction, having the maximum effect observed 4 weeks after the streptozotocin injection. This mechanical allodynia was significantly improved by intrathecal treatment with ACEA or JWH 133 (only at the higher dose of 100 ?g). Pre-treatment with AM251 or AM630 significantly reverted the anti-allodynic effect of the ACEA or JWH 133, respectively. Considering the clinical challenge that the treatment of DPN represents, this study showed for the first time, that the intrathecal cannabinoid receptors agonists may represent an alternative for the treatment of DNP.

Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease.
McKallip, R. J.
Blood, 100(2), 627–634.(2002).
doi:10.1182/blood-2002-01-0098
In the current study, we examined whether ligation of CB2 receptors would lead to induction of apoptosis in tumors of immune origin and whether CB2 agonist could be used to treat such cancers. Exposure of murine tumors EL-4, LSA, and P815 to delta-9-tetrahydrocannabinol (THC) in vitro led to a significant reduction in cell viability and an increase in apoptosis. Exposure of EL-4 tumor cells to the synthetic cannabinoid HU-210 and the endogenous cannabinoid anandamide led to significant induction of apoptosis, whereas exposure to WIN55212 was not effective. Treatment of EL-4 tumorbearing mice with THC in vivo led to a significant reduction in tumor load, increase in tumor-cell apoptosis, and increase in survival of tumor-bearing mice. Examination of a number of human leukemia and lymphoma cell lines, including Jurkat, Molt-4, and Sup-T1, revealed that they expressed CB2 receptors but not CB1. These human tumor cells were also susceptible to apoptosis induced by THC, HU-210, anandamide, and the CB2-selective agonist JWH-015. This effect was mediated at least in part through the CB2 receptors because pretreatment with the CB2 antagonist SR144528 partially reversed the THC-induced apoptosis. Culture of primary acute lymphoblastic leukemia cells with THC in vitro reduced cell viability and induced apoptosis. Together, the current data demonstrate that CB2 cannabinoid receptors expressed on malignancies of the immune system may serve as potential targets for the induction of apoptosis. Also, because CB2 agonists lack psychotropic effects, they may serve as novel anticancer agents to selectively target and kill tumors of immune origin.

Targeting the cannabinoid receptor CB2 in a mouse model of l-dopa induced dyskinesia.
Rentsch, P., Stayte, S., Egan, T., Clark, I., & Vissel, B.
Neurobiology of Disease, 104646.(2019).
doi:10.1016/j.nbd.2019.104646
L-dopa induced dyskinesia (LID) is a debilitating side -effect of the primary treatment used in Parkinson’s disease (PD), l -dopa. Here we investigate the effect of HU -308, a cann abinoid CB2 receptor agonist, on LIDs. Utilizing a mouse model of PD and LIDs, induced by 6 -OHDA and subsequent l -dopa treatment, we show that HU -308 reduce d LIDs as effectively as amantadine, the current frontline treatment. Furthermore , treatment with HU -308 plus amantadine resulted in a greater anti - dyskinetic effect than maximally achieved with HU -308 alone , potentially suggesting a synergistic effect of these two treatments. Lastly, we demonstrate d that treatment with HU -308 and amantadine either alone, or in combination, decrease d striatal neuroinflammation, a mechanism which has been suggested to contribute to LIDs . Taken together, our results suggest pharmacological treatments with CB2 agonists merit further investigation as therapies for LID s in PD patients. Furthermore, since CB2 receptors are thought to be primarily expressed on, and signal through, glia, our data provide weight to suggestion that neuroinflammation, or more specifically, altered glial function , plays a role in development of LIDs.

Targeting the cannabinoid system for pain relief?
Lih-Chu Chiou, Sherry Shu-Jung Hu, Yu-Cheng Ho
Acta Anaesthesiologica Taiwanica 51 (2013) 161e170
DOI: 10.1016/j.aat.2013.10.004
Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB1) receptors, as well as peripheral CB1 and CB2 receptors. CB2-selective agonists have the benefit of lacking CB1 receptor-mediated CNS side effects. Anandamide and 2- arachidonoylglycerol (2-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, we also review recent studies that revealed a novel analgesic mechanism, involving 2-AG in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. This GqPCR-PLC-DAGL-2-AG retrograde disinhibition mechanism in the PAG can be initiated by activating type 5 metabotropic glutamate receptor (mGluR5), muscarinic acetylcholine (M1/M3), and orexin (OX1) receptors. mGluR5-mediated disinhibition can be initiated by glutamate transporter inhibitors, or indirectly by substance P, neurotensin, cholecystokinin, capsaicin, and AM404, the bioactive metabolite of acetaminophen in the brain. The putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed.

The Bivalent Rewarding and Aversive properties of ?9-tetrahydrocannabinol are Mediated Through Dissociable Opioid Receptor Substrates and Neuronal Modulation Mechanisms in Distinct Striatal Sub-Regions
Christopher Norris, Hanna J. Szkudlarek, Brian Pereira, Walter Rushlow & Steven R. Laviolette
https://www.nature.com/articles/s41598-019-46215-7.pdf
une 2019 Scientific Reports IN PRESS
DOI: 10.1038/s41598-019-46215-7
The primary psychoactive compound in cannabis, ?9-tetrahydrocannabinol (THC), is capable of producing bivalent rewarding and aversive affective states through interactions with the mesolimbic system. However, the precise mechanisms underlying the dissociable effects of THC are not currently understood. In the present study, we identify anatomically dissociable effects of THC within the rat nucleus accumbens (NAc), using an integrative combination of behavioral pharmacology and in vivo neuronal electrophysiology. We report that the rewarding vs. aversive stimulus properties of THC are both anatomically and pharmacologically dissociable within distinct anterior vs. posterior sub-regions of the NAc. While the rewarding effects of THC were dependent upon local ?-opioid receptor signaling, the aversive effects of THC were processed via a ?-opioid receptor substrate. Behaviorally, THC in the posterior NASh induced deficits in social reward and cognition whereas THC in the anterior NAc, potentiated opioid-related reward salience. In vivo neuronal recordings demonstrated that THC decreased medium spiny neuron (MSN) activity in the anterior NAc and increased the power of gamma (?) oscillations. In contrast, THC increased MSN activity states in the posterior NASh and decreased ?-oscillation power. These findings reveal critical new insights into the bi-directional neuronal and pharmacological mechanisms controlling the dissociable effects of THC in mesolimbic-mediated affective processing

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABAA receptors
R Baur, J Gertsch and E Sigel
British Journal of Pharmacology (2012) 165 2479–2484
DOI:10.1111/j.1476-5381.2011.01405.x
BACKGROUND AND PURPOSE Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB1 antagonists / inverse agonists. Concentrations of 0.5–10 mM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABAA receptors but found a significant positive allosteric modulation instead.
EXPERIMENTAL APPROACH Recombinant GABAA receptors were expressed in Xenopus oocytes. Receptors were exposed to AM251 or rimonabant in the absence and presence of GABA. Standard electrophysiological techniques were used to monitor the elicited ionic currents.
KEY RESULTS AM251 dose-dependently potentiated responses to 0.5 mM GABA at the recombinant a1b2g2 GABAA receptor with an EC50 below 1 mM and a maximal potentiation of about eightfold. The Hill coefficient indicated that more than one binding site for AM251 was located in this receptor. Rimonabant had a lower affinity, but a fourfold higher efficacy. AM251 potentiated also currents mediated by a1b2, axb2g2 (x = 2,3,5,6), a1b3g2 and a4b2d GABAA receptors, but not those mediated by a1b1g2. Interestingly, the CB1 receptor antagonists LY320135 and O-2050 did not significantly affect a1b2g2 GABAA receptor-mediated currents at concentrations of 1 mM.
CONCLUSIONS AND IMPLICATIONS This study identified rimonabant and AM251 as positive allosteric modulators of GABAA receptors. Thus, potential GABAergic effects of commonly used concentrations of these compounds should be considered in in vitro experiments, especially at extrasynaptic sites where GABA concentrations are low.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain
Klauke, A.-L., Racz, I., Pradier, B., Markert, A., Zimmer, A. M., Gertsch, J., & Zimmer, A. (2014). European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology 24(4)
Doi: 10.1016/j.euroneuro.2013.10.008
The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent
manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

The cannabinoid receptors.
Howlett, A. C.
Prostaglandins & Other Lipid Mediators, 68-69, 619–631.(2002).
doi:10.1016/s0090-6980(02)00060-6
Cannabinoid receptors were named because they have affinity for the agonist 9-tetrahydrocannabinol (9-THC), a ligand found in organic extracts from Cannabis sativa. The two types of cannabinoid receptors, CB1 and CB2, are G protein coupled receptors that are coupled through the Gi/o family of proteins to signal transduction mechanisms that include inhibition of adenylyl cyclase, activation of mitogen-activated protein kinase, regulation of calcium and potassium channels (CB1 only), and other signal transduction pathways. A class of the eicosanoid ligands are relevant to lipid-mediated cellular signaling because they serve as endogenous agonists for cannabinoid receptors, and are thus referred to as endocannabinoids. Those compounds identified to date include the eicosanoids arachidonoylethanolamide (anandamide), 2-arachidonoylglycerol and 2-arachidonylglyceryl ether (noladin ether). Several excellent reviews on endocannabinoids and their synthesis, metabolism and function have appeared in recent years [1–4]. This paper will describe the biological activities, pharmacology, and signal transduction mechanisms for the cannabinoid receptors, with particular emphasis on the responses to the eicosanoid ligands. © 2002 Elsevier Science Inc. All rights reserved.

The case for peripheral CB1 receptor blockade in the treatment of visceral obesity and its cardiometabolic complications
George Kunos, Joseph Tam
British Journal of Pharmacology (2011) 163 1423–143
doi: 10.1111/j.1476-5381.2011.01352.x
In this review, we consider the role of endocannabinoids and cannabinoid?1 (CB1) cannabinoid receptors in metabolic regulation and as mediators of the thrifty phenotype that underlies the metabolic syndrome. We survey the actions of endocannabinoids on food intake and body weight, as well as on the metabolic complications of visceral obesity, including fatty liver, insulin resistance and dyslipidemias. Special emphasis is placed on weighing the relative importance of CB1 receptors located in peripheral tissues versus the central nervous system in mediating the metabolic effects of endocannabinoids. Finally, we review recent observations that indicate that peripherally restricted CB1 receptor antagonists retain efficacy in reducing weight and improving metabolic abnormalities in mouse models of obesity without causing behavioural effects predictive of neuropsychiatric side effects in humans.

The CB1 cannabinoid receptor C-terminus regulates receptor desensitization in autaptic hippocampal neurones
Alex Straiker, Jim Wager-Miller and Ken Mackie
British Journal of Pharmacology (2012) 165 2652–2659
DOI:10.1111/j.1476-5381.2011.01743.x
BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is the chief mediator of the CNS effects of cannabinoids. In cell culture model systems, CB1 receptors both desensitize and internalize on activation. Previous work suggests that the extreme carboxy-terminus of this receptor regulates internalization via phosphorylation of residues clustered within this region. Mutational analysis of the carboxy-terminus of CB1 receptors has demonstrated that the last six serine/threonine residues are necessary for agonist-induced internalization. However, the structural determinants of CB1 receptor internalization are also dependent on the local cellular environment. The importance of cell context on CB1 receptor function calls for an investigation of the functional roles of these residues in neurones.
EXPERIMENTAL APPROACH To determine the structural requirements of CB1 internalization in neurones, we evaluated the signalling properties of carboxyterminal mutated CB1 receptors expressed in cultured autaptic hippocampal neurones, using electrophysiological methods.
KEY RESULTS CB1 receptors transfected into CB1 knockout neurones signalled and desensitized as did wild-type neurones, allowing us to test specific CB1 receptor mutations. Deletion of the last 13 residues yielded a CB1 receptor that inhibited excitatory postsynaptic currents but did not desensitize. Furthermore, mutation of the final six serine and threonine residues to alanines resulted in a non-desensitizing receptor. In contrast, CB1 receptors lacking residues 419–460, leaving the last 14 residues intact, did desensitize.
CONCLUSIONS AND IMPLICATIONS The distal thirteen residues of CB1 receptors are crucial for their desensitization in cultured neurones. Furthermore, this desensitization is likely to follow phosphorylation of serines and threonines within this region.

The CB1 Receptor Differentially Regulates IFN-c Production In Vitro and in Experimental Autoimmune Encephalomyelitis
James M. Nichols and Barbara L.F. Kaplan
Cannabis and Cannabinoid Research Volume X, Number X, 2020
https://www.liebertpub.com/doi/pdf/1.../can.2020.0046
DOI: 10.1089/can.2020.0046
Introduction: Activation of the peripheral immune system and the infiltration of immune cells into the central nervous system are both key features of the experimental autoimmune encephalomyelitis (EAE) model. By exploring how the endocannabinoid system works to modulate this response, we can better understand how exogenous cannabinoids, such as THC, might be used to modulate the immune responses of multiple sclerosis patients. Materials and Methods: In this study, we examined the role of the CB1 receptor in IFN-c and IL-17A production in the EAE model and in vitro stimulations of naive splenocytes using Cnr1/ mice and wild-type (WT) littermates. We also introduce a novel method of scoring spinal cord histological sections to show the differences in disease severity between Cnr1/ and WT mice with EAE. Results: Clinical scores of Cnr1//EAE and WT/EAE mice showed more severe disease progression in Cnr1/ mice, which was confirmed using our new histological scoring method. In the peripheral immune system, IFN-c production by restimulated splenocytes from Cnr1//EAE mice, compared with WT/EAE mice, was increased and the primary source of IFN-c was a CD3 cell population; however, IFN-c production by Cnr1/ splenocytes was decreased compared with WT splenocytes when the primary source of IFN-c was CD3 + T cells in cultures from naive mice stimulated by either anti-CD3/anti-CD28 antibodies or Staphylococcal superantigens. Conclusion: These findings suggest a duality to the CB1 receptor’s effects on the peripheral immune response, which varies based on the specific cell types stimulated. Knowledge of the complex nature of a receptor is an important part of determining its potential usefulness as a therapeutic target, and these findings further define the role of CB1 in IFN-c responses.

The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: ?9-tetrahydrocannabinol, cannabidiol and ?9-tetrahydrocannabivarin
R G Pertwee
r J Pharmacol. 2008 Jan; 153(2): 199–215.
Cannabis sativa is the source of a unique set of compounds known collectively as plant cannabinoids or phytocannabinoids. This review focuses on the manner with which three of these compounds, (?)-trans-?9-tetrahydrocannabinol (?9-THC), (?)-cannabidiol (CBD) and (?)-trans-?9-tetrahydrocannabivarin (?9-THCV), interact with cannabinoid CB1 and CB2 receptors. ?9-THC, the main psychotropic constituent of cannabis, is a CB1 and CB2 receptor partial agonist and in line with classical pharmacology, the responses it elicits appear to be strongly influenced both by the expression level and signalling efficiency of cannabinoid receptors and by ongoing endogenous cannabinoid release. CBD displays unexpectedly high potency as an antagonist of CB1/CB2 receptor agonists in CB1- and CB2-expressing cells or tissues, the manner with which it interacts with CB2 receptors providing a possible explanation for its ability to inhibit evoked immune cell migration. ?9-THCV behaves as a potent CB2 receptor partial agonist in vitro. In contrast, it antagonizes cannabinoid receptor agonists in CB1-expressing tissues. This it does with relatively high potency and in a manner that is both tissue and ligand dependent. ?9-THCV also interacts with CB1 receptors when administered in vivo, behaving either as a CB1 antagonist or, at higher doses, as a CB1 receptor agonist. Brief mention is also made in this review, first of the production by ?9-THC of pharmacodynamic tolerance, second of current knowledge about the extent to which ?9-THC, CBD and ?9-THCV interact with pharmacological targets other than CB1 or CB2 receptors, and third of actual and potential therapeutic applications for each of these cannabinoids.

The endocannabinoid pathway in Huntingtons disease: a comparison with other Neurodegenerative diseases
M. Maccarrone, N. Battista, D. Centonze
Progress in Neurobiology 1 April 2007
DOI:10.1016/j.pneurobio.2006.11.006
Endocannabinoids are endogenous agonists of cannabinoid receptors, and comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol are the best-studied members of this class of lipid mediators, and it is now widely accepted that their in vivo concentration and biological activity are largely dependent on a "metabolic control." Therefore, the proteins that synthesize, transport and degrade endocannabinoids, and that together with the target receptors form the so-called "endocannabinoid system," are the focus of intense research. This new system will be presented in this review, in order to put in a better perspective the impact of its modulation on Huntington's disease. In particular, the effect of agonists/antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, will be discussed in the context of onset and progression of Huntington's disease, and will be compared with other neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotropic lateral sclerosis. Also the plastic changes of endocannabinoids in multiple sclerosis will be reviewed, as a paradigm of their impact in neuroinflammatory disorders.

The endocannabinoid system: an overview
Natalia Battista, Monia Di Tommaso, Monica Bari and Mauro Maccarrone
Front. Behav. Neurosci., 14 March 2012 |
Doi: 10.3389/fnbeh.2012.00009
https://www.frontiersin.org/articles...article_inline
Upon the identification of anandamide (AEA) in the porcine brain, numerous studies contributed to the current state of knowledge regarding all elements that form the “endocannabinoid system (ECS).”How this complex system of receptors, ligands, and enzymes is integrated in helping to regulate fundamental processes at level of central nervous and peripheral systems and how its regulation and dysregulation might counteract disturbances of such functions, is nowadays still under investigation. However, the most recent advances on the physiological distribution and functional role of ECS allowed the progress of various research tools aimed at the therapeutic exploitation of endocannabinoid (eCB) signaling, as well as the development of novel drugs with pharmacological advantages. Here, we shall briefly overview the metabolic and signal transduction pathways of the main eCBs representatives, AEA, and 2-arachidonoylglycerol (2-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs.

The Endocannabinoid System and the Therapeutic Potential of Cannabinoids
Billy R. Martin
In Marijuana and the Cannabinoids, Chapter 6, 125–143.
doi:10.1007/978-1-59259-947-9_6
Much has been written about the history of the medical uses of cannabis (1). In the past two centuries, there have been numerous references to the use of cannabis extracts for a wide range of disorders (2). In the early part of the 20th century, a standardized cannabis elixir was marketed in the United States. Following the introduction of synthetic drugs such as barbiturates and opioids into medicine, interest in cannabis elixir declined. The discovery of the primary active constitutent in marijuana, '9 -tetrahydrocannabinol (THC), in 1964 (3) rekindled interest in the area. However, the emphasis shifted to synthetic cannabinoids rather than the plant or plant extracts. For example, in the 1970s, clinical studies were conducted in an effort to determine the efficacy of THC as an analgesic (4), antiemetic (5), antidepressant (6,7), appetite stimulant (7), and for treatment of glaucoma (8). These efforts resulted in the approval of THC (dronabinol, Marinol™) for treatment of chemotherapy-induced nausea and vomiting in 1985 and for appetite stimulation in 1992. There have been several attempts to develop THC derivatives for medical uses. Nabilone was found to have anxiolytic (9) and antiemetic properties (10) and is presently marketed as Cesamet™. Levonantradol was evaluated as an antiemetic (11) and analgesic (12) but was never approved for clinical use. Nabitan was studied clinically as an analgesic in cancer pain (13) but, like levonantradol, was never approved for use. However, the emphasis shifted back to cannabis in the early 1990s following the HIV epidemic. The lack of effective treatments for HIV led the advocacy community to demand more effective treatments and greater access to any material that might be beneficial for symptoms management. Hence, there has been increased attention to smoked marijuana not only for HIV patients, but also for a wide range of diseases. During this same period it became obvious that THC and marijuana were producing their effects through a newly discovered endocannabinoid system. The discovery of this biological system has provided opportunities for developing new medications that were not possible previously.

THE ENDOCANNABINOID SYSTEM: PHYSIOLOGY AND PHARMACOLOGY
FERNANDO RODRÍGUEZ DE FONSECA, IGNACIO DEL ARCO, FRANCISCO JAVIER BERMUDEZ-SILVA, AINHOA BILBAO, ANDREA CIPPITELLI and MIGUEL NAVARRO
Alcohol & Alcoholism Vol. 40, No. 1, pp. 2–14, 2005
doi:10.1093/alcalc/agh110
https://academic.oup.com/alcalc/arti...402?login=true
The endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early in evolution and which has important regulatory functions throughout the body in all vertebrates. The main endocannabinoids (endogenous cannabis-like substances) are small molecules derived from arachidonic acid, anandamide (arachidonoylethanolamide) and 2-arachidonoylglycerol. They bind to a family of G-protein-coupled receptors, of which the cannabinoid CB1 receptor is densely distributed in areas of the brain related to motor control, cognition, emotional responses, motivated behaviour and homeostasis. Outside the brain, the endocannabinoid system is one of the crucial modulators of the autonomic nervous system, the immune system and microcirculation. Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner and serve as retrograde signalling messengers in GABAergic and glutamatergic synapses, as well as modulators of postsynaptic transmission, interacting with other neurotransmitters, including dopamine. Endocannabinoids are transported into cells by a specific uptake system and degraded by two well-characterized enzymes, the fatty acid amide hydrolase and the monoacylglycerol lipase. Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and antagonists, anandamide uptake blockers and potent, selective inhibitors of endocannabinoid degradation. These new tools have enabled the study of the physiological roles played by the endocannabinoids and have opened up new strategies in the treatment of pain, obesity, neurological diseases including multiple sclerosis, emotional disturbances such as anxiety and other psychiatric disorders including drug addiction. Recent advances have specifically linked the endogenous cannabinoid system to alcoholism, and cannabinoid receptor antagonism now emerges as a promising therapeutic alternative for alcohol dependence and relapse.

The Endogenous Cannabinoid System Regulates Seizure Frequency and Duration in a Model of Temporal Lobe Epilepsy
Melisa J. Wallace, Robert E. Blair, Katherine W. Falenski, Billy R. Martin and Robert J. DeLorenzo
Journal of Pharmacology and Experimental Therapeutics October 2003, 307 (1) 129-137;
DOI: 10.1124/jpet.103.051920
Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract ?9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.

The phytocannabinoid, ?9-tetrahydrocannabivarin, can act through 5-HT1A receptors to produce anti-psychotic effects
Maria Grazia Cascio1, Erica Zamberletti, Pietro Marini, Daniela Parolaro2,3 and Roger G. Pertwee
Br J Pharmacol. 2015 Mar;172(5):1305-18.
doi: 10.1111/bph.13000
BACKGROUND AND PURPOSE
To address the questions of whether ?9-tetrahydrocannabivarin (THCV) can (a) enhance activation of 5-HT1A receptors in vitro and (b) induce any apparent 5-HT1A receptor-mediated anti-psychotic effects in vivo.
EXPERIMENTAL APPROACH
In vitro studies investigated the effect of THCV on targeting by 8-hydroxy-2-(di-npropylamino)tetralin (8-OH-DPAT) of 5-HT1A receptors in membranes obtained from rat brainstem or human 5-HT1A CHO cells, using [35S]GTP?S and 8-[3H]-OH-DPAT binding assays. In vivo studies investigated whether THCV induces signs of 5-HT1A receptor-mediated antipsychotic effects in rats.
KEY RESULTS
We found that THCV (a) potently, albeit partially, displaced 8-[3H]-OH-DPAT from specific binding sites in rat brainstem membranes, (b) at 100 nM, significantly enhanced 8-OH-DPATinduced activation of receptors in these membranes, (c) produced concentration-related increases in 8-[3H]-OH-DPAT binding to specific sites in membranes of human 5-HT1A receptortransfected CHO cells, and (d) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of these human 5-HT1A receptors. In phencyclidine-treated rats, THCV, like clozapine, (a) reduced stereotyped behavior, (b) decreased time spent immobile in the forced swim test, and (c) normalized hyperlocomotor activity, social behaviour and cognitive performance. Some of
these effects were counteracted by the 5-HT1A receptor antagonist, WAY100635, or could be produced by the CB1 antagonist, AM251.
CONCLUSIONS AND IMPLICATIONS
Our findings suggest that THCV can enhance 5-HT1A receptor activation, and that some of its apparent anti-psychotic effects may depend on this enhancement. We conclude that THCV has
therapeutic potential for ameliorating some of the negative, cognitive and positive symptoms of schizophrenia.

The Structure–Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation
Bow, E. W., & Rimoldi, J. M. otoxicity
Perspectives in Medicinal Chemistry, 8, PMC.S32171. (2016).
doi:10.4137/pmc.s32171
The cannabinoids are members of a deceptively simple class of terpenophenolic secondary metabolites isolated from Cannabis sativa highlighted by (?)-?9-tetrahydrocannabinol (THC), eliciting distinct pharmacological effects mediated largely by cannabinoid receptor (CB1 or CB2) signaling. Since the initial discovery of THC and related cannabinoids, synthetic and semisynthetic classical cannabinoid analogs have been evaluated to help define receptor binding modes and structure–CB1/CB2 functional activity relationships. This perspective will examine the classical cannabinoids, with particular emphasis on the structure–activity relationship of five regions: C3 side chain, phenolic hydroxyl, aromatic A-ring, pyran B-ring, and cyclohexenyl C-ring. Cumulative structure–activity relationship studies to date have helped define the critical structural elements required for potency and selectivity toward CB1 and CB2 and, more importantly, ushered the discovery and development of contemporary nonclassical cannabinoid modulators with enhanced physicochemical and pharmacological profiles.

Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis.
Di Giacomo D, De Domenico E, Sette C, Geremia R, Grimaldi P.
FASEB J. 2016 Apr;30(4):1453-63.
doi: 10.1096/fj.15-279034.
Type 2 cannabinoid receptor (CB2) has been proposed to play a pivotal role in meiotic entry of male germ cells, similar to retinoic acid (RA). In this study, we showed that activation of CB2with the specific agonist JWH133 [3-(1',1'-dimethylbutyl)-1-deoxy-8-THC] (IC5010(-6)M) mimics epigenetic events induced by RA (IC5010(-7)M) in spermatogonia. Both JWH133 and RA treatments stimulate the expression of the meiotic genes c-KitandStra8, by up-regulating H3K4me3 and down-regulating H3K9me2 levels in genomic regions flanking the transcription start site. Moreover, both agents increase the expression ofPrdm9, the gene encoding a meiosis-specific histone, H3K4me3 methyltransferase, which marks hotspots of recombination in prophase I, thus resulting in a global increase in H3K4me3. Notably, prolonged administration of JWH133 to immature 7 dpp CD-1 mice induced an acceleration of the onset of spermatogenesis, whereas the specific CB2antagonist delayed germ cell differentiation. Thus, both hyper- and hypostimulation of CB2disrupted the temporal dynamics of the spermatogenic cycle. These findings highlight the importance of proper CB2signaling for the maintenance of a correct temporal progression of spermatogenesis and suggest a possible adverse effect of cannabis in deregulating this process.-Di Giacomo, D., De Domenico, E., Sette, C., Geremia, R., Grimaldi, P. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis.

Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid,cannabidiol,in a rat model of acute inflammation
Barbara Costa, Gabriella Giagnoni, Chiara Franke, Anna Elisa Trovato & Mariapia Colleoni
British Journal of Pharmacology (2004) 143, 247–250
DOI: 10.1038/sj.bjp.0705920
Cannabidiol (CBD), a nonpsychoactive marijuana constituent, was recently shown as an oral antihyperalgesic compound in a rat model of acute inflammation. We examined whether the CBD antihyperalgesic effect could be mediated by cannabinoid receptor type 1 (CB1) or cannabinoid receptor type 2 (CB2) and/or by transient receptor potential vanilloid type 1 (TRPV1). Rats received CBD (10 mg kg?1) and the selective antagonists: SR141716 (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) for CB1, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3 carboxamide) for CB2 and capsazepine (CPZ) for TRPV1 receptors. The intraplantar injection of carrageenan in rats induced a time-dependent thermal hyperalgesia, which peaked at 3 h and decreased at the following times. CBD, administered 2 h after carrageenan, abolished the hyperalgesia to the thermal stimulus evaluated by plantar test. Neither SR141716 (0.5 mg kg?1) nor SR144528 (3 and 10 mg kg?1) modified the CBD-induced antihyperalgesia; CPZ partially at the lowest dose (2 mg kg?1) and fully at the highest dose (10 mg kg?1) reversed this effect. These results demonstrate that TRPV1 receptor could be a molecular target of the CBD antihyperalgesic action.

Variously substituted 2-oxopyridine derivatives: Extending the structure-activity relationships for allosteric modulation of the cannabinoid CB2 receptor
Francesca Gado, Kawthar Mohamed. Serena Meini, Clementina Manera
European Journal of Medicinal Chemistry 211(2):113116 Feb 2021
DOI: 10.1016/j.ejmech.2020.113116
We previously reported the 2-oxopyridine-3-carboxamide derivative EC21a as the first small synthetic CB2R positive allosteric modulator which displayed antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Herein, we extended the structure-activity relationships of EC21a through structural modifications regarding the p-fluoro benzyl moiety at position 1 and the amide group in position 3 of the central core. The characterization in vitro was assessed through radioligand binding experiments and functional assays (GTPγS, cAMP, βarrestin2). Among the new compounds, the derivatives A1 (SV-10a) and A5 (SB-13a) characterized respectively by fluorine atom or by chlorine atom in ortho position of the benzylic group at position 1 and by a cycloheptane-carboxamide at position 3 of the central core, showed positive allosteric behavior on CB2R. They enhanced the efficacy of CP55,940 in [³⁵S]GTPγS assay, and modulated CP55,940-dependent βarrestin2 recruitment and cAMP inhibition. The obtained results extend our knowledge of the structural requirements for interaction with the allosteric site of CB2R.
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Mustafunk

Brand new oldschool
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Thanks as usual. Most people are busy collecting genetics, while some of us are collecting rare papers!

Some of those are jewels indeed. Sci Hub mades my day everytime! Respect to this guys, they're real heros.

Peace.

Both seeds and the knowledge to use them are needed.
-SamS
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
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IC Cannabis Analysis

A brief history of cannabinoid and endocannabinoid pharmacology as inspired by the work of British scientists

Vincenzo Di Marzo
(2006) TRENDS in Pharmacological Sciences Vol.27 No.3 March 2006
DOI: 10.1016/j.tips.2006.01.010
British scientists have played a leading role in the long history of cannabinoid and endocannabinoid research. Such research has progressed from the first crucial evaluation of the medicinal properties of Cannabis sativa in the Western world to pioneering studies of the chemical constituents of this plant, the development of in vitro biological assays to study cannabinoids, the identification of the mechanism of action of cannabinoids, the discovery of endocannabinoids and the assessment of their therapeutic implications. Stemming from the many innovative ideas and achievements of these researchers, I provide a personal view of where these studies have led us thus far and where they are ikely to take us in the future.

A Case of Status Epilepticus and Transient Stress Cardiomyopathy Associated with Smoking the Synthetic Psychoactive Cannabinoid, UR-144
Sarah Al Fawaz, Mohammad Al Deeb, James L. Huffman, Naji A. Al Kholaif, Fiona Garlich, Ryan Chuang
Am J Case Rep 2019; 20:1902-1906
DOI: 10.12659/AJCR.918918
https://www.researchgate.net/publica...abinoid_UR-144
BACKGROUND: Synthetic cannabinoids have a higher affinity for the cannabinoid receptors CB1 and CB2 than natural cannabinoids. Their use can be associated with cardiovascular disease and neurological complications. A case is reported of status epilepticus and stress cardiomyopathy following the recreational use of the synthetic cannabinoid, UR-144.
CASE REPORT: A 19-year-old woman presented to the emergency department in status epilepticus after smoking the synthetic cannabinoid known as ‘space’. Recurring seizure activity was controlled after three hours. On hospital day 3, the patient developed severe biventricular failure. Cardiac magnetic resonance imaging (MRI) confirmed the diagnosis of stress cardiomyopathy. A comprehensive urine drug screen was performed using gas chromatography-mass spectrometry (GC-MS), which was positive for UR-144, or (1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)-methanone, and negative for all other illicit recreational drugs. The patient improved at one week following admission, with a left ventricular ejection fraction (LVEF) of 40%. She was discharged home on hospital day 10.
CONCLUSIONS: The use of the synthetic cannabinoid, UR-144, may be associated with prolonged status epilepticus and stress cardiomyopathy. Physicians should be aware of these potentially lethal complications associated with the recreational use of this and other illicit synthetic cannabinoids.

A Clinical Framework for Evaluating Cannabis Product Quality and Safety
Caroline A. MacCallum, Lindsay A. Lo, Carly A. Pistawka, and Michael Boivin Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0137
Increase in medical cannabis use, along products, warrants the need for clinicians to be knowledgeable in evaluating the quality of any cannabis product presented in clinical practice. Determining whether a product is regulated within the region is key in assessing overall quality and safety. Regulated products are held to a higher standard including independent testing, contamination mitigation, and concentration limits. Here, we present a clinical framework in evaluating cannabis products to ascertain the quality and regulation level of the product. Evaluation includes assessing the source company, reviewing product details (e.g., type, cannabinoid content, and labeling), and assessing quality control variables such as manufacturing and decontamination processes. The quality of products patients use is an important part of mitigating cannabis-related harms, especially in medically vulnerable patients. Currently, there is a great need to implement widespread standardization and regulations to ensure product quality and safety.
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A Comprehensive Phytochemical Analysis of Terpenes, Polyphenols and Cannabinoids, and Micromorphological Characterization of 9 Commercial Varieties of Cannabis sativa L.

Eugenia Mazzara, Jacopo Torresi, Gelsomina Fico, Alessio Papini, Nicola Kulbaka, Stefano Dall’Acqua, Stefania Sut, Stefania Garzoli, Ahmed M. Mustafa, Loredana Cappellacci, Dennis Fiorini, Filippo Maggi, Claudia Giuliani And Riccardo Petrell

Plants 2022
New hemp (Cannabis sativa L.) strains developed by crossbreeding selected varieties represent a novel research topic worthy of attention and investigation. This study focused on the phytochemical characterization of nine hemp commercial cultivars. Hydrodistillation was performed in order to collect the essential oils (EO), and also the residual water and deterpenated biomass. The volatile fraction was analyzed by GC-FID, GC-MS, and SPME-GC-MS, revealing three main chemotypes. The polyphenolic profile was studied in the residual water and deterpenated biomass by spectrophotometric assays, and HPLC-DAD-MSn and 1H-NMR analyses. The latter were employed for quali–quantitative determination of cannabinoids in the deterpenated material in comparison with the one not subjected to hydrodistillation. In addition, the glandular and non-glandular indumentum of the nine commercial varieties was studied by means of light microscopy and scanning electron microscopy in the attempt to find a possible correlation with the phytochemical and morphological traits. The EO and residual water were found to be rich in monoterpene and sesquiterpene hydrocarbons, and flavonol glycosides, respectively, while the deterpenated material was found to be a source of neutral cannabinoids. The micromorphological survey allowed us to partly associate the phytochemistry of these varieties with the hair morphotypes. This research sheds light on the valorization of different products from the hydrodistillation of hemp varieties, namely, essential oil, residual water, and deterpenated biomass, which proved to be worthy of exploitation in industrial and health applications. .

A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass.
Krill, C., Rochfort, S., & Spangenberg, G.
Metabolites, 10(7), 276. (2020).
doi:10.3390/metabo10070276
Cannabis and its secondary metabolite content have recently seen a surge in research interest. Cannabis terpenes and terpenoids in particular are increasingly the focus of research efforts due to the possibility of their contribution to the overall therapeutic effect of medicinal cannabis. Current methodology to quantify terpenes in cannabis biomass mostly relies on large quantities of biomass, long extraction protocols, and long GC gradient times, often exceeding 60 min. They are therefore not easily applicable in the high-throughput environment of a cannabis breeding program. The method presented here, however, is based on a simple hexane extract from 40 mg of biomass, with 50 µg/mL dodecane as internal standard, and a gradient of less than 30 min. The method can detect 48 individual terpenes and terpenoids and was validated for selectivity, linearity, LOD/LOQ, precision, intermediate precision, and accuracy (recovery) for 22 terpenes and terpenoids. The validation parameters are comparable to previously published studies that employ significantly longer runtimes and/or more complex extraction protocols. It is currently being applied to medicinal cannabis precision breeding programs.

A Metabolomic Approach Applied to a Liquid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry Method (HPLC-ESI-HRMS/MS): Towards the Comprehensive Evaluation of the Chemical Composition of Cannabis Medicinal Extracts.
Citti, C., Battisti, U. M., Braghiroli, D., Ciccarella, G., Schmid, M., Vandelli, M. A., & Cannazza, G.
Phytochemical Analysis, 29(2), 144–155.(2017).*
doi:10.1002/pca.2722*
Introduction – Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pathologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition, can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical preparation has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extraction procedure. Objective – This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extraction is carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and 120 min for olive oil). Methodology. Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC–MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariate analysis. Results – Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramatically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this difference are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC–MS and LC-UV method. Conclusions – Notwithstanding the use of a standardised starting plant material, great changes are caused by different extraction procedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts

A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis.
Berman, P., Futoran, K., Lewitus, G. M., Mukha, D., Benami, M., Shlomi, T., & Meiri, D.
Scientific Reports, 8(1).(2018).
doi:10.1038/s41598-018-32651-4
Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-?9- trans-tetrahydrocannabinol (?9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic efects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufcient to comprehend the medicinal and pharmacological efects of the whole plant. In this study we suggest a new ESI-LC/ MS/MS approach to identify phytocannabinoids from 10 diferent subclasses, and comprehensively profle the identifed compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identifed and used for profling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant efects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same efects. This study’s approach for phytocannabinoid profling can enable researchers and physicians to analyze the efects of specifc Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis

A PHYSIOLOGICALLY ACTIVE PRINCIPLE FROM CANNABIS SATIVA (MARIHUANA).
HAAGEN-SMIT, A. J., WAWRA, C. Z., KOEPFLI, J. B., ALLES, G. A., FEIGEN, G. A., & PRATER, A. N.
Science, 91(2373), 602–603. (1940).
doi:10.1126/science.91.2373.602
While it has long been known that the physiological activity of Cannabis sativa (marihuana or hashish) is associated with its contained resins, no physiologically active crystalline material has heretofore been isolated.
We report in this note the isolation of such a substance. The hydrocarbon nonacosane and an oily product termed canabinol were first isolated by Wood, Spivey and Easterfie1d In 1938 Bergel, Todd and Work reported the preparation of a crystallilie p-nitro benzoate of cannabinol which could be used to separate the cannabinol from the oil by chromatographic adsorption methods. Recently an oily product which was named cannabidiol was isolated by Adams, Hunt and Clark. None of these well-defined products has eshibited the characteristic physiological activities that are shown by the crude drug though canabinol was found to be quite toxic. Reviews of the earlier work on the chemistry of Cannabis have been published by Walton and by Blatt.
Work on the separation of physiologically active fractions from alcoholic extracts of Cannabis sativa has been in progress for the past year in our laboratories. The extracts of Minnesota wild hemp used for the work were generously supplied by the Narcotics Laboratory, United States Treasury Department, and we are indebted to Nessrs. H. J. Anslinger and H. J. Wollner for their collaboration which made this work possible. The alcohol extract of the crude drug was diluted with water to yield a seventy per cent, alcohol solution, and this vas partitioned into petroleum ether. Salt forming compounds were extracted and then colored substances were largely removed by adsorption on zinc carbonate. The resultant resinous material was fractionally precipitated from methanol with water and there was obtained a physiologically active fraction of about one twentieth the weight of the crude resin material. This purified product 1~as fractionally distilled under 0.005 mm pressure, with the most active fraction distilling at 128O-135O C. This fraction is a red-colored oil which shows typical activity in dogs following an oral dose of 1.0 mg per kg. By cooling a solution of this oil in a methanol-acetic acid mixture, some crystalline material was obtained. This was then recrystallized several times from methanol to yield colorless needles melting at 128O-129O C.

A Principal Components Analysis-Based Method for the Detection of Cannabis Plants Using Representation Data by Remote Sensing
Carmine Gambardella, Rosaria Parente, Alessandro Ciambrone and Marialaura Casbarra
Data (2021), 6, 108.
DOI: 10.3390/data6100108
Integrating the representation of the territory, through airborne remote sensing activities with hyperspectral and visible sensors, and managing complex data through dimensionality reduction for the identification of cannabis plantations, in Albania, is the focus of the research proposed by the multidisciplinary group of the Benecon University Consortium. In this study, principal components analysis (PCA) was used to remove redundant spectral information from multiband datasets. This makes it easier to identify the most prevalent spectral characteristics in most bands and those that are specific to only a few bands. The survey and airborne monitoring by hyperspectral sensors is carried out with an Itres CASI 1500 sensor owned by Benecon, characterized by a spectral range of 380–1050 nm and 288 configurable channels. The spectral configuration adopted for the research was developed specifically to maximize the spectral separability of cannabis. The ground
resolution of the georeferenced cartographic data varies according to the flight planning, inserted in the aerial platform of an Italian Guardia di Finanza’s aircraft, in relation to the orography of the sites under investigation. The geodatabase, wherein the processing of hyperspectral and visible images converge, contains ancillary data such as digital aeronautical maps, digital terrain models, color orthophoto, topographic data and in any case a significant amount of data so that they can be processed synergistically. The goal is to create maps and predictive scenarios, through the applicationof the spectral angle mapper algorithm, of the cannabis plantations scattered throughout the area. The protocol consists of comparing the spectral data acquired with the CASI1500 airborne sensor and the spectral signature of the cannabis leaves that have been acquired in the laboratory with ASD Fieldspec PRO FR spectrometers. These scientific studies have demonstrated how it is possible to achieve ex ante control of the evolution of the phenomenon itself for monitoring the cultivation of cannabis plantations.

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.
Fischedick, J. T., Glas, R., Hazekamp, A., & Verpoorte, R.
Phytochemical Analysis, 20(5), 421–426 (2009).
doi:10.1002/pca.1143
Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development
as medicinal agents, necessitating new low-cost, high-throughput analytical methods for quality control.
Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of D9-tetrahydrocannabinol (D9-THC) and qualitative analysis of other main neutral cannabinoids found
in cannabis.
Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method.
Results – D9-THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r2 = 0.9968.
Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of D9-THC in cannabis.
The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples
Fidelia Cascini, Simona Martello
Forensic Science International 217 (2012) 134–138
DOI: 10.1016/j.forsciint.2011.10.041
In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and
storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drugtype from the fibre-type varieties.
A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency
plants, we chose the DDCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished.
As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested.

A Retrospective Analysis of Chemical Constituents in Regulated and Unregulated E-Cigarette Liquids
Alaina K. Holt, Justin L. Poklis and Michelle R. Peace
Front. Chem., 28 October 2021
DOI: 10.3389/fchem.2021.752342
http://www.frontiersin.org/articles/...21.752342/full
E-cigarette or vaping use-associated lung injury (EVALI) was identified with the incidents of a multi-state outbreak of acute lung injuries associated with the use of electronic cigarettes (e-cigs) and attributed to vitamin E acetate in off-market cannabis-based e-liquids. Aside from EVALI, hypersecretion of mucus, irritated nasal passages, and watery, red eyes have been defined as complaints associated with vaping standard nicotine-based e-liquids. The chemical composition of e-liquids varies between manufacturers and robust oversight of ingredients is lacking. Manufacturers use chemicals deemed “generally recognized as safe” (GRAS) by the FDA, a designation for chemicals used in foodstuffs to be ingested. Most “GRAS” chemicals are associated with at least one Global Harmonization System (GHS) warning class, ranging from irritant to toxic. Untargeted chemical analysis is critical to evaluate e-liquid products to determine chemical composition; equally important is the quantitation of components to help elucidate the potential harms from exceeding recommended exposure limits. Untargeted screening of e-liquids was accomplished using gas chromatography-mass spectrometry (GC-MS) and Direct Analysis in Real Time-AccuTOF™ mass spectrometry (DART-ToF-MS) and has identified 350 chemical constituents from 241 products analyzed. Nicotine, caffeine, menthol, and vitamin E were confirmed and quantitated by GC-MS, ethanol was confirmed and quantitated by headspace-gas chromatography-dual flame ionization detection (HS-GC-FID), and olivetol and cannabinoids were confirmed and quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Maximum identified concentrations of nicotine, caffeine, menthol, vitamin E, ethanol, olivetol, Δ9-tetrahydrocannabinol, and cannabidiol were 56.4, 26.9, 4.28, 307.9, 217.2, 399.6, 497.7, and 332.6 mg/ml, respectively. Evaluation of untargeted analysis and quantitation of unlabeled chemical components of e-liquids is essential to improving etiology of acute lung injury and less severe impacts of vaping, both short-term and long-term. The historical documentation of unlabeled ingredients can provide some insight for a retrospective analysis of health consequences and inform policy discussions.

A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010–2019)
Lutfun Nahar | Alev Onder | Satyajit D. Sarker
Phytochemical Analysis. 2019;1–45
DOI: 10.1002/pca.2906
Abstract Introduction: Organic molecules that bind to cannabinoid receptors are called cannabinoids, and they have similar pharmacological properties like the plant, Cannabis sativa L. Hyphenated liquid chromatography (LC), incorporating high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC, also known as ultrahigh-performance liquid chromatography, UHPLC), usually coupled to an ultraviolet (UV), UV-photodiode array (PDA) or mass spectrometry (MS) detector, has become a popular analytical tool for the analysis of naturally occurring cannabinoids in various matrices.
Objective: To review literature on the use of various LC-based analytical methods for the analysis of naturally occurring cannabinoids published since 2010. Methodology: A comprehensive literature search was performed utilising several databases, like Web of Knowledge, PubMed and Google Scholar, and other relevant published materials including published books. The keywords used, in various combinations, with cannabinoids being present in all combinations, in the search were Cannabis, hemp, cannabinoids, Cannabis sativa, marijuana, analysis, HPLC, UHPLC, UPLC, quantitative, qualitative and quality control.
Results: Since 2010, several LC methods for the analysis of naturally occurring cannabinoids have been reported. While simple HPLC-UV or HPLC-UV-PDA-based methods were common in cannabinoids analysis, HPLC-MS, HPLC-MS/MS, UPLC (or UHPLC)-UV-PDA, UPLC (or UHPLC)-MS and UPLC (or UHPLC)-MS/MS, were also used frequently. Applications of mathematical and computational models for optimisation of different protocols were observed, and pre-analyses included various environmentally friendly extraction protocols.
Conclusions: LC-based analysis of naturally occurring cannabinoids has dominated the cannabinoids analysis during the last 10 years, and UPLC and UHPLC methods have been shown to be superior to conventional HPLC methods.

A study of infraspecific flavonoid variation of Cannabis sativa L. (Cannabaceae)
Murry Nelson Clarke
Thesis Brandon Thesis 1978
831-UBC_1978_A6_7 C59.pdf
https://open.library.ubc.ca/cIRcle/c...tems/1.0094357
The genus Cannabis has been treated taxonomical ly by several authors, but two main hypotheses predominate. Small and Cronquist (1976) concluded that the genus is monotypic and contains only Cannabis sativa L. Subspecies were named according to the economic purpose of the plants, i.e. fibre or drug, and varieties were assigned according to habitat (wild or cultivated). Schultes (1974) believed the genUs is polytypic, with Cannabis sativa L. representing the fibre plants, C_. indica Lam, the drug plants, and C_. ruderalis Jam's, the wild and weedy plants. A study of flavonoid variation was undertaken to determine which of the taxonomic schemes would be supported by this variation. Individual plants were examined by thin-layer chromatography and presence/absence data for nine flavonoids were analyzed by three types of computer programs. The infraspecific variation of flavonoids in the genus Cannabis supports the monotypic theory of Small and Cronquist (1976). No consistent, distinguishable groups were noticeable in the data. Wild taxa were more closely related to their cultivated progenitors than they were to each other. A division of the genus Cannabis into several species would not be supported by flavonoid variation

A validated method for the simultaneous quantification of CBD, THC, and their metabolites in human plasma, and application to plasma samples from an oral CBD open label trial.
Kevin, R. C., Vogel, R., Doohan, P., Berger, M., Amminger, G. P., & McGregor, I. S.
Drug Testing and Analysis. (2020).
doi:10.1002/dta.2947
Cannabidiol (CBD) and ? 9 -tetrahydrocannabinol (THC) are the two best known and most extensively studied phytocannabinoids within Cannabis Sativa. An increasing number of preclinical studies and clinical trials have been conducted with one or both compounds, often probing their therapeutic effects in conditions such as paediatric epilepsy, anxiety disorders or chronic pain. Accurate monitoring of THC and CBD and their metabolites is essential for tracking treatment adherence and pharmacokinetics. However, fully validated methods for the comprehensive analysis of major phase I CBD metabolites are yet to be developed due to a historical lack of commercially available reference material. In the present study, we developed, optimized, and validated a method for the simultaneous quantification of CBD, THC, and their major phase I metabolites 6-hydroxy-cannabidiol (6-OH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 7-carboxy-cannabidiol (7-COOH-CBD), 11-hydroxytetrahydrocannabinol (11-OH-THC), and 11-carboxy-tetrahydrocannabinol (11-COOH-THC) as per Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The method is accurate, reproducible, sensitive, and can be carried out in high-throughput 96 well formats, ideal for larger-scale clinical trials. Deuterated internal standards for each analyte were crucial to account for variable matrix effects between plasma lots. Application of the method to plasma samples, taken from people who had been administered oral CBD as part of an open label trial of CBD effects in anxiety disorders, demonstrated its immediate utility in ongoing and upcoming clinical trials. The method will prove useful for future studies involving CBD and/or THC and can likely accommodate the inclusion of additional metabolites as analytical reference materials become commercially available.

Accumulation of bioactive metabolites in cultivated medical Cannabis.
Richins, R. D., Rodriguez-Uribe, L., Lowe, K., Ferral, R., & O’Connell, M. A.
PLOS ONE, 13(7), e0201119.(2018).
doi:10.1371/journal.pone.0201119
There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

An Analysis of Over-the-Counter Cannabidiol Products in the United Kingdom
Jonathan Paul Liebling, Nicholas James Clarkson, Blair William Gibbs, Andrew Stephen Yates, and Saoirse Elizabeth O’Sullivan
Cannabis and Cannabinoid Research Volume X, Number X, 2020
DOI: 10.1089/can.2019.0078
Introduction: Over-the-counter cannabidiol (CBD) products have seen unprecedented recent growth in the United Kingdom. However, analysis of these predominantly unregulated products from other countries tells us that they are often mislabeled or contain unlabeled and potentially dangerous chemicals. Thus, the aim of the present study was to analyze CBD oils available in the United Kingdom. Materials and Methods: Phytocannabinoids, residual solvent, and heavy metals were measured blinded in 29 widely available CBD products by an independent testing facility using high-performance liquid chromatography with diode-array detection for cannabinoids, Headspace-gas chromatography-flame ionization detector and gas chromatography–mass spectrometry for residual solvents, and inductively coupled plasma–mass spectrometry for heavy metals. Results: The mean advertised CBD content was 4.5%, and the actual mean measured CBD content of products was 3.2% ( p = 0.053, Mann–Whitney test). Only 11/29 (38%) products were within 10% of the advertised CBD content. Fifty five percent of products had measurable levels of the controlled substances D9 - tetrahydrocannabinol (mean content 0.04%) or cannabinol (mean content 0.01%), as well as most other phytocannabinoid compounds including cannabidiolic acid (CBDA), cannabidivarin (CBDV), and cannabidivarin acid (CBDVA). Detectable levels of N-pentane, ethanol, isopropanol, heptane, lead, and arsenic were found in many of the CBD products, but these were within acceptable levels. Conclusions: As demonstrated in other countries, the quality of over-the-counter CBD products in the United Kingdom can be substandard, particularly with regard to CBD content, and often contains levels of controlled substances. We recommend that these products be more strictly regulated for consumer welfare.

An Approach to Biomarker Discovery of Cannabis Use Utilizing Proteomic, Metabolomic, and Lipidomic Analyses.
Hinckley, J. D., Saba, L., Raymond, K., Bartels, K., Klawitter, J., Christians, U., & Hopfer, C.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0002
Introduction: Relatively little is known about the molecular pathways influenced by cannabis use in humans. We used a multi-omics approach to examine protein, metabolomic, and lipid markers in plasma differentiating between cannabis users and nonusers to understand markers associated with cannabis use.
Methods: Eight discordant twin pairs and four concordant twin pairs for cannabis use completed a blood draw, urine and plasma toxicology testing, and provided information about their past 30-day cannabis use and other substance use patterns. The 24 twins were all non-Hispanic whites. Sixty-six percent were female. Median age was 30 years. Fifteen participants reported that they had used cannabis in the last 30 days, including eight participants that used every day or almost every day (29–30 of 30 days). Of these 15 participants, plasma 11-nor-9-carboxy-D9 -tetrahydrocannabinol (THC-COOH) and total tetrahydrocannabinol (THC) concentrations were detectable in 12 participants. Among the eight ‘‘heavy users’’ the amount of total THC (sum of THC and its metabolites) and plasma THC-COOH concentrations varied widely, with ranges of 13.1– 1713 ng/mL and 2.7–284 ng/mL, respectively. A validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay measured plasma THC-COOH, THC, and other cannabinoids and metabolites. Plasma THCCOOH was used as the primary measure. Expression levels of 1305 proteins were measured using SOMAScan assay, and 34 lipid mediators and 314 metabolites were measured with LC-MS/MS. Analyses examined associations between markers and THC-COOH levels with and without taking genetic relatedness into account.
Results: Thirteen proteins, three metabolites, and two lipids were identified as associated with THC-COOH levels. Myc proto-oncogene was identified as associated with THC-COOH levels in both molecular insight and potential marker analyses. Five pathways (interleukin-6 production, T lymphocyte regulation, apoptosis, kinase signaling pathways, and nuclear factor kappa-light-chain-enhancer of activated B cells) were linked with molecules identified in these analyses.
Conclusions: THC-COOH levels are associated with immune system-related pathways. This study presents a feasible approach to identify additional molecular markers associated with THC-COOH levels.

An Update on Plant Photobiology and Implications for Cannabis Production
[/COLOR]Mark Lefsrud, Samuel Eichhorn Bilodeau, Bo-Sen Wu, Anne-Sophie Rufyikiri, Sarah MacPherson
Plant Sci. 10:296.
DOI: 10.3389/fpls.2019.00296
This review presents recent developments in plant photobiology and lighting systems for horticultural crops, as well as potential applications for cannabis (Cannabis sativa and C. indica) plant production. The legal and commercial production of the cannabis plant is a relatively new, rapidly growing, and highly profitable industry in Europe and North America. However, more knowledge transfer from plant studies and horticultural communities to commercial cannabis plant growers is needed. Plant photosynthesis and photomorphogenesis are influenced by light wavelength, intensity, and photoperiod via plant photoreceptors that sense light and control plant growth. Further, light properties play a critical role in plant vegetative growth and reproductive (flowering) developmental stages, as well as in biomass secondary metabolite synthesis and accumulation. Advantages and disadvantages of widespread greenhouse lighting systems that use high pressure sodium lamps or light emitting diode (LED) lighting are known. Some artificial plant lighting practices will require improvements for cannabis production. By manipulating LED light spectra and stimulating specific plant photoreceptors, it may be possible to minimize operation costs while maximizing cannabis biomass and cannabinoid yield, including tetrahydrocannabinol (or ? 9 -tetrahydrocannabinol) and cannabidiol for medicinal and recreational purposes. The basics of plant photobiology (photosynthesis and photomorphogenesis) and electrical lighting systems are discussed, with an emphasis on how the light spectrum and lighting strategies could influence cannabis production and secondary compound accumulation.

Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA).
Citti, C., Pacchetti, B., Vandelli, M. A., Forni, F., & Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis, 149, 532–540. (2018).
doi:10.1016/j.jpba.2017.11.044*
Hemp seed oil from*Cannabis sativa*L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.

Analysis of cannabis components by growth stage using solid-phase microextraction and solvent extraction
Japanese Journal of Forensic Science and Technology June 2019
DOI: 10.3408/jafst.758
Tomoya Kudo, Akio Kiguchi, Hiroshi Fujii
Illegal distribution of cannabis products, such as cannabis-infused foods and e-liquids, are increasing in Japan. The main component of cannabis is tetrahydrocannabinol (THC). THC extracted from cannabis is regulated by the Cannabis Control Act, and chemically synthesized THC is regulated by the Narcotics and Psychotropics Control Act; therefore, it is important to identify the source of THC for judicial appraisal. Recently, the crime of cultivating cannabis has occurred frequently in Japan. Illegal cultivation is difficult to discover because there are cases in which cannabis is cultivated secretly in a room in apartments, etc. Cannabis is a plant that emits unique odors. Detection of the odor constituents can provide a breakthrough for the criminal investigation of cannabis cultivation. The purpose of this study was to identify cannabis products and utilize them for the criminal investigation of cannabis cultivation. Cannabis plants were cultivated from seeds and analyzed using both solid-phase microextraction (SPME) and solvent extraction. Fifty kinds of volatile components were detected by SPME and 21 kinds of refractory components were detected by solvent extraction in fresh cannabis plants. We found that terpenes were detected more strongly in cannabis buds than in cannabis leaves. Furthermore, sesquiterpenes were strongly detected from cannabis leaves and monoterpenes were strongly detected from cannabis buds. The most abundant components were ?-myrcene, cis-?-ocimene, terpinolene, ?-caryophyllene, trans-?-bergamotene, humulene and ?-farnesene. Additionally, THC, cannabichromene, cannabigerol and tetrahydrocannabivarin known as cannabinoids, were detected in all the samples. For the identification of cannabis products, 7 kinds of terpenes and 4 kinds of cannabinoids are promising combinations. Additionally, these terpenes may be useful in the criminal investigation of cannabis cultivation.

Analysis of Cannabis Seizures in NSW, Australia: Cannabis Potency and Cannabinoid Profile.
Swift, W., Wong, A., Li, K. M., Arnold, J. C., & McGregor, I. S.
PLoS ONE, 8(7), e70052.(2013).
doi:10.1371/journal.pone.0070052
Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of D9 -tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales ‘‘Cannabis Cautioning’’ scheme. A further 26 ‘‘Known Provenance’’ samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The ‘‘Cannabis Cautioning’’ samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (,0.1%). ‘‘Known Provenance’’ samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed.

Analysis of Fatty Acids in Marijuana (Cannabis Sativa Leaf)
Nadia Fucci
Microgram Journal, Volume 5, Numbers 1-4 (January - December 2007)
https://forendex.safs1966.org/upload....1-4.20.25.pdf
Various fatty acids (palmitic, myristic, oleic, and stearic acids) were identified in 20 marijuana (cannabis leaf) samples recently seized on the illicit market in Rome, Italy. Samples were analyzed by gas chromatography/mass spectrometry to determine delta-9-tetrahydrocannabinol, other minor cannabinoid congeners, and fatty acids. Although cannabis seeds and the oil derived from those seeds are known to be rich in fatty acids, this is believed to be the first study demonstrating the presence of fatty acids in marijuana. The potential value of the results in source determination and comparative analyses is discussed

Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog.
Citti, C., Linciano, P., Forni, F., Vandelli, M. A., Gigli, G., Laganà, A., & Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis. (2019). doi:10.1016/j.jpba.2019.06.049
Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use.

Analysis of Phenolic Compounds in Commercial Cannabis sativa L. Inflorescences Using UHPLC-Q-Orbitrap HRMS.
Izzo, L., Castaldo, L., Narváez, A., Graziani, G., Gaspari, A., Rodríguez-Carrasco, Y., & Ritieni, A.
Molecules, 25(3), 631. (2020).
doi:10.3390/molecules25030631
Industrial hemp (Cannabis sativa L. Family Cannabaceae) contains a vast number of bioactive relevant compounds, namely polyphenols including flavonoids, phenolic acids, phenol amides, and lignanamides, well known for their therapeutic properties. Nowadays, many polyphenols-containing products made of herbal extracts are marketed, claiming to exert health-promoting effects. In this context, industrial hemp inflorescence may represent an innovative source of bioactive compounds to be used in nutraceutical formulations. The aim of this work was to provide a comprehensive analysis of the polyphenolic fraction contained in polar extracts of four different commercial cultivars (Kompoti, Tiborszallasi, Antal, and Carmagnola Cs) of hemp inflorescences through spectrophotometric (TPC, DPPH tests) and spectrometry measurement (UHPLC-Q-Orbitrap HRMS). Results highlighted a high content of cannflavin A and B in inflorescence analyzed samples, which appear to be cannabis-specific, with a mean value of 61.8 and 84.5 mg/kg, meaning a ten-to-hundred times increase compared to other parts of the plant. Among flavonols, quercetin-3-glucoside reached up to 285.9 mg/kg in the Carmagnola CS cultivar. Catechin and epicatechin were the most representative flavanols, with a mean concentration of 53.3 and 66.2 mg/kg, respectively, for all cultivars. Total polyphenolic content in inflorescence samples was quantified in the range of 10.51 to 52.58 mg GAE/g and free radical-scavenging included in the range from 27.5 to 77.6 mmol trolox/kg. Therefore, C. sativa inflorescence could be considered as a potential novel source of polyphenols intended for nutraceutical formulations


Analysis of the cannabinoid content of strains available in the New Jersey Medicinal Marijuana Program
Thomas A. Coogan
Journal of Cannabis Research 1, Article number: 11 (2019)
https://jcannabisresearch.biomedcent.. .238-019-0011-z
Background
The six licensed operators in the New Jersey Medicinal Marijuana Program submit their strains of cannabis flower to a single laboratory, administered by the state’s Department of Health, for testing. The results of these tests are made available by the State on a web page for patients, allowing a study of the range of cannabinoid profiles available in the program.
Methods
Reports on cannabinoid concentrations were collected from 245 test reports released by the State lab; the relative quantities of cannabinoids on all strains was evaluated, as well as trends in the strain types being tested.
Results
The collection of strain profiles available in New Jersey conforms to results of other population studies, revealing three broad classification of strains based on their relative concentration of cannabinoids: the overwhelmingly majority of strains contain only trace (<?1%) CBDA but high THCA concentration; a handful are balanced in CBDA and THCA content; and a very few strains have a high concentration of CBDA and minimal THCA (<?1%). In those strains that contain more than 1% of both THCA and CBDA, those two substances are present in comparable quantities. The concentration of CBGA is higher in those strains that have the highest THCA concentration, though there are strains that have high THCA (>?20%) with CBGA concentrations at the low end of the range (<?0.5%). In the high CBD strains, the concentration of CBGA is positively correlated with CBDA, but the CBGA concentrations are several fold less in CBD-dominant strains than in THC-dominant strains: the highest measured CBGA concentration in a CBD-dominant strain is only at the average value of CBGA concentration in THC-dominant strains. The most-recently tested strains are overwhelmingly of the THC-dominant type.
Conclusions
Though some high CBD strains are available in the New Jersey medical marijuana program, the vast majority of strains that have been tested are the THC-dominant strains which contain less than 1% CBDA. The data available from the State does not include any information on how well the different strains sell, but it can be inferred from the trend in strain types tested that the demand in the New Jersey medical market is for THC-dominant strains.

Analytical and Pharmacological Challenges in Cannabis Research.
Gertsch, J.
Planta Medica, 84(04), 213–213. (2018).
doi:10.1055/s-0044-101051
The millennial history of the use of cannabis as crop, medicinal and recreational plant reads like a novel in which the most recent chapter is dedicated to the current worldwide transformation of Cannabis sativa L. flos into a modern evidence-based botanical drug. Although ?9-tetrahydrocannabinol (?9-THC) (dronabinol) is an approved monosubstance that can be prescribed to treat anorexia in AIDS patients and nausea during chemotherapy, muscle spasms and certain forms of chronic pain, Sativex® (Nabiximols®) is the only standardized cannabis extract approved to reduce symptoms in Multiple Sclerosis. Despite a lack of clinical trials for most of the indications in which cannabis is used medically, cannabis products are increasingly demanded by patients. Like a molecular fountain, cannabis pours bioactive cannabinoids into the toolbox of pharmacologists, as illustrated by the recent pharma enterprise to develop cannabidiol (CBD) and cannabidiverin (CBDV) to treat certain forms of epilepsy. While it is recognized that the major psychoactive cannabinoid ?9-THC and the major non-psychoactive cannabinoid CBD, which occur in different ratios in distinct cannabis chemotypes, are of pharmaceutical relevance; minor cannabinoids, acid precursors and terpenes are believed to also play a role in the action of the diverse botanical formulations of medicinal cannabis. Importantly, increasing evidence suggests that CBD, though rather poorly bioavailable to the brain, can decrease the adverse effects of ?9-THC by acting as a negative allosteric CB1 receptor modulator. Thus, in addition to the different worldwide and regional legal situations, numerous analytical and pharmacological questions arise. The aim of this Planta Medica special issues is to increase the awareness in the natural product research community regarding challenges in cannabinoid analytics and pharmacology, both of which are fundamental for the urgently required standardization of medical cannabis. Clearly, standardization in the context of cannabis implies improved pharmacological and clinical insights regarding the different forms of administration and chemotypes employed. Intriguingly, less than 2% of the published papers on cannabis currently deal with analytics. Here, a state of the art LC MS/MS method as well as an UV MS method were developed and validated for quantitative determination of major and minor cannabinoids of pharmacological relevance in different C. sativa samples including medicinal chemovars. Moreover, a gas-chromatography with flame ionization detector (GC FID) method was developed and validated for the versatile qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts

Anti-Inflammatory Activity in Colon Models Is Derived from D9-Tetrahydrocannabinolic Acid That Interacts with Additional Compounds in Cannabis Extracts
Rameshprabu Nallathambi, Moran Mazuz, Aurel Ion, Gopinath Selvaraj, Smadar Weininger, Marcelo Fridlender, Ahmad Nasser, Oded Sagee, Puja Kumari, Diana Nemichenizer, Maayan Mendelovitz, Nave Firstein, Orly Hanin, Fred Konikoff, Yoram Kapulnik, Timna Naftali, and Hinanit Koltai
Cannabis and Cannabinoid Research, 2(1), 167–182.
DOI: 10.1089/can.2017.0027
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner. Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell cultureand colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

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Markus Roggen
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Association of Naturalistic Administration of Cannabis Flower and Concentrates With Intoxication and Impairment.
Bidwell, L. C., Ellingson, J. M., Karoly, H. C., YorkWilliams, S. L., Hitchcock, L. N., Tracy, B. L., … Hutchison, K. E. ).
JAMA Psychiatry. (2020) doi:10.1001/jamapsychiatry.2020.0927
IMPORTANCE The rapidly growing legal cannabis market includes new and highly potent products, the effects of which, to our knowledge, have not previously been examined in biobehavioral research studies because of federal restrictions on cannabis research. OBJECTIVE To use federally compatible, observational methods to study high-?9- tetrahydrocannabinol (THC) legal market forms of cannabis. DESIGN, SETTING, AND PARTICIPANTS In this cohort study with a between-groups design that was conducted in a community and university setting, cannabis flower users and concentrate users were randomly assigned to higher- vs lower-THC products within user groups. Participants completed a baseline and an experimental mobile laboratory assessment that included 3 points: before, immediately after, and 1 hour after ad libitum legal market flower and concentrate use. Of the 133 individuals enrolled and assessed, 55 regular flower cannabis users (41.4%) and 66 regular concentrate cannabis users (49.6%) complied with the study’s cannabis use instructions and had complete data across primary outcomes. EXPOSURES Flower users were randomly assigned to use either 16% or 24% THC flower and concentrate users were randomly assigned to use either 70% or 90% THC concentrate that they purchased from a dispensary. MAIN OUTCOMES AND MEASURES Primary outcome measures included plasma cannabinoids, subjective drug intoxication, and neurobehavioral tasks testing attention, memory, inhibitory control, and balance. RESULTS A total of 121 participants completed the study for analysis: 55 flower users (mean [SD] age, 28.8 [8.1] years; 25 women [46%]) and 66 concentrate users (mean [SD] age, 28.3 [10.4] years; 30 women [45%]). Concentrate users compared with flower users exhibited higher plasma THC levels and 11-hydroxy?9 -THC (THC’s active metabolite) across all points. After ad libitum cannabis administration, mean plasma THC levels were 1016 (SE = 1380) ?g/mL in concentrate users (to convert to millimoles per liter, multiply by 3.18) and 455 (SE = 503) ?g/mL in flower users. Most neurobehavioral measures were not altered by short-term cannabis consumption. However, delayed verbal memory (F1,203 = 32.31; P < .001) and balance function (F1,203 = 18.88; P < .001) were impaired after use. Differing outcomes for the type of product (flower vs concentrate) or potency within products were not observed. CONCLUSIONS AND RELEVANCE This study provides information about the association of pharmacological and neurobehavioral outcomes with legal market cannabis. Short-term use of concentrates was associated with higher levels of THC exposure. Across forms of cannabis and potencies, users’ domains of verbal memory and proprioception-focused postural stability were primarily associated with THC administration.

Bioactive products from singlet oxygen photooxygenation of cannabinoids.
Galal Osman, A., Elokely, K. M., Yadav, V. K., Carvalho, P., Radwan, M., Slade, D. ElSohly, M. A.
European Journal of Medicinal Chemistry, 143, 983–996. (2018).
doi:10.1016/j.ejmech.2017.11.043
Photooxygenation of D8 tetrahydrocannabinol (D8 -THC), D9 tetrahydrocannabinol (D9 -THC), D9 tetrahydrocannabinolic acid (D9 -THCA) and some derivatives (acetate, tosylate and methyl ether) yielded 24 oxygenated derivatives, 18 of which were new and 6 were previously reported, including allyl alcohols, ethers, quinones, hydroperoxides, and epoxides. Testing these compounds for their modulatory effect on cannabinoid receptors CB1 and CB2 led to the identification of 7 and 21 as CB1 partial agonists with Ki values of 0.043 mM and 0.048 mM, respectively and 23 as a cannabinoid with high binding affinity for CB2 with Ki value of 0.0095 mM, but much less affinity towards CB1 (Ki 0.467 mM). The synthesized compounds showed cytotoxic activity against cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3) with IC50 values ranging from 4.2 to 8.5 mg/mL. Several of those compounds showed antimicrobial, antimalarial and antileishmanial activities, with compound 14 being the most potent against various pathogens.

Bioavailability and Bioefficacy of Hemp By-Products in Ruminant Meat Production and Preservation: A Review
Farouk Semwogerere, Chenaimoyo L. F. Katiyatiya, Obert C. Chikwanha, Munyaradzi C. Marufu and Cletos Mapiye
Front. Vet. Sci. 7:572906. (2020)
doi: 10.3389/fvets.2020.572906
Plant by-products obtained from agro-industrial processes require valorisation to demonstrate their potential for enhancing animal health, meat production, and shelf life extension. One example is the fast-growing hemp industry, which produces seeds, leaves, seed oil, and cake. Studies on the nutritional value of hempseed cake have shown it can be a valuable source of protein in ruminant diets. However, there is limited documentation on the bioavailability and bioefficacy of hemp phytochemicals for improving ruminant health, production, and extending meat shelf life. The current review provides an overview of existing information on nutrient and phytochemical composition of hemp by-products, their bioavailability, and bioefficacy, and explores current limitations and prospects regarding their valorisation

Biochemical aspects of seeds from Cannabis sativa L. plants grown in a mountain environment
Chiara Cattaneo, Annalisa Givonetti, Valeria Leoni, Nicoletta Guerrieri, Marcello Manfredi, Annamaria Giorgi, & Maria Cavaletto
Scientifc Reports | Nature (2021) 11:3927
https://www.ncbi.nlm.nih.gov/pmc/art...icle_83290.pdf
Cannabis sativa L. (hemp) is a versatile plant which can adapt to various environmental conditions. Hempseeds provide high quality lipids, mainly represented by polyunsaturated acids, and highly digestible proteins rich of essential aminoacids. Hempseed composition can vary according to plant genotype, but other factors such as agronomic and climatic conditions can afect the presence of nutraceutic compounds. In this research, seeds from two cultivars of C. sativa (Futura 75 and Finola) grown in a mountain environment of the Italian Alps were analyzed. The main purpose of this study was to investigate changes in the protein profle of seeds obtained from such environments, using two methods (sequential and total proteins) for protein extraction and two analytical approaches SDS-PAGE and 2D-gel electrophoresis, followed by protein identifcation by mass spectrometry. The fatty acids profle and carotenoids content were also analysed. Mountain environments mainly afected fatty acid and protein profles of Finola seeds. These changes were not predictable by the sole comparison of certifed seeds from Futura 75 and Finola cultivars. The fatty acid profle confrmed a high PUFA content in both cultivars from mountain area, while protein analysis revealed a decrease in the protein content of Finola seeds from the experimental fields

Biosynthesis of Cannabinoids
Tamir Bresler
https://terpenesandtesting.com/categ...-cannabinoids/
Or, How Plants Build up Terpenoids in the Cells
Any time a living organism synthesizes a chemical compound on its own, we term that activity ‘biosynthesis.’ These chemical reactions are different than ones we do intest tubes because biological systems have a host of systems in place to drive energetically unfavorable reactions to completion, including enzyme catalyzers and energy currency in the form of adenosine triphosphate (ATP). Chemists mimic these processes by imbuing the system with heat, mechanical agitation, metal catalyzers and more, but these are mere shadows on the wall compared to the vast beauty and efficiency of a biological synthetic pathway. Biosynthesis of cannabinoids generally takes place in the plant cells of leaves, which makes sense. Building up these complex molecules involves a large amount of energy expenditure, and leaves are the main powerhouse of the plant. Cannabinoids are also big and bulky; THC alone requires 21 carbon atoms. All this carbon matter has to come from somewhere, and in the case of plants, this matter comes right from the air—from carbon dioxide (CO2). Plants take up CO2 in their leaves, some of it becoming photosynthesized into molecular oxygen (O2) and some being catabolized into simple organic compounds for use throughout the plant.
Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR
Kostas Ioannidis, Evangelos Dadiotis , Vangelis Mitsis, Eleni Melliou and Prokopios Magiatis
Molecules 2020, 25, 5928;
DOI: 10.3390/molecules25245928
High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or ?-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ? 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

Cannabidiol and Cannabol, Constituents of Cannabis indica Resin.
JACOB, A., & TODD, A. R.
Nature, 145(3670), 350–350. (1940).
doi:10.1038/145350a0

III.—Cannabinol. Part I
Thomas Barlow Wood, M.A., W. T. Newton Spivey, M.A. and Thomas Hill Easterfield, M.A., Ph.D. (1899)
J. Chem. Soc., Trans., 75(0), 20–36.
DOI: 10.1039/ct8997500020
IN a paper communicated to the Society in 1896 (Trans., 1896, 88, 539) the authors, under the name of “cannabinol,” described a physiologically active substance which they had isolated from "charas,” the exuded resin of Indian hemp. From the constancy of composition of a number of preparations of this substance obtained from different samples of “charas,” it was believed to be a definite chemical compound of the formula C18H,,02 ; this conclusion seemed to be justified by the determination of the molecular weight, and by the examination of several derivatives. Since then, the authors have further examined cannabinol, and have found that it is a mixture of at least two compounds having similar physical characters. One of these, of the formula C21H2602, has been isolated, and it is proposed to retain the name cannabinol for this compound. During the progress of this investigation, a note on oxycannabin by Messrs. Dunstan and Henry appeared in the Proceedings of the Society (Proc., 1898, p. 44), in which the formulae C,,H,,,NO, and C18H,0Ac were assigned to oxycannabin and acetylcannabinol respectively. From the description of these substances, there can be no doubt that they are identical with those we have obtained, but the results of our analyses correspond with the formulae Cl,H,,NO, for oxycannabin and C2,H2,02*C,H,0 for acetylcannabinol. These formulae are confirmed by molecular weight determinations, and by analyses of many derivatives. Dunstan and Henry (Zoc. cit.) state that, on oxidising cannabinol with nitric acid, normal butyric acid is formed ; we can confirm this statement, with the addition that larger quantities of normal valeric and caproic acids are produced at the same time.

III.—Cannabinol. Part I
Thomas Barlow Wood, M.A., W. T. Newton Spivey, M.A. and Thomas Hill Easterfield, M.A., Ph.D. (1899)
DOI: 10.1039/ct8997500020
IN a paper communicated to the Society in 1896 (Trans., 1896, 88,539) the authors, under the name of “cannabinol,” described a physiologically active substance which they had isolated from (‘ charas,” the exuded resin of Indian hemp. From the constancy of composition of a number of preparations of this substance obtained from different samples of “charas,” it was believed to be a definite chemical compound of the formula C18H,,02 ; this conclusion seemed to be justified by the determination of the molecular weight, and by the examination of several derivatives. Since then, the authors have further examined cannabinol, and have found that it is a mixture of at least two compounds having similar physical characters. One of these, of the formula C21H2602, has been isolated, and it is proposed to retain the name cannabinol for this compound. During the progress of this investigation, a note on oxycannabin by Messrs. Dunstan and Henry appeared in the Proceedings of the Society (Proc., 1898, p. 44), in which the formulae C,,H,,,NO, and C18H,0Ac were assigned to oxycannabin and acetylcannabinol respectively. From the description of these substances, there can be no doubt that they are identical with those we have obtained, but the results of our analyses correspond with the formulae Cl,H,,NO, for oxycannabin and C2,H2,02*C,H,0 for acetylcannabinol. These formulae are confirmed by molecular weight determinations, and by analyses of many derivatives. Dunstan and Henry (Zoc. cit.) state that, on oxidising cannabinol with nitric acid, normal butyric acid is formed ; we can confirm this statement, with the addition that larger quantities of normal valeric and caproic acids are produced at the same time. As the present paper deals mainly with the substances produced by the breaking down of the cannabinol molecule, the authors have only described such of its reactions as suffice to show that it is a true chemical compound. An account of the reactions of cannabinol, together with a more complete examination of several of the compounds described below, will shortly be brought before the Society.

Cannabinoid Quantitation via Rugged and Adaptable HPLC/UHPLC Method Development
The Analytical Scientist & phenomenex 04/23/2020 WebVideo
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Potency testing demands in the cannabis industry have exploded and are continuing to expand in product formulations, applications, and plant cultivations. Of the hundreds of known cannabinoid molecules, six have been of primary interest to quantitate (THC, THC-A, CBD, CBD-A, CBG, and CBG-A). With various regulations, such as the Farm Bill, distinguishing between Marijuana and Hemp through levels of intoxicating cannabinoids, specific and accurate quantitation of these cannabinoids is ever more critical and even more challenging with a rising number and level of minor cannabinoids potentially present from chemical reactions or novel cultivars. Along with the sheer number of potentially interfering cannabinoids, their chromatographic profiles are a challenge to maintain as their retention profiles can have significant shifts from minute changes in common chromatographic conditions. Here we’ll cover the key underlying chromatographic factors to ensure robust and rugged potency method development that can be adapted readily to accommodate the dynamic landscape.
Learning Objectives
Mobile phase factors applied to method accuracy and ruggedness.
Instrument and method transfer considerations in robustness.
Critical cannabinoids resolved chromatographically.

Cannabinoid, Terpene, and Heavy Metal Analysis of 29 Over-the-Counter Commercial Veterinary Hemp Supplements
Wakshlag, J. J., Cital, S., Eaton, S. J., Prussin, R., & Hudalla, C.
Veterinary Medicine: Research and Reports, Volume 11, 45–55.(2020).
doi:10.2147/vmrr.s248712
Purpose: The use of veterinary low tetrahydrocannabinol (THC) Cannabis sativa (ie, hemp) products has increased in popularity for a variety of pet ailments. Low-THC Cannabis sativa is federally legal for sale and distribution in the USA, and the rise in internet commerce has provided access to interested consumers, with minimal quality control. Materials and Methods: We performed an internet word search of “hemp extract and dog” or “CBD product and dog” and analyzed 29 products that were using low-THC Cannabis sativa extracts in their production of supplements. All products were tested for major cannabinoids including cannabidiol (CBD), ?9-tetrahydrocannabinol (THC), cannabigerol (CBG), and other minor cannabinoids, as well as their carboxylic acid derivatives (CBDA, THCA, CBGA) using an ISO/IEC 17025 certified laboratory. Products were also tested for major terpenes and heavy metals to understand constituents in the hemp plants being extracted and distributed. Results: All products were below the federal limit of 0.3% THC with variable amounts of CBD (0–88 mg/mL or g). Only two products did not supply a CBD or total cannabinoid concentration on their packaging or website, while 22/29 could supply a certificate of analysis (COA) from a third-party laboratory. Ten of the 27 products were within 10% of the total cannabinoid concentrations of their label claim with a median concentration of 93% of claims (0–154%). Heavy metal contamination was found in 4/29 products, with lead being the most prevalent contaminant (3/29). Conclusion: The products analyzed had highly variable concentrations of CBD or total cannabinoids with only 18 of 29 being appropriately labeled according to current FDA nonmedication, non-dietary supplement or non-food guidelines. Owners and veterinarians wanting to utilize CBD-rich Cannabis sativa products should be aware of low-concentration products and should obtain a COA enabling them to fully discuss the implications of use and calculated dosing before administering to pets

Cannabinoids analysis: Analytical methods for different biological specimens.
ElSohly, M. A., & Salem, M.
Forensic Science, 163–193. (2000).
doi:10.1016/s1567-7192(00)80055-8
The development of methods to determine cannabinoids is an area of increasing interest, and a large number of publications appear every year describing a variety of analytical techniques that vary in sensitivity, specificity, and instrumentation. This chapter discusses the methods published in the recent past with special emphasis on those methods that appear to be more practical and feasible for routine analysis of these compounds in various types of biological specimens. Various types of biological samples can be analyzed for cannabinoids to test for marijuana use. Immunoassays are the most widely used screening methods for cannabinoids in urine. These methods are based on developing antibodies specific to the drug to be tested and/or one or more of its major urinary metabolites. Chromatographic methods can be used for qualitative and quantitative screening and/or confirmation of cannabinoids in biological specimens. Thin layer chromatography (TLC) has been used for the screening and identification of cannabinoids for many years. Immunoassays have almost replaced TLC as a screening method. Immunoassay methods for screening blood samples for cannabinoids are now widely used. The methods employed are often based on the use of those tests primarily developed for use with urine samples.

Cannabinoids Analysis of Laser-Microdissected Trichomes of Cannabis sativa L. by LC-MS and Cryogenic NMR
Nizar Happyana, Remco Muntendam, Sara Agnolet, Annie Van Dam, Bernd Schneider, Oliver Kayser
Tu technische universitat Dortmund (poster) Pdf can be obtained by contacting Remco Muntendam, at Reseachgate.net
Trichomes of Cannabis sativa have been reported as the main site of cannabinoids production. A comprehensive study of cannabinoids was performed on capitate-stalked and capitate-sessile trichomes, and on capitate-stalked glands and stems harvested by laser microdissection (LMD) during flowering time (week 4 - 8). LC-MS and cryogenicNMR analysis were used for qualitative and quantitative assessment of cannabinoids in the collected cells. 9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabigerolic acid (CBCA) were identified as the major constituents in the all tested samples, while 9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabigerol (CBG) were present in less quantity. Cannabichromene (CBC) and cannabinol (CBN) were detected as minor compounds only in intact capitate-stalked trichomes in week 8. Based on the cannabinoids levels, discrimination of capitate-stalked and capitate-sessile trichomes at flowering time was possible. The study demonstrated the possibility of other spots for cannabinoids production besides the gland of the capitate-stalked trichomes. In particular the presence of cannabinoids in the stem of capitate-stalked trichomes is reported for the first time. The combined use of LMD, LC-MS and cryogenic NMR constitutes a valuable method for the comprehensive assessment of plant metabolites at the cellular level

Cannabinomics: application of metabolomics in Cannabis (Cannabis sativa L.) research and development
Konstantinos A. Aliferis, David Bernard-Perron
Front. Plant Sci. 11:554.
doi:10.3389/fpls.2020.00554
Cannabis (Cannabis sativa L.) is a complex, polymorphic plant species, which produces a vast array of bioactive metabolites, the two major chemical groups being cannabinoids and terpenoids. Nonetheless, the psychoactive cannabinoid tetrahydrocannabinol (?9-THC) and the non-psychoactive cannabidiol (CBD), are the two major cannabinoids that have monopolized the research interest. Currently, more than 600 Cannabis varieties are commercially available, providing access to a multitude of potent extracts with complex compositions, whose genetics are largely inconclusive. Recently introduced legislation on Cannabis cultivation in many countries represents a
great opportunity, but at the same time, a great challenge for Cannabis research and development (R&D) towards applications in the pharmaceutical, food, cosmetics, and agrochemical industries. Based on its versatility and unique capabilities in the deconvolution of the metabolite composition of complex matrices, metabolomics represents an ideal bioanalytical tool that could greatly assist and accelerate Cannabis 38 R&D. Among others, Cannabis metabolomics or cannabinomics can be applied in the taxonomy of Cannabis varieties in chemovars, the research on the discovery and assessment of new Cannabis-based sources of bioactivity in medicine, the development of new food products, and the optimization of its cultivation, aiming for improvements in yield and potency. Although Cannabis research is still in its infancy, it is highly
foreseen that the employment of advanced metabolomics will provide insights that will assist the sector to face the aforementioned challenges. Within this context, here, the current state-of-the-art and conceptual aspects of cannabinomics are presented.

Cannabis Analysis Challenges: One Size Does Not Fit All

07/30/2020 Cannabis Scientist & UCT WebVideo​

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UCT and AltaSci have partnered together in presenting the vast analytical challenges faced in the cannabis and hemp marketplace. Specifically, the diversity of matrix encountered does not afford a one size fits all approach. Today’s webinar will explore various sample clean-up options for flower, edibles, and concentrates and will also showcase how each matrix requires its own tailor-made extraction to ensure overall data accuracy and repeatability. By tapping on UCT’s diverse sorbent chemistry offerings in addition to AltaSci’s experience in full pharmaceutical grade testing for 75% of the licensed production facilities in Connecticut, an exhaustive cannabis analysis will be co-presented.
Learning Objectives of Webinar

Sample clean-up is highly dependent upon the matrix of analysis.

Different sorbent chemistries can provide varying levels of sample clean-up and analyte recovery.



Cannabis and Cannabis Resin Pre-Review Report A document prepared for the World Health Organization Expert Committee on Drug Dependence Thirty-eight Meeting Geneva, 14 – 18 November 2016
H. Valerie Curran Philip Wiffen David J. Nutt Willem Scholten
https://www.drugsandalcohol.ie/26377...abisreport.pdf
With this report, DrugScience provides a unique opportunity for the World Health Organization (WHO) and its Expert Committee on Drug Dependence to initiate a critical review process of cannabis and cannabis resin for their scheduling under the Single Convention on Narcotic Drugs as amended by the 1972 Protocol. The process could ultimately open the discussion on the scheduling of cannabis and cannabis resin and lead to recommendations to the Commission on Narcotic Drugs within two years.
In the eighty years since cannabis and cannabis resin were last reviewed by the Health Committee of the League of Nations in 1935, both the social context of cannabis use and the science of drug dependence have dramatically changed. Yet, cannabis and cannabis resin continue to remain under the strictest control regime possible under the Single Convention, without a valid scientific re-assessment of this decision. Cannabis and cannabis resin are listed in Schedule I and Schedule IV respectively, which means that both remain strictly prohibited worldwide.
This also means that the Committee implicitly continues to recommend that cannabis is not to be used medically despite growing evidence of medical use worldwide and despite the availability of pharmaceutical preparations with a marketing authorization in multiple countries. Many countries are struggling with the impact of the prohibition of cannabis, be it the negative impact of prohibition on society, including over-incarceration and disproportionate sentencing, or impact on drug markets (including synthetic cannabinoids) and drug use.
The current scheduling of cannabis is in marked divergence with the Convention’s principle that scheduling of substances should be based on a scientific assessment by WHO. In the absence of a recent assessment, the continued prohibition of cannabis appears completely illegitimate even though it may be legal.
A scientific review by the WHO, the only authoritative global body to make such an assessment, would greatly legitimize international policies and their national implementation. A scientific assessment of cannabis and cannabis resin appears most timely given the many debates that have emerged on this issue across the world in recent years.
I trust therefore, that the Thirty-eighth Meeting of the Expert Committee on Drug Dependence will act responsibly and will adopt, on the basis of this strong Pre-Review Report, the Pre-review of Cannabis and Cannabis Resin as an agenda item.
Geneva, September 2016


Cannabis and the Anxiety of Fragmentation—A Systems Approach for Finding an Anxiolytic Cannabis Chemotype.
Kamal, B. S., Kamal, F., & Lantela, D. E.
Frontiers in Neuroscience, 12. (2018).
doi:10.3389/fnins.2018.00730
Cannabis sativa is a medicinal herb with a diverse range of chemotypes that can exert both anxiolytic and anxiogenic effects on humans. Medical cannabis patients receiving organically grown cannabis from a single source were surveyed about the effectiveness of cannabis for treating anxiety. Patients rated cannabis as highly effective overall for treating anxiety with an average score of 8.03 on a Likert scale of 0 to 10 (0=not effective,10=extremely effective). Patients also identified which strains they found the most or least effective for relieving their symptoms of anxiety. To find correlations between anxiolytic activity and chemotype, the top four strains voted most and least effective were analyzed by HPLC-MS/MS to quantify cannabinoids and GC-MS to quantify terpenes. Tetrahydrocannabinol and trans-nerolidol have statistically significant correlations with increased anxiolytic activity. Guiaol, eucalyptol, ?-terpinene, ?-phellandrene, 3-carene and sabinene hydrate all have significant correlations with decreased anxiolytic activity. Further studies are needed to better elucidate the entourage effects that contribute to the anxiolytic properties of cannabis varieties.

Cannabis Contamination Netherlands 2015 RIVM Briefrapport
https://www.rijksoverheid.nl/binarie...anten/rivm.pdf
Cannabis contaminants
Traces of pesticides were found in Cannabis samples (nederwiet) from different coffee shops in the Netherlands. The levels are very low and of no consequence to the uses health. This applies to the samples used as tea or smoked. Aflatoxines produced by fungi commonly found on plants were not found.
In this exploratory study RIVM analysed nederwiet samples from 25 coffee shops for the presence of contaminates, specifically aflatoxines and residues of pesticides.
This study was commissioned by the Ministry of Health, Wellfare and Sports. It is important that Cannabis does not contain significant levels of pesticides because it may harm the user, particularly when already in poor health. This is relevant because people that take Cannabis for medical reasons do not always turn to a pharmacy. Only the Cannabis available at pharmacies (mediwiet) is produced under quality conditions.
In 23 of the 25 samples one or more pesticides were identified. In 11 samples the obserbed levels exceded the limits set for herbal medicines.One sample contained a banned pesticide but at a level that is of no consequence to the users health.

Cannabis Contaminants: Regulating Solvents, Microbes, and Metals in Legal Weed.
Seltenrich, N.
Environmental Health Perspectives, 127(8), 082001. (2019).
doi:10.1289/ehp5785
In June 2019, Illinois became the 11th U.S. state—plus the District of Columbia—to legalize recreational cannabis sales or use.1 Many more permit medical cannabis.2 Tax collectors, entrepreneurs, and law enforcement officials have all watched closely as legalization has hopscotched across the United States since 2014. So have some environmental health experts. That’s because products derived from cannabis can deliver a number of contaminants to the user, including pesticides, molds, bacteria, metals, and solvents.3,4 Although many of these contaminants are also found in our food, water, and air, the potential exposures and health consequences are less well understood in the context of cannabis use. EHP previously reported on the regulation of pesticides on cannabis, including the difficulty of setting specific residue limits.5 But solvent residues, microbes, and heavy metals each pose regulatory challenges of their own

Cannabis Indica.
Wallich, G. C. (1883).
BMJ, 1(1173), 1224–1224.
doi:10.1136/bmj.1.1173.1224
THE following. facts, though bearing but indirectly upon those special medical uses of Indian hemp to which attention has been drawn in recent Issues of the BRITISH MEDICAL JOURNAL, may, serve in some measure to explain the conflicting estimate that have' been formed of the general value of the drug byjjjffqrent observers. My knowledge of Indian hemp as a therapeutic; agent dates from the year 1838, ,when... its properties were commhunicated to me -in Calcutta by my friend Dr. W.lB. O'Shaughnessy and, at his -request, I made the sketch of thq plant which is appeaded to !his earliest memoir on the subject. During my subseqgent- service in. India, I had many opportunities of testing the efflcacy of jndian- heixp in cases of cholera, tetanus, hydrophobia, and various minor disorders. But, though, fully convinced of its value, like many other medical men, I soon became aware that the action of the drug was singularly uncertain, and that doses which in one locality produced a given effect, frequently. failed to produce the same effect in another.' Eventually it became manifest that this uncertain action was due, to inherent differences in the composition of the resinous extract of: the plant, incident on its being grown either in the plains or in the hill-districts of. India. -The. drug bad, not, at this periods been supplied on indent to military hospitals at a distance from. the Ptesidency; and those-who.wanted to. employ it ezperimnentally in their practice were, therefore,,obliged to procure it for -themselves from the native drugAdealers, eitheriAn, the form of the Acrde dried plant or of -the inspissated extractvwhidh.went by the native name of " churrus.

Cannabis indica
E.V. Ratnam
FIND DOI OR LINK

Cannabis indica. Part II. Isolation of cannabidiol from Egyptian hashish. Observations on the structure of cannabinol.
Jacob, A. and Todd, A.R.
J. Chem. Soc., 649–653 5 (1940)
DOI: 10.1039/jr9400000649
Cannabidiol, C21H3002, a typical constituent of American hemp resin (Adams,Hunt, and Clark, J. Amer. Chem. SOG., 1940, 62, 196), has been isolated from hashish of Egyptian origin, where it was accompanied by an approximately equal amount of cannabinol.
The simultaneous occurrence of these substances and their resemblance in many reactions lend support to the view that cannabidiol has a structure of the type suggested by Adams, Hunt, and Clark (ZOG. cit.). Cannabidiol contains two double bonds and its absorption spectrum indicates that neither double bond is conjugated with the aromatic nucleus. The structure of cannabinol is discussed ; of four possible structures, (I) and (111) seem most in keeping with the available evidence.

Cannabis indica. Part X. The essential oil from Egyptian hashish
J. L. SIMONSEN and A. R. TODD (1942)
DOI: 10.1039/jr9420000188
Examination of the essential oil from Egyptian hashish shows that the lower-boiling “ terpenefraction ” consists mainly of p-cymene with small amounts of 1-methyl-4-isopropenylbenzene and unidentified optically active material. From the higher-boiling fraction of the oil, humulene (?-caryophyllene) has been isolated. A hypothetical scheme for the biogenesis of cannabinol and related compounds is discussed in the light of these findings.

CXXI.—Cannabis Indica resin. Part I. The constitution of nitrocannabinolactone (oxycannabin).
Cahn, R. S. (
J. Chem. Soc., 0(0), 986–992. 1930).
doi:10.1039/jr9300000986
The general problem presented by Cannabis Indica resin, the essential portion of the drug known as hashish, bhang, etc., will be considered in a later communication. The present paper is confined to oxycannabin, which is a product of oxidation by concentrated nitric acid of the higher-boiling portions of the resin. Oxycannabin has been assigned various formula, viz., C,Hm07N, (Bolas and Francis, J., 1860, 22, 417; Chem. News, 1871, 24, 77), C,,HllO,N (Dunstan and Henry, P., 1898, 44), and CllHI,O,N (Wood, Spivey, and Easterfield, J., 1899, 75, 20), the last of which has been confirmed in the present investigation. Wood, Spivey, and Easterfield removed the nitro-group, via the amino- and iodogroups,to obtain the substance C,,M,,O,; this they termed cannabinolactone, renaming oxycannabin as nitrocannabinolactone. Among other results they showed that cannabinolactone on fusion with potassium hydroxide yielded m-toluic acid, and as, further, it contains an excessively stable lactone group, they concluded that it must be represented by one of the y-lactonic formuh (I)-III).

LXXXVI.—Cannabis Indica resin. Part II.
Cahn, R. S. (1931).
J. Chem. Soc., 0(0), 630–638.
doi:10.1039/jr9310000630
INVESTIGATIONS of Cannabis Indim resin (hashish, etc.), though numerous, are to a large extent contradictory. It is agreed that the active principle is contained in a high-boiling resin and that it is not alkaloidal (T. and H. Smith, J. Pharm., 1857, 21, 47), but it has never been isolated. From this high-boiling oil four substances, namely, cannabinol, trinitrocannabinol, a hydrocarbon, and nitrocannabinolactone, have been prepared by the use either of solvents or of reagents. The present communication records a preliminary investigation of the first three, concerning which the evidence hitherto available is highly conflicting. The fourth formed the subject of Part I of this series (J., 1930, 986). Wood, Spivey, and Easterfield (J., 1896, 69, 539) obtained from five different samples of Cannabis Indica resin a fraction boiling apparently constantly at 265'/20 mm. and in four cases giving on analysis results required for the formula C,,H,O,; it was not unnaturally assumed to be homogeneous a,nd given the name cannabinol. Later, however, they (J., 1899, 75, 20) and independently Dunstan and Henry (P., 1898, 44) prepared from this fraction a crystalline acetyl derivative, m. p. 75". The former authors, who only succeeded in obtaining the crystalline derivative in small yield (20-25%. Dunstan and Henry give no details), concluded correctly that their resin, b. p. 265"/20 mm., was a mixture and transferred the name cannabinol to the resin obtained on hydrolysis of the crystalline acetyl derivative. Later investigators have, however, consistently failed to repeat this work; they have obtained apparently constant-boiling resins and, although these yielded only oily derivatives, have claimed homogeneity for each product, appropriated the name cannabinol, and variously assigned to it the formulze C20H3002 (Casparis, Pharm. HeEv. Acta, 1926,1,210 ; Bergel, Annalen, 1930,482,55) and C,,H,,O, (Frankel, Arch. exp. Path. Pharm., 1903, 49, 266; Czerkis, Annalen, 1907, 351, 467; also considered possible by Bergel, Zoc. cit.).

174. Cannabis indica resin. Part III. The constitution of cannabinol.
Cahn, R. S. (1932).
Journal of the Chemical Society (Resumed), 1342.
doi:10.1039/jr9320001342
Ow~xa to the improved method (J., 1931, 630) of isolating cannabinol from the high-boiling, pharmacologically active fraction of Cannabis indica resin (hashish, etc.), it has become possible to examine the reactions of this substance in greater detail and to suggest tentatively a formula for it.
Cannabinol has the molecular formula C,,H,,O,. The formula C,,H,,O,, which is not conclusively excluded by the analytical data, is shown below to be inconsistent with chemical evidence. Cannabinol readily forms a trinitro-derivative, which on oxidation affords nitrocannabinolactone, C,,HllO,~NO, (loc. cit.). In a previous paper (J., 1930, 986) it was shown that the last substance is probably represented by formula (I) or (11). It can be converted into the lactonic acid, cannabinolactonic acid (Wood, Spivey, and Easterfield, J., 1899, '75, 20), which is represented by (111) or the corresponding formula derived from (11). This acid has now been oxidised to trimcllitic acid (IV), and it follows that cannabinolactonic acid must be represented by formula (111) and nitrocannabinolactone by formula (I). This has been confirmed by a repetition of the synthetic acid is identical with cannabinolactonic acid. Dr. Bergel has informed the author that he has synthesised cannabinolactone and cannabinolactonic acid, and is now in agreement with the views here put forward.

326. Cannabis indica resin. Part IV. The synthesis of some 2?: 2-dimethyldibenzopyrans, and confirmation of the structure of cannabinol.
Cahn, R. S.
Journal of the Chemical Society (Resumed), 1400. (1933) doi:10.1039/jr9330001400
IT was suggested recently (J., 1932,1342) that cannabinol was ( ?)3”-hydroxy-2 : 2 : 5‘-trimethyl-(?)5”-n-amyldibenzopyran (I). Since the methods of degradation hitherto attempted brought no further evidence of structure, it was decided to synthesise the parent substance, 2 : 2-dimethyldibenzopyran (IV), and a few simple derivatives as a preliminary to further experiments with the natural product. A study of this ring system appeared to have an enhanced interest, since, although the analogous 2 : 2-dimethylcoumaran system, containing one benzene ring fused to a 1 : 2-pyran ring, is present in tephrosin and deguelin, two of the constituents of derris root, cannabinol is the first substance shown to have a dibenzo-1 : 2-pyran structure.

Cannabis Oil: chemical evaluation of an upcoming cannabis-based medicine
Luigi L Romano, Arno Hazekamp
Cannabinoids 2013;1(1):1-11
https://www.researchgate.net/publica...based_medicine
Concentrated cannabis extracts, also known as Cannabis oils because of their sticky and viscous appearance, are becoming increasingly popular among self-medicating patients as a claimed cure for cancer. In general, preparation methods for Cannabis oils are relatively simple and do not re-quire particular instruments. The most well-known example of such a product is called ‘Simpson oil’. The purpose of the extraction, often followed by a solvent evaporation step, is to make canna-binoids and other beneficial components such as terpenes available in a highly concentrated form. Although various preparation methods have been recommended for Cannabis oils, so far no stud-ies have reported on the chemical composition of such products.
Recognizing the need for more information on quality and safety issues regarding Cannabis oils, an analytical study was performed to compare several generally used preparation methods on the basis of content of cannabinoids, terpenes, and residual solvent components. Solvents used include ethanol, naphtha, petroleum ether, and olive oil. The obtained results are not intended to support or deny the therapeutic properties of these products, but may be useful for better understanding the experiences of self-medicating patients through chemical analysis of this popular medicine.

Cannabis plant and cannabis resin Section 1: Chemistry
WHO Expert Committee on Drug Dependence Pre-Review
https://www.who.int/medicines/access...istry.pdf?ua=1
The dried cannabis inflorescence (the complete flower head) is one of the most commonly encountered formulations for administration of cannabinoids. Cannabis can be grown and marketed for either medicinal or recreational purposes. Medical cannabis is produced in several countries. For example, Aurora Cannabis Inc. is one of 26 authorized producers in Canada
that also exports its products abroad. In the Netherlands, medicinal cannabis is grown and marketed by Bedrocan B.V. under contract to the Dutch Ministry of Health and made available as the herbal inflorescence in Dutch pharmacies on prescription. Bedrocan B.V. grows six varieties according to a controlled regimen and with a standardized level of three cannabinoids: (?)-transdelta-9-tetrahydrocannabinol (?9-THC), cannabidiol (CBD) and cannabinol (CBN). The Ministry also exports cannabis for medicinal use to Canada, the Czech Republic, Finland, Germany and Italy, and to authorized researchers around the world. The Italian Stabilimento Chimico Farmaceutico Militare developed the strain FM2, dispensed for medicinal purposes in the national territory.
The flowering tops or buds of a wide range of varieties are also available through dispensaries and medicinal programmes in the United States of America and elsewhere and are sold under popular names such as “skunk”, “kush”, “diesel”, and “haze”. In contrast to medicinal cannabis, the chemical content, the pharmacological and organoleptic effects of these products are not often readily discernible from the label and, in some instances, even through rigorous quantitative analysis of cannabinoids, can be unpredictable.
Recently, a series of recreational varieties of cannabis with low ?9 -THC content (<0.2% in Europe and <1% in Switzerland), called “cannabis light”, have come on to the popular market and are sold as a smoking product (e.g. Artur, CannLab, Hempy, etc.).

Cannabis potency in North-East Italy: A ten-year study (2010–2019)
Luca Zamengoa, Chiara Bettina, Denis Badoccob, Giampietro Frison
Forensic Science International 317 (2020)
10.1016/j.forsciint.2020.110556 0379-0738
This paper presents data about potency of herbal and resin cannabis products seized during 2010–2019 in north-east Italy. More than 12,000 cannabis samples were analyzed and concentrations of THC, CBD and CBN were collected. The results of our study provided clear evidence for an increase in the potency of cannabis products across the study period, which is consistent with other studies. Globally, the median THC concentrations increased from about 6%–11%, but differences were found between herbal and resin materials. THC potency in resin materials increased more consistently across the study period with a dramatic raise during 2018–2019, with median THC contents around 17%. CBD concentrations were found to decrease constantly over the study period, especially in herbal materials, which had a mean CBD concentration of 0.3%. In particular, about 75% of the analyzed herbal samples had a CBD concentration which was less than 3% of the corresponding THC concentration. In contrast, more than 50% of the analyzed resin materials had a CBD concentration which was about 30% of the corresponding THC concentration. This is consistent with the increase in prevalence of high-potency seedless female herbal products observed in the same period and indicates that herbal and resin materials were produced from different varieties of cannabis plants. However, resin materials derived from high THC/low CBD cannabis plants were recently found. Different routes (e.g. northern Europe) or different modalities of distribution were assumed for these products. CBN concentrations were also considered and found to be very low and consistent across the study period indicating reliability of THC values used in statistical analysis. In conclusion, this study provided an accurate picture of cannabis products seized over a decade over a definite geographical area which can be extremely helpful for comparative purposes and for national and international statistical analyses on cannabis products.

Cannabis resin in the region of madrid: adulteration an contamination
Authors: P´erez-Moreno M, P´erez-Lloret P, Gonz´alez-Soriano
J, Santos-A´ lvarez I
Forensic Science International (2019),
doi.org/10.1016/j.forsciint.2019.02.049
The aim of this study is to analyze the adulteration and contamination of cannabis resin obtained on the streets of Madrid, in order to establish whether it is suitable for human consumption. A total of 90 samples obtained through street vending in the Region of Madrid (CAM) were analyzed. Our results showed a direct relationship between the shape of the samples (acorn or ingot) and the presence of foreign elements, adulterants and microbiological contamination. Foreign elements were found in 64.7% of the ingot-shaped samples and in 30.2% of the acorn-shaped samples (p<0.01); 25% of the samples were deliberately adulterated, 66.7% of which had an ingot shape. With regard to microbiological contamination, 93% of acorns were contaminated by E. coli, compared to 29.4% of ingots (p<0.0001). In addition, all samples with fecal odor were acorns and were contaminated by E. coli. Ten per cent of the samples were contaminated by Aspergillus; of these, 66.7% had the shape of an acorn. Overall, our results showed that most (88.3%) of the hashish samples were not suitable for consumption. This percentage was significantly higher (p <0.0001) in acorn than in ingot samples (100% vs. 58.8%). Hence, illegal street vending of hashish constitutes a public health issue.

Cannabis Sativa L.: a comprehensive review on the analytical methodologies for cannabinoids and terpenes characterization.
Micalizzi G, Vento F, Alibrando F, Donnarumma D, Dugo P, Mondello L.
J Chromatogr A. 2020 Dec 30;1637:461864.
doi: 10.1016/j.chroma.2020.461864.
Undoubtedly, the enormous interest about cannabis cultivation mainly derives from the well-known pharmacological properties of cannabinoids and terpenes biosynthesized by the plants. ...Lastly, GC GC techniques are also reported for accurate identification and quantificatification of terpenes in complex cannabis matrices.

Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy: An Untargeted Chemical Characterization from Early Flowering to Ripening.
Ingallina, C., Sobolev, A. P., Circi, S., Spano, M., Fraschetti, C., Filippi, A., … Mannina, L.
Molecules, 25(8), (2020).
doi:10.3390/molecules25081908
The chemical composition of the inflorescences from four Cannabis sativa L. monoecious cultivars (Ferimon, Uso-31, Felina 32 and Fedora 17), recently introduced in the Lazio Region, was monitored over the season from June to September giving indications on their sensorial, pharmaceutical/nutraceutical proprieties. Both untargeted (NMR) and targeted (GC/MS, UHPLC, HPLC-PDA/FD and spectrophotometry) analyses were carried out to identify and quantify compounds of different classes (sugars, organic acids, amino acids, cannabinoids, terpenoids, phenols, tannins, flavonoids and biogenic amines). All cultivars in each harvesting period showed a THC content below the Italian legal limit, although in general THC content increased over the season. Citric acid, malic acid and glucose showed the highest content in the late flowering period, whereas the content of proline drastically decreased after June in all cultivars. Neophytadiene, nerolidol and chlorogenic acid were quantified only in Felina 32 cultivar, characterized also by a very high content of flavonoids, whereas alloaromadendrene and trans-cinnamic acid were detected only in Uso-31 cultivar. Naringenin andnaringin were present only in Fedora 17 and Ferimon cultivars, respectively. Moreover, Ferimon had the highest concentration of biogenic amines, especially in July and August. Cadaverine was present in all cultivars but only in September. These results suggest that the chemical composition of Cannabis sativa L. inflorescences depends on the cultivar and on the harvesting period. Producers can use this information as a guide to obtain inflorescences with peculiar chemical characteristics according to the specific use.

Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes
Leonardo Anabalon, Jaime Solano, Francisco Encina-Montoya, Marco Bustos, Alejandra Figueroa, and David Gangitano
Cannabis and Cannabinoid Research Volume X, Number X, June 2021
DOI: 10.1089/can.2020.0168
Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market.
Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation.
Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2C on average.
Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.

Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performancesupercritical fluid chromatography
Mazzoccanti, O. H. Ismail, I. D’Acquarica, C. Villani, a C. Manzo,
M. Wilcox, A. Cavazzini and F. Gasparrini
Chem. Commun., 2017, 53, 12262
DOI:10.1039/c7cc06999e
By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (_)-D9-THC in medicinal marijuana (Bedrocans). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity.

Cannflavins from hemp sprouts, a novel cannabinoid-free hemp food product, target microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase.
Werz, O., Seegers, J., Schaible, A. M., Weinigel, C., Barz, D., Koeberle, A., … Appendino, G.
PharmaNutrition, 2(3), 53–60. (2014).
doi:10.1016/j.phanu.2014.05.001
Hemp seeds are of great nutritional value, containing all essential amino acids and fatty acids in sufficient amount and ratio to meet the dietary human demand. Hemp seeds do not contain cannabinoids, and, because of their high contents of ?-3 fatty acids, are enjoying a growing popularity as a super-food to beneficially affect chronic inflammation. Seeds also lack the typical phenolics of hemp leaves and inflorescences, but we found that sprouting, while not triggering the production of cannabinoids, could nevertheless induce the production of the anti-inflammatory prenylflavonoids cannflavins A and B. This effect was especially marked in Ermo, a cannabinoid-free variety of
Cannabis sativa L.. Microsomal prostaglandin E2 synthase (mPGES-1) and 5-lipoxygenase (5-LO) were identified as the molecular targets of cannflavins A and B, solving an almost three-decade old uncertainty on the mechanism of their the anti-inflammatory activity. No change on the fatty acid profile was observed during sprouting, and the presence of lipophilic flavonoids combines with the high concentration of ?-3 essential acids to qualify sprouts from Ermo as a novel anti-inflammatory hemp food product worth considering for mass production and commercial development.

Carbonyl Compounds in Mainstream Smoke of Hemp Cigarettes
Alexandra M. Ward and Jon O. Ebbert
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0039
Introduction: CBD is a major phytocannabinoid in hemp (Cannabis sativa containing less than 0.3% THC). Hemp cigarettes are a combustible form of hemp consisting of dried and smokable flowers, which represent 2% of the overall CBD market, and the market is expected to grow. Combustion and pyrolysis of organic material are associated with the production of carbonyl compounds, which are known toxicants and are associated with adverse health outcomes. Concentrations of carbonyl compounds in mainstream hemp cigarette smoke are unknown. Materials and Methods: We analyzed and compared carbonyl concentrations in the mainstream smoke produced by a hemp cigarette (Brand B), a premium hemp cigarette (Brand A), Marlboro Red tobacco cigarette, and a research reference tobacco cigarette using high-performance liquid chromatography. We measured carbonyl concentrations in lg per puff and mg per cigarette. Carbonyls investigated were formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, and butyraldehyde. Significance was determined using Tukey’s test. Results: We observed that Brand B had significantly higher butyraldehyde than any cigarette. No significant differences were observed in crotonaldehyde concentration in the cigarettes. For the remaining carbonyls, Brand A had consistently lower concentrations in mainstream smoke than tobacco cigarettes. Hemp cigarettes emit carbonyls in a lower concentration in lg/puff than tobacco cigarettes, but the magnitude of significance generally decreases when normalized to mg/cigarette. Conclusions: Smoke from hemp cigarettes contains carbonyls at biologically significant concentrations. Opportunities may exist to reduce carbonyl production in these products, and identified potential risks must be considered when balancing the harms and benefits of hemp cigarettes when used for therapeutic purposes.

CBN and Δ9-THC concentration ratio as an indicator for the age of stored marijuana samples.
Ross SA, and ElSohly MA
Bull Narcotics, 49-50(1-2):139-147, 1997-1998.
https://extractionmagazine.com/wp-co...na-samples.pdf
The concentration of d~9-tetrahydrocannabinol (THC) and cannabinol (CBN) in cannabis plant material (marijuana) of different varieties stored at room temperature (20-22° Celsius (e)) over a four-year period was determined. The percentage loss of THC was proportional to the storage time. On average, the concentration of THC in the plant material decreased by 16.6% ±7.4 of its original value after one year and 26.8% ±7.3, 34.5% ±7.6 and 41.4% ±6.5 after two, three and four years, respectively. A relationship between the concentration ratio of CBN to THC and the storage time was developed and could serve as a guide in determining the approximate age of a given marijuana sample stored at room temperature.

Changes in Cannabis Potency Over the Last 2 Decades (1995–2014): Analysis of Current Data in the United States.
ElSohly, M. A., Mehmedic, Z., Foster, S., Gon, C., Chandra, S., & Church, J. C.
Biological Psychiatry, 79(7), 613–619.(2016).
doi:10.1016/j.biopsych.2016.01.004
BACKGROUND: Marijuana is the most widely used illicit drug in the United States and all over the world. Reports indicate that the potency of cannabis preparation has been increasing. This report examines the concentration of cannabinoids in illicit cannabis products seized by DEA (drug and enforcement administration) over the last two decades, with particular emphasis on ? 9 - THC and cannabidiol (CBD).
METHODS: Samples in this report are received over time from DEA confiscated materials and processed for analysis using a validated ‘gas chromatograph with flame ionization detector (GC/FID)’ method.
RESULTS: A total of 38,681samples of cannabis preparations were received and analyzed between January 1, 1995 and December 31, 2014. The data showed that, while the number of marijuana samples seized over the last four years has declined, the number of sinsemilla samples has increased. Overall, the potency of illicit cannabis plant material has consistently risen over time since 1995 from approximately 4% in 1995 to approximately 12% in 2014. On the other hand, the CBD content has fallen on average from approximately 0.28% in 2001 to <0.15% in 2014, resulting in a change in the ratio of THC to CBD from 14 times in 1995 to approximately 80 times in 2014.
CONCLUSION: It is concluded that there is a shift in the production of illicit cannabis plant material from regular marijuana to sinsemilla. This increase in potency poses higher risk of cannabis use, particularly among adolescents.

Changes in delta?9?tetrahydrocannabinol (THC) and cannabidiol (CBD) concentrations in cannabis over time: systematic review and meta?analysis
Tom P. Freeman, Sam Craft, Jack Wilson, Stephan Stylianou, Mahmoud ElSohly, Marta Di Forti & Michael T. Lynskey
Addiction 2020
doi:10.1111/add.15253
Background and aims Cannabis products with high delta?9?tetrahydrocannabinol (THC) concentrations carry an increased risk of addiction and mental health disorders, while it has been suggested that cannabidiol (CBD) may moderate the effects of THC. This study aimed to systematically review and meta?analyse changes in THC and CBD concentrations in cannabis over time (PROSPERO registration: CRD42019130055). Design Embase, MEDLINE® and Epub Ahead of Print, In?Process and Other Non?Indexed Citations and Daily, Global Health, PsycINFO and Scopus were searched from inception to 27/03/2019 for observational studies reporting changes in mean THC and/or CBD concentration in cannabis over at least three annual time points. Searches and extraction were conducted by two independent reviewers. Random effects meta?regression models estimated annual changes in THC and CBD for each product within each study; these estimates were pooled across studies in random effects models. Results We identified 12 eligible studies from the USA, UK, Netherlands, France, Denmark, Italy and New Zealand. For all herbal cannabis, THC concentrations increased by 0.29% each year (95% CI: 0.11, 0.47), P < 0.001 based on 66747 cannabis samples from eight studies, 1970–2017. For cannabis resin, THC concentrations increased by 0.57% each year (95% CI: 0.10, 1.03), P = 0.017 based on 17 371 samples from eight studies, 1975–2017. There was no evidence for changes in CBD in herbal cannabis [0.01% (95% CI: 0.02, 0.01), P = 0.280; 49434 samples from five studies, 1995–2017] or cannabis resin [0.03% (95% CI: 0.11, 0.18), P = 0.651; 11382 samples from six studies, 1992–2017]. Risk of bias was low apart from non?random sampling in most studies. There was evidence of moderate to substantial heterogeneity. Conclusions Concentrations of delta?9?tetrahydrocannabinol (THC) in international cannabis markets increased from 1970 to 2017 while cannabidiol (CBD) remained stable. Increases in THC were greater in cannabis resin than herbal cannabis. Rising THC in herbal cannabis was attributable to an increased market share of high?THC sinsemilla relative to low?THC traditional herbal cannabis.

Changes in the composition of cannabis from 2000-2017 in Denmark: Analysis of confiscated samples of cannabis resin
Kristine Rømer Thomsen, Birgitte Thylstrup, Christian Lindholst, Sinikka Kvamme, Lotte Ask Reitzel, Martin Worm-Leonhard, Amir Englund, Tom P. Freeman, and Morten Hesse
Experimental and Clinical Psychopharmacology · June 2019
DOI: 10.1037/pha0000303
Globally, recent studies report increases in -9-tetrahydrocannabinol (THC) concentration in seized samples of cannabis for human consumption. This is important, because use of cannabis with a high concentration of THC has been linked to a number of adverse health outcomes. The objective of this study was to assess recent changes in the composition of seized cannabis resin in Denmark by (a) examining THC concentration in samples from Danish forensic laboratories from 2000 to 2017 (N 430) and (b) examining cannabidiol (CBD) concentration and the THC:CBD concentration ratio in samples from the forensic laboratory in Western Denmark from 2008 to 2017 (N 147). Cannabis resin samples were analyzed using a gas chromatographic analysis with flame ionization detection quantifying the total THC and CBD concentration. Results showed that the THC concentration increased 3-fold from 2000 (mean: 8.3%) to 2017 (mean: 25.3%). Significant increases occurred in all areas of Denmark. After 2011, we found a dramatic increase in cannabis resin samples with high THC concentration and the near disappearance of cannabis resin samples with medium- and low THC concentration. Furthermore, the THC:CBD concentration ratio increased significantly from 1.4 in 2008 to 4.4 in 2017. Whereas THC concentration increased, CBD concentration remained stable at 6%. In conclusion, the THC concentration of cannabis resin, and THC:CBD concentration ratio, have increased dramatically in Denmark, potentially leading to higher risk of harm to users. Policymakers, treatment professionals, and educators should be aware of this change

Characterisation of cannabinoid composition in a diverse Cannabis sativa L. germplasm collection
Matthew T. Welling, Lei Liu. Tim Shapter, Carolyn A. Raymond,
Graham J. King
Euphytica vol. 208. no. 3, pp. 463-475. (2015)
DOI 10.1007/s10681-015-1585-y
The ability to characterise cannabinoid chemical phenotype (chemotype) accurately is important for the development of Cannabis sativa L. cultivars specific for pharmacological, hemp fibre, or seed end use. Although a number of chemotyping and genotyping methods have previously been developed to predict and characterise cannabinoid composition, only a subset of the gene pool has been examined. A representative survey from a wide range of geographically and genetically diverse C. sativa accessions using liquid chromatography–mass spectrometry (LC–MS) cannabinoid profiling together with dominant and co-dominant DNA marker assays was performed. Overall variability of chemotype across the gene pool was found to be three-fold greater within heterozygote genotypes than previously reported. Interestingly, an individual plant of East Asian origin was found to exhibit a rare propyl alkyl cannabinoid homologue and a chemotype inconsistent with the predicted genotype. We propose that in order to carry out comprehensive screening of genetic resource collections and to identify chemotypic variants specific for end-use pharmacological applications, a strategy which adopts both cannabinoid profiling and the co-dominant DNA marker assay is required. Further research with consideration of propyl-alkyl-cannabinoid homologues should explore the relationship between chemotype and genotype in greater detail.

XL.- Charas. The Resin of Indian Hemp. T. BARLOW WOOD M. A.
W. T. NEWTON SPIVEY, M. A., and THOMAS HILL EASTERFIELD . M.A., Ph. D. (1896)
DOI: 10.1039/ct8966900539
SEVERAL preparations of Canwubis indica (syn. sativa), differing in physiological activity and in the manner of application to the human subject, are familiar to the Eastern nations. Of these,.‘‘ charas” is an Indian drug, and is regarded by the natives as the most potetit substance obtainable from the plant. It is chiefly used in the Punjab, and consists essentially of the resinous exudation from the sterms, leaves, and flower heads of the unfertilised female plant. By the kindness of Mr. Charles A. Silberrad, B.A., I.C.S., at present resident at Etawah, N.W.P., the authors have been provided with
several pounds of the resin, the examination of which has furnished the materials for the present communication. Cannabis indica has been employed medicinally, both in this country and on tlhe Continent, but the uncertain action of the drug has prevented its use from becoming general, different preparations having apparently very different efYects on the organism. It was with the object of isolating the physiologically active constituent of the hemp plant that tbe present research was undertaken, the importance of cannabis in veterinary medicine having. been poiiited out to the
authors by Professor El. R. Hobday, of the Royal Veterinary College, and Mr. W. A. Wood, N.R.C.V.S. Charas was examined rather than any other preparation because the active principle appeared to be most highly concentrated in this natriral exudation, and also because it contains no chlorophyll, a substance which causes much inconvenience in the examination of extracts made from the plant itself. Now that the characteristic compound has been isolated by the authors and placed in the hands of experienced pharmacologists, the difficulties which have hitherto attended the pharmaceutical use of Indian hemp will probably disappear.

Chemataxonomic researches in higher plants. XV Carotenoid and chlorophyll pigments in the leaves of Cannabis sativa L.
NEAMTU Gavrila, ILLYES Gh, POP Ecaterina
Notulae Botanicae Horti Agrobotanici Cluj-Napoca 11(1) 1981
10.15835/nbha111144 https://www.researchgate.net/publica...nabis_sativa_L
In the leaves of Cannabis sativa L. were found the following carotenoid pigments: ?-carotene, lutein, zeaxanthin, violaxanthin, neoxanthin and ?-cryptoxanthin. The ratio between the content of ?-carotene and lutein - which are the principal foliar carotenoids - was supraunitary, a feature that might be used as a chemotaxonomic criterion. It is worth to be mentioned the relatively high content of zeaxanthin and ?-cryptoxanthin in comparison with the level of these pigments in the leaves of most higher plants. The ratio between the content of chlorophyll a and b was of 2,17.

Chemical and physical variations of cannabis smoke from a variety of cannabis samples in New Zealand
Thomas J. Sheehan, Hilary J. Hamnett, Richard Beasley & Paul S. Fitzmaurice
FORENSIC SCIENCES RESEARCH, 2018
DOI: 10.1080/20961790.2018.1445937
Studies have compared the chemical properties of tobacco smoke to those of cannabis smoke, with the objective of identifying the chemical attributes responsible for the mutagenicity and carcinogenicity of cannabis smoke. Comparative studies have included small sample sizes and produced conflicting results. The aim of this study was to assess the major chemical and
physical variations of cannabis smoke across a range of cannabis samples of different potencies and origins, sourced from the illegal market in New Zealand. Twelve cannabis samples were studied ranging from 1.0% to 13.4% delta-9-tetrahydrocannabinol (D9THC) content. A smoking machine was used to smoke “joints” (cannabis cigarettes) and the chemical/physical properties
of the smoke assessed. The chemical constituents of the smoke extracts were analysed by gas chromatography/mass spectrometry. A range of different chemical constituents (in addition to D9THC) were identified and their concentrations estimated. Terpenoids were identified as the major variable in cannabis smoke, showing a 40-fold range in total terpenoid content. Analysis
of the total particulate matter showed that significantly different levels of particulate matter were produced between the different cannabis samples, ranging from 14.6 to 66.3 mg/g of cannabis smoked. The D9THC delivery efficiency during smoking was also investigated and produced consistent results showing a mean and median of 12.6% and 10.8%, respectively of the theoretically available D9THC (ranging from 7.2% to 28.0%).

Chemical characterization of leaves, male and female flowers from spontaneous cannabis (Cannabis sativa var. spontanea) growing in Hungary.
Nagy DU, Cianfaglione K, Maggi F, Sut S, Dall'Acqua S.
Chem Biodivers. 2018 Dec 13.
doi: 10.1002/cbdv.201800562.
Cannabis sativa var. spontanea is a spontaneous form of hemp with a low content of psychoactive cannabinoids and can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this paper we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa var. spontanea growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analysed using GC-MS, NMR and LC-DAD-MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists (E)-caryophyllene (28.3%) and cannabidiol (CBD) (24.9%), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC-MS allowed to quantify CBD and CBD-A in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9%, and 0.2, 0.3 and 0.4%, respectively. Flavonoids were formed by C-glycosides and glucuronic acids of kaempferol and apigenin, with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, C. sativa var. spontanea may represent an important source of CB2 agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.

Chemical Characterization of Lignin and Lipid Fractions in Industrial Hemp Bast Fibers Used for Manufacturing High-Quality Paper Pulps
ANA GUTIEÄ RREZ, ISABEL M. RODRIÄGUEZ, AND JOSEÄ C. DEL RIÄO
J. Agric. Food Chem. 2006, 54, 2138-2144
DOI: 10.1021/jf052935a
The chemical composition of lignin and lipids of bast fibers from industrial hemp (Cannabis sativa) used for high-quality paper pulp production was studied. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of fibers showed a lignin with a p-hydroxyphenyl:guaiacyl:syringy l unit (H:
G:S) molar proportion of 13:53:34 (S/G ratio of 0.64). p-Hydroxycinnamic acids, namely, p-coumaric and ferulic acids, were found in only trace amounts. Among the lipids, the main compounds identified by GC/MS of the hemp fibers extracts were series of n-alkanes, free and esterified sterols and
triterpenols, waxes, and long-chain n-fatty acids. Other compounds such as n-aldehydes, n-fatty alcohols, steroid hydrocarbons, and steroid and triterpenoid ketones as well as steryl glycosides were also found.

Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells
Lisa Anceschi, Alessandro Codeluppi, Virginia Brighenti, Riccardo Tassinari, Valentina Taglioli, Lucia Marchetti, Luca Roncati, Andrea Alessandrini, Lorenzo Corsi, Federica Pellati
Pharmacological Research 174 (2021)
https://onlinelibrary.wiley.com/doi/....1002/ptr.7357
DOI: 10.1002/ptr.7357
In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

Chemical Composition and Antioxidants of Cannabis Roots
Petar Petrov
Extraxtion Magazine
https://extractionmagazine.com/2021/...annabis-roots/

Chemical composition and antioxidant potential of Cannabis sativa roots.
Christoph Kornpointner, Aitor Sainz Martinez, Silvija Marinovic, Christian Haselmair-Gosch, Polona Jamnik, Katharina Schroder, Christian Lofke, Heidi Halbwirth,
Industrial Crops and Products. 2021;165
https://www.sciencedirect.com/scienc...26669021001862
Cannabis sativa L. has long been exploited for multiple purposes. Whereas all parts of the shoots are extensively
used and well investigated, the roots have always received less attention. The phytochemical spectrum of the
roots differs significantly from the rest of the plant, as no significant amounts of cannabinoids are found, whereas
triterpenes as well as phytosterols are abundantly present. To shed light on the unique phytochemistry of hemp
roots and the related industrial potential, three chemovars were investigated for the secondary metabolite
composition and antioxidant activities by using in vitro and in vivo methods. Five triterpenes, ten phytosterols and
five aliphatic compounds were identified by GC–MS analysis. Glutinol, ß-amyrone, stigmastanol, fucosterol,
stigmasta-3,5-diene, stigmasta-3,5,22-triene, and oleamide were described for the first time in cannabis root
extracts. The predominant triterpenoids friedelin (0.100− 0.709 mg/g) and epifriedelinol (0.059− 0.205 mg/g)
were quantified in dependence of chemovar, harvest times, drying conditions, and extraction efficiency with
ethanol, n-hexane, and supercritical CO2.

Chemical Constituents of Cannabis sativa L. Root.
Journal of Pharmaceutical Sciences, 60(12), 1891–1892.(1971).
Slatkin, D. J., Doorenbos, N. J., Harris, L. S., Masoud, A. N., Quimby, M. W., & Schiff, P. L.
doi:10.1002/jps.2600601232
An extract of Cannabis satiua L. root yielded two pentacyclic triterpenes, friedelin and epifriedelanol, and N-(p-hydroxy- 8-phenylethy1)-phydroxy-trans-cinnamamide. Structures of the triterpenes were confirmed by preparation of derivatives and com- parison to authentic friedelin. Epifriedelanol was also oxidized to friedelin. The amide was confirmed by synthesis.

Chemical constituents of marijuana: The complex mixture of natural cannabinoids
Mahmoud A. ElSohly, Desmond Slade
Life Sciences 78 (2005) 539 – 548
doi:10.1016/j.lfs.2005.09.011
The cannabis plant (Cannabis sativa L.) and products thereof (such as marijuana, hashish and hash oil) have a long history of use both as a medicinal agent and intoxicant. Over the last few years there have been an active debate regarding the medicinal aspects of cannabis. Currently cannabis products are classified as Schedule I drugs under the Drug Enforcement Administration (DEA) Controlled Substances act, which means that the drug is only available for human use as an investigational drug. In addition to the social aspects of the use of the drug and its abuse potential, the issue of approving it as a medicine is further complicated by the complexity of the chemical make up of the plant. This manuscript discusses the chemical constituents of the plant with particular emphasis on the cannabinoids as the class of compounds responsible for the drug’s psychological properties.

Chemical Fingerprinting of Cannabis as a Means of Source Identification.
ElSohly, M. A., Stanford, D. F., & Murphy, T. P.
Marijuana and the Cannabinoids, 51–66.
doi:10.1007/978-1-59259-947-9_3
Marijuana is the most widely abused and readily available illicit drug in the United States, with an estimated 11.5 million current users annually purchasing more than $10 billion of the drug (1). Drug enforcement agencies are therefore keenly interested in trafficking routes of both foreign and domestically grown supplies of marijuana. From confidential sources to satellites, these agencies employ a multitude of methods to gather intelligence to direct resources, plan control operations, and develop policies. A practical means to recognize the source of seized marijuana would be a valuable tool for those purposes. Based on findings from 1990 to 1992 and described here, one way to determine origin is by using a chemical fingerprint system, a method that has shown promise as an effective intelligence tool to ascertain the geographic origin of confiscated marijuana samples. Of the many factors that affect the chemical constituents of marijuana, it is apparent that environmental factors consistently induce profiles unique to each environ. An “environ of origin” as broad as a continent or as small as an indoor garden may be differentiated based on the chemical fingerprint, or “signature,” of marijuana cultivated there—if a statistically significant number of samples grown in that environ are available for comparison. However, because all environs are not unique, the chemical fingerprint of cannabis is not considered to be an ultimate tool for forensic applications, although the technique may effectively support other types of evidence and is certainly of particular value in intelligence operations.

*Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents
From: Forensic Science and Medicine: Marijuana and the Cannabinoids
Rudolf Brenneisen
DOI: 10.1007/978-1-59259-947-9_2
The Cannabis plant and its products consist of an enormous variety of chemicals. Some of the 483 compounds identified are unique to Cannabis, for example, the more than 60 cannabinoids, whereas the terpenes, with about 140 members forming the most abundant class, are widespread in the plant kingdom. The term “cannabinoids” represents a group of C21 terpenophenolic compounds found until now uniquely in Cannabis sativa L. As a consequence of the development of synthetic cannabinoids (e.g., nabilone, HU-211 [dexanabinol; ref., or ajulemic acid [CT-3; ref.] and the discovery of the chemically different endogenous cannabinoid receptor ligands (“endocannabinoids,” e.g., anandamide, 2-arachidonoylglycerol) the term
“phytocannabinoids” was proposed for these particular Cannabis constituents

Chemometric Analysis of Cannabinoids: Chemotaxonomy and Domestication Syndrome
E. M. Mudge, S. J. Murch & P. N. Brown
Scientific Reports, 8(1).
doi:10.1038/s41598-018-31120-2
Cannabis is an interesting domesticated crop with a long history of cultivation and use. Strains have been selected through informal breeding programs with undisclosed parentage and criteria. The term “strain” refers to minor morphological differences and grower branding rather than distinct cultivated varieties. We hypothesized that strains sold by different licensed producers are chemotaxonomically indistinguishable and that the commercial practice of identifying strains by the ratio of total THC and
CBD is insufficient to account for the reported human health outcomes. We used targeted metabolomics to analyze 11 known cannabinoids and an untargeted metabolomics approach to identify 21 unknown cannabinoids. Five clusters of chemotaxonomically indistinguishable strains were identified from the 33 commercial products. Only 3 of the clusters produce CBDA in significant quantities while the other 2 clusters redirect metabolic resources toward the THCA production pathways. Six unknown metabolites were unique to CBD-rich strains and/or correlated to CBDA and 3 unknowns were found only in THCrich strains. Together, these data indicate the domestication of the cannabis germplasm has resulted in a loss of the CBDA pathway in some strains and reallocation of resources between CBDA and THCA pathways in others. The impact of domestication is a lack of chemical diversity and loss of biodiversity in modern cannabis strains

Chemotyping and Determination of Antimicrobial, Insecticidal, and Cytotoxic Properties of Wild-Grown Cannabis sativa from Nepal
Prabodh Satyal, William N. Setzer
Journal of Medicinally Active Plants 3, (1):9-16 2014
doi: 10.7275/R58W3B8V
Cannabis sativa was collected from a wildgrowing population in Biratnagar, Nepal. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography – mass spectrometry. A total of 107 constituents were identified in the oil accounting for 94.2% of the composition. This Nepalesechemotype is characterized by a predominance of sesquiterpenoids (68.1%) dominated by (E)-caryophyllene (20.4%), ?-humulene (7.0%), and ?-bisabolol (5.8%), but a paucity of monoterpene hydrocarbons (0.9%). In particular, neither myrcene nor terpinolene were detected. The oil in the Nepalese Cannabis plants did contain small amounts of cannabidiol (1.6%), cannabichromene (0.2%) and ?9-tetrahydrocannabinol (0.4%). The essential oil from Nepalese Cannabis sativa was screened for antimicrobial, cytotoxic, larvicidal, and insecticidal activity, and it appears as though C. sativa is relatively non-toxic.

Chemotyping and Determination of Antimicrobial, Insecticidal, and Cytotoxic Properties of Wild-Grown Cannabis sativa from Nepal
Prabodh Satyal, William N. Setzer
Journal of Medicinally Active Plants 3, (1):9-16 2014
doi: 10.7275/R58W3B8V
Cannabis sativa was collected from a wildgrowing population in Biratnagar, Nepal. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography – mass spectrometry. A total of 107 constituents were identified in the oil accounting for 94.2% of the composition. This Nepalesechemotype is characterized by a predominance of sesquiterpenoids (68.1%) dominated by (E)-caryophyllene (20.4%), ?-humulene (7.0%), and ?-bisabolol (5.8%), but a paucity of monoterpene hydrocarbons (0.9%). In particular, neither myrcene nor terpinolene were detected. The oil in the Nepalese Cannabis plants did contain small amounts of cannabidiol (1.6%), cannabichromene (0.2%) and ?9-tetrahydrocannabinol (0.4%). The essential oil from Nepalese Cannabis sativa was screened for antimicrobial, cytotoxic, larvicidal, and insecticidal activity, and it appears as though C. sativa is relatively non-toxic.

Chirality in Cannabinoid Research.
Filer, C. N.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0027
Mankind has long utilized Cannabis for diverse purposes. However, it has only been since the late 19th century that its individual cannabinoids began to be isolated, analyzed, and synthesized. By the mid-20th century it was discovered that many cannabinoids were asymmetric, with chirality often controlling their pharmacology. Increasingly accurate measurement and understanding of cannabinoid chirality will facilitate their synthesis and accelerate their medicinal applications.

Chromatographic characterisation of 11 phytocannabinoids:
Quantitative and fit?to?purpose performance as a function of

extra?column variance
Matthew Noestheden,| Gareth Friedlander | Jason Anspach | Scott Krepich |
K. C. Hyland | Wesley F. Zandberg
DOI: 10.1002/pca.2761
Introduction: Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids.
Objective: To develop a rapid, quantitative and broadly applicable liquid chromatography–tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character.
Methodology: Bulk diffusion coefficients were calculated using the Taylor–Aris open tubular method, with four reference compounds used to validate the experimental set?up. Three columns were quantitatively evaluated using van Deemter plots and fit?to?purpose performance metrics. Low (1.2 ?L2) and standard (3.6 ?L2) extra?column variance ultra?high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts.
Results: Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging ?8/?9?tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids.
Conclusions: The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra?column variance UPLC configurations.

Classification of Cannabis Plants Grown in Northern Thailand using Physico-Chemical Properties
Prapatsorn Tipparat, Weerapun Kunkaew, Suthat Julsrigival, Sarita Pinmanee, Surapol Natakankitku
Journal of Natural Sciences Research Vol.4, No.4, 2014
https://www.researchgate.net/publica...cal_properties
The Thai government has recognized that hemp may make useful contributions to the economy as an alternative crop. This study was conducted to provide information in both chemical and physical characters of Thai-grown cannabis for breeders to discriminate their phenotypes and accessions in order to select the low intoxicant with high fiber producing cultivars. The cannabinoids on the basis of THC, CBD and CBN content of 750 plants from eight accessions derived from five local cannabis cultivars were analyzed individually and their morphological features were also determined. According to the individual plants belonging to the same accessions showing distinct THC/CBD ratios were classified into different phenotypes, it is impossible to classify only single plant for defining the phenotype or determine cannabinoids content on the single analysis. THC content is found to correlate negatively to their physical characters such as plant height, stem diameter and fiber weight. Principal component analysis showed that the fiber weight, cork weight and stem diameter of the plant as well as chemical features such a THC content, CBD content, THC/CBD ratio and log10(THC/CBD ratio) explained most of the total variation which could distinguish accession and phenotype of the cannabis plants. Stepwise discriminant analysis confirmed that cannabinoids and some physical properties could be used to classify the phenotype of cannabis plants into drug, intermediate and fiber types as well, whereas the accessions of cannabis could not be discriminated clearly by using only their physico-chemical parameters. The genetic characteristics which affect the chemical patterns and morphological traits among cannabis accessions grown in northern Thailand should be considerable to investigate in further study

Classification of cannabis strains in the Canadian market with discriminant analysis of principal components using genome-wide single nucleotide polymorphisms
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
DOI: 10.1371/journal.pone.0253387
https://journals.plos.org/plosone/ar...l.pone.0253387
The cannabis community typically uses the terms “Sativa” and “Indica” to characterize drug strains with high tetrahydrocannabinol (THC) levels. Due to large scale, extensive, and unrecorded hybridization in the past 40 years, this vernacular naming convention has become unreliable and inadequate for identifying or selecting strains for clinical research and medicinal production. Additionally, cannabidiol (CBD) dominant strains and balanced strains (or intermediate strains, which have intermediate levels of THC and CBD), are not included in the current classification studies despite the increasing research interest in the therapeutic potential of CBD. This paper is the first in a series of studies proposing that a new classification system be established based on genome-wide variation and supplemented by data on secondary metabolites and morphological characteristics. This study performed a whole-genome sequencing of 23 cannabis strains marketed in Canada, aligned sequences to a reference genome, and, after filtering for minor allele frequency of 10%, identified 137,858 single nucleotide polymorphisms (SNPs). Discriminant analysis of principal components (DAPC) was applied to these SNPs and further identified 344 structural SNPs, which classified individual strains into five chemotype-aligned groups: one CBD dominant, one balanced, and three THC dominant clusters. These structural SNPs were all multiallelic and were predominantly tri-allelic (339/344). The largest portion of these SNPs (37%) occurred on the same chromosome containing genes for CBD acid synthases (CBDAS) and THC acid synthases (THCAS). The remainder (63%) were located on the other nine chromosomes. These results showed that the genetic differences between modern cannabis strains were at a whole-genome level and not limited to THC or CBD production. These SNPs contained enough genetic variation for classifying individual strains into corresponding chemotypes. In an effort to elucidate the confused genetic backgrounds of commercially available cannabis strains, this classification attempt investigated the utility of DAPC for classifying modern cannabis strains and for identifying structural SNPs.


Comparative Proteomics of Cannabis sativa Plant Tissues
Tri Joko Raharjo, Ivy Widjaja, Sittiruk Roytrakul, Robert Verpoorte
Journal of biomolecular techniques: JBT 15(2):97-106 July 2004
https://www.researchgate.net/publica..._Plant_Tissues
Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue-expressed proteins. These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Proteins extracted from glands, flowers, and leaves were separated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimensional gels from leaves and flowers. The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching. Flower and gland proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified: F1D9.26-unknown prot. (Arabidopsis thaliana), phospholipase D beta 1 isoform 1a (Gossypium hirsutum), and PG1 (Hordeum vulgare). Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.

Comprehensive characterization of mainstream marijuana and tobacco smoke.
Graves, B. M., Johnson, T. J., Nishida, R. T., Dias, R. P., Savareear, B., Harynuk, J. J., … Boies, A. M.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-63120-6
Recent increases in marijuana use and legalization without adequate knowledge of the risks necessitate the characterization of the billions of nanoparticles contained in each puf of smoke. Tobacco smoke ofers a benchmark given that it has been extensively studied. Tobacco and marijuana smoke particles are quantitatively similar in volatility, shape, density and number concentration, albeit with diferences in size, total mass and chemical composition. Particles from marijuana smoke are on average 29% larger in mobility diameter than particles from tobacco smoke and contain 3.4× more total mass. New measurements of semi-volatile fractions determine over 97% of the mass and volume of the particles from either smoke source are comprised of semi-volatile compounds. For tobacco and marijuana smoke, respectively, 4350 and 2575 diferent compounds are detected, of which, 670 and 536 (231 in common) are tentatively identifed, and of these, 173 and 110 diferent compounds (69 in common) are known to cause negative health efects through carcinogenic, mutagenic, teratogenic, or other toxic mechanisms. This study demonstrates striking similarities between marijuana and tobacco smoke in terms of their physical and chemical properties.

Comprehensive chromatographic profiling of cannabis from 23 USA States marketed for medical purposes
RAMIA Z. AL BAKAIN, YAHYA S. AL-DEGS, JAMES V. CIZDZIEL and MAHMOUD A. ELSOHLY
Acta Chromatographica (2020)
DOI: 10.1556/1326.2020.00767
In this research, cannabis varieties represent 23 USA States were assayed by GC-FID to generate their complex chemical profiles informative for plants clustering. Results showed that 45 cannabinoids and terpenoids were quantified in all plant samples, where 8 cannabinoids and 18 terpenoids were identified. Among organics, ?9 -THC, CBN (cannabinoids) and Fenchol (terpenoid) not only showed the highest levels overall contents, but also were the most important compounds for cannabis clustering. Among States, Washington, Oregon, California and Hawaii have the highest cannabis content. GC-FID data were subjected to PCA and HCA to find (1) the variations among cannabis chemical profiles as a result of growing environment, (2) to reveal the compounds that were responsible for grouping cultivars between clusters and (3) finally, to facilitate the future profile prediction and States clustering of unknown cannabis based on the chemical profile. The 23 cannabis USA States were grouped into three clusters based on only ?9 -THC, CBN, C1 and Fenchol content. Cannabis classification based on GCprofile will meet the practical needs of cannabis applications in clinical research, industrial production, patients’ self-production, and contribute to the standardization of commercially-available cannabis cultivars in USA.

Comprehensive classification of USA cannabis samples based on chemical profiles of major cannabinoids and terpenoids.
Albakain, R. Z., Al-Degs, Y. S., Cizdziel, J. V., & Elsohly, M. A.
Journal of Liquid Chromatography & Related Technologies, 1–13.
( 2019).
doi:10.1080/10826076.2019.1701015
Different USA-origin cannabis samples were analyzed by GC-FID to quantify all possible cannabinoids and terpenoids prior to their clustering. Chromatographic analysis confirmed the presence of seven cannabinoids and sixteen terpenoids with variable levels. Among tested cannabinoids, D9 -Tetrahydrocannabinol D9 -THC and cannabinol CBN were available in excess amounts (1.2–8.0 wt%) and (0.22–1.1 wt%), respectively. Fenchol was the most abundant terpenoid with a range of (0.03–1.0 wt%). The measured chemical profile was used to cluster 23 USA states and to group plant samples using different unsupervised multivariate statistical tools. Clustering of plant samples and states was sensitive to the selected cannabinoids/terpenoids. Principal component analysis (PCA) indicated the importance of D9 -THC, CBN, CBG, CBC, THCV, D8 -THC, CBL, and fenchol for samples clustering. D9 -THC was significant to separate California-origin samples while CBN and fenchol were dominant to separate Oregon-origin samples away from the rest of cannabis samples. A special PCA analysis was performed on cannabinoids after excluding D9 -THC (due to its high variability in the same plant) and CBN (as a degradation byproduct for THC). Results indicated that CBL and D8 -THC were necessary to separate Nevada and Washington samples, while, CBC was necessary to isolate Oregon and Illinois plant samples. PCA based on terpenoids content confirmed the significance of caryophyllene, guaiol, limonene, linalool, and fenchol for clustering target. Fenchol played a major role for clustering plant samples that originated from Washington and Nevada. k-means method was more flexible than PCA and generated three different classes; samples obtained from Oregon and California in comparison to the rest of other samples were obviously separated alone, which attributed to their unique chemical profile. Finally, both PCA and k-means were useful and quick guides for cannabis clustering based on their chemical profile. Thus, less effort, time, and materials will be consumed in addition to decreasing operational conditions for cannabis clustering.

Concentrations of ?9-Tetrahydrocannabinol and 11-Nor-9-Carboxytetrahydrocannabinol in Blood and Urine After Passive Exposure to Cannabis Smoke in a Coffee Shop
J. Röhrich1, I. Schimmel, S. Zörntlein, J. Becker, S. Drobnik, T. Kaufmann, V. Kuntz, and R. Urban
Journal of Analytical Toxicology, Vol. 34, (4):196-203 May 2010
DOI: 10.1093/jat/34.4.196
Cannabinoid concentrations in blood and urine after passive exposure to cannabis smoke under real-life conditions were investigated in this study. Eight healthy volunteers were exposed to cannabis smoke for 3 h in a well-attended coffee shop in Maastricht, Netherlands. An initial blood and urine sample was taken from each volunteer before exposure. Blood samples were taken 1.5, 3.5, 6, and 14 h after start of initial exposure, and urine samples were taken after 3.5, 6, 14, 36, 60, and 84 h. The samples were subjected to immunoassay screening for cannabinoids and analyzed using gas chromatography–mass spectrometry (GC–MS) for ?9-tetrahydrocannabinol (THC), 11-nor-hydroxy-?9- tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-?9- tetrahydrocannabinol (THC-COOH). It could be demonstrated that all volunteers absorbed THC. However, the detected concentrations were rather small. None of the urine samples produced immunoassay results above the cutoff concentration of 25 ng/mL. THC-COOH concentrations up to 5.0 and 7.8 ng/mL before and after hydrolysis, respectively, were found in the quantitative GC–MS analysis of urine. THC could be detected in trace amounts close to the detection limit of the used method in the first two blood samples after initial exposure (1.5 and 3.5 h). In the 6 h blood samples, THC was not detectable anymore. THC-COOH could be detected after 1.5 h and was still found in 3 out of 8 blood samples after 14 h in concentrations between 0.5 and 1.0 ng/mL.

Confirmatory non-invasive and non-destructive differentiation between hemp and cannabis using a hand-held Raman spectrometer
Lee Sanchez, Conor Filter, David Baltensperger, and Dmitry Kurouski
RSC Adv., 2020, 10, 3212–3216
DOI: 10.1039/C9RA08225E
Cannabis is a generic term that is used to denote hemp plants (Cannabis sativa) that produce delta-9- tetrahydrocannabinolic acid (THCA) in amounts higher than industrial hemp. While THCA itself is not considered psychoactive, it is the source of the psychoactive delta-9 tetrahydrocannabinol (THC) that forms from its oxidation. About 147 million people, which is around 2.5% of the world population, consume cannabis. This makes cannabis by far the most widely cultivated and trafficked illicit drug in the world. Such enormous popularity of cannabis requires substantial effort by border control and law enforcement agencies to control illegal trafficking and distribution. Confirmatory diagnostics of cannabis is currently done by high pressure liquid chromatography (HPLC), which requires sample transportation to a certified laboratory, making THC diagnostics extremely time and labor consuming. This catalyzed a push towards development of a portable, confirmatory, non-invasive and non-destructive approach for cannabis diagnostics that could be performed by a police officer directly in the field to verify illicit drug possession or transport. Raman spectroscopy (RS) is a modern analytical technique that meets all these strict expectations. In this manuscript, we show that RS can be used to determine whether plant material is hemp or cannabis with 100% accuracy. We also demonstrate that RS can be used to probe the content of THCA in the analyzed samples. These findings suggest that a hand-held Raman spectrometer can be an ideal tool for police officers and hemp breeders to enable highly accurate diagnostics of THCA content in plants

Constituents of Cannabis sativa
Carolina Echeverry, Miguel Reyes-Parada, Cecilia Scorza
Advances in Experimental Medicine and Biology 1297:1-9 Feb 2021
In book: Cannabinoids and Sleep
DOI: 10.1007/978-3-030-61663-2_1
Cannabis sativa L. is a psychoactive plant that contains more than 500 chemical components. Even though the consumption (in the form of marijuana, hashish, or hashish oil) for recreational purposes, is the most popular way of using the plant, the knowledge of its components has also led to classify Cannabis sativa L. is a plant with medicinal or therapeutical use. Several comprehensive reviews have already been published focused on the chemical composition of Cannabis sativa. In this chapter, we will summarize relevant information about those components, which may help to understand its biological actions that will be described in the following chapters.

Constructing a Draft Map of the Cannabis Proteome
Conor Jenkins, Benjamin C Orsburn
March 2019
DOI: 10.1101/577635
Recently we have seen a relaxing on the historic restrictions on the use and subsequent research on the cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. In this study we present our initial findings consisting of the identification of 17,269 unique proteins identified from Cannabis plant materials, as well as 6,110 post-translational modifications identified on these proteins. The results presented demonstrate the first steps toward constructing a complete draft map of the Cannabis proteome.

Contaminants of Concern in Cannabis: Microbes, Heavy Metals and Pesticides
John McPartland, Kevin McKernan
In book: Cannabis sativa L. - Botany and Biotechnology
DOI: 10.1007/978-3-319-54564-6_22
Microbiological contaminants pose a potential threat to cannabis consumers. Bacteria and fungi may cause opportunistic infections in immunocompromized individuals. Even dead organisms may trigger allergies and asthma. Toxins from microbial overloads, such as Shigla toxin and aflatoxins, may pose a problem—unlikely, but possible. The Cannabis plant hosts a robust microbiome; the identification of these organisms is underway. Cannabis bioaccumulates heavy metals in its tissues, so avidly that hemp crops have been used for bioremediation. Heavy metals cause myriad human diseases, so their presence in crops destined for human consumption must be minimized. Pesticide residues in cannabis pose a unique situation among crop plants—the Environmental Protection Agency (EPA) will not propose pesticides guidelines, because Cannabis is illegal on the federal level. The use of illegal pesticides is a rising crisis, and a breakdown in ethics. Testing for pesticide residues and maximal limits are proposed.

Costs of cannabis testing compliance: Assessing mandatory testing in the California cannabis market.
Valdes-Donoso, P., Sumner, D. A., & Goldstein, R.
PLOS ONE, 15(4), (2020).
doi:10.1371/journal.pone.0232041
Most U.S. states that have regulated and taxed cannabis have imposed some form of mandatory safety testing requirements. In California, the country’s largest and oldest legal cannabis market, mandatory testing was first enforced by state regulators in July 2018, and additional mandatory tests were introduced at the end of 2018. All cannabis must be tested and labeled as certified by a state-licensed cannabis testing laboratory before it can be legally marketed in California. Every batch that is sold by licensed retailers must be tested for more than 100 contaminants, including 66 pesticides with tolerance levels lower than the levels allowable for any other agricultural product in California. This paper estimates the costs of compliance with mandatory cannabis testing laws and regulations, using California’s testing regime as a case study. We use state government data, data collected from testing laboratories, and data collected from lab equipment suppliers to run a set of Monte Carlo simulations and estimate the cost per pound of compliance with California’s new cannabis testing regulations. We find that cost per pound is highly sensitive to average batch size and testing failure rates. We present results under a variety of different assumptions about batch size and failure rates. We also find that under realistic assumptions, the loss of cannabis that must be destroyed if a batch fails testing accounts for a larger share of total testing costs than does the cost of the lab tests. Using our best estimates of average batch size (8 pounds) and failure rate (4%) in the 2019 California market, we estimate testing cost at $136 per pound of dried cannabis flower, or about 10 percent of the reported average wholesale price of legal cannabis in the state. Our findings explain effects of the testing standards on the cost of supplying legal licensed cannabis, in California, other U.S. states, and foreign jurisdictions with similar testing regimes.

D9-Tetrahydrocannabinol Content in Cannabis Plants of Greek Origin
Maria STEFANIDOU, Sotiris ATHANASELIS, Giorgos ALEVISOPOULOS, John PAPOUTSIS, and Antonis KOUTSELINIS Chem. Pharm. Bull. 48(5) 743—745 (2000) DOI: 10.1248/cpb.48.743 The D9-tetrahydrocannabinol (D9-THC) content was identified and determined quantitatively using a Gas Chromatography Detector (Gas Chromatography - Electron Ion Detector) instrument in samples of illicit herbal cannabis. Law enforcement authorities sent the samples to the Department of Forensic Medicine and Toxicology, University of Athens, for toxicological analysis. The concentrations of D9-THC in these samples ranged from 0.08% to 4.41%. Such concentrations suggest that Greece might be at high risk, as an area for the illicit cultivation of “pedigree” cannabis plants. The forensic aspects of cannabis classification are discussed.

Δ9-Tetrahydrocannabivarin testing may not have the sensitivity to detect marijuana use among individuals ingesting dronabinol Frances R. Levin, John J. Mariani, Daniel J. Brooks, Shan Xie, and Kathleen A. Murray Drug Alcohol Depend. 2010 January 1; 106(1): 65.
doi:10.1016/j.drugalcdep.2009.07.021
https://www.ncbi.nlm.nih.gov/labs/pm...ihms138956.pdf
The purpose of this study was to determine whether Δ 9 -tetrahydrocannabivarin (THCV), a plant cannabinoid, is a sensitive measure to detect recent marijuana use in cannabis dependent patients. It has been purported that smoking an illicit plant cannabis product will result in a positive THCV urinalysis, whereas the oral ingestion of therapeutic THC such as dronabinol will result in a negative THCV urinalysis, allowing for discrimination between pharmaceutical THC products and illicit marijuana products. In a double-blind placebo-controlled trial to determine the efficacy of dronabinol in cannabis dependence, all 117 patients produced a positive urine for the marijuana metabolite 11- nor-Δ 9 -THC-9-carboxylic acid; THC-COOH, but 50% had an undetectable (< 1 ng/ml) THCVCOOH test. This suggests that THCV may not be a sensitive enough measure to detect recent marijuana use in all heavy marijuana users or that its absence may not discriminate between illicit marijuana use and oral ingestion of THC products such as dronabinol. We propose that the lack of THCV detection may be due to the variability of available cannabis strains smoked by marijuana users in community settings.

Novel Δ8‑Tetrahydrocannabinol Vaporizers Contain Unlabeled Adulterants, Unintended Byproducts of Chemical Synthesis, and Heavy Metals
Jiries Meehan-Atrash and Irfan Rahman Chem. Res. Toxicol. (2022) DOI: 10.1021/acs.chemrestox.1c00388 Supporting Information: separate Pdf Cannabis e-cigarettes containing Δ8 –tetrahydrocannabinol (Δ8 -THC) produced synthetically from hemp-derived cannabidiol (CBD) have recently risen in popularity as a legal means of cannabis consumption, but questions surrounding purity and unlabeled additives have created doubts of their safety. Herein, NMR, GC-MS, and ICP-MS were used to analyze major components of 27 products from 10 brands, and it was determined none of these had accurate Δ8 -THC labeling, 11 had unlabeled cutting agents, and all contained reaction side-products including olivetol, Δ4(8) -iso tetrahydrocannabinol, 9-ethoxyhexahydrocannabinol, Δ9 –tetrahydrocannabinol (Δ9 -THC), heavy metals, and a novel previously undescribed cannabinoid, iso -tetrahydrocannabifuran.

Detectability of cannabinoids in the serum samples of cannabis users: Indicators of recent cannabis use? A follow-up study
Michael Kraemer, Martin Schaeper, Kristina Duecker, Alexandra Philipsen, Moritz Losacker, Nadine Dreimueller, Jan Engelmann, Burkhard Madea, Cornelius Hess
Drug Test Anal. 2021 Jun 11.
doi: 10.1002/dta.3110
https://analyticalsciencejournals.on.. ..1002/dta.3110
Forensic toxicologists are frequently required to predict the time of last cannabis consumption. Several studies suggested the utility of minor cannabinoids as indicators of recent cannabis use. Because several factors influence blood cannabinoid concentrations, the interpretation of serum cannabinoid concentrations remains challenging. To assess the informative value of serum cannabinoid levels in cannabis users (in total N = 117 patients, including 56 patients who stated an exact time of last cannabis use within 24 h before blood sampling), the detectability of cannabinoids, namely delta-9-tetrahydrocannabinol (delta-9-THC), 11-hydroxy-delta-9-THC, 11-nor-9-carboxy-delta-9-THC, cannabichromene (CBC), cannabidiol (CBD), cannabinol (CBN), cannabidivarin, tetrahydrocannabivarin, cannabigerol (CBG), cannabicyclol, delta-8-THC, tetrahydrocannabinolic acid A, cannabichromenic acid, cannabidiolic acid (CBDA), cannabigerolic acid, cannabicyclolic acid (CBLA), 11-nor-9-carboxy-THCV (THCVCOOH), and 11-nor-CBN-9-COOH, was investigated. Excluding CBDA and CBLA, all investigated cannabinoids were detected in at least one analyzed sample. The interval between cannabis consumption and sample collection (reported by the patients) was not correlated with cannabinoid concentrations. Minor cannabinoids tended to be more easily detected in samples obtained shortly after consumption. However, some samples tested positive for minor cannabinoids despite an interval of several hours or even days between consumption and sampling (according to patients' statements). For instance, CBC, CBG, THCVCOOH, CBD, and CBN in certain cases could be detected more than 24 hours after the last consumption of cannabis. Thus, findings of minor cannabinoids should always be interpreted with caution.
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Detectability of various cannabinoids in plasma samples of cannabis users: Indicators of recent cannabis use?
Michael Krämer, Burkhard Madea, Cornelius Hess
Drug Testing and Analysis 11(10) August 2019
DOI: 10.1002/dta.2682
Despite many studies on cannabinoid pharmacokinetics, the proposals of marker cannabinoids for recent cannabis use, and the introduction of mathematical models estimating the time frame between consumption and blood sampling, it is still challenging for forensic toxicologists to estimate the last time of cannabis exposure. To assess the informative value of determining (minor) cannabinoids in plasma of cannabis users, detection rates of 14 cannabinoids next to Δ9 ‐THC and THC‐COOH (11‐OH‐THC, CBC, CBD, CBN, CBDV, THCV, CBG, CBL, Δ8 ‐THC, THCA, CBDA, CBGA, THCV‐ COOH, CBN‐COOH) were determined. Three hundred fifty‐five plasma samples, previously tested positive for cannabinoids (Δ9 ‐THC: approximately 0.4 ng/mL – 125 ng/mL (range), mean: 10.1 ng/mL; THC‐COOH: approximately 3.8 ng/mL – 457 ng/mL (range), mean: 71.6 ng/mL) were analyzed by means of liquid chromatography−tandem mass spectrometry (LC–MS/MS). All analyzed cannabinoids could be detected in plasma samples with varying incidence. 11‐OH‐THC, THCA, CBC, CBN, and CBD were the most frequent detectable cannabinoids (next to Δ9 ‐THC and THC‐ COOH). The dependency of cannabinoid detectability on the plasma Δ9 ‐THC concentration and on the probable time of consumption (estimated by a model of Huestis and coworkers) was examined. Detection incidences (eg, 11‐OH‐THC, CBC) often increased with increasing Δ9 ‐THC concentration but not for all cannabinoids (eg, CBD, THCA). The presented data for minor cannabinoid findings in plasma can be helpful for a comprehensive interpretation of cannabinoid findings in plasma samples of cannabis users

Detection of Cannabinoids by LC-MS-MS and ELISA in Breast Milk
Sempio C, Wymore E, Palmer C, Bunik M, Henthorn TK, Christians U, Klawitter J.
J Anal Toxicol. 2020 Oct 2;bkaa142.
doi: 10.1093/jat/bkaa142
Cannabis is the most commonly used drug of abuse in pregnancy and after delivery. However, little is known regarding the disposition of cannabinoids in breast milk, although delta-9-tetrahydrocannabinol (THC), the main psychoactive component, is highly lipophilic. Quantification of cannabinoids in breastmilk is essential for clinical monitoring and research studies and breastmilk banks mainly rely on ELISA in terms of screening for cannabinoids. To support clinical studies on disposition of cannabinoids in breastmilk, we validated a high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay for the simultaneous quantification of 12 cannabinoids and their metabolites in human breast milk. Said assay was based upon a simple one-step protein precipitation, online column extraction and detection in the positive multiple reaction monitoring mode. After successful validation, the assay was used to analyze 30 samples from a clinical research study that had tested negative using an ELISA kit that is commonly used by breastmilk banks. In human breast milk, depending on the analyte, the lower limits of quantification of the LC-MS-MS assay ranged from 0.39 to 7.81 ng/mL. Acceptance criteria for intra- and inter-batch accuracy (85-115%) and imprecision (<15%) were met for all compounds. Mean extraction efficiencies were above 60% for all analytes. Mean matrix effect ranged from -12.5% to 44.5% except of THC- glucuronide for which significant matrix effects were noted. No carry-over was detected. Although cannabinoid-negative based on the ELISA, all 30 samples tested positive for THC using LC-MS-MS (0.8-130 ng/mL) and several also for 11-OH-THC, THCCOOH, CBD and CBG. We validated a sensitive and specific assay for the quantification of 12 cannabinoids in human breastmilk that outperformed an ELISA commonly used by breastmilk banks.

(?)-trans-?9-Tetrahydrocannabiphorol Content of Cannabis sativa Inflorescence from Various Chemotypes
Justin Bueno and Eric A. Greenbaum
J. Nat. Prod. 2021,
DOI: 10.1021/acs.jnatprod.0c01034
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01034
The (?)-trans-?9-tetrahydrocannabiphorol (?9-THCP, 1) content of the inflorescence from sixCannabis sativa chemotypes, including 14 plants of distinct genotypes, and two extracts was determined quantitatively via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This represents the first comprehensive quantitative screening for 1 from various C. sativa chemotypes. Compound 1 was detected in all 13 inflorescence samples originating from “(?)-trans-?9-tetrahydrocannabinol (?9-THC, 2) dominant” C. sativa chemotypes, but was not detected in a “cannabidiol (CBD, 3) dominant” chemotype. The inflorescence content of 1 ranged approximately from 0.0023% to 0.0136% (w/w). Comprehensive inflorescence sampling was performed for each specimen investigated. A trend between inflorescence cannabinoid potency and the location of which the inflorescence was sampled on the C. sativa plant was observed for the three cannabinoids tested (1–3). The preliminary results obtained indicate ?9-THCP (1) may have a higher degree of prevalence in C. sativa inflorescence than previously estimated.

Determination and distribution of cannabinoids in nail and hair samples.
Cobo-Golpe, M., de-Castro-Ríos, A., Cruz, A., López-Rivadulla, M., & Lendoiro, E.
Journal of Analytical Toxicology.
doi:10.1093/jat/bkaa164
Hair has been used for decades in toxicology as a biological matrix for long-term detection of substances. Nails are another keratinized matrix that is being studied as an alternative when hair cannot be obtained. Although cannabis is the most prevalent illicit drug in the world, cannabinoid distribution in nails compared to hair has been scarcely studied. In this work, we described two methods for the determination of cannabidiol (CBD), cannabinol (CBN) and ? 9 -Tetrahydrocannabinol (THC), and THC’s main metabolites (11-nor-9-carboxy-THC (THCCOOH), 11-hydroxy-THC (OHTHC) and 8- ?-11-dihydroxyTHC (diOHTHC)) in nail and hair samples. After an alkaline hydrolysis, samples were submitted to solid-phase extraction and analysed by LC-MS/MS. The methods were fully validated, with good linearity (r2 >0.99) in the range of 20-100 to 20000 pg/mg. No endogenous or exogenous interferences were found. Accuracy was from 99.5 to 109.8% and imprecision was

Determination of Acid and Neutral Cannabinoids in Extracts of Different Strains of Cannabis sativa Using GC-FID.
Ibrahim, E., Gul, W., Gul, S., Stamper, B., Hadad, G., Abdel Salam, R., … ElSohly, M.
Planta Medica, 84(04), 250–259. (2017).
doi:10.1055/s-0043-124088
Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-?9-tetrahydrocannabinol (?9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and ?9-THC, have been tissue cultured via micropropagation and cultivated. A GC FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-?8-tetrahydrocannabinol, ?9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and ?9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ? 15% and ± 5%, respectively. The limit of detection range was 0.11–0.19 µg/mL, and the limit of quantitation was 0.34–0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).

Determination of Cannabinoid Vapor Pressures to Aid in Vapor Phase Detection of Intoxication
Tara M. Lovestead, Thomas J. Bruno
Forensic Chemistry (2017)
Doi: 10.1016/j.forc.2017.06.003
The quest for a reliable means to detect cannabis intoxication with breathalyzer is ongoing. To design such a device, it is important to understand the fundamental thermodynamics of the compounds of interest. The vapor pressures of two important cannabinoids, cannabidiol (CBD) and ?9-tetrahydrocannabinol (?9-THC), are presented, as well as the predicted normal boiling temperature (NBT) and the predicted critical constants (these predictions are dependent on the vapor pressure data). The critical constants are typically necessary to develop an equation of state (EOS). EOS-based models can provide estimations of thermophysical properties for compounds to aid in designing processes and devices. An ultra-sensitive, quantitative, trace dynamic headspace analysis sampling called porous layered open tubular-cryoadsorption (PLOT-cryo) was used to measure vapor pressures of these compounds. PLOT-cryo affords short experiment durations compared to more traditional techniques for vapor pressure determination (minutes versus days). Additionally, PLOT-cryo has the inherent ability to stabilize labile solutes because collection is done at reduced temperature. The measured vapor pressures are approximately 2 orders of magnitude lower than those measured for n-eicosane, which has a similar molecular mass. Thus, the difference in polarity of these molecules must be impacting the vapor pressure dramatically. The vapor pressure measurements are presented in the form of
Clausius-Clapeyron (or van’t Hoff) equation plots. The predicted vapor pressures that would be expected at near ambient conditions (25 ?C) are also presented.

Determination of cannabinoids in urine, oral fluid and hair samples after repeated intake of CBD-rich cannabis by smoking.
Gerace, E., Bakanova, S. P., Di Corcia, D., Salomone, A., & Vincenti, M.
Forensic Science International, 110561.(2020).
doi:10.1016/j.forsciint.2020.110561
Cannabidiol prevalent (CBD-rich) cannabis derivatives are increasingly popular and widely available on the market as replacement of THC, tobacco substitutes or therapeutics for various health conditions. In this paper, we evaluate the impact of a repeated CBD-rich cannabis intake on levels of cannabinoids in biological samples. Urine, oral fluid and hair (pubic and head) samples were obtained from a naive user during a 26-day smoking period of one 250-mg CBD-rich cannabis joint/day containing 6.0% cannabidiol (CBD; 15 mg) and 0.2% delta-9-tetrahydrocannabinol (THC; 0.5 mg). In total, 35 urine, 8 oral fluid and 4 hair sample were collected. Cannabinoids concentrations were quantified by a UHPLC/MSn technique. The results suggested that the repeated exposure to CBDrich cannabis (containing small amounts of THC) can generate positive results in biological samples. Urinary concentrations of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) were quantitatively detected after 8 days from the smoking start and exceeded the 15 ng/mL cut-off limit on day-15 even in the urine sample collected 12 hours after the last intake. In the oral fluid collected on day-26, no cannabinoids were found before the cannabis intake, thus excluding accumulation, while THC was detectable up to 3 h after the cannabis intake, at concentrations progressively decreasing from about 18 to 6 ng/mL. Hair samples collected one week after the end of the study turned out negative for THC and THC-COOH, suggesting that this matrix is suitable to discriminate the chronic consumption of CBD-rich cannabis from THC-prevalent products. The obtained findings are relevant for the interpretations of cannabinoids levels in biological fluids, also in light of the legal implications of a positive result.

Determination of eighty-two pesticides and application to screening pesticides in cannabis growing facilities.
Craven, C. B., Birjandi, A. P., Simons, B., Jiang, P., & Li, X.-F.
Journal of Environmental Sciences, 104, 11–16.(2021).
doi:10.1016/j.jes.2020.11.004
Determination of pesticides in cannabis facilities is increasingly important as medicinal and recreational uses of cannabis products expand rapidly. We report a method involving wipe sampling, liquid chromatography separation, and tandem mass spectrometry, which enables determination of 82 pesticides out of the 96 regulated by Health Canada. To demonstrate an application of the method, we sampled and measured pesticides in two cannabis growing facilities, representing a non-certified and a certified site. We detected 41 pesticides in surface wipe samples at the non-certified site and 6 at the certified site. This study provides the first evidence showing pesticide occurrence on common surfaces in cannabis growing facilities and points to a need for routine monitoring and strict control of pesticide use in cannabis facilities

Determination of Heavy Metals in Cannabis and Hemp Products Following AOAC Method for ICP-MS
Jenny Nelson, Craig Jones, Sam Heckle, Leanne Anderson,
https://www.agilent.com/en/solutions...g-for-cannabis
Routine monitoring of As, Cd, Hg, Pb, and other elements using an Agilent 7850 ICP-MS
The analysis of metals in cannabis has been difficult for many labs because of a lack of official methods in the industry. In August 2021, AOAC adopted an ICP-MS method for the determination of arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) in a variety of cannabis and cannabis-derived products. The new method is adopted as an Official Method of Analysis in First Action status (1) and will be tracked for a maximum of two years. If the method is shown to be reproducible, it will be recommended for Final Action status. This AOAC method has undergone rigorous assessment by the AOAC Expert Review Panel (ERP) and achieved consensus through AOAC members.

Determination of Herbicides Paraquat, Glyphosate, and Aminomethylphosphonic Acid in Marijuana Samples by Capillary Electrophoresis
Rafael Lanaro, Jos_e L. Costa, Silvia O. S. Cazenave, Luiz A. Zanolli-Filho, Marina F. M. Tavares, and Alice A. M. Chasin
J Forensic Sci, January 2015, Vol. 60, No. S1
doi: 10.1111/1556-4029.12628
In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7).
The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to
25.1 mg/g. Three samples were positive for glyphosate (0.15–0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.


Determination of melatonin content of diferent varieties of hemp (Cannabis sativa L.) by liquid chromatography tandem mass spectrometry
Gianna Allegrone · Federica Razzano · Federica Pollastro1 · Gianpaolo Grassi
SN Appl. Sci. 1, 720 (2019).
Doi: 10.1007/s42452-019-0759-y
Melatonin (N-acetyl-5-methoxytryptamine), was first isolated in the bovine pineal gland, and then was discovered in bacteria, algae and higher plants. Melatonin concentrations in plants differ from species to species, among varieties within the same species and also within different organs or tissues of a given plant as roots, leaves, fruits, flowers, and seeds. Although the presence of melatonin in plants seems to be a universal trait, there is still lack of information on its occurrence in several plants, in particular in hemp (Cannabis sativa L.). The aim of this study was to develop a method to quantitate melatonin in the aerial parts of the plant, constituted of leaves and flower heads, and in the seeds, of four different hemp varieties using liquid chromatography–tandem mass spectrometry (LC–ESI/MS/MS). A sample preparation procedure with methanol extraction followed by solid-phase clean-up protocol was set up for melatonin extraction and a LC–ESI/MS/MS method in single reaction monitoring (SRM) was used for its determination. Chromatographic separation was achieved on a C18 column using a gradient elution with acetonitrile–buffer ammonium formate/formic acid system. The developed method, validated as per International Conference on Harmonization guidelines, was successfully applied to analyze hemp samples varieties belonging to fiber-type or drug-type chemotypes. The melatonin was found to accumulate highly in seeds in the range 13.43–30.40 ng g?1 while the content in the aerial parts was assessed in the range 1.16–4.85 ng g?1. No correlation was found between the melatonin levels detected in aerial parts and seeds in each hemp variety and the concentration of specific cannabinoids in the same variety.

Determination of Pesticide Residues in Cannabis Smoke
Nicholas Sullivan, Sytze Elzinga, and Jeffrey C. Raber
Journal of Toxicology
Volume 2013, Article ID 378168, 6 pages
doi: 10.1155/2013/378168
The present study was conducted in order to quantify to what extent cannabis consumers may be exposed to pesticide and other chemical residues through inhaled mainstream cannabis smoke. Three different smoking devices were evaluated in order to provide a generalized data set representative of pesticide exposures possible for medical cannabis users. Three different pesticides,
bifenthrin, diazinon, and permethrin, along with the plant growth regulator paclobutrazol, which are readily available to cultivators in commercial products,were investigated in the experiment. Smoke generated from the smoking devices was condensed in tandem chilled gas traps and analyzed with gas chromatography-mass spectrometry (GC-MS). Recoveries of residues were as high as 69.5% depending on the device used and the component investigated, suggesting that the potential of pesticide and chemical residue
exposures to cannabis users is substantial and may pose a significant toxicological threat in the absence of adequate regulatory frameworks.

Determination of the relative percentage distribution of THCA and D9-THC in herbal cannabis seized in Austria – Impact of different storage temperatures on stability
Magdalena Taschwer, Martin G. Schmid
Forensic Science International 254 (2015) 167–171
Doi: 10.1016/j.forsciint.2015.07.019
Cannabis is globally by far the most widespread illicit drug of abuse. Especially since its legalization in some of the US, controversies about the legal status of cannabis for recreational and medical use have come up. D9-Tetrahydrocannabinol (D9-THC), which is the major active ingredient in cannabis products, is mainly responsible for the psychoactive effects. Its inactive biosynthetic precursor tetrahydrocanna- binolic acid (THCA) is present in different quantities in fresh and undried cannabis plants. Under influence of drying, temperature and UV exposure it decomposes to D9-THC. In this study, a quantification of D9-THC and THCA was carried out to check the stability of cannabis samples. The determination of the degradation of THCA to D9-THC in 29 cannabis products seized in Austria was monitored by HPLC-UV. Mobile phase consisted of a 25 mM triethylammoniumphosphate buffer (pH 3.0) and acetonitrile (36:64). A common LiChrospher1 100 RP-18 column was utilized as stationary phase. To check the influence of low as well as high temperature on the degradation process of the cannabinoid THCA to D9-THC, samples were stored in a freezer or in a drying cabinet for a specified time period. It was shown successfully that high storage temperatures led to a more rapid and complete decomposition of THCA to D9-THC while at low temperatures only slight or no changes of the percentage distribution were determined.

Developing Robust Standardised Analytical Procedures for Cannabinoid Quantification: Laying the Foundations for an Emerging Cannabis-Based Pharmaceutical Industry
Matthew T. Welling, Lei Liu, Arno Hazekamp, Ashley Dowella, Graham J. King
Med Cannabis Cannabinoids
Published online: February 25, 2019
DOI: 10.1159/000496868
The plant genus Cannabis is a prolific producer of unique pharmaceutically relevant metabolites, commonly referred to as cannabinoids. Robust and standardised methods for the quantification of cannabinoids within botanical and drug forms is a critical step forward for an emerging Cannabis-based pharmaceutical industry, which is poised for rapid expansion. Despite a growing body of analytical methods for the quantification of cannabinoids, few have been validated using internationally accredited guidelines. Moreover, standardised methods have yet to be developed for application at various stages of manufacture as well as for different levels of processing and refinement. Validation parameters for establishing robust standardised methods for cannabinoid quantification within Cannabis-based drug forms are critically discussed. Determining an appropriate level of specificity (discrimination) among heterogeneous botanical matrices as well as evaluating accuracy (recovery) and inter-laboratory precision (reproducibility) within strict and volatile regulatory environments are potential obstacles to the establishment of robust analytical procedures. We argue that while some of these challenges remain unique to Cannabis, others are common to botanical-based drug development and manufacture. In order to address potential barriers to analytical method standardisation, a collaborative research initiative inclusive of academic and commercial stakeholders is proposed.

*Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis.
Giese MW, Lewis MA, Giese L, Smith KM.
J AOAC Int.
DOI: 10.5740/jaoacint.15-116
The requirements for an acceptable cannabis assay have changed dramatically over the years resulting in a large number of laboratories using a diverse array of analytical methodologies that have not been properly validated. Due to the lack of sufficiently validated methods, we conducted a single- laboratory validation study for the determination of cannabinoids and terpenes in a variety of commonly occurring cultivars. The procedure involves high- throughput homogenization to prepare sample extract, which is then profiled for cannabinoids and terpenes by HPLC-diode array detector and GC-flame ionization detector, respectively. Spike recovery studies for terpenes in the range of 0.03-1.5% were carried out with analytical standards, while recovery studies for ?9-tetrahydrocannabinolic acid, cannabidiolic acid, ?9-tetrahydrocannabivarinic acid, and cannabigerolic acid and their neutral counterparts in the range of 0.3-35% were carried out using cannabis extracts. In general, accuracy at all levels was within 5%, and RSDs were less than 3%. The interday and intraday repeatabilities of the procedure were evaluated with five different cultivars of varying chemotype, again resulting in acceptable RSDs. As an example of the application of this assay, it was used to illustrate the variability seen in cannabis coming from very advanced indoor cultivation operations.

Development of a new method for the analysis of cannabinoids in honey by means of high-performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry detection.
Brighenti, V., Licata, M., Pedrazzi, T., Maran, D., Bertelli, D., Pellati, F., & Benvenuti, S.
Journal of Chromatography A.(2019).
doi:10.1016/j.chroma.2019.03.034
Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male flowers. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its physiological effects, attributable to its antioxidant, neuroprotective and anti-inflammatory properties. The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources, including hemp. In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards the detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products. The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance

Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of cannabidiol (CBD), ? 9 -tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD.
Palazzoli, F., Citti, C., Licata, M., Vilella, A., Manca, L., Zoli, M., … Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis, 150, 25–32. (2018).*
doi:10.1016/j.jpba.2017.11.054
The investigation of the possible conversion of cannabidiol (CBD) into ?9-tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50mg/kg) was administered orally to rats and their blood was collected after 3 and 6h. A highly sensitive and selective LC-MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

Development of extraction technique and GC/FID method for the analysis of cannabinoids in Cannabis sativa L. spp. santicha (hemp).
Baranauskaite, J., Marksa, M., Ivanauskas, L., Vitkevicius, K., Liaudanskas, M., Skyrius, V., & Baranauskas, A.
Phytochemical Analysis. (2020).
doi:10.1002/pca.2915
Abstract Introduction: Nowadays, the interest in industrial Cannabis sativa L. herb has been increasing in the world. As a result, it is becoming one of the most studied plants due to its multifunctional benefits. Objectives: To the best of our knowledge, no study has been conducted so far to determine the impact of extraction methods and conditions on the extraction yields of CBD and CBG from the Cannabis sativa L. ssp. Santhica. Therefore, we aimed to investigate a simple and sensitive GC-FID method to determine CBD and CBG in hemp extract. Methods: As regards sample preparation, three extraction techniques were compared, including maceration (ME), ultrasound-assisted extraction (UAE) and refluxheat extraction (RHE), in order to obtain a high recovery of the CBD of interest from the plant material. The GC-FID method developed in this study represents a powerful tool for the extraction and analysis of non-psychoactive cannabinoids from hemp varieties to be used for the preparations of extracts with a high content of bioactive compounds for both pharmaceutical and nutraceutical applications. Results: A simple extraction procedure for CBD and CBG from hemp was also optimized in this work, by using ultrasound assisted extraction method with 96% ethanol, material/solvent ratio 1:10 and extraction time 10 min at room temperature. Conclusion: The overall analytical method was fully validated in agreement with international guidelines. Therefore, proving a powerful and reliable tool for both the selection of hemp varieties with a high content of bioactive compounds and the quality control of its derivatives.

Development of Gas-Chromatographic Method for Simultaneous Determination of Cannabinoids and Terpenes in Hemp
Jure Zekic , and Mitja Križman
Molecules 2020, 25, 5872;
doi:10.3390/molecules25245872
An original gas chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimised, also by avoiding any analyte derivatisation. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.

Dispersive liquid–liquid microextraction of 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid applied to urine testing
Leonardo C Rodrigues, Júlia MM Kahl, Kauê O de Chinaglia , Eduardo G de Campos & José Luiz Costa
Doi: 10.4155/bio-2021-0237
https://www.future-science.com/doi/e.../bio-2021-0237
Aim: THC-COOH is the major metabolite of Δ9-tetrahydrocannabinol commonly tested in urine to determine cannabis intake. In this study, a method based on dispersive liquid–liquid microextraction was developed for testing THC-COOH in urine. Materials & methods: Hydrolyzed urine specimens were extracted via dispersive liquid–liquid microextraction with acetonitrile (disperser solvent) and chloroform (extraction solvent). Derivatization was performed with N,O-Bis(trimethylsilyl)trifluoroac etamide with 1% trichloro(chloromethyl)silane. Analysis was performed by GC–MS/MS.Results: The method showed acceptable linearity (5–500 ng/ml), imprecision (<10.5%) and bias (<4.9%). Limits of detection and quantitation were 1 and 5 ng/ml, respectively. Twenty-four authentic samples were analyzed, with 22 samples being positive for THC-COOH. Conclusion: The proposed method is more environmentally friendly and provided good sensitivity, selectivity and reproducibility.
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Does Cannabis Extract Obtained From Cannabis Flowers With Maximum Allowed Residual Level of Aflatoxins and Ochratoxin a Have an Impact on Human Safety and Health?

Tijana Serafimovska, Sasho Stefanovski, Joachim Erler, Zlatko Keskovski, Gjoshe Stefkov, Marija Mitevska, Marija Darkovska Serafimovska, Trajan Balkanov and Jasmina Tonic Ribarska
Front. Med. 8:759856.
doi: 10.3389/fmed.2021.759856
https://www.frontiersin.org/articles...21.759856/full
Aim: The aim of this study was to investigate whether the cannabis extract obtained from cannabis flowers that contain the maximum allowed level of mycotoxins affects human safety and health. For that purpose, a novel liquid chromatography with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of aflatoxins and ochratoxin A (OchA) in cannabis extracts to demonstrate that this analytical method is suitable for the intended experimental design.
Methods: Experimental design was done by adding maximum allowed concentration of aflatoxins (B1, B2, G1, G2) and OchA according to the European Pharmacopeia related to cannabis flowers. The concentration of aflatoxins and OchA was determined using the same LC/MS/MS analytical method in the starting material (dry flower) before preparing the spiked sample and after obtaining decarboxylated extract with ethanol 96%.
Results: The results obtained indicate that aflatoxins and OchA, primarily added to the cannabis dried flowers, were also determined into the obtained final extract in amounts much higher (m/m) than in the starting plant material.
Conclusion: With this experiment, we have shown that mycotoxins, especially aflatoxins, which are extremely toxic secondary metabolites, can reach critical values in cannabis extracts obtained from dry cannabis flowers with the maximum allowed quantity of mycotoxins. This can pose a great risk to consumers and their health especially to those with compromised immune systems.

Driving Performance and Cannabis Users’ Perception of Safety Thomas D. Marcotte, Anya Umlauf, David J. Grelotti, Emily G. Sones, Philip M. Sobolesky, Breland E. Smith, Melissa A. Hoffman, Jacqueline A. Hubbard, Joan Severson, Marilyn A. Huestis, Igor Grant, Robert L. Fitzgerald doi:10.1001/jamapsychiatry.2021.4037 https://jamanetwork.com/journals/jam...rticle/2788264
IMPORTANCE Expanding cannabis medicalization and legalization increases the urgency to understand the factors associated with acute driving impairment.
OBJECTIVE To determine, in a large sample of regular cannabis users, the magnitude and time course of driving impairment produced by smoked cannabis of different Δ9-tetrahydrocannabinol (THC) content, the effects of use history, and concordance between perceived impairment and observed performance.
DESIGN, SETTING, AND PARTICIPANTS This double-blind, placebo-controlled parallel randomized clinical trial took place from February 2017 to June 2019 at the Center for Medicinal Cannabis Research, University of California San Diego. Cannabis users were recruited for this study, and analysis took place between April 2020 and September 2021.
INTERVENTIONS Placebo or 5.9% or 13.4%THC cannabis smoked ad libitum. MAIN OUTCOMES AND MEASURES The primary end pointwas the Composite Drive Score (CDS), which comprised key driving simulator variables, assessed prior to smoking and at multiple time points post smoking. Additional measures included self-perceptions of driving impairment and cannabis use history.
RESULTS Of 191 cannabis users, 118 (61.8%) were male, the mean (SD) age was 29.9 (8.3) years, and the mean (SD) days of use in the past month was 16.7 (9.8). Participants were randomized to the placebo group (63 [33.0%]), 5.9% THC (66 [34.6%]), and 13.4%THC (62 [32.5%]). Compared with placebo, the THC group significantly declined on the Composite Drive Score at 30 minutes (Cohen d = 0.59 [95%CI, 0.28-0.90]; P < .001) and 1 hour 30 minutes (Cohen d = 0.55 [95%CI, 0.24-0.86]; P < .001), with borderline differences at 3 hours 30 minutes (Cohen d = 0.29 [95%CI, –0.02 to 0.60]; P = .07) and no differences at 4 hours 30 minutes (Cohen d = –0.03 [95%CI, –0.33 to 0.28]; P = .87). The Composite Drive Score did not differ based on THC content (likelihood ratio χ24 = 3.83; P = .43) or use intensity (quantity Å~ frequency) in the past 6 months (likelihood ratio χ24 = 1.41; P = .49), despite pos tsmoking blood THC concentrations being higher in those with the highest use intensity. Although there was hesitancy to drive immediately post smoking, increasing numbers (81 [68.6%]) of participants reported readiness to drive at 1 hour 30 minutes despite performance not improving from initial post smoking levels.
CONCLUSIONS AND RELEVANCE Smoking cannabis ad libitum by regular users resulted in simulated driving decrements. However, when experienced users control their own intake, driving impairment cannot be inferred based on THC content of the cigarette, behavioral tolerance, or THC blood concentrations. Participants’ increasing willingness to drive at 1 hour 30 minutes may indicate a false sense of driving safety.Worse driving performance is evident for several hours post smoking in many users but appears to resolve by 4 hours 30 minutes in most individuals. Further research is needed on the impact of individual biologic differences, cannabis use history, and administration methods on driving performance

Effect-directed analysis of bioactive compounds in Cannabis sativa L. by high-performance thin-layer chromatography
Giulia Corni, Virginia Brighenti, Fredrica Pelliti, Gertrud E.Morlock
Journal of Chromatography A Volume 1629, 11 October 2020, 461511
https://www.sciencedirect.com/scienc...2196732030786X
DOI: 10.1016/j.chroma.2020.461511
The scientific interest on the plant Cannabis sativa L., and in particular on its non-psychoactive or fibre-type variety (hemp), has been highly increasing in recent years, due to the pharmaceutical and nutraceutical potential of its bioactive compounds. This plant is indeed characterized by a very rich chemical composition, which encompasses different classes of constituents, such as cannabinoids and terpenes. In this context, the bioanalytical testing of hemp extracts can be difficult and time-consuming. Effect-directed analysis (EDA) by the combination of high-performance thin-layer chromatography (HPTLC) with biological and enzymatic assays represents one of the latest tools available for the rapid bioprofiling of complex matrices, such as plant extracts. In this ambit, the aim of this project was the non-targeted screening of inflorescence extracts from ten different hemp varieties for components exhibiting radical scavenging, antibacterial, enzyme inhibiting and estrogen-like effects. By HPTLC-EDA, the hemp samples exhibited strong antibacterial activities against both Gram-positive Bacillus subtilis and especially Gram-negative Aliivibrio fischeri bacteria, and also estrogen-like activity. They also inhibited ?- and ?-glucosidase, tyrosinase and acetylcholinesterase. The characterization of two prominently multipotent bioactive compound zones was finally achieved by HPTLC-HRMS and preliminary assigned as cannabidiolic acid and cannabidivarinic acid.

Effect of Soil Contamination on Some Heavy Metals Content of Cannabis sativa
Murad Ali Khan
Journal- Chemical Society of Pakistan 30(6):805-809 December 2008
https://www.researchgate.net/publica...annabis_sativa
Heavy metals were investigated in the medicinal plant Cannabis sativa and the soil of the area from where the plant was collected using atomic absorption spectrophotometer. The plant samples were collected from five different locations of N.W.F.P, Pakistan. The plant parts including roots, stem and leaves were found to have the quantity of heavy metals corresponding to their contents in the soil. The purpose of the study is to make awareness among the people about the proper use and collection of medicinal plants, containing high level of heavy metals and their adverse health effects.

Effects of long term storage on secondary metabolite profiles of cannabis resin.
Grafström K, Andersson K, Pettersson N, Dalgaard J, Dunne S
Forensic Sci Int. 2019 Aug;301:331-340.
doi: 10.1016/j.forsciint.2019.05.035
The structural identification and the monitoring of the relative concentrations of a wide range of major (3) and minor secondary (16) metabolites used as marker substances for profiling of cannabis resin using GC-FID at the Swedish National Forensic Centre (NFC) has facilitated the mapping of their chemical and physical behaviors over a period of 48months whilst stored under different conditions (exposure to light, exposure to air, temperature). In all cases the behavior of this group of sesquiterpenes, sesquiterpenoids, cannabinoids and waxes could be directly related to their chemical lability/functionality. In particular, the identification of homologue triads for both ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD) together with a group of seemingly chemically inert substances (for example, cannabicyclol(CBL) and the waxes (n-alkanes)) has created new tools for the establishment of common origins between samples of cannabisresins aged under different conditions. Since sampling of the resin blocks in NFC's method for profiling of cannabis resin is made below the surface, the effects of light incursion were found to be negligible. The effects of exposure to air (and indirectly temperature) were found to be more significant, not unexpectedly as many of the observed transformations were based on oxidation or rearrangement processes.

Elevated Phosphorus Fertility Impact on Cannabis sativa ‘BaOx_’ Growth and Nutrient Accumulation
Patrick Veazie, Paul Cockson, Dylan Kidd, and Brian Whipker
IJISET - International Journal of Innovative Science, Engineering & Technology, Vol. 8 Issue 2, February 2021
http://ijiset.com/vol8/v8s2/IJISET_V8_I02_32.pdf
Limited research exists on the fertility requirements for industrial hemp (Cannabis sativa) and the impact of fertility on plant growth and cannabinoids. Optimizing floral production for cannabinoid production and especially cannabidiol (CBD) production, is an economic goal for growers. Phosphorus (P) is an essential nutrient for plant growth and plays many key roles in plant growth and when deficient leads to suboptimal plant growth. Grower P fertility rate recommendations vary greatly, with suggestions of up to 196 mg·L-1 P over part of the production cycle. Four P fertility rates (15, 60, 120, and 180 mg·L-1 P) were evaluated to determine the optimal fertility for C. sativa on a high CBD-type cultivar ‘BaOx’. Plant height, diameter, and total biomass were similar across all examined P fertility rates. Foliar P concentrations increased linearly, with the greatest P accumulation occurring in plants that received the highest fertility rate of 180 mg·L-1 P. Given no differences in biomass production were found, and the luxury uptake of P as fertilization rates increased, the results indicate that rates above 15 mg·L-1 P are not beneficial for plant growth and only add economic cost to the grower and potentially cause waste and pollution to the environment.

Emerging challenges in the extraction, analysis and bioanalysis of cannabidiol and related compounds.
Virginia, B., Protti, M., Lisa, A., Chiara, Z., Laura, M., & Federica, P.
Journal of Pharmaceutical and Biomedical Analysis, 113633.(2020).
doi:10.1016/j.jpba.2020.113633
Cannabidiol (CBD) is a bioactive terpenophenolic compound isolated from Cannabis sativa L. It is known to possess several properties of pharmaceutical interest, such as antioxidant, antiinflammatory,
anti-microbial, neuroprotective and anti-convulsant, being it active as a multi-target compound. From a therapeutic point of view, CBD is most commonly used for seizure disorder in children. CBD is present in both medical and fiber-type C. sativa plants, but, unlike ?9 tetrahydrocannabinol (THC), it is a non-psychoactive compound. Non psychoactive or fiber-type C. sativa (also known as hemp) differs from the medical one, since it contains only low levels of THC and high levels of
CBD and related non-psychoactive cannabinoids. In addition to medical Cannabis, which is used for many different therapeutic purposes, a great expansion of the market of hemp plant material and related products has been observed in recent years, due to its usage in many fields, including food, cosmetics and electronic cigarettes liquids (commonly known as e-liquids). In this view, this work is focused on recent advances on sample preparation strategies and analytical methods for the chemical analysis of CBD and related compounds in both C. sativa plant material, its derived products and biological samples. Since sample preparation is considered to be a crucial step in the development of reliable analytical methods for the determination of natural compounds in complex matrices, different extraction methods are discussed. As regards the analysis of CBD and related compounds, the application of both separation and non-separation methods is discussed in detail. The advantages, disadvantages and applicability of the different methodologies currently available are evaluated. The scientific interest in the development of portable devices for the reliable analysis of CBD in vegetable and biological samples is also highlighted.

Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis.
Nardo, F. D., Cavalera, S., Baggiani, C., Chiarello, M., Pazzi, M., & Anfossi, L.
Toxins, 12(4), 265. (2020).
doi:10.3390/toxins12040265
The diffusion of the legalization of cannabis for recreational, medicinal and nutraceutical uses requires the development of adequate analytical methods to assure the safety and security of such products. In particular, aflatoxins are considered to pose a major risk for the health of cannabis
consumers. Among analytical methods that allows for adequate monitoring of food safety, immunoassays play a major role thanks to their cost-effectiveness, high-throughput capacity, simplicity and limited requirement for equipment and skilled operators. Therefore, a rapid and
sensitive enzyme immunoassay has been adapted to measure the most hazardous aflatoxin B1 in cannabis products. The assay was acceptably accurate (recovery rate: 78–136%), reproducible (intraand inter-assay means coefficients of variation 11.8% and 13.8%, respectively), and sensitive (limit of detection and range of quantification: 0.35 ng mL?1 and 0.4–2 ng mL?1, respectively corresponding to 7 ng g?1 and 8–40 ng g-1 ng g?1 in the plant) and provided results which agreed with a HPLCMS/MS method for the direct analysis of aflatoxin B1 in cannabis inflorescence and leaves. In addition, the carcinogenic aflatoxin B1 was detected in 50% of the cannabis products analyzed (14 samples collected from small retails) at levels exceeding those admitted by the European Union in commodities intended for direct human consumption, thus envisaging the need for effective surveillance of aflatoxin contamination in legal cannabis.

Evaluating the effects of gamma-irradiation for decontamination of medicinal cannabis
Arno Hazekamp
Front. Pharmacol. 7:108.
doi:10.3389/fphar.2016.00108
In several countries with a National medicinal cannabis program, pharmaceutical regulations specify that herbal cannabis products must adhere to strict safety standards regarding microbial contamination. Treatment by gamma irradiation currently seems the only method available to meet these requirements. We evaluated the effects of irradiation treatment of four different cannabis varieties covering different chemical compositions. Samples were compared before and after standard gamma-irradiation treatment by performing quantitative HPLC analysis of major cannabinoids, as well as qualitative GC analysis of full cannabinoid and terpene profiles. In addition, water content and microscopic appearance of the cannabis flowers was evaluated. This study found that treatment did not cause changes in the content of THC and CBD, generally considered as the most important therapeutically active components of medicinal cannabis. Likewise, the water content and the microscopic structure of the dried cannabis flowers were not altered by standard irradiation protocol in the cannabis varieties studied. The effect of gamma-irradiation was limited to a reduction of some terpenes present in the cannabis, but keeping the terpene profile qualitatively the same. Based on the results presented in this report, gamma irradiation of herbal cannabis remains the recommended method of decontamination, at least until other more generally accepted methods have been developed and validated. Because medicinal cannabis is often used by chronically ill patients affected by a weakened immune system, pharmaceutical regulations in countries such as The Netherlands and Canada specify that these products must adhere to strict safety standards regarding microbial contamination. When harmful microbes or fungal spores are inhaled during e.g. vaporizing or smoking, they may directly enter the bloodstream and cause opportunistic infections. Such contamination risks are not merely hypothetical: cases of chronic pulmonary aspergillosis associated with smoking unsafe cannabis are well established in the scientific literature (Ruchlemer et al. 2015; Bal et al. 2010; Cescon et al. 2008; Kouevidjin et al. 2003; Szyper-Kravitz et al. 2001; Marks et al. 1996; Sutton et al. 1986; Llamas et al. 1978). For those with compromised immune systems, such lung diseases could be even fatal (Hamadeh et al. 1988).

Evaluation of Cannabinoid and Terpenoid Content: Cannabis Flower Compared to Supercritical CO2 Concentrate.
Michelle Sexton, Kyle Shelton, Pam Haley, Mike West
Planta Med
DOI: 10.1055/s-0043-119361
A recent cannabis use survey revealed that 60% of cannabis users rely on smelling the flower to select their cannabis. Olfactory indicators in plants include volatile compounds, principally represented by the terpenoid fraction. Currently, medicinal- and adult-use cannabis is marketed in the United States with relatively little differentiation between products other than by a common name, association with a species type, and ?-9 tetrahydrocannabinol/cannabidiol potency. Because of this practice, how terpenoid compositions may change during an extraction process is widely overlooked.
Here we report on a comparative study of terpenoid and cannabinoid potencies of flower and supercritical fluid CO2 (SC CO2) extract from six cannabis chemovars grown in Washington State. To enable this comparison, we employed a validated high-performance liquid chromatography/diode array detector methodology for quantification of seven cannabinoids and developed an internal gas chromatography-mass spectrometry method for quantification of 42 terpenes. The relative potencies of terpenoids and cannabinoids in flower versus concentrate were significantly different. Cannabinoid potency increased by factors of 3.2 for ?-9 tetrahydrocannabinoland 4.0 for cannabidiol in concentrates compared to flower. Monoterpenes were lost in the extraction process; a ketone increased by 2.2; an ether by 2.7; monoterpene alcohols by 5.3, 7 and 9.4; and sesquiterpenes by 5.1, 4.2, 7.7, and 8.9. Our results demonstrate that the product of SC CO2 extraction may have a significantly different chemotypic fingerprint from that of cannabis flower. These results highlight the need for more complete characterization of cannabis and associated products, beyond cannabinoid content, in order to further understand health-related consequences of inhaling or ingesting concentrated forms.

Evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil by ultra-high performance liquid chromatography tandem mass spectrometry
Roberta Pacifici, Emilia Marchei, Francesco Salvatore, Luca Guandalini, Francesco Paolo Busard and Simona Pichini
Clinical Chemistry and Laboratory Medicine 55(10) 2017
DOI 10.1515/cclm-2016-1060
Background: Cannabis has been used since ancient times to relieve neuropathic pain, to lower intraocular pressure, to increase appetite and finally to decrease nausea and vomiting. The combination of the psychoactive cannabis alkaloid ?9-tetrahydrocannabinol (THC) with the nonpsychotropic
alkaloids cannabidiol (CBD) and cannabinol (CBN) demonstrated a higher activity than THC alone. The Italian National Institute of Health sought to establish conditions and indications on how to correctly use nationally produced cannabis to guarantee therapeutic continuity in individuals treated with medical cannabis.
Methods: The evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil was conducted using an easy and fast ultra-high performance liquid chromatography tandem
mass spectrometry (UHPLC-MS/MS) assay.
Results: Extraction efficiency of oil was significantly higher than that of water with respect to the different cannabinoids. This was especially observed in the case of the pharmacologically active THC, CBD and their acidic precursors. Fifteen minutes boiling was sufficient to achieve the highest concentrations of cannabinoids in the cannabis tea solutions. At ambient temperature, a significant THC and CBD decrease to 50% or less of the initial concentration was observed over 3 and 7 days, respectively. When refrigerated at 4 ÅãC, similar decreasing profiles were observed for the two compounds. The cannabinoids profile in cannabis oil obtained after preheating the flowering tops at 145 ÅãC for 30 min in a static oven resulted in a complete decarboxylation of cannabinoid acids CBDA and THCA-A. Nevertheless, it was apparent that heat not only decarboxylated acidic compounds, but also significantly increased the final concentrations of cannabinoids in oil. The stability of cannabinoids in oil samples was higher than that in tea samples since the maximum decrease (72% of initial concentration) was observed in THC coming from unheated flowering tops at ambient temperature. In the case of the other cannabinoids, at ambient and refrigerated temperatures, 80%– 85% of the initial concentrations were measured up to 14 days after oil preparation.
Conclusions: As the first and most important aim of the different cannabis preparations is to guarantee therapeutic continuity in treated individuals, a strictly standardized preparation protocol is necessary to assure the availability of a homogeneous product of defined stability.

Evaluation of elemental profiling methods, including laser-induced breakdown spectroscopy (LIBS), for the differentiationof Cannabis plant material grown in different nutrient solutions
Moteaa M. El-Deftara, , , James Robertsona, , Simon Fosterb, , Chris Lennardc,
Forensic Science International Volume 251, June 2015, Pages 95–106
Doi: 10.1016/j.forsciint.2015.03.029
Application of LIBS, ICP-MS, LA-ICP-MS and ?XRF for the analysis of Cannabis plant.

Discrimination of Cannabis is evaluated based on their elemental composition data.

It is possible to correctly associate Cannabis with a particular nutrient.

ICP-MS, LA-ICP-MS and LIBS are suitable methods for the analysis of Cannabis plant.
Abstract
Laser-induced breakdown spectroscopy (LIBS) is an emerging atomic emission based solid sampling technique that has many potential forensic applications. In this study, the analytical performance of LIBS, as well as that of inductively coupled plasma mass spectrometry (ICP-MS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray microfluorescence (?XRF), was evaluated for the ability to conduct elemental analyses on Cannabis plant material, with a specific investigation of the possible links between hydroponic nutrients and elemental profiles from associated plant material. No such study has been previously published in the literature. Good correlation among the four techniques was observed when the concentrations or peak areas of the elements of interest were monitored. For Cannabis samples collected at the same growth time, the elemental profiles could be related to the use of particular commercial nutrients. In addition, the study demonstrated that ICP-MS, LA-ICP-MS and LIBS are suitable techniques for the comparison of Cannabis samples from different sources, with high discriminating powers being achieved. On the other hand, ?XRF method was not suitable for the discrimination of Cannabis samples originating from different growth nutrients.

Evaluation of Pesticides Found in Oregon Cannabis from 2016 to 2017
Richard Evoy, and Laurel Kincl
Annals of Work Exposures and Health, 2019, 1–5
doi: 10.1093/annweh/wxz075
Pesticide use and worker exposures to pesticides in the US cannabis industry have not been studied due to cannabis being illegal at the federal level. Without knowing the types of pesticides being used in this industry, it is difficult to assess whether workers are being exposed to potentially dangerous pesticides. When recreational cannabis became legal in the state of Oregon in 2014, the Oregon Department of Agriculture (ODA) published a list of recommended pesticides for use in the cannabis industry and developed the state's pesticide testing regulations for all cannabis products, medicinal and recreational. Using the state's pesticide testing data, the aim of this study was to investigate the types of pesticides being used in the Oregon cannabis industry and if they present a hazard to cannabis workers. Both recreational and medicinal cannabis samples contained high levels of residual pesticides and pesticides not legally allowed to be used on cannabis products. Medicinal cannabis products were found to have mean levels of residual pesticides that were 3–12 times higher than recreational products. Nine of the 50 pesticides identified were classified highly or extremely hazardous by the World Health Organization.

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes
Oier Aizpurua-Olaizola, Umut Soydaner, Ekin Öztürk, Daniele Schibano, Yilmaz Simsir, Patricia Navarro, Nestor Etxebarria, and Aresatz Usobiaga
J. Nat. Prod., Article ASAP
DOI: 10.1021/acs.jnatprod.5b00949
Publication Date (Web): February 2, 2016
The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one fromchemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

Extraction of cannabinoids from hemp (Cannabis sativa L.) using high pressure solvents: an overview of different processing options.
Moreno, T., Montanes, F., Tallon, S. J., Fenton, T., & King, J. W.
The Journal of Supercritical Fluids, 104850. (2020).
doi:10.1016/j.supflu.2020.104850
Hemp varieties of Cannabis Sativa L. contain low levels of 9-tetrahydrocannabinol (THC) and can be used to produce therapeutic extracts rich in cannabidiol (CBD). In this work, extracts containing up to 449 mg/g CBD were obtained from New Zealand industrial hemp varieties by extraction of flower buds with supercritical CO2. The composition of the extracts and the influence of different processing parameters (extraction pressure up to 1300 bar, use of ethanol co-solvent, decarboxylation of feed) were determined. The apparent solubility ofthe extractin CO2 at different pressures was measured. Extractions using near-critical propane and dimethyl-ether were also performed. Total extraction yields reached 12.0 wt% with CO2 and 8.2 wt% with propane, whereas total cannabinoid yield ranged from 51 to 100 % with CO2 and from 74 to 99 wt% with propane. Addition of 5 % ethanol co-solvent enhances the extraction of cannabinoid acid forms, as does an increase in extraction pressure.

Fast and Sensitive Quantification of ?9-Tetrahydrocannabinol and Its Main Oxidative Metabolites by Liquid Chromatography/Tandem Mass Spectrometry.
Vozella, V., Zibardi, C., Ahmed, F., & Piomelli, D.
Cannabis and Cannabinoid Research. (2019).
doi:10.1089/can.2018.0075
Introduction: Few animal studies have evaluated the pharmacological effects of D9-tetrahydrocannabinol (THC) in relation to its pharmacokinetic properties. Understanding this relationship is essential, however, if comparisons are to be drawn across conditions—such as sex, age, and route of administration—which are associated with variations in the absorption, metabolism, and distribution of THC. As a first step toward addressing this gap, in this report, we describe a rapid, sensitive, and accurate method for the quantification of THC and its main oxidative metabolites, and apply it to representative rodent tissues.
Materials and Methods: The sample workup procedure consisted of two steps: bulk protein precipitation with cold acetonitrile (ACN) followed by phospholipid removal by elution through Captiva-Enhanced Matrix Removal cartridges. The liquid chromatography/tandem mass spectrometry (LC/MS-MS) protocol utilized a commercially available C18 reversed-phase column and a simple methanol/water gradient system. The new method was validated following Food and Drug Administration (FDA) guidelines, and was applied to the quantification of THC and its main oxidative metabolites—11-hydroxy-D9 -tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxyD9 -tetrahydrocannabinol (11-COOH-THC)—in plasma and brain of mice treated with a single intraperitoneal
dose of THC (10 mg/kg).
Results: ACN precipitation and column elution effectively depleted matrix constituents—most notably cholinecontaining phospholipids—which are known to interfere with THC analysis, with average recovery values of >85% for plasma and >80% for brain. The LC conditions yielded baseline separation of all analytes in a total run time of 7 min (including re-equilibration). The 10–point calibration curves showed excellent linearity (R2 > 0.99) over a wide range of concentrations (1–1000 pmol/100 lL). Lowest limit of quantification was 2 pmol/100 lL for all analytes, and lowest limits of detection were 0.5 pmol/100 lL for THC and 11-OH-THC, and 1 pmol/100 lL for 11-COOH-THC. Intraday and interday accuracy and precision values were within the FDA-recommended range (–15% of nominal concentration). An application of the method to adult male mice is presented. Conclusions: We present a fast and sensitive method for the analysis of THC, which should facilitate
studies aimed at linking the pharmacokinetics and pharmacodynamics of this compound in animal models.

First systematic evaluation of the potency of Cannabis sativa plants grown in Albania.
Bruci, Z., Papoutsis, I., Athanaselis, S., Nikolaou, P., Pazari, E., Spiliopoulou, C., & Vyshka, G.
Forensic Science International, 222(1-3), 40–46. (2012).
doi:10.1016/j.forsciint.2012.04.032
Cannabis products (marijuana, hashish, cannabis oil) are the most frequently abused illegal substances worldwide. Delta-9-tetrahydrocannabinol (THC) is the main psychoactive component of Cannabis sativa plant, whereas cannabidiol (CBD) and cannabinol (CBN) are other major but no psychoactive constituents. Many studies have already been carried out on these compounds and chemical research was encouraged due to the legal implications concerning the misuse of marijuana. The aim of this study was to determine THC, CBD and CBN in a significant number of cannabis samples of Albanian origin, where cannabis is the most frequently used drug of abuse, in order to evaluate and classify them according to their cannabinoid composition. A GC–MS method was used, in order to assay cannabinoid content of hemp samples harvested at different maturation degree levels during the summer months and grown in different areas of Albania. This method can also be used for the determination of plant phenotype, the evaluation of psychoactive potency and the control of material quality. The highest cannabinoid concentrations were found in the flowers of cannabis. The THC concentrations in different locations of Albania ranged from 1.07 to 12.13%. The influence of environmental conditions on cannabinoid content is discussed. The cannabinoid content of cannabis plants were used for their profiling, and it was used for their classification, according to their geographical origin. The determined concentrations justify the fact that Albania is an area where cannabis is extensively cultivated for illegal purposes.

Flavanone synthase: simple and rapid assay for the key enzyme of flavonoid biosynthesis
Joachim Dipl Ing Schroeder, Werner Heller, Klaus Hahlbrock
Published 1979
Plant Science Letters Volume 14, Issue 3, March 1979, Pages 281-286
DOI:10.1016/0304-4211(79)90083-X
It is suggested that flavanone synthase activity should be measured when the key reaction of flavonoid biosynthesis is to be tested. A simple and rapid procedure for the determination of flavanone synthase activity, based on extraction of the 14 C-labelled product(s) into ethylacetate, is described. The enzyme can be stored under appropriate conditions for several weeks without significant loss of activity. Results obtained with cell suspension cultures of parsley indicate that the activity of flavanone synthase is regulated differently from the activity of phenylalanine ammonia-lyase, an enzyme frequently referred to as a key enzyme of flavonoid biosynthesis

Flavonoid variation in Cannabis L.
M. N. Clark, B. A. Bohm
Boyanical Journal of the Linnean Society, Volume 79, Issue 3, October 1979, Pages 249-257
doi: 10.1111/j.1095-8339.1979.tb01517.x
Fifty- three individual plants of Cannabis sativa L., grown from seed obtained from nine countries, were analyzed for their flavonoid constituents. Compounds present were the C-glycoflavones vitexin, orientin, cytisoside, and an 0"-glucoside of each. A C-glycuronide of luteolin was detected in srvrral plants. Karmpferol and quercetin-3-0-diglucosides were also seen in many specimens. The distribution of compounds supported the subspecific treatment of the genus suggested by Small & Crouquist i 1976). The data were less helpful in resolving trends amongst the varieties recognized by those authors. A considerable amount of plant to plant variation was observed.

Forensic laboratory backlog: The impact of inconclusive results of marijuana analysis and the implication on analytical routine
Ana Flavia Belchior de Andrade, Lívia Barros Salum, Ettore Ferrari Júnior
Science & Justice Volume 61, Issue 6, November 2021, Pages 755-760
DOI: 10.1016/j.scijus.2021.09.005
https://www.sciencedirect.com/scienc...26X?via%3Dihub
Forensic laboratories worldwide are struggling to keep up with the increasing number of cases submitted for analysis, regardless of the reasons, backlog of controlled substances cases is a reality in many countries. In this paper we analyse the number of petitioned examinations (from 2016 to 2020) and the data from 11,655 marijuana TLC results from the Forensic Laboratory in the Federal District Civil Police in Brazil. Data demonstrates that backlog increases inconclusive results, with storage and light playing a crucial role in the process. Additionally we explored the repercussions of delayed forensic results for controlled substances and propose an approach to overcome waiting time in this context.
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Free and Glucuronide Urine Cannabinoids after Controlled Smoked, Vaporized, and Oral Cannabis Administration in Frequent and Occasional Cannabis Users.
Huestis, M. A., Sempio, C., Newmeyer, M. N., Andersson, M., Barnes, A. J., Abulseoud, O. A., … Smith, M. L.
Journal of Analytical Toxicology.(2020).
doi:10.1093/jat/bkaa046
BACKGROUND: Total urinary 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) concentrations are generally reported following cannabis administration. Few data are available for glucuronide and minor cannabinoid metabolite concentrations.
METHODS: All urine specimens from 11 frequent and 9 occasional cannabis users were analyzed for 11 cannabinoids for up to 85 h by LC–MS-MS following controlled smoked, vaporized or oral 50.6 mg ? 9 - tetrahydrocannabinol (THC) in a randomized, placebo-controlled, within-subject dosing design.
RESULTS: No cannabidiol, cannabinol, cannabigerol, tetrahydrocannabivarin (THCV), THC, 11-OH-THC, ? 9 -tetrahydrocannabinolic acid were detected in urine. Median THCCOOH-glucuronide maximum concentrations (Cmax) following smoked, vaporized and oral routes were 68.0, 26.7 and 360 ?g/L for occasional and 378, 248 and 485 ?g/L for frequent users, respectively. Median time to specific-gravity normalized Cmax (Tmax) was 5.1–7.9 h for all routes and all users. Median Cmax for THCCOOH, THCglucuronide and 11-nor-9-carboxy-? 9 -THCV (THCVCOOH) were less than 7.5% of THCCOOHglucuronide Cmax concentrations. Only THC-glucuronide mean Tmax differed between routes and groups, and was often present only in occasional users’ first urine void. Multiple THCCOOH-glucuronide and THCCOOH peaks were observed. We also evaluated these urinary data with published models for determining recency of cannabis use.
CONCLUSIONS: These urinary cannabinoid marker concentrations from occasional and frequent cannabis users following three routes of administration provide a scientific database to assess single urine concentrations in cannabis monitoring programs. New target analytes (CBD, CBN, CBG, THCV and phase II metabolites) were not found in urine. The results are important to officials in drug treatment, workplace and criminal justice drug monitoring programs, as well as policy makers with responsibility for cannabis regulations.

GCMS analysis of Cannabis sativa L. from four different areas of Pakistan
Muhammad Tayyab, , Durre Shahwar
Egyptian Journal of Forensic Sciences
Doi: 10.1016/j.ejfs.2014.07.008
Cannabis is most frequently used drug of abuse not only in Pakistan but also in the whole world. Its use is mounting drastically every year. GCMS allows analysis of Cannabis sativa which shows divergence of the constituents of this plant. Prevalence of this plant can be identified through knowledge of its constituents. In this way we can obstruct the production if we know the region in which it is produced. GCMS is a useful technique for the comparison of constituents of this drug of abuse which will assist the investigator concerning the origin of plant. Comparison also aids in the understanding and acquaintance of similarities of different samples of cannabinoids.

GC-MS Analysis of the Total THC Content of Both Drug- and Fiber-Type Cannabis Seeds
Samir A. Ross, Zlatko Mehmedic, Timothy P. Murphy, and Mahmoud A. EISohly
Journal of Analytical Toxicology, Vol. 24, November/December 2000
DOI: 10.1093/jat/24.8.715
A GC-MS method was performed to determine the total delta9-THC content in both drug- and fiber-type cannabis seeds. Drug-type seeds were found to contain much higher levels of delta9-THC (35.6-124 microg/g) than fiber (hemp) seeds (0-12 microg/g). The majority of delta9-THC was found to be located on the surface of the seeds. Approximately 90% of the total delta9-THC was removed by a simple, quick wash with chloroform. Washed drug-type seeds contained less than 10 microg/g. Separation of the seeds into the kernel and testa showed that the bulk of delta9-THC is located in the testa, mainly on the outside. The kernels of drug- and fiber-type cannabis seeds contained less than 2 and 0.5 microg delta9-THC/g seeds, respectively. Fluctuations in the delta9-THC content of different replicates of the same type of seeds could be the result of the degree of contamination on the outside of the seeds.

Green extraction of cannabidiol from industrial hemp (Cannabis sativa L.) using deep eutectic solvents coupled with further enrichment and recovery by macroporous resin
Changyong Cai, Wang Yu, Chaoyun Wang, Lianglei Liu, Fenfang Li, Zhijian Tan
Journal of Molecuar liquids Volume 287, 1 August 2019, 110957
DOI: 10.1016/j.molliq.2019.110957
Deep eutectic solvents (DESs) are good alternatives to conventional organic solvents and ionic liquids (ILs) in the extraction and separation field. In this study, DESs were developed as the media for the green extraction of cannabidiol (CBD) from industrial hemp (Cannabis sativa L.). The factors influencing the extraction, including the type and concentration of DESs, solid-liquid ratio, extraction temperature, extraction time, and system pH were investigated in the single factor experiments. Then, the major factors of DESs concentration, solid-liquid ratio, extraction temperature, and extraction time were optimized by response surface methodology (RSM). Under the optimized conditions, the highest extraction yield for CBD was 12.22mg/g. The Kamlet-Taft polarity parameters were studied to analyze the effect of polarity and hydrogen-bond ability to the extractability of DESs. The extraction kinetics was also studied and the kinetics parameters were analyzed. CBD was further enriched and recovered from the crude extract by macroporous resins, and the recovery yield and the purity reached 81.46% and 28.93%, respectively. This extraction of CBD using DESs has the advantages of high extraction yield, simple operation, low cost and good environmental friendliness, which is potential for the scale-up production.

Handbook of phytochemical constituents of GRAS herbs and other economic plants.
Duke, James A.
1992 Database
https://phytochem.nal.usda.gov/phyto...320&max=20&et=
Cannabis Terpenes, Cannabioids, and other compounds found in Cannabis, Cannabis seeds, roots, pollen.

Hashish.1 A Simple One-Step Synthesis of ( —) Delta 1 Tetrahydrocannabinol (THC) from p-Mentha-2,8-dien-1 -ol and Olivetol
Raj K. Razdan, Haldean C. Dalzell, and G. Richard Handrick Journal of the American Chemical Society 96:18 Sept 4, 1974 DOI: 10.1021/ja00825a026
Optically pure (—j-A+THC (7) was produced in 50% yield (glc; isolated yield 31%) in a single-step synthesis from dí/rra/ií-(+)-p-mentha-2,8-dien-l-ol (1) and olivetol (2) in the presence of 1% boron trifluoride etherate and anhydrous magnesium sulfate in methylene chloride at 0°. The product was readily separated by column chromatography. The other major product formed was /r««s-A8-iso-THC (8). By the same procedure (—)-cannabidiol (3) was obtained on a preparative scale when <0.5% boron trifluoride etherate or wet ^-toluenesulfonic acid was used. A mechanistic scheme is presented for this reaction. It is shown that cannabidiols (3 and 4) are the key intermediates in this reaction and abnormal cannabidiol (4) undergoes a retro-Friedel-Crafts reaction followed by recombination to normal cannabidiol (3). This retroreaction of 4 is rationalized on steric arguments. The isolation and study of products from this reaction give a much clearer understanding of the factors which control the outcome of acid-catalyzed reactions of p-mentha-2,8-dien-l-ol and olivetol and have provided three new cannabinoids, 9,10, and 12.

Hemp seed (Cannabis sativa L.) enriched pasta: Physicochemical properties and quality evaluation
Dorota Teterycz, Aldona Sobota, Dominika Przygodzka, Paulina Łysakowska
Plos One March 2021
DOI: 10.1371/journal.pone.0248790
https://journals.plos.org/plosone/ar...8790&utm_sourc e=The+Cannabis+%2B+Cannabinoid +Curator&utm_campaign=cd2a62ba 57-EMAIL_CAMPAIGN_2020_03_13_07_5 9_COPY_01&utm_medium=email&utm _term=0_47f6c7c552-cd2a62ba57-365750754
Hemp seed (Cannabis sativa L.) contain large amounts of nutrients, e.g. protein, dietary fiber, minerals, and unsaturated fatty acids, which make them a good fortifying component in food production. The aim of the present study was to determine the effect of hemp addition on the physicochemical properties, cooking quality, texture parameters and sensory properties of durum wheat pasta. The samples were fortified with 5–40% of commercially available hemp flour or 2.5–10% of hemp cake obtained from hemp seed oil pressing. Our study showed that the addition of hemp seed raw materials led to an increase in the protein, total dietary fiber (TDF), ash and fat content in the pasta samples. Due to its lower granulation and higher nutritional value, hemp flour was found to be a better raw material for the fortification of pasta than hemp cake. Pasta enriched with hemp flour at the level of 30–40% contains 19.53–28.87% d.m. of protein and 17.02–21.49% d.m. of TDF and according to the EU, a definition can be described as a high-protein and high-fiber products. All enriched pasta samples were also characterized by safe Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) content, and their sensory properties were accepted by consumers.

Highly sensitive screening and analytical characterization of synthetic cannabinoids in nine different herbal mixtures.
Alves, V.L., Gonçalves, J.L., Aguiar, J. et al.
Anal Bioanal Chem (2021).
DOI: https://doi.org/10.1007/s00216-021-03199-6
The popularity of new psychoactive substances among drug users has become a public health concern worldwide. Among them, synthetic cannabinoids (SCs) represent the largest, most diversified and fastest growing group. Commonly known as ‘synthetic marijuana’ as an alternative to cannabis, these synthetic compounds are easily accessible via the internet and are sold as ‘herbal incenses’ under different brand names with no information about the chemical composition. In the present work, we aim to integrate gas chromatography-tandem mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) data as useful strategy for the identification and confirmation of synthetic cannabinoids present in nine seized herbal incenses. The analysis of all samples allowed the initial identification of 9 SCs, namely 5 napthoylindoles (JWH-018, JWH-073, JWH-122, JWH-210, MAM-2201), APINACA, XLR-11 and CP47,497-C8 and its enantiomer. JWH-018 was the most frequently detected synthetic compound (8 of 9 samples), while APINACA and XLR-11 were only identified in one herbal product. Other non-cannabinoid drugs, including oleamide, vitamin E and vitamin E acetate, have also been detected. Oleamide and vitamin E are two adulterants, frequently added to herbal products to mask the active ingredients or added as preservatives. However, to our knowledge, no analytical data about vitamin E acetate was reported in herbal products, being the first time that this compound is identified on this type of samples. The integration data obtained from the used analytical technologies proved to be useful, allowing the preliminary identification of the different SCs in the mixture. Furthermore, the examination of mass spectral fragment ions, as well as the results of both 1D and 2D NMR experiments, enabled the identification and confirmation of the molecular structure of SCs.

1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification
Wieland Peschel, Matteo Poli
Talanta Volume 140, 1 August 2015, Pages 150–165
doi: 10.1016/j.talanta.2015.02.040

1H NMR (DMSO-d6) key signals allow distinction of THC, CBD and CBG type cannabis.

We detect neutral/acidic cannabinoids and phenolics co-compounds via HPLC/DAD.

Non-heated extract profiles suggest consideration of cannabinoid acids.

Cell viability reduction correlated with the total cannabinoid content but not THC.

We suggest markers for drug distinction and pharmaceutical specification.
Abstract
The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ?9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of 1H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide together with two new validated HPLC/DAD methods used for identification and extract profiling based on the main pattern of cannabinoids and other phenolics alongside the assayed content ofTHC, cannabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid pairs allowed chemotyperecognition via assignment of selective proton signals and via HPLC even in cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid acids in non-heated extracts suggest their consideration for total values in chemotype distinction and specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile. Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2–4 lead cannabinoids are considered essential. The suitability of analytical methods and the range of compound groups summarized in group and ratio markers are discussed regarding plant classification and pharmaceutical specification.

HPLC Method to Differentiate Four THC Stereoisomers: (6aR,9R)-Δ10-THC, (6aR,9S)-Δ10-THC, 9(R)-Δ6a,10a-THC, and 9(S)-Δ6a,10a-THC
Jeffrey B. Williams, Kathleen B. Calati, Kirk W. Hering, Roxanne E. Franckowski, and Donna M. Iula.
Cayman Chemical
https://thecannabisscientist.com/fil...rs_2021MAR.pdf
· Δ10-THC and Δ6a,10a-THC isomers produced from Δ9-THC degradation have stereochemical similarities that are challenging to differentiate.
· Traditional reversed-phase (C18) HPLC analysis is not suitable for accurate determination of these isomers.
· A method using a chiral HPLC column under normal-phase conditions offers reliable and robust identification of the four stereoisomers.
· Use of a chiral HPLC stationary phase for characterization of these phytocannabinoid degradants is an essential tool for the determination of potency and safety
As the primary phytocannabinoid associated with psychoactive properties, Δ9-tetrahydrocannabinol (Δ9-THC) is crucial to analytical Cannabis testing. Degradation of Δ9-THC, from the processing of inflorescences for Cannabis isolates or other products, may lead to isomers that can be misidentified and provide invalid potency claims (Figure 1). Δ10-THC* and Δ6a,10a-THC† isomers are of particular interest because of their stereochemical similarities, which makes traditional reversed-phase liquid chromatography (RPLC) analysis unreliable. Under strongly alkaline conditions Δ9 -THC is known to isomerize to two diastereomers, (6aR,9S)-Δ10-THC and (6aR,9R)-Δ10-THC.‡ These two distinct stereoisomers may undergo additional isomerization, even under mildly acidic conditions to form a pair of enantiomers, with the (6aR,9S)-Δ10-THC providing the 9(S)-Δ6a,10a-THC, and (6aR,9R)-Δ10-THC providing the opposite enantiomer, 9(R)-Δ6a,10a-THC. Only the 9(S)-Δ6a,10a-THC isomer has been found to have psychoactive effects in animals, similar to Δ9-THC.§

HRMS Detector for the New HILIC CBD Method Development in Hemp Seed Oil.
Wang, Y., Hao, Z., & Pan, L.
Journal of the American Society for Mass Spectrometry.(2020).
doi:10.1021/jasms.0c00331
The cannabis analysis has gained new importance worldwide due to the rapid expansion of the hemp global market. Many reverse phase high performance liquid chromatography (HPLC) methods have been developed to analyze cannabidiol (CBD) and its analogues due to the nice fit with their log P values at around 6. However, when CBD was blended with hemp seed oil in consumer, cosmetic, and food industries, the high content of triacylglycerides (TAGs) from hemp seed oil were retained and accumulated inside C18 columns with the common mobile phases and caused a column pressure increase and ghost peaks after continuous sample injections. Coupled with the chemical profile from high resolution mass spectrometry (HRMS) detection, a novel hydrophilic interaction liquid chromatography (HILIC) CBD quantitation method was developed, specifically for CBD blended hemp seed oil materials, which can overcome the matrix buildup in reverse phase columns. The zwitterionic (ZIC)-cHILIC column containing a dense water layer on the stationary phase surface provided a stable partitioning separation mechanism to separate the CBD from TAGs in hemp seed oil. This water layer favors the sustaining NH4F buffer ions, which can maximize the salting-out action and help reduce the adsorptive interaction between TAGs and stationary phase sulfobetaine materials. The high percentage of acetonitrile (99%) contributed to method sensitivity and reduced instrument maintenance time. The method was developed and validated for the first time. It has been successfully applied to quantify CBD content in hemp seed oil samples, thus demonstrating it to be a useful tool for both quality control and safety assurance in CBD hemp seed oil raw materials and related products

Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography-mass spectrometry
Oier Aizpurua-Olaizola & Jone Omar & Patricia Navarro & Maitane Olivares & Nestor Etxebarria & Aresatz Usobiaga
Anal Bioanal Chem (2014) 406:7549–7560
DOI 10.1007/s00216-014-8177-x
High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to cannabis plant extracts in order to identify cannabinoid compounds after their quantitative isolation by means of supercritical fluid extraction (SFE). MS conditions were optimized by means of a central composite design (CCD) approach, and the analysis method was fully validated. Six major cannabinoids [tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabigerol (CBG), and cannabinol (CBN)] were quantified (RSD < 10%), and seven more cannabinoids were identified and verified by means of a liquid chromatograph coupled to a quadrupole-time-of-flight (Q-ToF) detector. Finally, based on the distribution of the analyzed cannabinoids in 30 Cannabis sativa L. plant varieties and the principal component analysis (PCA) of the resulting data, a clear difference was observed between outdoor and indoor grown plants, which was attributed to a higher concentration of THC, CBN, and CBD in outdoor grown plants.

Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol.
Linciano, P., Citti, C., Russo, F., Tolomeo, F., Laganà, A., Capriotti, A. L., … Cannazza, G.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-79042-2
The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and ?9 -tetrahydrocannabinol (?9 -THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and ?9 -THCB) and a heptyl-(in CBDP and ?9 -THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that flls the gap between the pentyl and heptyl homologs of CBD and ?9 -THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and ?9 -tetrahydrocannabihexol (?9 -THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and ?9 -THCH have been already identifed and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and ?9 -tetrahydrocannabinol monomethyl ether (?9-THCM). The unambiguously identifcation in cannabis extract of the n-hexyl homologues of CBD and ?9 -THC diferent from the corresponding methylated isomers (CBDM, CBGM and ?9 -THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantifcation of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of ?9 -THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confrmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and ?9 -THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware.
Angelini R, Argueta DA, Piomelli D, DiPatrizio NV.
Cannabis Cannabinoid Res. 2017 Jun 1;2(1):123-132.
doi: 10.1089/can.2017.0019.
Introduction: Fatty acid ethanolamides (FAEs) are a family of lipid mediators that participate in a host of biological functions. Procedures for the quantitative analysis of FAEs include organic solvent extraction from biological matrices (e.g., blood), followed by purification and subsequent quantitation by liquid chromatography-mass spectrometry (LC/MS) or gas chromatography-mass spectrometry. During the validation process of a new method for LC/MS analysis of FAEs in biological samples, we observed unusually high levels of the FAE, palmitoylethanolamide (PEA), in blank samples that did not contain any biological material. Materials and Methods: We investigated a possible source of this PEA artifact via liquid chromatography coupled to tandem mass spectrometry, as well as accurate mass analysis.Results: We found that high levels of a contaminant indistinguishable from PEA is present in new 5.75? glass Pasteur pipettes, which are routinely used by laboratories to carry out lipid extractions. This artifact might account for discrepancies found in the literature regarding PEA levels in human blood serum and other tissues. Conclusions: It is recommended to take into account this pitfall by analyzing potential contamination of the disposable glassware during the validation process of any method used for analysis of FAEs.

Identification of Cannabis sativa using morpho-anatomical and microscopic characteristics
Conference: XII Brazilian Symposium of Pharmacognosy and the XVII Latin American Symposium on Pharmacobotany At: Petrópolis, RJ, Brazil
Vijayasankar Raman, Hemant Lata, Jane M. Budel, Mahmoud A Elsohly,
https://www.researchgate.net/publica...haracteristics
Cannabis sativa L. (Cannabaceae), commonly known by various names such as cannabis, marijuana, grass, weed and pot, is a taxonomically complex species widely considered to be indigenous to Central Asia. Cannabis is one of the earliest domesticated plants in the history of mankind and has a long history of widespread cultivation hence the original distribution of the species is unclear. Three forms of plants, namely sativa, indica and ruderalis were recognized by different authors at varying taxonomic levels such as types/forms, varieties, subspecies or as three different species. However, it is currently widely accepted that the genus is represented by one highly polymorphic species, C. sativa

Identification of novel phytocannabinoids from Ganoderma by label-free dynamic mass redistribution assay.
Zhou, H., Peng, X., Hou, T., Zhao, N., Qiu, M., Zhang, X., & Liang, X. 112218.
Journal of Ethnopharmacology (2019)
doi:10.1016/j.jep.2019.112218
Ethnopharmacological relevance: Located throughout the body, cannabinoid receptors (CB1 and CB2) are therapeutic targets for obesity/metabolic diseases, neurological/mental disorders, and immune modulation. Phytocannabinoids are greatly important for the development of new medicines with high efficacy and/or minor side effects. Plants and fungi are used in traditional medicine for beneficial effects to mental and immune system. The current research studied five fungi from the genus Ganoderma and five plants: Ganoderma hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang; Ganoderma capense (Lloyd) Teng, Zhong Guo De Zhen Jun; Ganoderma cochlear (Blume & T. Nees) Bres., Hedwigia; Ganoderma resinaceum Boud.; Ganoderma applanatum (Pers.) Pat.; Carthamus tinctorius L. (Compositae); Cynanchum otophyllum C. K. Schneid. (Asclepiadaceae); Coffea arabica L. (Rubiaceae); Prinsepia utilis Royle (Rosaceae); Lepidium meyenii Walp. (Brassicaceae). They show immunoregulation, promotion of longevity and maintenance of vitality, stimulant effects on the central nervous system, hormone balance and other beneficial effects. However, it remains unclear whether cannabinoid receptors are involved in these effects. Aim of the study: This work aimed to identify components working on CB1 and CB2 from the above plants and fungi, as novel phytocannabinoids, and to investigate mechanisms of how these compounds affected the cells. By analyzing the structure-activity relationship, we could identify the core structure for future development. Materials and methods: Eighty-two natural compounds were screened on stably transfected Chinese hamster ovary (CHO) cell lines, CHO-CB1 and CHO-CB2, with application of a label-free dynamic mass redistribution (DMR) technology that measured cellular responses to compounds. CP55,940 and WIN55,212–2 were agonist probe molecules, and SR141716A and SR144528 were antagonist probes. Pertussis toxin, cholera toxin, LY294002 and U73122 were signaling pathway inhibitors. The DMR data were acquired by Epic Imager software (Corning, NY), processed by Imager Beta 3.7 (Corning), and analyzed by GraphPad Prism 6 (GraphPad Software, San Diego, CA). Results: Transfected CHO-CB1 and CHO-CB2 cell lines were established and characterized. Seven compounds induced responses/activities in the cells. Among the seven compounds, four were purified from two Ganoderma species with potencies between 20 and 35 μM. Three antagonists: Kfb68 antagonized both receptors with a better desensitizing effect on CB2 to WIN55,212–2 over CP55,940. Kga1 and Kfb28 were antagonists selective to CB1 and CB2, respectively. Kfb77 was a special agonist and it stimulated CB1 in a mechanism different from that of CP55,940. Another three active compounds, derived from the Lepidium meyenii Walp. (Brassicaceae), were also identified but their effects were mediated through mechanisms much related to the signaling transduction pathways, especially through the stimulatory Gs protein. Conclusions: We identified four natural cannabinoids that exhibited structural and functional diversities. Our work confirms the presence of active ingredients in the Ganoderma species to CB1 and CB2, and this finding establishes connections between the fungi and the cannabinoid receptors, which will serve as a starting point to connect their beneficial effects to the endocannabinoid system. This research will also enrich the inventory of cannabinoids and phytocannabinoids from fungi. Yet due to some limitations, further structure-activity relationship studies and mechanism investigation are warranted in future.

Identification of Phenotypic Characteristics in Three Chemotype Categories in the Genus Cannabis
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
HORTSCIENCE
DOI: 10.21273/HORTSCI15607-20
https://journals.ashs.org/hortsci/vi...e=The+Cannabis +%2B+Cannabinoid+Curator&utm_c ampaign=70b2ae2af2-EMAIL_CAMPAIGN_2020_03_13_07_5 9_COPY_01&utm_medium=email&utm _term=0_47f6c7c552-70b2ae2af2-365750754
Modern Cannabis cultivars are morphologically distinguished by their leaflet shapes (wide for “Indica” and narrow for “Sativa”) by users and breeders. However, there are no scientific bases or references for determining the shape of these leaflets. In addition, these two categories contained mostly THC dominant (high THC) cultivars while excluded CBD dominant (high CBD) and intermediate (intermediate level of both THC and CBD) cultivars. This study investigated the phenotypic variation in 21 Cannabis cultivars covering three chemical phenotypes, referred to as chemotypes, grown in a commercial greenhouse. Thirty morphological traits were measured in the vegetative, flowering, and harvest stages on live plants and harvested inflorescences. The collected data were subjected to correlation analysis, hierarchical clustering, principal component analysis, and canonical correlation analysis with preassigned chemotypes. Canonical correlation analysis assigned individual plants to their chemotypes with 92.9% accuracy. Significant morphological differences were identified. Traits usable as phenotype markers for CBD dominant cultivars included light-green and narrow leaflets, a greater number of primary and secondary serrations, loose inflorescences, dense and resinous trichomes, and Botrytis cinerea resistance. Traits for intermediate cultivars included deep-green and medium-wide leaflets, more primary and secondary serrations, medium compact inflorescences, trichomes that are less dense and less resinous, and Botrytis cinerea resistance. Traits for THC dominant cultivars included deep-green and wide leaflets, large and compact inflorescences, dense and resinous trichomes, and Botrytis cinerea susceptibility. The results of this study provide a comprehensive profile of morphological traits of modern Cannabis cultivars and provides the first such profile for CBD dominant and intermediate cultivars. Additionally, this study included the traits of inflorescences, which have not been compared between three chemotypes in the literature. Phenotype markers identified in this study can facilitate preliminary cultivar identification and selection on live plants before or as a supplement to chemical and genetic analysis.

Identification of Psychoactive Metabolites from Cannabis sativa, Its Smoke, and Other Phytocannabinoids Using Machine Learning and Multivariate Methods.
Jagannathan, R.
ACS Omega. (2020).
doi:10.1021/acsomega.9b02663
Cannabis sativa is a medicinal plant having a very complex matrix composed of mainly cannabinoids and terpenoids. The literature has numerous reports, which indicate that tetrahydrocannabinol (THC) is the only major psychoactive metabolite in C. sativa. It is important to explore other metabolites having the possibility of exhibiting the psychoactive character of various degrees and also to identify metabolites targeting other receptors such as opioid, ? amino butyric acid (GABA), glycine, serotonin, and nicotine present in C. sativa, the smoke of C. sativa, and other phytocannabinoid matrices. This article aims to achieve this goal by application of batteries of computational tools such as machine learning tools and multivariate methods on physiochemical and absorption, distribution, metabolism, excretion, and toxicity (ADMET) descriptors of 468 metabolites from C. sativa, its smoke and, other phytocannabinoids. The structure? activity relationship (SAR) showed that 54 metabolites from C. sativa have high scaffold homology with THC. Its implications on the route of administration and factors affecting the SAR are discussed. C. sativa smoke has metabolites that have possibility of interacting with GABA, and glycine receptors.

Immunoassays for the Detection of Cannabis Abuse
Jane S C Tsai
In book Marijuana and the Cannabinoids 2007
DOI: 10.1007/978-1-59259-947-9_7
https://www.researchgate.net/publica...assays_for_the _Detection_of_Cannabis_Abuse
The power of molecular recognition and effective interaction of specific binding partners have been exploited to develop assay technologies for diverse biochemical analysis. The unique features of immunoglobulins and technological advancement in antibody engineering and manipulation have made antibodies the most versatile binding reagents for detecting analytes of interest in a variety of matrices. The term immunoassay is customarily used to denote antibody-mediated analytical procedures; however, there are assortments of nomenclature for various immunoassay techniques that usually are named after the reaction principle of the particular immunoassay format.

Improved identification of phytocannabinoids using a dedicated structure-based workflow.
Montone, C. M., Cerrato, A., Botta, B., Cannazza, G., Capriotti, A. L., Cavaliere, C., … Laganà, A.
Talanta, 121310.(2020).
doi:10.1016/j.talanta.2020.121310
Phytocannabinoids are a broad class of compounds uniquely synthesized by the various strains of Cannabis sativa. Up to date, most investigation on phytocannabinoids have been addressed to the most abundant species, Δ9-tetrahydrocannabinol and cannabidiol, for their well-known wide range of pharmaceutical activities. However, in the recent years a large number of minor constituents have been reported, whose role in cannabis pharmacological effects is of current scientific interest. With the purpose of gaining knowledge on major and minor species and furnishing a strategy for their untargeted analysis, in this study we present an innovative approach for comprehensively identifying phytocannabinoids based on high-resolution mass spectrometry in negative ion mode, which allows discrimination of the various isomeric species. For a faster and more reliable manual validation of the tandem mass spectra of known and still unknown species, an extensive database of phytocannabinoid derivatives was compiled and implemented on Compound Discoverer software for the setup of a dedicated data analysis tool.
The method was applied to extracts of the Italian FM-2 medicinal cannabis, resulting in the identification of 121 phytocannabinoids, which is the highest number ever reported in a single analysis. Among those, many known and still unknown unconventional phytocannabinoids have been tentatively identified, another piece in the puzzle of unravelling the many uncharted applications of this matrix

Investigation of aflatoxin and ochratoxin A contamination of seized cannabis and cannabis resin samples Laetitia Buchicchio, Laurent Asselborn, Serge Schneider, Claude Schummer,
https://www.researchgate.net/publica..._resin_samples Recreational cannabis is being legalized in more and more countries, and methods for the determination of contaminants, thereunder mycotoxins, start to emerge in scientific literature. On the other hand, cannabis continues being available on the illegal market without any quality control at all. Today, no information about mycotoxin contamination of illegal cannabis is available in literature. Therefore, in order to increase knowledge about mycotoxin contamination of cannabis, aflatoxins (AF) and ochratoxin A (OTA) were analyzed in 142 samples of illegal cannabis seized on the local market using a method based on HPLC-FLD detection, after clean up with immuno-affinity cartridges. AF were derivatized prior to detection with a Kobra cell. No AF contamination (LOD = 0.04 µg/kg) was detected in any of the samples analyzed. OTA however was detected in about one-third of the samples with an average concentration of 4.30 µg/kg (range from 1.02 to 16.21 µg/kg). No significant difference was observed between resin and herbal samples. Overall, the concentrations remain low and do not suggest an issue to human health if the cannabis consumption remains moderate.
Find PDF .

Investigation of Chocolate Matrix Interference on Cannabinoid Analytes.
Dawson, D., & Martin, R.
Journal of Agricultural and Food Chemistry.(2020).
doi:10.1021/acs.jafc.0c01161
The first known findings of chocolate matrix interference on cannabinoid analytes is reported. Stock solutions of four biogenic cannabinoids (Δ9 -tetrahydrocannabinol, cannabidiol, cannabinol, and cannabigerol) and one synthetic cannabinoid (cannabidiol dimethyl ether) are subjected to milk chocolate, dark chocolate, and cocoa powder. A clear trend of matrix interference is observed, which correlates to several chemical factors. The amount of chocolate present is directly proportional to the degree of matrix interference, which yields lower percent recovery rates for the cannabinoid analyte. Structural features on the cannabinoid analytes are shown to affect matrix interference, because cannabinoids with fewer phenolic −OH groups suffer from increased signal suppression. Additionally, aromatization of the p-menthyl moiety appears to correlate with enhanced matrix effects from chocolate products high in cocoa solids. These findings represent the first known documentation of chocolate matrix interference in cannabinoid analysis, which potentially has broad implications for complex matrix testing in the legal Cannabis industry.

Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography
Chiara De Luca · Alessandro Buratti · Yannick Krauke · Svea Stephan · Kate Monks · Virginia Brighenti · Federica Pellati · Alberto Cavazzini · Martina Catani · Simona Felletti
Analytical and Bioanalytical Chemistry February 2022 DOI: 10.1007/s00216-021-03862-y https://link.springer.com/content/pd...21-03862-y.pdf
This work reports about a screening of four adsorbents with different polarity employed for the separation of the main phytocannabinoids contained in Cannabis sativa L., under normal phase liquid chromatography (NPLC). The effect of polarity and type of interaction mechanisms of the adsorbents (namely Si-, CN-, Diol-, and NH 2 -based SPs) on retention has been investigated under a variety of conditions either by using different combinations of apolar solvents (heptane or hexane) and alcohols (ethanol or isopropanol). The columns have also been employed for the separation of a real cannabis sample.

Isolation, structure and partial synthesis of an active constituent of hashish.
Gaoni Y, Mechoulam R (1964).
Journal of the American Chemical Society 86 (8): 1646–1647.
DOI: 10.1021/ja01062a046
Hashish (marihuana) ~ the psychotomimetically active resin of the female flowering tops of Cannabis sativa L. is one of the most widely used illicit narcotic drugs. A number of groups have reported the isolation of active constituents.2 Most of these substances are not fully characterized, and comparisons with or between them are difficult.

Isolation, Synthesis And Structure Determination Of Cannabidiol Derivatives And Their Cytotoxic Activities
Yedukondalu Nalli, Suraya Jan, Gianluigi Lauro, Javeed Ur Rasool, Waseem I. Lone, Aminur R. Sarkar, Junaid Banday, Giuseppe Bifulco, Hartmut Laatsch, Sajad H. Syed & Asif Ali
Natural Product Research,
DOI: 10.1080/14786419.2019.1638381
In a continuing effort to explore the structural diversity and pharmacological
activities of natural products based scaffolds, herein, we report the isolation, synthesis, and structure determination of cannabidiol and its derivatives along with their cytotoxic activities. Treatment of cannabidiol (1) with acid catalyst POCl3 afforded a new derivative 6 along with six known molecules 2_5, 7 and, 8. The structure of 6 was elucidated by extensive spectroscopic analyses and DFT calculations of the NMR and ECD data. All the compounds (2 – 8)
were evaluated for their cytotoxic potential against a panel of eight cancer cell lines. Compounds 4, 5, 7, and 8 showed pronounced in vitro cytotoxic activity with IC50 values ranging from 5.6 to 60 lM. Out of the active molecules, compounds 4, and 7 were found to be comparable to that of the parent molecule 1 on the inhibition of almost all the tested cancer cell lines.

ITS barcoding using high resolution melting analysis of Cannabis sativa drug seizures in Chile: A forensic application.
Solano, J., Anabalón, L., Figueroa, A., & Gangitano, D.
Forensic Science International, 110550.(2020).
doi:10.1016/j.forsciint.2020.110550
Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine, and intoxicant. Traditionally, C. sativa is divided into two main types: fiber type (hemp) and drug type. Drug-type C. sativa differs from hemp by the presence of a high quantity of the psychoactive drug, ?9-tetrahydrocannabinol (?9 THC). Cannabis sativa is the most commonly used used illicit controlled substance in Chile. Chile is the third greatest consumer of Cannabis in South America. The objective of this study was to determine the genetic composition of ten drug seizures of Cannabis spp. in the south of Chile using a high resolution melting (HRM) strategy combined with a barcoding marker, ITS. C. sativa samples were selected from previously processed more than a thousand crime cases at the, Araucania region crime lab, National Dept. of Health. Ten cases were selected. Sample collection was based on the following: a) dry and fresh samples with no evidence of decomposition or degradation, b) defined plant fragments such as flowers
and leaves from individual plants and, c) samples with different content of THC, CBN and CBD. Five sub samples were randomly selected from each case (N=50). The commercial Silver Haze strain was used as a control. Two real-time PCR and HRM analyses were conducted. The first analysis was performed with a representative sample of each of the 10 cases studied. Then a second assay was performed with all subsamples of cases 1, 5 and 8.
Results showed that real-time PCR combined with HRM analysis using ITS allowed to determine the genetic composition of cannabis in all cases studied. The derivative of melting and the analysis of the shape of the curve and the peak of Tm, showed that three groups can be clearly distinguished. A first
group exhibited a peak of Tm close to 87.4 °C and includes cases 7 and 8. A second group had a peak of Tm close to 87.6 °C and includes case 5. A third group displayed a peak of Tm close to 87.9 °C and includes case 1, 6 and Silver Haze strain. A second experiment was performed using subsamples of cases 1, 5 and 8. Case 1 displayed a unique composition of the drug suggesting that this seizure contained cannabis clonally propagated. In case 5,
two genotypes were present, therefore this could be associated with two strain or two different origin. Case 8, was composed of a mixture of cannabis strains indicating the presence of various crop type and/or different biogeographic origin. In general, our results suggested genetically homogeneous seizures from Araucanía Region. The high latitude (37° 35’ and 39° 37’ South latitude) and the natural geographic borders that surround southern Chile helps the control of cannabis traffic into the country. Finally, HRM analysis coupled with the barcode ITS demonstrated to be a rapid and low-cost screening method.
Microbiological contaminants pose a potential threat to cannabis consumers. Bacteria and fungi may cause opportunistic infections in immunocompromized individuals. Even dead organisms may trigger allergies and asthma. Toxins from microbial overloads, such as Shigla toxin and aflatoxins, may pose a problem—unlikely, but possible. The Cannabis plant hosts a robust microbiome; the identification of these organisms is underway. Cannabis bioaccumulates heavy metals in its tissues, so avidly that hemp crops have been used for bioremediation. Heavy metals cause myriad human diseases, so their presence in crops destined for human consumption must be minimized. Pesticide residues in cannabis pose a unique situation among crop plants—the Environmental Protection Agency (EPA) will not propose pesticides guidelines, because Cannabis is illegal on the federal level. The use of illegal pesticides is a rising crisis, and a breakdown in ethics. Testing for pesticide residues and maximal limits are proposed.

Its Far More Than The Cannabinoids
James Freire
FIND DOI or LINK
Synergy is very important to the overall function of the Cannabis plant medicinally. We explored just some of the cannabinoids and looked briefly into their function and touched upon the synergy of THC (tetrahydrocannabinol) and CBD (cannabidiol), but these are just one system in a vastly complex biochemical engine. To complete the picture we must look at two other therapeutic groups of compounds found in whole Cannabis that are just as important medicinal compounds as the cannabinoids and work with them to produce the vastly therapeutic properties of whole cannabis. The compounds are the flavinoids and the terpenes and we shall take a brief look at each.

Keeping Cannabis Consumers Safe
The Cannabis Scientist
https://thecannabisscientist.com/fil...umers_Safe.pdf
Heavy Metal Measurement Made Light
Taking Back Cannabis Control
Gurus of Pesticide Residue Analysis
Making Cannabis Labs Accountable

Leaf Tissue Sampling for Floral Hemp Monitor in‐season fertility by testing leaf samples for nutrient analysis June 2020
Michelle McGinnis,
NC Dept of Agriculture & Consumer Services
https://hemp.ces.ncsu.edu/wp-content...une.pdf?fwd=no
Foliar nutrient analysis is an excellent tool to monitor in‐season fertility and to troubleshoot plant growth problems (such as identifying a specific nutrient imbalance or ruling out fertility as the source of the problem). It is KEY to collect a representative sample of the most recently matured leaves (MRML) at the correct growth stage to compare with survey ranges that correspond to nutrient concentrations found within MRMLs. If the incorrect leaf is sampled, comparison of analytical results to the survey ranges has little to no meaning and can result in both incorrect interpretations and poor nutrient application recommendations.

Leaner and greener analysis of cannabinoids
Elizabeth M. Mudge, & Susan J. Murch & Paula N. Brown
Anal Bioanal Chem
DOI 10.1007/s00216-017-0256-3
There is an explosion in the number of labs analyzing cannabinoids in marijuana (Cannabis sativa L., Cannabaceae) but existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging solvents. The objective of this work was to
develop and validate an accurate method for analyzing cannabinoids in cannabis raw materials and finished products that is more efficient and uses fewer toxic solvents. An HPLC-DAD method was developed for eight cannabinoids in cannabis flowers and oils using a statistically guided optimization plan based on the principles of green chemistry. A single laboratory validation determined the linearity, selectivity, accuracy,
repeatability, intermediate precision, limit of detection, and limit of quantitation of the method. Amounts of individual cannabinoids above the limit of quantitation in the flowers ranged from 0.02 to 14.9% w/w, with repeatability ranging from 0.78 to 10.08% relative standard deviation. The intermediate precision determined using HorRat ratios ranged from 0.3 to 2.0. The LOQs for individual cannabinoids in flowers ranged from 0.02 to 0.17% w/w. This is a significant improvement over previous methods and is suitable for a wide range of applications including regulatory compliance, clinical studies, direct patient medical services, and commercial suppliers.

LightLab Interlaboratory Test Comparison - Preliminary Results
https://www.orangephotonics.com/
LightLab a portable cannabis potency analyzer manufactured by Orange Photonics Inc enrolled in the 2016 Emerald Test, which is an inter-laboratory proficiency test for marijuana potency analysis. LightLab™ is the first portable instrument to participate in the Emerald Test. Preliminary results were obtained by completing the Emerald Practice Test. The LightLab results for the Emerald Practice Test were as accurate as a proficient laboratory. Results for full proficiency test are pending as of the date of this paper
Method:
An Emerald Practice Test was ordered from Emerald Scientific (part number #38417-IN) which contains 5 cannabinoids of varying concentration (THCA, D9THC, CBDA, CBD, CBN) in acetonitrile. The concentrations of the cannabinoids were not known to the operator prior to submitting test results. The sample was first adjusted with buffered water to ensure the sample was consistent with typical LightLab samples. After adjustment, the sample was injected into the analyzer and results were calculated in milligrams of cannabinoid per milliliter of solvent (Note: typically, a laboratory instrument reports mg/ml which is subsequently converted to a % cannabinoid content by weight. LightLab operates in accordance with a similar process). After calculation, results were compared with practice test proficiency results.
Results:
The Emerald Practice Test provides a range of results for each cannabinoid. If a test result falls within this range, the laboratory may be considered proficient. LightLab results fell within the proficiency range for all 5 cannabinoids, with 4 of the five close to the center of the range. The LightLab performed as well as a proficient laboratory.

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urine Specimens for K2 (JWH-018) Metabolites.
ElSohly, M. A., Gul, W., ElSohly, K. M., Murphy, T. P., Madgula, V. L. M., & Khan, S. I.
Journal of Analytical Toxicology, 35(7), 487–495.(2011).
doi:10.1093/anatox/35.7.487
Marijuana is the most widely used drug of abuse all over the world. The major active constituent of the drug is ∆9- tetrahydrocannabinol (∆9-THC). ∆9-THC exerts its psychological activities by interacting with the cannabinoid receptors (CB1 and CB2) in the brain. JWH-018, HU-210, and CP-47497, with CB1 agonist activity (similar to ∆9-THC), have been used by the drug culture to spike smokable herbal products to attain psychological effects similar to those obtained by smoking marijuana. The products spiked with these CB1 agonists are commonly referred to as “Spice” or “K2”. The most common compound used in these products is JWH-018 and related compounds (JWH-073 and JWH- 250). Little work has been done on the detection of these synthetic cannabimimetic compounds in biological specimens. This report investigated the metabolism of JWH-018 by human liver microsomes, identification of the metabolites of JWH-018 in urine specimen of an individual who admitted use of the drug, and reports on the quantitation of three of its urinary metabolites, namely the 6-OH-, the N-alkyl OH (terminal hydroxyl)-, and the N-alkyl terminal carboxy metabolites using liquid chromatography–tandem mass spectrometry. The concentrations of these metabolites are determined in several forensic urine specimens.

Long - term Storage and Cannabis Oil Stability
IRENNE GABRIELA TROFIN1, GABRIEL DABIJA1, DANUT–IONEL VAIREANU, LAURENTIU FILIPESCU
REV. CHIM. (Bucharest) ? 63 ? No. 3 ? 2012
https://www.researchgate.net/publica..._Oil_Stability
The paper presents the results of an experimental study regarding the stability of cannabis oil during its longterm storage in different conditions. The content of major cannabinoids, namely tetrahydrocannabinol (?9–THC), cannabinol (CBN), and cannabidiol (CBD) contained in two batch samples of cannabis oil seizures made by criminal prosecution authorities from Romania was measured during their storage over a period of four years in darkness at 4°C and in laboratory light at 22°C. The results revealed a steadily decay of ?9–THC over the entire storage period from a very high initial content up to a relatively low final content. A slight difference regarding the degree of decay of ?9–THC between the two storage conditions was recorded, meaning that this is more pronounced when the samples were exposed to light at 22°C. The same trend was recorded for CBD. As expected, the content of CBN increases during storage and the increase is higher when
the samples were exposed to light at 22°C.

Magnesium’s Impact on Cannabis sativa ‘BaOx’ and ‘Suver Haze’ Growth and Cannabinoid Production
Patrick Veazie , Paul Cockson , David Logan , Brian Whipker
Journal of Agricultural Hemp Research: Vol. 2 : Iss. 2 , Article 1.
https://digitalcommons.murraystate.edu/jahr/vol2/iss2/1
Limited research exists on the fertility needs for industrial hemp (Cannabis sativa) and the impact of fertility on plant growth and cannabinoids. Optimizing floral production for cannabinoid production and especially cannabidiol (CBD) production, is an economic goal for growers. Magnesium (Mg) is an essential nutrient for plant growth and plays many key roles in plant growth and when deficient leads to suboptimal plant growth. Six Mg fertility rates (0.0, 12.5, 25.0, 50.0, 75.0, and 100.0 mg·L-1) were evaluated to determine the optimal fertility for C. sativa on two High CBD-type cultivars ‘BaOx; and ‘Suver Haze’. Foliar Mg concentrations increased linearly for all life stages with the greatest foliar Mg concentrations being in the highest rate of 100.0 mg·L-1 Mg. Of the six rates, 50.0 and 75.0 mg·L-1 Mg optimized plant height, diameter, and plant total dry weight as well as having similar cannabinoid concentrations during the three life stages.

Managing Floral Hemp Fertility in North Carolina
Jeanine Davis
Horticultural Science NC State Extension, NC State University
https://hemp.ces.ncsu.edu/2020/05/ma...orth-carolina/
There are many questions coming in this spring on how to fertilize floral hemp (hemp grown for CBD, CBG, CBN, etc.). Research to develop those recommendations is still ongoing in North Carolina, but Dr. Michelle McGinnis with the N.C. Department of Agriculture and Consumer Services, Agronomic Division has prepared guidelines for us on leaf tissue sampling and soil fertility. (live links in the PDFs at the end). This article and recommendations were originally posted in late May. These are the revised/updated documents.

Managing Hemp Soil Fertility in NC June 2020
Michelle McGinnis,
NC Dept of Agriculture & Consumer Services
https://hemp.ces.ncsu.edu/wp-content...une.pdf?fwd=no
Soil fertility guidelines for floral, fiber, and seed/grain hemp are included in Table 1. The fiber and seed/grain hemp recommendations are based on peer reviewed published research. The floral hemp guidelines are developed from the fiber and seed/grain recommendations, on‐going NCSU and NCDA floral hemp research, and common practices of NC farmers. Base actual nutrient applications on a recent soil test report (NCDA&CS Soil Test Crop Code 310) as well as soil type, cropping history, and environmental conditions. The target pH for most North Carolina soil is 6.2. (http://www.ncagr.gov/agronomi/sthome.htm).

Mapping cannabis potency in medical and recreational programs in the United States
Mary Catherine Cash, Katharine Cunnane, Chuyin Fan, E. Alfonso Romero Sandova
PLoS ONE 15(3):
doi: 10.1371/journal.pone.0230167
https://journals.plos.org/plosone/ar...type=printable
Cannabis related online searches are associated with positive attitudes toward medical cannabis, particularly when information is obtained from dispensaries. Since pain is the main reason for medicinal cannabis use, information from dispensary websites has the potential to shape the attitude of pain patients towards cannabis. This is relevant because cannabis has demonstrated efficacy in neuropathic pain with low tetrahydrocannabinol (THC) concentrations (< 5–10%), in contrast to potent cannabis (>15% THC), which is highly rewarded in the recreational realm. The role of CBD in pain is not clear, however it has gained popularity. Thus, we hypothesize that the potency of medical cannabis that is advertised online is similar to the cannabis advertised for recreational purposes, which would potentially create a misconception towards medical cannabis. The current lack of knowledge surrounding advertised potencies in the legal cannabis market limits the ability to generate clear policies regarding online advertising to protect patients that are willing to use cannabis for their condition. Thus, we evaluated the advertised THC and CBD content of cannabis products offered online in dispensaries in the United States to determine products’ suitability to medicinal use and compare the strength of products offered in legal medical and recreational programs. We recorded THC and CBD concentrations for all herb cannabis products provided by dispensary websites and compared them between or within states. Four Western states (CA, CO, NM, WA) and five Northeastern states (ME, MA, NH, RI, VT) were included. A total of 8,505 cannabis products across 653 dispensaries were sampled. Despite the clear differences between medicinal and recreational uses of cannabis, the average THC concentration advertised online in medicinal programs was similar (19.2% ±6.2) to recreational programs (21.5% ±6.0) when compared between states with different programs, or between medicinal and recreational programs within the same states (CO or WA). Lower CBD concentrations accompanied higher THC products. The majority of products, regardless of medicinal or recreational programs, were advertised to have >15% THC (70.3% - 91.4% of products). These stated concentrations seem unsuitable for medicinal purposes, particularly for patients with chronic neuropathic pain. Therefore, this information could induce the misconception that high potency cannabis is safe to treat pain. This data is consistent with reports in which THC and CBD in products from legal dispensaries or in nationwide products from the illegal market were actually measured, which indicates that patients consuming these products may be at risk of acute intoxication or long-term side effects. Our study offers grounds to develop policies that help prevent misconceptions toward cannabis and reduce risks in pain patients.

MARIHUANA, BULLETIN OF THE NEW YORK ACADEMY OF MEDICINE NOVEMBER, 1941
Roger Adams
https://www.ncbi.nlm.nih.gov/pmc/art...00560-0006.pdf
The facts on marihuana which I shall present to you this evening comprise the results of the cooperative efforts of three laboratories-the chemical investigations at the University of Illinois, the pharmacology at Cornell Medical College under the direction of Dr. S. Loewe, and the clinical experiments at Welfare Island Hospital under the auspices of the Mayor's Committee on Marihuana and under the immediate direction of Dr. Samuel Allentuck. All three laboratories acquired their supplies of raw materials from Dr. H. J. Wollner of the Narcotics Laboratory of the Treasury Department, and received much encouragement and stimulation from him. Dr. J. R. Matchett of the same laboratory contributed significantly to the general problem by devising a method whereby a fraction of the red oil of hemp, containing a very high concentration of the active principle, could be obtained.

Marijuana Smoke Condensate.
ElSohly, H. N., & ElSohly, M. A.
Marijuana and the Cannabinoids, 67–96.
doi:10.1007/978-1-59259-947-9_4
Cannabis sativa is one of the oldest plants known to medicine and one of the most thoroughly studied plants today. Much knowledge has been gained about the chemistry, pharmacology, metabolism, and pharmacokinetics of pure compounds from Cannabis, as well as the chemical and biological analysis of marijuana smoke condensate (MSC). In this chapter, we review data related to the preparation of MSC, the composition and analysis of MSC, and the pharmacological and toxicological effects of MSC.

Measuring the bioactivity of phytocannabinoid cannabidiol from cannabis sources, and a novel non-cannabis source.
D. Cushing, S. Kristipati, R. Shastri, & B. Joseph
Journal of Medical Phyto Research Vol. 1, Article 2, (8-23)
Doi: 10.31013/1002b
Phytocannabinoid Cannabidiol (CBD) has been shown to elicit a great many immunological benefits. It acts on the endocannabinoid system, namely through interactions with cannabinoid receptor 2 (CB2). CBD-CB2 affinity, which we refer to as bioactivity, is rarely tested for clinical samples. We believe that uncontrolled variation in bioactivity levels have been silently confounding many CBD experiments. In our four-part study, we validate an efficient bioactivity test that can enable greater scientific control over CBD studies. We use it to compare the bioactivity of CBD obtained from different plant organs, and we also studied whether processing methods play a role in determining bioactivity. We also examine the bioactivity and processing factors of a novel non-cannabis plant capable of producing CBD in commercial quantities, named Humulus Kriya (H. Kriya, U.S. Patent No. 15/932,529, 2018). We also test the bioactivity of some CBD isolates/extracts currently sold in the market, and compare them with a CBD product called ImmunAG, which was extracted from the inflorescence of H. Kriya. We find that the CBD from the inflorescence of the plant produces the highest bioactivity, followed by the apical buds/leaves, the petioles, and finally the stalk. We find that H. Kriya has a bioactivity profile similar to Cannabis Sativa. We find that the bioactivity levels among cannabis-based commercial CBD products are quite low, and variable. We find significantly higher bioactivity levels in ImmunAG.

Menthol-based hydrophobic deep eutectic solvents: Towards greener and efficient extraction of phytocannabinoids
Tomas Krízek, Miroslava Bursova, Rachel Horsley, Martin Kuchar, Petr Tuma, Radomír Cabala, Tomas Hlozek
Journal of Cleaner Production 193 (2018) 391e396
DOI: 10.1016/j.jclepro.2018.05.080
As the demand for medical cannabis preparations increases, so does the use of the common organic solvents that are used in the extraction and quantification of phytocannabinoids. Since common organic solvents are typically hazardous to the environment and to human health, it is vital to identify safer, greener, and more efficient alternatives. The aim of the present research was to develop a series of hydrophobic deep eutectic solvents (DESs) based on terpenes and natural organic acids and to establish whether these might be potential substitutes for the extraction of phytocannabinoids (tetrahydrocannabinol, cannabidiol, and their carboxylated homologues) from raw cannabis plant material. Data were obtained using capillary electrophoresis with diode array detection (DAD). Initial screening showed that the DES composed of a menthol: acetic acid (1:1 M ratio) mixture showed the greatest extraction efficiency (of all the DESs that were tested), with yields ranging from 118.6% to 132.6% (compared to a methanol: chloroform mixture). In conclusion, menthol: acetic acid DES extraction is efficient, as well as non-toxic and biodegradable. As such it has applications within the pharmaceutical industry and represents a greener alternative organic solvent for the extraction of phytocannabinoids.

Metabolic profiling of Cannabis secondary metabolites for evaluation of optimal postharvest storage conditions
Looz Milay, Paula Berman, Anna Shapira, Ohad Guberman, David Meir
Front. Plant Sci. 11:1556. (2020)
doi:10.3389/fpls.2020.583605
The therapeutic use of medical Cannabis is growing, and so is the need for standardized and therapeutically stable Cannabis products for patients. The therapeutic effects of Cannabis largely depend on the content of its pharmacologically active secondary metabolites and their interactions, mainly terpenoids and phytocannabinoids. Once harvested and during storage, these natural compounds may decarboxylate, oxidize, isomerize, react photochemically, evaporate and more. Despite its widespread and increasing use, however, data on the stability of most of the plant's terpenoids and phytocannabinoids during storage is scarce. In this study, we therefore aimed to determine postharvest optimal storage conditions for preserving the composition of naturally biosynthesized secondary metabolites in Cannabis inflorescences and Cannabis extracts. To this end, Cannabis inflorescences (whole versus ground samples) and Cannabis extracts (dissolved in different solvents) from (-)-?9-trans-tetrahydrocannabinol- or cannabidiol-rich chemovars, were stored in the dark at various temperatures (25, 4, -30 and -80 °C), and their phytocannabinoid and terpenoid profiles were analyzed over the course of one year.

Metabolomic Differentiation of Cannabis sativa Cultivars Using1H NMR Spectroscopy and Principal Component Analysis.
Choi, Y. H., Kim, H. K., Hazekamp, A., Erkelens, C., Lefeber, A. W. M., & Verpoorte, R.
Journal of Natural Products, 67(6), 953–957.(2004).
doi:10.1021/np049919c
The metabolomic analysis of 12 Cannabis sativa cultivars was carried out by 1H NMR spectroscopy and multivariate analysis techniques. Principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by principal component 1 (PC1) and principal component 3 (PC3) in cannabinoid fraction. The loading plot of PC value obtained from all 1H NMR signals shows that ?9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are important metabolites to differentiate the cultivars from each other. The discrimination of the cultivars could also be obtained from a water extract containing carbohydrates and amino acids. The level of sucrose, glucose, asparagine, and glutamic acid are found to be major discriminating metabolites of these cultivars. This method allows an efficient differentiation between cannabis cultivars without any prepurification steps.

Metabolism of Fenhexamid, Metalaxyl-M, Tebuconazole, Flurtamone, and Spirodiclofen in Cannabis sativa L. (hemp) Plants
Leonie Hillebrands, Marc Lamshoeft, Andreas Lagojda, Andreas Stork, and Oliver Kayser
Doi: 10.1021/acsagscitech.1c00010
The consumption of and demand for Cannabis sativa L. (C. sativa , hemp) have been increasing because of its legalization in several parts of the world. Pesticides could be applied to manage the fi tness of C. sativa , but their application has not been studied in detail from a metabolic perspective. This study examined the metabolic degradation of fi ve pesticides after hydroponic root uptake: fenhexamid, metalaxyl-M, tebuconazole, fl urtamone, and spirodiclofen. Young plants were hydroponically exposed to 10 μ M solutions of the selected pesticides for 14 days. Plant extracts were analyzed by a high-resolution mass spectrometer. Known mechanisms degraded the pesticides in C. sativa . For example, hydroxylation of all pesticides was followed by glycosylation and malonic acid conjugation. In addition, desaturation, demethylation, and non-enzymatic degradation were detected and are in good agreement with the known pathways in regulatory guideline data of the investigated pesticides in traditional crop plants such as, for example, Triticum aestivum L. (wheat), Vitis vinifera L. (wine), and Lactuca sativa L. (lettuce). Therefore, young
plants make for a reliable and fast model system for elucidating the metabolism of pesticides in C. sativa .

Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests.
Kevin McKernan, Jessica Spangler, Yvonne Helbert, Ryan C. Lynch , Adrian Devitt-Lee, Lei Zhang, Wendell Orphe, Jason Warner, Theodore Foss, Christopher J. Hudalla, Matthew Silva, Douglas R. Smith.
DOI: 10.12688/f1000research.9662.1
Abstract
Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation.
Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media.
Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample.
Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

Methods for quantification of cannabinoids: a narrative review.
Pourseyed Lazarjani, M., Torres, S., Hooker, T., Fowlie, C., Young, O., & Seyfoddin, A.
Journal of Cannabis Research, 2(1). (2020).
doi:10.1186/s42238-020-00040-2
Background: Around 144 cannabinoids have been identified in cannabis plant, among them tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most prominent ones. Because of the legal restrictions on cannabis in many countries, it is difficult to obtain standards to use in research; nonetheless, it is important to develop a cannabinoid quantification technique with pharmaceutical applications for quality control of future therapeutic cannabinoids.
Method: To find relevant articles for this narrative review paper, a combination of keywords such as medicinal cannabis, analytical, quantification and cannabinoids were searched for in PubMed, EMBASE, MEDLINE, Google Scholar and Cochrane Library (Wiley) databases.
Results: The most common cannabinoid quantification techniques include gas chromatography (GC) and highperformance liquid chromatography (HPLC). GC is often used in conjunction with mass spectrometry (MS) or flame ionization detection (FID). The major advantage of GC is terpenes quantification however, for evaluating acidic cannabinoids it needs to be derivatised. The main advantage of HPLC is the ability to quantify both acidic and neutral forms of cannabinoids without derivatisation which is often with MS or ultraviolet (UV) detectors.
Conclusion: Based on the information presented in this review, the ideal cannabinoid quantification method is HPLC- MS/MS for the cannabinoids.

Methods to assay anandamide hydrolysis and transport in synaptosomes
Filomena Fezza, Natalia Battista, Monica Bari, Mauro Maccarrone
https://www.researchgate.net/publica...n_synaptosomes
DOI: 10.1385/1-59259-999-0:163
https://www.researchgate.net/publica...to_assay_anand amide_hydrolysis_and_transport _in_synaptosomes

Anandamide (AEA) is the most studied member of a new class of lipid mediators, collectively called endocannabinoids. The biological activity of AEA at cannabinoid and noncannabinoid receptors depends on its life span in the extracellular space, which is regulated by a rapid cellular uptake, followed by intracellular degradation by the enzyme AEA hydrolase (fatty acid amide hydrolase). Here, we present the methodological details of the procedures that we have developed to assay fatty acid amide hydrolase activity and to characterize AEA transport through cell membranes in a new ideal ex vivo system like brain synaptosomes.


Moisture Content Determination in Hemp and Hemp Flower

The Cannabis Scientist. & METTLER TOLEDO 11/16/2020 Webvideo

https://thecannabisscientist.com/webinar/moisture-content-determination-in-hemp-and-hemp-flower?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email

https://view6.workcast.net/register...F84nwIAFgoRQ5rb0NBG2tKifZdeUmS37SdvIwCej7of2K

https://view6.workcast.net/AuditoriumAuthenticator.aspx?cpak=5312466848601485&pak=6239340224033044

Moisture content determination techniques and their implication on potency based on dry weight calculations in cannabis and hemp industry. Discussion of use cases for Karl Fischer Titration, Thermogravimetric Halogen Moisture Analysis and Conventional Loss on Drying (drying oven and balance).
Learning Objectives:

Moisture content determination techniques

Use cases for various instruments


Molecular neuroscience at its “high”: bibliometric analysis of the most cited papers on endocannabinoid system, cannabis and cannabinoids
Andy Wai Kan Yeung1*, Nikolay T. Tzvetkov2,3, Nicolas Arkells4 , Luigi Milella5 , Adrian M. Stankiewicz6 , ?ukasz Huminiecki6 , Olaf K. Horbanczuk7 and Atanas G. Atanaso
Journal of Cannabis Research (2019) 1:4
Doi: 10.1186/s42238-019-0004-y
Background: Cannabis, cannabinoids and endocannabinoids are heavily investigated topics with many articles published every year. We aimed to identify the 100 most cited manuscripts among the vast literature and analyze their contents.
Methods: Web of Science (WoS) Core Collection was searched to identify the 100 most cited relevant manuscripts, which were analyzed with reference to (1) authorship, (2) institution, (3) country, (4) document type, (5) journal, (6) publication year, (7) WoS category, and (8) citation count. Semantic content and citation data of the manuscripts were analyzed with VOSviewer.
Results: The most cited manuscripts were published between 1986 and 2016, with the majority being published in the 2000s (n = 51). The number of citations for the top 100 articles ranged from 469 to 3651, with a median citation count of 635.5. The most prolific authors were Vincenzo Di Marzo (n = 11) and Daniele Piomelli (n = 11). The major contributing countries were USA (n = 49), Italy (n = 22), UK (n = 19), and France (n = 11). The most prolific institutions were University of California (n = 14), National Research Council of Italy (n = 12) and National Institutes of Health USA (n = 12). The manuscripts consisted of original articles (n = 75), reviews (n = 24) and a note (n = 1). The most dominant journal was Nature (n = 15). The major WoS categories associated were Multidisciplinary sciences (n = 31), Neurosciences (n = 20), Pharmacology / Pharmacy (n = 16), and General / Internal Medicine (n = 11).
Conclusions: The top-ranked manuscripts among the 100 were concerning analgesia, weight loss, long-term potentiation, depolarization-induced suppression of inhibition, opiates and other topics. Cannabinoid type 1 (CB1) receptor was studied by more of the top 100 papers in comparison to cannabinoid type 2 (CB2) receptor. The most frequently mentioned chemicals in these publications were 2-arachidonoylglycerol, tetrahydrocannabinol, and anandamide. Together, these manuscripts comprise the most highly cited publications in the topic, literally the molecular neuroscience at its “high”.

Not Cannabis specific
Multispecies Identification of Oilseed- and Meat-Specific Proteins and Heat-Stable Peptide Markers in Food Products
Klaudia Kotecka-Majchrzak, Natalia Kasałka-Czarna, Agata Sumara, Emilia Fornal, and Magdalena Montowska
Molecules 2021, 26, 1577.
DOI: 10.3390/ molecules26061577
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-01577.pdf
Consumer demand for both plant products and meat products enriched with plant raw materials is constantly increasing. Therefore, new versatile and reliable methods are needed to find and combat fraudulent practices in processed foods. The objective of this study was to identify oilseed species-specific peptide markers and meat-specific markers that were resistant to processing, for multispecies authentication of different meat and vegan food products using the proteomic LC-MS/MS method. To assess the limit of detection (LOD) for hemp proteins, cooked meatballs consisting of three meat species and hemp cake at a final concentration of up to 7.4% were examined. Hemp addition at a low concentration of below 1% was detected. The LOD for edestin subunits and albumin was 0.9% (w/w), whereas for 7S vicilin-like protein it was 4.2% (w/w). Specific heat-stable peptides unique to hemp seeds, flaxseed, nigella, pumpkin, sesame, and sunflower seeds, as well as guinea fowl, rabbit, pork, and chicken meat, were detected in different meat and vegan foods. Most of the oilseed-specific peptides were identified as processing-resistant markers belonging to 11S globulin subunits, namely conlinin, edestin, helianthinin, pumpkin vicilin-like or late embryogenesis proteins, and sesame legumin-like as well as 2S albumins and oleosin isoforms or selected enzymic proteins.

Near infrared spectroscopy combined with chemometrics for growth stage classification of cannabis cultivated in a greenhouse from seized seeds
Bruna Tassi Borille, Marcelo Caetano Alexandre Marcelo, Rafael Scorsatto Ortiz, Kristiane de Cássia Mariotti, Marco Flôres Ferrão, Renata Pereira Limberger
DOI: 10.1016/j.saa.2016.09.040
Cannabis sativa L. (cannabis, Cannabaceae), popularly called marijuana, is one of the oldest plants known to man and it is the illicit drug most used worldwide. It also has been the subject of increasing discussions from the scientific and political points of view due to its medicinal properties. In recent years in Brazil, the form of cannabis drug trafficking has been changing and the Brazilian Federal Police has exponentially increased the number of seizures of cannabis seeds sent by the mail. This new form of trafficking encouraged the study of cannabis seeds seized germinated in a greenhouse through NIR spectroscopy combined with chemometrics. The plants were cultivated in a homemade greenhouse under controlled conditions. In three different growth periods (5.5 weeks, 7.5 weeks and 10 weeks), they were harvested, dried, ground and directly analyzed. The iPCA was used to select the best NIR spectral range (4000–4375 cm?1) in order to develop unsupervised and supervised methods. The PCA and HCA showed a good separation between the three groups of cannabis samples at different growth stages. The PLS-DA and SVM-DA classified the samples with good results in terms of sensitivity and specificity. The sensitivity and specificity for SVM-DA classification were equal to unity. This separation may be due to the correlation of cannabinoids and volatile compounds concentration during the growth of the cannabis plant. Therefore, the growth stage of cannabis can be predicted by NIR spectroscopy and chemometric tools in the early stages of indoor cannabis cultivation.

New insights in hemp chemical composition: a comprehensive polar lipidome characterization by combining solid phase enrichment, high-resolution mass spectrometry, and cheminformatics
Michela Antonelli & Barbara Benedetti & Giuseppe Cannazza & Andrea Cerrato & Cinzia Citti & Carmela Maria Montone & Susy Piovesana & Aldo Laganà
Analytical and Bioanalytical Chemistry 2019
10.1007/s00216-019-02247-6
The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to highresolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles.

New Methods for the Comprehensive Analysis of Bioactive Compounds in Cannabis sativa L. (hemp).
Pellati, F., Brighenti, V., Sperlea, J., Marchetti, L., Bertelli, D., & Benvenuti, S.
Molecules, 23(10), 2639. (2018).
doi:10.3390/molecules23102639
Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS2 . The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while ?-myrcene and ?-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety

Nitrogen and Potassium Fertilizer Rates for Floral Hemp in North Carolina: Research Summary
Maggie Short, David Suchoff
Department of Crop and Soil Sciences
https://hemp.ces.ncsu.edu/hemp-ferti...earch-summary/
North Carolina leads the nation in flue-cured tobacco production, but market decline has left many growers seeking other opportunities to make up for lost revenue. Floral hemp (Cannabis sativa L.) grown for biomass and cannabidiol (CBD) extraction fits nicely in a tobacco production model. Growers found early on that much of their knowledge (ex: obtaining and managing large labor crews), equipment (transplanters, spray equipment, etc.; Fig. 1), and infrastructure (curing barns) can be used in the production of floral hemp.

Non-cannabinoid constituents from a high potency Cannabis sativa variety,
Mohamed M. Radwan, Mahmoud A. ElSohly, Desmond Slade, Safwat A. Ahmed, Lisa Wilson, Abir T. El-Alfy, Ikhlas A. Khan, Samir A. Ross,
Phytochemistry, 69(2008), 2627-2633.
http://europepmc.org/backend/ptpmcre...?accid=PMC4888 767&blobtype=pdf
Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6- trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10- dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and β-sitosteryl-3-O-β-D-glucopyranoside-2'-O-palmitate (6). In addition, five known compounds, α- cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and antioxidant activities. Compounds 2–4 were inactive as analgesics.


Non-Cannabinoid Metabolites of Cannabis sativa with Therapeutic Potential
Henry Lowe1,2,3,4, Blair Steele1*, Joseph Bryant1, Ngeh Toyang2,3 and Wilfred F. Ngwa
doi:10.20944/preprints202010.0551.v1
The Cannabis plant (Cannabis sativa L.) produces an estimated 545 chemical
compounds of different biogenetic classes. In addition to economic value, many of these phytochemicals have medicinal and physiological activity. The plant is most popularly known for its two most prominent and most studied secondary metabolites—?9-Tetrahydrocannabinol (?9-THC) and Cannabidiol (CBD). Both ?9-THC and CBD have a wide therapeutic window across many ailments and form part of a class of secondary metabolites called cannabinoids of which approximately over 104 exist. This review will focus on noncannabinoid metabolites of Cannabis sativa that also have therapeutic potential, some of which share medicinal properties similar to those of cannabinoids. The most notable of these noncannabinoid phytochemicals are flavonoids and terpenes. We will also discuss future directions in cannabis research and development of cannabis-based pharmaceuticals. Caflanone, a
flavonoid molecule with selective activity against the human viruses including the coronavirus SARS-COV2, and certain cancers, is one of the most promising non-cannabinoid molecules that is being advanced into clinical trials. As validated by thousands of years of the use of cannabis for medicinal purposes, vast anecdotal evidence abounds on the medicinal benefits of the
plant. These benefits are attributed to the many phytochemicals in this plant, including noncannabinoids. The most promising non-cannabinoids with potential to alleviate global disease burdens are discussed.

Non-Smoker Exposure to Secondhand Cannabis Smoke. I. Urine Screening and Confirmation Results.
Cone, E. J., Bigelow, G. E., Herrmann, E. S., Mitchell, J. M., LoDico, C., Flegel, R., & Vandrey, R
Journal of Analytical Toxicology, 39(1), 1–12. . (2014).
doi:10.1093/jat/bku116
Increased cannabis potency has renewed concerns that secondhand exposure to cannabis smoke can produce positive drug tests. A systematic study was conducted of smoke exposure on drug-free participants. Six experienced cannabis users smoked cannabis cigarettes (5.3% THC in Session 1 and 11.3% THC in Sessions 2 and 3) in a sealed chamber. Six non-smokers were seated with smokers in an alternating manner. Sessions 1 and 2 were conducted with no ventilation and ventilation was employed in Session 3. Non-smoking participant specimens (collected 0 – 34 h) were analyzed with four immunoassays at different cutoff concentrations (20, 50, 75 and 100 ng/mL) and by GC-MS (LOQ 5 0.75 ng/mL). No presumptive positives occurred for non-smokers at 100 and 75 ng/mL; a single positive occurred at 50 ng/mL; and multiple positives occurred at 20 ng/ mL. Maximum THCCOOH concentrations by GC-MS for non-smokers ranged from 1.3 to 57.5 ng/mL. THCCOOH concentrations generally increased with THC potency, but room ventilation substantially reduced exposure levels. These results demonstrate that extreme cannabis smoke exposure can produce positive urine tests at commonly utilized cutoff concentrations. However, positive tests are likely to be rare, limited to the hours immediately post-exposure, and occur only under environmental circumstances where exposure is obvious.

Not So Volatile

Low VOC emissions from indoor cannabis cultivation facilities – good news for people and the planet?
Margot Lespade | 04/20/2022

https://thecannabisscientist.com/testing-processing/not-so-volatile?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS%20Newsletter%20Week%2016%202022&utm_medium=email

As cannabis legalization becomes increasingly common, there is growing concern over its environmental impact, particularly in indoor cultivation settings. In an effort to explore these effects, researchers from the Colorado Department of Public Health and Environment (CDPHE) examined air quality samples from three indoor cultivation facilities in Colorado (1). Despite these facilities’ high nuisance odors, they found the volatile organic compounds (VOCs) emissions rate to be low – even at large cultivation facilities. We spoke with the paper’s first author, Kaitlin Urso, to find out more.

Nutrient concentrations, digestibility, and cannabinoid concentrations of industrial hemp plant components.
Kleinhenz, M. D., Magnin, G., Ensley, S. M., Griffin, J. J., Goeser, J., Lynch, E., & Coetzee, J. F.
Applied Animal Science, 36(4), 489–494.(2020).
doi:10.15232/aas.2020-02018
Objective: The cultivation and production of industrial hemp [Cannabis sativa containing <0.3% tetrahydrocannabinol (THC)] has increased. Information regarding the nutrient composition and cannabinoid concentration of different plant parts are deficient. Materials and Methods: Single lots of industrial hemp plants and by-products of cannabinoid production were obtained from a licensed research station located in south-central Kansas. Samples obtained were (1) whole industrial hemp plants (no roots), (2) stalks remaining after seed harvesting, (3) unprocessed female flowers intended for cannabinoid extraction, (4) whole seed heads for seed production, (5) leaves obtained from the drying process, (6) chaff obtained after seed harvesting and cleaning, and (7) processed female flowers after cannabinoid extraction. Plant materials were submitted for nutrient concentration and digestibility analysis, and for cannabinoid concentration determination, to a commercial nutrition laboratory. Results and Discussion: Dry matter of the plant material ranged from 65 to 96.6%. Crude protein ranged from 5.3 to 24.5%. Calcium concentration was from 1.0 to 5.7% DM. The plants tested had high fiber concentrations, with NDF ranging from 28 to 80% and ADF ranging from 18 to 65% DM. Total digestible nutrients was 19.8 to 61.5. Six of the 10 cannabinoids tested were detected in all samples. Cannabidiolic acid, cannabidiol, and ?9-tetrahydrocannabinolic acid A were detected in all samples at the highest concentrations. Implications and Applications: These findings will assist livestock producers in using industrial hemp in animal feeds through consideration of both the nutritional and cannabinoid concentrations in the ration.

Objective Identification of Cannabis Use Levels in Clinical Populations Is Critical for Detecting Pharmacological Outcomes
Weize Huang, Lindsay C. Czuba, Jennifer A. Manuzak, Jeffrey N. Martin, Peter W. Hunt, Nichole R. Klatt, and Nina Isoherranen
Cannabis and Cannabinoid Research Nov 2021
DOI: 10.1089/can.2021.0068
https://www.liebertpub.com/doi/10.1089/can.2021.0068
Introduction: Cannabis is widely used for recreational and medical purposes, but its therapeutic efficacy remains unresolved for many applications as data from retrospective studies show dramatic discrepancy. We hypothesized that false self-reporting of cannabis use and lack of differentiation of heavy users from light or occasional users contribute to the conflicting outcomes.
Objective: The goal of this study was to develop an objective biomarker of cannabis use and test how application of such biomarker impacts clinical study outcomes and dose–response measures.
Methods and Analysis: Population pharmacokinetic (PK) models of (–)-trans-Δ9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC) were developed based on published studies reporting cannabinoid disposition in individual subjects following intravenous administration or smoking of cannabis. Plasma 11-COOH-THC concentration distributions in different cannabis user groups smoking cannabis were generated via Monte Carlo simulations, and plasma concentration cutoff values of 11-COOH-THC were developed to differentiate light and heavy daily cannabis users in clinical studies. The developed cutoff value was then applied to a retrospective study that assessed the impact of cannabis use on T cell activation in subjects with HIV who self-reported as either nonuser or daily user of cannabis.
Results: The developed population PK models established plasma 11-COOH-THC concentration of 73.1 μg/L as a cutoff value to identify heavy daily users, with a positive predictive value of 80% in a mixed population of equal proportions of once daily and three times a day users. The stratification allowed detection of changes in T cell activation in heavy users which was not detected based on self-reporting or detectability of plasma cannabinoids. A proof-of-concept power analysis demonstrated that implementation of such cutoff value greatly increases study power and sensitivity to detect pharmacological effects of cannabis use.
Conclusions: This study shows that the use of plasma 11-COOH-THC concentration cutoff value as an objective measure to classify cannabis use in target populations is critical for study sensitivity and specificity and provides much needed clarity for addressing dose–response relationships and therapeutic effects of cannabis.
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On the preparations of the Indian hemp (Cannabis indica); their effects on the animal system in health, and their utility in the treatment of tetanus and other convulsive diseases.
Transactions of the Medical and Physical Society, Bengal 71– 102, 421–426 (1843) O’Shaughnessy, W.B.
DOI: 10.1136/bmj.s1-5.123.363
Such was the amount of preliminary information before me, by which I was guided in my subsequent attempts to gain more accurate knowledge of the action, powers, and possible medicinal applications of this extraordinary agent. There was sufficient to show that hemp possesses, in small doses, an extraordinary power of stimulating the digestive organs, exciting the cerebral system, of acting also on the generative apparatus. Larger doses, again, were shown by the historical statements to induce insensibility or to act as a powerful sedative. The influence of the drug in allaying pain was equally manifest in all the memoirs referred to. As to the evil sequels so unanimously dwelt on by all writers, these did not appear to me so numerous, so immediate, or so formidable, as many which may be clearly traced to over-indulgence in other powerful stimulants or narcotics-viz, alcohol, opium, or tobacco. The dose in which the hemp preparations might be administered, constituted, of course, one of the first objects of inquiry. Ibn Beitar had mentioned adirem, or forty-eight grains of churrmu; but this dose seemed to me so enormous, that I deemed it expedient to proceed
with much smaller quantities. How fortunate was this caution, the sequel will sufficiently denote. An extensive series of experiments on animals was in the first place undertaken, among which the following may be cited:

XXXV.—On the products of the action of nitric acid on the resinous extract of Indian hemp.
Bolas, T., & Francis, E. E. H. (1869).
. Chem. Soc., 22(0), 417–419.
doi:10.1039/js8692200417
FOUR ounces of the commercial resinous extract of Indian hemp (Cannabis indica) were treated with 30 02. of nitiic acid, sp. gr. 1-32 ; a violent action accompanied by a copious evolution of nitrous fumes took place. When the action had in a great measure subsided, the flask was heated for five hours in a waterbath; it was then taken out and allowed to cool ; when cold, the acid liquor was decanted from a resinous substance, and evaporated to dryness on the water-bath. The resinous substance which remained in the flask was treated with 20 oz. of nitric acid, sp. gr. 1.42, and boiled for eight hours in a longnecked flask. After having stood all night, the clear acid liquor was poured into the basin containing the residue from the first evaporation ; it wag then evaporated to dryless on the waterbath. A reainous portion remaining in the flnfilk was again treated with nitric acid, and so on until the whole of the resinous substance well dissolved. The substance obtained by. the evaporation of the acid liquors was diasolved in warm nitric acid, sp. gr. 1.42, evaporated a little, and allowed to cool. The substance which was deposited was washed with cold water and dissolved in hot methylated spirit, &om which it crystallised in long flat prisme. It was purified by two recrystallisations from spirit.

Optical and spectroscopic characterization of crystalline structures in cannabis extracts
Otyllia R. Abraham, Ruth Waddell Smith
Journal of Forensic Sciences Nov 2021
DOI: 10.1111/1556-4029.14940
https://onlinelibrary.wiley.com/doi/...556-4029.14940
Marijuana and hemp represent two broad classes of Cannabis sativa plants that are distinguished based on the concentration of the psychoactive cannabinoid delta-9-tetrahydrocannabinol (Δ9-THC). In this work, solvent extracts derived from marijuana and hemp were characterized using optical and spectroscopic techniques. The crystalline components of the solvent extracts were first analyzed using polarized light microscopy to determine optical properties, namely, crystal system, optical sign, and principle refractive indices. Crystals from the marijuana-derived extracts exhibited an orthorhombic crystal system and were optically negative, with nβ between 1.6320 and 1.6330 ± 0.0002. In contrast, crystals from hemp-derived extracts exhibited a monoclinic crystal system and were optically positive, with nβ between 1.600 and 1.6040 ± 0.0002. Crystals were further distinguished through infrared spectroscopy, which highlighted structural differences between the two sample types, primarily based on differences in O-H stretching. Finally, single-crystal X-ray diffraction was used to definitively identify the crystalline components, confirming the presence of tetrahydrocannabinolic acid in marijuana-derived extracts and cannabidiol in hemp-derived extracts. Given the differences in crystal structure identified between marijuana-derived and hemp-derived solvent extracts, optical characterization provides a screening method to differentiate visually similar samples prior to confirmatory analysis

Optimal condition of cannabis maceration to obtain the high cannabidiol and ?9 -tetrahydrocannabinol content
CHAOWALIT MONTON, FAMEERA MADAKA, SUKANYA SETTHARAKSA, THANIYA WUNNAKUP, JIRAPORNCHAI SUKSAEREE and THANAPAT SONGSAK
Anais da Academia Brasileira de Ciências (2019) 91(3)
The aim of this work was to optimize a maceration condition of cannabis (Cannabis sativa L.). A circumscribed central composite experimental design was applied in this work. Temperature and time were varied from 40-80 °C and 30-90 min, respectively. The three responses (i.e., extraction yield, cannabidiol content, and ?9- tetrahydrocannabinol content) were predicted by computer software. The yield was high when cannabis was macerated using ethanol at high temperature and long duration time. While cannabidiol and ?9- tetrahydrocannabinol content was high when macerating at a low heating temperature and short duration time. The optimal condition provided the simultaneous high of cannabidiol and ?9- tetrahydrocannabinol content was 40 °C for 30 min. The prediction was accurate due to low percent error. This optimal condition could be used as a guide for maceration of cannabis to obtain the extract containing a high content of cannabidiol and ?9- tetrahydrocannabinol.

Optimisation and characterisation of marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction and retention time locking GC-MS
Jone Omar, Maitane Olivares, Mikel Alzaga, Nestor Etxebarria
J. Sep. Sci. 2013, 36, 1397–1404
doi: 10.1002/jssc.201201103
The optimisation of focused ultrasound extraction and supercritical fluid extraction of volatile oils and cannabinoids from marihuana has been accomplished by experimental design approach. On the one hand, the focused ultrasound extraction method of volatile compounds and cannabinoids was studied based on the optimisation of cyclohexane and isopropanol solvent mixtures, and the instrumental variables. The optimal working conditions were finally fixed at isopropanol/cyclohexane 1:1 mixture, cycles (3 s?1), amplitude (80%) and sonication time (5 min). On the other hand, the supercritical fluid extraction method was optimised in order to obtain a deterpenation of the plant and a subsequent cannabinoid extraction. For this purpose, pressure, temperature, flow and co-solvent percentage were optimised and the optimal working conditions were set at 100 bar, 35C, 1 mL/min, no co-solvent for the terpenes and 20% of ethanol for the cannabinoids. Based on the retention time locking GC-MS analysis of the supercritical fluid extracts the classification of the samples according to the type of plant, the growing area and season was attained. Finally, three monoterpenes and three cannabinoids were quantified in the ranges of 0.006–6.2 g/g and 0.96–324 mg/g, respectively.

Optimization of Cannabis Grows Using Fourier Transform Mid-Infrared Spectroscopy
Brian C. Smith, Mark A. Lewis, Judah Mendez
Perkin-Elmer Inc. 2016
https://cdn.technologynetworks.com/t...ectroscopy.pdf

Origin of ?9-Tetrahydrocannabinol Impurity in Synthetic Cannabidiol.
Citti, C., Russo, F., Linciano, P., Strallhofer, S. S., Tolomeo, F., Forni, F., … Cannazza, G.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0021
Introduction: Cannabidiol (CBD), the nonintoxicating constituent of cannabis, is largely employed for pharmaceutical and cosmetic purposes. CBD can be extracted from the plant or chemically synthesized. Impurities of psychotropic cannabinoids D9 -tetrahydrocannabinol (D9 -THC) and D8 -THC have been found in extracted CBD, thus hypothesizing a possible contamination from the plant. Materials and Methods: In this study, synthetic and extracted CBD samples were analyzed by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry and the parameters that can be responsible of the conversion of CBD into THC were evaluated by an accelerated stability test. Results: In synthetic and extracted CBD no trace of THC species was detected. In contrast, CBD samples stored in the dark at room temperature on the benchtop for 3 months showed the presence of such impurities. Experiments carried out under inert atmosphere in the absence of humidity or carbon dioxide led to no trace of THC over time even at high temperature. Conclusions: The results suggested that the copresence of carbon dioxide and water from the air could be the key for creating the acidic environment responsible for the cyclization of CBD. These findings suggest that it might be appropriate to review the storage conditions indicated on the label of commercially available CBD.

Over eight hundred cannabis strains characterized by the relationship between their psychoactive effects, perceptual profiles, and chemical compositions
Alethia de la Fuente, Federico Zamberlan, Andrés Sánchez Ferrán, Facundo Carrillo, Enzo Tagliazucchi, Carla Pallavicini
bioRxiv.org
DOI: 10.13140/RG.2.2.19760.92165
Commercial cannabis strains have multiplied in recent years as a consequence of regional changes in legislation for medicinal and recreational use. The lack of a standardized system to label plants and seeds hinders the consistent identification of particular strains with their elicited psychoactive effects. We analyzed a large publicly available dataset where users freely reported their experience with different strains, including subjective effects and flavour associations. Metrics of strain similarity based on self-reported effects and flavour tags allowed machine learning classification into three major clusters associated with species (Cannabis sativa, Cannabis indica, and hybrids). Synergy between terpene and cannabinoid content was suggested by significative correlations between psychoactive effects and flavour tags. The use of predefined tags was validated by the application of Latent Semantic Analysis (LSA) to unstructured written reviews, also providing breed-specific topics consistent with their purported medicinal and subjective effects. While cannabinoid content was variable even within individual strains, terpene profiles matched the perceptual characterizations made by the users and could be used to predict psychoactive effects. Our work represents the first data-driven synthesis of self-reported and chemical information on a large number of cannabis strains. Since terpene content is robustly inherited and less influenced by environmental factors, flavour perception could represent a reliable marker for the prediction of psychoactive effects of cannabis. Our novel methodology contributes towards the demands for reliable strain classification and characterization in the context of an ever-growing market for medicinal and recreational cannabis.

Pesticide Analysis at Pace
Lauren Robertson | 03/31/2022 | Interview
https://thecannabisscientist.com/tes...alysis-at-pace
Application chemist Kirk Jensen tells us how a low-pressure gas chromatography kit and short collision cell technology helped him measure 244 pesticides in 11 minutes
Speed is king in the world of pesticide analysis; as many lab managers know, being able to process more samples in a shorter time frame – while maintaining data quality – can be invaluable in high-throughput applications. Current gas chromatography-tandem mass spectrometry (GC-MS/MS) methods are certainly sensitive, but they require longer analysis times to effectively separate complex mixtures. Feeling the need for speed, Kirk Jensen, Robert “Chip” Cody, and John Dane from JEOL decided to test an approach using a low-pressure GC (LPGC) kit (Restek) with the enhanced selected reaction monitoring (SRM) switching speed of the short collision cell in a GC-triple quadrupole MS system (JMS-TQ4000GC, JEOL)
The result? Three transitions for each of 244 pesticides were measured in a standard mixture in just 11 minutes. We spoke to Kirk Jensen to find out a little more about his work.

Pesticides in cannabis: A review of analytical and toxicological considerations
Amelia Taylor BSc, & Jason W. Birkett
Drug Testing and Analysis December 2019
DOI: 10.1002/dta.2747
A review of the literature surrounding the use, analysis and detection of pesticide material for cannabis cultivation is presented. The use of pesticides in crop cultivation is not new, and cannabis crops are no exception. Studies have found that the use of these are common and that high levels of the pesticides are transferred into the cannabis smoke. The most common pesticides classes associated with cannabis are insecticides, acaricides and fungicides. Over 350 different pesticide products may be used on cannabis materials and of these, 16 pesticides and 3 plant growth regulators (PGR) are considered to be the main candidates. Many of the pesticides found in cannabis samples destined for consumption are classed as moderately hazardous by the World Health Organisation. Analytical methods for pesticide detection on cannabis are being developed with a view to implement quality control to cannabis where it is legal before being sold. However, no standardised protocol exists. The pesticide levels found in the cannabis samples tested were generally low (less than ?g·g-1 ), these results do not however provide information on chronic low-dose adverse effects of pesticides in relation to cannabis consumption. Currently no research exists on the toxicity of pyrolysed pesticides in humans from smoking cannabis. More studies are needed to further understand this potentially harmful health threat.

Pharmaceutical and biomedical analysis of cannabis
extracts: a critical review
Cinzia Citti, Daniela Braghiroli, Maria Angela
Vandelli, Giuseppe Cannazza
Journal of Pharmaceutical and Biomedical Analysis 147, June 2017
doi:10.1016/j.jpba.2017.06.003
Cannabis products have recently regained much attention due to the high pharmacological potential of their cannabinoid content. In this review, the most widely used sample preparation strategies for the extraction of cannabinoids are described for the specific application to either plant materials or biological matrices. Several analytical techniques are described pointing out their respective advantages and drawbacks. In particular, chromatographic methods, such as TLC, GC and HPLC, are discussed and compared in terms of selectivity and sensitivity. Various detection methods are also presented based on the specific aim of the cannabinoids analysis. Lastly, critical considerations are mentioned with the aim to deliver useful suggestions for the selection of the optimal and most suitable method of analysis of cannabinoids in either biomedical or cannabis derived samples.

Pharmacokinetic Profile of Oral Cannabis in Humans: Blood and Oral Fluid Disposition and Relation to Pharmacodynamic Outcomes
Ryan Vandrey, Evan S. Herrmann, John M. Mitchell, George E. Bigelow, Ronald Flegel, Charles LoDico, and Edward J. Cone
J Anal Toxicol. 2017 Mar; 41(2): 83–99.
doi: 10.1093/jat/bkx012
Most research on cannabis pharmacokinetics has evaluated inhaled cannabis, but oral (“edible”) preparations comprise an increasing segment of the cannabis market. To assess oral cannabis pharmacokinetics and pharmacodynamics, healthy adults (N = 6 per dose) were administered cannabis brownies containing 10, 25 or 50 mg 9-tetrahydrocannabinol (THC). Whole blood and oral fluid specimens were obtained at baseline and then for 9 days post-exposure; 6 days in a residential research setting and 3 days as outpatients. Measures of subjective, cardiovascular and performance effects were obtained at baseline and for 8 h post-ingestion. The mean Cmax for THC in whole blood was 1, 3.5 and 3.3 ng/mL for the 10, 25 and 50 mg THC doses, respectively. The mean maximum concentration (Cmax) and mean time to maximum concentration (Tmax) of 11-OH-THC in whole blood were similar to THC. Cmax blood concentrations of THCCOOH were generally higher than THC and had longer Tmax values. The mean Tmax for THC in oral fluid occurred immediately following oral dose administration, and appear to reflect local topical residue rather than systemic bioavailbility. Mean Cmax oral fluid concentrations of THCCOOH were lower than THC, erratic over time and mean Tmax occurred at longer times than THC. The window of THC detection ranged from 0 to 22 h for whole blood (limit of quantitation (LOQ) = 0.5 ng/mL) and 1.9 to 22 h for oral fluid (LOQ = 1.0 ng/mL). Subjective drug and cognitive performance effects were generally dose dependent, peaked at 1.5–3 h post-administration, and lasted 6–8 h. Whole blood cannabinoid concentrations were significantly correlated with subjective drug effects. Correlations between blood cannabinoids and cognitive performance measures, and between oral fluid and all pharmacodynamic outcomes were either non-significant or not orderly by dose. Quantitative levels of cannabinoids in whole blood and oral fluid were low compared with levels observed following inhalation of cannabis. The route of administration is important for interpretation of cannabinoid toxicology.

Photochemical efficiency is negatively correlated with the ?9- tetrahydrocannabinol content in Cannabis sativa L.
Khajuria, M., Rahul, V. P., & Vyas, D.
Plant Physiology and Biochemistry.(2020).
doi:10.1016/j.plaphy.2020.04.003
Cannabis sativa L is an important plant, which is a source of durable fibers, nutritious seeds, and medicinally important phytocannabinoids including ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Light has shown to be a key modulator of biomass and cannabinoid yield
suggesting responsive photochemical machinery. The present study was envisaged to understand the effect of the increasing levels of metabolic THC on the photochemical efficiency in Cannabis. The chlorophyll a fluorescence kinetics, photosynthetic pigments, immuno-detection of the photosynthetic machinery was analyzed on seven accessions from different environments, in conjunction with the cannabinoid content. All the accessions were clearly divided into three groups based on their relative content of CBD and THC. Group I with (CBD/THC > 1) had a clear advantage in terms of the damage to the D1, RbCL and Lhc1 protein
holo-complex. Performance indicators of photochemistry based on the OJIP kinetics suggested a stoichiometrically negative correlation with the THC content. Zeaxanthin-dependent quenching is primarily responsible for lower NPQ in Group III with high THC content (THC > 6%).
The THC treatement on Arabisopsis thaliana also suggested dose-dependent decrease in the photochemical efficiency suggesting the exclusivity of THC in causing the response. This resulted in the damage of photosynthetic machinery and the generation of free radicals, thereby compromising the yield. The study opens a new screening method for Cannabis, based on cannabinoid content.

Phytocannabinomics: Untargeted metabolomics as a tool for cannabis chemovar differentiation
Andrea Cerratoa1Cinzia Cittibc1Giuseppe CannazzabcAnna Laura Capriottia Chiara Cavalierea Giampaolo Grassid Federico Marinia Carmela Maria Montonea Roberta Parise, Aldo Laganà
Talanta Volume 230, 1 August 2021, 122313
DOI: 10.1016/j.talanta.2021.122313
Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.
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Phytocannabinoids beyond the Cannabis plant - do they exist?
Gertsch, J., Pertwee, R. G., & Di Marzo, V.
British Journal of Pharmacology, 160(3), 523–529.(2010).
doi:10.1111/j.1476-5381.2010.00745.x
It is intriguing that during human cultural evolution man has detected plant natural products that appear to target key protein receptors of important physiological systems rather selectively. Plants containing such secondary metabolites usually belong to unique chemotaxa, induce potent pharmacological effects and have typically been used for recreational and medicinal purposes or as poisons. Cannabis sativa L. has a long history as a medicinal plant and was fundamental in the discovery of the endocannabinoid system. The major psychoactive Cannabis constituent D9 -tetrahydrocannabinol (D9 -THC) potently activates the G-protein-coupled cannabinoid receptor CB1 and also modulates the cannabinoid receptor CB2. In the last few years, several other non-cannabinoid plant constituents have been reported to bind to and functionally interact with CB receptors. Moreover, certain plant natural products, from both Cannabis and other plants, also target other proteins of the endocannabinoid system, such as hydrolytic enzymes that control endocannabinoid levels. In this commentary we summarize and critically discuss recent findings.

Phytochemical and ecological analysis of two varieties of hemp (Cannabis sativa L.) grown in a mountain environment of Italian Alps
Radmila Pavlovic, Sara Panseri, Luca Giupponi, Valeria Leoni, Cinzia Citti, Chiara Cattaneo, Maria Cavaletto, Anna M. Giorgi
Plant Sci. 10:1265. doi:10.3389/fpls.2019.01265
Hemp (Cannabis sativa L.) is a multifunctional crop that is capable of prompt environmental adaptation. In this study, a monoecious cultivar (Futura 75) and a dioecious one (Finola) were tested in a mountain area in Valsaviore (Rhaetian Alps, Italy; elevation: 1.100 m a.s.l.) during the growing season 2018. Phytochemical behaviour was evaluated by different analytical approaches: HPLC-high-resolution mass spectrometry, SDS-PAGE LC-MS/MS, HS-SPME GC-MS and GC-FID in order to obtain complete profile of two varieties cultivated in altitude. CSR functional strategy used for ecological evaluation revealed that both genotypes are mainly competitors although Finola is more stress tolerator (C:S:R = 57:26:17%) than Futura (C:S:R = 69:15:16%). The Finola inflorescences were characterised by higher quantities of ?-ocimene and ?-terpinolene, while ?- and ?-pinene accompanied by extremely high ?-myrcene were found as predominant in Futura. Both varieties were rich in sesquiterpenes (45 recognized) among which trans-caryophyllene and ?-humulene were the most abundant. Total tetrahydrocannabinol level was lower than 0.1% while the most abundant cannabinoid was cannabidiolic acid (CBDA): 2.3% found in Finola vs 2.7% revealed for Futura. The level of corresponding neutral form, cannabidiol, varied drastically: 0.27% (Finola) vs 0.056% (Futura). Finola showed the unique cannabinoid profile with unexpectedly high cannabidivarin, 2-fold higher that corresponding acidic analogue, whereas the particularity of Futura 75 was the occurrence of cannabigerolic acid (CBGA) in the quantities that was double than those exposed for Finola. The seeds from both chemovars proved to be rich in polyunsaturated fatty acids and Finola showed a higher ratio ?6/?3. No difference was found in the protein content and the SDS-PAGE profile was similar. The most abundant protein was edestin, followed by heat shock protein 70, ?-conglycinin and vicilin. In conclusion, comprehensive phytochemical and ecological study of two fibre-type varieties cultivated in Italian Alps displayed specific, legal and safe, cannabinoids profile, followed by particular terpene composition, polyunsaturated fatty acids content and favourable protein profile. This postulates that geographical provenience of hemp should be considered in selecting a variety that would be suitable for a specific end-use nutraceutical application

Pitfalls in the analysis of phytocannabinoids in cannabis inflorescence.
Cinzia Citti, Fabiana Russo, Salvatore Sgrò, Alfonso Gallo, Antonio Zanotto, Flavio Forni, Maria Angela Vandelli, Aldo Laganà, Carmela Maria Montone, Giuseppe Gigli, Giuseppe Cannazza
Analytical and Bioanalytical Chemistry.(2020).
doi:10.1007/s00216-020-02554-3
The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: ?9 -tetrahydrocannabinol (?9 -THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist.

Plasma concentrations of eleven cannabinoids in cattle following oral administration of industrial hemp (Cannabis sativa).
Kleinhenz, M. D., Magnin, G., Lin, Z., Griffin, J., Kleinhenz, K. E., Montgomery, S., … Coetzee, J. F.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-69768-4
Cannabinoid production for medicinal purposes has renewed interest in utilizing byproducts of industrial hemp (IH) as a feed source for livestock. However, the presence of bioactive residues in animal tissues may pose a risk to consumers. The purpose of this study was to characterize the plasma pharmacokinetics (PK) of cannabinoids and their metabolites in cattle after a single oral exposure to IH. Eight castrated male Holstein calves received a single oral dose of 35 g of IH to achieve a target dose of 5.4 mg/kg cannabidiolic acid (CBDA). Blood samples were collected for 96 h after dosing. Plasma cannabinoid concentrations were profled using liquid chromatography coupled with massspectroscopy (UPLC) and PK parameters were calculated using noncompartmental methods. The cannabinoids CBDA, tetrahydrocannabinolic acid-A (THCA-A), cannabidivarinic acid (CBDVA), and cannabichromenic acid (CBCA) were detected in all cattle after IH dosing. The geometric mean maximum concentration of CBDA of 72.7 ng/mL was observed at 14 h after administration. The geometric mean half-life of CBDA was 14.1 h. No changes in serum biochemistry analysis were observed following IH dosing compared to baseline values. These results show acidic cannabinoids, especially CBDA, are readily absorbed from the rumen and available for distribution throughout the body.

Preliminary data on the potential for unintentional anti?doping rule violations by permitted CBD use.
Mareck, U., Fusshöller, G., Geyer, H., Huestis, M. A., Scheiff, A. B., & Thevis, M.
Drug Testing and Analysis.(2020).
doi:10.1002/dta.2959
According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9- carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/mL. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA’s Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of anti-doping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography / tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the Minimum Required Performance Level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially-available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete´s in-competition urine sample represents a substantial risk of an anti-doping rule violation.

PROCEEDINGS OF THE CANNABIS CHEMISTRY SUBDIVISION
Assembled and Edited by Nigam B. Arora
https://cdn.technologynetworks.com/a...sfp=3419431105
Pg 5 Degradant Formation in Cannabis Concentrate Aerosols
Pg 6 Combined LC-DAD-MS/MS and FT-IR Platform for the Analysis of Cannabis and Cannabis-related Products
Pg 8 Homogeneity, Formulation, and Shelf Life of Cannabis-infused Beverage Emulsions
Pg 9 FEE, QbD, HACCP, GMP, OSHA: Industry Jargon or Hidden Treasures
Pg 10 Raising the Bar for Cannabis Extraction Methods: Introducing a Novel, Safe, Efficient, and Environmentally Friendly Approach to Extracting High Quality Cannabis Resins
Pg 11 HPLC-UV Method Development for the Baseline Resolution of 17 Cannabinoids
Pg 12 Cannabis Product Critical Safety Attributes, Critical Quality Attributes, and Good Distribution Practice
Pg 13 How to Create a Consistent Water Soluble Cannabinoid Bulk Ingredient
Pg 14 Fundamental Research for In-process Analytics to Control Cannabis Formulations
Pg 18 Scaling Production for a GMP Cannabis Facility
Pg 19 AuditProHBX, a Tool for Regulatory Compliance in a Field That Needs Modern Tools to Flourish
Pg 20 Extraction Methods and Their Effect on Terpene Retention
Pg 21 Establishing Robust Quality-based Systems for Cannabis Production
Pg 22 Investigation of Matrix Effects in Cannabis-infused Chocolates

Quality Control of Traditional Cannabis Tinctures: Pattern, Markers, and Stability
Wieland Peschel
Sci. Pharm. 2016, 84, 567–584
DOI: 10.3390/scipharm84030567
Traditional tinctures of Cannabis sativa L. became obsolete before elucidation of the main cannabinoids and routine quality testing for medicines. In view of increasing medicinal use of cannabinoids and associated safety concerns, tinctures from a ?9-tetrahydrocannabinol (THC)-type chemovar were studied. High-performance liquid chromatography with diode-array detection (HPLC/DAD) was used to determine THC, ?9-tetrahydrocannabinolic acid A (THCA), cannabinol (CBN), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannflavin A/B, and total phenolics. Derived group and ratio markers describe absolute and relative profiles when varying plant part (flos, folium), extraction solvent (EtOH percentage), storage conditions (‘shelf’ or ‘fridge’ up to 15 months), and pasteurization (2 h 70 ?C, 20 min 80 ?C). Tinctures from female flowering tops contained ten-fold more cannabinoids than tinctures from leaves; tinctures (80%–90% EtOH) contained ten-fold more cannabinoids than tinctures (40% EtOH). The analysis of CBGA + CBG, the main co-cannabinoids aside from THCA + THC, appears more relevant than CBDA + CBD. The decarboxylation of THCA to THC—the main change during storage of freshly prepared tinctures—is after 15 months in the ‘fridge’ comparable to 3 months on the ‘shelf’. Minimally increased CBN totals did not correlate to diminished totals of THCA and THC (up to 15% after 3 months ‘shelf’, 45% after 15 months ‘fridge’). Instead, total cannabinoids or acidic/neutral cannabinoid ratios are better stability markers. Moderate changes after pasteurization and partial losses below 10% for total cannabinoids after 9 months ‘fridge’ indicate possibilities for a reasonable shelf life. Yet storage and use of non-stabilized tinctures remain critical without authorized specification and stability data because a consistent cannabinoid content is not guaranteed.

Quantitative analysis of cannabinoids using benchtop NMR instruments.
Araneda, J. F., Chu, T., Leclerc, M. C., Riegel, S. D., & Spingarn, N.
Analytical Methods.(2020).
doi:10.1039/d0ay01511c
The quantification of cannabinoids is an essential part of cannabis profiling and testing, whether for medical or recreational use. As regulatory bodies continue to increase testing requirements for these products, it is crucial that alternative and effective analytical methods be developed. Herein, we describe the use of benchtop NMR instruments for the quantification of D9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) in a variety of cannabis concentrates and compare the values to those obtained using HPLC, the most common approach for the quantification of cannabinoids. Based on the discrepancies observed in test values from different laboratories using only HPLC, the value of orthogonal testing methods has been identified and is increasingly desired.

Quantitative GC-MS Analysis of Delta9-Tetrahydrocannabinol in Fiber Hemp Varieties
A.K. Hewavitharana, G. Golding, G. Tempany, G. King, and N. Holling
Journal of Analytical Toxicology, Vol. 29, May/June 2005
DOI: 10.1093/jat/29.4.258
A sensitive and specific method for the determination of total available Ag-tetrahydrocannabinol in fiber hemp varieties is described. The method was used for the regulatory purposes in which the detection of higher than the maximum allowed concentration of the psychoactive cannabinoid, Ag-tetrahydrocannabinol, in industrial fiber hemp would result in cancellation of the grower's license. Cannabinoids were extracted from dry leaf powder into hexane containing internal standard chrysene-dl~ using sonication. Ag-Tetrahydrocannabinoi in the extract was separated by gas chromatography and quantitated by mass spectroscopy. A linear calibration range extending to 40 ppm and a limit of detection of 0.2 ng were obtained by using the total ion current mode of detection.

Regulatory sampling of industrial hemp plant samples (Cannabis sativa L.) using UPLC-MS/MS method for detection and quantification of twelve cannabinoids
Erin C. Berthold, Rui Yang, Abhisheak Sharma, Shyam H. Kamble, Siva R. Kanumuri, Tamara I. King, Raluca Popa, Joshua H. Freeman, Zachary T. Brym, Bonnie A. Avery, and Christopher R. McCurdy
Journal of Cannabis Research (2020) 2:42
DOI: 10.1186/s42238-020-00050-0
https://jcannabisresearch.biomedcentra l.com/track/pdf/10.1186/s42238-020-00050-0.pdf
Background: In 2018, the Farm Bill mandated the United States Department of Agriculture to develop regulations governing the cultivation, processing, and marketing of industrial hemp. Industrial hemp is defined as Cannabis sativa L. with a total Δ-9-tetrahydrocannabinol (Δ-9-THC) content ≤0.3%. Therefore, for hemp to become an agricultural commodity, it is important to regulate production by developing standard methods for sampling and testing of the plant material.
Methods: An ultra-performance liquid chromatography-tandem mass spectrometry analytical method for the quantification of twelve cannabinoids was developed. The method was applied to a regulatory sampling trial of three hemp varieties cultivated for cannabidiol (CBD) production. Two samples were taken from 28 plants with one sample being flower only while the other was a composite sample that included flowers, leaves, and stems.
Results: The assay method was validated for specificity, range, repeatability, reproducibility, and recovery in accordance with all applicable standards for analytical methods. The results of the regulatory study indicated a significant decrease in the concentration of total Δ-9-THC and total CBD of 0.09% and 1.32%, respectively, between a flower only and a composite sample.
Conclusions: There are many factors that may influence reported total Δ-9-THC content in industrial hemp. A robust analytical method was developed to analyze hemp samples in a trial regulatory study. The results indicate that the way hemp is sampled and analyzed may influence the legality of a crop, which could have negative economic and legal consequences.

Rapid front End Cleanup of Cannabis-Infused Edibles using Automated Flash Column Chromatography.
Marcu J, Wilcox M.
American Chemical Society 251st National Meeting & Exposition. 2016.
https://www.eurekalert.org/pub_relea...-ntc021916.php
https://bit.ly/ACSliveSanDiego.
FIND DOI or LINK

Regulatory status of pesticide residues in cannabis: Implications
to medical use in neurological diseases
Dorina V. Pinkhasova, Laura E. Jameson, Kendra D. Conrow, Michael P. Simeone, Allan Peter Davis, Thomas C. Wiegers, Carolyn J. Mattingly, Maxwell C.K. Leung
Current Research in Toxicology 2 (2021) 140–148
DOI: 10.1016/j.crtox.2021.02.007
Medical cannabis represents a potential route of pesticide exposure to susceptible populations. We compared the qualifying conditions for medical use and pesticide testing requirements of cannabis in 33 states and Washington, D.C. Movement disorders were the most common neurological category of qualifying conditions, including epilepsy, certain symptoms of multiple sclerosis, Parkinson’s Disease, and any cause of symptoms leading to seizures or spasticity. Different approaches of pesticide regulation were implemented in cannabis and cannabisderived products. Six states imposed the strictest U.S. EPA tolerances (i.e. maximum residue levels) for food commodities on up to 400 pesticidal active ingredients in cannabis, while pesticide testing was optional in three states. Dimethomorph showed the largest variation in action levels, ranging from 0.1 to 60 ppm in 5 states. We evaluated the potential connections between insecticides, cannabinoids, and seizure using the Comparative Toxicogenomics Database. Twentytwo insecticides, two cannabinoids, and 63 genes were associated with 674 computationally generated chemicalgenephenotypedisease (CGPD) tetramer constructs. Notable functional clusters included oxidationreduction process (183 CGPDtetramers), synaptic signaling pathways (151), and neuropeptide hormone activity (46). Cholinergic, dopaminergic, and retrograde endocannabinoid signaling pathways were linked to 10 genetic variants of epilepsy patients. Further research is needed to assess human health risk of cannabinoids and pesticides in support of a national standard for cannabis pesticide regulations.

Relationship among subjective responses, flavor, and chemical composition across more than 800 commercial cannabis varieties.
De la Fuente, A., Zamberlan, F., Sánchez Ferrán, A., Carrillo, F., Tagliazucchi, E., & Pallavicini, C.
Journal of Cannabis Research, 2(1). (2020).
doi:10.1186/s42238-020-00028-y
Background: Widespread commercialization of cannabis has led to the introduction of brand names based on users’ subjective experience of psychological effects and flavors, but this process has occurred in the absence of agreed standards. The objective of this work was to leverage information extracted from large databases to evaluate the consistency and validity of these subjective reports, and to determine their correlation with the reported cultivars and with estimates of their chemical composition (delta-9-THC, CBD, terpenes).
Methods: We analyzed a large publicly available dataset extracted from Leafly.com where users freely reported their experiences with cannabis cultivars, including different subjective effects and flavour associations. This analysis was complemented with information on the chemical composition of a subset of the cultivars extracted from Psilabs.org. The structure of this dataset was investigated using network analysis applied to the pairwise similarities between reported subjective effects and/or chemical compositions. Random forest classifiers were used to evaluate whether reports of flavours and subjective effects could identify the labelled species cultivar. We applied Natural Language Processing (NLP) tools to free narratives written by the users to validate the subjective effect and flavour tags. Finally, we explored the relationship between terpenoid content, cannabinoid composition and subjective reports in a subset of the cultivars.
Results: Machine learning classifiers distinguished between species tags given by “Cannabis sativa” and “Cannabis indica” based on the reported flavours: = 0.828 ± 0.002 (p < 0.001); and effects: = 0.9965 ± 0.0002 (p < 0.001). A significant relationship between terpene and cannabinoid content was suggested by positive correlations between subjective effect and flavour tags (p < 0.05, False-Discovery-rate (FDR)-corrected); these correlations clustered the reported effects into three groups that represented unpleasant, stimulant and soothing effects. The use of predefined tags was validated by applying latent semantic analysis tools to unstructured written reviews, also providing breed-specific topics consistent with their purported subjective effects. Terpene profiles matched the perceptual characterizations made by the users, particularly for the terpene-flavours graph (Q = 0.324).
Conclusions: Our work represents the first data-driven synthesis of self-reported and chemical information in a large number of cannabis cultivars. Since terpene content is robustly inherited and less influenced by environmental factors, flavour perception could represent a reliable marker to indirectly characterize the psychoactive effects of cannabis. Our novel methodology helps meet demands for reliable cultivar characterization in the context of an ever-growing market for medicinal and recreational cannabis.

Not Cannabis specific
Selectivity of Current Extraction Techniques for Flavonoids from Plant Materials.
Tzanova, M.; Atanasov, V.; Yaneva, Z.; Ivanova, D.; Dinev, T.
Processes 2020, 8, 1222.
DOI: 10.3390/pr8101222
Flavonoids have a broad spectrum of established positive effects on human and animal health. They find an application in medicine for disease therapy and chemoprevention, whence the interest in flavonoids increases. In addition, they are used in food and cosmetic industries as pigments and biopreservatives. Plants are an inexhaustible source of flavonoids. The most important step of plant raw material processing is extraction and isolation of target compounds. The quality of an extract and efficiency of a procedure are influenced by several factors: Plant material and pre-extracting sample preparation, type of solvent, extraction technique, physicochemical conditions, etc. The present overview discusses the common problems and key challenges of the extraction procedures and the different mechanisms for selective extraction of flavonoids from different plant sources. In summary, there is no universal extraction method and each optimized procedure is individual for the respective plants. For an extraction technique to be selective, it must combine an optimal solvent or mixture of solvents with an appropriate technique. Last but not least, its optimization is important for a variety of applications. Moreover, when the selected method needs to be standardized, it must achieve acceptable degree of repeatability and reproducibility.

Screening for More than 1,000 Pesticides and Environmental Contaminants in Cannabis by GC/Q-TOF.
Wylie, P. L., Westland, J., Wang, M., Radwan, M. M., Majumdar, C. G., & ElSohly, M. A.
Medical Cannabis and Cannabinoids, 1–11. (2020).
doi:10.1159/000504391
A method has been developed to screen cannabis extracts for more than 1,000 pesticides and environmental pollutants using a gas chromatograph coupled to a high-resolution accurate mass quadrupole time-of-flight mass spectrometer (GC/Q-TOF). An extraction procedure was developed using acetonitrile with solid phase extraction cleanup. Before analysis, extracts were diluted 125:1 with solvent. Two data mining approaches were used together with a retention-timelocked Personal Compound Database and Library (PCDL) containing high-resolution accurate mass spectra for pesticides and other environmental pollutants. (1) A Find-byFragments (FbF) software tool extracts several characteristic exact mass ions within a small retention time window where the compound elutes. For each compound in the PCDL, the software evaluates the peak shape and retention time of each ion as well as the monoisotopic exact mass, ion ratios, and other factors to decide if the compound is present or not. (2) A separate approach used Unknowns Analysis (UA) software with a peak-finding algorithm called SureMass to deconvolute peaks in the chromatogram. The accurate mass spectra were searched against the PCDL using spectral matching and retention time as filters. A subset PCDL was generated containing only pesticides that are most likely to be found on foods in the US. With about 250 compounds in the smaller PCDL, there were fewer hits for non-pesticides, and data review was much faster. Organically grown cannabis was used for method development. Twenty-one confiscated cannabis samples were analyzed and ten were found to have no detectable pesticides. The remaining 11 samples had at least one pesticide and one sample had seven detectable residues. Quantitative analysis was run on the confiscated samples for a subset of the pesticides found by screening. Two cannabis samples had residues of carbaryl and malathion that were estimated to be about 10 times greater than the highest US Environmental Protection Agency tolerance set for food and about 4,000 times greater than the Canadian maximum residue limits for dried cannabis flower.

Simultaneous determination of terpenes and cannabidiol in hemp ( Cannabis sativa L.) by fast Gas Chromatography with Flame Ionization Detection.
Bakro, F., Jedryczka, M., Wielgusz, K., Sgorbini, B., Inchingolo, R., & Cardenia, V.
Journal of Separation Science. (2020).
doi:10.1002/jssc.201900822
Hemp (Cannabis sativa L.) has become widely used in several sectors due to the presence of various bioactive compounds such as terpenes and cannabidiol. In general, terpenes and cannabidiol content is determined separately which is time-consuming. Thus, a fast Gas Chromatography with Flame Ionization Detection method was validated for simultaneous determination of both terpenes and cannabidiol in hemp. The method enabled a rapid detection of 29 different terpenes and cannabidiol within a total analysis time of 16 min, with satisfactory sensitivity (LOD = 0.03 – 0.27 ?g/mL, LOQ = 0.10 – 0.89 ?g/mL). The interday and intraday precision (RSD) was <7.82% and <3.59% respectively. Recoveries at two spiked concentration levels (low, 3.15 µg/mL; high, 20.0 µg/mL) were determined on both apical leaves (78.55 – 101.52 %) and inflorescences (77.52 - 107.10 %). The reproducibility (RSD) was <5.94% and <5.51% in apical leaves and inflorescences, respectively. The proposed and validated method is highly sensitive, robust, fast, and accurate for determination of the main terpenes and cannabidiol in hemp and could be routinely used for quality control.

Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzymealkaline hydrolysis
Oier Aizpurua-Olaizola, Iratxe Zarandona, Laura Ortiz, Patricia Navarro,
Nestor Etxebarria and Aresatz Usobiaga
Drug Test. Analysis 2017, 9, 626–633
DOI 10.1002/dta.1998
A high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of ?9-tetrahydrocannabinol (THC), its two metabolites 11-hydroxy-?9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-?9-tetrahydrocannabinol (THC-COOH), and four additional cannabinoids (cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), and cannabinol (CBN)) in 1mL of human urine and plasma was developed and validated.
The hydrolysis process was studied to ensure complete hydrolysis of glucuronide conjugates and the extraction of a total amount
of analytes. Initially, urine and plasma blank samples were spiked with THC-COOH-glucuronide and THC-glucuronide, and four
different pretreatment methods were compared: hydrolysis-free method, enzymatic hydrolysis with Escherichia Coli
?-glucuronidase, alkaline hydrolysiswith 10MNaOH, and enzyme-alkaline tandemhydrolysis. The last approach assured themaximum
efficiencies (close to 100%) for both urine and plasmamatrices. Regarding the figures of merit, the limits of detection were
below 1 ng/mL for all analytes, the accuracy ranged from 84% to 115%, and both within-day and between-day precision were
lower than 12%. Finally, the method was successfully applied to real urine and plasma samples from cannabis users.

Stability of cannabinoids in dried samples of cannabis dating from around 1896–1905.
Harvey, D. J.
Journal of Ethnopharmacology, 28(1), 117–128. (1990).
doi:10.1016/0378-8741(90)90068-5
Cannabinoids from three samples of cannabis obtained from the Pitt-Rivers Museum, Oxford, and dating from the turn of the century were examined by gas chromatography and mass spectometry for the presence of cannabinoids. Although the samples were from different geographical locations, the profiles of constituent cannabinoids were similar. In common with other aged material, most of the cannabinoid content was present as cannabinol (CBN), the main chemical degradation product of the major psychoactive constituent, delta-9-tetrahydrocannabinol (delta-9-THC). However, a
substantial concentration of CBN acid-A was also present; this compound is unstable to heat and readily undergoes decarboxylation to CBN. Methyl and propyl homologues of CBN, together with delta-9-THC and its naturally occurring acid-A were also found at low concentrations in all samples. Intermediates in the formation of CBN from delta-9-THC, previously identified in aged solutions of the drug, were absent or present in only trace concentrations. However, oxidation products involving hydroxylation at the benzylic positions, C-11 and C-l’, not seen in solution, were identified in substantial abundance. The results suggest that decomposition of cannabis samples may proceed more slowly than originally thought.

Step toward Roadside Sensing: Noninvasive Detection of a THC Metabolite from the Sweat Content of Fingerprints
Erica Brunelle, Brenna Thibodeau, Alyssa Shoemaker, Jan Halámek
ACS Sens. 2019, 4, 12, 3318-3324
10.1021/acssensors.9b02020
The sudden increase in states legalizing marijuana has forced law enforcement into a situation where the use and consumption are legal, but there are no limitations for what is acceptable for driving or operating machinery. Using ultraviolet–visible (UV–vis) spectroscopy, fingerprints from volunteers who had used marijuana were analyzed via a competitive immunoassay for the detection of ?9-tetrahydrocannabinol (?9-THC), the main psychoactive component of marijuana, and 11-nor-9-carboxy-THC (THC-COOH), one of the main metabolites produced in the body following the use/consumption of THC-related products. In this research, the THC-COOH metabolite and the enzyme-labeled conjugate compete against each other as the antigens for the system. The antibody used in this assay has a greater affinity for the metabolite; so, as its concentration increases, the absorbance of the system decreases due to less binding of the enzyme-labeled conjugate.

Structure elucidation of the tetrahydrocannabinol complex with randomly methylated -cyclodextrin
Arno Hazekamp, Rob Verpoorte
european journal of pharmaceutical sciences 29 (2006) 340–347
Doi: 10.1016/j.ejps.2006.07.001
The low aqueous solubility of the bioactive cannabinoid tetrahydrocannabinol (THC) is a serious obstacle for the development of more efficient administration forms. In this study the aqueous solubility of THC was tested in the presence of -, - and -CD, and randomly methylated -CD (RAMEB). It was found that only RAMEB was able to increase the aqueous solubility of THC to a significant level. A THC concentration of about 14mg/ml was reached by using a 24% (187mM) RAMEB solution, which means an increase in solubility of four orders of magnitude. The resulting THC/RAMEB complex was investigated through phase-solubility analysis, complemented by 1H NMR, NOESY- and UV-studies in order to obtain details on the stoichiometry, geometry and thermodynamics of the complexation. The binding ratio of THC to CD was found to be 2:1, with the second THC molecule bound by non-inclusion interactions. Based on the obtained results a model for the complex structure is presented. Stability of the complex under laboratory room conditions was tested up to 8 weeks. Results show that complexation with RAMEB seems to be promising for the development of waterbased THC formulations

Structure of cannabinol. III Synthesis of cannabinol, 1-hydroxy-3-n-amyl-6,6,9-trimethyl-dibenzopyran.
Adams, R. et al. (1940)
J. Am. Chem. Soc. 62, 2204–2207
DOI: 10.1021/ja01865a083
'The synthesis of 1 -hydroxy-3-n-amyl-6,6,9-trimethyl-6-dibenzopyran (I), the previously postulated formula for cannabinol,la has now been accomplished by an unequivocal method. It proved to be cannabinol and its derivatives were identical with those obtained from cannabinol. These results also establish with certainty that the product obtained by the condensation of 4-methyl-2-bromobenzoic acid and olivetol followed by conversion of the pyrone to the pyran must have structure II

Study of cannabinoids content in marihuana samples (Cannabis sativa L.) cultivated in several regions of Colombia
VITAE, REVISTA DE LA FACULTAD DE QUÍMICA FARMACÉUTICA ISSN 0121-4004 Volumen 16 número 2, año 2009. Universidad de Antioquia, Medellín, Colombia. págs. 237-244
https://www.scielo.org.co/scielo.php...42009000200008
SPANISH
ESTUDIO DEL CONTENIDO DE CANABINOIDES EN MUESTRAS DE MARIHUANA (Cannabis sativa L.) CULTIVADAS EN VARIAS REGIONES DE COLOMBIA
Néstor M. FLORIAN R. 1,2*, Fabián PARADA A. 2 y William F. GARZÓN M. 2,3 Recibido: Febrero 12 de 2009 Aceptado: Mayo 21 de 2009 RESUMEN El presente estudio analiza el contenido de cannabinoides en muestras de Cannabis sativa L. cultivadas ilícitamente en Colombia. En primer término se optimizan las condiciones para la extracción y cuantificación de Cannabidiol (CBD), ?9-tetrahidrocannabinol (THC) y Cannabinol (CBN) a partir de una muestra vegetal mediante cromatografía de gases con detector de ionización de llama (CG-FID), validando el respectivo método analítico. Se analizan muestras procedentes de cuatro regiones colombianas (región de los Llanos Orientales, LL; región del Cauca, CA; región de la Sierra Nevada de Santa Marta, SN; región del Eje Cafetero, EC) , determinando cuantitativamente la presencia de CBD, THC y CBN. El contenido promedio de THC en las muestras de la región LL es de 15.74 % ± 2.92, en la región CA de 10.98% ± 6.70, en la región SN de 2.81% ± 1.72 y en la región EC de 1.87% ± 1.25. El alto contenido de THC en las muestras vegetales de Cannabis de los Llanos Orientales y el Cauca podría ser indicativo del empleo de variedades mejoradas, lo cual genera una gran preocupación en torno a los mayores efectos potenciales de la droga entre los consumidores. Palabras clave: Cannabis sativa, marihuana, cannabidiol, cannabinol. ABSTRACT The present study analyzes the cannabinoids content in samples of Cannabis sativa L. cultivated illicitly in Colombia. The physicochemical conditions are optimized for the extraction and quantification of Cannabidiol (CBD), 9-Tetrahidrocannabinol (THC) and Cannabinol (CBN) starting from a vegetable sample using gas chromatography with flame ionization detector (GC-FID), validating the respective analytic method. Samples coming from four diferent Colombian regions are analyzed (Llanos Orientales, LL; Cauca, CA; Sierra Nevada de Santa Marta, SN; Eje Cafetero, EC). The study let determination of CBD, THC and CBN samples quantitatively. The average content of THC in the samples of the region LL is of 15.74% ± 2.92, in the region CA is of 10.98% ± 6.70, in the region SN is of 2.81% ± 1.72 and in the region EC of 1.87 % ± 1.25. The higher content of THC in vegetable samples of Cannabis from the Llanos Orientales (LL) and Cauca (CA) could be indicative of the employment of improved varieties. This fact generates a great concern about the potential effects produced by the drug in consumers. Keywords: Cannabis sativa, marihuana, cannabidiol, cannabinol. V

Study of Pesticides in Clones
Anthony Torres, Wilson Linker, Donald Land Ph.D, Reggie Gaudino, Ph.D
https://www.nmlegis.gov/handouts/LHH...n%20Clones.pdf
https://landing.steephill.com/cleanclones
Premise: With increased testing of cannabis derived samples for pesticides, numerous detections of low-to-moderate levels of pesticide contamination (10’s of ppb or less) from a number of growers claiming to use “organic” or “clean green” growing methods prompted us to investigate possible sources. The most prevalent pesticide detected in these cases was Myclobutanil, most commonly used as a treatment for mold infestations. Numerous postings on message boards from growers and clone producers indicate frequent use of Myclobutanil-containing commercially available pesticide products (and others) and a common belief that such use many weeks prior to harvest would not lead to detectable residues in the harvested materials or products refined therefrom. We hypothesize that pesticides used in this manner would persist and, particularly, if the growing medium (soil, rock wool, etc.) were contaminated in the application process, levels of the contaminants in the plants may be present in significant amounts at harvest even without reapplication late in the process.
Study Goal: Determine whether and at what point in the supply chain pesticides are being introduced.
Conclusion: Pesticide use occurs during clone production, and that use remains present through the growth cycle of the plant, and can be tracked through at least 1 generation of passage (e.g., making a clone from the clone; longer term studies in progress). Purchasers of the treated clones likely do not know that they can lead to contamination in the harvested product, even if the grower does not apply pesticides in their practice.

Synthetic cannabinoids, Analysis and metabolites,
Mahmoud A. ElSohly, Waseem Gul, Amira S. Wanas, Mohamed M. Radwan
Life Science, 97(1), 78-90. (2014)
DOI: 10.1016/j.lfs.2013.12.212
Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a 23 wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity 24 toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that 25 could possess high therapeutic activity without many side effects. However, underground laboratories have 26 used the information generated by the scientific community to develop these compounds for illicit use as 27 marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular 28 emphasis on the methods used for their identification in the herbal products with which they are mixed and 29 identification of their metabolites in biological specimens

Targeted and untargeted characterization of underivatized policosanols in hemp inflorescence by liquid chromatography-high resolution mass spectrometry.
Montone, C. M., Aita, S. E., Cannazza, G., Cavaliere, C., Cerrato, A., Citti, C., … Capriotti, A. L.
Talanta, 235, 122778. (2021).
doi:10.1016/j.talanta.2021.122
The paper describes the development of a targeted quantitative method for the analysis of policosanols in hemp inflorescence. Policosanols are long chain aliphatic alcohols, with carbon chains typically in the range 20–36, with interesting biological activities. These compounds are typically separated by gas chromatography and only a few methods employ liquid chromatography for policosanols. In both cases, methods always include the derivatization of policosanols. In this study, policosanols were separated by ultra-high performance liquid chromatography without any derivatization and detected using high resolution mass spectrometry by formation of lithiated adducts. The procedure was optimized and a quantitative method was validated for the most abundant policosanols (with C24, C26, C27, C28, and C30 chain lengths) in industrial hemp inflorescence extracts. The method was used for the quantitative analysis of policosanols in five hemp types. Hemp wax was
found rich in these compounds, especially C26 and C28 policosanols, which may prove useful for revalorization of wax by-products. Finally, the acquired data were also used to expand the search to the untargeted qualitative analysis of policosanols using Compound Discoverer. The untargeted method allowed the annotation of underivatized policosanols up to C33.

Technical Note: Pine Rosin Identified as a Toxic Cannabis Extract Adulterant
Jiries Meehan-Atrash, Robert M. Strongin
Forensic Science International 312 (2020) 110301
DOI: 10.1016/j.forsciint.2020.110301
https://www.researchgate.net/publica...act_Adulterant
Pine rosin (colophony) has been identified as a potentially new adulterant in cannabis oil. Its inhalation toxicity poses a significant health concern to users. For example, pine rosin fumes are released during soldering, and have been cited as a causative agent of occupational asthma. Symptoms also include desquamation of bronchial epithelium, which has also been observed in e-cigarette or vaping product used-associated lung injury (EVALI) patients. The sample analyzed herein was acquired from a cannabis industry source, also contains medium chain triglycerides and oleamide, the latter of which is a hypnotic that is commonly found in the synthetic marijuana product Spice, or K2. A combination of proton nuclear magnetic resonance (¹H NMR) and high pressure liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) was used to unambiguously identify major pine rosin ingredients such as abietic and other resin acids. Comparison to commercial samples of pure pine rosin confirmed the assignment.

Tetrahydrocannabinol (THC) Detection using Semiconductor-enriched Single-Walled Carbon Nanotube Chemiresistors
Hwang, S. I., Franconi, N. G., Rothfuss, M. A., Bocan, K., Bian, L., White, D. L., … Star, A.
ACS Sensors. (2019). 4, 2084?2093
doi:10.1021/acssensors.9b00762
Semiconductor-enriched single-walled carbon nanotubes (s-SWCNTs) have potential for application as a chemiresistor for the detection of breath compounds, including tetrahydrocannabinol (THC), the main psychoactive compound found in the marijuana plant. Herein we show that chemiresistor devices fabricated from s-SWCNT ink using dielectrophoresis can be incorporated into a hand-held breathalyzer with sensitivity toward THC generated from a bubbler containing analytical standard in ethanol and a heated sample evaporator that releases compounds from steel wool. The steel wool was used to capture THC from exhaled marijuana smoke. The generation of the THC from the bubbler and heated breath sample chamber was confirmed using ultraviolet?visible absorption spectroscopy and mass spectrometry, respectively. Enhanced selectivity toward THC over more volatile breath components such as CO2, water, ethanol, methanol, and acetone was achieved by delaying the sensor reading to allow for the desorption of these compounds from the chemiresistor surface. Additionally, machine learning algorithms were utilized to improve the selective detection of THC with better accuracy at increasing quantities of THC delivered to the chemiresistor.

THE ABSOLUTE CONFIGURATION OF DELTA 1-TETRAHYDROCANNABINOL, THE MAJOR ACTIVE CONSTITUENT OF HASHISH.
Raphael Mechoulam, Yehiel Gaoni
DOI: 10.1016/S0040-4039(00)90646-4
We have recently the reported the isolation, structure elucidation and partial synthesis of (-)- A’-tetrahydrocannabinol the major active principle in hashish, as well as a total
reported synthesis of the racemate of I. We wish now to present data showing that the absolute configuration of the naturally occuring (-)-I is D. relative to D-(+)-glyceraldehyde.
Adams has reported that tetrahydrocannabidiol (III) obtained by reduction of cannabidiol which has since been shown to possess structure II, can be oxidized to menthane carboxylic acid (IVa). The anilide of IVa, thus obtained did not depress the melting point of the anilide of IVa prepared from menthol (V) through the menthyl chloride (VI) followed by carbonation of the Grignard derivative of VI. Unfortunately the rotation of the anilide of IVa prepared by the degradation of the natural product was not reported.

XIV. THE ACTIVE PRINCIPLES OF CANNABIS INDICA RESIN.
BY THOMAS SPENCE WORK, FRANZ BERGEL AND ALEXANDER ROBERTUS TODD (1939)
Biochem J. 1939 Jan; 33(1): 123–127.
doi: 10.1042/bj0330123
THE resinous exudate of the female flowers of Cannabis indica (C. sativa) forms the essential constituent of the drug variously known as hashish, bhang, charas, ganja and marihuana according to the country of origin and mode of pre- paration. Extracts of the flowers have been used in European medicine to some extent but were found to be so variable in activity that C. indica has been removed from the British Pharmacopoeia. The plant is, however, well known through its use as a drug in oriental countrie s and recently, under the name of "marihuana", its use has assumed dangerous proportions in America. Cannabis resin has been the subject of many investigations in the past but much of the earlier work is contradictory. The active principle is contained in a high-boiling resin and is not an alkaloid [Smith, 1857]. The active fraction isolated from the crude resin by Wood Spivey & Easterfield [1896] and given by them the name cannabinol was later found by the same workers [1899]to be a mixture, and the name cannabinol wast ransferred to that portion of the active resin yielding acrystalline acetate,M.P.75°. A good deal of confusion was introduced by later workers, who, although failing to obtain a crystalline acetate, nevertheless applied the name cannabinol to their products. The situation was largely clarified by Cahn [1931], who confirmed the observations of Wood Spivey & Easterfield and established for cannabinol the formula C21H2,O2.The con- stitution of cannabinol has been investigated by Cahn [1930-33] and by Bergel [1932], and for it the former proposed structure (I) in which only the positions of the hydroxyl and n-amyl groups are to be regarded as uncertain.

*The Analytical Chemistry of Cannabis Quality Assessment, Assurance, and Regulation of Medicinal Marijuana and Cannabinoid Preparations
Brian F. Thomas, Mahmoud A. ElSohly
Emerging Issues in Analytical Chemistry 2016
doi: I cant find it, please send to me if anyone finds the DOI
This book is very interesting to say the least, check out the references....
-SamS

CHEMICAL CONSTITUENTS AND PHENOTYPES OF C. SATIVA L.
CBN was the first cannabinoid to be isolated and identified from C. sativa. The elucidation of CBN led to speculation that the
psychotropically active constituents of cannabis could be THCs. The nonpsychotropic compound CBD was subsequently isolated from Mexican marijuana and the structure was determined. Gaoni and Mechoulam, two pioneers of cannabis research, determined the structureof ?9-THC after finally succeeding in isolating and purifying this elusive compound (see Mechoulam Close-up: How to Pamper an Idea). Since then, the number of cannabinoids and other compounds isolated from cannabis has increased continually, with 545 now reported. Of these, 104 are phytocannabinoids (Table 1.3). From the isolation and structural elucidation of ?9-THC in 1964 until 1980, 61 phytocannabinoids were isolated and reported. Only nine new ones were characterized between 1981 and 2005, but 31 were reported between 2006 and 2010. The 13 chemical constituent type groups shown in Table 1.3 suggests the chemical complexity of the cannabis plant.
Table 1.3 Constituents of Cannabis sativa L.
No. Groups Number of Known Compounds
1 CBG type 17
2 CBC type 8
3 CBD type 8
4 ?9-THC type 18
5 ?8-THC type 2
6 CBL type 3
7 CBE type 5
8 CBN type 10
9 CBND type 2
10 CBT type 9
11 Miscellaneous 22
12 Total cannabinoids 104

13 Total noncannabinoids 441
Total 545


The analytical landscape of cannabis compliance testing
Stephen Goldman, Julia Bramante, Gordon Vrdoljak, Weihong Guo, Yun Wang, Olivera Marjanovic, Sean Orlowicz, Robert Di Lorenzo, Matthew Noestheden
Journal of Liquid Chromatography & Related Technologies Dec 2021
Doi: 10.1080/10826076.2021.1996390
https://www.tandfonline.com/doi/epub...eedAccess=true
Owing to the lack of federal oversight of recreational and medical cannabis in the United States, a patchwork of regulatory guidelines exists for compliance testing. Adding to this complexity is the fact that Canadian cannabis regulations differ from those in any of the state mandated regulatory jurisdictions and, at the time of writing, cannabis was only recently legalized in Mexico. Therefore, from a North American perspective, cannabis testing represents a significant regulatory landscape to navigate. This not only makes things confusing for those involved in cannabis production and processing, it also creates challenges for those in the analytical testing world when they have to understand and develop methods to be compliant with these various regulatory jurisdictions. In this review article, the current state of analytical chemistry knowledge for cannabis compliance testing is summarized, with an emphasis on suitable techniques and some common problems to avoid. This includes summaries of analytical methods for potency, terpenes, pesticides, mycotoxins, residual solvents, heavy metals and microbiology.

The art and science of Cannabis powder LE HERB https://leherbe.com/images/pdf/the_a...bis_powder.pdf

The Cannabinoid Content of Legal Cannabis in Washington State Varies Systematically Across Testing Facilities and Popular Consumer Products
Nick Jikomes & Michael Zoorob
Scientific REPORTS | (2018) 8:4519
DOI:10.1038/s41598-018-22755-2
The majority of adults in the U.S. now have state-legal access to medical or recreational cannabis products, despite their federal prohibition. Given the wide array of pharmacologically active compounds in these products, it is essential that their biochemical profile is measured and reported to consumers, which requires accurate laboratory testing. However, no universal standards for laboratory testing protocols currently exist, and there is controversy as to whether all reported results are legitimate. To investigate these concerns, we analyzed a publicly available seed-to-sale traceability dataset from Washington state containing measurements of the cannabinoid content of legal cannabis products from state-certified laboratories. Consistent with previous work, we found that commercial Cannabis
strains fall into three broad chemotypes defined by the THC:CBD ratio. Moreover, we documented systematic differences in the cannabinoid content reported by different laboratories, relative stability in cannabinoid levels of commercial flower and concentrates over time, and differences between popular commercial strains. Importantly, interlab differences in cannabinoid reporting persisted even after controlling for plausible confounds. Our results underscore the need for standardized laboratory methodologies in the legal cannabis industry and provide a framework for quantitatively assessing laboratory quality.

The Cannabis Scientist Journal ( you need to sign up for free)
https://thecannabisscientist.com/issues

The case for analyzing whole Cannabis sp., it’s extracts and preparations for terpenoid and “bio-flavinoid” components
James Freire
Applied Synergy Botanicals
AAMC Newsletter Vol. 6, Number 7, July 2014
https://www.letfreedomgrow.com/newsletter/2014/7/
With an emerging body of knowledge proving the efficacy of whole plant Cannabis in a wide range of medicinal applications, much of the present focus is on cannabinoids and the emerging data is seriously challenging reductionist thinking which spurred the “single compound” approach taken by most mainstream pharmaceutical companies. Find a molecule that works, derivatize it by SAR and patent. This approach has been marked by failure to replicate the broad therapeutic range of whole Cannabis in the “single ompound” approach. Further research has shown that within the cannabinoids is a synergistic system which ,in concert, produces impressive results. A prime example is the simplistic relationship between THC and CBD. Where research is lagging seriously behind is in the elucidation of the two other significant therapeutic species, the terpenoids and the “bio-flavinoids”. I hope to provide a case for the importance of analysis for these compounds in characterizing the therapeutic nature/value of a strain, extract or preparation. It is my assertion that terpenoids , as precursors to the cannabinoids and as known therapeutic compounds structurally related to cannabinoids, are as important as the ’cannabinoid profile” in determining medicinal efficacy. The group of substances collectively known as “bio-flavinoids” are also vital and instrumental synergistic
components to the overall effects of Cannabis as a medicinally important substance.

The Conversion and Transfer of Cannabinoids from Cannabis to Smoke Stream in Cigarettes
Sytze Elzinga, Oscar Ortiz and Jeffrey C Raber
Nat Prod Chem Res 2015, 3:1
DOI: 10.4172/2329-6836.1000163
Cannabis cigarettes (100% cannabis) with various percentages of cannabinoids were smoked through an experimental setup, and cannabinoids in the mainstream, sidestream and ash were collected. The cannabis used for the experiments predominantly contained cannabinoid acids. Less than 0.5% of the originally present tetrahydrocannabinolic acid or cannabidiolic acid was recovered as carboxylic acids in the smoke stream, indicating almost complete decarboxylation of cannabinoid acids upon combustion. Recovery of the theoretically present amount of tetrahydrocannabinol from plant material in the smoke stream varied from 27.5-46.3% and an average over all the experiments of 36.9%. The recovery of the theoretically present amount of cannabidiol showed similar results with a range of recovery from 29.9-42.6%, with an average of 38.4%. On average over 50% of the cannabinoids originally present in the plant material could be recovered in the combined ash, mainstream and sidestream smoke. The missing mass balance is most likely caused by destruction of the cannabinoids upon combustion.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools
Julian Brose´ us, Fre´de´ ric Anglada 1, Pierre Esseiva
Forensic Science International 200 (2010) 87–92
doi:10.1016/j.forsciint.2010.03.034
Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant’s chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants’ maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for
the control of legal (fibre type) and illegal (drug type) Cannabis production.

The effect of cannabis dry flower irradiation on the level of cannabinoids, terpenes and anti-cancer properties of the extracts.
Kovalchuk, O., Li, D., Rodriguez-Juarez, R., Golubov, A., Hudson, D., & Kovalchuk, I.
Biocatalysis and Agricultural Biotechnology, 101736. (2020). doi:10.1016/j.bcab.2020.101736
Flowers of Cannabis sativa contain many potentially therapeutic ingredients, including phytocannabinoids and terpenes. Consumption of cannabis requires drying and processing flowers which can potentially lead to accumulation of pathogens, including fungi and bacteria. In many countries, the level of pathogens in dried flowers is strictly regulated and thus, licensed cannabis cultivators irradiate dry flowers to decrease the chance of accumulation of pathogens. We have hypothesized that irradiation of flowers may change the level of active ingredients in flowers. We have tested the effect of gamma-radiation on the level of cannabinoids and terpenes in four varieties of cannabis. Plants were grown to maturity and flowers were harvested. One flower sample per variety was irradiated with 5 kGy using electron beam, while another sample was mock-treated. Cannabinoids were analyzed using HPLC and terpenes using GC-FID. Ethyl acetate was used to prepare extracts from two sets of flowers and extracts were tested for anti-proliferative capacity using methyl-thiazolyl-tetrazolium (MTT) assay on four different cancer cell lines, BJ-5ta, BT16, HCC1806 and IMR5. Solvent-based extraction of irradiated flowers showed slight increase in the total amount of solute. Irradiation of flowers resulted in the increased level of cannabinoids in the extract, more prominent in delta-9-tetrahydrocannabinol. There was also an increase in terpenes content in some cultivars. MTT assay showed that irradiation of flowers changed anti-cancer properties of three out of four tested extracts. Our experiments demonstrated that irradiation of flowers changes the amount of active ingredients in the extract and anti-cancer properties of such extracts.

THE ITALIAN PANORAMA OF CANNABIS LIGHT PREPARATION: DETERMINATION OF CANNABINOIDS BY LC-UV.
Dei Cas, M., Casagni, E., Saccardo, A., Arnoldi, S., Young, C., Scotti, S., … Roda, G.
Forensic Science International, (2020).
doi:10.1016/j.forsciint.2019.110113
Cannabis light preparations are products derivedor containing driedfemale inflorescences of Cannabis sativabelonging to Chemotype III (THC/CBD ratio <<1) ; the total THC (THC+THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation. In Italy themost recentlaw for industrial hemp (242/2016) states that only for farmers this limit is extended to0.6 %. On the other hand, the Ministry of the Interiorpublished a notestatingthat the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5% constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determiningthe total amount of THC. To this end a reliable LC-UV analytical method was developedand validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD = 0.002% for all cannabinoids) and limit of quantification (LOQ=0.005% for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3% and Er% between ± 6% were obtained. The linearity was proven in the concentration range 0.005-1% for THC, THCA and CBN and 0.005%-50% for CBD and CBDA. The analytical method was applied to more than nine hundred cannabis light samples. Based on the law 242/2016, only 18% of the crops are to be considered legal for the market (total THC < 0.2%). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24%) would be considered illegal (total THC > 0.5%). On the other hand, the most of the inflorescences (58%) have a total THC content comprised between 0.2% and 0.5%, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.

The main cannabinoids content in hashish samples seized in Israel and Czech Republic
Lumír O. Hanuš, Rina Levy, Dafna De La Vega, Limor Katz, Michael Roman & Pavel Tomí?ek
Israel Journal of Plant Sciences Volume 63, 2016 - Issue 3 Pages 182-190
doi: 10.1080/07929978.2016.1177983
Cannabis, both herbal and resin, has been the most popular illicit drug in Israel in recent years. Until 2005, the main sources of cannabis resin (known also as hashish) to the Israeli drug market were Lebanon and India. Hashish from these sources can be distinguished by its external appearance. The aim of this study was to determine if there is any difference in the quality of the hashish from each source. For this purpose, we quantified the main cannabinoids, cannabidiol (CBD), ?9-tetrahydrocannabinol (?9-THC), and cannabinol (CBN) of hashish from different police seizures of known origins, Lebanon, India and Morocco, that had been submitted to the Analytical Chemistry Laboratory of the Division of Identification and Forensic Science at the Israeli National Police Headquarters and subsequently to the Hebrew University for quantitative analysis. The results, based on many different seizures, showed that the average CBD content of hashish from Lebanon varied from 5.69% to 12.79% (an average 8.98 ± 0.59%), THC of hashish from Lebanon varied from 0.93% to 4.20% (an average of 2.38 ± 0.27 %), CBD of hashish from Morocco varied from 1.52% to 5.14% (an average of 3.72 ± 0.19%), THC of hashish from Morocco varied from 5.08% to 13.41% (an average of 9.21 ± 0.40%), CBD of hashish from India varied from 0.78% to 13.13% (an average of 4.59 ± 1.07%), and THC of hashish from India varied from 0.53% to 16.45% (an average of 6.35 ± 1.50%), At the same time, several other cannabinoids present in the samples in lower amounts were identified (cannabidivarol, CBDV; cannabicitran; ?9-tetrahydrocannabivarol, ?9-THCV; cannabivarol, CBV; cannabicyclol, CBL; cannabichromene, CBC; cannabielsoin, CBE; ?8-THC; and cannabigerol, CBG). The samples, predominantly from Lebanon, Morocco, and India, were evaluated for chemical phenotype (drug type and fiber type) to determine the geographical origin of these samples.
Indian hashish, seized by Czech Republic authorities, was analyzed qualitatively for many cannabinoids and terpenes. In this sample three new cannabinoids were identified: cannabiorcochromene, cis-?9-tetrahydrocannabivarol, and cannabinerol.

The need to calibrate standardized cannabis measurements across cultures
Lauren Kuhns, Emese Kroon
Addiction. 2021;1–2.
DOI: 10.1111/add.15744
https://onlinelibrary.wiley.com/doi/...1111/add.15744
The iCann Toolkit is an important step towards standardized measurement of cannabis consumption in both clinical and research settings. However, there are still significant challenges caused by regional differences in cannabis potency and cultural differences in methods of administration that will need to be addressed.
Lorenzetti et al. [1] proposed the International Cannabis (iCann) Toolkit as a framework for international minimum standards for the measurement of cannabis use across multiple contexts based on agreement from international cannabis researchers. This is a crucial step forward to standardized measurements of cannabis consumption and has the potential to vastly improve comparability of evidence for the effects of cannabis consumption across contexts. As Lorenzetti et al. [1] point out, differences in legislation, cultural customs, cannabis products and patterns of consumption make the measurement of cannabis use particularly challenging. In a cannabis research setting, the proposed mid- and top layer measures are the most appropriate for quantifying cannabis use. However, challenges still lie ahead in how to integrate measurements across testing sites that often have distinct cannabis cultures. The proposed top layer biological measures include quantifying cannabis consumption or abstinence through analysis of tetrahydrocannabinol (THC) and metabolites in urine and blood plasma. Although this will constitute a gold standard for cannabis use quantification, the related cost, practical and even legislative issues limit the feasibility of using these measures, highlighting the importance of harmonizing mid-layer assessments such as the Timeline Followback (TLFB). However, as Lorenzetti et al. [1] acknowledge, cross-cultural differences in potency, unit of measurement and method of administration result in extra challenges to consider when using the TLFB method.

The Phytochemical Diversity of Commercial Cannabis in the United States
Christiana J. Smith, Daniela Vergara, Brian Keegan, Nick Jikomes
BioRxiv 2021
DOI: 10.1101/2021.07.05.451212
https://www.biorxiv.org/content/10.1...212v1.full.pdf
The legal status of Cannabis is changing, fueling an increased diversity of Cannabis-derived products. Because Cannabis contains dozens of chemical compounds with potential psychoactive or medicinal effects, understanding its phytochemical diversity is crucial. The legal Cannabis industry heavily markets products to consumers based on widely used labelling systems purported to predict the effects of different Cannabis “strains.” We analyzed the cannabinoid and terpene content of tens of thousands of commercial Cannabis samples across six US states, finding distinct chemical phenotypes (chemotypes) which are reliably present. After careful descriptive analysis of the phytochemical diversity and comparison to the commercial labels commonly attached to Cannabis samples, we show that commercial labels do not consistently align with the observed chemical diversity. However, certain labels are statistically over represented for specific chemotypes. These results have important implications for the classification of commercial Cannabis, the design of animal and human research, and the regulation of legal Cannabis marketing.

The study of chemical differences of hashish from different sources seized in Israel.
Lumir Hanus
Israel Anti Drug Authority grant (2014)
https://www.researchgate.net/publica...ized_in_Israel
Cannabis, both herbal and resin, has been the most popular illicit drug in Israel in recent years. Until 2005, the main sources of cannabis resin (known also as hashish) to the Israeli drug market were Lebanon and India. Hashish from these sources can be distinguished by its external appearance. The aim of this study was to find if there is any difference in the quality of the hashish from each source. For this purpose, we quantified the main cannabinoids, cannabidiol (CBD), ?9-
tetrahydrocannabinol (?9-THC), and cannabinol (CBN) of hashish from different police seizures of known origins, Lebanon, India and Morocco that had been submitted to the Analytical Chemistry Laboratory of the Division of Identification and Forensic Science (DIFS) at the Israeli National Police Headquarters and subsequently to the Hebrew University for quantitative analysis. The results, based on many different seizures showed that CBD of hashish from Lebanon varied from 5.69% to 12.79% (an average 8.98 ± 0.59%), THC of hashish from Lebanon varied from 0.93% to 4.20% (an average of 2.38 ± 0.27 %), CBD of hashish from Morocco varied from 1.52% to 5.14% (an average of 3.72 ± 0.19%), THC of hashish from Morocco varied from 5.08% to 13.41% (an average of 9.21 ± 0.40 %), CBD of hashish from India varied from 0.78% to 13.13% (an average of 4.59 ± 1.07%), and THC of hashish from India varied from 0.53% to 16.45% (an average of 6.35 ± 1.50 %). At the same time several other cannabinoids, present in the samples in lower amount, were identified (cannabidivarol - CBDV, ?9-tetrahydrocannabivarol - ?9-THCV, cannabivarol - CBV, cannabichromene - CBC, cannabielsoin – CBE, cannabigerol monomethyl ether - CBGM, ?8-tetrahydrocannabinol - ? 8-THC, and cannabigerol - CBG). The samples, predominantly from Lebanon, Morocco, and India were evaluated for chemical phenotype (drug type and fiber type) in aim to determine the geographical origin of these samples. Samples of false hashish seized in Israel and Czech Republic were worked-up for analysis and analyzed as usual for cannabis plant. Such “hashish” can endanger the health of a user. I also evaluated the way of use of medicinal cannabis in Israel. Homogeneity of the female flowering tops with or without surrounding small leaves and just only surrounding small leaves from different plants of the same strain of cannabis and inside one cannabis plant was studied. From the results it is justified concern that patient even when using the same strain and the same amount of medicinal cannabis can smoke different amounts of the active compound for treatment. It means that patient cannot use for smoking just one bud, but it is necessary to supply patients with homogenized plant material with constant content and ratio of important cannabinoids. If the patient cannot tolerate the cookies or the sublingual oily drops, the remaining methods are smoking or evaporation. The plant for inhalation must be for immunocompromised patients sterilized. The effectiveness of different kinds of sterilization was studied. Samples of the same homogenized cannabis strain revealed different concentration of cannabinoids after sieving with different mesh size sieves. Simple sieving can give material with almost twice higher amount of THC than in the flowering tops. Concentration of cannabinoids from the plant can be increased by preparation simple so called “bubble hash”. It was proved that with this simple mechanical method can be prepared in much more pure cannabinoids. Different strains of medicinal cannabis cultivated in Israel were evaluated according to the quantitative cannabinoid content (low CBD and high ? 9 -THC, high CBD and low ? 9 -THC, high CBD and high ? 9 -THC, and approximately the same content of CBD and ? 9 -THC). Review of all strains used for medicinal cannabis cultivation in Israel is presented. Results of analyses of different cannabis products for treatment from all growers are presented (female flowering tops, cannabis extract in plant oil, cannabis extract, cannabis cakes, cannabis butter for cakes baking, cannabis tincture, cannabis skin cream, and the other preparations).

Too Many Mouldy Joints – Marijuana aand Chronic Pulmonary Aspergillosis
Yousef Gargani, Paul Bishop and David W. Denning
Mediterr J Hematol Infect Dis 2011, 3; Open Journal System
DOI 10.4084/MJHID.2011.005
Chronic pulmonary aspergillosis is a progressive debilitating disease with multiple underlying pulmonary diseases described. Here we report the association of chronic pulmonary aspergillosis and long term marijuana smoking in 2 patients and review the literature related to invasive and allergic aspergillosis.

Not Cannabis specific:
Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening
Wangshu Mou, Dongdong Li, Zisheng Luo, Linchun Mao, Tiejin Ying
PLoS One. 2015 Jun 8;10(6)
doi: 10.1371/journal.pone.0129598
Abscisic acid (ABA) has been proven to be involved in the regulation of climacteric fruit ripening, but a comprehensive investigation of its influence on ripening related processes is still lacking. By applying the next generation sequencing technology, we conducted a comparative analysis of the effects of exogenous ABA and NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) on tomato fruit ripening. The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples, 10388 were identified as significantly differently expressed genes. Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these processes. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenousABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis. Our results highlighted the significance of ABA in regulating tomato ripening, which provided insight into the regulatory mechanism of fruit maturation and senescence process.

Understanding dabs: contamination concerns of cannabis concentrates and cannabinoid transfer during the act of dabbing.
Raber, J. C., Elzinga, S., & Kaplan, C.
The Journal of Toxicological Sciences, 40(6), 797–803.(2015).
doi:10.2131/jts.40.797
Cannabis concentrates are gaining rapid popularity in the California medical cannabis market. These extracts are increasingly being consumed via a new inhalation method called 'dabbing'. The act of consuming one dose is colloquially referred to as "doing a dab". This paper investigates cannabinoid transfer efficiency, chemical composition and contamination of concentrated cannabis extracts used for dabbing. The studied concentrates represent material available in the California medical cannabis market. Fifty seven (57) concentrate samples were screened for cannabinoid content and the presence of residual solvents or pesticides. Considerable residual solvent and pesticide contamination were found in these concentrates. Over 80% of the concentrate samples were contaminated in some form. THC max concentrations ranged from 23.7% to 75.9% with the exception of one outlier containing 2.7% THC and 47.7% CBD. Up to 40% of the theoretically available THC could be captured in the vapor stream of a dab during inhalation experiments. Dabbing offers immediate physiological relief to patients in need but may also be more prone to abuse by recreational users seeking a more rapid and intense physiological effect.

Untargeted characterization of extracts from Cannabis sativa L. cultivars by gas and liquid chromatography coupled to mass spectrometry in high resolution mode.
Delgado-Povedano, M. M., Sánchez-Carnerero Callado, C., Priego-Capote, F., & Ferreiro-Vera, C.
Talanta, 120384. (2019).
doi:10.1016/j.talanta.2019.120384
Elucidation of Cannabis composition is required to evaluate the potential of this plant for pharmacological uses, but also for implementation in breeding programs with agronomical purposes. The aim of the present study was to develop a method for untargeted analysis of polar and non-polar Cannabis extracts. For this purpose, extracts from 17 cultivars of Cannabis sativa L. were analyzed by gas chromatography–time-of-flight/mass spectrometry (GC–TOF/MS) and liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC–QTOF MS/ MS) in high resolution mode. One hundred sixty-nine compounds were identified in the extracts by searching MS and MS/MS information. Among identified families, there were mainly cannabinoids, terpenoids, lipids and flavonoids, but also some interesting compounds such as amino and organic acids, among others. Relative contents of terpenoids and cannabinoids in the same cultivars grown in greenhouse and field were compared. Compositional differences in the profile of terpenoids and cannabinoids between both types of grown conditions were found

Urinary Acrylonitrile Metabolite Concentrations Before and after Smoked, Vaporized, and Oral Cannabis in Frequent and Occasional Cannabis Users.
Ashley, D. L., De Jesús, V. R., Abulseoud, O. A., Huestis, M. A., Milan, D. F., & Blount, B. C.
International Journal of Environmental Research and Public Health, 17(18),(2020).
doi:10.3390/ijerph17186438
Cannabis use through smoking, vaping, or ingestion is increasing, but only limited studies have investigated the resulting exposure to harmful chemicals. N-acetyl-S-(2-cyanoethyl)-L-cysteine (2CYEMA), a urinary metabolite of acrylonitrile, a possible carcinogen, is elevated in the urine of past-30-day cannabis users compared to non-cannabis users. Five frequent and five occasional cannabis users smoked and vaped cannabis on separate days; one also consumed cannabis orally. Urine samples were collected before and up to 72 h post dose and urinary 2CYEMA was quantified. We compared 2CYEMA pre-exposure levels, maximum concentration, time at maximum concentration for occasional versus frequent users following different exposure routes, and measured half-life of elimination. Smoking cannabis joints rapidly (within 10 min) increased 2CYEMA in the urine of occasional cannabis users, but not in frequent users. Urine 2CYEMA did not consistently increase following vaping or ingestion in either study group. Cigarette smokers had high pre-exposure concentrations of 2CYEMA. Following cannabis smoking, the half-lives of 2CYEMA ranged from 2.5 to 9.0 h. 2CYEMA is an effective biomarker of cannabis smoke exposure, including smoke from a single cannabis joint, however, not from vaping or when consumed orally. When using 2CYEMA to evaluate exposure in cannabis users, investigators should collect the details about tobacco smoking, route of consumption, and time since last use as possible covariates.

Validation of the 4?aminophenol color test for the differentiation of marijuana?type and hemp?type cannabis.
Lewis, K., Wagner, R., Rodriguez?Cruz, S. E., Weaver, M. J., & Dumke, J. C.
Journal of Forensic Sciences. (2020).
doi:10.1111/1556-4029.14562
The analysis of cannabis plant material submitted to seized-drug laboratories was significantly affected by the signing of the Agricultural Improvement Act of 2018, which defined hemp and removed it from the definition of marijuana in the Controlled Substances Act. As a result, field law enforcement personnel and forensic laboratories now are in need of implementing new protocols that can distinguish between marijuana-type and hemp-type cannabis. Colorimetric tests provide a cost-effective and efficient manner to presumptively identify materials prior to submission to a laboratory for analysis. This work presents the validation of the 4-aminophenol (4-AP) color test and demonstrates its utility for discriminating between marijuana-type and hemptype cannabis (i.e., typification). Validation studies included the testing of numerous cannabinoid reference materials, household herbs, previously characterized cannabis plant samples, and real-case samples. The 4-AP test reliably produces a pink result when the level of ?9 -tetrahydrocannabinol (THC) is approximately three times lower than the level of cannabidiol (CBD). A blue result is generated when the level of THC is approximately three times higher than that of CBD. Inconclusive results are observed when the levels of THC and CBD are within a factor of three from each other, demonstrating the limitations of the test under those scenarios.

Variations of 9-THC content in single plants of hemp varieties
Klemens Mechtler, Josef Bailer, Karl de Hueber
Industrial Crops and Products. 19(1), 19-24
DOI: 10.1016/S0926-6690(03)00077-3
Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. To test the distribution of 9-tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of five well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30–61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribution of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single-plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC values far outside this bandwidth.

What are some of the chemicals in cannabis sativa?
Robert T. Gale
https://cannabisresearch.mcmaster.ca.. .annabis-sativa
Many natural compounds in Cannabis are synthesized via pathways that are not absolutely required for growth and development, but aid in the plant’s survival; i.e., through secondary metabolism. Amongst these compounds are the terpenes (volatile organic compounds also found in the essential oils of many plants) and the cannabinoids (or phytocannabinoids) that have pharmaceutical effects in humans[ 5 ]. Cannabis terpenes and cannabinoids are manufactured in the secretory cavity of specialized structures on the surface of the plant, called glandular trichomes. These crystal-like outgrowths are densely concentrated on female flowers and in other aerial parts of Cannabis.
While the exact biological function of terpenes and phytocannabinoids in Cannabis remains unclear, some hypothesize that increases in the production of these secondary metabolites might improve the plant’s chances of fertilization and bolster self-defenses against pests and microorganisms. In humans, the majority of Cannabis pharmacological activity – including the well-known psychoactive effects – are thought to be caused by the phytocannabinoids.
 
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IC Cannabis Art

Early Iconography of Cannabis sativa and Cannabis indica
John McPartland, Karl Hillig
October 2008 Journal of Industrial Hemp 13(2):189-203
DOI: 10.1080/15377880802393239
The purpose of this article was to track down the earliest known illustrations of Cannabis. Our in silico search methodology utilized computerized search engines and electronic databases for citation tracking. Many botanists prior to Linnaeus, beginning with Dioscorides, described more than one presumptive type of Cannabis. In some cases they were describing male and female plants of Cannabis sativa L., sometimes they were describing a species of a genus other than Cannabis, and in some cases they were describing a second putative species, Cannabis indica Lamarck. The oldest extant illustration we found of C. sativa dates to 512 AD in the Juliana Anicia Codex, although its authenticity is in question; the next oldest icon may date to ca. 650 AD, or possibly 800–900 AD. The
oldest illustration of Chinese hemp (a biotype of C. indica) that we know of in a mechanically printed book dates to 1248 AD. The first illustration of C. sativa in a mechanically printed book dates to 1484 AD, less than 30 years after the first printing of the Gutenberg Bible (although the Chinese invented movable type printing about 400 years earlier), and the first illustration of C. indica by a European dates to 1578 AD.


IC Cannabis Botany


A Dynamical Phyllotaxis Model to Determine Floral Organ Number

Miho S. Kitazawa and Koichi Fujimoto
PLoS Comput Biol. 2015 May; 11(5): e1004145.
doi: 10.1371/journal.pcbi.1004145
How organisms determine particular organ numbers is a fundamental key to the development of precise body structures; however, the developmental mechanisms underlying organ-number determination are unclear. In many eudicot plants, theprimordia of sepals and petals (the floral organs) first arise sequentially at the edge of a circular, undifferentiated region called the floral meristem, and later transition into a concentric arrangement called a whorl, which includes four or five organs. The properties controlling the transition to whorls comprising particular numbers of organs is little explored. We propose a development-based model of floral organ-number determination, improving upon earlier models of plant phyllotaxis that assumed two developmental processes: the sequential initiation of primordia in the least crowded space around the meristem and the constant growth of the tip of the stem. By introducing mutual repulsion among primordia into the growth process, we numerically and analytically show that the whorled arrangement emerges spontaneously from the sequential initiation of primordia. Moreover, by allowing the strength of the inhibition exerted by each primordium to decrease as the primordium ages, we show that pentamerous whorls, in which the angular and radial positions of the primordia are consistent with those observed in sepal and petal primordia in Silene coeli-rosa, Caryophyllaceae, become the dominant arrangement. The organ number within the outmost whorl, corresponding to the sepals, takes a value of four or five in a much wider parameter space than that in which it takes a value of six or seven. These results suggest that mutual repulsion among primordia during growth and a temporal decrease in the strength of the inhibition during initiation are required for the development of the tetramerous and pentamerous whorls common in eudicots.

A MULTIFACETED APPROACH TO ADDRESS VARIATION IN CANNABIS SATIVA
Anna Louise Schwabe
UNIVERSITY OF NORTHERN COLORADO Greeley, Colorado
DOI: 10.13140/RG.2.2.17042.81607
Available from Author via Facebook, just ask her
For thousands of years, humans have cultivated and dispersed Cannabis sativa L. across the globe. Although Cannabis has been largely illegal worldwide for decades, public perceptions and attitudes are changing. Increasing interest in potential Cannabis usage worldwide and nationwide is leading to less restrictions to make way for an expanding and lucrative industry with numerous applications. Although only one species is formally recognized in the Cannabis genus, thousands of years of artificial selection for diverse phenotypes and uses have resulted in two major usage groups; hemp-types which are defined worldwide as having very low levels of THC (< 1.0%), and drug-types which exceed a specified level of THC that varies among nations. The drug-type category includes three commonly used subcategories including Sativa, Indica and Hybrid types, and newly developed high CBD varieties that have more THC than hemp-types but are not bred for high THC. The quality of federally produced Cannabis for medical studies in the U.S. has recently been brought into question, and we included samples to determine the genetic relationship to these groups.
Phenotypic variation in Cannabis gives rise to commonly referenced categories, but sources of variation are unclear and understudied. Phenotypes are observable characteristics that results from a combination of both genotype and the environment. .
The preferred method of propagation for Cannabis is cloning, and therefore variation within varietals should be from differences in environmental factors. Ten microsatellite markers were developed de-novo to investigate four aims: (1) genetic variation within strains, (2) genetic relationships among the common categories, (3) if genetic variation is detectable through olfactory sensation, and (4) how genetic variation is reflected in phytochemical levels. This dissertation includes four manuscript chapters representing each aim and uses a genetic basis for a multifaceted approach to investigate variation in Cannabis sativa. Substantial genetic variation was found within strains from obtained from different facilities. Genetic divergence between hemp and drug-types was genetically supported, but the Sativa, Indica, and Hybrid subcategories were not genetically well defined. The high CBD strains appear to bridge the genetic gap between hemp and drug-types, and federally grown research grade marijuana was genetically more similar to hemp than Cannabis available through the legal cannabis market. Genetic imposters within a strain had measurable aromatic differences, but there was considerable variation in aromas among samples with identical genetic identity. Analyses of both terpene and cannabinoid profiles among individuals with identical genotypes acquired from different sources varied considerably indicating environmental variation has a substantial impact on phenotype in Cannabis.
Together these results show a need for the Cannabis industry to implement regulatory checks in the form of genetic testing in order to provide consistency, especially for medical applications. These results demonstrate the need for genotyping in order for phenotypic consistency to be achieved if standard growing conditions can be established. When genetic verification and standard protocols are established, deviations in phenotypic changes can be identified and disclosed to consumers so they are aware that there may be abnormal effects. This investigation highlights the need for additional research to provide consistent products, which is especially important for medical marijuana flower products. In order to provide consumers consistent products, it is imperative to understand sources of variation. Consumers deserve to be provided with quality consistent products as the industry continues to grow on a global scale.

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages
Mattia Spano 1, Giacomo Di Matteo 1, Cinzia Ingallina 1, Bruno Botta 1, Deborah Quaglio 1, Francesca Ghirga 1, Silvia Balducci 1, Silvia Cammarone 1, Enio Campiglia 2, Anna Maria Giusti 3, Giuliana Vinci 4, Mattia Rapa 4, Salvatore Ciano 4, Luisa Mannina 1, Anatoly P Sobolev
Molecules. 2021 May 14;26(10):2912.
doi: 10.3390/molecules26102912.
https://www.mdpi.com/1420-3049/26/10/2912
The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the beginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications.

A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana
Christopher J. Grassa, George D. Weiblen, Jonathan P. Wenger, Clemon Dabney, Shane G. Poplawski, S. Timothy Motley, Todd P. Mitchael, C.J. Schwartz
New Phytologist (2021)
DOI: 10.1111/nph.17243
https://nph.onlinelibrary.wiley.com/...1111/nph.17243
*Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance.
*A nanopore-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for CBDRx, a potent CBD-type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp x marijuana segregating population was also performed.
*Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and
lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD-type Cannabis, while both
CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol
(THC) : CBD ratio.
*Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

Not Cannabis specific
A Nodule-Specific Protein Secretory Pathway Required for Nitrogen-Fixing Symbiosis.
Wang, D., Griffitts, J., Starker, C., Fedorova, E., Limpens, E., Ivanov, S., … Long, S.
Science, 327(5969), 1126–1129.(2010).
doi:10.1126/science.1184096
The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified by DNF1

A Preliminary Study of Pollen Dispersal in Cannabis sativa in Relation to Wind Direction.
Small, E., & Antle, T.
Journal of Industrial Hemp, 8(2), 37–50.(2003).
doi:10.1300/j237v08n02_03
Pollen of Cannabis sativa is disseminated by wind in large amounts and for long distances, and regulations concerning the production of pedigreed seed of industrial hemp, therefore, often call for extremely large isolation distances to prevent unwanted pollination. In Europe and Canada, a standard distance of 5 km is required for the highest classes of hemp seed. This study examines the relative distribution of pollen from an isolated field over the 3-week maximum flowering period, with particular reference to wind direction. The amount of pollen distributed downwind was about six times the amount distributed upwind. In effect, this means that an isolation distance of 5 km on the downwind side is about equivalent to an isolation distance of 0.9 km on the upwind side. In theory, at the experimental site examined, the required isolation area could be reduced by about 58% while still achieving the equivalent of 5 km isolation in all directions. Given that weedy and illegally cultivated plants are widespread, making it difficult to ensure their absence over a distance of 5 km, it seems advisable, when possible, to take advantage of the considerably reduced isolation distance that is necessary on the upwind side. Pollen distribution appeared to follow the expected leptokurtic curve, reducing rapidly with initial distance from the source, but much more slowly with increasing distance. This makes it impossible to guarantee complete absence of potentially contaminating pollen in the field and, for practical purposes, a very low amount of undesired gene flow needs to be tolerated.

Not Cannabis specific
A Program for Estimating the Optimum Sample Size for Germplasm Conservation.
Hernandez, C. M., & Crossa, J.
Journal of Heredity, 84(1), 85–86.(1993).
doi:10.1093/oxfordjournals.jhered.a111284
The main objective of genetic resource conservation systems is to maintain genetic diversity from regeneration to regeneration by attempting to retain at least one copy of each allele present in the original population. The genetic diversity of a population depends on the number and frequency of alleles at a locus and across all loci. Marshall and Brown (1975) suggested that the most important measure of genetic diversity is the average number of alleles per locus. Weir (1990) defined genie diversity at a singe locus as one minus the sum of squares of the allelic frequencies. Managers of genetic resource conservation systems try to preserve the existing gene diversity of each accession by maintaining copies of those alleles that account for useful genie variation. For this reason, sample size is extremely important in regenerating an accession. Crossa (1989) pointed out that the effectiveness of regeneration for maintaining gene diversity is related to proper sampling procedures, random genetic drift due to sample size, and optimum seed viability. When sample sizes are large, regeneration is difficult and expensive. Because of random genetic drift, small sample sizes may result in the loss of alleles present at low frequency. Several probability models were used for outbreeding species to determine the sample size needed to maintain a specific number of desired rare alleles at a locus (Crossa 1989). This is achieved by ensuring that the sample contains at least one copy of the alleles.

A Study of Cotyledon Asymmetry in Cannabis sativa L.
Ernest Small, Tanya Antle
May 2007 Journal of Industrial Hemp 12(1):3-14
DOI: 10.1300/J237v12n01_02
Although it has been known that the two cotyledons of Cannabis sativa L. seedlings tend to differ in size, the development and magnitude of this phenomenon have not previously been described. In the dry seeds, the “outer cotyledon” (remote from the radicle) is about 50% heavier than the “inner cotyledon” (adjacent to the radicle). Two days after germination, the difference in fresh weight has been reduced considerably, but thereafter the outer cotyledon remains slightly more than 20% heavier than the inner. In about one in four seedlings, the tip of the outer (normally the larger) cotyledon is persistently covered by remnants of the seed coat, including a characteristic dark area which enclosed the tips of the cotyledons in the seed. In about one in ten seedlings, the achene shell (pericarp) remains up to several days on the tip of the outer cotyledon. These features serve to confirm the identity of very young seedlings of C. sativa, for which there has not previously been a reliable means of identification. Seeds from several sources did not differ significantly from each other with respect to weight ratio of the cotyledons, nor were there significant differences in cotyledon ratio found when seedlings were grown in darkness, a 16-hour light regime, and in continuous light. Microscopic connections of unknown significance were observed between the adpressed adaxial surfaces of the cotyledons in the seed.

Agricultural and agrochemical uses of natural zeolite of the clinoptilolite type
M. Rehakova, S. Cuvanova, M. Dzivak, J. Rimar, Z. Gavalova
Current Opinion in Solid State and Materials Science 8 (2004) 397–404
doi: 10.1016/j.cossms.2005.04.004
https://www.kmizeolite.com/wp-conten...-zeolite-2.pdf
The present paper deals with the agricultural and agrochemical uses of natural zeolite of the clinoptilolite type from the Nizˇny´ Hrabovec deposit in Eastern Slovakia. The structure of natural clinoptilolite is ideal for sorption and ion exchange processes. Due to its structure and properties this natural, inert and non-toxic material can be used as a slowly releasing carrier of fertilizer, as well as other agrochemically, pharmaceutically and biochemically active compounds including disinfectants. Natural zeolite can also be used to improve physical properties of soils and for treatment of contaminated soils. It is also suitable—in very small amounts—as additive to animal feed

Not Cannabis specific
Maybe this is why O Haze from seed has more females than normal?
Allelic incompatibility can explain female biased sex ratios in dioecious plants.
Pucholt, P., Hallingbäck, H. R., & Berlin, S.
BMC Genomics, 18(1). (2017).
doi:10.1186/s12864-017-3634-5
Background: Biased sex ratios are common among dioecious plant species despite the theoretical prediction of selective advantage of even sex ratios. Albeit the high prevalence of deviations from even sex ratios, the genetic causes to sex biases are rarely known outside of a few model species. Here we present a mechanism underlying the female biased sex ratio in the dioecious willow species Salix viminalis.
Results: We compared the segregation pattern of genome-wide single nucleotide polymorphism markers in two contrasting bi-parental pedigree populations, the S3 with even sex ratio and the S5 with a female biased sex ratio. With the segregation analysis and comparison between the two populations, we were able to demonstrate that sex determination and sex ratio distortion are controlled by different genetic mechanisms. We furthermore located the sex ratio distorter locus to a Z/W-gametologous region on chromosome 15, which was in close linkage with the sex determination locus. Interestingly, all males in the population with biased sex ratio have in this sex ratio distorter locus the same genotype, meaning that males with the Z1/Z3-genotype were missing from the population, thereby creating the 2:1 female biased sex ratio.
Conclusions: We attribute the absence of Z1/Z3 males to an allelic incompatibility between maternally and paternally inherited alleles in this sex ratio distorter locus. Due to the tight linkage with the sex determination locus only male individuals are purged from the population at an early age, presumably before or during seed development. We showed that such allelic incompatibility could be stably maintained over evolutionary times through a system of overdominant or pseudooverdominant alleles. Thus, it is possible that the same mechanism generates the female biased sex ratio in natural willow populations.

An Alternative In Vitro Propagation Protocol of Cannabis sativa L. (Cannabaceae) Presenting Efficient Rooting, for Commercial Production
Kostas Ioannidis, Ioanna Tomprou and Vangelis Mitsis Plants 2022, 11, 1333.
DOI: 10.3390/ plants11101333
https://www.mdpi.com/2223-7747/11/10/1333
An alternative in vitro propagation protocol for medical Cannabis sativa L. cultivars for pharmaceutical industrial use was established. The aim of the protocol was to reduce the culture time, offering healthy and aseptic propagating material, while making the whole process more economic for industrial use. The propagation procedure was performed using plastic autoclavable vented and non-vented vessels, containing porous rooting fine-milled sphagnum peat moss-based sponges, impregnated in 1 2 Murashige and Skoog liquid growth medium, supplemented with indole-3-butyric acid (IBA) at various concentrations (0, 2.46, 4.92, and 9.84 µM) or by dipping nodal cuttings into 15 mM IBA aqueous solution. The highest average root numbers per cutting, 9.47 and 7.79 for high cannabidiol (H_CBD) and high cannabigerol (H_CBG) varieties, respectively, were achieved by dipping the cuttings into IBA aqueous solution for 4 min and then placing them in non-vented vessels. The maximum average root length in H_CBD (1.54 cm) and H_CBG (0.88 cm) was ascertained using 2.46 µM filter sterilized IBA in non-vented vessels. Filter-sterilized IBA at concentrations of 2.46 µM in vented and 4.92 µM in non-vented vessels displayed the maximum average rooting percentages in H_CBD (100%) and H_CBG (95.83%), respectively. In both varieties, maximum growth was obtained in non-vented vessels, when the medium was supplemented with 4.92 µM filter-sterilized IBA. Significant interactions between variety and vessel type and variety and IBA treatments were observed in relation to rooting traits. Approximately 95% of plantlets were successfully established and acclimatized in field. This culture system can be used not only for propagating plant material at an industrial scale but also to enhance the preservation and conservation of Cannabis genetic material
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An endocannabinoid catabolic enzyme FAAH and its paralogs in an early land plant reveal evolutionary and functional relationship with eukaryotic orthologs.
Haq, I., & Kilaru, A.
Scientific Reports, 10(1).(2020).
doi:10.1038/s41598-020-59948-7
Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identifed nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37°C and pH 8.0, with higher specifcity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identifed that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

An Update on Plant Photobiology and Implications for Cannabis Production
Mark Lefsrud, Samuel Eichhorn Bilodeau, Bo-Sen Wu, Anne-Sophie Rufyikiri, Sarah MacPherson
Front. Plant Sci. 10:296. (2019)
doi:10.3389/fpls.2019.00296
This review presents recent developments in plant photobiology and lighting systems for horticultural crops, as well as potential applications for cannabis (Cannabis sativa and C. indica) plant production. The legal and commercial production of the cannabis plant is a relatively new, rapidly growing, and highly profitable industry in Europe and North America. However, more knowledge transfer from plant studies and horticultural communities to commercial cannabis plant growers is needed. Plant photosynthesis and photomorphogenesis are influenced by light wavelength, intensity, and photoperiod via plant photoreceptors that sense light and control plant growth. Further, light properties play a critical role in plant vegetative growth and reproductive (flowering) developmental stages, as well as in biomass secondary metabolite synthesis and accumulation. Advantages and disadvantages of widespread greenhouse lighting systems that use high pressure sodium lamps or light emitting diode (LED) lighting are known. Some artificial plant lighting practices will require improvements for cannabis production. By manipulating 1LED light spectra and stimulating specific plant photoreceptors, it may be possible to minimize operation costs while maximizing cannabis biomass and cannabinoid yield, including tetrahydrocannabinol (or ? 9 -tetrahydrocannabinol) and cannabidiol for medicinal and recreational purposes. The basics of plant photobiology (photosynthesis and photomorphogenesis) and electrical lighting systems are discussed, with an emphasis on how the light spectrum and lighting strategies could influence cannabis production and secondary compound accumulation.

Not Cannabis specific
Arabidopsis Paired Amphipathic Helix Proteins SNL1 and SNL2 Redundantly Regulate Primary Seed Dormancy via Abscisic Acid–Ethylene Antagonism Mediated by Histone Deacetylation
Zhi Wang, Hong Cao, Yongzhen Sun, Xiaoying Li, Fengying Chen, Annaick Carles, Yong Li, Meng Ding, Cun Zhang, Xin Deng, Wim J.J. Soppe, Yong-Xiu Liu
The Plant Cell, 25(1), 149–166.
doi:10.1105/tpc.112.108191
Histone (de)acetylation is a highly conserved chromatin modification that is vital for development and growth. In this study, we identified a role in seed dormancy for two members of the histone deacetylation complex in Arabidopsis thaliana, SIN3- LIKE1 (SNL1) and SNL2. The double mutant snl1 snl2 shows reduced dormancy and hypersensitivity to the histone deacetylase inhibitors trichostatin A and diallyl disulfide compared with the wild type. SNL1 interacts with HISTONE DEACETYLASE19 in vitro and in planta, and loss-of-function mutants of SNL1 and SNL2 show increased acetylation levels of histone 3 lysine 9/18 (H3K9/18) and H3K14. Moreover, SNL1 and SNL2 regulate key genes involved in the ethylene and abscisic acid (ABA) pathways by decreasing their histone acetylation levels. Taken together, we showed that SNL1 and SNL2 regulate seed dormancy by mediating the ABA-ethylene antagonism in Arabidopsis. SNL1 and SNL2 could represent a crosslink point of the ABA and ethylene pathways in the regulation of seed dormancy

Architecture and Florogenesis in Female Cannabis sativa Plants
April 2019 Frontiers in Plant Science 10
Ben Spitzer-RimonShai DuchinNirit BernsteinRina Kamenetsky
DOI: 10.3389/fpls.2019.00350
The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly
abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be “non-inductive” or “vegetative”, but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture, and to maximize plant productivity and uniformity in medical Cannabis.

Augmenting Nutrient Acquisition Ranges of Greenhouse Grown CBD (Cannabidiol) Hemp (Cannabis sativa) Cultivars
Jennifer Kalinowski, Keith Edmisten, Jeanine Davis, Michelle McGinnis, Kristin Hicks, Paul Cockson, Patrick Veazie and Brian E. Whipker
Horticulturae 2020, 6, 98;
doi:10.3390/horticulturae6040098
https://www.mdpi.com/2311-7524/6/4/98
There is a growing interest in the production of hemp for the extraction of cannabidiol (CBD) due to reported therapeutic benefits. Recent policy reform has permitted state hemp pilot programs, including the land grant research institutions, the ability to investigate the potential of growing and harvesting Cannabis sativa plants (_ 0.3% tetrahydrocannabinol) for these purposes in the U.S. There are vast gaps of knowledge regarding the fertility requirements of hemp cultivars grown in a horticultural production setting for floral attributes such as the cannabinoid constituents. Foliar tissue analysis provides an avenue to determine adequate ranges for nutrient uptake and estimating fertilizer requirements prior to visual symptoms of deficiency or toxicity. To facilitate a survey range of elemental nutrient acquisition in hemp cultivars propagated for CBD production, foliar analysis was executed using the most recently mature leaves (MRML) of mother stock plants. All plants were maintained in the vegetative stage for twelve weeks, prior to initiation of cutting for clone harvesting. A total of thirteen cultivars were utilized to broaden previously reported baseline survey ranges. Significant differences were found among all thirteen cultivars in accumulation of both micro and macro essential nutrients, widening the range of the fertility requirements of Cannabis plants grown in this production model for CBD harvesting.

Associations between cannabinoids and growth stages of twelve industrial hemp cultivars grown outdoors in Atlantic Canada
Nada Hammami, Jean-Pierre Privé, David L Joly, Gaétan Moreau Industrial Crops and Products 172(29):113997 Nov 2021 DOI: 10.1016/j.indcrop.2021.113997
https://www.researchgate.net/public...p_cultivars_grown_outdoors_in_Atlantic_Canada

Industrial hemp is increasingly grown and harvested for its cannabinoids of pharmaceutical interest. These compounds are generally obtained from plants harvested at maturity but not all cannabinoids are present or abundant during the last stage of hemp development. This study examined intraspecific cannabinoid variability during ontogenic development of hemp to identify growth stages and cultivars that optimize production of specific compounds. The cannabinoid content of twelve commercial industrial hemp (Cannabis sativa L. subsp. sativa) cultivars at three growth stages (i.e., flowering, grain filling and maturity) was determined by high performance liquid chromatography in an experimental field in Cocagne, New Brunswick, Canada. Most cannabinoids in acidic or neutral form were more abundant at maturity. However, cannabigerolic acid, a precursor to all acidic and neutral forms of cannabinoids mentioned in our study, was more abundant during the grain-filling stage. In contrast, cannabichromene was associated with the flowering stage and found in greater abundances in grain cultivars than in dual-purpose cultivars. The cultivar Katani exhibited higher concentrations of most cannabinoids while the cultivars Ferimon, Altair and Anka exhibited higher concentrations of cannabinoid acidic precursors. The current study could help optimize the targeted production of cannabinoids at specific growth stages and to identify the chemical phenotype of different hemp cultivars. . .


Not Cannabis Specific
AtMND1 is required for homologous pairing during meiosis in Arabidopsis
Panoli, A. P., Ravi, M., Sebastian, J., Nishal, B., Reddy, T. V., Marimuthu, M. P. A., … Siddiqi, I. (2006).
BMC Molecular Biology, 7(1), 24.
doi:10.1186/1471-2199-7-24
Background: Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1. Results: We have analyzed the function of the Arabidopsis orthologue of the budding yeast MND1 gene (AtMND1). Loss of AtMND1 did not affect normal vegetative development but caused fragmentation and missegregation of chromosomes in male and female meiosis, formation of inviable gametes, and sterility. Analysis of the Atmnd1 Atspo11-1 double mutant indicated that chromosome fragmentation in Atmnd1 was suppressed by loss of Atspo11-1. Fluorescence in situ hybridization (FISH) analysis showed that homologous pairing failed to occur and homologues remained apart throughout meiosis. AtMND1 showed strong expression in meiocytes as revealed by RNA in situs. Conclusion: We conclude that AtMND1 is required for homologous pairing and is likely to play a role in the repair of DNA double strand breaks during meiosis in Arabidopsis, thus showing conservation of function with that of MND1 during meiosis in yeast

Botany of Cannabis sativa L.: Identification, Cultivation and Processing
Suman Chandra, Hemant Lata, A. M. Galal, MA ElSohly
March 2011 Planta Medica 77(05)
DOI: 10.1055/s-0031-1273533
Cannabis sativa L. belongs to the family Cannabaceae. Indigenous to temperate regions of Asia, the plant is now distributed worldwide. It is a monospecific plant (Cannabis sativa L.) that is divided into several subspecies (C. sativa subsp. sativa, C. sativa subsp. indica, C. sativa subsp. ruderalis, C. sativa subsp. spontanea, C. sativa subsp. kafiristanca). Cannabis sativa is typically a dioecious species, i.e., male and female flowers develop on separate plants. Morphologically, it is difficult to differentiate between male and female plants at the vegetative stage since sexual dimorphism occurs late in plant development. However, after the onset of flowering, male plants can be differentiated from female ones. The sexual phenotype of cannabis occasionally shows flexibility that leads to differentiation of hermaphrodite flowers or bisexual inflorescences from other types, i.e., a monoecious phenotype. Due to the allogamous (cross fertilization) nature, this species is highly hybridized and found to be in different varieties/strains with variations in morphology, color and chemotypes. In the present study, a detailed monographic account of Cannabis sativa L. including taxonomy, geographical distribution, macro and microscopy of leaf and stem, powder analysis, indoor and outdoor cultivation, harvesting, processing, storage, its possible adulterants and biomass quality control is provided.

Botany of Natural Cannabis Medicines
Robert C. Clarke, David Paul Watson
In: Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential Ed. Franjo Grotenhermen, Ethan Russo pg1-15
doi:10.1007/978-1-59259-947-9_1
Cannabis is among the very oldest of economic plants, providing fiber, edible seed, and drug resin. Human selection for varying uses and natural selection pressures imposed by diverse climates have resulted in a wide variety of growth forms and chemical compositions. Innovative classical breeding techniques have been used to improve drug cannabis, resulting in many cannabinoid-rich cultivars suitable for medical use. The production of cannabinoids is unique to Cannabis, and cultivars with specific chemical profiles are being developed for diverse potential pharmaceutical uses.

Not Cannabis specific
Callus, Dedifferentiation, Totipotency, Somatic Embryogenesis: What These Terms Mean in the Era of Molecular Plant Biology?
Attila Fehér
Front. Plant Sci., 26 April 2019
DOI: 10.3389/fpls.2019.00536
https://www.frontiersin.org/articles...019.00536/full
Recent findings call for the critical overview of some incorrectly used plant cell and tissue culture terminology such as dedifferentiation, callus, totipotency, and somatic embryogenesis. Plant cell and tissue culture methods are efficient means to preserve and propagate genotypes with superior germplasm as well as to increase genetic variability for breading. Besides, they are useful research tools and objects of plant developmental biology. The history of plant cell and tissue culture dates back to more than a century. Its basic methodology and terminology were formulated preceding modern plant biology. Recent progress in molecular and cell biology techniques allowed unprecedented insights into the underlying processes of plant cell/tissue culture and regeneration. The main aim of this review is to provide a theoretical framework supported by recent experimental findings to reconsider certain historical, even dogmatic, statements widely used by plant scientists and teachers such as “plant cells are totipotent” or “callus is a mass of dedifferentiated cells,” or “somatic embryos have a single cell origin.” These statements are based on a confused terminology. Clarification of it might help to avoid further misunderstanding and to overcome potential “terminology-raised” barriers in plant research.


Can DEA-backed cannabis growers strike gold via drug development?

mar Sacirbey MJBizDaily​

https://mjbizdaily.com/dea-backed-cannabis-growers-seek-to-strike-gold-via-drug-development/?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email
The cannabis industry hit a possible milestone in March when Bright Green Corp., a Florida company with “conditional” approval from the U.S. Drug Enforcement Administration to grow marijuana for research purposes, applied to list on the Nasdaq stock exchange.
If successful, Bright Green would become the first U.S. plant-touching company to list on a major U.S. stock exchange.
But the move also highlights the lofty – some say unrealistic – financial ambitions of such companies.
And it raises the question of whether a business model based on DEA approval is realistic from a financial standpoint.
The five entities registered with the DEA as “Bulk Manufacturer Marihuana Growers” are allowed to grow and sell marijuana flower and extract to researchers registered with the federal agency.
Those researchers can be at universities, pharmaceutical companies and other entities.
The University of Mississippi received the first DEA cultivation registration in 1968, while four more entities received registrations in 2021.
Three of the other four entities that have secured DEA approval are private companies, while the Scottsdale Research Institute in Cave Creek, Arizona, is a nonproft focused on determining “the general medical safety and efficacy of cannabis and cannabis products.”
Other entities, such as Fort Lauderdale-based Bright Green, are vying for additional registrations, although it’s not clear if or when the DEA will issue any more.
In addition to making money through the cultivation of marijuana for scientific research, at last three of the DEA-approved companies hope to further capitalize on cannabis-based drug development.
They are:
Biopharmaceutical Research Co., in Castroville, California.
Groff North America, in Red Lion, Pennsylvania.
Royal Emerald Pharmaceuticals, in Desert Hot Springs, California.
How can such drug development be a money winner?
The answer? By partnering with pharmaceutical companies and multistate operators to develop drugs approved by the U.S. Food and Drug Administration that could be sold by prescription or even over the counter.
Each company attained DEA bulk cannabis manufacturing registration in 2021.
“Conceptually, the opportunity is phenomenal. If you total up all the pharmaceutical sales for the indications that cannabis can address, like sleep pain, nausea, etc., it’s a $330 billion-a-year market. There’s lots and lots of activity to be had in this pharmaceutical facilitator area,” said Joe Grzyb, CEO of Groff North America.
But he added: “You have to be patient, because it takes several years to get through the FDA process.”
Doubts over DEA-based business model
Sue Sisley, head of the Scottsdale Research Institute – which is among the five DEA cannabis cultivation registrants – said business models based on the DEA registrations are bound to fail.
“The entities who are trying to build a business model around these few research registrations won’t be successful. The demand for research cannabis is minimal,” Sisley said.
“This is not a lucrative business model and never will be. It takes over 10 years to develop drugs that get FDA approval – and is massively more complicated when it comes to agricultural products that have complex chemical composition with tons of different bioactive molecules.”
The University of Mississippi received the first DEA permit to grow cannabis for research in 1968.

Cannabinoid–hormone interactions in the regulation of motivational processes
Hassan H. LópezHormones and Behavior 58 (2010) 100–110
doi:10.1016/j.yhbeh.2009.10.005 https://d1wqtxts1xzle7.cloudfront.ne.. .ne%3B+filename %3DCannabinoid_hormone_interac tions_in_the.pdf&Expires=16488 46795&Signature=M6uyVrfsBxCNVQ X7Rn83jlahHuNeeggboBMUGOc-QmKt034vtUmssSJBYdOCUkK-YIZZhE~Z44rLDEkk8G7YrmnQJMXVnP hXBVQksS8m80EjtSzcIK8In3pRUkRu x85IB7wscY5bnRF5QHquW6Dct~8wQo m6jw6uRxmWkZKzeC89MAfZ~dBRslif hA51Pzrgdw6215fQx23tPvWGddB7fd FZsLZXtELqWC9uLkK4dfu0U81FMBqg wtU2FQBtbrVjO93D3e9vFX3RjWfSw2 yHf8FlSSBifv2k-NT2NqUIS5RR4W2je1VBQWDSr4g-sr~a0GNfWT-5sd0qb1PBdY65gg__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA
There is a bi-directionality in hormone–cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic–pituitary–gonadal ), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a “case study.” I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction

Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.
Livingston, S. J., Quilichini, T. D., Booth, J. K., Wong, D. C. J., Rensing, K. H., Laflamme?Yonkman, J., … Samuels, A. L.
The Plant Journal. (2019).
doi:10.1111/tpj.14516
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile-like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two-photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12–16 secretory disc cells and strongly monoterpene-dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed redshifted autofluorescence, eight secretory disc cells and less monoterpene-dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co-expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.

Cannabis Indoor Growing Conditions, Management Practices, and Post-Harvest Treatment: A Review
Dan Jin, Shengxi Jin, Jie Chen
American Journal of Plant Sciences, 2019, 10, 925-946
DOI: 10.4236/ajps.2019.106067
https://www.scirp.org/pdf/AJPS_2019061713561499.pdf
Cannabis has attracted a new wave of research attention as an herbal medicine. To deliver compliant, uniform, and safe cannabis medicine, growers should optimize growing environments on a site-specific basis. Considering that environmental factors are interconnected, changes in a factor prompts adjustment of other factors. This paper reviews existing work that considers indoor growing conditions (light, temperature, CO2 concentration, humidity, growing media, and nutrient supply), management practices (irrigation, fertilization, pruning & training, and harvest timing), and post-harvest treatment (drying and storage) for cannabis indoor production.

Cannabis Inflorescence Yield and Cannabinoid Concentration Are Not Increased With Exposure to Short-Wavelength Ultraviolet-B Radiation
Victoria Rodriguez-Morrison, David Llewellyn and Youbin Zheng
Front. Plant Sci. 12:725078. doi: 10.3389/fpls.2021.725078
https://www.ncbi.nlm.nih.gov/pmc/art...-12-725078.pdf
Before ultraviolet (UV) radiation can be used as a horticultural management tool in commercial Cannabis sativa (cannabis) production, the effects of UV on cannabis should be vetted scientifically. In this study we investigated the effects of UV exposure level on photosynthesis, growth, inflorescence yield, and secondary metabolite composition of two indoor-grown cannabis cultivars: ‘Low Tide’ (LT) and ‘Breaking Wave’ (BW). After growing vegetatively for 2 weeks under a canopy-level photosynthetic photon flux density (PPFD) of ≈225 µmol·m−2 ·s −1 in an 18-h light/6-h dark photoperiod, plants were grown for 9 weeks in a 12-h light/12-h dark “flowering” photoperiod under a canopy-level PPFD of ≈400 µmol·m−2 ·s −1 . Supplemental UV radiation was provided daily for 3.5 h at UV photon flux densities ranging from 0.01 to 0.8 µmol·m−2 ·s −1 provided by light-emitting diodes (LEDs) with a peak wavelength of 287 nm (i.e., biologically-effective UV doses of 0.16 to 13 kJ·m−2 ·d −1 ). The severity of UV-induced morphology (e.g., whole-plant size and leaf size reductions, leaf malformations, and stigma browning) and physiology (e.g., reduced leaf photosynthetic rate and reduced Fv/Fm) symptoms intensified as UV exposure level increased. While the proportion of the total dry inflorescence yield that was derived from apical tissues decreased in both cultivars with increasing UV exposure level, total dry inflorescence yield only decreased in LT. The total equivalent 19 -tetrahydrocannabinol (19 -THC) and cannabidiol (CBD) concentrations also decreased in LT inflorescences with increasing UV exposure level. While the total terpene content in inflorescences decreased with increasing UV exposure level in both cultivars, the relative concentrations of individual terpenes varied by cultivar. The present study suggests that using UV radiation as a production tool did not lead to any commercially relevant benefits to cannabis yield or inflorescence secondary metabolite composition

Cannabis Microbiome and the Role of Endophytes in Modulating the Production of Secondary Metabolites: An Overview
Suha Jabaji**and*Meysam Taghinasab
Microorganisms 2020, 8(3), 355?DOI:*10.3390/microorganisms8030355
Plants, including Cannabis (Cannabis sativa subsp. sativa) host distinct beneficial microbial communities on and inside their tissues, designated the plant microbiota from the moment that they are planted into the soil as seed. They contribute to plant growth promotion, facilitating mineral nutrient uptake, inducing defense resistance against pathogens, higher yield and modulating plant secondary metabolites. Understanding the microbial partnerships with Cannabis has the potential to affect agricultural practices by improving plant fitness and the production yield of cannabinoids. Much less is known about this beneficial Cannabis-microbe partnership, and the complex relationship between the endogenous microbes associated with various tissues of the plant, particularly, the role that cannabis may play in supporting or enhancing them. This review will focus on Cannabis microbiota studies and the effect of endophytes on the elicitation of secondary metabolites production in Cannabis plants. The aim of this review is to shed light on the importance of Cannabis microbiome and how cannabinoid compounds concentration can be stimulated through symbiotic and or mutualistic relationships with endophytes.

Cannabis Plants-Cultivation & Yield
Cultivation & Yield Classification and Overview
Find Pdf link I have Pdf .



Cannabis sativa: An ancient wild edible plant of India
Mohammed Kuddus, Ibrahim A. M. Ginawi and Awdah Al-Hazimi
Emir. J. Food Agric. 2013. 25 (10): 736-745
doi: 10.9755/ejfa.v25i10.16400
Cannabis sativa, also known as Cannabis indica or Indian hemp, is an annual herb of the family Cannabinaceae. It has been used by humans throughout recorded history for its food, fiber and medicine. It is a native to Central Asia, and long cultivated in Asia, Europe and China. Plants yielding the drug seem to have been discovered in India, cultivated for medicinal purposes as early as 900 BC. Hindu devotees offered Cannabis to Shiva during religious ceremonies, and the herb continues to have a religious association in India. The medicinal use of Cannabis has very long history. However, the sociopolitical pressure led to decline the medicinal use of Cannabis. But despite its illegality, people have continued to obtain Cannabis in black market for self-medication. Together with coffee and tobacco, Cannabis is the most commonly used psychoactive drug worldwide, and it is the single most popular illegal drug. This review analyzes the diversity, botanical description, consumption and pharmacological studies along with medicinal uses of Cannabis among the human being throughout the world

Cannabis sativa. (Quick Guide)
Schilling, S., Melzer, R., & McCabe, P. F.
Current Biology, 30(1), R8–R9.(2020).
doi:10.1016/j.cub.2019.10.039
Cannabis is the botanical name of a genus within the Cannabaceae, the same plant family that contains hops.
The genus includes three species, C. sativa, C. ruderalis and C. indica. However, the three species interbreed and species boundaries are fluid — therefore, it has been suggested that only a single Cannabis species, C. sativa, be recognised. One common trait of all Cannabis plants is the presence of secondary compounds called ‘cannabinoids’, or more precisely ‘phytocannabinoids’. There are over 100 different phytocannabinoids, which are predominantly produced in trichomes growing on female Cannabis inflorescences. However, between Cannabis accessions, the profile and quantity of specific phytocannabinoids varies enormously. To reflect this variation, it has been suggested to classify Cannabis strains according to their chemical phenotypes into ‘chemotypes’ with distinct cannabinoid profiles.

Cannabis, the multibillion dollar plant that no genebank wanted
Davoud Torkamaneh and Andrew Maxwell Phineas Jones
Genome Volume 65, Number 1, January 2022
https://cdnsciencepub.com/doi/10.1139/gen-2021-0016
Although cannabis is legalized and accepted as an agricultural commodity in many places around the world, a significant lack of public germplasm repositories remains an unresolved problem in the cannabis industry. The acquisition, preservation, and evaluation of germplasm, including landraces and ancestral populations, is key to unleashing the full potential of cannabis in the global marketplace. We argue here that accessible germplasm resources are crucial for long-term economic viability, preserving genetic diversity, breeding, innovation, and long-term sustainability of the crop. We believe that cannabis restrictions require a second look to allow genebanks to play a fuller and more effective role in conservation, sustainable use, and exchange of cannabis genetic resources.

Cannabis Yield, Potency, and Leaf Photosynthesis Respond Differently to Increasing Light Levels in an Indoor Environment
Victoria Rodriguez-Morrison, David Llewellyn and Youbin Zheng
Front. Plant Sci. 12:646020.
doi: 10.3389/fpls.2021.646020
https://www.frontiersin.org/articles...21.646020/full
Since the recent legalization ofmedical and recreational use of cannabis (Cannabis sativa ) in many regions worldwide, there has been high demand for research to improve yield and quality. With the paucity of scientific literature on the topic, this study investigated the relationships between light intensity (LI) and photosynthesis, inflorescence yield, and inflorescence quality of cannabis grown in an indoor environment. After growing vegetatively for 2 weeks under a canopy-level photosynthetic photon flux density (PPFD) of ≈ 425 μ mol. m−2. s−1 and an 18-h light/6-h dark photoperiod, plants were grown for 12 weeks in a 12-h light/12-h dark “flowering” photoperiod under canopy-level PPFDs ranging from 120 to 1,800 μ mol. m−2. s−1 provided by light emitting diodes. Leaf light response curves varied both with localized (i.e., leaf-level) PPFD and temporally, throughout the flowering cycle. Therefore, it was concluded that the leaf light response is not a reliable predictor of whole-plant responses to LI, particularly crop yield. This may be especially evident given that dry inflorescence yield increased linearly with increasing canopy-level PPFD up to 1,800 μ mol. m−2. s−1 , while leaf-level photosynthesis saturated well-below 1,800 μ mol. m−2. s−1 . The density of the apical inflorescence and harvest index also increased linearly with increasing LI, resulting in higher-quality marketable tissues and less superfluous tissue to dispose of. There were no LI treatment effects on cannabinoid potency, while there were minor LI treatment effects on terpene potency. Commercial cannabis growers can use these light response models to determine the optimum LI for their production environment to achieve the best economic return; balancing input costs with the commercial value of their cannabis products.

Challenges towards Revitalizing Hemp: A Multifaceted Crop
Craig Schluttenhofer, and Ling Yuan
Trends in Plant Science, November 2017, Vol. 22, No. 11
DOI: 10.1016/j.tplants.2017.08.004
https://www.cell.com/action/showPdf?...2817%2930177-2
Hemp has been an important crop throughout human history for food, fiber, and medicine. Despite significant progress made by the international research community, the basic biology of hemp plants remains insufficiently understood. Clear objectives are needed to guide future research. As a semi-domesticated plant, hemp has many desirable traits that require improvement, including eliminating seed shattering, enhancing the quantity and quality of stem fiber, and increasing the accumulation of phytocannabinoids. Methods to manipulate the sex of hemp plants will also be important for optimizing yields of seed, fiber, and cannabinoids. Currently, research into trait improvement is hindered by the lack of molecular techniques adapted to hemp. Here we review how addressing these limitations will help advance our knowledge of plant biology and enable us to fully domesticate and maximize the agronomic potential of this promising crop.

Not Cannabis specific
Chapter Seven - Cryptochrome-Mediated Light Responses in Plants
Xu Wang, Qin Wang, Paula Nguyen, Chentao Lin
https://doi.org/10.1016/B978-0-12-801922-1.00007-5Get rights and content
Cryptochromes (CRYs) are photolyase-like flavoproteins that have been found in all evolutionary lineages. Plant and animal CRYs are no longer DNA-repairing enzymes but they apparently gained other biochemical functions in evolution. Plant CRYs are UV-A/blue-light photoreceptors and play a pivotal role in plant growth and development, whereas animal CRYs act as either photoreceptors or transcription regulators. The first CRY gene was isolated from Arabidopsis thaliana, which regulates stem growth, flowering time, stomatal opening, circadian clock, and other light responses. CRYs are also found in all major crops investigated, with additional functions discovered, such as seed germination, leaf senescence, and stress responses. In this chapter, we will review some aspects of CRY-mediated light responses in plants. Readers are referred to other review articles for photochemistryand signal transduction mechanism of plant CRYs (Liu et al., 2010, 2011; Fankhauser and Ulm, 2011) [1], [2], [3].

Chemical and Physical Elicitation for Enhanced Cannabinoid Production in Cannabis.
Gorelick, J., & Bernstein, N. (2017).
Cannabis Sativa L. - Botany and Biotechnology, 439–456.
doi:10.1007/978-3-319-54564-6_21
Of the many medicinal plants with therapeutic potential, Cannabis sativa is, by far, the most promising in the near future for large scale utilization. However, the inherent chemical variability of plant based medicines must be addressed, before cannabis can be incorporated into modern medical practices. This chemical variability can only be controlled and potentially optimized if the underlying causes of the production of therapeutic compounds in cannabis is adequately understood. Many of the medically useful compounds produced by plants are the result of the plant stress response. Although not completely clear, there is a significant body of evidence suggesting a similar role for cannabinoids. Cannabinoids are implicating in both, biotic and abiotic stresses, including thermal, nutrient, and water stress, photoradiation, as well as bacterial and fungal pathogens. This chapter will explore the possible ecological roles of cannabinoids in cannabis and the potential utilization of these roles via biotic or abiotic elicitors.

Comparing hydroponic and aquaponic rootzones on the growth of two drug-type Cannabis sativa L. cultivars during the flowering stage
Brandon Yepa, Nigel V. Galeb, Youbin Zheng
Industrial Crops and Products 157:1122881 Dec 2020
DOI: 10.1016/j.indcrop.2020.1122881
https://www.researchgate.net/publica...ing_hydroponic _and_aquaponic_rootzones_on_th e_growth_of_two_drug-_type_Cannabis_sativa_L_cultiv ars_during_the_flowering_stage
The cultivation of drug-type Cannabis sativa Lin. (C. sativa) employs a variety of rootzone systems, yet little is understood about rootzone-effects on plant physiology, inflorescence biomass, and cannabinoid potency. To
determine if rootzone systems can affect C. sativa growth, physiology, and inflorescence biomass and phytochemicals, two C. sativa cultivars, colloquially known as ‘Nordle’ and ‘Sensi-Star’, were grown in three rootzone systems in a controlled environment. The three tested rootzone systems were: 1) an 11-litre pot containing a peat-based growing substrate, top fertigated with a synthetic fertilizer solution (“hydroponic”); 2) an 11-litre pot containing a peat-based growing substrate, top fertigated with aquaculture effluent solution (“aquaculture”); and 3) a 3-litre pot containing a custom made, predominantly peat-based growing substrate, with the lower portion of the roots submerged in a conventional aquaponics deep-water culture system (“aquaponics”). Throughout the experiment, plant growth (height, branch number, canopy volume), leaf-level physiological traits (e.g., chlorophyll) foliar mineral nutrition, and substrate physio-chemical properties were measured. At experimental completion, inflorescence yield (dry biomass) and potency (mass-based cannabinoid and terpene concentrations) were determined. Results revealed, significant rootzone-effects on most growth and physiological traits measured. Plants performed best in the in the hydroponic rootzone: e.g., producing 42–116% greater inflorescence biomass than the other rootzone systems. In contrast, however, Nordle plants in the aquaponics treatment had greater Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and the terpenes ß-pinene and limonene, relative to their congeners grown in the other rootzones investigated. Pronounced differences in substrate macro-nutrient availability and foliar nutrient content occurred among the rootzones, suggesting a potential mechanism to explain the observed growth, physiological, yield and potency responses. This work underscores the importance of the rootzone environment for the cultivation of C. sativa, specifically in indoor systems focused of medical or research production.

Comparing methods for controlled capture and quantification of pollen in Cannabis sativa.
Wizenberg, S. B., Weis, A. E., & Campbell, L. G.
Applications in Plant Sciences, 8(9). (2020).
doi:10.1002/aps3.11389
PREMISE: Precise pollen collection methods are necessary for crop breeding, but anemophilous pollen is notoriously difficult to capture and control. Here we compared a variety of methods for the controlled capture of cannabis pollen, intended to ease the process of cross-fertilization for breeding this wind-pollinated plant, and measured the utility of light spectroscopy for quantifying relative pollen yield. METHODS AND RESULTS: In two independent trials, we compared a control method of pollen collection (hand collection) to either vacuum-, water-, or bag-collection methods. We used visible light spectroscopy to quantify relative pollen yield, and validated this approach using microscopic pollen counts. We determined that pollen yield was highest when using hand collection or vacuum collection, but efficiency did not differ significantly among methods. CONCLUSIONS: To maximize yield, pollen should be collected by hand or vacuum, but all collection methods were equally efficient in a relative sense because yield increased with collection time. We also found that light spectroscopy is an accurate and rapid method of quantifying pollen abundance (R2 = 0.86) in a liquid suspension

Contents of Cannabinoids in Hemp Varieties Grown in Maryland
Xiaoyan Chen, Hua Deng, Janai A. Heise, David P. Puthoff, Nabeel Bou-Abboud, Hongtao Yu, and Jiangnan Peng
ACS Omega 2021, 6, 32186−32197
https://doi.org/10.1021/acsomega.1c04992
https://www.researchgate.net/public...nabinoids_in_Hemp_Varieties_Grown_in_Maryland
Coincident with the cannabis legalization and the increased interest in the medicinal use of the plant, the cannabis marketplace and farming have seen tremendous growth. It is reported that there are more than 2000 cannabis varieties available to customers. However, the data that is available to the growers and breeders regarding the cannabinoid contents of various varieties remains low. Here, a high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous separation and determination of 11 cannabinoids. A total of 104 hemp bud materials belonging to 20 varieties were collected from farms in the state of Maryland and analyzed with the HPLC method. The contents of the 11 cannabinoids in various varieties were compared and discussed, highlighting the varieties that showed a high yield of cannabinoids and good consistency that are more appropriate for cannabinoid production.

Not Cannabis specific
Control of flowering
Alon Samach, in Plant Biotechnology and Agriculture, 2012
Florigen and FLOWERING LOCUS T (FT)
Plants are subject to annual cycles in climate including temperature, light quality, light intensity, humidity, precipitation, and other environmental events; yet the timing and intensity of those phenomena are subject to change and are influenced by human impact. Changes in daylength (photoperiod) are a reliable annual clock, unlikely to be disrupted by human or other impact. Annual amplitude in daylength cycle is influenced by where organisms reside on Earth. In regions further from the equator, many plants use these predicted changes in photoperiod to correctly time the transition to flowering as well as other developmental processes. Some plants respond to shortening of daylength while others respond to increasing daylengths. The switch occurs in the apical meristem which starts making flowers instead of leaves. Scientists noted this photoperiodic affect on flowering time (Tournois, 1914; Klebs, 1918; Garner and Allard, 1920), and the need for leaves to perceive daylength
suggested the existence of a moving signal termed “Florigen” formed in leaves and reaching the meristem (Knott, 1934; Chailakhyan, 1936). By grafting between species with opposite daylength requirements, it was shown that Florigen could be a common mobile flowering chemical (Zeevaart, 1962).
Molecular genetic studies in Arabidopsis (Redei, 1962; Koornneef et al., 1991; Kobayashi et al., 1999; Kardailsky et al., 1999; Michaels et al., 2005; Yamaguchi et al., 2005; Jang et al., 2009) as well as rice (Kojima et al., 2002; Ishikawa et al., 2005), tomato (Lifschitz et al., 2006), and wheat (Yan et al., 2006) provided the identity of one, and perhaps the only, component of Florigen (Turck et al., 2008), which is a small (23 kDa) protein encoded by FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) in Arabidopsis, SINGLE FLOWER TRUSS (SFT) in tomato, HEADING DATE 3 (HD3A) in rice, and VRN3 in wheat. Here we will use the name FT to describe proteins of different species that are similar in structure and function to the protein encoded by FT of Arabidopsis. Lack of FT causes late flowering, and high levels of FT are correlated with early flowering. Many environmental cues regulate flowering time through regulating FT-encoding gene expression. Under conditions that promote flowering in Arabidopsis, FT is expressed in the phloem of leaves (Takada and Goto, 2003), and after translation, FT is loaded into the phloem and moves toward the meristem (Corbesier et al., 2007; Jaeger and Wigge, 2007; Lin et al., 2007; Mathieu et al., 2007; Tamaki et al., 2007). Once reaching the vegetative meristem, FT was shown to trigger a change in its identity, turning it into an inflorescence meristem producing flower rather than leaf primordia. Based on present knowledge, it is likely that FT-like proteins are formed in leaves of all plant species and once they reach the meristem they can potentially trigger the flowering response in any plant species (Figure 25.1). The timing of FT formation, together with internal signals that intervene in steps that occur after FT formation, will define the flowering behavior of each plant. Polycarpic plants require a mechanism preventing all meristems from reacting to the FT signal, once it is formed. This would allow certain meristems to remain vegetative, and to act as a source of a new round of meristems that will be induced in the next flowering cycle. There is no current evidence that FT-like proteins are DNA binding proteins, so they likely require other proteins to regulate changes in gene expression. One such candidate is the bZIP transcription factor FLOWERING LOCUS D (FD), expressed in meristems and young leaf primordia (anlagen), which interacts with FT-like proteins and in its absence, flowering is delayed (Abe et al., 2005; Wigge et al., 2005). The fact that a mutation in FD does not mimic the severity in delay of flowering shown by loss-of-function of both FT and TSF (Wang et al., 2009a), and that this mutation does not completely suppress early flowering caused by high expression of FT (Teper-Bamnolker and Samach, 2005) suggests that, in the absence of FD, paralogs or proteins of a different nature help FT turn on the flowering switch. Two proteins that negate FT function in what seems to be direct protein–protein interaction are FWA and 14-3-3. FWA is not usually expressed outside of the seed, but when it is, it causes a delay in flowering (Soppe et al., 2000; Ikeda et al., 2007). 14-3-3 proteins were shown to interact with FT-like proteins (Pnueli et al., 2001), and a role for such a protein in delaying flowering by interfering with FT entry into the nucleus was recently shown in rice (Purwestri et al., 2009). TERMINAL FLOWER1 (TFL1) encodes a protein similar to FT and TSF yet with an opposite role. TFL1 is expressed in the vegetative and inflorescence meristem, and in its absence plants are early flowering and the inflorescence meristem is rapidly transformed into a flower, causing a terminal flower phenotype (Shannon and Meeks-Wagner, 1991). The transformation of the inflorescence meristem into flower primordia is accompanied by the appearance in the meristem of flower-primodia-specific genes, such as APETALA1 and LEAFY (Liljegren et al., 1999). TFL1 over-expression causes late flowering (Ratcliffe et al., 1998; Kobayashi et al., 1999). The interaction between these two proteins (FT and TFL1) with negating affects has been nicely studied in tomato where the TFL1 ortholog is encoded by the SELF PRUNING (SP) gene (Pnueli et al., 1998; Krieger et al., 2010)

Cryopreservation of 13 Commercial Cannabis sativa Genotypes Using In Vitro Nodal Explants
Cassandra D. Downey, Gregory Golenia, Ekaterina A. Boudko and Andrew Maxwell P. Jones
Plants 2021, 10, 1794.
DOI: 10.3390/ plants10091794
https://mdpi-res.com/d_attachment/pl...0-01794-v2.pdf
Cannabis has developed into a multi-billion-dollar industry that relies on clonal prop- agation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 ◦C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3–80%) and regrowth of shoots (26.7–66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.

Not Cannabis specific
Cryopreservation of Pollen and Pollen Embryos, and the Establishment of Pollen Banks
Y.P.S.Bajaj
International Review of Cytology Volume 107, 1987, Pages 397-420
doi: 10.1016/S0074-7696(08)61083-9
The cryopreservation of pollen plays an important role in hybridization programs and in the conservation of genetic resources in agricultural and forest biotechnology. In addition to the several potentials outlined, the most important aspect is the storage of “recalcitrant” pollen of important fruit and forest trees and agricultural crops for overcoming geographic, seasonal, and physiological barriers in hybridization. Moreover, in some plant species the flowering period is very short and the pollen grains are very short-lived; thus the plant breeder is not able to handle it. In many fruit crops where yield is erratic, controlled pollination with stored pollen would help to stabilize production. It also avoids the interference of foreign or illegitimate pollen. The establishment of “pollen banks,” in addition to fulfilling various other needs, considerably reduces maintenance of orchards and nurseries for the regular supply of pollen.

Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa,
Peter V. Apicella et al
Plant Direct (2022). DOI: 10.1002/pld3.412
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Not Cannabis specific
Determinants of Genetic Diversity
Hans Ellegren and Nicolas Galtier
Nature Reviews Genetics volume17, pages422–433 (2016)
doi:10.1038/nrg.2016.58
Genetic polymorphism varies among species and within genomes, and has important implications for the evolution and conservation of species. The determinants of this variation have been poorly understood, but population genomic data from a wide range of organisms now make it possible to delineate the underlying evolutionary processes, notably how variation in the effective population size (Ne) governs genetic diversity. Comparative population genomics is on its way to providing a solution to ‘Lewontin’s paradox’ — the discrepancy between the many orders of magnitude of variation in population size and the much narrower distribution of diversity levels. It seems that linked selection plays an important part both in the overall genetic diversity of a species and in the variation in diversity within the genome. Genetic diversity also seems to be predictable from the life history of a species.

Development and optimization of a germination assay and long-term storage for Cannabis sativa pollen
Daniel Gaudet, Narendra Singh Yadav, Aleksei Sorokin, Andrii Bilichak, Igor Kovalchuk
bioRxiv preprint ( March 2020)
DOI: 10.1101/2020.03.19.999367
Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, we have deciphered the bicellular nature of cannabis pollen using DAPI staining. We have also standardized a pollen germination assay to assess the viability of cannabis pollen, and found pollen collected from different principal growth stages exhibits different longevity. Finally, we developed a long-term storage method which includes pollen combination with baked whole wheat flower and desiccation under vacuum for cryopreservation. By using this method, we were able to maintain germination viability in liquid nitrogen after 4 months, suggesting potentially indefinite preservation of cannabis pollen.

Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa
BIO-PROTOCOL 11(1) January 2021
DOI: 10.21769/BioProtoc.3875
https://www.researchgate.net/publica...annabis_sativa
Aleksei Sorokin, Narendra Singh Yadav, Daniel Gaudet, Igor Kovalchuk
Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H2O2) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H2O2 solution of different concentrations; 1% H2O2 solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it suitable for tissue culture and other sensitive applications.
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Development of a hemp (Cannabis sativa L.) simulation model 2.The flowering response of two hemp cultivars to photoperiod
Shaun Lisson, N. J. Mendham, P. S. Carberr
Australian Journal of Experimental Agriculture 40(3)January 2000
DOI: 10.1071/EA99059
The duration from sowing to flowering is an important determinant of fibre yield potential in hemp, since maximum stem yield occurs shortly after flowering. As a short-day plant, daylength has a key influence on the timing of flowering in hemp. This paper reports on studies into the effect of photoperiod on the thermal time duration from sowing to flowering for 2 hemp cultivars, and develops parameters to enable simulation of post-emergent phenology in the hemp model described in the final paper of this series. The hemp model divides the post-emergent period into a vegetative phase that ends at floral initiation, aflower development phase (FDP) between flower initiation and appearance, and a short phase between first flower appearance and harvest maturity (male anthesis). The vegetative phase is further divided into a temperature-dependent basic vegetative phase (BVP) and a daylength-dependent photoperiod induced phase (PIP). For a short-day plant, the duration of PIP is assumed to be zero degree days at daylengths below a base or maximum optimum photoperiod (MOP). Daylengths in excess of the MOP lead to an increase in thermal time within PIP, the duration of which is determined by a genotype’s photoperiod sensitivity (PS). Two hemp genotypes, Kompolti and Futura 77, were exposed to 6 different photoperiod regimes ranging from 8 to 16 h in a growth chamber. Thermal time durations from emergence to flower initiation and first flower formation (harvest) were calculated from thermograph plots. The flowering responses for the 2 cultivars were typical for a short-day plant, with flowering occurring rapidly in daylengths less than about 14 h and with increasing delay at longer photoperiods. With the exception of a longer thermal time duration from flower formation to harvest maturity in the case of Kompolti, the 2 cultivars had similar values for the key phenology parameters. Respectively, for Futura and Kompolti: BVP was 383?Cd and 390?Cd, MOP was 14 h and 13.8 h, PS was 266?Cd/h and 252?Cd/h, and FDP was 76.8?Cd and 80.2?Cd.

Not Cannabis specific
Developmental Signaling in Plants X. Liu, ... K. Wu, in The Enzymes, 2016 4.5 Regulation of Flowering Time
https://www.sciencedirect.com/topics...istry/florigen
The timing of the transition from a vegetative to reproductive phase is critical for reproductive success in the angiosperm life cycle. In Arabidopsis, flowering time is controlled by several pathways, including the photoperiod, gibberellin (GA), autonomous, and vernalization pathways [112]. FLOWERING LOCUS C (FLC) is a key negative regulator of flowering [113], whereas FLOWERING LOCUS T (FT) is a key component of florigen promoting flowering [114]. Recent studies indicated that the HDACs HDA6 and HDA5 are involved in the regulation of flowering through the autonomous pathway. hda6 mutants display a late-flowering phenotype both under long-day and short-day conditions in a FLC-dependent manner. Furthermore, HDA6 directly interacts with the histone demethylase FLD and represses the expression of FLC and two additional MADS-box genes, MADS AFFECTING FLOWERING 4 (MAF4) and MAF5, via histone deacetylation and demethylation [30]. HDA6 was also found to associate with MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4) and MSI5/FVE, two homologues of Retinoblastoma-Associated Protein 46/48 (RbAp46/48), to repress FLC expression in control of flowering time [31]. Similar to hda6 mutants, loss of function of HDA5 also results in a late-flowering phenotype [32]. It was found that HDA5, HDA6, FLD, and FVE are present in the same protein complex to repress FLC expression by histone deacetylation and H3K4 demethylation (Fig. 4). In contrast, hda9 mutants display an early flowering phenotype under short-day conditions [33]. HDA9 directly represses the expression of AGOMOUS-LIKE 19 (AGL19), an upstream activator of FT through histone deacetylation.

Distribution of trespass cannabis cultivation and its risk to sensitive forest predators in California and Southern Oregon
Greta M. Wengert, J. Mark Higley, Mourad W. Gabriel, Heather Rustigian-Romsos, Wayne D. Spencer, Deana L. Clifford, Craig Thompson
PLOS September 1, 2021
DOI: 10.1371/journal.pone.0256273
https://journals.plos.org/plosone/ar...l.pone.0256273
Illegal cannabis cultivation on public lands has emerged as a major threat to wildlife in California and southern Oregon due to the rampant use of pesticides, habitat destruction, and water diversions associated with trespass grow sites. The spatial distribution of cultivation sites, and the factors influencing where they are placed, remain largely unknown due to covert siting practices and limited surveillance funding. We obtained cannabis grow-site locality data from law enforcement agencies and used them to model the potential distribution of cultivation sites in forested regions of California and southern Oregon using maximum entropy (MaxEnt) methods. We mapped the likely distribution of trespass cannabis cultivation sites and identified environmental variables influencing where growers establish their plots to better understand the cumulative impacts of trespass cannabis cultivation on wildlife. We overlaid the resulting grow-site risk maps with habitat distribution maps for three forest species of conservation concern: Pacific fisher (Pekania pennanti), Humboldt marten (Martes caurina humboldtensis), and northern spotted owl (Strix occidentalis caurina). Results indicate that cannabis cultivation is fairly predictably distributed on public lands in low to mid-elevation (~800-1600m) forests and on moderate slopes (~30–60%). Somewhat paradoxically, results also suggest that growers either preferred sites inside of recently disturbed vegetation (especially those burned 8–12 years prior to cultivation) or well outside (>500m) of recent disturbance, perhaps indicating avoidance of open edges. We ground-truthed the model by surveying randomly selected stream courses for cultivation site presence in subsets of the modeling region and found previously undiscovered sites mostly within areas with predicted high likelihood of grow-site occurrence. Moderate to high-likelihood areas of trespass cultivation overlapped with 40 to 48% of modeled habitats of the three sensitive species. For the endangered southern Sierra Nevada fisher population, moderate-high likelihood growing areas overlapped with over 37% of modeled fisher denning habitat and with 100% of annual female fisher home ranges (mean overlap = 48.0% + 27.0 SD; n = 134) in two intensively studied populations on the Sierra National Forest. Locating and reclaiming contaminated cannabis grow sites by removing all environmental contaminants should be a high priority for resource managers.
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Not Cannabis Specific
Diverse cell signalling pathways regulate pollen-stigma interactions: the search for consensus.
Hiscock, S. J., & Allen, A. M.
New Phytologist, 179(2), 286–317 (2008).
.doi:10.1111/j.1469-8137.2008.02457.x
Siphonogamy, the delivery of nonmotile sperm to the egg via a pollen tube, was a key innovation that allowed flowering plants (angiosperms) to carry out sexual reproduction on land without the need for water. This process begins with a pollen grain (male gametophyte) alighting on and adhering to the stigma of a flower. If conditions are right, the pollen grain germinates to produce a pollen tube. The pollen tube invades the stigma and grows through the style towards the ovary, where it enters an ovule, penetrates the embryo sac (female gametophyte) and releases two sperm cells, one of which fertilizes the egg, while the other fuses with the two polar nuclei of the central cell to form the triploid endosperm. The events before fertilization (pollen–pistil interactions) comprise a series of complex cellular interactions involving a continuous exchange of signals between the haploid pollen and the diploid maternal tissue of the pistil (sporophyte). In recent years, significant progress has been made in elucidating the molecular identity of these signals and the cellular interactions that they regulate. Here we review our current understanding of the cellular and molecular interactions that mediate the earliest of these interactions between the pollen and the pistil that occur on or within the stigma – the ‘pollen–stigma interaction’.

Effect of seasonality and time after anthesis on the viability and longevity of Cannabis sativa pollen
Namrta Choudharya , M.B Siddiquia , Shazia Bia & Sayyada Khatoonb
Palynology (2014)
DOI: 10.1080/01916122.2014.892906
The aim of this study was to evaluate the pollen viability and longevity of Cannabis sativa, which is a significant economic crop. Viability and longevity are evaluated using different staining techniques: Alexander’s, Triphenyl Tetrazolium Chloride (TCT) and Fluorochromatic Reaction (FCR). The results of this study show that FCR is the most reliable and accurate for assessing viability and longevity in C. sativa. Viability was measured over days and across 2010. Viability was greatest between 0 – 3 days of anther dehiscence and during April, whilst December had the lowest pollen viability. For Cannabis sativa the first 3 days after anthesis are the most important for fertilization, following the 3rd day there is a rapid decline in viability.

Effect of Timing of Ethephon Treatment on the Formation of Female Flowers and Seeds from Male Plant of Hemp (Cannabis sativa L.
Youn-Ho Moon, Yoon Jeong Lee, Sung Cheol Koo, Mok Hur, Yun Chan Huh, Jae-Ki Chang and Woo Tae Park
Korean J. Plant Res. 33(6):682-688(2020)
DOI: 10.7732/kjpr.2020.33.6.682
http://203.250.217.22/article/JAKO202034965719736.pdf
Hemp (Cannabis sativa L.) is a dioecious plant, although monoecious plants are bred in some cultivars for fiber or seed production. Recently, hemp has received attention for medicinal use with some cannabinoids, including cannabidiol. Self-fertilization for breeding inbred lines is difficult because of dioeciousness and anemophily in hemp. This experiment was conducted to develop a self-fertilization method by forming female flowers and seeds from male plants of dioecious hemp. To induce the formation of female flowers on male plants, 500 ㎎ L-1 of ethephon was sprayed on plants at soon, seven and fourteen days after primordia formation. The plant ratio of female flowers formation and the number of harvested seeds were increased by ethephon treatment. Female flowers of male plants have 5 stigmas in contrast to the dual stigma of female 1plants. Male plant seeds were lighter and smaller than those from female plants. Although the germination rate was lower than that of normal seeds from female plants, the seeds from male plants germinated to grow seedlings. Thus, we suggest that hemp plants should be treated with ethephon at soon after primordia formation to induce the formation of more female flowers on the male plants. .

Effects of a Microbial Biostimulant, Mammoth PTM, on Cannabis sativa Bud Yield
Richard T Conant, Robert P Walsh, Michael Walsh, Colin W Bell, and Matthew D Wallenstein,
J Hortic 2017, 4:1
DOI: 10.4172/2376-0354.1000191
https://www.longdom.org/open-access/...54-1000191.pdf
High-value medicinal uses of non-psychoactive non-tetrahydrocannabinol (THC) hemp extracts like cannabidiol (CBD) are expanding to help reduce pain, severity of epileptic seizures and anxiety. This has stimulated the expansion of large indoor Cannabis grow operations in several markets. One key problem facing indoor cannabis producers is finding solutions to increase yield without significantly increasing operating costs. The purpose of this work was to evaluate how inoculation with a new sustainable microbial biostimulant, Mammoth PTM, developed at Colorado State University, affects plant growth rates and characteristics in Cannabis sativa. Treatment with Mammoth P led to significant increases in bud yields in Cannabis sativa by 16.5%. This increase in yield was accompanied by increases in plant height, and basal stem area. Increasing yield using this sustainable technology will help offset these capital expenses, reducing risk and likely increasing net income for indoor propagation facilities in this newly emerging market.

Effects of Cold Temperature and Acclimation on Cold Tolerance and Cannabinoid Profiles of Cannabis sativa L. (Hemp)
Andrei Galic, Heather Grab, Nicholas Kaczmar, Kady MaserWilliam B. Miller, And Lawrence B. Smart
https://www.mdpi.com/2311-7524/8/6/531/htm?xnpe_tifc=OfHpOf4_4kPsx.ss4dPN4ypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9X8OF4s4FH74.LNh.hLhF1J&utm_source=eNews&utm_campaign=TCS Newsletter Week 24 2022&utm_medium=email

Hemp (Cannabis sativa) is a multi-use crop garnering newfound attention from researchers and consumers. While interest has emerged, a lack of substantiated research still exists regarding effects of adverse weather events on physiological health and secondary metabolite production of hemp. The aim of this experiment was to assess cold tolerance of hemp using the cultivars ‘FINOLA’ and ‘AutoCBD’. Effects of cultivar, plant age, cold acclimation, frequency of cold treatments, and intensity of cold treatments were all considered in regard to their influence on physiological stress, biomass, and cannabinoid profile. Few effects of sequential cold treatments were noted, and they were not moderated by cold acclimation, which tended to have negative effects across many responses. This detrimental effect of cold acclimation conditions was further observed in decreased total CBD % and total THC % compared to non-acclimated plants. These findings bear consideration when assessing the unpredictability of a changing climate’s effects on the heath and cannabinoid profile of hemp. Effects of Light Spectra on Morphology, Gaseous Exchange, and Antioxidant Capacity of Industrial Hemp

Xia Cheng, Rong Wang, Xingzhu Liu, Lijuan Zhou, Minghua Dong, Muzammal Rehman, Shah Fahad, Lijun Liu and Gang Deng
Front. Plant Sci., 02 June 2022 |
https://doi.org/10.3389/fpls.2022.937436
https://www.frontiersin.org/articles/10.3389/fpls.2022.937436/full?xnpe_tifc=bIVpxdoJ4kodbIPNbfYj49pZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9XlhuhsxDbJx.xXb.1ZxdU_&utm_source=eNews&utm_campaign=TCS Newsletter Week 26 2022&utm_medium=email
One of the most important growth factors in cannabis cultivation is light which plays a big role in its successful growth. However, understanding that how light controls the industrial hemp growth and development is poor and needs advanced research. Therefore, a pot study was conducted to investigate the effects of different colors of light, that is, white light (WL), blue light (BL), red light (RL), and 50% red with 50% blue mix light (RBL) on morphology, gaseous exchange and antioxidant capacity of industrial hemp. Compared with WL, BL significantly increase hemp growth in terms of shoot fresh biomass (15.1%), shoot dry biomass (27.0%), number of leaves per plant (13.7%), stem diameter (10.2%), root length (6.8%) and chlorophyll content (7.4%). In addition, BL promoted net photosynthesis, stomatal conductance, and transpiration, while reduces the lipid peroxidation and superoxide dismutase and peroxidase activities. However, RL and RBL significantly reduced the plant biomass, gas exchange parameters with enhanced antioxidant enzymes activities. Thus, blue light is useful for large-scale sustainable production of industrial hemp.

Effects of X-ray treatment on Cannabis saliva pollen viability
Michela Zottini, Giuseppe Mandolino & Paolo Ranalli
Plant Cell, Tissue and Organ Culture volume 47, pages189–194(1997)
DOI: 10.1007/BF02318957
The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with X-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased ?-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum X-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.

Not directly Cannabis related
Evolution of Dioecy in Flowering Plants.
Bawa, K. S.
Annual Review of Ecology and Systematics, 11(1), 15–39.(1980).
doi:10.1146/annurev.es.11.110180.000311
Dioecy, characterized by the presence of distinct male and female plants, is widespread in angiosperms, being known in 37 out of Engler & Prantl's 51 orders (132). Yet botanists have paid little attention to dioecy, particularly to selective forces underlying its evolution. Several factors have contributed to this neglect. First, though there are many dioecious species, the proportion of such species in the world's flora is reputed to be quite small (37, 53, 132). The presumed rarity of the dioecious condition has led to the belief that it is not a particularly successful mode of reproduction (53, 125). Second, dioecy has been compared with self-incompatibility and, because 50% of the dioecious plants lose the capacity to bear seeds, it has been considered a poor substitute for self-incompatibility (3, 53). Third, selection for outcrossing has been almost universally proposed as the principal selective force responsible for the evolution of dioecy (3, 16-18, 26, 45, 67, 84-86, 98, 101, 103). The proposal has little empirical support, but one of its consequences was that biologists took for granted the outcrossing advantage as the principal factor and, until recently (10, 21, 128), did not consider other factors influencing the evolution of dioecy. I wish to argue that dioecy is not as rare as is generally assumed. In certain regions, among certain life forms, more than one fourth of all species may be dioecious (see below). In addition, I argue that the evolution of dioecy is not entirely due to selective pressure for increased outcrossing. By considering how such ecological factors as allocation of resources for male and female functions, sexual selection, seed dispersal, pollination, and predation may influence the evolution of dioecy, I attempt to counterbalance the widespread bias towards genetic models that assume outcrossing as the main selective force (18, 19, 67, 84, 101, 102, 104).Two related topics, sexual dimorphism and sex ratios, have been reviewed recently (71, 81, 91) and are not considered here.

Evolution of the Polyphenol and Terpene Content, Antioxidant Activity and Plant Morphology of Eight Different Fiber-Type Cultivars of Cannabis sativa L. Cultivated at Three Sowing Densities
Amandine André, Marianne Leupin, Markus Kneubühl, Vasilisa Pedan and Irene Chetschik
Plants 2020, 9, 1740;
doi:10.3390/plants9121740
The chemical composition of the inflorescences of eight different fibre-type Cannabis sativa L. cultivars grown in Switzerland was monitored for different sowing densities over the season 2019. HPLC-MS, GC-MS and GC-FID, as well as spectrophotometric techniques were used to measure the total phenolic content (TPC) and the antioxidative activity of the inflorescence extracts, and to characterise and quantify the flavonoids and terpenes produced by the different cultivars over different sowing densities from July to September 2019. The main finding of the present study is that the TPC, as well as the individual flavonoids and terpenes, were mainly influenced by the harvest period and the phenological stage of the plant. The content of polyphenols and flavonoids decrease during the flower development for all cultivars studied. The terpene content increased with maturation. The monoterpenes/sesquiterpenes ratio also changed between the early flowering (majority of sesquiterpenes) and the end of flowering (majority of monoterpenes). The sowing density showed an impact on plant morphology, a low density such as 30 seeds/m2 influencing the production of bigger flowers, thus increasing the yield of polyphenols and terpenes production. Therefore, hemp inflorescences can be regarded as valuable by-products of fibre production, for their valorisation in the food and beverage industry in addition to cosmetics and perfumery

Expanding Leaf Tissue Nutrient Survey Ranges for Greenhouse Cannabidiol-Hemp
Hunter Landis, Kristin Hicks, Paul Cockson, Josh B. Henry, James T. Smith, and Brian E. Whipker
Crop, Forage & Turfgrass Management Jan 24 2019
https://sci-hub.se/10.2134/cftm2018.09.0081
Recent legislation allows US growers to produce industrial hemp (Cannabis sativa L.) under State Industrial Hemp Pilot Programs. Hemp can be produced for seed, fiber, or cannabidiol (CBD) (Johnson, 2018). Cannabidiol is a non-intoxicating cannabinoid, and products from CBD-hemp are considered to have one of the greatest market potentials due to its pharmaceutical value (Cherney and Small, 2016). There are no researched nutrient recommendations specific to greenhouse CBD-hemp, few resources for diagnosing plant nutrient problems, and little scientific research supporting greenhouse production. While there are Cannabis nutrient survey tissue values reported by Bryson and Mills (2014), it is unknown if these values are applicable for both field and greenhouse CBD-hemp crops or if differences exist among Cannabis cultivars. The purpose of this study was to determine if there were leaf tissue nutrient differences among industrial hemp cultivars being grown as stock plants for CBD-hemp production. In addition, the goal was to provide more precise nutrient survey tissue values that would aid in diagnosing nutrient disorders.

Expansion of female sex organs in response to prolonged virginity in Cannabis sativa (marijuana).
Small, E., & Naraine, S. G. U.
Genetic Resources and Crop Evolution, 63(2), 339–348. (2015).
doi:10.1007/s10722-015-0253-3
Female flowers of Cannabis sativa in wild-growing populations and in hemp plantations are almost always well supplied with pollen. The style-stigma portion of the pistils of such plants was found to average only about 3 mm in length and to invariably be two-branched. By contrast, ‘‘buds’’ (congested female inflorescences), the standard form of marijuana now produced in the illicit and medicinal marijuana sectors, are protected against pollen. This report documents that in the absence of pollen, the style-stigma parts of virgin pistils expand notably, average over 8 mm in length, and tend to develop more than two branches and to increase in girth. From an evolutionary viewpoint, this expansion of pollenreceptive tissue is an apparent adaptation for increasing the probability of fertilizing the females when males are extremely scarce. From a practical viewpoint, the expanded presence of stigma tissues may be both advantageous and disadvantageous. The highTHC secretory gland heads of Cannabis tend to fall away from marijuana buds, significantly decreasing pharmacological potency, but many gland heads become stuck to the receptive papillae of the stigmas, reducing the loss. Although stigmas constitute a small proportion of marijuana, their distinctive chemistry could have health effects.

Not Cannabis Specific
Far-red radiation promotes growth of seedlings by increasing leaf expansion and whole-plant net assimilation.
Park, Y., & Runkle, E. S. (2017).
Environmental and Experimental Botany, 136, 41–49.
doi:10.1016/j.envexpbot.2016.12.013
By definition, photosynthetically active radiation (PAR) includes wavelengths between 400 and 700 nm and thus, far-red radiation (FR, 700 to 800 nm) is excluded when the photosynthetic photon flux density (PPFD) is measured and reported. However, FR radiation [and the ratio of red (R; 600 to 700 nm) to FR] regulates phytochrome-mediated morphological and developmental plant responses to promote radiation capture and survival under shade. We postulated that the inclusion of FR in a radiation spectrum would have little effect on photosynthesis but would increase radiation capture and plant growth, while accelerating the subsequent flowering of shade-avoiding species. Geranium (Pelargonium ×hortorum), petunia (Petunia ×hybrida), snapdragon (Antirrhinum majus), and impatiens (Impatiens walleriana) were grown at 20 °C under an 18-h photoperiod provided by sole-source lighting from lightemitting diodes that included 32 µmol?m–2 ?s –1 of blue and the following intensities of R and FR radiation: R128 (128 µmol?m–2 ?s –1 of R), R128+FR16, R128+FR32, R128+FR64, R96+FR32, and R64+FR64. Plant height in all species studied and total leaf area of geranium and snapdragon linearly decreased as the R:FR (or the estimated phytochrome photoequilibrium) of each treatment increased. In geranium and snapdragon, the increase in total leaf area (by 7 %) with the addition of FR to the same PPFD subsequently increased shoot dry weight (DW) (by 28-50%) while the increase in total leaf area (by 30-40%) with the partial substitution of R with FR partly compensated for the reduction in PPFD (by 40%), producing a similar shoot DW. Whole-plant net assimilation of geranium, snapdragon, and impatiens increased with additional FR radiation, showing a linear relationship with the calculated yield photon flux density of each radiation treatment. In addition, inclusion of FR during seedling growth promoted flowering in the longday plant snapdragon. We conclude that FR radiation increases plant growth indirectly through leaf expansion and directly through whole-plant net assimilation and in at least some species, promotes subsequent flowering. Abbreviations: B, blue radiation; FR, far-red radiation; LEDs, light-emitting diodes; PAR, photosynthetically active radiation; PPE, phytochrome photoequilibrium; PPFD, photosynthetic photon flux density; Pr, R-radiation-absorbing phytochrome; Pfr, FR-radiation-absorbing phytochrome; PSI, photosystem I; PSII, photosystem II; R, red; TPFD, total photon flux density; YPFD, yield photon flux density

Fertilization Following Pollination Predominantly Decreases Phytocannabinoids Accumulation and Alters the Accumulation of Terpenoids in Cannabis Inflorescences
Carni Lipson Feder, Oded Cohen, Anna Shapira, Itay Katzir, Reut Peer, Ohad Guberman, Shiri Procaccia, Paula Berman, Moshe Flaishman and David Meiri
Front. Plant Sci. 12:753847.
doi: 10.3389/fpls.2021.753847
https://www.frontiersin.org/articles...21.753847/full
In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-19 -trans -tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed
semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich
female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings
may point to the functional roles of secondary metabolites in Cannabis

Floral Biology and Pollination Biology of Cannabis sativa L.
Anita Rana and Namrta Choudhary
The International Journal of Plant Reproductive Biology 2 (2) pp. 191-195, 2010
https://pdfs.semanticscholar.org/737...3863957c09.pdf
Cannabis sativa L. commonly known as marijuana, hemp, bhang, charas is a dioecious, aromatic annual flowering herb native of Central Asia and belongs to an extremely small family Cannabiaceae (Urticaceae) of the order urticales containing only two genera Humulus and Cannabis . The genus Cannabis is monotypic with one species i.e. Cannabis sativa L. headache, flu, epilepsy, cough and pains. In modern medicine the crude drug and some pure chemical
derivatives are used for treating migraine, epilepsy, malaria, glaucoma, nausea from chemotherapy, for improving appetite in patients with cancer, AIDS, and anorexia nervosa and for suppressing muscular spasms in multiple sclerosis (Van Wyk 2000). The compounds which compress the active drug ingredients are Cannabinoids exist in form of carboxylic acids (Masoud & Doorenbos 1973, Small & Beckstead 1973, Turner et al . 1973). There are over 60 of these types of compounds present in the plant (Turner et al . 1980). flowering period. Floral morphology, floral biology, number of pollen grains/flower and number of ovules were studied by various methods given by Kearns & Inouye (1993). Total number of pollen/ anther and pollen/flower was measured by a hemocytometer (Barret 1985). Pollen viability was checked by 0.2% TTC solution (2, 3, 5-triphenyltetrazolium chloride). The staining solution was prepared in 10% sucrose solution. The pH was adjusted to 5.8 using 0.15 M Tris HCl buffer (Hauser & & Morrison 1964); by Fluorochromatic reaction (FCR) after Heslop-Harrison & Heslop-Harrison (1970). in vivo pollen germination on the stigmatic surface was also carried out by aniline blue fluorescence microscopic method (Martin 1959) was described by Shivanna & Rangaswamy (1992). Different pollinators, their population, types and visitation rates were recorded. The morphology of different floral parts was studied by scanning electron microscopy (SEM). The fresh anthers, pistils and other parts were fixed in 3% glutaraldehyde.

Not Cannabis specific
Florigen
Signaling Pathways in Plants Hiroyuki Tsuji, Ken-ichiro Taoka, in The Enzymes, 2014
https://www.sciencedirect.com/topics...y/florigen/pdf
Chapter Five - Florigen Signaling
10.1016/B978-0-12-801922-1.00005-1
Florigen is a systemic signal for the transition to flowering in plants. It is generated in leaves and transported to the shoot apical meristem to promote floral transition [1]. The molecular nature of florigen has long been a key question in the field of flowering research. Recently, florigen was identified as the protein product of the FLOWERING LOCUS T (FT) gene [2–4]. This finding facilitated new research aimed at understanding the mechanisms of florigen signaling in plants. In this review, we summarize recent advances in the understanding of florigen function.

Flowering Dynamics in Monoecious and Dioecious Hemp Genotypes
Stefano Amaducci, Michele Colauzzi, Alessandro Zatta, Gianpietro Venturi
April 2008 Journal of Industrial Hemp 13(1):5-19
DOI: 10.1080/15377880801898691
Flowering is a crucial phase in hemp cultivation. It influences both stem and seed yield. A uniform and short flowering duration in hemp is desirable, because it favors uniform crop development. Moreover, flowering is often taken as a reference point for harvesting, and very long durations of flowering might mislead the operator in judging the proper time for mowing. In this article, a large dataset of flowering time and duration for different monoecious and dioecious varieties was studied and the effect of sowing time and genotype on flowering duration is discussed. Minimal flowering duration was observed when the time from emergence to flowering was short. This, was related however, to low yields. Dynamics of flowering was accurately described by a bi-logistic curve that indicates the presence of two underlying logistic processes.

Not Cannabis specific
2.3.1 Flowering Locus T
Yin Wang, ... Toshinori Kinoshita,
A recent report indicated that Flowering Locus T (FT), which promotes flowering as florigen, is a positive regulator of the H+-ATPase and blue-light-induced stomatal opening [64]. Overexpression of FT in guard cells enhanced stomatal opening, and the loss-of-function FT mutant showed the repressed H+-ATPase activity, thereby inhibited blue-light-induced stomatal opening [64]. Most recently, Ando et al. [65] reported that the closest homolog of FT, Twin Sister of FT (TSF), is also a positive regulator in stomatal opening. Moreover, the floral regulators Gigantea (GI) and Constans (CO) that function upstream FT and TSF are also involved in promoting stomatal opening [65]. The loss-of-function mutants of GI, CO, and TSF repressed stomatal opening, but the treatment of all mutants with FC (an effective H+-ATPase activator) caused normal stomatal opening found in wild-type plants, indicating that the guard-cell H+-ATPase in these mutants works normally. Based on the photoperiodic pathway in the flowering, transcriptional regulation is possibly involved in downstream of FT/TSF for activation of the H+-ATPase (Fig. 8.1, orange arrows). Further studies are required to explore how they affect H+-ATPase

Foliar Sprays of Silver Thiosulfate Produce Male Flowers on Female Hemp Plants
Mark H Brand, Jessica D Lubell
HortTechnology 28(6):743-747 December 2018
DOI: 10.21273/HORTTECH04188-18
Interest in hemp ( Cannabis sativa ) for its medicinal compounds, cannabidiol (CBD), and ?-9-tetrahydrocannabidiol (THC), continues to increase. Maximizing yield of CBD and/or THC requires female plants because female inflorescences accumulate significantly greater concentrations of these compounds than male inflorescences. Production of all female seed requires induction of female plants to develop male flowers that produce genetically female pollen. Growers would like access to feminized seed to produce all-female crops. We evaluated the efficacy of 0, 0.3, and 3 m m silver thiosulfate (STS) applied as a foliar spray (on three occasions 7 days apart) to produce male flowers on four strains of female hemp (having a THC concentration of ?0.3%), designated CBD hemp A, CBD hemp B, CBD hemp C, and industrial hemp. Silver thiosulfate at 3 m m was the most efficacious treatment for all strains. The majority of inflorescences had 100% male flowers at 3 m m STS, and terminal inflorescences had ?95% conversion to male flowers. Silver thiosulfate at 0.3 m m produced partial conversion to male flowers, whereas most inflorescences had around 50% male flowers, except for CBD hemp A, which demonstrated greater levels of masculinization. At 0.3 m m STS, terminal inflorescences of CBD hemp A had 91% conversion to male flowers. This study demonstrates that male flowers can be produced easily and consistently on female plants through application of foliar sprays of STS under short-day conditions.

Not Cannabis Specific
Gamete formation without meiosis in Arabidopsis.
Ravi, M., Marimuthu, M. P. A., & Siddiqi, I.
Nature, 451(7182), 1121–1124.(2008).
doi:10.1038/nature06557
Apomixis, the formation of asexual seeds in plants, leads to populations that are genetically uniform maternal clones. The transfer of apomixis to crop plants holds great promise in plant breeding for fixation of heterozygosity and hybrid vigour because it would allow the propagation of hybrids over successive generations1,2. Apomixis involves the production of unreduced (diploid) female gametes that retain the genotype of the parent plant (apomeiosis), followed by parthenogenetic development of the egg cell into an embryo and the formation of functional endosperm3 . The molecular mechanisms underlying apomixis are unknown. Here we show that mutation of the Arabidopsis gene DYAD/SWITCH1 (SWI1) 4,5, a regulator of meiotic chromosome organization, leads to apomeiosis. We found that most fertile ovules in dyad plants form seeds that are triploid and that arise from the fertilization of an unreduced female gamete by a haploid male gamete. The unreduced female gametes fully retain parental heterozygosity across the genome, which is characteristic of apomeiosis. Our results show that the alteration of a single gene in a sexual plant can bring about functional apomeiosis, a major component of apomixis.

Not directly Cannabis related
Gender in plants: sex chromosomes are emerging from the fog.
Vyskot, B., & Hobza, R.
Trends in Genetics, 20(9), 432–438.(2004).
doi:10.1016/j.tig.2004.06.006
Although most plants have flowers with both male and female sex organs, there are several thousands of plant species where male or female flowers form on different individuals. Surprisingly, the presence of well-established sex chromosomes in these dioecious plants is rare. The best-described example is white campion, for which large sex chromosomes have been identified and mapped partially. A recent study presented a comprehensive genetic and physical mapping of the genome of dioecious papaya. It revealed a short male specific region on the Y chromosome (MSY) that does not recombine with the X chromosome, providing strong evidence that the sex chromosomes originated from a regular pair of autosomes. The primitive papaya Y chromosome thus represents an early event in sex chromosome evolution. In this article, we review the current status of plant sex-chromosome research and discuss the advantages of different dioecious models.

Greenhouse propagation of Cannabis Sativa L. by vegetative cuttings.
Coffman, C. B., & Gentner, W. A.
Economic Botany, 33(2), 124–127.(1979).
doi:10.1007/bf02858280
Previous work revealed significant variations in cannabinoid profiles of Cannabis sativa L. derived from a single seed source (P.I. 378939) and subjected to the same growth environment. Studies were conducted to evaluate the efficacy of propagation of C. sativa by vegetative cuttings in order to increase uniformity of cannabinoid concentrations within a given plant population. C. sativa was successfully propagated by vegetative cuttings. However, there were both morphological and biochemical differences between seed-derived plants and their
vegetative propagules. Delta-9-tetrahydrocannabinol concentrations were 4.1 times higher in vegetative propagules than in seed propagules. Vegetative cuttings also generally developed more profuse lateral branch growth; hence, foliage increased relative to their parent plants. Cannabinoid levels within the population of vegetative cuttings remained highly variable.

Heavy Metals Interaction in Soil-Plant System of Carmagnola cannabis Strain
Dani Fadel, Najoie Assaad, Ali Hachem, Ariadne Argyraki & Zacharenia Kypritidou
Journal of Agricultural Science; Vol. 12, No. 7; 2020
https://www.researchgate.net/publica...annabis_Strain
Evolution in the legislation of Cannabis in Lebanon regarding production and consumption of related products for medicinal and recreational uses is leading to emerging regulations regarding the potency and cannabinoid
profiles. On 21 April 2020, the Parliament passed a law legalizing cannabis cultivation for medical use. The objective of this work was to estimate the heavy metals accumulation in the soil-plant system and to help the governmental regulatory body on having also into account the impurities of metals within their rules of regulation. The impurity content of hyperaccumulating metals (zinc, chromium, arsenic, manganese, cadmium, barium, aluminum, iron, cobalt, nickel, copper and lead) was evaluated by inductively coupled plasma atomic emission spectrometry (ICP-OES) taking into account the pseudototal and mobilizable concentrations of the elements in the rhizosphere of plants and the total concentrations of their aerial parts and spikes from allotments in Kropia region-Athens, Greece. The main physicochemical assets of topsoil samples such as pH (7.99±0.05) and organic matter content (rich), the X-ray crystallography test (basically quartz, albite and vermiculite) and soil texture determination test (basically sandy loam soil) were also determined. The concentrations of most of our studied elements in soil plant system samples were recorded below or around the plant reference material concentrations used in our analysis. Results showed also that Al was highly toxic in soil and plant samples. In the plant samples, the arsenic was nearly absent and the lead, nickel, copper, chromium and cadmium contents were less than those found in the plant reference material. In the soil samples, only copper and zinc concentrations
were found to be within the accepted ranges. The maximum transfer factor is found in lead (Tf – Pb = 0.8223). Average transfer factor of elemental concentrations showed that heavy metals were not easily translocated in the soil-plant system (0.0514±0.0032). In addition, hemp plants that are considered as "hyper-accumulators" showed very acceptable results for industrial and other uses.

Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences – Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation.
Punja, Z. K., & Holmes, J. E.
Frontiers in Plant Science, 11.(2020).
doi:10.3389/fpls.2020.00718
Cannabis sativa L. (hemp, marijuana) produces male and female inflorescences on different plants (dioecious) and therefore the plants are obligatory out-crossers. In commercial production, marijuana plants are all genetically female; male plants are destroyed as seed formation reduces flower quality. Spontaneously occurring hermaphroditic inflorescences, in which pistillate flowers are accompanied by formation of anthers, leads to undesired seed formation; the mechanism for this is poorly understood. We studied hermaphroditism in several marijuana strains with three objectives: (i) to compare the morphological features of this unique phenotype with normal male flowers; (ii) to assess pollen and seed viability from hermaphroditic flowers; and (iii) to assess the effect of hermaphroditism on progeny male:female (sex) ratios and on genetic variation using molecular methods. The morphological features of anthers, pollen production and germination in hermaphroditic flowers and in staminate inflorescences on male plants were compared using light and scanning electron microscopy. Seeds produced on hermaphroditic plants and seeds derived from cross-fertilization were germinated and seedlings were compared for gender ratios using a PCR-based assay as well as for the extent of genetic variation using six ISSR primers. Nei’s index of gene diversity and Shannon’s Information index were compared for these two populations. The morphology of anthers and pollen formation in hermaphroditic inflorescences was similar to that in staminate flowers. Seedlings from hermaphroditic seeds, and anther tissues, showed a female genetic composition while seedlings derived from cross-fertilized seeds showed a 1:1 male:female sex expression ratio. Uniquely, hermaphroditic inflorescences produced seeds which gave rise only to genetically female plants. In PCR assays, a 540 bp size fragment was present in male and female plants, while a 390 bp band was uniquely associated with male plants. Sequence analysis of these fragments revealed the presence of Copia-like retrotransposons within the C. sativa genome which may be associated with the expression of male or female phenotype. In ISSR analysis, the percentage of polymorphic loci ranged from 44 to 72% in hermaphroditic and cross-fertilized populations. Nei’s index of gene diversity and Shannon’s Information index were not statistically different for both populations. The extent of genetic variation after one generation of selfing in the progeny from hermaphroditic seed is similar to that in progeny from cross-fertilized seeds

How different light spectra impact the metabolism of the cannabis plant
David Hawley
mmj daily mag
https://www.mmjdaily.com/article/922...R4051Vy0kFPWRE
With a plant intended for medical applications, the effects of that plant are dictated by its chemistry, which is itself dictated by its genetics and production environment. Some of the metabolites thought to be involved in medicinal applications can be increased or decreased in concentration by manipulating the production environment.

Identification of Phenotypic Characteristics in Three Chemotype Categories in the Genus Cannabis
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
HORTSCIENCE
DOI: 10.21273/HORTSCI15607-20
https://journals.ashs.org/hortsci/vi...e=The+Cannabis +%2B+Cannabinoid+Curator&utm_c ampaign=70b2ae2af2-EMAIL_CAMPAIGN_2020_03_13_07_5 9_COPY_01&utm_medium=email&utm _term=0_47f6c7c552-70b2ae2af2-365750754
Modern Cannabis cultivars are morphologically distinguished by their leaflet shapes (wide for “Indica” and narrow for “Sativa”) by users and breeders. However, there are no scientific bases or references for determining the shape of these leaflets. In addition, these two categories contained mostly THC dominant (high THC) cultivars while excluded CBD dominant (high CBD) and intermediate (intermediate level of both THC and CBD) cultivars. This study investigated the phenotypic variation in 21 Cannabis cultivars covering three chemical phenotypes, referred to as chemotypes, grown in a commercial greenhouse. Thirty morphological traits were measured in the vegetative, flowering, and harvest stages on live plants and harvested inflorescences. The collected data were subjected to correlation analysis, hierarchical clustering, principal component analysis, and canonical correlation analysis with preassigned chemotypes. Canonical correlation analysis assigned individual plants to their chemotypes with 92.9% accuracy. Significant morphological differences were identified. Traits usable as phenotype markers for CBD dominant cultivars included light-green and narrow leaflets, a greater number of primary and secondary serrations, loose inflorescences, dense and resinous trichomes, and Botrytis cinerea resistance. Traits for intermediate cultivars included deep-green and medium-wide leaflets, more primary and secondary serrations, medium compact inflorescences, trichomes that are less dense and less resinous, and Botrytis cinerea resistance. Traits for THC dominant cultivars included deep-green and wide leaflets, large and compact inflorescences, dense and resinous trichomes, and Botrytis cinerea susceptibility. The results of this study provide a comprehensive profile of morphological traits of modern Cannabis cultivars and provides the first such profile for CBD dominant and intermediate cultivars. Additionally, this study included the traits of inflorescences, which have not been compared between three chemotypes in the literature. Phenotype markers identified in this study can facilitate preliminary cultivar identification and selection on live plants before or as a supplement to chemical and genetic analysis.

Impact of N, P, K, and Humic Acid Supplementation on the Chemical Profile of Medical Cannabis
Bernstein, N., Gorelick, J., Zerahia, R., & Koch, S.
(Cannabis sativa L). Frontiers in Plant Science, 10 . (2019).
doi:10.3389/fpls.2019.00736
Mineral nutrition is a major factor affecting plant growth and function. Increasing evidence supports the involvement of macro and micronutrients in secondary metabolism. The use of the appropriate nutritional measures including organic fertilizers, supplements, and biostimulants is therefore a vital aspect of medicinal plant production including medical cannabis. Due to legal restriction on cannabis research, very little information is available concerning the effects of nutritional supplements on physiological and chemical properties of medical cannabis, and their potential role in standardization of the active compounds in the plant material supplied to patients. This study therefore evaluated the potential of nutritional supplementations, including humic acids (HAs) and inorganic N, P, and K to affect the cannabinoid profile throughout the plant. The plants were exposed to three enhanced nutrition treatments, compared to a commercial control treatment. The nutrition treatments were supplemented with HA, enhanced P fertilization, or enhanced NPK. The results demonstrate sensitivity of cannabinoids metabolism to mineral nutrition. The nutritional supplements affected cannabinoid content in the plants differently. These effects were location and organ specific, and varied between cannabinoids. While the P enhancement treatment did not affect THC, CBD, CBN, and CBG concentrations in the flowers from the top of the plants, a 16% reduction of THC concentration was observed in the inflorescence leaves. Enhanced NPK and HA treatments also produced organspecific and spatially specific responses in the plant. NPK supplementation increased CBG levels in flowers by 71%, and lowered CBN levels in both flowers and inflorescence leaves by 38 and 36%, respectively. HA was found to reduce the natural spatial variability of all of the cannabinoids studied. However, the increased uniformity came at the expense of the higher levels of cannabinoids at the top of the plants, THC and CBD were reduced by 37 and 39%, respectively. Changes in mineral composition were observed in specific areas of the plants. The results demonstrate that nutritional supplements influence cannabinoid content in cannabis in an organ- and spatial-dependent manner. Most importantly, the results confirm the potential of environmental factors to regulate concentrations of individual cannabinoids in medical cannabis. The identified effects of nutrient supplementation can be further developed for chemical control and standardization in cannabis

Impact of Three Different Light Spectra on the Yield, Morphology and Growth Trajectory of Three Different Cannabis sativa L. Strains
Philipp Rechel, Sebastian Munz, Jens Hartung, Achim Präger, Stiina Kotiranta, Lisa Burgel, Torsten Schober, Simone Graeff-Hönninger
Plants 10(9):1866 September 2021
DOI: 10.3390/plants10091866
https://www.researchgate.net/publica..._of_Three_Diff erent_Light_Spectra_on_the_Yie ld_Morphology_and_Growth_Traje ctory_of_Three_Different_Canna bis_sativa_L_Strains
https://www.researchgate.net/publica..._of_Three_Diff erent_Light_Spectra_on_the_Yie ld_Morphology_and_Growth_Traje ctory_of_Three_Different_Canna bis_sativa_L_Strains
Cannabis is one of the oldest cultivated plants, but plant breeding and cultivation are restricted by country specific regulations. Plant growth, morphology and metabolism can be manipulated by changing light quality and intensity. Three morphologically different strains were grown under three different light spectra with three real light repetitions. Light dispersion was included into the statistical evaluation. The light spectra considered had an influence on the morphology of the plant, especially the height. Here, the shade avoidance induced by the lower R:FR ratio under the ceramic metal halide lamp (CHD) was of particular interest. The sugar leaves seemed to be of elementary importance in the last growth phase for yield composition. Furthermore, the last four weeks of flowering were crucial to influence the yield composition of Cannabis sativa L. through light spectra. The dry flower yield was significantly higher under both LED treatments compared to the conventional CHD light source. Our results indicate that the plant morphology can be artificially manipulated by the choice of light treatment to create shorter plants with more lateral branches which seem to be beneficial for yield development. Furthermore, the choice of cultivar has to be taken into account when interpreting results of light studies, as Cannabis sativa L. subspecies and thus bred strains highly differ in their phenotypic characteristics.

Not Cannabis specific
Improving soil micronutrients availability under organic farming
Salwinder Singh Dhaliwal. Vivek Sharma, Agniva Mandal, Krishna Rama, Rk Naresh, Gayatri Verma
In book: Advances in Organic Farming Chapter: 7 Publisher: Elsevier August 2021
https://www.researchgate.net/publica...rganic_farming
Intensive agriculture has covered the way for “Green Revolution” with the use of high yielding varieties, chemical fertilizers and pesticides with sole objective of yield maximization. But in the same time, it deteriorated the soil health, macro and micronutrient deficiencies, low yields, poor quality and environmental hazards which resulted in serious health problems and disorders in animals and human beings. Globally, deficiencies of micronutrients in soils have emerged as a major limiting factor to higher crop yields and lowers the concentration of micronutrient in crops. One of the ways to cope with the problems of micronutrient deficiencies is to improve the soil micronutrient availability or uptake efficiency of plants. Micronutrient availability in soil is determined by the parent materials which is influenced by the edaphic and biological factors in soil such as pH, redox potential, soil minerals, organic matter and soil microbial activity. The improved agricultural practices such as soil organic amendments and soil water management play vital role in soil micronutrient availability. Under organic farming, soil health and nutrient availability is sustained by the addition of local organic inputs i.e. waste, dungs, biofertilizers, crop residues, green manures followed by crop rotation system. Effective management and recycling of available on-farm wastes helps to reduce the dependency on external chemical inputs and limits the environmental pollution arising out with burning of farm wastes. In this chapter, we summarize behaviour, availability and factors affecting of micronutrients availability in soils as well as detail about the different organic sources of micronutrients to improve the availability in soil.

Not Cannabis specific
Improving the Predictive Value of Phytochrome Photoequilibrium: Consideration of Spectral Distortion Within a Leaf
Paul Kusuma, Bruce Bugbee
Frontiers in Plant Science 12:596943 May 2021
DOI: 10.3389/fpls.2021.596943
The ratio of active phytochrome (Pfr) to total phytochrome (Pr + Pfr), called phytochrome photo-equilibrium (PPE; also called phytochrome photostationary state, PSS) has been used to explain shade avoidance responses in both natural and controlled environments. PPE is commonly estimated using measurements of the spectral photon distribution (SPD) above the canopy and photoconversion coefficients. This approach has effectively predicted morphological responses when only red and far-red (FR) photon fluxes have varied, but controlled environment research often utilizes unique ratios of wavelengths so a more rigorous evaluation of the predictive ability of PPE on morphology is warranted. Estimations of PPE have rarely incorporated the optical effects of spectral distortion within a leaf caused by pigment absorbance and photon scattering. We studied stem elongation rate in the model plant cucumber under diverse spectral backgrounds over a range of one to 45% FR (total photon flux density, 400–750 nm, of 400 μmol m–2 s–1) and found that PPE was not predictive when blue and green varied. Preferential absorption of red and blue photons by chlorophyll results in an SPD that is relatively enriched in green and FR at the phytochrome molecule within a cell. This can be described by spectral distortion functions for specific layers of a leaf. Multiplying the photoconversion coefficients by these distortion functions yields photoconversion weighting factors that predict phytochrome conversion at the site of photon perception within leaf tissue. Incorporating spectral distortion improved the predictive value of PPE when phytochrome was assumed to be homogeneously distributed within the whole leaf. In a supporting study, the herbicide norflurazon was used to remove chlorophyll in seedlings. Using distortion functions unique to either green or white cotyledons, we came to the same conclusions as with whole plants in the longer-term study. Leaves of most species have similar spectral absorbance so this approach for predicting PPE should be broadly applicable. We provide a table of the photoconversion weighting factors. Our analysis indicates that the simple, intuitive ratio of FR (700–750 nm) to total photon flux (far-red fraction) is also a reliable predictor of morphological responses like stem length.

Induction of female flowers on male plants of Cannabis Sativa L. by 2-chloroethanephos-phonic acid.
Ram, H. Y. M., & Jaiswal, V. S.
Experientia, 26(2), 214–216. (1970).
doi:10.1007/bf01895593
Sex expression in Cannabis sativa, a dioecious annual, can be modified by temperature, day length, as well as exogenous application of auxin. The present investigation was undertaken to test whether Ethrel (2-chloroethanephosphonic acid), a recently recommended source of ethylene, could induce femaleness in male plants of Cannabis sativa.

Induction of male flowers on female plants of Cannabis sativa by gibberellins and its inhibition by abscisic acid.
Ram, H. Y. M., & Jaiswal, V. S. (
Planta, 105(3), 263–266. (1972).
doi:10.1007/bf00385397
Gibberellins (GA3, GAt+~, GA 7 and GAg) induce male flowers on
female plants of Cannabis sativa. This is, depending on concentration, partially or fully inhibited by abscisie acid (ABA). The ABA effect can in turn be partially overcome by increasing the concentration of GA a .

Influence of Light Spectra on the Production of Cannabinoids
Pascal Amrein, Stefan Rinner, Tindaro Pittorino, Joan Espel, David Schmidmayr
Med Cannabis Cannabinoids 2020;3:103–110
DOI: 10.1159/000510146
In recent years, more attention has been paid to cannabis from both medical and political points of view. This study investigates the influence of 5 different light spectra on the active substance content in THC-poor hemp of the Alessia chemotype II variety. The focus is on comparing conventional growing under metal halide lamps with growing under high-pressure sodium (HPS) vapor lamps with regard to different spectra of LED lighting modules. Growing was carried out in 10 growing boxes under controlled and mostly identical conditions for all boxes. The photoperiod during the vegetative phase was 18 h light and photosynthetic photon flux density ∼ 520 μmol⋅ m−2 s−1 . The flowering phase was 12 h light and ∼ 540 μmol⋅ m−2 s−1 . During the experiment, CO2 , temperature, and humidity were measured and logged. Additionally, weekly measurements of chlorophyll, electric conductivity of the fertilizer, activity measurement (salt content) of the soil, and pH value of the soil were
checked. The content of cannabinoids was measured by high-performance liquid chromatography (HPLC). Plant height and growth were monitored during the whole experiment by cameras taking pictures every 30 min and loading them onto a cloud storage platform. Cannabinoid content was measured using HPLC. Plant wet weight was determined at the end of the experiment and showed that plants under the high pressure lamp treatment had less flower weight than those under the LED treatment. In conclusion, it could be shown that certain LED spectra can considerably increase the amount of cannabinoids with respect to conventional illumination (HPS)

Not Cannabis specific
INFLUENCE OF STORAGE AND MIXING FACTORS ON THE BIOLOGICAL ACTIVITY OF SILVER THIOSULFATE
ARTHUR C. CAMERON, ROYAL D. HEINS and HAROLD N. FONDA
DOI: 10.1016/0304-4238(85)90009-3
Scientia Horticulturae, 26 (1985) 167--174
Silver thiosulfate (STS) complex stability and degradation during formulation and storage were monitored indirectly by determining the effectiveness of treatment solutions in retarding flower petal abscission in geraniums (Pelargonium hortorum Baily). Freshly prepared solutions composed of Ag+:S2032- ratios from1:1 to 1:16 at constant silver concentration were all equally effective. There were no differences in effectiveness when a Ag+:S2032- solution of ratio 1:4 was formulated at 5, 25 or 50°C, when prepared at pH 4.01, 7.0 or 10.0, or when prepared in the presence of 10 mM KC1, Na2CO3, Ca(NO3) 2 or MgSO~. The ability of solutions to retard abscission was reduced when Ag + was substantially in excess of $2032-. Rapid mixing of AgNO3 and Na2S~O3 solutions yielded effectiw~ solutions, independent of mixing order. Complete loss of activity was observed when solutions were stored in contact with either tin or galvanized metal for 5 days, whereas there was no loss in activity after 3 months' storage in plastic or glass at 2°C. These results indicate that currently recommended formulation procedures are unnecessarily stringent, and that long-term cold storage of prepared STS solutions is feasible.

Interactions Between Bacillus Spp., Pseudomonas Spp. and Cannabis sativa Promote Plant Growth
Dominique Comeau, Carole Balthazar, Amy Novinscak, Nadia Bouhamdani, David L. Joly and Martin Filion
Front. Microbiol. 12:715758
doi: 10.3389/fmicb.2021.715758
https://www.frontiersin.org/articles...21.715758/full
Plant growth-promoting rhizobacteria (PGPR) deploy several mechanisms to improve plant health, growth and yield. The aim of this study was to evaluate the efficacy of two Pseudomonas spp. strains and three Bacillus spp. strains used as single treatments and in consortia to improve the yield of Cannabis sativa and characterize the impact of these treatments on the diversity, structure and functions of the rhizosphere microbiome. Herein, we demonstrate a significant C. sativa yield increase up to 70% when inoculated with three different Pseudomonas spp./Bacillus spp. consortia but not with single inoculation treatments. This growth-promoting effect was observed in two different commercial soil substrates commonly used to grow cannabis: Promix and Canna coco. Marker-based genomic analysis highlighted Bacillus spp. as the main modulator of the rhizosphere microbiome diversity and Pseudomonas spp. as being strongly associated with plant growth promotion. We describe an increase abundance of predicted PGPR metabolic pathways linked with growth-promoting interactions in C. sativa.

Intersex inflorescences of Rumex acetosa demonstrate that sex determination is unique to each flower.
Ainsworth, C., Rahman, A., Parker, J., & Edwards, G
New Phytologist, 165(3), 711–720. (2005).
doi:10.1111/j.1469-8137.2005.01281.x
A triploid intersex individual of the normally dioecious species Rumex acetosa showed extreme variability in gynoecium development. Analysis of the development and distribution of these flowers on inflorescences enabled insight to be gained into the mechanism of sex determination. • Floral phenotypes on intersex inflorescences were classified according to gynoecium development. Flower morphology was investigated by scanning electron and light microscopy. Organ identity gene expression in intersex floral primordia was assessed using in situ hybridization. • The distribution of the different floral phenotypes shows that each individual flower is determined separately, and that the phenotype of each flower is not influenced by its position on the inflorescence, or by the phenotype of neighbouring flowers. C-function gene expression persisted in gynoecia that had ceased development. • Gynoecium development in mutant flowers resembled the phenotype of the Arabidopsis mutant ettin and suggests that a hormone gradient may be involved. C-function expression does not appear to control the extent of female development, and indicates that genes which are downstream of the organ-identity genes must control organ suppression.

Not Cannabis specific
Callus, Dedifferentiation, Totipotency, Somatic Embryogenesis: What These Terms Mean in the Era of Molecular Plant Biology?
Attila Fehér
Front. Plant Sci., 26 April 2019
DOI: 10.3389/fpls.2019.00536
https://www.frontiersin.org/articles...019.00536/full
Recent findings call for the critical overview of some incorrectly used plant cell and tissue culture terminology such as dedifferentiation, callus, totipotency, and somatic embryogenesis. Plant cell and tissue culture methods are efficient means to preserve and propagate genotypes with superior germplasm as well as to increase genetic variability for breading. Besides, they are useful research tools and objects of plant developmental biology. The history of plant cell and tissue culture dates back to more than a century. Its basic methodology and terminology were formulated preceding modern plant biology. Recent progress in molecular and cell biology techniques allowed unprecedented insights into the underlying processes of plant cell/tissue culture and regeneration. The main aim of this review is to provide a theoretical framework supported by recent experimental findings to reconsider certain historical, even dogmatic, statements widely used by plant scientists and teachers such as “plant cells are totipotent” or “callus is a mass of dedifferentiated cells,” or “somatic embryos have a single cell origin.” These statements are based on a confused terminology. Clarification of it might help to avoid further misunderstanding and to overcome potential “terminology-raised” barriers in plant research.

Not Cannabis specific
Light-Controlled flavonoid biosynthesis in fruits
Laura Zoratti, Katja Karppinen, Ana Luengo Escobar, Laura Jaakola
Frontiers in Plant Science 5(534):534 Oct 2014
DOI: 10.3389/fpls.2014.00534
Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants. The absolute dependency of light to the plant development has driven evolvement of sophisticated mechanisms to sense and transduce multiple aspects of the light signal. Light effects can be categorized in photoperiod (duration), intensity (quantity), direction and quality (wavelength) including UV-light. Recently, new information has been achieved on the regulation of light-controlled flavonoid biosynthesis in fruits, in which flavonoids have a major contribution on quality. This review focuses on the effects of the different light conditions on the control of flavonoid biosynthesis in fruit producing plants. An overview of the currently known mechanisms of the light-controlled flavonoid accumulation is provided. R2R3 MYB transcription factors are known to regulate by differential expression the biosynthesis of distinct flavonoids in response to specific light wavelengths. Despite recent advances, many gaps remain to be understood in the mechanisms of the transduction pathway of light-controlled flavonoid biosynthesis. A better knowledge on these regulatory mechanisms is likely to be useful for breeding programs aiming to modify fruit flavonoid pattern.

Light dependence of photosynthesis and water vapor exchange characteristics in different high _9-THC yielding varieties of Cannabis sativa L.
Suman Chandraa, Hemant Lataa, Zlatko Mehmedica, Ikhlas A. Khana, Mahmoud A. ElSohly
Journal of Applied Research on Medicinal and Aromatic Plants 2(2) March 2015
Doi: 10.1016/j.jarmap.2015.03.002
The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600and 2000 _mol m?2s?1) on gas and water vapor characteristics of four high _9-tetrahydrocannabinol (_9-THC) yielding drug type varieties (HPM, K2, MX and W1) ofCannabis sativa was studied. Plants of each variety were grown from seeds. On flowering,male plants were removed and vegetatively propagated clones of selected female plantswere used for gas and water vapor studies at different photosynthetic photon flux densi-ties (PPFDs). Our data show an increasing trend in photosynthesis (PN), transpiration (E)and stomatal conductance (gCO2) with increase in PPFD up to 2000 _mol m?2s?1in allvarieties at optimum growth temperature (25 ± _3?C). However, the magnitude of increaseand maximum rate of PN(PN max) varied considerably with the varieties. Highest PNwasobserved in W1 followed by MX, K2 and HPM. Water use efficiency (WUE) in W1, MX andHPM increased with PPFDs up to the highest level tested, whereas, in K2 the highest WUEwas observed at 1600 _mol m?2s?1. Our results suggest that this species is able to use high level of PPFDs for its PNand therefore, may be cultivated in sun exposed areas in the field orunder high PPFDs using indoor grow lights for the optimum growth. Strict control of otherenvironmental factors, however, needs to be maintained while growing the plants indoor.

Lighting Strategies for the Flowering Stage of Indoor Cannabis Production
Victoria A. Rodriguez Morrison
A Thesis presented to The University of Guelph
https://atrium.lib.uoguelph.ca/xmlui...=7&isAllowed=y
Given the paucity of scientific research regarding lighting in cannabis production, this thesis investigated the effects of light intensity (LI) and ultraviolet (UV) radiation on indoor cannabis production during the flowering stage. When plants grew under LI ranging from 120 to 1800 μmol·m–2 ·s–1 provided by light emitting diodes (LEDs), inflorescence yield increased linearly as LI increased up to 1800 μmol·m–2 ·s–1 . When plants were grown under 400 μmol·m–2 ·s–1 supplemented with UV (peak wavelength of 287 nm) levels from 0.01 to 0.8 μmol·m–2 ·s–1 , for 3.5 h·d–1 , there were no changes in total Δ 9 -tetrahydrocannabinol or total cannabidiol concentrations. The severity of UV-induced cannabis morphology and physiology symptoms worsened as UV exposure level increased. The light response models developed in this thesis can be used to determine the optimum LI for a production environment, but caution should be used when exposing cannabis to UV radiation.

Not Cannabis Specific
Local maternal control of seed size by KLUH/CYP78A5-dependent growth signaling
Proceedings of the National Academy of Sciences 106(47):20115-20 2009
DOI: 10.1073/pnas.0907024106
Nikolai M Adamski, Elena Anastasiou, Sven Eriksson, Michael Lenhard
Seed development in plants involves the coordinated growth of the embryo, endosperm, and maternal tissue. Several genes have been identified that influence seed size by acting maternally, such as AUXIN RESPONSE FACTOR2, APETALA2, and DA1. However, given the lack of gain-of-function effects of these genes on seed size, it is unclear whether their activity levels are limiting in WT plants and whether they could thus be used to regulate seed size in development or evolution. Also, whether the altered seed sizes reflect local gene activity or global physiological changes is unknown. Here, we demonstrate that the cytochrome P450 KLUH (KLU) regulates seed size. KLU acts locally in developing flowers to promote seed growth, and its activity level is limiting for seed growth in WT. KLU is expressed in the inner integument of developing ovules, where it non-cell autonomously stimulates cell proliferation, thus determining the growth potential of the seed coat and seed. A KLU-induced increase in seed size leads to larger seedlings and higher relative oil content of the seeds. Genetic analyses indicate that KLU acts independently of other tested maternal factors that influence integument cell proliferation. Thus, the level of KLU-dependent growth factor signaling determines size in ovules and seeds, suggesting this pathway as a target for crop improvement.

Managing Plant Nutrition to Produce Elevated Cannabinoids
Ozadia Plant Science Sap & Leaf Analysis

In this webinar, Ozadia Plant Science Chief Science Officer, John Kempf, discusses the important factors surrounding cannabinoid levels, how to increase them with proper plant nutrition, and their relationships with microbial organisms and their environments. John and the listeners have a Q&A session at the end of the video that provides additional insights to compliment the webinar. Please watch this video to learn more about these topics and how the Ozadia system for holistic plant nutrition can help you!

Manipulation of thiol contents in plants
R. Höfgen, O. Kreft, L. Willmitzer, and H. Hesse
Amino Acids (2001) 20: 291–299
DOI: 10.1007/s007260170045
As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-59-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 39-phosphoadenosine-59-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and Jäger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are commonly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.

Not Cannabis Specific
Maternal control of seed size in plants.
Li, N., & Li, Y
Journal of Experimental Botany, 66(4), 1087–1097. (2015). doi:10.1093/jxb/eru549
Seed size is a key determinant of evolutionary fitness, and is also one of the most important components of seed yield. In angiosperms, seed development begins with double fertilization, which leads to the formation of a diploid embryo and a triploid endosperm. The outermost layer of the seed is the seed coat, which differentiates from maternal integuments. Therefore, the size of a seed is determined by the co-ordinated growth of the embryo, endosperm, and maternal tissue. Recent studies have identified several factors that act maternally or zygotically to regulate seed size, and revealed possible mechanisms that underlie seed size control in Arabidopsis and rice. In this review, we summarize current research progress in maternal control of seed size and discuss the roles of several newly identified regulators in maternal regulation of seed growth.

Not Cannabis Specific
Meiosis-Specific Loading of the Centromere-Specific Histone CENH3 in Arabidopsis thaliana.
Ravi, M., Shibata, F., Ramahi, J. S., Nagaki, K., Chen, C., Murata, M., & Chan, S. W. L.
PLoS Genetics, 7(6), e1002121.(2011).
doi:10.1371/journal.pgen.1002121
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior mising model to study sex chromosome evolution

Not Cannabis specific
Methodologies for estimating the sample size required for genetic conservation of outbreeding crops*
J. Crossa
Theor Appl Genet (1989) 77:153-161
doi: 10.1007/BF00266180.
The main purpose of germplasm banks is to preserve the genetic variability existing in crop species. The effectiveness of the regeneration of collections stored in gene banks is affected by factors such as sample size, random genetic drift, and seed viability. The objective of this paper is to review probability models and population genetics theory to determine the choice of sample size used for seed regeneration. A number of conclusions can be drawn from the results. First, the size of the sample depends largely on the frequency of the least common allele or genotype. Genotypes or alleles occurring at frequencies of more than 10% can be preserved with a sample size of 40 individuals. A sample size of 100 individuals will preserve genotypes (alleles) that occur at frequencies of 5%. If the frequency of rare genotypes (alleles) drops below 5%, larger sample sizes are required. A second conclusion is that for two, three, and four alleles per locus the sample size required to include a copy of each allele depends more on the frequency of the rare allele or alleles than on the number. Samples of 300 to 400 are
required to preserve alleles that are present at a frequency of 1%. Third, if seed is bulked, the expected number of parents involved in any sample drawn from the bulk will be less than the number of parents included in the bulk.
Fourth, to maintain a rate of breeding (F) of 1%, the effective population size (Ne) should be at least 150 for three alleles, and 300 for four alleles. Fifth, equalizing the reproductive output of each family to two progeny doubles the effective size of the population. Based on the results presented here, a practical option is considered for regenerating maize seed in a program constrained by limited funds.

Microbiome of Field Grown Hemp Reveals Potential Microbial Interactions With Root and Rhizosphere Soil
Bulbul Ahmed, Lawrence B. Smart and Mohamed Hijrl
Front. Microbiol. 12:741597.
doi: 10.3389/fmicb.2021.741597
https://www.frontiersin.org/articles...21.741597/full
Hemp (Cannabis sativa L.) is a crop bred and grown for the production of fiber, grain, and floral extracts that contribute to health and wellness. Hemp plants interact with a myriad of microbiota inhabiting the phyllosphere, endosphere, rhizoplane, and rhizosphere. These microbes offer many ecological services, particularly those of below ground biotopes which are involved in nutrient cycling, uptake, and alleviating biotic and abiotic stress. The microbiota communities of the hemp rhizosphere in the field are not well documented. To discover core microbiota associated with field grown hemp, we cultivated single C. sativa cultivar, “TJ’s CBD,” in six different fields in New York and sampled hemp roots and their rhizospheric soil. We used Illumina MiSeq amplicon sequencing targeting 16S ribosomal DNA of bacteria and ITS of fungi to study microbial community structure of hemp roots and rhizospheres. We found that Planctobacteria and Ascomycota dominated the taxonomic composition of hemp associated microbial community. We identified potential core microbiota in each community (bacteria: eight bacterial amplicon sequence variant – ASV, identified as Gimesia maris, Pirellula sp. Lacipirellula limnantheis, Gemmata sp. and unclassified Planctobacteria; fungi: three ASVs identified as Fusarium oxysporum , Gibellulopsis piscis , and Mortierella minutissima ). We found 14 ASVs as hub taxa [eight bacterial ASVs (BASV) in the root, and four bacterial and two fungal ASVs in the rhizosphere soil], and 10 BASV connected the root and rhizosphere soil microbiota to form an extended microbial communication in hemp. The only hub taxa detected in both the root and rhizosphere soil microbiota was ASV37 (Caulifigura coniformis ), a bacterial taxon. The core microbiota and Network hub taxa can be studied further for biocontrol activities and functional investigations in the formulation of hemp bioinoculants. This
study documented the microbial diversity and community structure of hemp grown in six fields, which could contribute toward the development of bioinoculants for hemp that could be used in organic farming.

*Morpho-Anatomy of Marijuana (Cannabis sativa L.)
Vijayasankar Raman, Hemant Lata, Suman Chandra, Ikhlas A. Khan, Mahmoud A Elsohly
In book: Cannabis sativa L. - Botany and Biotechnology May 2017
DOI: 10.1007/978-3-319-54564-6_5
Cannabis sativa is a complex species with highly variable morphological features. The present chapter provides detailed descriptions of morphological and anatomical characters of various parts of C. sativa plant and illustrated with bright-field and scanning electron micrographs. Male and female flowers occur in separate plants. Three types of glandular trichomes namely, glandular stalked, glandular sessile and bulbous glandular trichomes are found. Of these, glandular stalked trichomes are restricted to the floral bracts in pistillate plants and anthers in staminate plants. The other two types of glandular trichomes are found in various parts including bracts, leaves, stems and petioles. Two types of non-glandular trichomes namely, cystolith trichomes and slender covering trichomes, are present. Cystolith trichomes are primarily found on the adaxial leaf surface while the covering trichomes are commonly present on the abaxial leaf surface, stems, petioles and tepals. Cystolith crystals of calcium carbonate and cluster crystals of calcium oxalate are observed in the leaves. Anatomical features of various parts of the plant are described and illustrated.

Morphology of cannabis sativa L
Joyce Reed
State University of Iowa THESIS 1914
https://ir.uiowa.edu/cgi/viewcontent...98&context=etd
Cannabis sative L.,the common hemp,has long been of interest because of its commercial,agricultural,chemic al,and physiological importance and because of its dioecious character. The flower, fruit,and seed of Cannabis have been discussed by many writers. The dubiety regarding its floral structure,as to the correlation of the floral parts of the two sexes,and the nature of the organs that compose them,has been dealt with in an extensive literature. Since the plant is a strictly dioecious form the problem of its sexuality is of great importance and has been considered by numerous experimenters on diclinism. The nature,origin,ratio and significance of sex are questions which are being much investigated and discussed. Research on the subject of diclinism is going more and more into the inner morphology of plants in an attempt to solve the important question of sex determination and related problems of heredity. The critical morphology of the floral structures of Cannabis sativa L . ,especially that of the staminate flower,has been but briefly touched upon. Hence at the suggestion of Dr. R. B. Wylie this study was undertaken in the hope of contributing somewhat to the knowledge of the morphology of the plant,and perhaps aid in solving the great question of sexuality. Since the species is
included in the Urticales,one of the primitive orders of the Archichlaraydeae,
a brief discussion of the characters of this great group will be entered upon.

NOT CANNABIS SPECIFIC
Move on up, it’s time for change—mobile signals controlling photoperiod-dependent flowering
Yasushi Kobayashi and Detlef Weigel
Genes & Development, 21(19), 2371–2384.
doi:10.1101/gad.1589007
Plants do not bloom randomly—but how do they know when and where to make flowers? Here, we review molecular mechanisms that integrate spatial and temporal information in day-length-dependent flowering. Primarily through genetic analyses in two species, Arabidopsis thaliana and rice, we today understand the essentials of two central issues in plant biology: how the appropriate photoperiod generates an inductive stimulus based on an external coincidence mechanism, and the nature of the mobile flowering signal, florigen, which relays photoperiod-dependent information from the leaf to the growing tip of the plant, the shoot apex.

Not Cannabis specific
Naturally Occurring Anthocyanin, Structure, Functions and Biosynthetic Pathway in Fruit Plants.
Pervaiz, T., Songtao, J., Faghihi, F., Haider, M. S., & Fang, J.
Journal of Plant Biochemistry & Physiology, 05(02). (2017).
doi:10.4172/2329-9029.1000187
Anthocyanins are naturally occurring compounds, member of the flavonoid groups of photochemical, involved in defense against the damaging effects of UV irradiation in plants and protect from many oxidants. The anthocyanins, group of pigments are relatively small and diverse flavonoid family in nature, and responsible for the attractive colors, red and purple to blue in many plants. Presence of pigments in flowers and fruits seems to provide attraction for pollination and aiding seed distribution, it also provides antiviral and antimicrobial activities, however their occurrence in the vacuoles remains ambiguous. During the last decades, anthocyanin gene expression and many structural genes encoding enzymes has been extensively studied in fruits, flowers and leaves in many plants. In addition, the genetic regulating mechanism, their biosynthesis and other factors involved are well described. The biosynthesis pathway of anthocyanin is a complex with diverse branches responsible to produce variety of metabolites. In general Anthocyanins, production through the flavonoid path, are a class of vital phenolic compounds. Over six thousand diverse anthocyanins have been reported from various species. So far, the potential health benefits of anthocyanins have been reported in the contexts of their antioxidant properties. Anthocyanins are also extensively studied for their several positive effects on body. Based on these facts, the present review briefly summarizes recent advances, highlighting the importance of biosynthetic pathway of anthocyanins, thus will serve to encourage advance investigation in this field.

Night Interruption Lighting Equally Effective as Daylength Extension in Retaining the Vegetative State of Cannabis Mother Plants
Brian E. Whipker, Paul Cockson, and James T. Smith
AGROJNL:Agronomy Journal 2019
DOI: 10.1002/cft2.20001
Industrial hemp (Cannabis sativa) used for CBD (cannabidiol) production is a short-day (SD) plant that initiates inflorescence development when daylengths become clonally propagated CBD-strains of Cannabis, mother stock plants are grown in greenhouses to provide cuttings for field production. In order to retain the vegetative stage of mother plants, daylengths >16 hours must be provided to avoid flowering. The common practice used by Cannabis growers is daylength extension (DE), while in floriculture, night interruption (NI) lighting is preferred. Research conducted in growth chambers compared the effectiveness of 12+4-hour DE to a truncated 12+4-hour NI lighting regime, and 12-hour SD (flowering controls). As expected, plants grown under SD developed inflorescences. Both NI and DE were found to be equally effective management strategies in retaining the vegetative status of Cannabis plants. Plants grown with a NI were also more compact than DE plants. This offers an alternative growth control strategy for Cannabis since no plant growth regulators are registered.

Nitrogen deficiency stimulates cannabinoid biosynthesis in medical cannabis plants by inducing a metabolic shift towards production of low-N metabolites
Chao Song, Avia Saloner, Aaron Fait, Nirit Bernstein
Industrial Crops and Products, Volume 202, 15 October 2023, 116969
DOI: 10.1016/j.indcrop.2023.116969
Abstract: The primary and secondary metabolism of plants is closely connected to the resources supplied and obtained by the plant, including their mineral nutrition. We recently reported that nitrogen (N) deficiency enhances the production of terpenoids and cannabinoids, the unique biologically-active secondary metabolites in medical cannabis plants. Knowledge-gaps concerning effects of N supply on primary metabolism in cannabis hinder understanding of the interrelations between N inputs and biosynthesis of the therapeutic secondary metabolites. The present study therefore evaluated the hypothesis that a decrease in the plant’s C (carbon): N ratio by N-limitation, induces a shift in the plant metabolism towards lower production of N-containing metabolites and higher production of metabolites that do not contain N. Effects of N input levels (30, 80, 160, 240, and 320 mg L−1) on total C and N concentrations and C: N ratio in leaves and inflorescences of medical cannabis plants were studied; and gas chromatography-mass spectrometry (GC-MS) analysis of primary metabolites added to the analyses of secondary metabolites. Elevation of N supply resulted in an increase in total N and N-containing compounds (chlorophylls and most amino acids), and decreased total C and compounds that do not contain N, such as sugars (fructose, glucose, and xylose), and phosphates (phosphate and glucose-6-phosphate) in both inflorescences and leaves. In the inflorescences, the elevation of N input also decreased total cannabinoids, phenols, and flavonoids, that do not contain N. Integrating the metabolic datasets revealed positive correlations between C sources (fructose and glucose) and most of the cannabinoids and terpenoids; the latter were negatively correlated with N-compounds (most amino acids). Taken together, these results suggest that elevated N supply induce a metabolic shift in the inflorescences towards increased production of N-compounds via deflecting the C sources from the biologically active compounds. In addition, the cannabis leaf was found to be more sensitive than the inflorescence to N supply, presenting greater changes in primary metabolism and more coordinated metabolic associations. These findings highlight the importance of adequate and precise N nutrition for standardization of the therapeutic-metabolite profile and for preventing undesirable metabolic repartitioning in medical cannabis plants.

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Observation of parthenocarpy in Cannabis sativa L. (excerpt from ‘Cannabis’ ontologies I: Conceptual issues with Cannabis and cannabinoids terminology)
Kenzi Riboulet-Zemouli
Drug Science, Policy and Law 2020;6
DOI: 10.1177/2050324520945797
The mechanism of ‘parthenocarpy’ was first described (and named) more than a Century ago by Noll (1902; Gustafson, 1942). Widely spread among dicots (Vazart, 1955; Frankel and Galun, 1977; Dhatt and Kaur, 2016; Joldersma and Liu, 2018; Picarella and Mazzucato, 2019), Gustafson has described in-depth these counter-intuitive parthenocarpic fruits in 1942, referring to them as seedless fruits‘produced without pollination or other stimulation’ (Gustafson, 1942). A more recent definition describes ‘developing fruits from unfertilized ovaries; the resulting fruit is, therefore, usually seedless’ (Stern, 1997) Parthenocarpy is described as ‘the development of fruits without fertilisation’
(Scagel et al., 1967; Weier et al., 1970; Esau, 1977). Literature shows (Weier et al., 1970: 307; Stern, 1997: 408) that there are several kinds of biological mechanisms able to provoke the development of seedless fruits, and only those which are the result of the absence of fertilisation stimuli on female eggs are referred to as parthenocarpic. Because C. sativa naturally has both abilities to produce seeded and seedless fruits, according to Koltunow et al. (2002) the genus would be more precisely classified as a‘facultative parthenocarpic plant’. It is not surprising that research related to cannabis missed this element: parthenocarpy is a counterintuitive biological mechanism often subject to confusion, even among experts. Although, in cucumber like any other dicot, parthenocarpy is a phenomenon bound to fruits, the respected Frankel and Galun (1977) incoherently use ‘parthenocarpic flowers’ when discussing seedless cucumbers. USHP (Upton et al., 2014) mentions C. sativa ‘female inflorescence with senesced reddish-brown styles and stigmas, an indicator of inflorescence maturity’ but senesced inflorescences, i.e. flowers brought to maturity, is nowadays considered as the very definition of a fruit (Scagel et al., 1967; Weier et al., 1970; Esau, 1977; Stern, 1997). The very floral cluster involved, the inflorescence, is a compact agglomerate of flowers, turning into an infructescence, compact agglomerate of fruits, complicating observation.

Not directly Cannabis related
On the rarity of dioecy in flowering plants.
Käfer, J., Marais, G. A. B., & Pannell, J. R.
Molecular Ecology, 26(5), 1225–1241.(2017).
doi:10.1111/mec.14020
Dioecy, the co-existence of separate male and female individuals in a population, is a rare but phylogenetically widespread sexual system in flowering plants. While research has concentrated on why and how dioecy evolves from hermaphroditism, the question of why dioecy is rare, despite repeated transitions to it, has received much less attention. Previous phylogenetic and theoretical studies have suggested that dioecy might be an evolutionary dead end. However, recent research indicates that the phylogenetic support for this hypothesis is attributable to a methodological bias, and that there is no evidence for reduced diversification in dioecious angiosperms. The relative rarity of dioecy thus remains a puzzle. Here, we review evidence for the hypothesis that dioecy might be rare not because it is an evolutionary dead end, but rather because it easily reverts to hermaphroditism. We review what is known about transitions between hermaphroditism and dioecy, and conclude that there is an important need to consider more widely the possibility of transitions away from dioecy, both from an empirical and a theoretical point of view, and by combining tools from molecular evolution and insights from ecology.

Optimisation of Nitrogen, Phosphorus, and Potassium for Soilless Production of Cannabis sativa in the Flowering Stage Using Response Surface Analysis
Lewys Bevan, Max Jones and Youbin Zheng
Front. Plant Sci. 12:764103.
doi: 10.3389/fpls.2021.764103
https://www.frontiersin.org/articles...21.764103/full
Following legalisation, cannabis has quickly become an important horticultural crop in Canada and increasingly so in other parts of the world. However, due to previous legal restrictions on cannabis research there are limited scientific data on the relationship between nitrogen (N), phosphorus (P), and potassium (K) supply (collectively: NPK) and the crop yield and quality. This study examined the response of a high delta-9- tetrahydrocannabinol (THC) Cannabis sativa cultivar grown in deep-water culture with different nutrient solution treatments varying in their concentrations (mg L􀀀1 ) of N (70, 120, 180, 250, 290), P (20, 40, 60, 80, 100), and K (60, 120, 200, 280, 340) according to a central composite design. Results demonstrated that inflorescence yield responded quadratically to N and P, with the optimal concentrations predicted to be 194 and 59 mg L􀀀1 , respectively. Inflorescence yield did not respond to K in the tested range. These results can provide guidance to cultivators when formulating nutrient solutions for soilless cannabis production and demonstrates the utility of surface response design for efficient multi-nutrient optimisation

Overexpression of CsMIXTA, a Transcription Factor from Cannabis sativa, Increases Glandular Trichome Density in Tobacco Leaves, Samuel R. Haiden, Peter V. Apicella , Yi Ma and Gerald A. Berkowitz Plants (2022). DOI: 10.3390/plants11111519 GET LINK Cannabinoids are synthesized in glandular stalked trichomes on the female flowers of Cannabis sativa (cannabis). The regulation of glandular trichome development has not been characterized in cannabis. We recently identified an R2R3-MYB transcription factor, CsMIXTA, which could be involved in trichome morphogenesis in cannabis. Some homologous genes of CsMIXTA are known to function in glandular trichome initiation in other plant species. CsMIXTA is highly expressed in flower tissue compared to vegetative tissues. Interestingly, CsMIXTA is also highly expressed in trichomes isolated from female flower tissue. In addition, CsMIXTA is upregulated during the peak stages of female flower maturation in correlation with some cannabinoid biosynthetic genes. Transient expression in Nicotiana benthamiana showed that CsMIXTA is localized in the nucleus. Furthermore, yeast transcriptional activation assay demonstrated that CsMIXTA has transactivation activity. Overexpression of CsMIXTA in Nicotiana tabacum resulted in higher trichome density, larger trichome size, and more branching on stalked glandular trichomes. The results indicate that CsMIXTA not only promotes glandular trichome initiation in epidermal cells, but also regulates trichome development in tobacco leaves. In this report, we characterized the novel function of the first cannabis transcription factor that may be critical for glandular trichome morphogenesis.

Photoperiodic Flowering Response of Essential Oil, Grain, and Fiber Hemp (Cannabis sativa L.) Cultivars
Mengzi Zhang, Steven L. Anderson, Zachary T. Brym and Brian J. Pearson
Front. Plant Sci. 12:694153. (2021)
doi: 10.3389/fpls.2021.694153
https://www.frontiersin.org/articles...21.694153/full
Cultivation of hemp (Cannabis sativa L.) in tropical and subtropical regions can be challenging if the flowering behavior of a given cultivar is unknown, poorly understood, or not accurately selected for the photoperiod. Identifying cultivars adapted to
local environmental conditions is key to optimizing hemp vegetative and flowering performance. We investigated the effects of varying light cycles in regulating extension growth and flowering response of 15 essential oil and 12 fiber/grain hemp cultivars both indoors and outdoors. Plants were subjected to 11 photoperiods in the controlled rooms ranging from 12 to 18 h, and natural day length in the field. The critical photoperiod threshold was identified for seven essential oil cultivars and two fiber/grain cultivars. “Cherry Wine-CC,” “PUMA-3,” and “PUMA-4” had the shortest critical day length between 13 h 45min and 14 h. The flowering of essential oil cultivars was generally delayed by 1–2 days when the photoperiod exceeded 13 h compared with 12 h, and flowering was further delayed by 7–8 days when the photoperiod exceeded 14 h. In fiber/grain cultivars, flowering was generally delayed by 1–3 days when the day length exceeded 14 h. Flowering formost essential oil cultivars was delayed by 5–13 days under a 14-h photoperiod compared with 13 h 45min, suggesting a photoperiod difference as little as 15min can significantly influence the floral initiation of some essential oil cultivars. Cultivars represented by the same name but acquired from different sources can perform differently under the same environmental conditions, suggesting genetic variation among cultivars with the same name. Average days to flower of fiber/grain cultivars was correlated with reported cultivar origin, with faster flowering occurring among northern cultivars when compared with southern cultivars. Plant height generally increased as the day length increased in essential oil cultivars but was not affected in fiber/grain cultivars. In addition, civil twilight of ∼ 2 μ mol. m−2. s−1 was discovered to be biologically effective in regulating hemp flowering. Collectively, we conclude that most of the essential oil cultivars and some southern fiber/grain cultivars tested express suitable photoperiods for tropical and sub-tropical region cultivation.

Phenotypic plasticity influences the success of clonal propagation in industrial pharmaceutical Cannabis sativa.
Campbell, Lesley G.; Naraine, Steve G. U.; Dusfresne, Jaimie; Lightfoot, David A
PLOS ONE, 14(3), e0213434–. (2019).
doi:10.1371/journal.pone.0213434
https://www.researchgate.net/publica...annabis_sativa
The burgeoning cannabis market requires evidence-based science such that farmers can quickly and efficiently generate new plants. In part, horticultural operations are limited by the success of cloning procedures. Here, we measured the role of environmental conditions and cultivar identity on the success of generating long branch material with many meristems in planting stock (mothers) and in rooting success of stem-derived clones. To evaluate the influence of lighting treatments on the optimal production of branching mothers, four lighting conditions (Fluorescent High Output T5s [T5], Metal halide lamps [MH], Plasma lamps [PL], or Metal halide lamps augmented with far red LED lights [MH+FR]) were applied to two cultivars of container grown plants (Cannabis sativa L. ‘Bubba Kush’, ‘Ghost Train Haze’) grown in peat-based organic substrates in mylar grow tents. To evaluate the influence of lighting, cutting tool (secateurs or scalpels), and stem wounding (present/absent) on optimal rooting of stems, three lighting conditions (Fluorescent T8s, T5, PL) were applied to three cultivars of peat pellet grown plants (C. sativa L. ‘Bubba Kush’, ‘Ghost Train Haze’, ‘Headband’). Mothers grown under T5 and MH (vs MH+FR) produced ~30% more meristems. However, growing mothers under MH+FR were 19% taller than mothers under T5, with ~25% longer internodes on dominant stems than plants under any other lighting condition. Canopies were denser under T5 because petiole length was ~30% shorter under T5 and fan leaves were longer and narrower under MH+FR and MH+FR and PL, respectively, than under other lighting conditions. Cultivar Ghost Train Haze stems rooted most frequently and most quickly. Wounded stems were 162% more likely to root than unwounded stems and rooted 1.5 days earlier. Our results will guide producers attempting to maximize the rate of clone production in licensed facilities; although results may differ among cultivars, where cultivars differed in their average phenotype as mother plants, and their propensity to root from cuttings, and the speed with which they produced those roots.

Photoperiodic Flowering Response of Essential Oil, Grain, and Fiber Hemp (Cannabis sativa L.) Cultivars
Mengzi Zhang, Steven L. Anderson, Zachary T. Brym and Brian J. Pearson
Front. Plant Sci. 12:694153. (2021)
doi: 10.3389/fpls.2021.694153
https://www.frontiersin.org/articles...21.694153/full
Cultivation of hemp (Cannabis sativa L.) in tropical and subtropical regions can be challenging if the flowering behavior of a given cultivar is unknown, poorly understood, or not accurately selected for the photoperiod. Identifying cultivars adapted to
local environmental conditions is key to optimizing hemp vegetative and flowering performance. We investigated the effects of varying light cycles in regulating extension growth and flowering response of 15 essential oil and 12 fiber/grain hemp cultivars both indoors and outdoors. Plants were subjected to 11 photoperiods in the controlled rooms ranging from 12 to 18 h, and natural day length in the field. The critical photoperiod threshold was identified for seven essential oil cultivars and two fiber/grain cultivars. “Cherry Wine-CC,” “PUMA-3,” and “PUMA-4” had the shortest critical day length between 13 h 45min and 14 h. The flowering of essential oil cultivars was generally delayed by 1–2 days when the photoperiod exceeded 13 h compared with 12 h, and flowering was further delayed by 7–8 days when the photoperiod exceeded 14 h. In fiber/grain cultivars, flowering was generally delayed by 1–3 days when the day length exceeded 14 h. Flowering formost essential oil cultivars was delayed by 5–13 days under a 14-h photoperiod compared with 13 h 45min, suggesting a photoperiod difference as little as 15min can significantly influence the floral initiation of some essential oil cultivars. Cultivars represented by the same name but acquired from different sources can perform differently under the same environmental conditions, suggesting genetic variation among cultivars with the same name. Average days to flower of fiber/grain cultivars was correlated with reported cultivar origin, with faster flowering occurring among northern cultivars when compared with southern cultivars. Plant height generally increased as the day length increased in essential oil cultivars but was not affected in fiber/grain cultivars. In addition, civil twilight of ∼ 2 μ mol. m−2. s−1 was discovered to be biologically effective in regulating hemp flowering. Collectively, we conclude that most of the essential oil cultivars and some southern fiber/grain cultivars tested express suitable photoperiods for tropical and sub-tropical region cultivation.

Not Cannabis specific
Photosynthetic responses to altitude: an explanation based on optimality principles
Han Wang1, I. Colin Prentice, Tyler W. Davis, Trevor F. Keenan, Ian J. Wright and Changhui Peng
New Phytologist (2017) 213: 976–982
https://doi.org/10.1111/nph.14332
https://nph.onlinelibrary.wiley.com/...1111/nph.14332
Ecophysiologists have long been fascinated by the photosynthetic behaviour of alpine plants, which often have to withstand extreme environmental pressures (Gale, 1972; Friend & Woodward, 1990; Korner, 2003, 2007; Shi et al. , 2006). About8%of the world’s land surface is above 1500 maltitude (Korner, 2007). High altitudes can be climatically unusual, often with (for example) low temperatures, strong winds, and now high rates of warming (Korner, 2003; Pepin &Lundquist, 2008; Rangwala & Miller, 2012). Moreover, the low atmospheric pressure provides a set of environmental conditions unique on Earth (Table 1). There has been extensive speculation about altitudinal effects on photosynthesis and, in particular, how to account for the puzzling – but consistently observed – tendencies towards higher carbon dioxide (CO2 ) drawdown (low ratio of leaf internal to ambient CO2 partial pressures (ci : ca ; hereafter, v ), resulting in low carbon isotope discrimination) and higher carboxylation capacity (Vcmax ) with increasing altitude (Gale, 1972; Korner & Diemer, 1987; Friend et al. , 1989; Terashima et al. , 1995; Bresson et al. , 2009; Zhu et al. , 2010). At first glance, it might be expected that CO2 assimilation rates would be reduced at high altitudes due to the low partial pressure of CO2 (Friend & Woodward, 1990).However, actual measured photosynthetic rates are usually as high as, or even higher than, those at low altitudes (M€ achler & N€ osberger, 1977; K€ orner & Diemer, 1987; Cordell et al. , 1999; Shi et al. , 2006).

Not Cannabis specific
Physiological basis of UV-C induced resistance to Botrytis cinerea in tomato fruit.
Charles, M. T., Makhlouf, J., & Arul, J.
Postharvest Biology and Technology, 47(1), 21–26. (2008). doi:10.1016/j.postharvbio.2007.05.014
Effect of pre-storage treatment with hormic dose of UV-light and ripening on the changes in topography and fine structure of postharvest tomato fruit during storage was studied by scanning electron microscopy (SEM). Both ripening and UV-treatment induced significant structural modifications in tomato fruit surface. Flattening of cellular mounds associated with normal ripening process was more intense with UV-treatment, and the fruit surface was also more wrinkled with treatment. The formation of an operculum over broken trichomes was a common feature of ripened control fruit, while this structure was incompletely formed in the treated fruit. Surface of senescent control fruit was characterized by the presence of an amorphous epicuticular wax, which was quasi-absent on UV-treated fruit. Surface colonization of UV-treated fruit by Botrytis cinerea was also different from untreated control. Colonization was sparse on the treated fruit, although direct cuticle penetration as well as penetration through damaged trichomes was observed in both cases. Fewer adhesion structures (appressoria) were observed on UV-treated fruit than on non-irradiated control, suggesting that structural modification of the epicuticular wax induced by UV may be a factor affecting the ability of B. cinerea to attach to the treated fruit surface. This study shows that UV-treatment causes alteration in the amount of epicuticular wax and its ultrastructural arrangement, presumably due to changes in its chemical composition. These changes could affect light reflectance characteristics of the fruit surface, and possibly increase transpiration loss leading to changes in fruit appearance. Another consequence of UV-induced physical and chemical modifications of tomato fruit surface could be an improved ability of the tissue to resist infection by B. cinerea. However, the reduced colonization of the UV-treated fruit by the pathogen cannot be attributed solely to changes in surface topograph

FIND PDF of Book
Not directly Cannabis related
PLANT EPIGENETICS. Methods and Protocols by Igor Kovalchuk; Franz J. Zemp
Folia Geobotanica 48(2):288-289 Jan 2013
DOI: 10.2307/23486818 book review
Expert researchers explore the most recent developments, examining in great detail the contribution of epigenetic regulation to cell function in plants. Chapters include a variety of protocols for studying the function of small non-coding RNAs, DNA methylation, and histone modifications in plants, often in different degrees of complexity. Describes bioinformatic approaches to the analysis of high-throughput data, such as bisulfite sequencing and Chip-on-chip assays and features much-desired protocols for plant transgenesis and the analysis of genome stability, with a detailed discussion of their applications to epigenetic studies.
Contents
1. Analysis of DNA Methylation in Plants by Bisulfite Sequencing / Andrea M. Foerster and Ortrun Mittelsten Scheid
2. Analysis of bisulfite sequencing data from plant DNA with CyMATE / Andrea M. Foerster ... [et al.]
3. Analysis of locus-specific changes in methylation patterns using a COBRA (combined bisulfite restriction analysis) assay / Alex Boyko and Igor Kovalchuk
4. Detection of changes in global genome methylation using the cytosine-extension assay / Alex Boyko and Igor Kovalchuk
5. In situ analysis of DNA methylation in plants / Palak Kathiria and Igor Kovalchuk
6. Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using RFLP (restriction fragment length polymorphism) / Alex Boyko and Igor Kovalchuk
7. Isoschizomers and Amplified Fragment Length Polymorphism (AFLP) for the Detection of Specific Cytosine Methylation Changes / Leonor Ruiz-Garcia ... [et al.]
8. Analysis of small RNA populations using hybridization to DNA tiling arrays / Martine Boccara ... [et al.]
9. Northern blotting techniques for small RNAs / Todd Blevins
10. qRT-PCR of small RNAs / Erika Varkonyi-Gasic and Roger P. Hellens
11. Cloning new small RNA sequences / Yuko Tagami, Naoko Inaba and Yuichiro Watanabe
12. Genome-wide Mapping of Protein-DNA Interaction by Chromatin Immunoprecipitation and DNA Microarray Hybridization (ChIP-chip). Pt. A: ChIP-chip Molecular Methods / Julia J. Reimer and Franziska Turck
13. Genome-wide mapping of protein-DNA interaction by chromatin immunoprecipitation and DNA microarray hybridization (ChIP-chip). Pt. B: ChIP-chip data analysis / Ulrike Go?bel, Julia Reimer and Franziska Turck
14. Metaanalysis of ChIP-chip data / Julia Engelhorn and Franziska Turck
15. Chromatin Immunoprecipitation Protocol for Histone Modifications and Protein-DNA Binding Analyses in Arabidopsis / Stephane Pien and Ueli Grossniklaus
16. cDNA Libraries for Virus-Induced Gene Silencing / Andrea T. Todd, Enwu Liu and Jonathan E. Page
17. Detection and quantification of DNA strand breaks using the ROPS (random oligonucleotide primed synthesis) assay / Alex Boyko and Igor Kovalchuk 18. Reporter gene-based recombination lines for studies of genome stability / Palak Kathiria and Igor Kovalchuk
19. Plant transgenesis / Alicja Ziemienowicz.

Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa “Finola” cultivation: An alternative fertilization strategy to improve plant growth and quality characteristics.
Pagnani, G., Pellegrini, M., Galieni, A., D’Egidio, S., Matteucci, F., Ricci, A., … Del Gallo, M.
Industrial Crops and Products, 123, 75–83.(2018).*
doi:10.1016/j.indcrop.2018.06.033
The massive employment of chemical fertilizers entails substantial costs for agriculture and leads to significant environmental pollution, soils depletion and crop productivity declines. The aim of this preliminary study was to evaluate the suitability of plant growth-promoting rhizobacteria (PGPR) as an alternative fertilization approach in Cannabis sativa L. ‘Finola’, one of the low-psychoactive substances industrial hemp varieties cultivated in the Abruzzo territory. The PGPR inoculum was first studied in a model system by monitoring the colonization and survival of bacteria in roots of hemp seedling grown in vitro. Following a complete randomized block design with three replicates, female plants were also cultivated in greenhouse and subjected to different cultivation conditions: (i) two different PGPR inoculum concentrations, (ii) nitrogen fertilization, and (iii) unfertilized control. At the flowering stage, plant growth parameters, main cannabinoid content, antioxidant, and total phenolic content, were assessed. In the model system experiment, scanning electron microscope (SEM) imaging revealed an excellent ability of bacteria to adhere to the surface of roots, and to colonize root vascular tissues of hemp seedlings. Under greenhouse conditions PGPR favored plant growth and development as well as plant secondary metabolites accumulation and, consequently, antioxidant capacity. In particular, the lowest PGPR concentration allowed obtaining results comparable with those induced by the recommended nitrogen fertilization. These results underline the potentiality of PGPR application in hemp plants in terms of both higher biomass accumulation and chemical composition, also meeting environmental goals such as an increase in soil biodiversity and a reduction in chemical inputs. This study represents the first step toward the potential application of PGPR in hemp cultivation and could be the base for future extensive evaluations.

Plant-Growth Promoting Rhizobacteria in Soilless Cannabis Cropping Systems: Implications for Growth Promotion and Disease Suppression
Chaitanya Kumar Jha, Meenu Saraf
SLU Swedish University of Agricultural Sciences 2020
DOI: 10.13140/RG.2.1.5171.2164
https://stud.epsilon.slu.se/16079/11...m_l_200923.pdf
The microbial life associated with plants has been studied systematically for about a century (Ruzzi & Aroca, 2015), and much of the scientific literature agrees upon its pivotal role in supporting plant growth, development and overall health (Backer et al., 2018). Beneficial outcomes for plants are primarily a result of microbe’s nitrogen-fixation, inorganic nutrient solubilization, pathogen inhibition and stress amelioration through a wide array of biological, chemical and physical mechanisms (Pandey et al., 2019). These microbes are found within both the bacterial and fungal kingdom (Turner, James & Poole, 2013), and inhabit every part of the plant, albeit in different amounts (Backer et al., 2018; Compant et al., 2019).
The roots and their close vicinity have a rich phytomicrobiome (Vacherone et al., 2013), which in combination with the highly dynamic nature of roots makes the rhizosphere a very biologically and chemically active region (Jones, Nguyen & Finlay, 2009). As such, it influences factors essential to plant survival and health, like nutrient availability and presence of pathogens (Walker et al., 2003 ; Jones, Nguyen & Finlay, 2009). An interest in manipulating the composition of the rhizosphere has emerged, and along with it the notion of Plant-Growth Promoting Rhizobacteria (PGPR). Strains pertaining to this group have been shown to have a substantial impact on the growth, development, health, yield and quality of several commercially important crops, while at the same time decreasing the need for energy-intensive and environmentally detrimental inputs like mineral nutrition and
synthetic pesticides (Pandey et al., 2019). From a microbiological point of view, the roots and the rhizosphere are important since they serve as a great energy source, owing to their release of compounds rich in carbon and nitrogen.

Pollen Appearance and In Vitro Germination Varies for Five Strains of Female Hemp Masculinized Using Silver Thiosulfate
Jessica DiMatteo, Lauren Kurtz, Jessica D. Lubell-Brand
HortScience 2020
DOI: 10.21273/HORTSCI14842-20
Genetically female seed is sought for cannabidiol (CBD) hemp production because CBD is extracted from the flowers of female hemp plants. The production of all female seed requires masculinization of female plants to produce genetically female pollen that reliably generates female seed. Of the five female hemp genotypes that we masculinized using foliar sprays of silver thiosulfate (Abacus, Cherry Wine, Mountain Mango, Youngsim10, Wife), all genotypes produced fewer large and more irregular or misshapen pollen grains than genetically male plants. The masculinized female genotypes Wife and Cherry Wine produced pollen with germination rates similar to those of the male genotype Kentucky Sunshine. Female hemp genotypes vary in their ability to produce usable pollen that disperses well, is easily collected, and germinates as well as pollen from genetically male hemp plants.

Not Cannabis specific
Postharvest UV-C Treatment, Followed by Storage in a Continuous Low-Level Ethylene Atmosphere, Maintains the Quality of ‘Kensington Pride’ Mango Fruit Stored at 20 ?C
Penta Pristijono, John B. Golding, and Michael C. Bowyer
DOI: 10.3390/horticulturae5010001
Mature green ‘Kensington Pride’ mangoes (Mangifera indica L.) were treated with a short-term UV-C light at four different intensities (0, 4.0, 8.3 and 11.7 kJ m?2 ). After treatment, mangoes were stored for 12 d in air (<0.005 µL L?1 ethylene) or 0.1 µL L?1 ethylene at 20 ?C and 100% relative humidity (RH). Weight loss, peel colour, firmness, ethylene production, respiration rate, total soluble solids (TSS), titratable acidity (TA), total chlorophyll content, total phenolic content (TPC) and total antioxidant activity were assessed at 3-d intervals. The results showed that UV-C treatment delayed skin degreening, reduced endogenous ethylene production, suppressed respiration rate and lowered chlorophyll content compared to untreated control fruit. Fruit treated with UV-C had significantly higher TPC and total antioxidant activity at the end of the storage period than untreated fruits for both storage atmospheres. In addition, UV-C treated fruits remained significantly firmer than untreated fruits. UV-C treatment significantly affected TSS and TA levels in different ways. Storage of fruits in 0.1 µL L?1 ethylene significantly affected fruit firmness, respiration rate and ethylene production, while other fruit quality parameters were similar to fruit stored in air. These results indicated that UV-C irradiation could be used as an effective and rapid method to extend the postharvest life of mature green mangoes without adversely affecting certain quality attributes in the presence of low-level ethylene during storage.

Not Cannabis specific
Preventing seed quality loss during storage. The role of oxygen is underestimated
Steven P.C. Groot
https://www.researchgate.net/publica...underestimated
DOI: 10.13140/2.1.5013.3447
Crop seeds can often survive for a considerable time under dry conditions, but eventually they lose their germination capacity due to accumulating damage. There is a large variation in shelf life between seeds of different crops. Whereas primed seeds deteriorate relatively fast. To reduce the rate of ageing and prolong shelf life of the seeds, it is recommended to store the seeds dry and cool. Most if not all damage is related to oxidation of proteins, membranes, DNA, mRNA and lipids. Although the effect of both low temperatures and low moisture seed moisture content is through a reduction in the rate of oxidation, the role of oxygen in the storage environment is hardly or not considered. The deleterious effects of oxygen during dry seed storage are presented, as well methods to reduce oxygen levels during dry seed storage and prolong the shelf life of seeds.

Production of Feminized Seeds of High CBD Cannabis sativa L. by Manipulation of Sex Expression and Its Application to Breeding
Marko Flajšman, Miha Slapnik, and Jana Murovec
Front Plant Sci. 2021; 12: 718092.
doi: 10.3389/fpls.2021.718092
https://www.ncbi.nlm.nih.gov/pmc/art...-12-718092.pdf
The use of the cannabis plant as a source of therapeutic compounds is gaining great importance since restrictions on its growth and use are gradually reduced throughout the world. Intensification of medical (drug type) cannabis production stimulated breeding activities aimed at developing new, improved cultivars with precisely defined, and stable cannabinoid profiles. The effects of several exogenous substances, known to be involved in sex expressions, such as silver thiosulfate (STS), gibberellic acid (GA), and colloidal silver, were analyzed in this study. Various concentrations were tested within 23 different treatments on two high cannabidiol (CBD) breeding populations. Our results showed that spraying whole plants with STS once is more efficient than the application of STS on shoot tips while spraying plants with 0.01% GA and intensive cutting is ineffective in stimulating the production of male flowers. Additionally, spraying whole plants with colloidal silver was also shown to be effective in the induction of male flowers on female plants, since it produced up to 379 male flowers per plant. The viability and fertility of the induced male flowers were confirmed by fluorescein diacetate (FDA) staining of pollen grains, in vitro and in vivo germination tests of pollen, counting the number of seeds developed after hybridization, and evaluating germination rates of developed seeds. Finally, one established protocol was implemented for crossing selected female plants. The cannabinoid profile of the progeny was compared with the profile of the parental population and an improvement in the biochemical profile of the breeding population was confirmed. The progeny had a higher and more uniform total CBD (tCBD) to total tetrahydrocannabinol (tTHC) ratio (up to 29.6; average 21.33 ± 0.39) compared with the original population (up to 18.8; average 7.83 ± 1.03). This is the first comprehensive report on the induction of fertile male flowers on female plants from dioecious medical cannabis (Cannabis sativa L.).

NOT CANNABIS SPECIFIC
PROJECT 1: THE COMPLEX REGULATION OF FLOWERING LOCUS T
A lesson in Complexity:
Regulation of FLOWERING LOCUS T
Florigen and photoperiod
https://www.mpipz.mpg.de/turck/project_1
Flowering in many plant species is controlled by photoperiod, which represents the most reliable seasonal change in nature. The gene network controlling flowering is well studied in the model plant Arabidopsis thaliana, a facultative long day plant that flowers earlier if days are longer than a critical threshold of approximately 10 hours light per day. The FLOWERING LOCUS T (FT) gene is crucial for the accelerated flowering in response to long days [1]. Interestingly, relatives of FTare also important to accelerate flowering in short day plants such as rice indicating that the role of FT-related genes is very general in controlling flowering although it may be differently hardwired within the regulatory network.
FT is tissue-specifically expressed, an import aspect its role in regulating flowering [2]. Messenger RNA of FT is only produced and translated to protein in the phloem of leaves. The protein moves with the assimilate flow towards the shoot apex where it acts as transcriptional co-regulator to switch the developmental program from vegetative to reproductive [1, 3]. FT fulfills all criteria that were postulated for the flowering hormone Florigen in 1937 by Mikhail Chailakhyan.

Not Cannabis specific
Prolonging the longevity of ex situ conserved seeds by storage under anoxia.
Groot, S. P. C., de Groot, L., Kodde, J., & van Treuren, R.
Plant Genetic Resources, 13(01), 18–26.(2014).
doi:10.1017/s1479262114000586
Plant genetic resources are conserved by genebanks mainly in the form of seeds. In most of the cases, the dried seeds can be stored for a considerable period of time, but eventually seed deterioration results in the inability to generate healthy seedlings. Prolonging seed longevity during storage reduces the frequency of regeneration, which is beneficial from a genetic as well as a management point ofview. To reduce the rate ofdeterioration,cool and dry storage conditions are usually practised for long-term seed storage. In spite of the growing body of evidence that seed deterioration is predominantly caused by oxidative processes, the importance of seed storage under anoxic conditions has received little attention from the genebank community. Herein, we report on the effects of anoxia on seed viability, the oxygen uptake by dry seeds in closed containers and the permeability for oxygen of various seed storage containers. Our results confirm that the ageing of dry seeds is accelerated by the presence of oxygen in the storage environment. Therefore, we recommend that genebanks store dry seeds under anoxic conditions to prolong their longevity during ex situ conservation. To reduce the initial rate of viability loss, we further recommend that the period of temporary storage after seed harvest be minimized and also that the seeds are kept during this period under controlled conditions, including anoxia.

Radiometric Method for Determining Canopy Stomatal Conductance in Controlled Environments.
Monje, O., & Bugbee, B.
Agronomy, 9(3), 114. (2019).
doi:10.3390/agronomy9030114
Canopy stomatal conductance is a key physiological factor controlling transpiration from plant canopies, but it is extremely difficult to determine in field environments. The objective of this study was to develop a radiometric method for calculating canopy stomatal conductance for two plant species—wheat and soybean from direct measurements of bulk surface conductance to water vapor and the canopy aerodynamic conductance in controlled-environment chambers. The chamber provides constant net radiation, temperature, humidity, and ventilation rate to the plant canopy. In this method, stepwise changes in chamber CO2 alter canopy temperature, latent heat, and sensible heat fluxes simultaneously. Sensible heat and the radiometric canopy-to-air temperature difference are computed from direct measurements of net radiation, canopy transpiration, photosynthesis, radiometric temperature, and air temperature. The canopy aerodynamic conductance to the transfer of water vapor is then determined from a plot of sensible heat versus radiometric canopy-to-air temperature difference. Finally, canopy stomatal conductance is calculated from canopy surface and aerodynamic conductances. The canopy aerodynamic conductance was 5.5 mol m?2 s ?1 in wheat and 2.5 mol m?2 s ?1 in soybean canopies. At 400 umol mol?1 of CO2 and 86 kPa atmospheric pressure, canopy stomatal conductances were 2.1 mol m?2 s ?1 for wheat and 1.1 mol m?2 s ?1 for soybean, comparable to canopy stomatal conductances reported in field studies. This method measures canopy aerodynamic conductance in controlled-environment chambers where the log-wind profile approximation does not apply and provides an improved technique for measuring canopy-level responses of canopy stomatal conductance and the decoupling coefficient. The method was used to determine the response of canopy stomatal conductance to increased CO2 concentration and to determine the sensitivity of canopy transpiration to changes in canopy stomatal conductance. These responses are useful for improving the prediction of ecosystem-level water fluxes in response to climatic variables.

Not Cannabis Specific
Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping.
Seymour, D. K., Filiault, D. L., Henry, I. M., Monson-Miller, J., Ravi, M., Pang, A., … Maloof, J. N. ).
Proceedings of the National Academy of Sciences, 109(11), 4227–4232 (2012)
doi:10.1073/pnas.1117277109
Quantitative trait loci (QTL) mapping is a powerful tool for investigating the genetic basis of natural variation. QTL can be mapped using a number of different population designs, but recombinant inbred lines (RILs) are among the most effective. Unfortunately, homozygous RIL populations are time consuming to construct, typically requiring at least six generations of selfing starting from a heterozygous F1. Haploid plants produced from an F1 combine the two parental genomes and have only one allele at every locus. Converting these sterile haploids into fertile diploids (termed “doubled haploids,” DHs) produces immortal homozygous lines in only two steps. Here we describe a unique technique for rapidly creating recombinant doubled haploid populations in Arabidopsis thaliana: centromere-mediated genome elimination. We generated a population of 238 doubled haploid lines that combine two parental genomes and genotyped them by reduced representation Illumina sequencing. The recombination rate and parental allele frequencies in our population are similar to those found in existing RIL sets. We phenotyped this population for traits related to flowering time and for petiole length and successfully mapped QTL controlling each trait. Our work demonstrates that doubled haploid populations offer a rapid, easy alternative to RILs for Arabidopsis genetic analysis.

Not directly Cannabis related
Reduced Root-zone Phosphorus Concentration Decreases Iron Chlorosis in Maize in Soilless Substrates.
Parry, C., & Bugbee, B.
HortTechnology, 27(4), 490–493.(2017).
doi:10.21273/horttech03735-17
Maize (Zea mays) is increasingly grown in controlled environments to facilitate phenotypic analysis. Even with ample chelated iron (Fe), maize often develops interveinal chlorosis in soilless substrates or hydroponics because of inadequate bioavailable Fe in the plant. We hypothesized that the excessive phosphorus (P) in standard greenhouse fertigation solutions would accentuate the chlorosis. Here, we report that reducing the P concentration from 0.7 to 0.07 mmolLL1 (22 to 2 mgLL1 ) provided adequate P for rapid growth and increased chlorophyll concentration from 263 to 380 mmolmL2 . Restricted root-zones in containers require frequent watering and are often watered to excess, which flushes the root-zone with a high P solution. In a separate study, minimizing the leaching fraction increased leaf chlorophyll concentration from 123 to 508 mmolmL2 . The use of a ceramic substrate typically improves the green leaf color of maize plants. Consistent with this observation, we found no effect of high P concentration in the irrigation solution on growth or chlorophyll density in ceramic substrates because it strongly absorbs P from solution. These findings can significantly improve maize growth and nutrition in controlled environments.

Regeneration of shoots from immature and mature inflorescences of Cannabis sativa
Kevin F. Piunno, Gregory Golenia, Ekaterina A. Boudko, Cassandra Downey, A. Maxwell P. Jones
Canadian Journal of Plant Science Can. J. Plant Sci April 2019
DOI: 10.1139/CJPS-2018-0308
Cannabis sativa is usually clonally propagated from plants in the vegetative phase. However, phenotypic traits such as yield and chemical composition can only be assessed in unfertilized plants reaching the end of their life cycle and there are no peer reviewed methods to propagate flowering plants. In this study, immature (3 cultivars) and mature (1 cultivar) floral explants were cultured on thidiazuron and shoot development was observed from both immature and mature explants. This provides the first report of micropropagation from floral tissues in C. sativa and will enable plants to be clonally propagated up to the date of harvest.

Response of essential oil hemp (Cannabis sativa L.) growth, biomass, and cannabinoid profiles to varying fertigation rates
Steven L. Anderson II, Brian Pearson, Roger Kjelgren, Zachary Brym
Plos One July 29, 2021
DOI: 10.1371/journal.pone.0252985
https://journals.plos.org/plosone/ar...l.pone.0252985
Five essential oil hemp (Cannabis sativa L.) cultivars (Cherry Blossom, Cherry Blossom (Tuan), Berry Blossom, Cherry Wine, and Cherry Blossom × Trump) were treated with six fertigation treatments to quantify the effects of synthetic fertilizer rates and irrigation electrical conductivity on plant growth, biomass accumulation, and cannabinoid profiles. Irrigation water was injected with a commercial 20-20-20 fertilizer at rates of 0, 50, 150, 300, 450, and 600 ppm nitrogen equating to 0.33 (control), 0.54, 0.96, 1.59, 2.22, and 2.85 dS m−1, respectively. Plants were grown under artificial lighting (18 hr) to maintain vegetative growth for eight weeks, followed by an eight-week flowering period. High linear relationship between chlorophyll concentrations and SPAD-502 measurements validated the utilization of SPAD meters to rapidly identify nutrient deficiency in essential oil hemp. Cultivars expressed significant variation in plant height and cannabinoid profiles (% dry mass), in concurrence with limited biomass and cannabinoid (g per plant) yield variation. Cherry Blossom was the best performing cultivar and Cherry Wine was the least productive. Variation in plant growth, biomass, and cannabinoid concentrations were affected to a greater extent by fertilizer rates. Optimal fertilizer rates were observed at 50 ppm N, while increased fertilizer rates significantly reduced plant growth, biomass accumulation, and cannabinoid concentrations. Increased fertilizer rates (> 300 ppm N) resulted in compliant THC levels (< 0.3%), although when coupled with biomass reductions resulted in minimal cannabinoid yields. Additionally, CBD concentration demonstrated higher sensitivity to increased fertilizer rates (> 300 ppm N) compared to THC and CBG (> 450 ppm N). The results of this study can serve as a guide when using fertigation methods on essential oil hemp cultivars; although results may differ with cultivar selection, environmental conditions, and management practices.

Not Cannabis specific
Response of Grapevine Defenses to UV-C Exposure
Alexandra Bonomelli, Laurence Mercier, Jérôme Franchel, Fabienne Baillieul, Émile Benizri, Marie-Claude Mauro
American Society for Enology and Viticulture, 2004, 55 (1), pp.51-59.
The defense potential of a tolerant American Vitis rupestris cultivar (Rupestris du Lot) and a susceptible European Vitis vinifera cultivar (Chardonnay) in response to UV-C irradiation was investigated. The expression of eight defense-related genes coding for enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase and stilbene synthase), the octadecanoid pathway (lipoxygenase), and pathogenesis-related proteins (class I and III chitinases, ?-1,3-glucanase, class 6 pathogenesis-related protein, and class 10 pathogenesis-related protein) was followed by real-time reverse transcription polymerase chain reaction (RT-PCR). Phenolic compound accumulation was monitored by microscopic observation. Accumulation of resveratrol, a major grapevine phytoalexin, was evaluated by HPLC, and chitinase and ?-1,3-glucanase enzyme activities were measured. Both grapevine species responded to UV-C treatment by enhancement of defense mechanisms. Intensity of some defense responses was correlated with tolerance to diseases, as previously described for stilbene accumulation: the tolerant species responded more intensely to UV-C exposure than the susceptible one. UV-C irradiation is a practical and reproducible method for inducing grapevine defense responses and can be useful in determining the defense potential of grapevine cultivars.

Responses of Greenhouse-grown Cannabis sativa L. to Nitrogen, Phosphorus, and Potassium
C. B. Coffman, W. A. Gentner
Agronomy Journal (1977)
DOI 10.2134/agronj1977.0002196200690005002 6x
https://acsess.onlinelibrary.wiley.c.. .96200690005002 6x
Growers of illegal Cannabis sativa L. use various cultural practices to maximize crop production. The objective of this study was to evaluate the morphological and biochemical responses of greenhouse grown C. sativa to soil incorporated N, P, and K as they reflect the geographical origin of Cannabis derivatives. Fertilizers were blended with Ap horizon soil from a Gilpin silt loam before placement in 12-cm pots. NH4NO3-N was applied at 0, 25, and 125 ppm. Phosphorus and K from super-phosphate and KCI, respectively, were applied at 0, 50, and 150 ppm. Forty-five-day-old anthesic Cannabisplants were harvested and combined leaf and flower tissues were analyzed for cannabidiol (CBD) and Δ9-tetrahydrocanna-binol (Δ9THC). Nine essential elements were also measured in plant tissue. Plant growth, tissue yield, and concentration of CBD and Δ9THC were positively correlated with extractable P2O5 (p < 0.01). Phosphorus concentrations in tissue were similarly related to yield of dry matter and cannabinoid concentrations. Uptake of K was positively correlated with extractable K2O across all treatment levels (r=0.40**), but was negatively correlated with tissue yield (r=--0.36**). Growth and tissue yields were negatively related to total plant N (p< 0.01). Levels of extractable P2O5, Mn, B, and Mg were associated with specific concentration ranges for several plant elements plus Δ9THC. Thus, it was possible to partially characterize a soil by tissue analysis. For example, all of the plants grown on soil with less than 100 ppm of extractable P2O5 contained less than 8,000 ppm Δ9THC. Usefulness of such relationships will be dependent upon extensive evaluation of Cannabis on different soils under various cultural conditions. At this time, the reliability required for determination of origin ofCanabis derivatives via chemical analysis does not exist when only essential elements and cannabinoids are considered.

Response of Medical Cannabis (Cannabis sativa L.) Genotypes to K Supply Under Long Photoperiod
Avia Saloner, Mollie M. Sacks and Nirit Bernstein
Front. Plant Sci. 10:1369. (2019)
doi: 10.3389/fpls.2019.01369
https://www.frontiersin.org/articles...019.01369/full
Potassium is involved in regulation of multiple developmental, physiological, and metabolic processes in plants, including photosynthesis and water relations. We lack information about the response of medical cannabis to mineral nutrition in general, and K in particular, which is required for development of high-grade standardized production for the medical cannabis industry. The present study investigated the involvement of K nutrition in morphological development, the plant ionome, photosynthesis and gas-exchange, water relations, water use efficiency, and K use efficiency, comparatively for two genotypes of medical cannabis, under a long photoperiod. The plants were exposed to five levels of K (15, 60, 100, 175, and 240 ppm K). Growth response to K inputs varied between genotypes, revealing genetic differences within the Cannabis sativa species to mineral nutrition. Fifteen ppm of K was insufficient for optimal growth and function in both genotypes and elicited visual deficiency symptoms. Two hundred and forty ppm K proved excessive and damaging to development of the genotype Royal Medic, while in Desert Queen it stimulated rather than restricted shoot and root development. The differences between the genotypes in the response to K nutrition were accompanied by some variability in uptake, transport, and accumulation of nutrients. For example, higher levels of K transport from root to the shoot were apparent in Desert Queen. However, overall trends of accumulation were similar for the two genotypes demonstrating competition for uptake between K and Ca and Mg, and no effect on N and P uptake except in the K-deficiency range. The extent of accumulation was higher in the leaves > roots > stem for N, and roots > leaves > stem for P. Surprisingly, most micronutrients (Zn, Mn, Fe, Cu, Cl) tended to accumulate in the root, suggesting a compartmentation strategy for temporary storage, or for prevention of access concentrations at the shoot tissues. The sensitivity of net-photosynthetic rate, gas exchange, and water use efficiency to K supply differed as well between genotypes. The results suggest that growth reduction under the deficient supply of 15 ppm K was mostly due to impact of K availability on water relations of the tissue and transpiration in Royal Medic, and water relations and carbon fixation in Desert Queen.

Response of Medical Cannabis (Cannabis sativa L.) to Nitrogen Supply Under Long Photoperiod.
Saloner, A., & Bernstein, N.
Frontiers in Plant Science, 11. (2020).
doi:10.3389/fpls.2020.572293
The development progression of medical cannabis plants includes a vegetative growth phase under long photoperiod, followed by a reproductive phase under short photoperiod. Establishment of plant architecture at the vegetative phase affects its reproduction potential under short photoperiod. Nitrogen (N) is a main component of many metabolites that are involved in central processes in plants, and is therefore a major factor governing plant development and structure. We lack information about the influence of N nutrition on medical cannabis functional-physiology and development, and plant N requirements are yet unknown. The present study therefore investigated the developmental, physiological, and chemical responses of medical cannabis plants to N supply (30, 80, 160, 240, and 320 mgL?1 N) under long photoperiod. The plants were cultivated in an environmentally controlled growing room, in pots filled with soilless media. We report that the morpho-physiological function under long photoperiod in medical cannabis is optimal at 160 mgL?1 N supply, and significantly lower under 30 mgL?1 N, with visual deficiency symptoms, and 75 and 25% reduction in plant biomass and photosynthesis rate, respectively. Nitrogen use efficiency (NUE) decreased with increasing N supply, while osmotic potential, water use efficiency, photosynthetic pigments, and total N and N-NO3 concentrations in plant tissues increased with N supply. The plant ionome was considerably affected by N supply. Concentrations of K, P, Ca, Mg, and Fe in the plant were highest under the optimal N level of 160 mgL?1 N, with differences between organs in the extent of nutrient accumulation. The majority of the nutrients tested, including P, Zn, Mn, Fe, and Cu, tended to accumulate in the roots > leaves > stem, while K and Na tended to accumulate in the stem > leaves > roots, and total N, Ca, and Mg accumulated in leaves > roots > stem. Taken together, the results demonstrate that the optimal N level for plant development and function at the vegetative growth phase is 160 mgL?1 N. Growth retardation under lower N supply (30–80 mgL?1 ) results from restricted availability of photosynthetic pigments, carbon fixation, and impaired water relations. Excess uptake of N under supply higher than 160 mgL?1 N, promoted physiological and developmental restrictions, by ion-specific toxicity or indirect induced restrictions of carbon fixation and energy availability.

Not Cannabis Specific
Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant.
Wijnker, E., van Dun, K., de Snoo, C. B., Lelivelt, C. L. C., Keurentjes, J. J. B., Naharudin, N. S., … Dirks, R.
Nature Genetics, 44(4), 467–470.(2012).
doi:10.1038/ng.2203
Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.

Root Comparison: Seedlings vs Clones
Mick Donahue
North Carolina Farms
https://ncfarmsinc.com/articles/root...q_y6S13xrc_ssE
There’s an ongoing debate about which is superior: hemp clones or hemp seedlings. You’ll hear arguments that go something like this: clones are more uniform in the field, more true to type, and 100% female, but seeds are more easily stored, free from pests from the beginning, and–this is the big one–produce a taproot.
We decided to put that last one to the test. How would seedlings and clones compare in a side-by-side root test?

Root-TRAPR: a modular plant growth device to visualize root development and monitor growth parameters, as applied to an elicitor response of Cannabis sativa
Pipob Suwanchaikasem, Robert Walker, Alexander Idnurm, Jamie Selby-Pham, Berin A. Boughton
Research Square
DOI: 10.21203/rs.3.rs-764290/v1
https://www.researchsquare.com/article/rs-764290/v1
Background
Plant growth devices, for example rhizoponics, rhizoboxes, and ecosystem fabrication (EcoFAB) have been developed to facilitate studies of plant root morphology and plant-microbe interactions in controlled laboratory settings. However, several of these designs are suitable only for studying small model plants such as Arabidopsis thaliana and Brachypodium distachyon, and therefore require modification to be extended to larger plant species like crop plants. In addition, specific tools and technical skills required for fabricating these devices may not be available to researchers. Hence, this study aimed to establish an alternative protocol to generate a larger, modular and reusable plant growth device based on differently available resources.
Results
Root-TRAPR (Root-Transparent, Reusable, Affordable three-dimensional Printed Rhizo-hydroponic) system was successfully developed. It consists of two main parts, an internal root growth chamber and external structural frame. The internal root growth chamber is comprised of a polydimethylsiloxane (PDMS) gasket, microscope slide and acrylic sheet while the external frame is printed from a threedimensional (3D) printer and secured with nylon screws. To test the efficiency and applicability of the system, industrial hemp (Cannabis sativa) was grown with or without exposure to chitosan, a well-known plant elicitor used for stimulating plant defense. Plant root morphology was clearly detected in the system and plant tissues were easily collected and processed to examine plant biological responses. Upon chitosan treatment, chitinase and peroxidase activities increased in root tissues (1.7- and 2.3-fold, respectively) and exudates (7.2- and 21.6-fold, respectively). Phytohormones related to plant growth and defense response were higher in root tissues as compared to the shoots. Additionally, within two weeks of observation, hemp plants exhibited dwarf growth in Root TRAPR system, easing plant handling and allowing increased replication under limited growing space.
Conclusion
The Root-TRAPR system facilitates exploration of root morphology and root exudate of C. sativa under controlled conditions and at a smaller scale. The device is easy to fabricate and applicable for investigating plant responses toward elicitor challenge. This fabrication protocol is modifiable to suit
other plants and can be adapted to study plant physiology in other biological contexts, such as plant responses against biotic and abiotic stresses.

Sap & Leaf Analysis: Managing Plant Nutrition to Produce Elevated Cannabinoids
https://www.youtube.com/watch?v=x1hc4YmKqFg&t=723s
In this webinar, Ozadia Plant Science Chief Science Officer, John Kempf, discusses the important factors surrounding cannabinoid levels, how to increase them with proper plant nutrition, and their relationships with microbial organisms and their environments. John and the listeners have a Q&A session at the end of the video that provides additional insights to compliment the webinar. Please watch this video to learn more about these topics and how the Ozadia system for holistic plant nutrition can help you!

Not Cannabis specific
Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions.
Groot, S. P. C., Surki, A. A., de Vos, R. C. H., & Kodde, J. Annals of Botany, 110(6), 1149–1159.(2012).
doi:10.1093/aob/mcs198
† Background and Aims Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen. † Methods Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 8C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed. †Key Results The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under longterm dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 8C after equilibration at 85 % RH. †Conclusions Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.

Not directly Cannabis related
Sex-biased gene expression in dioecious garden asparagus (Asparagus officinalis)
Alex Harkess, Francesco Mercati, Hong-Yan Shan, Francesco Sunseri, Agostino Falavigna and Jim Leebens-Mack
New Phytologist (2015) 207: 883–892
doi: 10.1111/nph.13389
Sex chromosomes have evolved independently in phylogenetically diverse flowering plant lineages. The genes governing sex determination in dioecious species remain unknown, but theory predicts that the linkage of genes influencing male and female function will spur the origin and early evolution of sex chromosomes. For example, in an XY system, the origin of an active Y may be spurred by the linkage of female suppressing and male promoting genes.
Garden asparagus (Asparagus officinalis) serves as a model for plant sex chromosome evolution, given that it has recently evolved an XX/XY sex chromosome system. In order to elucidate the molecular basis of gender differences and sex determination, we used RNAsequencing (RNA-Seq) to identify differentially expressed genes between female (XX), male (XY) and supermale (YY) individuals.
We identified 570 differentially expressed genes, and showed that significantly more genes exhibited male-biased than female-biased expression in garden asparagus. In the context of anther development, we identified genes involved in pollen microspore and tapetum development that were specifically expressed in males and supermales.
Comparative analysis of genes in the Arabidopsis thaliana, Zea mays and Oryza sativa anther development pathways shows that anther sterility in females probably occurs through interruption of tapetum development before microspore meiosis.

Sex reversal in the male plants of Cannabis sativa L. by ethyl hydrogen-l-propylphosphonate.
Mohan Ram, H. Y., & Jaiswal, V. S.
Zeitschrift Für Pflanzenphysiologie, 68(2), 181–183. (1972).
doi:10.1016/s0044-32872)80168-5
Ethyl hydrogen-1-propylphosphonate causes retudation of growth, malformation of leaves, and formation of female and intersexual flowers when applied to male plants of Cannabis sativa L. in high concentrations (16,000 and 24,000 ppm).
Ethyl hydrogen-1-propylphosphonate (NIA 10637) retards growth of a wide range of herbaceous and woody species. It also causes increase in cold hardiness and drought tolerance, induction of seed germination, changes in root development, and alteration of flowering and fruiting patterns (Technical Report, Niagara Chemical Division, FMC Corporation, U.S.A.). DOLLWET and KUMAMOTO (1970) have recently demonstrated that this substance generates ethylene, which is probably responsible for the growth regulating properties observed.
We recently reported that 2-chloroethanephosphonic acid, which releases ethylene in the plant tissues (WARNER and LEOPOLD, 1969), was able to induce female flowers on male plants of Cannabis sativa (MOHAN RAM and ]AISWAL, 1970). The present work was undertaken to investigate whether NIA 10637 had a feminizing effect in this system.
A variety of strictly dioecious Cannabis sativa L. plants were grown in earthen pots, and their sex was determined after flowering. Female plants showed no change in sex expression when treated with NIA 10637. Male plants (Fig. 1 A) were, therefore, selected for further study. Ten plants were used for each treatment and NIA 10637 was applied in aqueous solution in one foliar spray at six concentrations (1,000, 2,000, 4,000, 8,000, 16,000 and 24,000 ppm) along with Triton X-114 at 0.10f0 as the wetting agent. The control plants received the wetting agent only. Morphological changes and flower sex were recorded at weekly intervals.

Not Cannabis Specific
Signaling and the Modulation of Pollen Tube Growth
Vernonica E. Franklin-Tong
The Plant Cell, Vol. 11, 727–738, April 1999,
DOI:10.1105/tpc.11.4.727
Pollination in flowering plants begins when pollen lands on a stigma. The process continues as the pollen germinates and grows through intercellular spaces in the pistil, and it culminates when the pollen reaches the ovary and fertilization occurs. This complex and hazardous process involves many interactions, including cell–cell recognition and intracellular and intercellular signaling, as well as many other factors that remain to be identified. Events occurring during pollination are assumed to be under tight genetic and cellular control because pollen tube growth needs to be regulated both spatially and temporally. Moreover, pollen tubes are a highly specialized cell type, comprising a generative cell, which contains the two sperm cells, and the vegetative nucleus. Thus, a pollen tube in fact contains a cell within a cell, which is itself haploid. This is a unique structure that is related to the singular biological role of pollen—to effect fertilization. The processes that control pollen tube growth and that regulate and guide pollen tubes during their journey toward the ovary are of considerable interest. These topics are regarded as important both for fundamental studies of the control of fertility and reproduction in plants and also as an attractive model system for the investigation of polarized tip growth, cell–cell interactions, and signal transduction. Furthermore, the potential future application of molecular tools in efforts to control some of these processes generates additional interest in pollination

Not Cannabis specific
Soil organic matter attenuates the efficacy of flavonoid-based plant-microbe communication.
Del Valle, I., Webster, T. M., Cheng, H.-Y., Thies, J. E., Kessler, A., Miller, M. K., … Lehmann, J.
Science Advances, 6(5), (2020).
doi:10.1126/sciadv.aax8254
Plant-microbe interactions are mediated by signaling compounds that control vital plant functions, such as nodulation, defense, and allelopathy. While interruption of signaling is typically attributed to biological processes, potential abiotic controls remain less studied. Here, we show that higher organic carbon (OC) contents in soils repress flavonoid signals by up to 70%. Furthermore, the magnitude of repression is differentially dependent on the chemical structure of the signaling molecule, the availability of metal ions, and the source of the plant-derived OC. Up to 63% of the signaling repression occurs between dissolved OC and flavonoids rather than through flavonoid sorption to particulate OC. In plant experiments, OC interrupts the signaling between a legume and a nitrogenfixing microbial symbiont, resulting in a 75% decrease in nodule formation. Our results suggest that soil OC decreases the lifetime of flavonoids underlying plant-microbe interactions.

Some aspects of sex determinism in hemp
ELENA TRU??, ZENOVIA OLTEANU, ? TEFANIA SURDU, MARIA-MAGDALENA ZAMFIRACHE, L? CR? MIOARA OPRIC?
Analele ?tiin?ifice ale Universit??ii „Alexandru Ioan Cuza”, Sec?iunea Genetic? ?i Biologie Molecular?, TOM VIII, 2007
https://www.researchgate.net/publica...minism_in_hemp
General considerations on hemp sexual phenotype. Most flowering plant species are hermaphrodite, but a small number of species, as Cannabis sativa L., are unisexual. Because the species with male individuals and female individuals have evolved repeatedly from hermaphroditic progenitors, the mechanisms for the control of sex determination in flowering plants are extremely diverse. The sex is principally determined by genotype in all species, but the mechanisms range from a single controlling locus to sex chromosomes bearing several linked loci required for sex determination.
Cannabis sativa L. is one of the best studied species under the aspect of genetic determinism of the sex, but the problem is not yet entirely deciphered. The opinions are different, sometimes contradictory. Thus, the hemp is included, according to some authors, in the category of plants with male heterogamy, whereas the others sustain the idea of a complex sex determinism, seen as resultant of interaction between individual hereditary potencies and the environmental factors. Although the hemp is a dioecious species, with sexual dimorphism occurring in a late stage of plant development, as a consequence of intensive improvement, many varieties with different sexual expressions were produced, and a large scale of sexualization types is observed in culture. The most frequent are the monoecious forms, classified in more categories, on a five-point scale, depending on female flowers/male flowers ratio. The sexual dimorphism in hemp is accompanied by a morphologic dimorphism, but this becomes evident only late in ontogeny, at anthesis. In early ontogenetic phases, the differences between plants are minor, from phenotypical point of view. The dimorphism also is manifest at anatomical level. For example, important differences between sexes were evidenced in respect of palisadic tissue, epidermal stomatic number, secretory hair distribution etc (Toma, 1975, 1977; Zanoschi and Toma 1985). In breeding activity, the early establishment of the sex would be necessary, imposed by the necessity to remove, from agronomic reasons, the male plants or the high masculinized monoecious plants.

Not Cannabis Specific
Spectral Effects of Three Types of White Light-emitting Diodes on Plant Growth and Development: Absolute versus Relative Amounts of Blue Light
Kevin R. Cope and Bruce Bugbee
HORTSCIENCE 48(4):504–509. 2013.
Doi: 10.21273/HORTSCI.48.4.504
Light-emitting diodes (LEDs) are a rapidly developing technology for plant growth lighting and have become a powerful tool for understanding the spectral effects of light on plants. Several studies have shown that some blue light is necessary for normal growth and development, but the effects of blue light appear to be species-dependent and may interact with other wavelengths of light as well as photosynthetic photon flux (PPF). We report the photobiological effects of three types of white LEDs (warm, neutral, and cool, with 11%, 19%, and 28% blue light, respectively) on the growth and development of radish, soybean, and wheat. All species were grown at two PPFs(200 and 500 mmol·mL2 ·s L1 ) under each LED type, which facilitated testing the effect of absolute (mmol photons per mL2 ·s L1 ) and relative (percent of total PPF) blue light on plant development. Root and shoot environmental conditions other than light quality were uniformly maintained among six chambers (three lamp types 3 two PPFs). All LEDs had similar phytochrome photoequilibria and red:far red ratios. Blue light did not affect total dry weight (DW) in any species but significantly altered plant development. Overall, the low blue light from warm white LEDs increased stem elongation and leaf expansion, whereas the high blue light from cool white LEDs resulted in more compact plants. For radish and soybean, absolute blue light was a better predictor of stem elongation than relative blue light, but relative blue light better predicted leaf area. Absolute blue light better predicted the percent leaf DW in radish and soybean and percent tiller DW in wheat. The largest percentage differences among light sources occurred in low light (200 mmol·mL2 ·sL1 ). These results confirm and extend the results of other studies indicating that light quantity and quality interact to determine plant morphology. The optimal amount of blue light likely changes with plant age because plant communities balance the need for rapid leaf expansion, which is necessary to maximize radiation capture, with prevention of excessive stem elongation. A thorough understanding of this interaction is essential to the development of light sources for optimal plant growth and development.

Not Cannabis Specific
Sperm Delivery in Flowering Plants: The Control of Pollen Tube Growth
KATHLEEN L. WILSEN
BioScience 57(Nov 2007):835-844 November 2007
DOI: 10.1641/B571006
Although most people think of pollen merely as an allergen, its true biological function is to facilitate sexual reproduction in flowering plants. The angiosperm pollen grain, upon arriving at a receptive stigma, germinates, producing a tube that extends through the style to deliver its cargo to the ovule, thereby fertilizing the egg, and completing the life cycle of the plant. The pollen tube grows rapidly, exclusively at its tip, and produces a cell that is highly polarized both in its outward shape and its internal cytoplasmic organization. Recent studies reveal that the growth oscillates in rate. Many underlying physiological processes, including ionic fluxes and energy levels, also oscillate with the same periodicity as the growth rate, but usually not with the same phase. Current research focuses on these phase relationships in an attempt to decipher their hierarchical sequence and to provide a physiological explanation for the factors that govern pollen tube growth.

Not Cannabis specific
Stressful ‘‘memories’’ of plants: Evidence and possible mechanisms
Toby J.A. Bruce, Michaela C. Matthes, Johnathan A. Napier, John A. Pickett
Plant Science 173 (2007) 603–608
DOI: 10.1016/j.plantsci.2007.09.002
A history of exposure to a range of different types of stress alters subsequent plant responses. The process of priming or hardening involves prior exposure to a biotic or an abiotic stress factor making a plant more resistant to future exposure. This feature, in higher plants, indicates some capacity for ‘‘memory’’. However, the molecular mechanism(s) by which this plant memory works must be entirely different from the memory in animals which is dependent on the nervous system. We therefore use the term ‘‘stress imprint’’ in this review to describe this plant-based phenomenon. Sustained alterations in levels of key signalling metabolites or transcription factors could provide an explanation for how plant metabolism is altered by exposure to various stresses. Alternatively epigenetic changes could play a role by enabling long-term changes in gene expression. Exposure to a priming agent could activate a gene or set of genes but instead of reverting to the transcriptionally silent state once the stimulus is removed, an epigenetic mark could perhaps be left, keeping the region in a ‘permissive’ state, facilitating quicker and more potent responses to subsequent attacks. Future research is needed to establish the molecular mechanism by which plants store information on stress exposure because biotic and abiotic stresses limit agricultural production and stress responses often lead to down-regulation of yield determining processes such as photosynthesis.

Not directly Cannabis related
Super Moisture Absorbent Gels for Sustainable Agriculture via Atmospheric Water Irrigation.
Zhou, X., Zhang, P., Zhao, F., & Yu, G.
ACS Materials Letters, 1419–1422.
doi:10.1021/acsmaterialslett.0c00439 (2020).
An atmospheric water irrigation system based on super moisture absorbent gels (SMAG) is developed for sustainable agriculture technology. The SMAG-soil can harvest water from the air and provide water to the plants upon solar heating regardless of the local accessibility to liquid water resources. As a benefit of this solar-powered atmospheric water irrigation system, agriculture can become geographically and hydrologically independent. Thus, crop planting in underdeveloped and drought areas can be liberated from the long-distance water and power supplies.

Not Cannabis specific
Suppression of Cucumber Powdery Mildew by Supplemental UV-B Radiation in Greenhouses Can be Augmented or Reduced by Background Radiation Quality.
Suthaparan, A., Stensvand, A., Solhaug, K. A., Torre, S., Telfer, K. H., Ruud, A. K., … Gislerød, H. R.
Plant Disease, 98(10), 1349–1357. (2014).
doi:10.1094/pdis-03-13-0222-re
This study demonstrates that the spectral quality of radiation sources applied with ultraviolet-B (UV-B; background radiation) affects the suppression of cucumber powdery mildew (Podosphaera xanthii) by UV-B. Suppression provided by daily UV-B exposure of 1 W/m2 for 10 min was greatest in the presence of red light or by a complete lack of background light, and powdery mildew suppression was least in the presence of ultraviolet-A (UV-A) or blue radiation compared with plants exposed only to 16 h of daily natural light supplemented with high-pressure sodium lamps that supply broad-spectrum radiation with peaks in the yellow-orange region. Exposure of powdery mildew-inoculated plants to supplemental red light without UV-B, beginning at the end of the daylight period, also reduced disease severity; however, supplemental blue light applied in the same fashion had no effect. Daily application of UV-B at 1 W/m2 beginning on the day of inoculation significantly reduced the severity of powdery mildew to 15% compared
with 100% severity on control plants. Maximum suppression of powdery mildew was observed following 15 min of exposure to UV-B (1.1% severity compared with 100% severity on control plants) but exposure time had to be limited to 5 to 10 min to reduce phytotoxicity. There was no additional disease suppression when plants were exposed to UV-B beginning 2 days prior to inoculation compared with plants exposed to UV-B beginning on the day of inoculation. UV-B inhibited
germination, infection, colony expansion, and sporulation of P. xanthii. The results suggest that efficacy of UV-B treatments, alone or in combination with red light, against P. xanthii can be enhanced by exposure of inoculated plants to these wavelengths of radiation during the night, thereby circumventing the counteracting effects of blue light and UV-A radiation. The effect of UV-B on powdery mildew seemed to be directly upon the pathogen, rather than induced resistance of the host. Night exposure of plants to 5 to 10 min of UV-B at 1 W/m2 and inexpensive, spectral-specific, light-emitting diodes may provide additional tools to suppress powdery mildews of diverse greenhouse crops.

Not Cannabis specific
Suppression of Powdery Mildews by UV-B: Application Frequency and Timing, Dose, Reflectance, and Automation.
Suthaparan, A., Solhaug, K. A., Bjugstad, N., Gislerød, H. R., Gadoury, D. M., & Stensvand, A.
Plant Disease, 100(8), 1643–1650.(2016).
doi:10.1094/pdis-12-15-1440-re
Disease-suppressive effects of nighttime applications of ultraviolet-B (UV-B) were investigated at two irradiance levels (0.8 or 1.6 W/m2) in strawberry and rosemary plants inoculated with Podosphaera aphanis or Golovinomyces biocellatus, respectively. Plants were exposed to each irradiance level every third night for 18 min; every night for 6 min; or three times every night for 2 minutes. Thus, over time, all plants received the same cumulative dose of UV-B, and severity of powdery mildew was reduced by 90 to 99% compared to untreated controls in both crops. Use of polished aluminum lamp reflectors and UV-B reflective surfaces on greenhouse benches significantly increased treatment efficacy. An automated apparatus consisting of an adjustable boom with directed airflow was used to move UV-B lamps over greenhouse benches at 25 or 50 cm/min. Directed airflow moved leaves on the subtending plants to better expose upper and lower surfaces to UV-B, but directed airflow actually decreased efficacy of UV-B treatments, possibly by dispersing conidia from lesions before they were exposed to a lethal dose of UV-B. Results indicate broad applicability of nighttime applications of UV-B to suppress powdery mildews, and that cumulative UV-B dose is an overriding factor determining efficacy. Finally, enhanced suppression on shaded or obscured tissues is more likely to be effected by reflective bench surfaces than through attempts to physically manipulate the foliage.

Tetrahydrocannabinolic Acid Synthase, the Enzyme Controlling Marijuana Psychoactivity, is Secreted into the Storage Cavity of the Glandular Trichomes.
Sirikantaramas, S., Taura, F., Tanaka, Y., Ishikawa, Y., Morimoto, S., & Shoyama, Y.
Plant and Cell Physiology, 46(9), 1578–1582.(2005).
doi:10.1093/pcp/pci166
Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription–PCR (RT–PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.

Not directly Cannabis related
The asparagus genome sheds light on the origin and evolution of a young Y chromosome.
Alex Harkess, Jinsong Zhou, Chunyan Xu, John E. Bowers, Ron Van der Hulst, Saravanaraj Ayyampalayam, Francesco Mercati, Paolo Riccardi, Michael R. McKain, Atul Kakrana, Haibao Tang, Jeremy Ray, John Groenendijk, Siwaret Arikit, Sandra M. Mathioni, Mayumi Nakano, Hongyan Shan, Alexa Telgmann-Rauber, Akira Kanno, Zhen Yue, Haixin Chen, Wenqi Li, Yanling Chen, Xiangyang Xu, Yueping Zhang, Shaochun Luo, Helong Chen, Jianming Gao, Zichao Mao, J. Chris Pires, Meizhong Luo, Dave Kudrna, Rod A. Wing, Blake C. Meyers, Kexian Yi, Hongzhi Kong, Pierre Lavrijsen, Francesco Sunseri, Agostino Falavigna, Yin Ye, James H. Leebens-Mack, Guangyu Chen
Nature Communications, 2017; 8 (1)
DOI:10.1038/s41467-017-01064-8
Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.

The Bee Community of Cannabis sativa and Corresponding Effects of Landscape Composition.
Flicker, N. R., Poveda, K., & Grab, H.
Environmental Entomology. (2019).
doi:10.1093/ee/nvz141
Industrial hemp, Cannabis sativa (Cannabaceae), is a newly introduced and rapidly expanding crop in the American agricultural landscape. As an exclusively wind-pollinated crop, hemp lacks nectar but produces an abundance of pollen during a period of floral dearth in agricultural landscapes. These pollen resources are attractive to a range of bee species but the diversity of floral visitors and their use of hemp across a range of agricultural contexts remains unclear. We made repeated sweep net collections of bees visiting hemp flowers on farms in New York, which varied in both landscape context and phenotypic traits of hemp varieties. We identified all bee visitors to the species level and found that hemp supported 16 different bee species. Landscape simplification negatively impacted the abundance of bees visiting hemp flowers but did not affect the species richness of the community. Plant height, on the other hand, was strongly correlated with bee species richness and abundance for hemp plots with taller varieties attracting a broader diversity of bee species. Because of its temporally unique flowering phenology, hemp has the potential to provide a critical nutritional resource to a diverse community of bees during a period of floral scarcity and thereby may help to sustain agroecosystem-wide pollination services for other crops in the landscape. As cultivation of hemp increases, growers, land managers, and policy makers should consider its value in supporting bee communities and take its attractiveness to bees into account when developing pest management strategies.


The biology of Canadian weeds. 119. Cannabis sativa L.
E. Small, T. Pocock, and P. B. Cavers
CANADIAN JOURNAL OF PLANT SCIENCE 83 (1):217–237.
DOI: 10.4141/P02-021
Cannabis sativa has been cultivated for millennia in Eurasia and for centuries in North America, as a source of a textile fibre, oilseed, and intoxicating drugs such as marijuana. Considerable literature is available on the agricultural and biological properties of these basic three cultigens, but relatively little is published on wild-growing plants of the species. Most weedy C. sativa differ
from the cultigens in a number of ecological properties, particularly with regard to reproductive biology. The species is the classical example of a “camp follower” that is exceptionally adapted to the habitat conditions around settlements: rich, highly manured, moist soils, and open areas resulting from recent removal or disturbance of the vegetation. In Canada, spontaneous populations have been found in all provinces, but forms that have re-evolved wild adaptations are concentrated along the St. Lawrence and lower Great Lakes. The ruderal plants pose a minor weed problem to agriculture but a major problem to law enforcement, and decades of eradication have exterminated many of the naturalized populations in Canada. With the recent re-authorization of hemp cultivation in Canada, it is inevitable that there will be additional escapes and a reinvigoration of the ruderal phase of the species. Mechanical eradication
for 2 or 3 yr is effective at destroying populations, and young plants are easily eliminated by herbicide applications.

The Botany of Cannabis sativa L
Andreu FV
https://www.academia.edu/25681288/Th...nabis_sativa_L (the s needs to be removed from https to get this)
Cannabis sativa L. is a widespread species in nature. It is found invarious habitats ranging from sea level to the temperate and alpinefoothills of the Himalayas, from where it was probably spread over thelast 10,000 years. The age-old cultivation makes its original distribu-tion difficult to pinpoint. Cannabis has a long history of medicinaluse in the Middle East and Asia, with references as far back as the 6thcentury BCE, and it was introduced in Western Europe as a medicinein the early 19th century to treat epilepsy, tetanus, rheumatism,migraine, asthma, trigeminal neuralgia, fatigue, and insomnia. As a plant, it is valued for its hallucinogenic and medicinal prop-erties, more recently being used for pain, glaucoma, nausea, asthma,depression, insomnia, and neuralgia. Derivatives are used inHIV/AIDS and multiple sclerosis. The pharmacology andtherapeutic efficacy of cannabis preparations and its main activeconstituen t?9-tetrahydrocannabinol (?9-THC) have been extensivelyreviewed. The other important cannabinoid constituent of current interest is cannabidiol (CBD). There has been a significantinterest in CBD over the last few years because of its reportedactivity as an antiepileptic agent, particularly its promise for thetreatment of intractable pediatric epilepsy. Other than ?9-THCand CBD, tetrahydrocannabivarin (THCV), cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC) are major isolates.Fig. 1.1 shows chemical structures.Cannabis is also one of the oldest sources of food and textilefiber. Hemp grown for fiber was introduced in Western Asia andEgypt and subsequently in Europe between 1000 and 2000 BCE.Cultivation of hemp in Europe became widespread after 500 CE.The crop was first brought to South America (Chile) in 1545, and toNorth America (Port Royal, Acadia) in 1606. Now its cultivation is prohibited or highly regulated in the United States.

The carbon footprint of indoor Cannabis production.
Mills, E.
Energy Policy, 46, 58–67.(2012).
doi:10.1016/j.enpol.2012.03.023
The emergent industry of indoor Cannabis production -- legal in some jurisdictions and illicit in others -- utilizes highly energy intensive processes to control environmental conditions during cultivation. This article estimates the energy consumption for this practice in the United States at 1% of national electricity use, or $6 billion each year. One average kilogram of final product is associated with 4600kg of carbon dioxide emissions to the atmosphere, or that of 3 million average U.S. cars when aggregated across all national production. The practice of indoor cultivation is driven by criminalization, pursuit of security, pest and disease management, and the desire for greater process control and yields. Energy analysts and policymakers have not previously addressed this use of energy. The unchecked growth of electricity demand in this sector confounds energy forecasts and obscures savings from energy efficiency programs and policies. While criminalization has contributed to the substantial energy intensity, legalization would not change the situation materially without ancillary efforts to manage energy use, provide consumer information via labeling, and other measures. Were product prices to fall as a result of legalization, indoor production using current practices could rapidly become non-viable.

The effect of N and P fertilization on growth, seed yield and quality of industrial hemp in the Parkland region of Saskatchewan
C. L. Vera, S. S. Malhi, J. P. Raney, and Z. H. Wang
Can. J. Plant Sci. 84: 939–947
https://cdnsciencepub.com/doi/pdf/10.4141/P04-022
Industrial hemp (Cannabis sativa L.) has sparked renewed interest in western Canada in recent years, and there is very little research information available on its fertilizer requirements. The objective of this study was to determine the effect of surface-broadcast ammonium nitrate and seedrow placed monoammonium phosphate fertilizers on the production and seed quality attributes of industrial hemp (cv. Fasamo and Finola). Field experiments were conducted on a Black Chernozem silty loam soil at Melfort, Saskatchewan, Canada, in 2000, 2001 and 2002. Increasing N rates significantly increased plant height, biomass, seed yield and seed protein content of hemp in all years. Seed-applied P fertilizer increased plant height in all years, and biomass in 2000, but reduced plant density, biomass and seed yield in 2001 and 2002. Finola consistently had lower plant height, earlier maturity, heavier seeds, and higher seed yield, seed protein content and seed oil content than Fasamo. The average amount of nitrate-N in the 0–60 cm soil was 40 kg N ha–1. Seed yield kg–1 of N was 9.4, 5.9, 4.5 and 3.7 kg ha–1 for Fasamo, and 10.6, 7.7, 6.0 and 4.5 kg ha–1 for Finola, respectively, at 40, 80, 120 and 160 kg ha–1 of soil plus fertilizer N.

Not Cannabis specific
THE EXPERIMENTAL MODIFICATION OF SEX EXPRESSION IN FLOWERING PLANTS.
HESLOP-HARRISON, J.
Biological Reviews, 32(1), 38–90. (1957).
doi:10.1111/j.1469-185x.1957.tb01576.x
Since the destiny of the spores of the angiosperm sporophyte-whether they shall form female or male gametophytes-is determined by the type of sporangia in which they are formed, the heterangy (Thomson, 1927) of the sporophyte is commonly interpreted as a form of sexual dimorphism, and by extension of usage, the staminate organs are termed male and the pistillate female. It is with the experimental control of diplophase sex expression in this sense that this article is concerned.
Notwithstanding the special genetical and evolutionary significance of sexual
phenomena, the differentiation of floral structures may be regarded simply as an example of morphogenesis and studied as such; in fact, as Went (1954) has pointed out, the flowering process, being under environmental control and thus accessible to the experimenter, represents a morphogenetic activity peculiarly suitable for physiological investigation. However, while the differentiation of the ‘ essential ’ organs, gynoecium and androecium, represents the indispensable part of the general process of flowering, this is an aspect little taken into consideration in the very numerous studies of flowering physiology of the last three decades. The bulk of the studies have been concerned with the factors which initiate flowering, flower formation itself being regarded as a single, indivisible process. It is certainly true that in the majority of plants which have been used in photoperiodic and vernalization experiments flowering represents an alternative mode of behaviour at the stem apex to vegetative growth, which, once initiated, usually proceeds through its various stages with a certain appearance of inevitability. It is perhaps a natural consequence of this that attention should have been focused principally upon the mechanism by which environmental factors determine which developmental path shall be followed at the apex. As Liverman (1955) has said, in the bulk of the research on the physiology of flowering the differentiation of the terminal meristem has been regarded as a form of ‘bioassay’ for inductive processes mostly taking place in the leaf.

The FAR-PAR Index is a better predictor of stem and leaf expansion than phytochrome photo-equilibrium
Paul Kusuma and Bruce Bugbee
Crop Physiology Laboratory
Dept of Plants, Soil & Climate Utha State University
https://digitalcommons.usu.edu/cgi/v...t=researchweek
The fraction of far-red photons (greater than 700 nm) is increased compared to shorter wavelengths under plant shade. The resulting spectral shift is sensed by the pigment phytochrome, which mediates stem and leaf expansion Phytochrome exists in one of two reversible forms, active (Pfr) and inactive (Pr ), that depend on the wavelengths it absorbs. Absorption and conversion rates of phytochrome have been measured by Butler et al. (1964), Gardner and Graceffo (1982) and Sager et al. (1988). All studies measured phytochrome from darkgrown tissue to minimize interference from chlorophyll. Phytochrome photo-equilibria (PPE), is the ratio of active phytochrome to total phytochrome: ?????? = ?????? ??????+ ?? ?? The red/far-red (R:FR) ratio is often used as a proxy for this ratio. Both PPE and R:FR have historically been used as predictors of stem elongation. They have both been adequate in sunlight and under electric light, but are not entirely reliable under a wide range of spectra, especially those produced by narrow bandwidth LEDs.The FAR-PAR Index is the ratio of far-red photons (700-750 nm) to total photosynthetic photons (PPFD), expressed as a percent.

The Globalization of Cannabis Cultivation
Gary R. Potter, Martin Bouchard and Tom Decorte
https://www.ararteko.eus/RecursosWeb/DOCUMENTOS/1/0_2563_1.pdf .
Chapter 1 in book: World Wide Weed 2011
https://www.taylorfrancis.com/chapte...rd-tom-decorte
This chapter examines the cannabis issue with a focus on the moral justifications for small-scale cannabis cultivation. In Finland, the cannabis available in the drug market has traditionally been imported hash. The share of marihuana in the market has been nearly nonexistent and it has likewise been imported. Drug crime is one of the most condemnable types of crime in modern culture, and people arrested, accused or sentenced for such crimes are heavily labeled and stigmatized by society. The most common consequence for small-scale cultivation is a day-fine, but production on a larger scale with intent to distribute may lead to imprisonment. Because of the criminalization of cannabis in the Penal Code, the good citizenship of cannabis growers is questioned in the civic world. Thus, in the market world the small-scale home growers are negotiating an agreement that is based on the values of traditional barter economy rather than on the values of modern capitalism.



The overview of existing knowledge on medical cannabis plants growing
Matěj Malík, Jiří Velechovský, Pavel Tlustoš
Plant, Soil and Environment, 67, 2021 (8): 425–442
DOI: 10.17221/96/2021-PSE
The use of cannabis for medicinal purposes dates back well before the era of modern medicine, but in recent years research into the use of medical cannabis in the medical and pharmaceutical sciences has grown significantly. In European countries, most cannabis plants have been and still are grown for industrial purposes. For this reason, hemp cultivation technology is relatively well researched, while little is known about the key factors affecting cannabis cultivation for medical purposes. The active substances of cannabis plant targeted by this review are called phytocannabinoids. The biosynthesis of phytocannabinoids is relatively well understood, but the specific environmental factors that influence the type and number of phytocannabinoids have been much less studied. Indoor or greenhouse cultivation, which uses automated lighting, ventilation, irrigation systems and complex plant nutrition has become much more sophisticated and appears to be the most effective method for producing medical cannabis. There are many different cultivation systems for cannabis plants, but one of the essential elements of the process is an optimal plant nutrition and selection of fertilisers to achieve it. This review summarises the existing knowledge about phytocannabinoid biosynthesis and the conditions suitable for growing plants as sources of medical cannabis. This review also attempts to delineate how nutrient type and bioavailability influences the synthesis and accumulation of specific phytocannabinoids based on contemporary knowledge of the topic.

Not Cannabis Specific
The plant adheri nAtSCC2 is required for embryogenesis and sister-chromatid cohesion during meiosis in Arabidopsis.
Sebastian, J., Ravi, M., Andreuzza, S., Panoli, A. P., Marimuthu, M. P. A., & Siddiqi, I.
The Plant Journal, 59(1), 1–13. (2009).
doi:10.1111/j.1365-313x.2009.03845.x
Adherin plays an important role in loading the cohesin complex onto chromosomes, and is essential for the establishment of sister-chromatid cohesion. We have identified and analyzed the Arabidopsis adherin homolog AtSCC2. Interestingly, the sequence analysis of AtSCC2 and of other putative plant adherin homologs revealed the presence of a PHD finger, which is not found in their fungal and animal counterparts. AtSCC2 is identical to EMB2773, and mutants show early embryo lethality and formation of giant endosperm nuclei. A role for AtSCC2 in sister-chromatid cohesion was established by using conditional RNAi and examining meiotic chromosome organization. AtSCC2-RNAi lines showed sterility, arising from the following defects in meiotic chromosome organization: failure of homologous pairing, loss of sister-chromatid cohesion, mixed segregation of chromosomes and chromosome fragmentation. The mutant phenotype, which included defects in chromosome organization and cohesion in prophase I, is distinct from that of the Arabidopsis cohesin mutant Atrec8, which retains centromere cohesion up to anaphase I. Immunostaining experiments revealed the aberrant distribution of the cohesin subunit AtSCC3 on chromosomes, and defects in chromosomal axis formation, in the meiocytes of AtSCC2-RNAi lines. These results demonstrate a role for AtSCC2 in sisterchromatid cohesion and centromere organization, and show that the machinery responsible for the establishment of cohesion is conserved in plants.

Not Cannabis specific
The Potential of Light Treatments to Suppress Certain Plant Pathogens and Pests
David M. Gadoury
Research News from Cornell’s Viticulture and Enology Program Research Focus 2019-3
https://grapesandwine.cals.cornell.e.. .s%202019-3.pdf
• Germicidal ultraviolet light, properly applied, can suppress powdery mildew on a variety of crops.
• Significant suppression has also been observed in laboratory and preliminary field studies of downy mildew and mites.
• Nighttime application is necessary to prevent reversal of the UV effect due to the blue light and UVA component of natural sunlight.
• Proper design of the light array is essential for proper dosing, and to reach the interior of the grapevine canopy.
• Lamp arrays are adaptable to a variety of carriages, including towable arrays and those moved by autonomous robotic devices.

Not Cannabis specific
The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana.
Ravi, M., Kwong, P. N., Menorca, R. M. G., Valencia, J. T., Ramahi, J. S., Stewart, J. L., … Chan, S. W.-L.
Genetics, 186(2), 461–471.(2010).
doi:10.1534/genetics.110.120337
Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain–CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3’s lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP–CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP–CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

The relationship between light intensity, cannabis yields, and profitability
James Eaves, Stephen Eaves, Chad Morphy, Chris Murray
doi: 10.1002/agj2.20008
The purpose of this study was to analyze the relationship between light intensity, cannabis yields, and profitability. We also look for evidence that spectrum differences across broad?spectrum, horticulture lights and general?purpose LEDs impact the relationship between yield and light intensity. Finally, we discuss the financial return of increasing light intensity in order to increase yields. We find that yields increase linearly with light intensity up to at least 1500 ?mols/m2 s, which is at least twice the intensity that is most commonly used by cannabis growers. That relationship did not appear to be influenced by spectrum quality differences across the lamps included in the study. Finally, for all the intensity ranges that we considered, the value of the gain in yields from increasing light intensity far exceeded the cost of using more electricity

The Role of Roots in Sex Expression in Hemp Plants
M.Kh. Chailakhyan and V.N. Khryanin
Planta 138, 185-187 (1978)
When the shoots of young hemp (Cannabis sativa L.) plants were cut off the roots, cultured as cuttings, and regenerating (adventitious) roots were removed as soon as appearing, ca. 80-90% of the
plants became male (had staminate flowers) whereas if the roots were allowed to develop a similar percentage became female (pistillate flowers). Treatment of de-rooted cuttings with 6-benzylaminopurine (15 mg/1) restored the percent of female plants to ca. 80. It is suggested'that the root system plays an essential role in sex expression in hemp and that this role is related to cytokinin synthesis in the root. The role of the various organs in sex expression of plants with unisexual flowers is not well understood. Minina (1952, pp. 150 152), working with cucumber, showed that treatment of the leaves with CO caused a shift in sex expression, favoring formation of female (pistillate) flowers. She concluded that the leaves played a decisive role in the determination of sex
expression, as sites of production of some "sexualizing" substance(s) (Minina and Kushnirenko, 1949). On the other hand, there are reports that the root system of adult female hemp plants is 3 times larger than that of male plants (see Senchenko et al., 1963, pp. 32-36) and that hemp plantlets with long roots produce more female individuals than do plantlets with short roots (Kubarev, 1966). The aim of the work reported in this paper was to study the role of the roots in sex expression in hemp (Cannabis sativa L.), by growing shoot cuttings in liquid culture and either allowing adventitious roots to develop, or removing them, and by studying the effect of cytokinin on de-rooted cuttings.

Not Cannabis specific
The small GTPase Rab11F represents a molecular marker within the secretory 4 pathway required for the nitrogen-fixing symbiosis
Prachumporn Nounurai, Holger Densow & Hanna Bednarz & Karsten Niehaus
Biorxiv June 19 2019
Doi: 10.1101/661835.
The nitrogen-fixing root nodule is generally derived through a successful symbiotic interaction between legume plants and bacteria of the genus Rhizobium. A root nodule shelter hundreds of Rhizobia, which are thought to invade into the plant cells through an endocytosis-like process despite the existence of turgor pressure. Each invading Rhizobium is surrounded by the peribacteroid membrane to form the symbiosome, which results in the higher acquisition of host membrane materials. In this study, we show the localization of Rab11F, a RabA6b homolog with the large Rab-GTPas family, which was highly expressed in root nodules of Medicago sativa and M. truncatula. Rab11F-labeled organelles accumulated the membrane specific dye FM4-64 and were sensitive to Brefeldin A by forming aggregates after treatment with this drug. By co-localization with the cis-Golgi marker, GmMan1-mCherry, Rab11F- organelles formed tri-colored organelles, whereby Rab11F was located to the opposite side of GmMan1-mCherry indicating that Rab11F-labeled structures were localized within the trans-Golgi network (TGN). In root nodules, Rab11F was localized transiently at the infection thread-covering membrane on the side of infection droplets and the peribacteroid membranes. The symbiosome acquires Rab11F during the entry process and differentiation. However, the symbiosome did not recruit Rab11F after cessation of division. In conclusion, the legume plant seemed to use a specialized secretion pathway from the TGN, which was marked by Rab11F, to proliferate the symbiosome membrane.

The state of the apex and the response to induction in Cannabis sativa.
Heslop-Harrison J, Heslop-Harrison Y. 1970 In: Bernier G, ed. Proceedings of the Symposium on Cellular and Molecular Aspects of Floral Induction, Liege, Belgium. Longmans Green, 3-26.
FIND DOI or LINK

Not Cannabis specific The Versatile Roles of Sulfur-Containing Biomolecules in Plant Defense—A Road to Disease Resistance
András Künstler, Gábor Gullner, Attila L. Ádám, Judit Kolozsváriné Nagy and Lóránt Király
Plants 2020, 9, 1705;
doi:10.3390/plants9121705
https://www.ncbi.nlm.nih.gov/labs/pm...s-09-01705.pdf
Sulfur (S) is an essential plant macronutrient and the pivotal role of sulfur compounds in plant disease resistance has become obvious in recent decades. This review attempts to recapitulate results on the various functions of sulfur-containing defense compounds (SDCs) in plant defense responses to pathogens. These compounds include sulfur containing amino acids such as cysteine and methionine, the tripeptide glutathione, thionins and defensins, glucosinolates and phytoalexins and, last but not least, reactive sulfur species and hydrogen sulfide. SDCs play versatile roles both in pathogen perception and initiating signal transduction pathways that are interconnected with various defense processes regulated by plant hormones (salicylic acid, jasmonic acid and ethylene) and reactive oxygen species (ROS). Importantly, ROS-mediated reversible oxidation of cysteine residues on plant proteins have profound effects on protein functions like signal transduction of plant defense responses during pathogen infections. Indeed, the multifaceted plant defense responses initiated by SDCs should provide novel tools for plant breeding to endow crops with efficient defense responses to invading pathogens.

Not directly Cannabis related
The X chromosome is necessary for ovule production in Silene latifolia.
Veltsos, P., & Delph, L. F. (2019).
PLOS ONE, 14(5), e0217558.
doi:10.1371/journal.pone.0217558
Sex chromosomes stop recombining and accumulate differences over time. In particular, genes on the chromosome restricted to the heterogametic sex degenerate and become non-functional. Here, we investigated whether or not the degeneration of a plant Y chromosome was sufficient to cause ovules containing a Y to fail to develop, thereby eliminating the possibility of YY individuals. We used two genotypic assays to determine the genotype— XX, XY, or YY—of offspring from a single fruit of an otherwise normal male XY plant of Silene latifolia. The fruit contained fewer ovules than normal pistillate flowers, produced an equal offspring sex ratio, and generated no YY offspring. The results indicate that ovaries must contain an X chromosome to develop properly. While haploid selection has slowed down Y-chromosome degeneration in S. latifolia, we find that it has progressed sufficiently to prevent the proper development of ovules, and hence prevent the presence of YY individuals.

Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L.
Lata, H., Chandra, S., Khan, I., & ElSohly, M. A.
In Vitro Cellular & Developmental Biology - Plant, 45(1), 12–19.(2008).
doi:10.1007/s11627-008-9167-5
Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yrold mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05– 5.0 ?M thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 ?M thidiazuron) than with benzyladenine or kinetin. Adding 7.0 ?M of gibberellic acid into a medium containing 0.5 ?M thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l?1 activated charcoal and 2.5 ?M indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 ?mol m?2 s ?1 ). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 ?mol m?2 s ?1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 ?mol m?2 s ?1 . Stomatal conductance and transpiration increased with light intensity up to highest level (2000 ?mol m?2 s ?1 ) tested. Intercellular CO2 concentration (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.

Not Cannabis specific
TMK-based cell surface auxin signaling activates cell wall acidification in Arabidopsis
Zhenbiao Yang, Wenwei Lin. Wenxin Tang, Koji Takahashi, Hong Ren, Songqin Pan, Haiyan Zheng, William Gray, Tongda Xu, Toshinori Kinoshita
Nature 2021
DOI: 10.1038/s41586-021-03976-4
https://assets.researchsquare.com/fi...f?c=1637594958
The phytohormone auxin controls a myriad of processes in plants, at least in part through its regulation of cell expansion. The "acid growth hypothesis" has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism underlying auxin-induced cell wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane (PM) H+- ATPase that pumps protons into the apoplast, yet how auxin activates its phosphorylation remains elusive. Here, we show that the transmembrane kinase (TMK) auxin signaling proteins interact with PM H+-ATPases and activate their phosphorylation to promote cell wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced TMK's interaction with H+-ATPase on the plasma
membrane within 1-2 minutes as well as TMK-dependent phosphorylation of the penultimate Thr residue. Genetic, biochemical, and molecular evidence demonstrates that TMKs directly phosphorylate PM H+-ATPase and are required for auxin-induced PM H+-ATPase activation, apoplastic acidification, and cell expansion. Thus, our findings reveal a crucial connection between auxin and PM H+-ATPase activation in regulating apoplastic pH changes and cell expansion via TMK-based cell surface auxin signaling.

Not Cannabis specific
Transcriptional Repression by Histone Deacetylases in Plants.
Liu, X., Yang, S., Zhao, M., Luo, M., Yu, C.-W., Chen, C.-Y., … Wu, K.
Molecular Plant, 7(5), 764–772. (2014).
doi:10.1093/mp/ssu033
Reversible histone acetylation and deacetylation at the N-terminus of histone tails play a crucial role in regulation of eukaryotic gene activity. Acetylation of core histones usually induces an “open” chromatin structure and is associated with gene activation, whereas deacetylation of histone is often correlated with “closed” chromatin and gene repression. Histone deacetylation is catalyzed by histone deacetylases (HDACs). A
growing number of studies have demonstrated the importance of histone
deacetylation/acetylation on genome stability, transcriptional regulation and development in plants. Furthermore, HDACs were shown to interact with various chromatin remolding factors and transcription factors involved in transcriptional repression in multiple developmental processes. In this review, we summarized recent findings on the transcriptional repression mediated by HDACs in plants.

USE OF NATURAL ZEOLITE (CLINOPTILOLITE) IN AGRICULTURE
Ersin Polat, Mehmet Karaca, Halil Demir and A. Naci Onus
Journal of Fruit and Ornamental Plant Research vol. 12, 2004
https://www.kmizeolite.com/wp-conten...-Agricultu.pdf
Zeolites have been increasingly used in various application areas such as industry, agriculture, environmental protection, and even medicine. Although, there are no certain figures on the total amount of these minerals the world, some countries e.g. Cuba, USA, Russia, Japan, Italy, South Africa, Hungary and Bulgaria, have important reserves and production potentials. According to reports of 2001, the total consumption of zeolites was 3.5 million tons of which 18% came from their natural resources and the rest from synthetics. More than forty naturally occurring zeolites were reported by different research groups, and clinoptilolite, erionite, chabazite, heulandite, mordenite, stilbit and philipsite are the most well-known. The most common for agricultural applications is clinoptilolite since it has high absorption, cation exchange, catalysis and dehydration capacities. Zeolites are, therefore, used as a promoter for better plant growth by improving the value of fertilizers; retaining valuable nitrogen and improving the quality of resulting manures and sludge. They can also be used as a molecular sieve or filter medium.

Not Cannabis specific
Use of UV-C irradiation to prevent decay and maintain postharvest quality of ripe “Tommy Atkins” mangoes.
González-Aguilar, G. A., Wang, C. Y., Buta, J. G., & Krizek, D. T.
International Journal of Food Science & Technology, 36(7), 767–773.
(2008).
doi:10.1111/j.1365-2621.2001.00522.x
Ripe mangoes `Tommy Atkins' were exposed to UV-C irradiation for 10 and 20 min, prior to storage for 14 days at 5 or 20 °C and a shelf-life period of 7 days at 20 °C. UV-C-treated fruit maintained better visual appearance than unirradiated controls. UV-C irradiation for 10 min was the most e?ective regime in suppressing decay symptoms and maintaining ®rmness during storage at 5 or 20 °C. Such fruit (treated with UV-C for 10 min) showed greater levels of putrescine and spermidine after cold storage than controls and those treated with UV-C for 20 min. Higher levels of sugars and lower levels of organic acids were observed in mangoes treated with UV-C for 20 min. However, the most e?ective UV-C treatment (10 min) for reducing decay maintained sugar and organic acid levels similar to the controls. No UV-damage was observed on treated fruits after storage. These results indicate that UV-C irradiation could be used as an e?ective and rapid method to preserve the postharvest life of ripe mangoes without adversely a?ecting certain quality attributes

Not Cannabis specific
Use of UV-C postharvest treatment for extending fresh whole tomato (Solanum lycopersicum, cv. Zinac) shelf-life.
Pinheiro, J., Alegria, C., Abreu, M., Gonçalves, E. M., & Silva, C. L. M.
Journal of Food Science and Technology, 52(8), 5066–5074.(2014).
doi:10.1007/s13197-014-1550-0
The effect of UV-C treatments (0.32, 0.97, 2.56, 4.16 and 4.83 kJ.m?2 at 254 nm) on the physical-chemical properties [colour, texture, total phenolic content (TPC), weight loss (WL)], and mesophylic counts of whole tomato, was evaluated during 15 days at 10 °C. During storage, the Ctr samples acquired faster red colour than all UV-C samples (higher a* and lower °h values). Comparing texture of Ctr and UV-C samples at 15th storage day, an increase of 9 and 8 % on firmness of treated samples at low UV-C intensities (0.32 and 0.97 kJ.m?2 , respectively) was observed. At the end of the storage, Ctr samples showed ca. 4 Log10 of mesophylic load, and the samples treated at 0.97 and 4.83 kJ.m?2 revealed the lowest microbial load (1.9 and 3.2 Log10, respectively). These results indicate that UV-C radiation, at an appropriate dose, combined with low storage temperature (10 °C) are an effective method to preserve the postharvest life of tomato, without adversely affecting quality parameters.

USU Cannabis Lab Tour with Dr Bruce Bugbee
YouTube
https://www.youtube.com/watch?v=9Z8S...lsOptCZfJvLVCI
Dr Bruce Bugbee gives a tour of the cannabis research he's conducting at the Crop Physiology Lab at Utah State University. The lab is one of the few in the world to have a cannabis license. The strains being researched are medical hemp with low THC and high CBD. The cultivation methods being developed apply to all types of cannabis. Please excuse the poor camera quality at the beginning of the video. This was a spur of the moment tour and the camera was not set to auto-focus and auto exposure at the beginning. it gets better later in the video.

Not Cannabis specific
UV-B as a means to control powdery mildew in strawberry
Aruppillai Suthaparan, Arne Stensvand, Knut Asbjørn Solhaug, Nils Bjugstad, David M. Gadoury, Hans Ragnar Gislerød
https://lightandplanthealth.org/pdf/...BStensvand.pdf
Powdery mildew (Podosphaera aphanis) is an important disease of strawberry (Fragaria × ananassa), especially if grown in high plastic tunnels or greenhouses. We have previously demonstrated suppression of powdery mildew in rose and cucumber strawberry following brief exposures to red light and UV-B radiation during night hours (Suthaparan et al. 2010a, 2010b, 2012, 2013). Here we report results from experiments with UV-B against strawberry powdery mildew.

Not Cannabis specific
UV-B Perceived by the UVR8 Photoreceptor Inhibits Plant
Hayes, S., Sharma, A., Fraser, D. P., Trevisan, M., Cragg-Barber, C. K., Tavridou, E., Franklin, K. A.
Thermomorphogenesis. Current Biology, 27(1), 120–127. (2017). doi:10.1016/j.cub.2016.11.004
Small increases in ambient temperature can elicit striking effects on plant architecture, collectively termed thermomorphogenesis [1]. In Arabidopsis thaliana, these include marked stem elongation and leaf elevation, responses that have been predicted to enhance leaf cooling [2–5]. Thermomorphogenesis requires increased auxin biosynthesis, mediated by the bHLH transcription factor PHYTOCHROMEINTERACTING FACTOR 4 (PIF4) [6–8], and enhanced stability of the auxin co-receptor TIR1, involving HEAT SHOCK PROTEIN 90 (HSP90) [9]. High-temperature-mediated hypocotyl elongation additionally involves localized changes in auxin metabolism, mediated by the indole-3-acetic acid (IAA)-amido synthetase Gretchen Hagen 3 (GH3).17 [10]. Here we show that ultraviolet-B light (UV-B) perceived by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8) [11] strongly attenuates thermomorphogenesis via multiple mechanisms inhibiting PIF4 activity. Suppression of thermomorphogenesis involves UVR8 and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)-mediated repression ofPIF4 transcript accumulation, reducing PIF4 abundance. UV-B also stabilizes the bHLH protein LONG HYPOCOTYL IN FAR RED (HFR1), which can bind to and inhibit PIF4 function. Collectively, our results demonstrate complex crosstalk between UV-B and high-temperature signaling. As plants grown in sunlight would most likely experience concomitant elevations in UV-B and ambient temperature, elucidating how these pathways are integrated is of key importance to the understanding of plant development in natural environments.

Not Cannabis specific
UV-C radiation as an alternative tool to control powdery mildew on apple and strawberry.
Van Hemelrijck, W., Van Laer, S., Hoekstra, S., Aiking, A. and Creemers, P.
https://www.jenit.dk/files/files/UV-...ew_article.pdf
The most used and effective strategy to control fungal diseases in fruit growing is the application of fungicides. The use of fungicides , however, can have a negative impact on the environment and its frequent use enhances the risk of fungicide resistance. Moreover, stronger limits concerning the maximum residue levels (MRLs) on fruits are set up by retailers and the export industry. In the future it will be a great challenge to produce fruits of high quality with a minimal input of pesticides. As such, a search for alternative methods, like biological control agents or physical treatments, which are able to efficiently reduce fruit diseases is required. To this end a physical technique, ultraviolet light in particular, was tested for its capacity to reduce powdery mildew infestation on apple and strawberry leaves. The applied exposure doses ranged from 5 to 60mJ/cm2. The effect of UV-c radiation was tested on the establishment of powdery mildew on leaves of apple seedlings and strawberry plants. For both apple and strawberry the powdery mildew infestation was significantly reduced by exposing the leaves of both plant species to UV-c with a dose of 30mJ/cm², without negative effects on plant tolerance. The obtained efficacy levels were comparable with specific fungicide treatments. A regular application in the time was better than using higher dose rates with longer intervals between applications. Results of these trials under controlled conditions will be presented.

Not Cannabis specific
UV-C technology to control postharvest diseases of fruits and vegetables
Rosalba Troncoso-Rojas, Martín E. Tiznado-Hernández and Alberto González-León
https://www.researchgate.net/publica...and_vegetables
In the last decades considerable efforts have been sustained to develop alternative strategies to chemical methods to control postharvest diseases. One of such strategies has been the use of UV-C radiation. Postharvest UV-treatments consist in exposing the commodities for a certain period of time under a bank of UV lamps, with a maximal emission at 254 nm. UV-C treatments have been shown to be effective to control the most common postharvest pathogens.

Not Cannabis specific
UV light can kill fungi that causes powdery mildew
Sarah Thompson
https://vegetablegrowersnews.com/new...owdery-mildew/
Growers may no longer have to rely on fungicides to control powdery mildew (PM) – a rampant fungal disease that affects nearly all plant species. The international, multidisciplinary Light and Plant Health team of researchers led by Cornell AgriTech’s David Gadoury has spent the last five years refining the science and applied technology behind using ultraviolet (UV) light to kill the fungi that cause PM.
“In more than three years of trials, UV light applications worked as well or better than available fungicides, killing 95 percent of PM in field strawberries. We’ve seen similar results in field and greenhouse trials of basil, roses, grapes and cucumbers,” said Gadoury, senior research associate in the Section of Plant Pathology and Plant Microbe-Biology in the School of Integrative Plant Science.

Vegetative propagation of cannabis by stem cuttings: effects of leaf number, cutting position, rooting hormone and leaf tip removal.
Caplan, D., Stemeroff, J., Dixon, M., & Zheng, Y.
Canadian Journal of Plant Science. (2018).
doi:10.1139/cjps-2018-0038
This study evaluated the influence of the several factors and their interactive effects on propagation success using stem cuttings of cannabis (Cannabis sativa L.). Factors included: (i) Leaf number (two or three) (ii) leaf tip removal (1/3 of leaf tips removed) (iii) basal/apical position of stem cutting on the stock plant, and (iv) rooting hormone (0.2% indole-3-butyric
(IBA) acid gel or 0.2% willow (Salix alba L.) extract gel). Cuttings were placed in a growth chamber for twelve days then assessed on their rooting success rate and root quality using a relative root quality scale. The IBA gel delivered a 2.1 times higher rooting success rate and 1.6 times higher root quality than the willow extract. Removing leaf tips reduced rooting success rate from 71% to 53% without influencing root quality. Cuttings with three leaves had 15% higher root quality compared to those with two, but leaf number did not influence rooting success rate. Position of cutting had little effect on rooting success or quality. To achieve maximum rooting success and root quality, cuttings from either apical or basal positions should have at least three fully expanded, uncut leaves and the tested IBA rooting hormone is preferential to the willowbased product.

Vegetative propagation of cannabis sativa
dabina sage, larry rupp, bruce bugbee.
Utah State University. Plants, Soils, and Climate Dept
Find DOI of the Poster Asked Bruce for a copy.

Not Cannabis specific
Whole Irradiated Plant Leaves Showed Faster Photosynthetic Induction Than Individually Irradiated Leaves via Improved Stomatal Opening
Shunji Shimadzu, Mitsunori Seo, Ichiro Terashima and Wataru Yamori
Front. Plant Sci. 10:1512. (2019)
doi: 10.3389/fpls.2019.01512
Rapid photosynthetic induction is crucial for plants under fluctuating light conditions in a crop canopy as well as in an understory. Most previous studies have focused on photosynthetic induction responses in a single leaf, whereas the systemic responses of the whole plant have not been considered. In a natural environment, however, both single leaves and whole plants are exposed to sunlight, since the light environment is not uniform even within a given plant. In the present study, we examined whether there is any difference between the photosynthetic induction response of a leaf of a whole irradiated plant and an individually irradiated leaf in Arabidopsis thaliana to consider photosynthetic induction as the response of a whole plant. We used two methods, the visualization of photosynthesis and direct measurements of gas-exchange and Chl fluorescence, to demonstrate that whole irradiated plant promoted its photosynthetic induction via improved stomatal opening compared with individually irradiated leaf. Furthermore, using two Arabidopsis knockout mutants of abscisic acid transporter, abcg25 and abcg40, the present study suggests that abscisic acid could be involved in this systemic response for stomatal opening, allowing plants to optimize the use of light energy at minimal cost in plants in a dynamic light environment.

Why the concept of terroir matters for drug cannabis production Pierre-Arnaud Chouvy GeoJournal (2022) DOI: 10.1007/s10708-022-10591-x
http://www.geopium.org/wp-content/up...g_cannabis.pdf
This article questions how the concepts of terroir and landrace are relevant for the drug cannabis industry at a time when cannabis legalisation and its associated ‘‘green rush’’ pose a growing threat to both the genetic and cultural diversity that is associated with historical small cannabis farming. The article draws on a multidisciplinary approach based on both extensive secondary sources and primary research. A large and detailed definition work first informs what terroir and landrace are and most especially what they have in common, from the typicity of their end products, to how they owe their existence to geographic remoteness and isolation, and to how tradition and change (or modernity) affect their development and conservation. Defining and connecting terroirs and landraces in historical, anthropological, environmental, and of course chemical terms, makes it possible to determine how cannabis terroirs compare with and differ from other terroirs and plants, based on the rare dual qualities of the plant (being both a food and a drug) but also, given the illegality of its cultivation, on the specific territorial characteristics of its production areas, notably their geographic remoteness and isolation, their politico-territorial control deficits, etc. The article concludes that acknowledging and protecting cannabis terroirs and landraces matters because it favours the conservation and the promotion of a biological, cultural, and sensorial diversity that has endured illegality and repression but is now threatened by legalisation.

Yield, Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation
Fabio Ferrini, Daniele Fraternale, Sabrina Donati Zeppa, Giancarlo Verardo, Andrea Gorassini, Vittoria Carrabs, Maria Cristina Albertini and Piero Sestili
Molecules 2021, 26, 4889.
DOI: 10.3390/ molecules26164889
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-04889.pdf
Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C. sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, β-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth—particularly of the roots—and a significant increase (up to 20-fold in the case of β-sitosterol) in the total content of the aforementioned roots’ bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Yield of Illicit Indoor Cannabis Cultivation in The Netherlands.
Toonen, M., Ribot, S., & Thissen, J.
Journal of Forensic Sciences, 51(5), 1050–1054.(2006).
doi:10.1111/j.1556-4029.2006.00228.x
To obtain a reliable estimation on the yield of illicit indoor cannabis cultivation in The Netherlands, cannabis plants confiscated by the police were used to determine the yield of dried female flower buds. The developmental stage of flower buds of the seized plants was described on a scale from 1 to 10 where the value of 10 indicates a fully developed flower bud ready for harvesting. Using eight additional characteristics describing the grow room and cultivation parameters, regression analysis with subset selection was carried out to develop two models for the yield of indoor cannabis cultivation. The median Dutch illicit grow room consists of 259 cannabis plants, has a plant density of 15 plants/m2 , and 510 W of growth lamps per m2 . For the median Dutch grow room, the predicted yield of female flower buds at the harvestable developmental stage (stage 10) was 33.7 g/plant or 505 g/m2 .


IC Cannabis Collectables


Hemp Stamps Bring Record Prices
Don Wirtshafter, Michael Krawitz
January 2006 Journal of Industrial Hemp 10(2):67-74
DOI: 10.1300/J237v10n02_06
The artifacts of hemp and marijuana’s rich histories are some of the hottest articles on the secondary market today. Revenue stamps issued under the Marihuana Tax Act of 1937 were recently auctioned by the Smithsonian Institute to raise money for future acquisitions. The prices received shattered all pre-auction estimates. Other collectable stamps have been issued by various states of the United States (US). The high values of these items and other collectibles reflects the huge investment the US government has expended in trying to eradicate the use of Cannabis. Collectors should be aware that the high value of these stamps reflects on the current hot market for collectibles of all things Cannabis.


IC Cannabis Hormones


Auxin and Sexuality in Cannabis sativa.
Heslop-Harrison, J. (1956).
Physiologia Plantarum, 9(4), 588–597.
doi:10.1111/j.1399-3054.1956.tb07821.x
It is now well established that sex expression in various monoecious Cucurbitaceae may be modified by auxin treatment. Laibach and Kribben (1950 a, 1950 b. 1950 c and 1950 d) bave shown that the proportion of female flowersproduced by the cucumber (Cucumis sativus) may be substantially increased by treatment during early growtb witb [3-indoIeacetic acid flAA) and a-naplitbaleaeacotic acid (NAA). applied either in lanolin paste medium or by spraying in aqueous solution. Nilsch. Kurtz. Liverman and Went (l952) have reporled a similar result witb squash (Cucurbita pepo var. Table Queen), in which spraying with NAA in aqueous solution al 100 p.p.m. al the two leaved stage induced the formation of the first temiilc flower bud al about the ninth node, the first female flowers in control plinits not being formed until the twentieth node or later. Corresponding results have been obtained by Wittwer and Hillyer (1954) with the cucumber varieties National Pickeling, and Burpee Hybrid, as well as with squash. Arguing from their findings witb cucumber, Laibach and Kribben (1950d) bave suggested tbal tbe sexuality of flowers is dependeut upon the concentration of native auxin available in the leaf axil during the period of flower formation. Were this to be eslablisbed as generally true for the flowering plants, it would constitute an important step forward in our understandingof the control of flowering aud flower morphogenesis. Wbilst Ibe
results so tar reported for the uioiioecious cucurbits are themselves unequivocal, it would obviously be valuable to have a demonstration Ithat auxin levels govern flower sexuality in dioecious plants also. The present paper provides evidence that tbis may indeed be so in the dioecious species Cannabis satiua (hemp)

NOT Cannabis specific
?-Cyclocitral is a conserved root growth regulator
Alexandra J. Dickinsona, Kevin Lehnera, Jianing Mid, Kun-Peng Jiad, Medhavinee Mijara, José Dinnenyc, Salim Al-Babilid, and Philip N. Benfeya
PNAS May 21, 2019 116 (21) 10563-10567; first published May 8, 2019
Doi: 10.1073/pnas.1821445116
Natural compounds capable of increasing root depth and branching are desirable tools for enhancing stress tolerance in crops. We devised a sensitized screen to identify natural metabolites capable
of regulating root traits in Arabidopsis. ?-Cyclocitral, an endogenous root compound, was found to promote cell divisions in root meristems and stimulate lateral root branching. ?-Cyclocitral rescued
meristematic cell divisions in ccd1ccd4 biosynthesis mutants, and ?-cyclocitral–driven root growth was found to be independent of auxin, brassinosteroid, and reactive oxygen species signaling
pathways. ?-Cyclocitral had a conserved effect on root growth in tomato and rice and generated significantly more compact crown root systems in rice. Moreover, ?-cyclocitral treatment enhanced
plant vigor in rice plants exposed to salt-contaminated soil. These results indicate that ?-cyclocitral is a broadly effective root growth promoter in both monocots and eudicots and could be a valuable
tool to enhance crop vigor under environmental stress.

Cannabis sativa
Mohan Ram H Y and Sett R 1985
in CRC handbook of flowering (ed. Halevy A H) (Boca Raton: CRC Press) Vol. II, pp 131–139
FIND DOI or link

Comparative effect of silver ion and gibberelic acid on the induction of male flower in female Cannabis plants
G. Sarath, Hassan Ram
March 1979Experientia 35(3):333-334
DOI: 10.1007/BF01964334
G. SarathHassan RamHassan Ram
Silver ion applied as AgNO3 to the shoot tip of female plants ofCannabis induces male flowers. It is more effective than gibberellic acid (GA3) in maintaining the induced state.

Development of indole sulfonamides as cannabinoid receptor negative allosteric modulators
Iain R. Greiga, Gemma L. Bailliea, Mostafa Abdelrahmanc, Laurent Trembleauc, Ruth A. Rossa (2016)
Bioorganic & Medicinal Chemistry Letters, 26(18), 4403–4407.
DOI: 10.1016/j.bmcl.2016.08.018
Existing CB1 negative allosteric modulators (NAMs) fall into a limited range of structural classes. In spite of the theoretical potential of CB1 NAMs, published in vivo studies have generally not been able to demonstrate the expected therapeutically-relevant CB1-mediated effects. Thus, a greater range of molecular tools are required to allow definitive elucidation of the effects of CB1 allosteric modulation. In this study, we show a novel series of indole sulfonamides. Compounds 5e and 6c (ABD1075) had potencies of 4 and 3 nM respectively, and showed good oral exposure and CNS penetration, making them highly versatile tools for investigating the therapeutic potential of allosteric modulation of the cannabinoid system

Effect of induced polyploidy on some biochemical parameters in Cannabis sativa L.
Mahsa Bagheri & Hakimeh Mansouri
Appl Biochem Biotechnol
DOI 10.1007/s12010-014-1435-8
This study is aimed at testing the efficiency of colchicine on inducing polyploidy in Cannabis sativa L. and investigation of effects of polyploidy induction on some primary and secondary metabolites. Shoot tips were treated with three different concentrations of colchicine (0, 0.1, 0.2 %w/v) for 24 or 48 h. The biggest proportion of the almost coplanar tetraploids (43.33 %) and mixoploids (13.33 %) was obtained from the 24-h treatment in 0.2 and 0.1 %w/v, respectively. Colchicine with 0.2 % concentration and 48 h duration was more destructive than 24 h. The ploidy levels were screened with flow cytometry. The biochemical analyses showed that reducing sugars, soluble sugars, total protein, and total flavonoids increased significantly in mixoploid plants compared with tetraploid anddiploid plants. Tetraploid plants had a higher amount of total proteins, total flavonoids, and starch in comparison with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabinol in mixoploid plants only, but tetraploid plants had lower amounts of this substance in comparison with diploids. Also, we found such changes in protein concentration in electrophoresis analysis. In overall, our study suggests that tetraploidization could not be useful to produce tetrahydrocannabinol for commercial use, and in this case, mixoploids are more suitable.

Effects of ABA on primary terpenoids and ?9-tetrahydrocannabinol in Cannabis sativa L. at flowering stage.
Mansouri, H., Asrar, Z., & Szopa, J. (2009).
Plant Growth Regulation, 58(3), 269–277.
doi:10.1007/s10725-009-9375-y
This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, a-tocopherol, squalene, phytosterols, D9 -tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l-1 ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in a-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l-1 ABA in male plants. Treatment with 1 and 10 mg l-1 ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-D-erythritol-4- phosphate (MEP) pathway secondary metabolites accumulation (a-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophy

Effects of abscisic acid on content and biosynthesis of terpenoids in Cannabis sativa at vegetative stage.
Mansouri, H., & Asrar, Z.
Biologia Plantarum, 56(1), 153–156. (2012).
doi:10.1007/s10535-012-0033-2
The influence of abscisic acid (ABA) on plastidial and cytosolic terpenoids and on two key enzymes for terpenoid biosynthesis was determined in vegetative stage of Cannabis sativa L. Low concentration of ABA (1 µM) increased 1-deoxy-D-xylulose 5-phosphate synthase (DXS) activity in treated plants in comparison to control plants. The amounts of chlorophyll a and carotenoids increased in response to ABA treatment but chlorophyll b content declined. The accumulation of ?-tocopherol was stimulated only by 10 µM ABA. The ABA-treated plants showed a decline in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity which was followed by a decrease in squalene and phytosterol content. ABA also decreased tetrahydrocannabinol (THC) and cannabidiol (CBD) contents. The essential oil had higher ratios of monoterpenes to sesquiterpenes as ABA-treated plants had less numbers of sesquiterpenes in comparison with control plants. Influence of ABA on the amounts of sesquiterpenes was different, some of them showed decrease of content and others increase of content

Endogenous synthesis of pyrethrins by cannabis
Adrian Devitt-Lee, Douglas Smith, David Chen, Kevin McKernan, Simon Groves, Ciaran McCarthy
BioRxiv Preprint
doi: /10.1101/169417
Pyrethrins are a class of natural terpenoid pesticides produced by Tanacetum cinerariifolium, commonly known as chrysanthemum. Here we present evidence that cannabis may be able to produce pyrethrins endogenously. Flower from a cannabis plant grown in a closed hydroponic environment contained 2.48 parts per million pyrethrin I by weight. A comparison of the genetics of T. cinerariifolium and Cannabis demonstrates Cannabis homologues of the genes that contribute to pyrethrins production in T. cinerariifolium. This provides a plausible pathway for the biosynthesis of pyrethrins in cannabis. Although preliminary, these data indicate a potentially significant confounding variable in both cannabis research and regulations on allowable pyrethrins residues in cannabis products.

Feminization of male flowers of Cannabis sativa L. by a morphactin
Hassan Ram, V. S. Jaiswal
March 1971The Science of Nature 58(3):149-150
DOI: 10.1007/BF00593110
The dioecious nature of Cannabis sativa can be altered by environmental conditions and plant hormones [t] and by Ethrel (2-ckloroethanephosphonic acid) [2J. Morphactins have been shown to modify significantly the morphology of plant organs. We became interested in studying the effects of morphactin on the developmental morphology of C. sativa flowers.

Induction of male flowers on female plants of Cannabis sativa by gibberellins and its inhibition by abscisic acid
H. Y. Mohan Ram, V. S. Jaiswal
September 1972, Planta Volume 105, Issue 3, pp 263–266|
https://www.researchgate.net/publica..._abscisic_acid
DOI: 10.1007/BF00385397
Gibberellins (GA3, GA4+7, GA7 and GA9) induce male flowers on female plants of Cannabis sativa. This is, depending on concentration, partially or fully inhibited by abscisic acid (ABA). The ABA effect can in turn be partially overcome by increasing the concentration of GA3.

*Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex.
Mohan Ram, H. Y., & Sett, R. (1982).
Theoretical and Applied Genetics, 62(4), 369–375.
doi:10.1007/bf00275107
Apical application of silver nitrate (AgNO~ ;50 and 100 ~tg per plant) and silver thiosulphate anionic complex (Ag(S203)~-; STS; 25, 50 and 100 ~tg per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 ~tg per plant of AgNO3 was ineffective and 150 ~tg treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 ~tg STS. The induced male flowers produced pollen grains that germinated on
stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO3 in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.

This book chapter by the same authors of the Effect of Induced Polyplodiy, a few years later, more references and a bit updated with additional info. Also the whole book is great info about Cannabis, it is online for free by people that have it and posted it
Induction of Polyploidy and Its Effect on Cannabis sativa L.
Hakimeh Mansouri, Mahsa Bagheri
chapter in Cannabis sativa L. - Botany and Biotechnology pp 365-383
First Online: 24 May 2017 "book"
DOI: 10.1007/978-3-319-54564-6_17
Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants, and is an important mechanism of speciation. Polyploids can be formed in various ways. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits. We reported the production of putative tetraploid plants of Cannabis sativa L., with the ultimate aim of improving the medicinal and physiological traits of this widely distributed cultivated plant. The production of tetraploid plant was improved with colchicine at different concentrations and time through dropping method. Flow cytometry analysis was used to confirm the ploidy level. Morphologic, anatomic and biochemical characteristics were compared between tetraploid and diploid control plants. The results showed that 0.2% colchicine for 24 h was the most efficient for production of polyploid plants. The percentage of tetraploid plants and the survival rate were lowered by the increasing the treatment time. In addition, the leaf index and height of tetraploid plants exhibited a significant decrease compared to the diploid plants. The size of stomata on epidermis of leaves were larger in tetraploid plant compared to the diploid ones, in spite of the tetraploid plants have less stomata density. However, the amount of total chlorophyll and carotenoids were almost the same in both tetraploid and diploid plants. In addition, some differences were also observed in the cross section of stem of these plants from a descriptive structural point of view. Overall, the results introduced usage of the stomata parameters as an effective, fast and convenient method for detecting the tetraploid plants. We also investigated polyploidy effects on some primary and secondary metabolites. The results of biochemical analyzes showed that soluble sugars and total protein content increased significantly into mixoploid plants compared to tetraploid and diploid plants. Tetraploid plants had higher amount of total proteins compared with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabionol only in mixoploid plants but tetraploid plants had lower amounts of this substance in comparison with diploids. Our results suggest that tetraploidization was not useful for production of tetrahydrocannabinol for commercial use but mixoploids were found suitable

INFLUENCE OF CULTIVAR, EXPLANT SOURCE AND PLANT GROWTH REGULATOR ON CALLUS INDUCTION AND PLANT REGENERATION OF CANNABIS SATIVA L.
AURELIA S´ LUSARKIEWICZ-JARZINA, ALEKSANDRA PONITKA, AND ZYGMUNT KACZMAREK
https://pdfs.semanticscholar.org/c22...1560054937ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 145–151, 2005
The effects of different combinations of plant growth regulators (PGRs) on callus induction and plant regeneration were investigated in five cultivars of Cannabis sativa L. Callus was induced from different explant sources (young leaves, petioles, internodes, axillary buds) on MS basal medium with various concentrations of PGRs (2,4-D,
DICAMBA, KIN, NAA). The highest frequency of callus induction (avg. 82.7% of eight medium combinations) was exhibited by petiole explants of cv. Fibrimon-24. Plant regeneration was obtained from all studied cultivars. The highest number of plants was regenerated from callus tissue of petiole explants on MS medium containing DICAMBA.
A total of 46 plants (1.35% of callus) were regenerated: 16 (0.47%) from cv. Silesia petioles, 7 (0.20%) from cv. Novosadska petioles, 6 (0.18%) from cv. Fedrina-74 petioles, 12 (0.35%) from cv. Fibrimon-24 axillary buds, and 5 (0.15%) from cv. Juso 15 internodes. Significant improvement of hemp plant regeneration in vitro was achieved.

Modification of Growth and Sex Expression in Cannabis sativa by Aminoethoxyvinylglycine and Ethephon
H.Y.Mohan Ram, RinaSett
Zeitschrift für Pflanzenphysiologie
Volume 105, Issue 2, January 1982, Pages 165-172
DOI: 10.1016/S0044-328X(82)80008-1
Apical application of aminoethoxyvinylglycine (AVG) to female plants (5, 10, 25, 50 and 75 ?g per plant) of Cannabis sativa induced the formation of fertile male flowers on the newly formed primary lateral branches (PLBs). 1 ?g per plant was found to be ineffective and 100 ?g treatment proved inhibitory. In response to 5 to 50 ?g treatments the PLBs bore reduced male, intersexual and male flowers, whereas with 75 ?g they formed only male flowers. When AVG (25, 50 ?g) was applied to the shoot tips of male plants sprayed with ethephon (1920 mg · 1 -1), the feminization effect caused by the latter was markedly curtailed

Photosynthetic response of Cannabis sativa L., an important medicinal plant, to elevated levels of CO2
Suman Chandra & Hemant Lata & Ikhlas A. Khan & Mahmoud A. ElSohly
July 2011 Physiology and Molecular Biology of Plants 17(3):291-5
DOI: 10.1007/s12298-011-0066-6
The effect of elevated CO2 concentrations (545 and 700 ?mol mol?1) on gas exchange and stomatal response of four high ?9-THC yielding varieties of Cannabis sativa (HPM, K2, MX and W1) was studied to assess their response to the rising atmospheric CO2 concentration. In general, elevated CO2 concentration (700 ?mol mol?1) significantly (p<0.05) stimulated net photosynthesis (PN), water use efficiency (WUE) and internal CO2 concentration (Ci), and suppressed transpiration (E) and stomatal conductance (gs) as compared to the ambient CO2 concentration (390 ?mol mol?1) in all the varieties whereas, the effect of 545 ?mol mol?1 CO2 concentration was found insignificant (p<0.05) on these parameters in most of the cases. No significant changes (p<0.05) in the ratio of internal to the ambient CO2 concentration (Ci/Ca) was observed in these varieties under both the elevated CO2 concentrations (545 and 700 ?mol mol?1). An average increase of about 48 %, 45 %, 44 % and 38 % in PN and, about 177 %, 157 %, 191 % and 182 % in WUE was observed due to elevated CO2 (700 ?mol mol?1) as compared to ambient CO2 concentration in HPM, K2, MX and W1 varieties, respectively. The higher WUE under elevated CO2 conditions in Cannabis sativa, primarily because of decreased stomatal conductance and subsequently the transpiration rate, may enable this species to survive under expected harsh greenhouse effects including elevated CO2 concentration and drought conditions. The higher PN, WUE and nearly constant Ci/Ca ratio under elevated CO2
concentrations in this species reflect a close coordination between its stomatal and mesophyll functions

Possible role of ethylene and gibberellins in flower sex differentiation in Cannabis sativa [1974]
Mohan-Ram, H.Y.Jaiswal, V.S.
FIND DOI or LINK

Production of ?1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris
Futoshi Taura, Emi Dono, Supaart Sirikantaramas, Kohji Yoshimura, Yukihiro Shoyama, Satoshi Morimoto
Biochemical and Biophysical Research Communications 10/2007; 361(3):675-80.
DOI: 10.1016/j.bbrc.2007.07.079
Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported nativeenzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Not directly Cannabis related
Rapid Sex-typing of Asparagus for Male Hybrid Seed Production using n-Propyl N-(3,4-dichlorophenyl)carbamate (NPC)
M. Aneja, T.J. Gianfagna, S.A. Garrison, and E.F. Durner
HORTSCIENCE 34(6):1090–1094. 1999
doi: 10.21273/HORTSCI.34.6.1090
Precocious flowering can be induced in asparagus (Asparagus officinalis L.) seedlings with N-phenylcarbamate herbicides, such as n-propyl N-(3,4-dichlorophenyl) carbamate (NPC); however, only ?50% of the treated seeds produce flowering plants because these compounds inhibit germination and seedling emergence. We have improved the treatment method by determining the environmental conditions, timing, dose, and duration needed to maximize the percentage of germination, emergence, and flowering. Imbibing seeds in water for 5 days, and then treating germinated seeds with 0.4 mM NPC for 5 days after radicle emergence, with seedling aeration in the light, resulted in the production of flowering seedlings from >90% of the treated seeds. For freshly harvested seeds, in which germination rates are more variable than aged seeds, individual seedlings must be transferred to NPC within 1 day after radicle emergence to produce a high percentage of flowering plants. For seven male asparagus cultivars, chemical induction of flowering in seedlings with NPC produced a sex ratio similar to that of field-grown plants, demonstrating that NPC induces flowering without altering floral differentiation or sex expression. This method can be used for rapidly and accurately identifying the percentage of females in “male” cultivars.

Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology.
Supaart Sirikantaramas, Futoshi Taura, Satoshi Morimoto, Yukihiro
Shoyama
Current pharmaceutical biotechnology 09/2007; 8(4):237-43.
DOI: 10.2174/138920107781387456
Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate:eek:livetola te geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

Sex reversal in the female plants of Cannabis sativa by cobalt ion
July 1979 Proceedings of the Indian Academy of Sciences - Section A Part 3 Mathematical sciences 88(4):303-308
DOI: 10.1007/BF03046194
Apical application of cobalt chloride to female plants (50 or 100 ?g/ plant) ofCannabis sativa caused drying of the shoot tip and formation of axillary branches. The latter bore male, reduced male and intersexual flowers, in addition to female flowers. Pollen in the flowers of altered sex were viable.

XXI.—Studies on Flowering-Plant Growth and Organogenesis. I. Morphogenetic Effects of 2, 3, 5-Triiodobenzoic Acid on Cannabis sativa
January 1957 Volume 66, Issue 41957 , pp. 409-423
Proceedings of the Royal Society of Edinburgh (1844 - 1940),
J. Heslop-Harrison, Y. Heslop-Harrison
DOI: 10.1017/S0080455X0000059X
Not withstanding its reported effect upon the levels of native auxin in plant tissues, 2, 3, 5~triiodobenzoic acid (TIBA) does not influence sex expression in Cannabis sativa, a dioecious species in -which there is evidence that flower sexuality may be regulated by auxin levels in the neighbourhood of developing primordia. The total production of flowers is, however, increased in both sexes by TIBA, an effect possibly resulting from an alleviation of the correlative inhibition of lateral buds. Whilst hardly influencing morphogenetic activity at a cellular level at all, TIBA treatment leads to gross ontogenetic abnormalities, of which the common feature is a tendency for the elimination of localized growth and the substitution instead of generalized growth fields, resulting in the formation of infundibuliform and cylindrical foliar and floral structures. It seems probable that the bulk of the effects of TIBA in the plant may be due to an influence on auxin distribution, perhaps through interference with the mechanism of polar transport.

The hormonal control of sex differenyiation in dioecious plants of hemp (Cannabis sativa)
Elzbieta Galoch
Acta Societatis Botanicorum Poloniae Vol. XLVII, nr 1-2 1978
DOI: 10.5586/asbp.1978.013
The influence of GA3, IAA, ethrel, ABA and kinetin on sex expression in male and female plants of hemp (Cannabis sativa cultivar LKC SD) was investigated. The growth regulators were applied separately and in combinations on stem apices of plant determined sexually and having the first flower primordia. Gibberellic acid promoted masculinization, whereas IAA, ethylene and kinetin had a feminization effect on sex of hemp. Abscisic acid did not exert any direct effect on sex determination, it however acted antagnisticaly in relation to the effect exerted by GA3 and IAA. The results of combined application of IAA and ethrel with other growth regulators suggest, that the mechanism of action of auxin and ethylene in the control of sex expression in hemp is different. Auxins in this process cannot be regarded only as causing agents of ethylene production increase.

The influence of growth regulators absorbed by the root on sex expression in hemp plants.
Chailakhyan, M. K., & Khryanin, V. N.
Planta, 138(2), 181–184. (1978).
doi:10.1007/bf00391176
Application, through the root system, of growth regulators to hemp (Cannabis sativa L.) plants having ~3 pairs of visible leaves caused pronounced shifts of sex expression in the adult individuals. Treatment
with gibberellic acid (25 mg/1) resulted in more than 80% of the plants being male, i.e. having staminate flowers (controls, ca. 30%). Treatment with 6-benzylaminopurine and with indole-3-acetic acid (in either case, 15 mg/1) resulted in all plants being either female (pistillate flowers) or intersexes (bisexual flowers); treatment with abscisic acid (10 mg/1) had a similar but somewhat less pronounced effect.

The possible role of ethylene and gibberellins in flower sex expression of Cannabis sativa
in Proceedings of 8th International Conference on Plant Growth Substances (Tokyo: Hirokawa Publishing Co)
pp 987–996172
FIND DOI or LINK

The response of terpenoids to exogenous gibberellic acid in Cannabis sativa L. at vegetative stage.
Mansouri, H., Asrar, Z., & Amarowicz, R.
Acta Physiologiae Plantarum, 33(4), 1085–1091. (2010).
doi:10.1007/s11738-010-0636-1
In this study the influence of gibberellic acid (GA3) on plastidic and cytosolic terpenoids and on two key enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), for terpenoid biosynthesis was compared in vegetative cannabis plants. Treatment with GA3 resulted in a decrease of DXS activity in comparison with the control plants. The amount of chlorophylls a, b and total carotenoids declined when plants treated by GA3 in a concentration dependent manner. The a-tocopherol content of cannabis plants decreased in 50 lM GA3 treatment and increased in 100 lM GA3 treatment. Exogenous GA3 caused an increase in HMGR activity. Concomitant with this result, the amount of squalene and phytosterols increased with GA3 treatment. The amount of THC and CBD did not change at 50 lM GA3 treatment, but applying of 100 lM GA3 increased THC and CBD content in leaf plant in comparison with control plants. GA3 treatment declined number and percentage of monoterpenes in treated plants. Also the number of sesquiterpenes decreased in response to GA3 treatment but among the remainder of them, the amount of some sesquiterpenes decreased and some sesquiterpenes increased with GA3 treatment. Our results showed that GA3 treatment had opposite effect on primary terpenoid biosynthesis by the plastidic 2C-methylD-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways. But secondary terpenoids showed different response to GA3 treatment probably due to interference of two biosynthetic pathways in their formation.


IC Cannabis R&D


Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures
Imane Wahby, Juan Manuel Caba, Francisco Ligero
Journal of Plant Interactions 8(4):312-320 December 2013
DOI: 10.1080/17429145.2012.746399
Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed normal pattern of ?-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.

Biosynthesis of cannabinoids
Incorporation experiments with 13C?labeled glucoses
Volume 268, Issue 6 March 2001 Pages 1596-1604
doi: 10.1046/j.1432-1327.2001.02030.x
Monika Fellermeier, Wolfgang Eisenreich, Adelbert Bacher, Meinhart H. Zenk
The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1?13C]glucose or [U?13C6]glucose. 13C?labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10?terpenoid moiety is biosynthesized entirely or predominantly (>?98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide?type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.

Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway
Futoshi Taura, Shinji Tanaka, Chiho Taguchi, Tomohide Fukamizu, Hiroyuki Tanaka,Yukihiro Shoyama, Satoshi Morimoto
FEBS Letters 583 (2009) 2061–2066
doi:10.1016/j.febslet.2009.05.024
Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally
expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

Coming soon to a lab near you? Genetically modified cannabis
Narure 559, 162 (2018)
doi: 10.1038/d41586-018-05659-z

Not Cannabis specific
Cryopreservation of Tropical Plant Germplasm. Current Research Progress and Application.
Edited by F. Englemann and H. Takagi
Expl Agric. Volume 37, Issue 1 January 2001 , pp. 125-134
DOI: 10.1017/S0014479701261055
Cryopreservation, i.e. the storage of biological material at ultra-low temperature, usually that of liquid nitrogen (–196°C), is the only method currently available to ensure the safe and cost-effective long-term conservation of genetic resources of species that have recalcitrant seeds or are vegetatively propagated. Dramatic progress has been made over the last 10 years in this area with the development of cryopreservation techniques for well over 100 plant species. Cryopreservation protocols are increasingly becoming available for routine application in genebanks. However, much of the work to date has been done on temperate species, with research on tropical and subtropical species lagging behind. This is of particular concern given the large number of tropical species that are either vegetatively propagated or that produce recalcitrant seeds. Both JIRCAS and IPGRI are heavily involved in cryopreservation research. In the framework of its Visiting Fellowship Programme, JIRCAS has carried out a project specifically to develop techniques for the long-term preservation of vegetatively propagated crop germplasm. During the project, visiting scientists from developing countries have developed cryopreservation protocols for selected tropical crops. For more than 15 years, IPGRI and its predecessor IBPGR has supported cryopreservation research in collaboration with partners in Asia, the Pacific and Oceania, Africa, the Americas and Europe. As a result of their experience in the field of cryopreservation, JIRCAS and IPGRI, in October 1998, jointly organized an international workshop to assess the current state of the science, to explore cryopreservation applications and to examine outstanding problems. The focus of the workshop was on the use of cryopreservation to conserve the germplasm of tropical plant species. An additional objective was to identify priority areas for collaborative research, technology development, transfer and application. The workshop was attended by a large number of cryopreservation experts from both developing and developed countries who presented their latest research results and contributed to the discussions. This publication of the proceedings of the workshop thus presents a comprehensive overview of current knowledge concerning the biological and physical mechanisms involved in cryopreservation, and the status of the development of protocols for new species and their application in genebanks. We trust that it will help to stimulate further collaborative research and thus contribute to the wider application of cryopreservation for the safe long-term and cost-effective conservation of genetic resources of tropical species.

Development of indole sulfonamides as cannabinoid receptor negative allosteric modulators
Iain R. Greiga, Gemma L. Bailliea, Mostafa Abdelrahmanc, Laurent Trembleauc, Ruth A. Rossa (2016)
DOI: 10.1016/j.bmcl.2016.08.018
Existing CB1 negative allosteric modulators (NAMs) fall into a limited range of structural classes. In spite of the theoretical potential of CB1 NAMs, published in vivo studies have generally not been able to demonstrate the expected therapeutically-relevant CB1-mediated effects. Thus, a greater range of molecular tools are required to allow definitive elucidation of the effects of CB1 allosteric modulation. In this study, we show a novel series of indole sulfonamides. Compounds 5e and 6c (ABD1075) had potencies of 4 and 3 nM respectively, and showed good oral exposure and CNS penetration, making them highly versatile tools for investigating the therapeutic potential of allosteric modulation of the cannabinoid system.

Does unit-dose packaging influence understanding of serving size information for cannabis edibles?
SAMANTHA GOODMAN, DAVID HAMMOND
https://www.lisbonaddictions.eu/lisb...%20Goodman.pdf POSTER
DOI: 10.15288/jsad.2020.81.173 Article
BACKGROUND Edible cannabis products have increased in popularity, particularly in jurisdictions that have legalized non-medical cannabis. Rates of adverse events from cannabis edibles have also increased, in part due to difficulties identifying and titrating THC levels in edible products. The current study tested whether packaging cannabis in separate units (‘unit-dose’ packaging) can enhance consumer understanding of serving sizes.
METHODS An experimental task was conducted as part of the 2018 International Cannabis Policy Study online survey, recruited from the Nielsen Global Insights Consumer Panel. A total of 26,894 respondents aged 16-65 years from Canada and the USA were randomly assigned to view an image of cannabis brownie packaged according to 1 of 3 experimental conditions: 1. multi-serving edible (‘control’ condition); 2. single-serving edibles; 3. separately packaged single-serving edibles (‘unit-dose packaging’). Participants were asked to identify a standard serving based on information on the product label. Logistic regression was used to test the influence of packaging condition on the likelihood of a correct response. Models were adjusted for key sociodemographic covariates (age, sex, visible minority status, education level, jurisdiction, self-reported THC knowledge, and past 12-month use of cannabis edibles). Adjusted odds ratios (AORs) are reported.
SUMMARY OF FINDINGS Compared to the multi-serving edible ‘control’ condition in which a whole brownie had to be divided into servings, participants were significantly more likely to identify the correct serving size in the single-serving edible condition (55.3%) and the ‘unit-dose packaging’ condition (54.3%).
CONCLUSIONS Packaging cannabis brownies such that each product ‘unit’ contained one dose of THC enhanced consumers’ ability to identify how much of a product constitutes a standard serving, or ‘dose’. Packaging edibles as individual doses eliminates the need for mental math and could reduce the risk of accidental over-ingestion of cannabis.

Drying of cannabis—state of the practices and future needs.
Challa, S. K. R., Misra, N. N., & Martynenko, A.
Drying Technology, 1–10. (2020).
doi:10.1080/07373937.2020.1752230
Cannabis is an important source of several bio molecules that possess medical applications. With the growing interest in cannabis processing in Canada, there is a need for innovation in this sector. At present, the cannabis industry relies on slow and inefficient drying practices that result in poor quality product. This review examines the state of the practices and challenges in cannabis drying, and the recent developments. Additionally, some prospective low temperature drying technologies of significance to cannabis industry are discussed. Non-isothermal, microwave-vacuum, electrohydrodynamic, radio-frequency, and freeze drying have been identified as potential candidates for industrial drying of cannabis.

Effect of induced polyploidy on some biochemical parameters in Cannabis sativa L.
Mahsa Bagheri & Hakimeh Mansouri
Appl Biochem Biotechnol
DOI 10.1007/s12010-014-1435-8
This study is aimed at testing the efficiency of colchicine on inducing polyploidy in Cannabis sativa L. and investigation of effects of polyploidy induction on some primary and secondary metabolites. Shoot tips were treated with three different concentrations of colchicine (0, 0.1, 0.2 %w/v) for 24 or 48 h. The biggest proportion of the almost coplanar tetraploids (43.33 %) and mixoploids (13.33 %) was obtained from the 24-h treatment in 0.2 and 0.1 %w/v, respectively. Colchicine with 0.2 % concentration and 48 h duration was more destructive than 24 h. The ploidy levels were screened with flow cytometry. The biochemical analyses showed that reducing sugars, soluble sugars, total protein, and total flavonoids increased significantly in mixoploid plants compared with tetraploid anddiploid plants. Tetraploid plants had a higher amount of total proteins, total flavonoids, and starch in comparison with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabinol in mixoploid plants only, but tetraploid plants had lower amounts of this substance in comparison with diploids. Also, we found such changes in protein concentration in electrophoresis analysis. In overall, our study suggests that tetraploidization could not be useful to produce tetrahydrocannabinol for commercial use, and in this case, mixoploids are more suitable.

NOT CANNABIS SPECIFIC
Effects of ultraviolet-B radiation on the growth and yield of crop plants.
Teramura, A. H.
Physiologia Plantarum, 58(3), 415–427. (1983).
doi:10.1111/j.1399-3054.1983.tb04203.x
This paper reviews growth chamber, greenhouse, and field studies on the effects of ultraviolet-B (UV-B, between 280 and 320 nm) radiation on agricultural crop plants. Our understanding of the physiological effects of UV-B radiation comes primarily from growth chamber studies, where UV-B is artificially supplied via filtered lamps. Both photosystems 1 and II, as well as carboxylating enzymes, are sensitive to UV-B radiation. Ultraviolet-B radiaiion also affects stomatal resistance, chlorophyll concentration, soluble leaf proteins, lipids, and carbohydrate pools. In general, the effects
of UV-B radiation are accentuated by the low levels of visible radiation typically found inside growth chambers. Ultraviolet-B radiation has also been shown to affect anatomical and morphological plant characteristics. Commonly observed UV-B induced changes include plant stunting, reductions in leaf area and total biomass, and alterations in the pattern of biomass partitioning into various plant organs. In sensitive plants, evidence of cell and tissue damage often appears on the upper leaf epidermis as bronzing, glazing, and chlorosis. Epidermal transmission in the UV region decreases in irradiated leaves. This decrease is primarily associated with a stimulation in flavonoid biosynthesis and is thought to be a protective, screening response to the deleterious effects of UV-B. A considerable degree of variability exists in sensitivity to UV-B radiation between different species. Approximately 30% of the species tested were resistant, another 20% were extremely sensitive, and the remainder were of intermediate sensitivity, in terms of reductions in total dry weight. In addition to this sizable interspecific variability, there appears to be a similarly wide intraspecific variability in UV-B response. The effects of UV-B radiation on crop yield have only been examined in a limited number of field studies, with ambient levels of UV-B radiation being supplemented with fluorescent sun lamps. Due to various deficiencies, all these field experiments to date have only limited utility for assessing the potential impact of enhanced levels of UV-B on crop productivity.

Effects of ultraviolet-B radiation on the growth, physiology and cannabinoid production of Cannabis sativa L
J. Lydon
January 1986 Thesis (Ph. D.)--University of Maryland, College Park, 1985. Vita. Includes bibliographical references (leaves 78-95).
GET DOI OR LINK
The concentration of cannabinoids in Cannabis sativa L. is correlated with high ultraviolet-B (UV-B) radiation environments. ..delta../sup 9/-Tetrahydrocannabinolic acid and cannabidiolic acid, both major secondary products of C. sativa, absorb UV-B radiation and may function as solar screens. The object of this study was to test the effects of UV-B radiation on the physiology and cannabinoid production of C. sativa. Drug and fiber-type C. sativa were irradiated with three levels of UV-B radiation for 40 days in greenhouse experiments. Physiological measurements on leaf tissues were made by infra-red gas analysis. Drug and fiber-type control plants had similar CO/sub 2/ assimilation rates from 26 to 32/sup 0/C. Drug-type control plant had higher dark respiration rates and stomatal conductances than fiber-type control plants. The concentration of ..delta../sup 9/-THC, but not of other cannabinoids) in both vegetative and reproductive tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation. The increased level of ..delta../sup 9/-THC found in leaves after irradiation may account for the physiological and morphological insensitivity to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidoil (CBD). Resin stripped form fresh fiber-type floral tissue by sonication was spotted on filter paper and irradiated continuously for 7 days. Cannabidiol (CBD) gradually decreased when irradiated but ..delta../sup 9/-THC and cannabichromene did not.

Endogenous synthesis of pyrethrins by cannabis
Adrian Devitt-Lee, Douglas Smith, David Chen, Kevin McKernan, Simon Groves, Ciaran McCarthy
BioRxiv Preprint
doi.org/10.1101/169417
Pyrethrins are a class of natural terpenoid pesticides produced by Tanacetum cinerariifolium, commonly known as chrysanthemum. Here we present evidence that cannabis may be able to produce pyrethrins endogenously. Flower from a cannabis plant grown in a closed hydroponic environment contained 2.48 parts per million pyrethrin I by weight. A comparison of the genetics of T. cinerariifolium and Cannabis demonstrates Cannabis homologues of the genes that contribute to pyrethrins production in T. cinerariifolium. This provides a plausible pathway for the biosynthesis of pyrethrins in cannabis. Although preliminary, these data indicate a potentially significant confounding variable in both cannabis research and regulations on allowable pyrethrins residues in cannabis products.

FLUSHING TRIAL IMPACT OF DIFFERENT FLUSHING TIMES ON QUALITY AND TASTE IN CANNABIS SATIVA L.
https://www.rxgreentechnologies.com/...rialReport.pdf
• Rx Green Technologies evaluated the effects of different flushing times on chemical profile, flavor, and smoking characteristics in Cannabis flower.
• Flushing periods of 14, 10, 7 and 0 days were imposed on Cherry Diesel.
• No differences were detected between flush treatments for yield, potency, or terpenes.
• Analysis of mineral content of leaves indicated small changes in content of iron and zinc.
• Taste test panelists tended to prefer flower flushed for 0 days.

Not Cannabis Specific
How Do Flowers Know When to Bloom?
https://www.esalq.usp.br/lepse/imgs/...n-to-Bloom.pdf
How do flowers know when to bloom? The secret is in their genes.
Flower petals breaking through the snow, an early hint of spring's arrival, hides a very complex genetic process behind its floral façade. Flowers know when to bloom because of a gene named Apetala1. A lone master gene, Apetala1 triggers the reproductive development of a plant, telling it when it's time to start blossoming. Yes, a single gene is all it takes to make a plant start producing flowers. A plant blooming with flowers has an active Apetala1, while a plant carrying inactive Apetala1 genes has very few flowers, if any, with leafy shoots growing in place of blossoms.

*Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex.
Mohan Ram, H. Y., & Sett, R. (1982).
Theoretical and Applied Genetics, 62(4), 369–375.
doi:10.1007/bf00275107
Apical application of silver nitrate (AgNO~ ;50 and 100 ~tg per plant) and silver thiosulphate anionic complex (Ag(S203)~-; STS; 25, 50 and 100 ~tg per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 ~tg per plant of AgNO3 was ineffective and 150 ~tg treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 ~tg STS. The induced male flowers produced pollen grains that germinated on
stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO3 in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.

This book chapter by the same authors of the Effect of Induced Polyplodiy, a few years later, more references and a bit updated with additional info. Also the whole book is great info about Cannabis, it is online for free by people that have it and posted it.
Induction of Polyploidy and Its Effect on Cannabis sativa L.
Hakimeh Mansouri, Mahsa Bagheri
chapter in Cannabis sativa L. - Botany and Biotechnology pp 365-383
First Online: 24 May 2017 "book"
DOI: 10.1007/s12010-014-1435-8

Induction of mutants in Cannabis Sativa L. by ? radiation
Fuochi, P., Lavalle, M., Di Candilo, M., Ranalli, P.
Proceedings of INC 02. International Nuclear Conference 2002: Global Trends and Perspectives, Seminar I: Agriculture and Biosciences
https://inis.iaea.org/search/search...._q=RN:34030228
Find DOI or LINK
A description is given of the mutations induced in a well known cultivar of fibre hemp belonging to the species Cannabis sativa in order to obtain new phenotypic characters that could unequivocally differentiate it from drug hemp. Two new dioecious hemp cultivars, characterized by clear morphological markers and low (?0.2%)?-9-tetrahydrocannabinol (THC) content, have been obtained by mutagenesis of seeds irradiated with 60Co ? rays(optimum dose 300-350 Gy) and selection of the segregant progenies. These new cultivars, named red petiole (exhibiting anthocyanin leaf petioles) and yellow apex (exhibiting yellow distal leaflets), have been compared with varieties C.S., carmagnola and fibranova by growing them in three different areas (centre-north and south) of Italy. These agronomical trials highlighted the good performance of both new cultivars as for yield in dry matter and in fibre, not significantly different from the results of carmagnola. Besides, the new cultivars showed THC contents (0.09 and 0.17%) lower than the threshold set by the E.U. (0.2%).

MARIJUANA AND CANNABINOID RESEARCH: METHODS AND PROTOCOLS
Ed. by Emmanuel S. Onaivi
https://epdf.pub/marijuana-and-canna...protocols.html
Marijuana has remained one of the most widely used and abused drugs in the world. Research on the biological basis of the effects of marijuana, and therefore its usefulness as medicine, may have been hampered by several decades of irrational prejudice and also by the lack of specific molecular tools and technology. But the discovery of specific genes coding for cannabinoid receptors (CBrs) that are activated by smoking marijuana, and that the human body and brain makes its own marijuana-like substances called endocannabinoids that also activate CBrs, has transformed marijuana–cannabinoid research into mainstream science. An overwhelming body of scientific evidence now indicates the existence of an elaborate, and previously unknown but ubiquitous, endocannabinoid physiological control system (EPCS) whose fundamental role in human development, health, and disease is unfolding. This system appears to exert a powerful modulatory action on retrograde signaling associated with cannabinoid inhibition of synaptic transmission. The promiscuous action and distribution of CBrs in most biological systems provides the EPCS limitless signaling capabilities of crosstalk within, and possibly between, receptor families that may explain the numerous behavioral effects associated with smoking marijuana. Advances in marijuana–cannabinoid research have already resolved the issue that marijuana use can be addicting in vulnerable individuals and that a missense in human fatty acid amide hydrolase, which inactivates endocannabinoids (anandamide) and related lipids, is associated with drug and alcohol dependence problems. These and other remarkable advances in understanding the biological actions of marijuana and cannabinoids have provided a much richer than previously appreciated cannabinoid genomics and raised a number of critical issues on the molecular mechanisms of cannabinoid-induced behavioral and biochemical alterations. Although these advances have enhanced our understanding of the molecular mechanisms associated with the behavioral effects of marijuana use, the molecular identity of other cannabinoid receptor subtypes and transporters (if any), along with the growing number of endocannabinoids, will allow specific therapeutic targeting of different components of the EPCS in health and disease

MetaCyc Pathway: cannabinoid biosynthesis
https://biocyc.org/META/NEW-IMAGE?ty...detail-level=1
https://biocyc.org/META/NEW-IMAGE?ty...detail-level=2
https://biocyc.org/META/NEW-IMAGE?ty...detail-level=3
The cannabinoid biosynthesis pathway remains a source of debate. Most cannabinoids are derived from the condensation product of olivetolate with geranylpyrophosphate. The exact route of synthesis of olivetolic acid remains to be confirmed. To date, two potential routes have been proposed: the first occurs through the action of a polyketide synthase, termed olivetolic acid synthase, catalyzing the condensation of n-hexanoyl-CoA with three molecules of malonyl-CoA to yield a tetraketide that would form olivetolic acid via a Claisen condensation reaction [Raharjo04]. No such enzyme has yet been identified. Alternatively, a recent NMR study have supported the existence of route for olivetolic acid formation though the condensation of acetyl-CoA with five molecules of malonyl-CoA [Shoyama75, Fellermeier01]. The next step in the pathway involves the prenylation of olivetolic acid with geranylpyrophosphate (GPP). A hemp enzyme ( NPP:eek:livetolate neryltransferase / GPP:eek:livetolate geranyltransferase) has been identified that can perform this reaction. The product of the reaction is the central branch-point intermediate: cannabigerolate (CBGA). The same enzyme was also shown to be able to use nerylpyrophosphate instead of GPP as prenyl chain although it prefers GPP. Enzymes have been identified that can catalyze the cyclization of CBGA, each forming a different cannabinoid cyclization product. Each of these enzymes was shown to be able to use cannabinerolate instead of CBGA, leading to the same cyclization product as CBGA [Morimoto98, Taura96]. The last step (decarboxylation) of this pathway occurs spontaneously in a non-enzymatic reaction during storage or smoking but is also found in vivo [Baker81, Bosy00].

Not Cannabis Specific
New Technology for Separating Resin Powder and Fiberglass Powder from Fiberglass–Resin Powder of Waste Printed Circuit Boards.
Li, J., Gao, B., & Xu, Z.
Environmental Science & Technology, 48(9), 5171–5178.(2014).
doi:10.1021/es405679n
New recycling technologies have been developed lately to enhance the value of the fiberglass powder?resin powder fraction (FRP) from waste printed circuit boards. The definite aim of the present paper is to present some novel methods that use the image forces for the separation of the resin powder and fiberglass powder generated from FRP during the corona electrostatic separating process. The particle shape charactization and particle trajectory simulation were performed on samples of mixed non-metallic particles. The simulation results pointed out that particles of resin powder and particles of fiberglass powder had different detach trajectories at the conditions of the same size and certain device parameters. An experiment carried out using a corona electrostatic separator validated the possibility of sorting these particles based on the differences in their shape characteristics. The differences in the physical properties of the different types of particles provided the technical basis for the development of electrostatic separation technologies for the recycling industry

Olivetol as product of a polyketide synthase in Cannabis sativa L.
Raharjo, T. J., Chang, W.-T., Choi, Y. H., Peltenburg-Looman, A. M. ., & Verpoorte, R.
Plant Science, 166(2), 381–385.(2004).
doi:10.1016/j.plantsci.2003.09.027
A polyketide synthase (PKS) was suggested to catalyze the first step of cannabinoid biosynthesis, leading to olivetolic acid. An activity of a PKS was detected in the protein extract of Cannabis sativa flowering top. The enzyme converts one molecule of n-hexanoyl-CoA and three molecules of malonyl-CoA to olivetol. The product was identified by its UV-spectrum, mass spectrometry analysis and comparison with reference compound. The activity of the enzyme was also found in the upper leaves, but the activity occurring there is lesser than in the one occurring in the flowers. The activity of chalcone synthase (CHS), another PKS enzyme, was also found in the protein extract.

Patty's Industrial Hygiene, Cannabis
Robert N. Phalen
DOI: 10.1992/0471435139.hyg142
Onlinelibrary.wiley.com/doi/epdf/10,1002/0471435139.hyg142
This chapter is designed to address the needs and interests of practicing industrial hygienists who may work within or with the cannabis industry. It is also intended to assist those working with law enforcement and government agencies in the protection of officers and agents who may enter these facilities for inspection or legal purposes. The focus of the chapter is to provide sufficient background information on the production of cannabis products from the Cannabis plant, as well as the recognition, evaluation, and control of various known biological, chemical, and physical hazards associated within the industry. It is not a review of all aspects of the cannabis industry, nor does it cover significant aspects of consumer use, public health, or legal ramifications. It is a starting point for industrial hygienists working to protect workers in and around this ancient but currently expanding industry.
This chapter is divided into four main sections addressing the production of cannabis, recognition of hazards associated with its production, evaluation of those hazards, and lastly common control measures. The cited literature provides valuable resources for learning more about the recognition, evaluation, and control of hazards associated with cannabis production.

Photosynthetic response of Cannabis sativa L., an important medicinal plant, to elevated levels of CO2
Suman Chandra & Hemant Lata & Ikhlas A. Khan & Mahmoud A. ElSohly
DOI 10.1007/s12298-011-0066-6

Polyketide synthases in Cannabis sativa L.
Isvett Josefina Flores Sanchez THESIS
https://openaccess.leidenuniv.nl/bit...pdf?sequence=2
Cannabis sativa L. is an annual dioecious plant from Central Asia.
Cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans
are some of the secondary metabolites present in C. sativa. Earlier
reviews focused on isolation and identification of more than 480 chemical
compounds; this review deals with the biosynthesis of the secondary
metabolites present in this plant. Cannabinoid biosynthesis and some
closely related pathways that involve the same precursors are discussed.

Polyploidization for the Genetic Improvement of Cannabis sativa
April 2019 Frontiers in Plant Science 10
Jessica L. Parsons, Tracey James, Shelley R Hepworth, Sara Lauretta Martin
DOI: 10.3389/fpls.2019.00476
Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square metre. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square metre and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis and biocontrol.
Cannabis sativa L. is a diploid species, cultivated throughout the ages as a source of fiber, food, and secondary metabolites with therapeutic and recreational properties. Polyploidization is considered as a valuable tool in the genetic improvement of crop plants. Although this method has been used in hemp-type Cannabis, it has never been applied to drug-type strains. Here, we describe the development of tetraploid drug-type Cannabis lines and test whether this transformation alters yield or the profile of important secondary metabolites: ?9-tetrahydrocannabinol (THC), cannabidiol (CBD), or terpenes. The mitotic spindle inhibitor oryzalin was used to induce polyploids in a THC/CBD balanced drug-type strain of Cannabis sativa. Cultured axillary bud explants were exposed to a range of oryzalin concentrations for 24 h. Flow cytometry was used to assess the ploidy of regenerated shoots. Treatment with 20-40 ?M oryzalin produced the highest number of tetraploids. Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30% larger and about half as dense compared to diploids. Trichome density was increased by about 40% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9% increase in CBD that was significant in buds. No significant increase in yield of dried bud or THC content was observed. This research lays important groundwork for the breeding and development of new Cannabis strains with diverse chemical profiles, of benefit to medical and recreational users.

Possible role of ethylene and gibberellins in flower sex differentiation in Cannabis sativa
[1974] Mohan-Ram, H.Y.Jaiswal, V.S.
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Possible Role of Ultraviolet Radiation in Evolution of Cannabis Chemotypes

DAVID W. PATE
Economic Botany October 1983, 37:396
DOI:10.1007/BF02904200
The damaging effects of UV-B radiation have apparently affected the amounts
of ultraviolet-absorbing secondary compounds in some plants. A similar role.for
A 9 tetrahydrocannabinol may explain the high levels of this compound in Cannabis from areas of intense ambient UV-B. Further research is needed to determine whether UV-B radiation serves only as a selection pressure or if UV-B-induced stress may also directly stimulate production.

Production of ?1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris
Futoshi Taura, Emi Dono, Supaart Sirikantaramas, Kohji Yoshimura, Yukihiro Shoyama, Satoshi Morimoto
Biochemical and Biophysical Research Communications 10/2007; 361(3):675-80.
DOI: 10.1016/j.bbrc.2007.07.079
Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported nativeenzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology.
Supaart Sirikantaramas, Futoshi Taura, Satoshi Morimoto, Yukihiro
Shoyama
Current pharmaceutical biotechnology 09/2007; 8(4):237-43.
DOI: 10.2174/138920107781387456
Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate:eek:livetola te geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

Regeneration of shoots from immature and mature inflorescences of Cannabis sativa
Kevin F. Piunno, Gregory Golenia, Ekaterina A. Boudko, Cassandra Downey, A. Maxwell P. Jones
Canadian Journal of Plant Science Can. J. Plant Sci April 2019
DOI: 10.1139/CJPS-2018-0308
Cannabis sativa is usually clonally propagated from plants in the vegetative phase. However, phenotypic traits such as yield and chemical composition can only be assessed in unfertilized plants reaching the end of their life cycle and there are no peer reviewed methods to propagate flowering plants. In this study, immature (3 cultivars) and mature (1 cultivar) floral explants were cultured on thidiazuron and shoot development was observed from both immature and mature explants. This provides the first report of micropropagation from floral tissues in C. sativa and will enable plants to be clonally propagated up to the date of harvest.

Removal of a cannabis metabolite from human urine in microbial fuel cells generating electricity
Murat Ozdemira, Vildan Enisoglu-Atalayb, Hakan Bermek, Selma Ozilhana, Nevzat Tarhane, Tunc Catalf,
Bioresource Technology Reports 5 (2019) 121–126
Electricity was generated directly from synthetic or human urine containing
11?nor?9?carboxy??9?tetrahydro cannabinol in air-cathode microbial fuel cells. Synthetic urine contained sodium acetate as carbon source, while actual human urine was used neat, without further supplements. Microbial fuel cells were capable of degrading more than 60% of the cannabis metabolite from human urine, while generating electricity. With synthetic urine, voltage generation reached 0.33 V, however the addition of 300 ng/mL of 11?nor?9?carboxy??9?tetrahydro cannabinol decreased the peak voltage to 0.27 V. This loss in power generation was nevertheless reversible when 11?nor?9?carboxy??9?tetrahydro cannabinol was removed from the media.
Human urine containing 170 ng/mL 11?nor?9?carboxy??9-tetrahydrocannabinol produced 0.23 V of continuous electricity in the microbial fuel cells. The mechanism for degradation of cannabis metabolites in microbial fuel cells was discussed according to the results of the computational studies. In conclusion, wastewaters contaminated with a urine-based cannabis major metabolite could be treated in microbial fuel cells along with voltage generation as added-value.

Not Cannabis specific
Selective Chlorophyll Removal Method to “Degreen” Botanical Extracts.
Kim, S. B., Bisson, J., Friesen, J. B., Pauli, G. F., & Simmler, C.
Journal of Natural Products.(2020).
doi:10.1021/acs.jnatprod.0c00005
Chlorophylls are present in all extracts from the aerial parts of green plant materials. Chlorophylls may act as in vitro bioassay nuisance compounds, possibly preventing the reproducibility and accurate measurement of readouts due to their UV/vis absorbance, fluorescence properties, and tendency to precipitate in aqueous media. Despite the diversity of methods used traditionally to remove chlorophylls, details about their mode of operation, specificity, and reproducibility are scarce. Herein, we report a selective and efficient 45 min liquid?liquid/countercurrent chlorophyll cleanup method using Centrifugal Partition Chromatography (CPC) with a solvent system composed of hexanes?EtOAc? MeOH?water (5:5:5:5, v/v) in elution-extrusion mode. The broader utility of the method was assessed with four different extracts prepared from three well-characterized plant materials: Epimedium sagittatum (leaves), Senna alexandrina (leaves), and Trifolium pratense (aerial parts). The reproducibility of the method, the selectivity of the chlorophyll removal, as well as the preservation of the phytochemical integrity of the resulting chlorophyll-free (“degreened”) extracts were evaluated using HPTLC, UHPLC-UV, 1 H NMR spectroscopy, and LC-MS as orthogonal phytochemical methods. The cleanup process adequately preserves the metabolomic diversity as well as the integrity of the original extracts. This method was found to be sufficiently rapid for the “degreening” of botanical extracts in higher-throughput sample preparation for further biological screening.

https://www.medicinalgenomics.com/cannmed2016/
You can download each presentation, check out at least: RYAN C. LYNCH. MEDICINAL GENOMICS, KEVIN MCKERNAN COURTAGEN LIFE SCIENCES, MARK A. LEWIS PRESIDENT OF NAPRO RESEARCH, ETHAN RUSSO MEDICAL DIRECTOR PHYTECS, JOSH WURZER SC LABORATORIES, RAPHAEL MECHOULAM

SEQUENCING OF THREE MALE CANNABIS GENOMES AND DEVELOPMENT OF MULTIPLEX QPCR ASSAYS FOR RAPID MALE SEX DETERMINATION
Kevin McKernan, Vasisht Tadigotla, Yvonne Helbert, Jessica Spangler, Lei Zhang, Douglas Smith
https://www.medicinalgenomics.com/wp...ation_sbmt.pdf
Male associated DNA Cannabis (MADC2) markers have previously been described by Mandolino et al1 . These markers were reported to target the MADC2 repeat and generate many bands with gel electrophoresis of which the male cannabis plant delivered a unique band. While these markers are reported to accurately detect male plants, the use of gels and visual inspection of banding patterns could be improved with more scalable quantitative PCR (qPCR) methods. Towards this end, we cloned and sequenced these bands to over 1000x coverage using next generation sequencing. We discovered highly variable MADC2 sequences complicating qPCR assay design

Sex reversal in the female plants of Camutbis sativa by cobalt ion
H Y MOHAN RAM and RINA SETT
Proc. Indian Acad. Sci., Vol. 88 B, Part II, Number 4, July 1979, pp. 303-308,
https://www.ias.ac.in/public/Volumes.../0303-0308.pdf
DOI:10.1007/BF03046194
Apical application of cobalt chloride to female plants (50 or 100/~g/ plant) of Cannabis sativa caused drying of the shoot tip and formation of axillary
branches. The latter bore male, reduced male and intersexual flowers, in addition to female flowers. Pollen in the flowers of altered sex were viable.

Not directly Cannabis related
Super Moisture Absorbent Gels for Sustainable Agriculture via Atmospheric Water Irrigation.
Zhou, X., Zhang, P., Zhao, F., & Yu, G.
ACS Materials Letters, 1419–1422.
doi:10.1021/acsmaterialslett.0c00439 (2020).
An atmospheric water irrigation system based on super moisture absorbent gels (SMAG) is developed for sustainable agriculture technology. The SMAG-soil can harvest water from the air and provide water to the plants upon solar heating regardless of the local accessibility to liquid water resources. As a benefit of this solar-powered atmospheric water irrigation system, agriculture can become geographically and hydrologically independent. Thus, crop planting in underdeveloped and drought areas can be liberated from the long-distance water and power supplies.

The arbuscular mycorrhizal fungus Glomus mosseae induces growth and metal accumulation changes in Cannabis sativa L.
Citterio, S., Prato, N., Fumagalli, P., Aina, R., Massa, N., Santagostino, A., Berta, G.
Chemosphere, 59(1), 21–29 (2005).
doi:10.1016/j.chemosphere.2004.10.
Hemp was grown in the presence and absence of 100 lg g1 Cd and Ni and 300 lg g1 Cr(VI), and inoculated or not with the arbuscular mycorrhizal fungus Glomus mosseae. In our experimental condition, hemp growth was reduced in inoculated plants and the reduction was related to the degree of mycorrhization. The percentage of mycorrhizal colonisation was 42% and 9% in plants grown in non-contaminated and contaminated soil, suggesting a significant negative effect of high metal concentrations on plant infection by G. mosseae. Soil pH, metal bioavailability and plant metal uptake were not influenced by mycorrhization. The organ metal concentrations were not statistically different between inoculated and non-inoculated plants, apart from Ni which concentration was significantly higher in stem and leaf of inoculated plants grown in contaminated soil. The distribution of absorbed metals inside plant was related to the soil heavy metal concentrations: in plant grown in non-contaminated soil the greater part of absorbed Cr and Ni was found in shoots and no significant difference was determined between inoculated and non-inoculated plants. On the contrary, plants grown in artificially contaminated soil accumulated most metal in root organ. In this soil, mycorrhization significantly enhanced the translocation of all the three metals from root to shoot. The possibility to increase metal accumulation in shoot is very interesting for phytoextraction purpose, since most high producing biomass plants, such as non-mycorrhized hemp, retain most heavy metals in roots, limiting their application.

3.1 The Plant Cannabis sativa L
Pawe? Rodziewicz, Oliver Kayser
IN:
Medicinal, Aromatic and Stimulant Plants. Handbook of Plant Breeding. (2020).
Novak, J., & Blüthner, W.-D. (Eds.).
doi:10.1007/978-3-030-38792-1
Cannabis sativa L. is a remarkable herbaceous species with a long and interesting history of its use for medicinal, recreational, and religious purposes (Kalant 2001; Zuardi 2006). Although this species is best known for its unique pharmacological properties, it is also a source of high-quality fber and valuable seed oil (Andre et al. 2016). Cannabis sp. originates from Central Asia (Kazakhstan/Mongolia), but it grows in most of the regions of the world. It is also one of the earliest cultivated
plants with frst documented evidence of its use tracing back to ancient civilizations of China, Egypt, Mesopotamia, and Summers (Long et al. 2017; Russo 2007). Roughly, Cannabis sativa L. grows in the subtropical areas; and in northern and southern temperate zones, Cannabis indica Lam. is present. In the past, the plant’s healing properties were mostly recognized among South Asian, Middle Eastern, and African cultures, whereas the Europeans and East Asians utilized this species mainly to produce strong fbers and seeds for nutritional purposes (Clarke and Watson 2007). As early as 2600 BC, cannabis preparations were recommended by Chinese Emperor Huang Ti as a remedy for cramps and rheumatic and menstrual pain. The frst written evidence of the medicinal use of cannabis recorded in Chinese pharmacopeia comes from the ffteenth century BC (Brand and Zhao 2017). In Western medicine, cannabis was introduced and popularized in the mid of nineteenth century by Irish physician William Brooke O’Shaughnessy. During his fellowship in India, he validated the folk use of cannabis – one of the sacred plants of that country – and also found new therapeutic applications for the extracted cannabis resin (O’Shaughnessy 1843). Shortly after, the herb was adopted as medicine in British and other European pharmacopeias, as well as in the US Dispensary in the form of dried fowers, extracts, and tinctures (Fig. 3.1), where it remained for over 100 years (Piomelli 2000). However, in the mid of twentieth century and starting in the USA, the use of cannabis fowers became controversial, and ultimately, its possession and cultivation for various purposes (including medicinal use) was penalized in most countries. After the US government introduced the Marihuana Tax Act in 1937 (Marihuana Tax Act 1937), cultivation of cannabis became economically unjustifed, and therefore, also its use as a medicine greatly diminished. Ultimately, and against the advice of the American Medical Association, the prohibition led to the classifcation of Cannabis sativa L.-derived products as containing substances of high potential for abuse and without any therapeutic effects (Aggarwal et al. 2009). The factors that played the main role in establishing the US federal cannabis prohibition policy were mostly related to the end of prohibition, ethnic and social problems in the USA, and the expansion of polymer and synthetic fber industry at that time.

NOT CANNABIS SPECIFIC, but also works with Cannabis
Thidiazuron Induces High-frequency Shoot Regenerationin Intact Seedlings of Pea (Pisum sativum), Chickpea (Cicer arietinum) and Lentil (Lens culinaris)
Kamal A. Malik and Praveen K. Saxena
Aust. J. Plant Physiol., 1992, 19, 731-40
DOI:10.1071/pp9920731
Axenic seedling cultures of chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.) and garden pea (Pisum sativum L.) were established by culturing mature seeds on Murashige and Skoog medium (MS) supplemented with thidiazuron (TDZ). Of various cytokinins or compounds with cytokinin-like activity (Kinetin, TDZ, Zeatin) tested for inducing shoot formation in pea seeds cultures, TDZ was found to be most effective. Pea seedlings exhibited a unique pattern of shoot formation which was accomplished in two distinct phases. Multiple shoots developed within a week, from the nodal and basal regions of the primary epicotyl in a medium that contained 5-50 I(M TDZ. When these seedlings were exposed for a prolonged time period (3-4 weeks), to the same medium, numerous shoot buds emerged de novo from the base and/or from the upper part of multiple shoots. These shoots had no apparent vascular connection with parent tissues. The inductive capability of TDZ was then tested in several other genotypes of Pisum sativum and two other large-seeded grain legumes, Cicer arietinum, and Lens culinaris. In Cicer arietinum, and Lens culinaris, multiple shoots developed after 1 week of seed culture on media that contained 1-50 I(M TDZ. However, de novo differentiation of shoot buds occurred in cultures exposed to TDZ for 4-6 weeks, only from nodal and subjacent areas. Secondary shoot formation occurred frequently in all of the species tested. Developing shoots were able to form roots and eventually whole plants on a modified MS medium containing 2.5 I(M NAA. No genotypic difference for morphogenesis was observed.

USE OF NATURAL ZEOLITE (CLINOPTILOLITE) IN AGRICULTURE
Ersin Polat, Mehmet Karaca, Halil Demir and A. Naci Onus
Journal of Fruit and Ornamental Plant Research vol. 12, 2004
https://www.kmizeolite.com/wp-conten...-Agricultu.pdf
Zeolites have been increasingly used in various application areas such as industry, agriculture, environmental protection, and even medicine. Although, there are no certain figures on the total amount of these minerals the world, some countries e.g. Cuba, USA, Russia, Japan, Italy, South Africa, Hungary and Bulgaria, have important reserves and production potentials. According to reports of 2001, the total consumption of zeolites was 3.5 million tons of which 18% came from their natural resources and the rest from synthetics. More than forty naturally occurring zeolites were reported by different research groups, and clinoptilolite, erionite, chabazite, heulandite, mordenite, stilbit and philipsite are the most well-known. The most common for agricultural applications is clinoptilolite since it has high absorption, cation exchange, catalysis and dehydration capacities. Zeolites are, therefore, used as a promoter for better plant growth by improving the value of fertilizers; retaining valuable nitrogen and improving the quality of resulting manures and sludge. They can also be used as a molecular sieve or filter medium.

UVB lights to increase terpenes and secondary metabolites
Peter Barber
GreenTech Amsterdam Rai 2018
https://issuu.com/amsterdamrai/docs/peter_barber.pptx

UV-B RADIATION EFFECTS ON PHOTOSYNTHESIS, GROWTH and CANNABINOID PRODUCTION OF TWO Cannabis sativa CHEMOTYPES.
Lydon J., Teramura, A. H., & Coffman, C. B.
(1987).
Photochemistry and Photobiology, 46(2), 201–206. (1987).
doi:10.1111/j.1751-1097.1987.tb04757.x
The effects of UV-B radiation on photosynthesis, growth and cannabinoid production of two greenhouse-grown C. sativa chemotypes (drug and fiber) were assessed. Terminal meristems of vegetative and reproductive tissues were irradiated for 40 days at a daily dose of 0, 6.7 or 13.4 kJ m-2 biologically effective UV-B radiation. Infrared gas analysis was used to measure the physiological response of mature leaves, whereas gas-liquid chromatography was used to determine the concentration of cannabinoids in leaf and floral tissue.
There were no significant physiological or morphological differences among UV-B treatments in either drug- or fiber-type plants. The concentration of A'-tetrahydrocannabinol (A"-THC), but not of other cannabinoids, in both leaf and floral tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation.
The increased levels of A'-THC in leaves after irradiation may account for the physiological and morphological tolerance to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidiol (a cannabinoid with UV-B absorptive characteristics similar to A' THC). Thus the contribution of cannabinoids as selective UV-B filters in C. Sutiva is equivocal.

Variation of Cannabis with reference to stem quality for paper pulp production
EPM. de Meijer
Industrial Crops and Products 3 (1994) 201-211
doi:10.1016/0926-6690(94)90067-1
Ca. 160 Cannabis accessions were evaluated for stem quality characteristics. Stems from field trials in two years were analysed. The mass fractions of woody core, bark and primary and secondary bark fibres in the stem dry weight, and the dimensions of the wood fibres were considered most relevant for the evaluation of Cannabis germplasm as a raw material for paper pulp.
The accession means for the woody core fraction ranged from 50% in fibre cultivars to 78% in wild populations, drug strains and fibre landraces. Consequently, the bark tissue, being the complementary stem fraction, ranged from 50% down to 22%. The ranges of the accession means were 8 to 27% and 0 to 14% for the fractions of primary and secondary bark fibres, respectively. The total bark fibre fraction ranged from 9 to 34%. Fibre cultivars had strongly increased fractions of primary and secondary bark fibres in comparison to wild populations, drug strains and fibre landraces. The levels of the assessed stem fractions were stable over years. Significant differences in wood fibre length among accessions were only detected in one of the two trials. The accession means in this trial ranged from 433 to 613 pm. For wood fibre width the accession means ranged from 24 to 37 km and from 25 to 41 ,um in the two trials, respectively. The stability of wood fibre dimensions over years was low. Within accessions, the wood fibres of male plants were usually somewhat shorter and wider than those of females. Apart from this difference between sexes there was little variation for wood fibre dimensions among
individual plants within accessions. Breeding for improved woody core quality is considered not very promising. The best way for genetic improvement of hemp pulping quality is continuous selection for increased bark fibre content, which implies a reduction of the woody core fraction
 
Last edited:

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Cannabis Seeds


A multivariate analysis of morphological divergence of “seeds” (achenes) among ruderal, fibre, oilseed, dioecious/monoecious and marijuana variants of Cannabis sativa L.
Steve G. U. Naraine, Ernest Small, Andrew E. Laursen, Lesley G. Campbell
Genetic Resources and Crop Evolution Oct 2019
DOI: 10.1007/s10722-019-00848-9
Cannabis sativa has been domesticated for stem fibre and oilseed (the two classes are both low in the euphoric cannabinoid THC and called “hemp”), and marijuana (high in THC), and also occurs as weedy, ruderal plants. Achenes (“seeds”) from herbarium collections representative of these classes were assessed for morphological characters and pericarp resistance to fracture. In contrast to ruderal plants, domesticated plants (both hemp and marijuana) possessed achenes that were significantly longer, heavier, covered with a less adherent perianth, and lacking a pronounced basal attenuation. All of these characteristics reflect traits that are advantageous in domesticated plants and are consistent with the “domestication syndrome” found in propagules of other crops. Marijuana achenes, in comparison with hemp achenes, tended to be about 26% shorter and about 32 shades darker (on a 256-bit grayscale). Achenes of fibre cultivars proved to be about 19% longer than the achenes of oilseed cultivars. Achenes of dioecious oilseed cultivars proved to be about 6% longer than the achenes of monoecious oilseed cultivars. The pericarps of hemp seeds were about 26% and about 15% more resistant to fracture than those of ruderal and marijuana plants, respectively.

NOT CANNABIS SPECIFIC
A new regulator of seed size control in Arabidopsis identified by a genome-wide association study
Diqiu Ren, Xuncheng Wang, Mei Yang, Li Yang, Guangming He and Xing Wang Deng
New Phytologist 222(2) December 2018
doi:10.1111/nph.15642
Organ size in plants is controlled by the interaction between genotype and the
environment. Seed size, an important agronomic trait, largely determines yield and is an important focus of research. However, the genetic components underpinning natural variation of seed size in undomesticated species remain largely unidentified.
? Here we report a genome wide association study (GWAS) of seed size in Arabidopsis thaliana, which identified 38 significantly associated loci, including one locus associated with CYCB1;4. Natural variations in CYCB1;4, which encodes a cyclin protein involved in the cell cycle, significantly influence seed size in A. thaliana .
? Transgenic plants with enhanced CYCB1;4 expression show normal development, exhibit increased seed size due to an accelerated cell cycle progression, and tend to produce higher yields. In contrast, cycb1;4 mutants have smaller seeds, and the effect is especially pronounced in a large-seed accession. The temporal and spatial expression pattern of CYCB1;4 suggests that this gene may function in both maternal tissues and zygotic tissues to
coordinate the final size of seeds.
? Taken together, our results provide genetic insight into natural variation in seed size in Arabidopsis. Moreover, CYCB1;4 homologs in other crops may have great potential as targets for efforts aimed at yield improvement.


A Review of Hemp As Food and Nutritional Supplement.
Cerino, P., Buonerba, C., Cannazza, G., D’Auria, J., Ottoni, E., Fulgione, A., … Gallo, A.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2020.0001
The term ‘‘hemp’’ refers to Cannabis sativa cultivars grown for industrial purposes that are characterized by lower levels of tetrahydrocannabinol (THC), the active principle responsible for Cannabis psychotropic effects. Hemp is an extraordinary crop, with enormous social and economic value, since it can be used to produce food, textiles, clothing, biodegradable plastics, paper, paint, biofuel, and animal feed, as well as lighting oil. Various parts of the hemp plant represent a valuable source of food and ingredients for nutritional supplements. While hemp inflorescence is rich in nonpsychoactive, yet biologically active cannabinoids, such as cannabidiol (CBD), which exerts potent anxiolytic, spasmolytic, as well as anticonvulsant effects, hempseed has a pleasant nutty taste and represents a valuable source of essential amino acids and fatty acids, minerals, vitamins, and fibers. In addition, hempseed oil is a source of healthy polyunsaturated fatty acids, and hemp sprouts are rich in antioxidants. This review article aims to provide a comprehensive outlook from a multidisciplinary perspective on the scientific evidence supporting hemp beneficial properties when consumed as food or supplement. Marketing of hemp-derived products is subjected to diversified and complex regulations worldwide for several reasons, including the fact that CBD is also the active principal of pharmaceutical agents and that regulatory bodies in some cases ban Cannabis inflorescence regardless of its THC content. Some key regulatory aspects of such a complex scenario are also analyzed and discussed in this review article.

Advanced Characterization of Hemp Flour (Cannabis sativa L.) from Dacia Secuieni and Zenit Varieties, Compared to Wheat Flour
Iulian-Eugen Rusu, Romina Alina Marc (Vlaic), Crina Carmen Murejsan , Andruta Elena Murejsan,
Miu¸ta Rafila Filip, Bogdan-Mihai Onica, Kádár Balázs Csaba , Ersilia Alexa , Lidia Szanto and Sevasti¸ta Muste
Plants 2021, 10,
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-01237.pdf
The advanced characterization of flour from hemp seeds (edible fruits of Cannabis sativa L.) from the Dacia Secuieni and Zenit varieties, compared to wheat flour, was studied in this research. The aim was to present the characterization of 2 varieties, out of the 70 accepted in Europe, for human consumption. The varieties selected from hemp meet the THC level requirement (0.3 or 0.2% of the dry weight of the reproductive part of the female flowering plant) in seeds. Hemp flour was obtained by grinding. The flour samples were evaluated for physicochemical parameters (moisture, crude protein, lipids, ash, crude fiber), the content of micro and macro elements, fatty acids, amino acids, and carbohydrates. The total proteins in hemp flour are found in larger quantities by over 35% compared to wheat flour, and the lipids reach the threshold of 28%. The amount of mineral substances exceeds 3% in hemp flour, and the fibers exceed 26%, compared to 0.61% for wheat flour. The predominant mineral substances were K, Ca, Mg, p, Fe, and Mn. The predominant fatty acids were the unsaturated ones, predominantly being linoleic acid, followed by oleic and gamma-linoleic acid. In the case of amino acids, the highest amount is found in glutamic acid for hemp flours. As for carbohydrates, sucrose is found in the largest amount, followed by glucose and fructose. In conclusion, hemp flours have superior non-traditional characteristics to wheat flour, being a potential raw material for fortifying food or using them as such, having beneficial effects of consumption on the proper functioning of the human body

Alternations in Cholesterol and Fatty Acids Composition in Egg Yolk of Rhode Island Red x Fyoumi Hens Fed with Hemp Seeds (Cannabis sativaL.).
Shahid, S., Chand, N., Khan, R. U., Suhail, S. M., & Khan, N. A.
Journal of Chemistry, 2015, 1–6. (2015).
doi:10.1155/2015/362936
The present study was designed to evaluate the influence of hemp seed (HS) supplementation on egg yolk cholesterol and fatty acid composition in laying hens. Sixty hens (Rhode Island Red x Fyoumi) were evenly distributed into four groups (three replicates per group) at the peak production (34 weeks). HS was included into the ration at the level of 0.0 (HS-0), 15 (HS-15), 20 (HS-20), and 25% (HS-25) and continued the supplementation for consecutively three weeks. At the end of the experiment, three eggs per replicate were randomly collected and analyzed for egg yolk fatty acids and cholesterol profile. The statistical analysis of the result revealed that supplementation of HS-25 significantly (?? < 0.05) decreased egg yolk total cholesterol, myristic (C14:0), palmitic (C16:0), and stearic (C18:0). Similarly, total as well as individual monounsaturated fatty acids decreased significantly (?? < 0.05) while total and individual polyunsaturated fatty acids increased significantly in the HS-25. In addition, total omega-3 and omega-6 increased significantly in the HS-25 group. From the present result, we concluded that addition of HS at the rate of 25% to the diet of laying hens augmented the cholesterol and fatty acids profile in egg yolk.

Not Cannabis specific
Arabidopsis Paired Amphipathic Helix Proteins SNL1 and SNL2 Redundantly Regulate Primary Seed Dormancy via Abscisic Acid–Ethylene Antagonism Mediated by Histone Deacetylation
Zhi Wang, Hong Cao, Yongzhen Sun, Xiaoying Li, Fengying Chen, Annaick Carles, Yong Li, Meng Ding, Cun Zhang, Xin Deng, Wim J.J. Soppe, Yong-Xiu Liu
The Plant Cell, 25(1), 149–166.
doi:10.1105/tpc.112.108191
Histone (de)acetylation is a highly conserved chromatin modification that is vital for development and growth. In this study, we identified a role in seed dormancy for two members of the histone deacetylation complex in Arabidopsis thaliana, SIN3- LIKE1 (SNL1) and SNL2. The double mutant snl1 snl2 shows reduced dormancy and hypersensitivity to the histone deacetylase inhibitors trichostatin A and diallyl disulfide compared with the wild type. SNL1 interacts with HISTONE DEACETYLASE19 in vitro and in planta, and loss-of-function mutants of SNL1 and SNL2 show increased acetylation levels of histone 3 lysine 9/18 (H3K9/18) and H3K14. Moreover, SNL1 and SNL2 regulate key genes involved in the ethylene and abscisic acid (ABA) pathways by decreasing their histone acetylation levels. Taken together, we showed that SNL1 and SNL2 regulate seed dormancy by mediating the ABA-ethylene antagonism in Arabidopsis. SNL1 and SNL2 could represent a crosslink point of the ABA and ethylene pathways in the regulation of seed dormancy

Biochemical Characteristics of Hemp Seeds from Various Regions of Russia
S. V. Grigor’ev, T. V. Shelenga, V. S. Baturin, and Yu. V. Sarana
Russian Agricultural Sciences, 2010, Vol. 36, No. 4, pp. 262–264.
DOI: 10.3103/S1068367410040105
The composition and content of fatty acids in oil of ruderal hemp seeds collected in the Volga, Southern, Urals, Siberian, and Far Eastern federal districts are studied. The content of tetrahydrocannabinol is assessed.

Cannabis sativa (Hemp) Seeds, D9-Tetrahydrocannabinol, and Potential Overdose
Yi Yang, Melissa M. Lewis, Angelica M. Bello, Ewa Wasilewski, Hance A. Clarke, and Lakshmi P. Kotra
Cannabis and Cannabinoid Research Volume 2.1, 2017
DOI: 10.1089/can.2017.0040
Introduction: Cannabis sativa (hemp) seeds are popular for their high nutrient content, and strict regulations are in place to limit the amount of potentially harmful phytocannabinoids, especially D9-tetrahydrocannabinol (D9-THC). In Canada, this limit is 10 lg of D9-THC per gram of hemp seeds (10 ppm), and other jurisdictions in the world follow similar guidelines.
Materials and Methods: We investigated three different brands of consumer-grade hemp seeds using four different procedures to extract phytocannabinoids, and quantified total D9-THC and cannabidiol (CBD).
Discussion: We discovered that D9-THC concentrations in these hemp seeds could be as high as 1250% of the legal limit, and the amount of phytocannabinoids depended on the extraction procedure employed, Soxhlet
extraction being the most efficient across all three brands of seeds. D9-THC and CBD exhibited significant variations in their estimated concentrations even from the same brand, reflecting the inhomogeneous nature of seeds and variability due to the extraction method, but almost in all cases, D9-THC concentrations were higher than the legal limit. These quantities of total D9-THC may reach as high as 3.8mg per gram of hemp seeds, if one were consuming a 30-g daily recommended amount of hemp seeds, and is a cause for concern for potential toxicity. It is not clear if these high quantities of D9-THC are due to contamination of the seeds, or any other reason.
Conclusion: Careful consideration of the extraction method is very important for the measurement of cannabinoids in hemp seeds.

Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes
Leonardo Anabalon, Jaime Solano, Francisco Encina-Montoya, Marco Bustos, Alejandra Figueroa, and David Gangitano
Cannabis and Cannabinoid Research Volume X, Number X, June 2021
DOI: 10.1089/can.2020.0168
Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market.
Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation.
Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2C on average.
Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.

Cannabis Varieties Can Be Distinguished by Achene Shape Using Geometric Morphometrics
Federico Márquez, Mariana Lozada, Yanina L. Idaszkin, Rolando González-José, and Gregorio Bigatti
Cannabis and Cannabinoid Research 2021
DOI: 10.1089/can.2020.0172
https://www.liebertpub.com/doi/pdf/1.../can.2020.0172
Introduction: Cannabis plant uses are widespread across human cultures. The current tendency is to classify Cannabis varieties into chemovars upon their chemical fingerprint, mainly cannabinoids and terpenoids content. The identification of chemovars has important medical implications; however, their pharmacological characterization is costly and time consuming. The goal of this study was to assess whether achene shape variation could be related to Cannabis varieties with contrasting cannabinoid concentrations, as a first approach to chemovar identification.
Methods: We used two-dimensional geometric morphometrics (GM) of the achenes and multivariate statistical analysis. We used achenes from 􀂦ve varieties, two from Type II chemotype (expressing both tetrahydrocannabinol [THC] and cannabidiol [CBD]), two Type I (THC-only), and one Type III (CBD-only).
Results: The achenes from the different chemotypes were clearly distinguishable. No significant differences between
varieties from the same chemotype were observed. The varieties with high THC concentration (Type I) were rounded and bigger, whereas achene from varieties containing only CBD (Type III) had a slender shape with smaller size.
Conclusion: Achene shape variation is a potential biomarker of cannabinoid content in the plant flowers. Further studies are needed to confirm the suitability of GM methods for high-throughput screening of Cannabis cultivars, including larger diversity of varieties, and taking into account growth conditions, which can also in􀂧uence plant chemical fingerprint.


Cannßavins from hemp sprouts, a novel cannabinoid-free hemp food product, target microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase
Oliver Werz, Julia Seegers, Anja Maria Schaible, Christina Weinigel,
Dagmar Barz, Andreas Koeberle, Gianna Allegrone, Federica
Pollastro, Lorenzo Zampieri, Gianpaolo Grassi, Giovanni Appendino
PharmaNutrition 2 (2014) 53-60
DOI: 10.1016/j.phanu.2014.05.001
Hemp seeds are of great nutritional value, containing all essential amino acids and fatty acids in sufficient amount and ratio to meet the dietary human demand. Hemp seeds do not contain cannabinoids, and, because of their high contents of ?-3 fatty acids, are enjoying a growing popularity as a super-food to beneficially affect chronic inflammation. Seeds also lack the typical phenolics of hemp leaves and inflorescences, but we found that sprouting, while not triggering the production of cannabinoids, could nevertheless induce the production of the anti-inflammatory prenylflavonoids cannflavins A and B. This effect was especially marked in Ermo, a cannabinoid-free variety of
Cannabis sativa L.. Microsomal prostaglandin E2 synthase (mPGES-1) and 5-lipoxygenase (5-LO) were identified as the molecular targets of cannflavins A and B, solving an almost three-decade old uncertainty on the mechanism of their the anti-inflammatory activity. No change on the fatty acid profile was observed during sprouting, and the presence of lipophilic flavonoids combines with the high concentration of ?-3 essential acids to qualify sprouts from Ermo as a novel anti-inflammatory hemp food product worth considering for mass production and commercial development

Characterization of bioactive compounds in defatted hempseed (Cannabis sativaL.) by UHPLC-HRMS/MS and anti-inflammatory activity in primary human monocytes.
Rea Martinez, J., Montserrat-de la Paz, S., De la Puerta, R., Garcia-Gimenez, M. D., & Fernandez-Anche, A.
Food & Function. (2020).
doi:10.1039/d0fo00066c
Hempseed (Cannabis sativa L.) has beneficial impact on human health mainly because of its wide variability of bioactive compounds. However, many of them are not fully characterized yet. In this work, hempseed was defatted and through a bio-guided studied, two fractions (F03 and F05) with the highest content of phenols, flavonoids and antioxidant capacity were selected. Fractions were chemically analyzed by UHPLC HRMS/MS. The anti-inflammatory capacities of these compounds were evaluated on human monocytes using flow cytometry, RT-qPCR and Elisa procedures. A high amount of phenolic compounds were identified, with the major compound being: N-trans-caffeoyltyramine (6.36 mg/g in F05 and 1.28 mg/g in F03). Both, F03 and F05 significantly reduced the inflammatory competence of LPS- treated human primary monocytes, decreasing TNF-? and IL-6 gene expression and secretion. These findings indicate that in the defatted fraction of the hempseed there are a wide number of compounds with beneficial potential to prevent and treat inflammatory disorders, as well as other processes caused by oxidative stress.

Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity
Jae Kwon Kim, Hee-Young Heo, SeonJu Park, Haheon Kim, Jeong Ju Oh, Eun-Hwa Sohn, Se-Hui Jung, and Kooyeon Lee
ACS Omega 2021, 6, 47, 31945–31954 Publication Date:November 18, 2021
DOI: 10.1021/acsomega.1c04727
https://pubs.acs.org/doi/pdf/10.1021/acsomega.1c04727
Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α- MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 μM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-transcaffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skinwhitening agent

COMPARISON OF PROTOCOLS FOR DNA EXTRACTION FROM CANNABIS SATIVA SEEDS
Salvador SOLER, Alicia SIFRES, Ernesto R LLOSA, Matìas LLAMAS, Santiago VILANOVA, Jaime PROHENS
BULLETIN OF UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA. HORTICULTURE Vol 70, No 1 (2013)
https://riunet.upv.es/bitstream/hand...=1&isAllowed=y
DOI: 10.15835/buasvmcn-hort:9255
Cannabis sativa is an important crop cultivated both for industrial (var. sativa) and medicinal and recreational (var. indica) purposes. Cultivation of var. indica plants is generally forbidden and this difficults genetic studies. An alternative is the extraction of DNA from seed embryos. In order to develop an efficient protocol for DNA extraction of C. sativa seeds we have tested six DNA extraction methods in seeds and leaves of a C. sativa var. sativa accession. We found that the best protocol is the CTAB-modified with phenol:chlorophorm:isoamyl washing, which allowed a large quantity of high quality DNA per seed. This method was tested in seeds of three C. sativa var. indica accessions and seven SSR markers tested could be amplified successfully. In summary, we have developed a highly efficient method for DNA extraction of individual seeds of C. sativa. This method will be useful for genetic studies in this species.

Conversion of a low protein hemp seed meal into a functional protein concentrate through enzymatic digestion of fibre coupled with membrane ultrafiltration
Sunday A. Malomo, Rotimi E. Aluko
Innovative Food Science & Emerging Technologies Volume 31, October 2015, Pages 151-159
doi.org/10.1016/j.ifset.2015.08.004
Industrial hemp seed meal (HPM) containing ~37% protein content was digested with carbohydrases and a phytase. The digest was then passed through a 10 kDa ultrafiltration membrane and the retentate freezedried as the membrane protein concentrate (mHPC), which had 74% protein content. Protein digestibility (89%) was significantly (P b0.05) higher than the ~85% obtained for HPM, traditional isoelectric protein isolate (iHPI), and a commercial protein concentrate (cHPC). The mHPC also had significantly (P b0.05) higher protein solubility than other hemp products in the pH 3–9 range with a minimum of 76% at pH 4.0. In contrast the HPM and iHPI had b25%maximum protein solubility. Foaming capacitywas highest formHPC (55–98%) when compared to 10–70% for the other protein products. However, mHPC and cHPC emulsions had bigger oil droplet sizes (4.5–15.5 ?m) when compared with b1 ?m for HPM and iHPI emulsions. Industrial relevance: Hemp seed oil remains the most valuable product of the hemp seed industry but the proteins are also becoming very popular due to the high arginine content. Arginine is perceived as a heart-healthy amino acid because it serves as a precursor of the vasodilatory agent, nitric oxide. The traditional method of protein isolation involves isoelectric precipitation, which damages protein functionality and reduces performance as an ingredient below the required level for high quality food products manufacture. Therefore, in this researchwork, a novel, industrially scalable method that uses food-grade enzymes to digest most of the polysaccharides in a defatted low protein hemp seed meal was developed. The digested polysaccharide fragments were then removed by ultrafiltration/diafiltration to leave behind a protein product with twice the original protein content of the starting raw material. Most importantly, the work confirmed that the membrane-isolated protein possessed superior functionality, especially protein solubility and digestibility in comparison with existing similar hemp seed protein products. Since the membrane ultrafiltration setup is available on a commercial scale, work could be adapted by the hemp seed processing industry to manufacture a new line of high protein products with desirable functionality in food systems.
However, spray-drying must be used instead of freeze-drying in order to make the process economically feasible.

Determination of melatonin content of diferent varieties of hemp (Cannabis sativa L.) by liquid chromatography tandem mass spectrometry
Gianna Allegrone · Federica Razzano · Federica Pollastro1 · Gianpaolo Grassi
SN Appl. Sci. 1, 720 (2019).
Doi: 10.1007/s42452-019-0759-y
Melatonin (N-acetyl-5-methoxytryptamine), was first isolated in the bovine pineal gland, and then was discovered in bacteria, algae and higher plants. Melatonin concentrations in plants differ from species to species, among varieties within the same species and also within different organs or tissues of a given plant as roots, leaves, fruits, flowers, and seeds. Although the presence of melatonin in plants seems to be a universal trait, there is still lack of information on its occurrence in several plants, in particular in hemp (Cannabis sativa L.). The aim of this study was to develop a method to quantitate melatonin in the aerial parts of the plant, constituted of leaves and flower heads, and in the seeds, of four different hemp varieties using liquid chromatography–tandem mass spectrometry (LC–ESI/MS/MS). A sample preparation procedure with methanol extraction followed by solid-phase clean-up protocol was set up for melatonin extraction and a LC–ESI/MS/MS method in single reaction monitoring (SRM) was used for its determination. Chromatographic separation was achieved on a C18 column using a gradient elution with acetonitrile–buffer ammonium formate/formic acid system. The developed method, validated as per International Conference on Harmonization guidelines, was successfully applied to analyze hemp samples varieties belonging to fiber-type or drug-type chemotypes. The melatonin was found to accumulate highly in seeds in the range 13.43–30.40 ng g?1 while the content in the aerial parts was assessed in the range 1.16–4.85 ng g?1. No correlation was found between the melatonin levels detected in aerial parts and seeds in each hemp variety and the concentration of specific

Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa
Aleksei Sorokin, Narendra Singh Yadav, Daniel Gaudet and Igor Kovalchuk
Bio-protocol 11(01): e3875. 2021
DOI:10.21769/BioProtoc.3875
https://www.researchgate.net/publica...annabis_sativa
Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H2O2) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H2O2 solution of different concentrations; 1% H2O2 solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it suitable for tissue culture and other sensitive applications.
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Effect of Dietary Hemp Seed on Oxidative Status in Sows during Late Gestation and Lactation and Their Offspring.
Palade, L. M., Habeanu, M., Marin, D. E., Chedea, V. S., Pistol, G. C., Grosu, I. A., … Taranu, I.
Animals, 9(4), 194. (2019).
doi:10.3390/ani9040194
This study shows the antioxidant effect of a dietary hemp seed diet rich in ?-6 polyunsaturated fatty acid (PUFA) on oxidative status in sows during late gestation and lactation and their offspring. Ten pregnant sows were divided into two groups and fed either a control diet (CD) or a hemp diet (HD) containing 2% hemp seed meal for a period of 10 days before farrowing and 5% throughout the lactation period (21 d). After farrowing, 16 of their resulting piglets were divided into two groups: control group CD (eight piglets derived from control sows) and HD group (eight piglets derived from HD sows), respectively. Blood collected from sows and piglets at day 1, 7 and 21 was used for the measurement of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), glutathione (GPx)), nitric oxide production (NO), lipid peroxidation (thiobarbituric acid reactive substances—TBARS), reactive oxygen species (ROS) generation and total antioxidant capacity (TAC) in plasma. The results showed a significant improvement in the oxidative status of sows fed HD throughout lactation compared with CD. Similarly, in piglets, HD positively influenced the activities of antioxidant enzymes, TAC and NO levels and significantly decreased lipid peroxidation in plasma until weaning, in comparison with the CD grou

Effect of feeding hemp seed and hemp seed oil on laying hen performance and egg yolk fatty acid content: Evidence of their safety and efficacy for laying hen diets.
Gakhar, N., Goldberg, E., Jing, M., Gibson, R., & House, J. D.
Poultry Science, 91(3), 701–711. (2012).
doi:10.3382/ps.2011-01825
Forty-eight 19-wk-old Bovan White laying hens were fed 1 of 5 diets containing either hemp seed (HS) or hemp seed oil (HO). The level of HO was 4, 8, or 12%, whereas the level was 10 or 20% for the HS. A set of 8 birds fed wheat-, barley-, and corn oil-based diets served as the control. Performance was monitored over 12 wk. Average hen-day egg production was not affected upon feeding of either HS or HO diets. Egg weight was higher than that of the controls for hens consuming the 20% HS diet (P < 0.05). Feed intake was lower than that of the controls for birds consuming the 4% HO diet but similar across other treatments. Final BW were not affected by diet, with the exception of being lower than that of the controls (P < 0.05) in hens consuming the 12% HO diet. The total egg yolk n-3 fatty acid content increased linearly (P < 0.05) with increasing dietary a-linolenic acid provision with the HSor HO-based diets. A quadratic response (P < 0.05) was observed for docosahexaenoic acid levels in egg yolk in response to increasing dietary a-linolenic acid supply. The expression of hepatic fatty acid desaturase 1 and 2, key genes for the desaturation of long-chain polyunsaturated fatty acids, was significantly decreased (50-60% of controls; P < 0.05) as a result of feeding HS or HO diets. Based on the results from the current study, the inclusion of the hemp products HS or HO in the diets of laying hens up to a maximum level of 20 and 12%, respectively, does not adversely effect the performance of laying hens and leads to the enrichment of the n-3 fatty acid content of eggs.

Effect of heating on the digestibility of isolated hempseed (Cannabis sativa L.) protein and bioactivity of its pepsin-pancreatin digests.
Lin, Y., Pangloli, P., Meng, X., & Dia, V. P.
Food Chemistry,.(2020).
doi:10.1016/j.foodchem.2020.126198
The objective was to investigate the effects of heat pretreatment and simulated gastrointestinal digestion on potential antioxidant, anticancer and anti-inflammatory activities of hempseed (Cannabis sativa L.) proteins. Unheated isolated hempseed protein (IHP) and its heated counterparts (100 °C, 15 min and 30 min, termed as HP15D and HP30D) were hydrolyzed sequentially with pepsin and pancreatin before and analyzed for digestibility and bioactivity (antioxidant, anti-proliferative and anti- inflammatory properties). Heat pretreatment lead to an increase of low molecular weight proteins and degree of hydrolysis, and decrease of concentration of soluble protein, which means heat pretreated can significantly improve the digestibility of IHP. Pepsin-pancreatin digests released from heat pretreated IHP possessed less antioxidant, antiproliferative and anti-inflammatory properties than digests from unheated IHP. In conclusion, heat pre-treatment improved the digestibility of IHP but the resulting digests from heated IHP had lower bioactivity.

Effect of malting on nutritional and antioxidant properties of the seeds of two industrial hemp (Cannabis sativa L.) cultivars.
Barbara Farinon a Lara Costantini a Romina Molinari a Giacomo Di Matteo b Stefania Garzoli b Serena Ferri c Brunella Ceccantoni c Luisa Mannina b Nicolò Merendino
Food Chemistry Volume 370, 15 February 2022, 131348
DOI: 10.1016/j.foodchem.2021.131348
The impact of malting on antioxidant, nutritional, and antinutritional features of two industrial hemp cultivars was investigated. The seeds were steeped (5 h; RT), germinated (3-days; 24 °C), and kilned at different temperatures (6 h; 50 °C or 70 °C). The following determinations were performed on malted and unmalted samples: total phenolic content, polyphenol profile, total antioxidant capacity, tocopherol composition, proximate analysis, fatty acids profile, trypsin inhibitors and phytate content. The results showed that malting increased the protein content up to 9%, without affecting the fat amount, and the fatty acids profile. Total phenolic content, tocopherol profile and total antioxidant capacity were also improved. 70 °C kilning temperature resulted effective to reduce the trypsin inhibitors (up to −27%), increase the reducing power and the level of N-trans-caffeoyltyramine and cannabisin A. Based on this, malting using 3 germination days and 70 °C as kilning temperature could be considered suitable transformation process for improving hempseeds quality.
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Effect of seed moisture content and storage temperature on seed longevity of hemp (Cannabis sativa)
S S PARIHAR, M DADLANI, S K LAL, V A TONAPI, P C NAUTIYAL and SUDIPTA BASU
December 2014 Indian Journal of Agricultural Sciences https://www.academia.edu/25507089/Ef...annabis_sativa
Hemp (Cannabis sativa L.) is one of the earliest domesticated plants grown for its protein and oil rich seed, fiber and psychoactive substances and it is one of the earliest known medicinal plants in human history. Studies were conducted on seed germinability (germination test) and viability (topographical tetrazoliun chloride test) in three seed lots to determine the seed quality. Studies conducted on effect of five seed moisture contents (5, 7, 8, 10 and 12 % on fresh weight basis), three storage temperature (ambient, 15°C and -20°C) and eight storage periods (0, 3, 6, 9, 12, 18, 24 and 36 months) on seed longevity revealed that the critical moisture content (moisture content required in seeds for retaining initial germination after storage of seeds up to 36 months) of seeds for ambient storage condition of Delhi was 5 %, which increased to 7 % in 15°C and 12 % at -20°C storage temperature. The seeds are desiccation as well as chilling tolerant, therefore, exhibit orthodox storage behavior and are ideal for ex-situ conservation of seeds in seed/gene banks.

Effects of Temperature during Moist Heat Treatment on Ruminal Degradability and Intestinal Digestibility of Protein and Amino Acids in Hempseed Cake
L. Karlsson1, M. Ruiz-Moreno, M. D. Stern and K. Martinsson
Asian-Aust. J. Anim. Sci. Vol. 25, No. 11 : 1559-1567 November 2012
Doi: 10.5713/ajas.2012.12213
The objective of this study was to evaluate ruminal degradability and intestinal digestibility of crude protein (CP) and amino acids (AA) in hempseed cake (HC) that were moist heat treated at different temperatures. Samples of cold-pressed HC were autoclaved for 30 min at 110, 120 or 130°C, and a sample of untreated HC was used as the control. Ruminal degradability of CP was estimated, using the in situ Dacron bag technique; intestinal CP digestibility was estimated for the 16 h in situ residue using a three-step in vitro procedure. AA content was determined for the HC samples (heat treated and untreated) of the intact feed, the 16 h in situ residue and the residue after the three-step procedure. There was a linear increase in RUP (p = 0.001) and intestinal digestibility of RUP (p = 0.003) with increasing temperature during heat treatment. The 130°C treatment increased RUP from 259 to 629 g/kg CP, while intestinal digestibility increased from 176 to 730 g/kg RUP, compared to the control. Hence, the intestinal available dietary CP increased more than eight times. Increasing temperatures during heat treatment resulted in linear decreases in ruminal degradability of total AA (p = 0.006) and individual AA (p<0.05) and an increase in intestinal digestibility that could be explained both by a linear and a quadratic model for total AA and most individual AA (p<0.05). The 130°C treatment decreased ruminal degradability of total AA from 837 to 471 g/kg, while intestinal digestibility increased from 267 to 813 g/kg of rumen undegradable AA, compared with the control. There were differences between ruminal AA degradability and between intestinal AA digestibility within all individual HC treatments (p<0.001). It is concluded that moist heat treatment at 130°C did not overprotect the CP of HC and could be used to shift the site of CP and AA digestion from the rumen to the small intestine. This may increase the value of HC as a protein supplement for ruminants

Evaluating the Quality of Protein from Hemp Seed (Cannabis sativa L.) Products Through the use of the Protein Digestibility-Corrected Amino Acid Score Method
JAMES D. HOUSE, JASON NEUFELD, AND GERO LESON
J. Agric. Food Chem. 2010, 58, 11801–11807
DOI:10.1021/jf102636b
The macronutrient composition and the quality of protein of hemp seed and products derived from hemp seed grown in Western Canada were determined. Thirty samples of hemp products (minimum 500 g), including whole hemp seed, hemp seed meal from cold-press expelling, dehulled, or shelled, hemp seed and hemp seed hulls, were obtained from commercial sources. Proximate analysis,
including crude protein (% CP), crude fat (% fat) and fiber, as well as full amino acid profiles, were determined for all samples. Protein digestibility-corrected amino acid score (PDCAAS) measurements, using a rat bioassay for protein digestibility and the FAO/WHO amino acid requirement of children (2-5 years of age) as reference, were conducted on subsets of hemp products. Mean ((SD) percentage CP and fat were 24.0(2.1) and 30.4(2.7) for whole hemp seed, 40.7(8.8) and 10.2(2.1) for hemp seed meal, and 35.9(3.6) and 46.7(5.0) for dehulled hemp seed. The percentage protein digestibility and PDCAAS values were 84.1-86.2 and 49-53% for whole hemp seed, 90.8-97.5 and 46-51% for hemp seed meal, and 83.5-92.1 and 63-66% for dehulled hemp seed. Lysine was the first limiting amino acid in all products. Removal of the hull fraction improved protein digestibility and the resultant PDCAAS value. The current results provide reference data in support of protein claims for hemp seed products and provide evidence that hemp proteins have a PDCAAS equal to or greater than certain grains, nuts, and some pulses.

Experimental Endozoochory of Cannabis sativa Achenes
John M. McPartland, Steve G. Naraine
Med Cannabis Cannabinoids Published online: October 8, 2018
DOI: 10.1159/000492971
The mechanism by which Cannabis sativa dispersed from its center of origin remains an open question. The literature provides many hypotheses, which we review for the first time, but experiments are few. Darwin was interested in zoochory – the transport of plants by animals. He demonstrated endozoochory (transport of seeds via animal digestive systems) of C. sativa achenes (seeds) by carrier pigeons, but he did not quantify achene survival rates. We assessed mammalian endozoochory in a triplicate experiment: feeding C. sativa achenes into a simulated gastrointestinal system,
a dog, and a human. The in vitro system subjected achenes to sequential digestive enzymes. Achenes were planted in potting soil and monitored for emergence under growroom conditions. The in vivo experiments added achenes to a normal morning meal (dog food or granola). Feces were collected for daily instillation into an outdoor garden and monitored for seedling emergence for 16 days.
Control achenes were planted directly into soil without ingestion. In the in vitro study, 34.7% of the digested achenes emerged as seedlings. The in vivo emergence rates were 10.3, 1.3, and 76.0% for the dog, human, and control conditions. The three groups differed significantly (?2 = 1,264.93, p < 0.0001). Achene survival was greatest under in vitro conditions, which lacked a mastication step, compared to dog (minimal chewing) and human (maximal chewing) conditions. Although C. sativa lacks evolutionary traits for classic endozoochory (i.e., a fleshy fruit), it seems well adapted to this manner of seed dispersal.

Fatty acid profile and health lipid indices in the raw milk of ewes grazing part-time and hemp seed supplementation of lactating ewes
D. Mierlita
South African Journal of Animal Science 2016, 46 (No. 3)
https://www.scielo.org.za/pdf/sajas/v46n3/03.pdf
This experiment was designed to evaluate the effects of the feeding system (indoor or part-time grazing) of ewes and dietary supplementation with hemp seed (non-drug varieties of Cannabis sativa L.) on milk production and the composition of milk fatty acids (FAs). The experimental diets were constituted as a 2 x 2 factorial arrangement of feeding system (I: indoors and receiving 1400 g dry matter (DM) grass hay/d or pasture grazing (PG)–4 h/day + 700 g DM grass hay/d), and with or without hemp seed (Hs) supplementation (-Hs = no hemp seed vs. +Hs = 175 g/d hemp seed). All animals received 700 g DM/day of a concentrate for lactation. Feeding the PG diets increased raw milk production and yields of fat, protein and lactose. Feeding +Hs increased fat-corrected milk, milk fat content and fat yield significantly while the combination of +Hs and PG increased fat-corrected milk and fat yield significantly. Milk protein content was not affected by dietary factors. Feeding the I diets increased the proportion of short-chain FAs (C4:0 - C10:0), at the expense of medium-chain FAs (12 to 17 C units). Feeding +Hs decreased the proportion of short-chain FAs, medium-chain FAs and hypercholesterolaemic FA (sum of C12:0, C14:0, C16:0) and increased the trans-vaccenic acid (C18:1 t11, VA), ?-linolenic acid (C18:3 n-3, ALA), conjugated linoleic acid (CLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in milk fat significantly. The combination of hemp seed and part-time grazing produced the highest proportion of c9,t11 CLA, t10,c12 CLA and n-3 FA (sum of ALA, EPA, DHA) in milk fat. The polyunsaturated fatty acid (PUFA)/saturated fatty acid (SFA) ratios in the milk from ewes grazing part time were significantly higher, whereas the atherogenicity index (AI), thrombogenic index (TI) and linoleic acid (LA)/ALA ratio were significantly lower compared with the milk of indoor-fed ewes. Supplementing with +Hs has a similar effect of increasing the PUFA/SFA ratio and lowering of the AI, TI and LA/ALA ratio. It was concluded that pasture grazing combined with hemp seed had a synergistic effect on milk production and fat yield without altering the milk protein content in ewes. This combination has favourable effects on milk FA profile from the human consumer’s point of view by increasing the nutritional quality of milk.

GC-MS Analysis of the Total ?9-THC Content of Both Drug- and Fiber-Type Cannabis Seeds.
Ross, S. A., Mehmedic, Z., Murphy, T. P., & ElSohly, M. A.
Journal of Analytical Toxicology, 24(8), 715–717. (2000).
doi:10.1093/jat/24.8.715
A GC-MS method was performed to determine the total L~%THC content in both drug- and fiber-type cannabis seeds. Drug-type seeds were found to contain much higher levels of Ag-THC (35.6-124 pg/g) than fiber (hemp) seeds (0-12 pg/g). The majority of A%THC was found to be located on the surface of the seeds.
Approximately 90% of the total Ag-THC was removed by a simple, quick wash with chloroform. Washed drug-type seeds contained less than 10 pg/g. Separation of the seeds into the kernel and testa showed that the bulk of Ag-THC is located in the testa, mainly on the outside. The kernels of drug- and fiber-type cannabis seeds contained less than 2 and 0.5 pg A%THC/g seeds, respectively. Fluctuations in the A%THC content of different replicates of the same type of seeds could be the result of the degree of contamination on the outside of the seeds.

Hemp as Food at High Latitudes
J. C. Callaway PhD
Journal of Industrial 2002 Hemp 7(1):105-117
DOI: 10.1300/J237v07n01_09
Hempseed offers a unique nutritional package, in terms of dietary oil, protein, vitamins and minerals, which can be produced at high latitudes ( > 50° latitude). Hempseed oil is highly unsaturated and contains both essential fatty acids (linoleic acid and alpha-linolenic acid) in a nutritionally balanced ratio, in addition to considerable amounts of biochemically important gamma-linolenic acid (GLA) and stearidonic acid (SDA). The protein in hempseed is complete, in that it contains all of the essential amino acids in nutritionally significant amounts, and lacks the nutritional inhibiting factors found in soya. Hempseed could become a viable replacement for imported soya in Northern Europe, particularly as feed stock for animals

Hemp (Cannabis sativa L.) Protein Hydrolysates Promote Anti-Inflammatory Response in Primary Human Monocytes.
Rodriguez-Martin, N. M., Montserrat-de la Paz, S., Toscano, R., Grao-Cruces, E., Villanueva, A., Pedroche, J., … Millan-Linares, M. C.
Biomolecules, 10(5), 803. (2020).
doi:10.3390/biom10050803
Hemp seeds have a wide variety of chemical compounds which present biological activity. Specifically, the focus on proteins and bioactive peptides are increasing as alternative sources of nutraceutical uses. In the literature, hemp protein products (HPPs) have reported antioxidant and anti-inflammatory properties. This study aimed to determine the inflammation-related modulatory effects of HPPs on lipopolysaccharide (LPS)-activated primary human monocytes. CD14+ cells were immunomagnetically isolated from buffy coats and the anti-inflammatory activity of hemp protein isolate (HPI) and hydrolysates (HPHs) was evaluated on LPS-stimulated human primary monocytes. The specific markers of inflammation, polarization, and chemoattraction were measured by RT-qPCR and ELISA assays. Our results showed that HPPs decreased the pro-inflammatory mediators (TNF-?, IL-1?, and IL-6) and increased the anti-inflammatory mediators (IL-10 and IL-4). In addition, M1 polarization marker gene expression (CCR7 and iNOS) was downregulated by HPPs and, M2 polarization marker gene expression (CD200R and MRC1) was upregulated. Finally, the mRNA expression of chemotaxis genes (CCR2 and CCL2) was downregulated by HPPs. In conclusion, this study suggests that HPPs may improve chronic inflammatory states and promote regenerative processes by reprogramming monocytes toward M2 polarization phenotype.

Hemp (Cannabis sativa L.) Seed Protein–EGCG Conjugates: Covalent Bonding and Functional Research
Xin-Hui Pang, Yang Yang, Xin Bian, Bing Wang, Li-Kun Ren, Lin-Lin Liu, De-Hui Yu, Jing Yang, Jing-Chun Guo, Lei Wang, Xiu-Min Zhang, Han-Song Yu, and Na Zhang
Foods 2021 10, 1618.
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-01618.pdf
In order to make HPI have a wide application prospect in the food industry, we used EGCG to modify HPI. In this study, we prepared different concentrations (1, 2, 3, 4, and 5 mM) of (−)-epigallocatechin gallate (EGCG) covalently linked to HPI and use methods such as particle size analysis, circular dichroism (CD), and three-dimensional fluorescence spectroscopy to study the changes in the structure and functional properties of HPI after being covalently combined with EGCG. The particle size data indicated that the covalent HPI-EGCG complex was larger than native HPI, and the particle size was mainly distributed at about 200 µm. CD and three-dimensional fluorescence spectroscopy analyses showed that the conformation of the protein was changed by conjugation with EGCG. The β-sheet content decreased from 82.79% to 66.67% after EGCG bound to the protein, and the hydrophobic groups inside the protein were exposed, which increased the hydrophobicity of the protein and changed its conformation. After HPI and 1 mM of EGCG were covalently bonded, the solubility and emulsifying properties of the covalent complex were improved compared with native HPI. These results indicated that HPI-EGCG conjugates can be added in some foods.

Hemp globulin forms colloidal nanocomplexes with sodium caseinate during pH-cycling
Chih-ChiehChuang, Aiqian Ye, Skelte G. Anema, Simon M. Loveday
Food Research International Volume 150, Part B, December 2021, 110810
DOI: 10.1016/j.foodres.2021.110810
https://www.sciencedirect.com/scienc...109?via%3Dihub
Seed from industrial hemp (Cannabis sativa L.) contains around 25% protein (mainly globulins) which is easily digested, but the low solubility of hemp globulins (HG) limits their application in many food systems. In this study, the solubility of HG was improved by blending HG with sodium caseinate (SC) and treating with a pH-cycling process. The pH-cycling involved adjusting the pH to 12 and reacting for 1 hr, followed by neutralisation to pH 7. Nanoparticles composed of HG and SC (Z-average diameter ≈ 130 nm) were formed after the pH-cycling, and the solubility of HG increased to > 80% when there was more than 1% of SC for 1% of HG. These HG|SC nanoparticles were monodisperse (PDI < 0.17) and ζ-potential was ≈ −17 mV. Hydrogen bonding is the main forces that assembles HG|SC nanoparticles because the nanoparticles dissociated by heat treatment (up to 60 °C) or urea, which is an effective hydrogen bond breaker. HG|SC nanoparticles will aggregate irreversibly above 60 °C, possibly due to thiol-disulphide exchange. The nanoparticles were heat-stable as the Z-average diameter was only 229 nm after heating (90 °C, 30 min). N-ethylmaleimide blocked free thiol groups on HG and resulted in less disulphide-linked HG aggregation after pH- cycling, which in turn lead to smaller HG|SC nanoparticles and a bimodal particle size distribution, indicating the importance of disulphide bond for the formation of monodisperse HG|SC nanoparticles. The soluble and heat-stable HG|SC nanoparticles could be used to increase the hemp protein content in beverages and emulsions.
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Hempseed as a nutritional resource: An overview
Euphytica 140: 65–72, (2004).
J.C. Callaway
DOI: 10.1007/s10681-004-4811-6
The seed of Cannabis sativa L. has been an important source of nutrition for thousands of years in Old World cultures. Non-drug varieties of Cannabis, commonly referred to as hemp, have not been studied extensively for their nutritional potential in recent years, nor has hempseed been utilized to any great extent by the industrial processes and food markets that have developed during the 20th century. Technically a nut, hempseed typically contains over 30% oil and about 25% protein, with considerable amounts of dietary fiber, vitamins and minerals. Hempseed oil is over 80% in polyunsaturated fatty acids (PUFAs), and is an exceptionally rich source of the two essential fatty acids (EFAs) linoleic acid (18:2 omega-6) and alpha-linolenic acid (18:3 omega-3). The omega-6 to omega-3 ratio (n6/n3) in hempseed oil is normally between 2:1 and 3:1, which is considered to be optimal for human health. In addition, the biological metabolites of the two EFAs, gamma-linolenic acid (18:3 omega-6; ‘GLA’) and stearidonic acid (18:4 omega-3; ‘SDA’), are also present in hempseed oil. The two main proteins in hempseed are edestin and albumin. Both of these high-quality storage proteins are easily digested and contain nutritionally significant amounts of all essential amino acids. In addition, hempseed has exceptionally high levels of the amino acid arginine. Hempseed has been used to treat various disorders for thousands of years in traditional oriental medicine. Recent clinical trials have identified hempseed oil as a functional food, and animal feeding studies demonstrate the long-standing utility of hempseed as an important food resource.

Hemp seed (Cannabis sativa L.) enriched pasta: Physicochemical properties and quality evaluation
Dorota Teterycz, Aldona Sobota, Dominika Przygodzka, Paulina
Łysakowska
Plos One March 2021
DOI: 10.1371/journal.pone.0248790
https://journals.plos.org/plosone/ar...8790&utm_sourc e=The+Cannabis+%2B+Cannabinoid +Curator&utm_campaign=cd2a62ba 57-EMAIL_CAMPAIGN_2020_03_13_07_5 9_COPY_01&utm_medium=email&utm _term=0_47f6c7c552-cd2a62ba57-365750754
Hemp seed (Cannabis sativa L.) contain large amounts of nutrients, e.g. protein, dietary fiber, minerals, and unsaturated fatty acids, which make them a good fortifying component in food production. The aim of the present study was to determine the effect of hemp addition on the physicochemical properties, cooking quality, texture parameters and sensory properties of durum wheat pasta. The samples were fortified with 5–40% of commercially available hemp flour or 2.5–10% of hemp cake obtained from hemp seed oil pressing. Our study showed that the addition of hemp seed raw materials led to an increase in the protein, total dietary fiber (TDF), ash and fat content in the pasta samples. Due to its lower granulation and higher nutritional value, hemp flour was found to be a better raw material for the fortification of pasta than hemp cake. Pasta enriched with hemp flour at the level of 30–40% contains 19.53–28.87% d.m. of protein and 17.02–21.49% d.m. of TDF and according to the EU, a definition can be described as a high-protein and high-fiber products. All enriched pasta samples were also characterized by safe Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) content, and their sensory properties were accepted by consumers.

Hempseed (Cannabis sativa) Peptides WVSPLAGRT and IGFLIIWV Exert Anti-inflammatory Activity in the LPS-Stimulated Human Hepatic Cell Line
Ivan Cruz-Chamorro, Guillermo Santos-Sánchez, Carlotta Bollati, Martina Bartolomei, Jianqiang Li, Anna Arnoldi, and Carmen Lammi J. Agric. Food Chem. 2022, 70, 577−583 DOI: 10.1021/acs.jafc.1c07520 https://www.ncbi.nlm.nih.gov/labs/pm...es/PMC8778635/
WVSPLAGRT (H2) and IGFLIIWV (H3) are two transepithelial transported intestinal peptides obtained from the hydrolysis of hempseed protein with pepsin, which exert antioxidant activity in HepG2 cells. Notably, both peptides reduce the H2 O2 -induced reactive oxygen species, lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells via the modulation of the nuclear factor erythroid 2-related factor 2 and the inducible nitric oxide synthase (iNOS) pathways, respectively. Due to the close link between inflammation and oxidative stress and with the objective of fostering the multifunctional behavior of bioactive peptides, in this study, the molecular characterization of the anti-inflammatory and immunomodulatory properties of H2 and H3 was carried out in HepG2 cells. In fact, both peptides were shown to modulate the production of pro (IFN-γ : − 33.0 ± 6.7% H2, p = 0.011; − 13.1 ± 2.0% H3, p = <0.0001; TNF: − 17.6 ± 1.7% H2, p = 0.0004; − 20.3 ± 1.7% H3, p = <0.0001; and IL-6: − 15.1 ± 6.5% H3, p = 0.010)- and anti (IL-10: +9.6 ± 3.1% H2, p = 0.010; +26.0 ± 2.3% H3, p = < 0.0001)-inflammatory cytokines and NO (− 9.0 ± 0.7% H2, p = <0.0001; − 7.2 ± 1.8% H3, p = <0.0001) through regulation of the NF-κ B and iNOS pathways, respectively, in HepG2 cells stimulated by lipopolysaccharides.

Hempseed Products Fed to Hens Effectively Increased n-3 Polyunsaturated Fatty Acids in Total Lipids, Triacylglycerol and Phospholipid of Egg Yolk.
Neijat, M., Suh, M., Neufeld, J., & House, J. D.
Lipids, 51(5), 601–614. (2015).
doi:10.1007/s11745-015-4088-7
Hempseed products represent potential alternative feed ingredients for poultry. However, their usage is not currently approved due to a lack of data to support their safety and efficacy. In this regard, the current study was conducted to assess the impact of dietary concentration of hempseed (HS) products and duration of their feeding to hens on the polyunsaturated fatty acid (PUFA) composition of egg yolk lipids. In the current study, 48 Lohmann LSL-Classic hens were individually housed in metabolism cages, in a completely randomized design, and provided one of six diets (wheat-barley-soybean-based) containing either HS (10, 20 and 30 %), hempseed oil (HO; 4.5 and 9.0 %) or no hempseed product (control) over 12 weeks. Increasing alpha-linolenic acid (ALA) intake via increasing dietary hempseed product inclusion, significantly (p < 0.0001) increased the n-3 PUFA contents of yolk total lipid. The values of ALA increased by 12-fold (152 ± 3.56 and 156 ± 2.42 mg/yolk) and docosahexaenoic acid (DHA) by twofold to threefold (41.3 ± 1.57 and 43.6 ± 1.61 mg/ yolk) over the control, for the highest levels of HS and HO inclusion, respectively. Increasing levels of hemp products in laying hen diets proved effective in manipulating the fatty acid profile of the total lipid, triacylglycerol (TAG) and total phospholipid (PL) fractions of yolks, enhancing the n-3 fatty acids and reducing the n-6/n-3 ratio. The latter benefit was achieved within 4 weeks of feeding hens either HS- or HO-containing diets.

High Fiber Cakes from Mediterranean Multipurpose Oilseeds as Protein Sources for Ruminants
Francesco Serrapica, Felicia Masucci, Emiliano Raffrenato, Maura Sannino, Alessandro Vastolo, Carmela Maria Assunta Barone and Antonio Di Franci
Animals 2019, 9, 918;
doi:10.3390/ani9110918
Fifteen oilseed cakes from sunflower, pomegranate, cardoon, tobacco and hemp were characterized with regard to chemical composition, Cornell Net Carbohydrate and Protein System (CNCPS) fractionation, in vitro digestibility of dry matter, neutral detergent fiber, and crude protein. All the cakes presented low moisture, rather variable ether extract contents and medium to high levels of crude protein and neutral detergent fiber. The cakes significantly differed in terms of CNCPS partitioning and in vitro digestibility. Tobacco and hemp cakes presented high contents of slow degradable fractions of crude protein and carbohydrate joined to good post-ruminal protein digestibility. Cardoon cakes presented the highest rumen protein degradability. Based on crude protein content and intestinal digestibility of rumen undegraded protein, cakes of tobacco and hemp showed the better potential as alternative protein supplements for ruminants, while pomegranate appears to be the least suitable for ruminant feeding.
p. This study suggests the potential of hemp seed diet to improve the overall antioxidant status of the lactating sows and their progeny.

Identification and Characterization of the Seed Storage Proteins and Related Genes of Cannabis sativa L.
Xin Sun, Yao Sun, Yao Li, Qiong Wu and Lei Wang
Front. Nutr. June 2021
doi: 10.3389/fnut.2021.678421
https://www.ncbi.nlm.nih.gov/pmc/art...-08-678421.pdf
Hemp (Cannabis sativa L.) seed is emerging as a novel source of plant protein owing to its rich protein content and reasonable nutritional structure. In the current study, the storage proteins of hemp seed were extracted using different methods. The modified Osborne method yielded maximum extraction of the hemp seed storage proteins, while degreasing had little effect on the hemp seed protein (HSP) extraction. Protein identification results revealed that 11S globulin (edestin) was the most abundant protein in hemp seed, and the molecular weights of the two subunits of this protein were ∼35 and 20 kDa, respectively. The second most abundant protein was 2S albumin (Cs2S), with a molecular weight of ∼14–15 kDa. The least abundant protein was 7S vicilin-like protein (Cs7S), with a molecular weight of ∼47 kDa. Subsequently, gene families encoding these three storage protein classes, including three genes for edestin, two for Cs2S, and one for Cs7S, were cloned and then analyzed for amino acid composition and structure. The three edestins were different in their amino acid sequences and calculated molecular weights. The analysis of coding sequences revealed a higher percentage of similarity (62.7%) between Edestin1 and Edestin3, while the similarity decreased significantly to ∼57% between Edestin1 and Edestin2, and 58% between Edestin2 and Edestin3. The calculated protein molecular weight was the highest for the protein encoded by Edestin1 and the smallest for the protein encoded by Edestin2. All three edestins were rich in arginine, while Edestin3 had a higher methionine content relative to that in the other two, which proved that Edestin3 had a better nutritional value. Cs2S and Cs7S were different from those reported in previous studies. Therefore, it could be inferred that amino acid composition varies with different hemp cultivars. The current research brought significant theoretical advance in illuminating the understanding of hemp seed storage protein and would have significance for future research on improving the nutritional quality of hemp seed and developing bioactive peptides.

Not Cannabis specific
Imprinting and Seed Development
Mary Gehring, Yeonhee Choi, and Robert L. Fischer1
The Plant Cell, Vol. 16 (2004)
doi:10.1105/tpc.017988
Imprinted genes are expressed predominantly from one allele in a parent-of-origin–specific manner. The endosperm, a seed tissue that mediates the transfer of nutrients from the maternal parent to the embryo, is an important site of imprinting in flowering plants. Imprinted genes have been identified in maize and Arabidopsis thaliana, but crosses in a variety of species suggest that the effect of imprinting on seed development is widespread throughout the angiosperms. The mechanism of imprinting in plants, as yet only partially understood, likely involves differences in DNA methylation and chromatin structure between differentially expressed maternal and paternal alleles.

Isolation and Primary Structure of a Methionine- and Cystine-rich Seed Protein of Cannabis sativa.
ODANI, S., & ODANI, S.
Bioscience, Biotechnology, and Biochemistry, 62(4), 650–654. (1988)
doi:10.1271/bbb.62.650
A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

Local knowledge held by farmers in Eastern Tyrol (Austria) about the use of plants to maintain and improve animal health and welfare
Christian R. Vogl, Brigitte Vogl-Lukasser & Michael Walkenhorst
Journal of Ethnobiology and Ethnomedicine volume 12, Article number: 40 (2016)
https://ethnobiomed.biomedcentral.co.. .002-016-0104-0
Background
The sustainable management of animal health and welfare is of increasing importance to consumers and a key topic in the organic farming movement. Few systematic studies have been undertaken investigating farmers’ local knowledge related to this issue. Ethnoveterinary medicine (EVM) is a discipline focusing on local knowledge and folk methods in veterinary medicine, however most ethnoveterinarian studies primarily address the treatment of animal diseases. Very few studies have explored prophylactic methods.
Methods
An ethnoveterinary research project in Eastern Tyrol (Austria) was conducted in 2004 and 2005 to gather information about local knowledge of animal husbandry from 144 informants, with the emphasis on plants that maintain livestock health and welfare.
Results
Informants mentioned a total of 87 plants and 22 plant-based generic terms in the context of maintaining and improving livestock health and welfare. The most important preventive measures for maintaining and improving animal health and welfare were practices related to “fodder” and “feeding”. In this category the plants mentioned could be grouped according to three different perceptions about their effect on animals: “Good or bad fodder”, “Functional fodder” and “Fodder medicine”. In addition to fodder, environmental management, the human-animal relationship, household remedies and cultural/religious activities were also mentioned. When asked about practices in the past that maintained animal health and well-being, interviewees mentioned, for example, the importance of the diversity of sources that used to be available to obtain feed and fodder.
Conclusions
The informants’ approach that feeding is central to livestock welfare is in line with the standard scientific literature on animal health, including in organic farming. Various scientific studies into common fodder evaluate the nutritive and dietary value, efficiency and safety of fodder. Future studies also have to consider the evaluation of traditional, local fodder resources. In fact, the value of ‘food as medicine’ for humans in the context of local knowledge has been widely assessed, but the potential health benefits of fodder and nutraceuticals in local and traditional ethnoveterinary methods require further attention.

Machine Learning-Mediated Development and Optimization of Disinfection Protocol and Scarification Method for Improved In Vitro Germination of Cannabis Seeds.
Marco Pepe, Mohsen Hesami, Andrew Maxwell Phineas Jones
Plants 2021, 10, 2397.
DOI: 10.3390/plants10112397
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-02397.pdf
In vitro seed germination is a useful tool for developing a variety of biotechnologies, but cannabis has presented some challenges in uniformity and germination time, presumably due to the disinfection procedure. Disinfection and subsequent growth are influenced by many factors, such as media pH, temperature, as well as the types and levels of contaminants and disinfectants, which contribute independently and dynamically to system complexity and nonlinearity. Hence, artificial intelligence models are well suited to model and optimize this dynamic system. The current study was aimed to evaluate the effect of different types and concentrations of disinfectants (sodium hypochlorite, hydrogen peroxide) and immersion times on contamination frequency using the generalized regression neural network (GRNN), a powerful artificial neural network (ANN). The GRNN model had high prediction performance (R > 0.91) in both training and testing. Moreover, a genetic algorithm (GA) was subjected to the GRNN to find the optimal type and level of disinfectants and immersion time to determine the best methods for contamination reduction. According to the optimization process, 4.6% sodium hypochlorite along with 0.008% hydrogen peroxide for 16.81 min would result in the best outcomes. The results of a validation experiment demonstrated that this protocol resulted in 0% contamination as predicted, but germination rates were low and sporadic. However, using this sterilization protocol in combination with the scarification of in vitro cannabis seed (seed tip removal) resulted in 0% contamination and 100% seed germination within one week.

Molecular characterization of edestin gene family in Cannabis sativa L.
Teresa Docimo, Immacolata Caruso, Elena Ponzoni, Monica Mattana, Incoronata Galasso
Plant Physiology and Biochemistry 84 (2014) 142e148
https://doi.org/10.1016/j.plaphy.2014.09.011
Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded bygene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98–99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11–12%), but CsEde2 resulted particularly rich inmethionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs.

New Technology of Harvesting Hemp Grown for Seed
Henryk Burczyk, Ryszard Kaniewski
June 2005 Journal of Industrial Hemp 10(1):49-60
DOI: 10.1300/J237v10n01_05
The expected growth of hemp cultivation world wide for industrial purposes will clearly cause a growing demand for sowing material. Hence, there is a need for the development of a new, more efficient method for harvesting hemp seed. The technology described in this paper, elaborated at the Institute of Natural Fibres (INF), presents the new concept of a panicle cutting device that involves mowing and cutting stems into smaller sections. The cut-off panicles are collected on a trailer hooked behind the mower. The panicles harvested during the flowering phase are used for the production of essential oils, while those harvested in full maturity are the source for sowing seed. In the latter case, the panicles–before ginning–must be initially dried in conditions allowing for preservation of good germination capacity. The seed must be cleaned immediately and dried further to 12% moisture content and protected with fungicidal treatments. After mowing and cutting stems are swathed in the field and left for retting or collected–after drying–using agricultural presses and then decorticated.

Occurrence of Endosperm Haustorium in Cannabis sativa L.: With thirteen Figures in the Text.
RAM, M.
Annals of Botany, 24(1), 79–82.(1960).
doi:10.1093/oxfordjournals.aob.a083690
The development and structure of the chalazal endosperm haustorium in Cannabis sativa are described. The endosperm is nuclear and a haustorium is formed at the chalazal end. The latter remains free nuclear throughout. Enucleate vesicles appear in the upper part of the endosperm but finally they merge with the cytoplasm of the haustorium. As the embryo reaches maturity it occupies the whole seed cavity, the haustorium collapses and the endosperm persists only as a thin layer.

Omega-3 and Omega-6 Fatty Acids in Poultry Nutrition: Effect on Production Performance and Health.
Alagawany, M., Elnesr, S. S., Farag, M. R., Abd El-Hack, M. E., Khafaga, A. F., Taha, A. E., … Dhama, K.
Animals, 9(8), 573. (2019).
doi:10.3390/ani9080573
Omega-3 (?-3) and omega-6 (?-6) fatty acids are important components of cell membranes. They are essential for health and normal physiological functioning of humans. Not all fatty acids can be produced endogenously owing to the absence of certain desaturases; however, they are required in a ratio that is not naturally achieved by the standard diet of industrialized nations. Poultry products have become the primary source of long-chain polyunsaturated fatty acids (LC-PUFA), with one of the most effective solutions being to increase the accretion of PUFAs in chicken products via the adjustment of fatty acids in poultry diets. Several studies have reported the favorable effects of ?-3 PUFA on bone strength, bone mineral content and density, and semen quality. However, other studies concluded negative effects of LC-PUFA on meat quality and palatability, and acceptability by consumers. The present review discussed the practical application of ?-3 and ?-6 fatty acids in poultry diets, and studied the critical effects of these fatty acids on productive performance, blood biochemistry, immunity, carcass traits, bone traits, egg and meat quality, and semen quality in poultry. Future studies are required to determine how poultry products can be produced with higher contents of PUFAs and favorable fatty acid composition, at low cost and without negative effects on palatability and quality.

Optimization of ultrasound?assisted solvent extraction of hemp ( Cannabis sativa L.) seed oil using RSM: Evaluation of oxidative stability and physicochemical properties of oil.
Esmaeilzadeh Kenari, R., & Dehghan, B.
Food Science & Nutrition.(2020).
doi:10.1002/fsn3.1796
In this study, the effect of solvent ratio of hexane to isopropanol (0:100, 50:50, and 100:0 v/v%), extraction temperature (30, 45, and 60°C), and time (30, 60, and 90 min) were investigated on the oil extraction efficiency, total phenol content, DPPH radical scavenging, peroxide value, and oil color index. Extraction conditions were optimized by response surface methodology and Box–Behnken design. The optimal conditions were obtained as hexane-to-isopropanol ratio of approximately 3:2, temperature of 40.26°C, and ultrasonication time of 54.40 min. Then, the hemp seed oil was extracted under the optimal conditions. The optimal predicted contents for oil yield (31.22%), total phenolics (3.19 mg GA/g oil), DPPH inhibition (73.86%), peroxide (4.62 meq/kg), and color index (28.2) were agreed with the predicted conditions because the RSE values were less than 5%. Hemp seed oil can be used extensively due to its high nutritional values, and antioxidant potential and ultrasound can improve oil extraction as a simple and fast method.

Selective Extraction of Cannabinoid Compounds from Cannabis Seed Using Pressurized Hot Water Extraction
Yannick Nuapia, Hlanganani Tutu , Luke Chimuka and Ewa Cukrowska
Molecules 2020, 25, 1335;
doi:10.3390/molecules25061335
Phytochemicals of Cannabis sativa mainly for the use in the different industries are that of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Pressurized hot water extraction (PHWE) is seen as an efficient, fast, green extraction technique for the removal of polar and semi-polar compounds from plant materials. The PHWE technique was applied to extract cannabinoid compounds from Cannabis sativa seed. Response surface methodology was used to investigate the influence of extraction time (5–60 min), extraction temperature (50–200 ?C) and collector vessel temperature (25–200 ?C) on the recovery of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), cannabichromene (CBG) and cannabigerol (CBC) from Cannabis sativa seed by PHWE. The identification and semi quantification of cannabinoid compounds were determined using GCXGC-TOFMS. The results obtained from different extractions show that the amount of THC and CBN was drastically decreasing in the liquid extract when the temperature rose from 140 to 160 ?C in the extraction cell and the collector0 s vessel. The optimal conditions to extract more CBD, CBC, and CBG than THC and CBN were set at 150 ?C, 160 ?C and 45 min as extraction temperature, the temperature at collector vessel, and the extraction time, respectively. At this condition, the predicted and experimental ratio of THCt (THC + CBN)/CBDt (CBD + CBC+ CBG) was found to be 0.17 and 0.18, respectively. Therefore, PHWE can be seen as an alternative to the classic extraction approach as the efficiency is higher and it is environmentally friendly

Soil Fertilization with Urea has Little Effect on Seed Quality but Reduces Soil N2O Emissions from a Hemp Cultivation
Anna Tedeschi, Franca Polimeno, Grazia Maria, Francesco Siano
Preprint in Agriculture · June 2020
DOI: 10.3390/agriculture10060240
https://www.researchgate.net/publica...mp_Cultivation
Multipurpose production of hemp has led to renewed interest for this crop cultivation, especially for human nutrition. To date, no information about the influence of nitrogen source on hemp seed quality is available. Hemp is also used for food and beverages due to its nutritional characteristics. This further use of hemp has led to an increase in hemp-grown areas. Therefore, it is important to get more information on the role of nitrogen on the quality production as well as to evaluate the environmental impact of the cultivation technique. In this work, we evaluate the influence of nitrogen source (i.e., NH4NO3 and urea) on the seed fatty acid composition of an edible hemp as well as on the environment in terms of soil N2O emission. Nitrogen source modified seed quality very little. Even if characterized by a lower acidic profile, seed from plants grown under urea and NH4NO3 had a ?-3/?-6 ratio (0.3) within the optimal range from the nutritional standpoint, being considered as the optimal proportion for human metabolism and health. Urea fertilization reduced soil N2O emission. Our findings suggest that nitrogen source seems not to influence seed quality and that urea fertilizer might be more climate-friendly than NH4NO3 in terms of greenhouse gas emissions, in an extensive cultivation of hemp for industrial use.

Synthetic Clonal Reproduction Through Seeds.
Marimuthu, M. P. A., Jolivet, S., Ravi, M., Pereira, L., Davda, J. N., Cromer, L., Mercier, R.
Science, 331(6019), 876–876.(2011)
doi: 10.1126/science.1199682
Engineering cloning through seeds (apomixis) in food crops would revolutionize agriculture by fixing hybrid vigor and allowing the perpetuation of any elite heterozygous genotype (1). The first component of apomixis, the formation of diploid clonalgametes, can beintroduced in a sexual plant, with Arabidopsis mutants (MiMe and dyad) (2, 3). However these clonal gametes participate in normal fertilization, giving rise to progeny with more chromosomes than their parent. Clonal gametes would be turned into clonal seeds if fertilized by a strain whose chromosomes are eliminated from the resultant progeny (Fig. 1A). Genome elimination can be induced by manipulating the centromere-specific histone CENH3 (4). By co-expressing green fluorescent protein (GFP)–CENH3 and GFP-tailswap variants in the cenh3-1 mutant, we made a line with increased fertility that causes genome elimination when crossed to a parent with diploid gametes (5) [supporting online material (SOM) text]. We named this line GEM for Genome Elimination induced by a Mix of CENH3 variants. When GEM was crossed to a tetraploid wild type (which has diploid gametes) or to osd1, a diploid mutant that produces a very high proportion of diploid male and female gametes (2), it induced conversion of diploid gametes into uniparental diploid progeny in addition to giving triploid and aneuploid progeny (table S1). However, uniparental diploid plants from osd1 crosses are not clones, because the diploid gametes produced by osd1 are recombined (2, 5) (SOM text and fig. S2). We then crossed dyad and MiMe plants as female to the GEM line and screened for clones in the F1 generation, which comprised diploids, triploids, and aneuploids (table S2). Because the genetic background of the parents differed, we could trace the origin of the chromosomes. MiMe × GEM gave an average of 14 viable (i.e., able to germinate) seeds per fruit; 34% (53/155) were diploid (table S2). Among these diploid plants, 98% (52/53) had only maternal chromosomes, lacking any paternal contribution for the eight diagnostic loci tested (Fig. 1B). Furthermore, these maternal diploids (named diploid eliminants) retained the heterozygosity of the mother plant at all tested loci (Fig. 1B). Likewise, dyad × GEM resulted in 0.9 viable seeds per fruit, 13% (29/220) of which were diploid eliminants retaining full maternal heterozygosity (table S2 and Fig. 1C). Although not fully penetrant, this demonstrates clonal propagation through seed in a manner akin to the

The variety, terroir, and harvest types affect the yield and the phenolic and sterolic profiles of hemp seed oil
Davide Calzolari, Gabriele Rocchetti, Luigi Lucini, Stefano Amaducci
Food Research International January 2021
DOI: 10.1016/j.foodres.2021.110212
In this work, considering the rising interest towards the exploitation of hemp seed oil in human nutrition, 45 hemp seeds from mono-variety fields were analyzed for their yield, oil content, in vitro antioxidant activity, followed by a comprehensive assessment of phenolic and sterolic composition. The results demonstrated that seed dimension is inversely correlated to total oil content, thus being a potential reference for quality assessment of seeds and for further improvement of hemp varieties. The UHPLC-QTOF metabolomic analysis revealed a large abundance of phytosterols, lower-molecular-weight phenolic acids, and lignanamides. Differences across varieties could be described, with Diana Hemp seed oil having the highest cumulative abundance of phytochemicals, recording 6.04 mg/g. Overall, the in vitro antioxidant activity results indicated that hemp seed oil antioxidants have a low potential for preventing oil rancidity, with phenolic acids being the most active radical scavengers. Besides, in the group of Futura 75 samples cultivated across Italy, the type of harvesting affected the acidity value significantly as a consequence of mechanical harvest and post-harvest handling. Finally, multivariate statistics following untargeted metabolomic analysis showed that variety, geographical origin, and harvest-type were able to affect the phytochemical profiles with different incidences, with some phytochemocals proposed for the first time as potential discriminant markers.
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Ttextual research of Cannabis sativa varieties and medicinal part
Yingfang Wei, Huadong Wang, Shanshan Guo, Fei Long
China journal of Chinese materia medica 35(13):1773-6 (2010) (Zhongguo Zhong yao za zhi)
(In Chinese)
https://www.researchgate.net/publica...medicinal_part
To determine the medicinal part and varieties of Cannabis Sativa through herbal textual research to Provide bibliographic reference for clinical application. Herbal textual research of C. Sativa from ancient herbal works and modern data analysis. Through the herbal textual research, the plant of the C. sativa, for Fructus Cannabis used now is identical with that described in ancient herbal literatures. People did not make a sharp distinction on medicinal part of C. sativa in the early stage literatures, female inflorescence and unripe fruit, fruit and kernel of seed were all used. Since Taohongjing realized the toxicity ofpericarp, all the herbal and prescription works indicate that the pericarp shall be removed before usage and only the kernel can be used. However, in modem literatures, both fruit and kernel can be used as medicinal part. The plants for Fructus Cannabis described in modern and ancient literatures are identical. The base of the original plant is the same either in ancient or modern. And the toxicity of the fruit is more than that of the kernel. The kernel is the exact medicinal part of C. Sativa.

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications.
Soler, S., Borràs, D., Vilanova, S., Sifres, A., Andújar, I., Figàs, M. R., … Prohens, J.
Journal of Forensic Sciences, 61(2), 494–500.(2015).
doi:10.1111/1556-4029.12995
Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.

Whole blood transcriptome analysis in ewes fed with hemp seed supplemented diet.
Iannaccone, M., Ianni, A., Contaldi, F., Esposito, S., Martino, C., Bennato, F., … Martino, G.
Scientific Reports, 9(1). (2019).
doi:10.1038/s41598-019-52712-6
The hemp plant (Cannabis sativa L.) has a long tradition of being used for many diferent purposes such as industry, medicine and nutrition. In particular, because hemp seed (HS) is rich in oil protein and considerable amounts of dietary fber, vitamins and minerals that are particularly suitable also for animal nutrition. Diferent studies have evaluated HS on qualitative and quantitative properties of livestock products but as of today, nobody has investigated the molecular pathway behind HS supplementation in farm animals. Thus, in this study, we will report the frst RNA sequencing of the whole-blood transcriptome of ewes fed either with a controlled diet (CTR, n=5) or with a diet supplemented with 5% of hemp seed (HSG, n=5). Applying a false discovery rate (FDR)


IC Cannabis Seed Oil

Aesculus hippocastanum L. as a Stabilizer in Hemp Seed Oil Nanoemulsions for Potential Biomedical and Food Applications
Maciej Jarz ˛ebsk, Wojciech Smułek, Przemysław Siejak, Ryszard Rezler, Jarosław Pawlicz , Tomasz Trzeciak, Małgorzata Jarz ˛ebska, Oliwia Majchrzak, Ewa Kaczorek, Pardis Kazemian, Marta Poniewaz-Pawlicz ˙and Farahnaz Fathordoobady
Int. J. Mol. Sci. 2021, 22, 887.
DOI: 10.3390/ ijms22020887
https://www.ncbi.nlm.nih.gov/pmc/art...s-22-00887.pdf
Nanoemulsion systems receive a significant amount of interest nowadays due to their promising potential in biomedicine and food technology. Using a two-step process, we produced a series of nanoemulsion systems with different concentrations of hemp seed oil (HSO) stabilized with Aesculus hippocastanum L. extract (AHE). Water and commercially-available low-concentrated hyaluronic acid (HA) were used as the liquid phase. Stability tests, including an emulsifying index (EI), and droplet size distribution tests performed by dynamic light scattering (DLS) proved the beneficial impact of AHE on the emulsion’s stability. After 7 days of storage, the EI for the waterbased system was found to be around 100%, unlike the HA systems. The highest stability was achieved by an emulsion containing 5% HSO and 2 g/L AHE in water, as well as the HA solution. In order to obtain the detailed characteristics of the emulsions, UV-Vis and FTIR spectra were recorded, and the viscosity of the samples was determined. Finally, a visible microscopic analysis was used for the homogeneity evaluation of the samples, and was compared with the DLS results of the water system emulsion, which showed a desirable stability. The presented results demonstrate the possible use of oil emulsions based on a plant extract rich in saponins, such as AHE. Furthermore, it was found that the anti-inflammatory properties of AHE provide opportunities for the development of new emulsion formulations with health benefits.

Analytical characterization of hemp (Cannabis sativa) seed oil from different agro-ecological zones of Pakistan.
Anwar, F., Latif, S., & Ashraf, M.
Journal of the American Oil Chemists’ Society, 83(4), 323–329. (2006).
doi:10.1007/s11746-006-1207-x
Cold-pressed oil content of Cannabis sativa (hemp) seeds from three different agro-ecological zones of Pakistan ranged from 26.90 to 31.50%. Protein, fiber, ash, and moisture content were found to be 23.00–26.50, 17.00–20.52, 5.00–7.60, and 5.60–8.50%, respectively. Results of some other physical and chemical parameters of the oil were as follows: iodine value, 154.00–165.00; refractive index (40°C), 1.4698–1.4750; density (24°C), 0.9180–0.9270 mg mL?1; saponification value, 184.00– 190.00; unsaponifiable matter, 0.70–1.25%; and color (1-in. cell), 0.50–0.80 R + 27.00–32.00 Y. The induction period (Rancimat, 20 L h?1, 120°C) of the nondegummed and degummed oils ranged from 1.35 to 1.72 h and from 1.20 to 1.49 h, respectively. Specific extinctions at 232 and 270 nm were 3.50–4.18 and 0.95–1.43, respectively. The hemp oils investigated were found to contain high levels of linoleic acid, 56.50–60.50%, followed by ?-linolenic, oleic, palmitic, stearic, and ?-linolenic acids: 16.85–20.00, 10.17– 14.03, 5.75–8.27, 2.19–2.79, and 0.63–1.65%, respectively. Tocopherols (?, ?, and ?) in the nondegummed oils were found to be 54.02–60.40, 600.00–745.00, 35.00–45.60, respectively, and were reduced to 29.90–50.00, 590.00–640.00, and 30.40–39.50 mg kg?1, respectively, after degumming. The results of the present analytical study, compared with those found in the typical literature on hempseed oils, showed C. sativa indigenous to Pakistan to be a potentially valuable nonconventional oilseed crop of comparable quality

Antioxidant Activity of Hemp (Cannabis sativa L.) Seed Oil in Drosophila melanogaster Larvae under Non-Stress and H2O2-Induced Oxidative Stress Conditions
Jelena Vitorovi´c, Nataša Jokovi´c, Niko Radulovi´c, Tatjana Mihajilov-Krstev, Vladimir J. Cvetkovi´c, Nikola Jovanovi´c, Tatjana Mitrovi´c, Ana Aleksi´c, Nemanja Stankovi´c and Nirit Bernstein
Antioxidants 2021, 10, 830.
DOI: 10.3390/antiox10060830
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-00830.pdf
The oil extracted from hemp seeds has significant nutritional and biological properties due to the unique composition of polyunsaturated fatty acids and various antioxidant compounds. The potential of this oil for the prevention of oxidative stress and for the treatment of oxidative-stressinduced ailments is of increasing interest. Most studies of hemp seed oil were conducted in-vitro, meaning we lack information about effects and activity in vivo. In the present study, we evaluated the hypothesis that hemp seed oil at different concentrations improves the oxidative state of D. melanogaster, under non-stress as well as hydrogen-peroxide-induced stress. We analyzed the effects of hemp seed oil on oxidative stress markers and on the life cycle of D. melanogaster under non-stress and hydrogen-peroxide-induced stress conditions. D. melanogaster larvae were exposed to hemp seed oil concentrations ranging from 12.5 to 125 µL/mL. The results revealed that under non-stress conditions, oil concentrations up to 62.5 µL/mL did not induce negative effects on the life cycle of D. melanogaster and maintained the redox status of the larval cells at similar levels to the control level. Under oxidative stress conditions, biochemical parameters were significantly affected and only two oil concentrations, 18.7 and 31.2 µL/mL, provided protection against hydrogen peroxide stress effects. A higher oil concentration (125 µL/mL) exerted negative effects on the oxidative status and increased larval mortality. The tested oil was characterized chemically by NMR, transesterification, and silylation, followed by GC-MS analyses, and was shown to contain polyunsaturated fatty acid triglycerides and low levels of tocopherols. The high levels of linoleic and linolenic acids in the oil are suggested to be responsible for the observed in vivo antioxidant effects. Taken together, the results show that hemp seed oil is effective for reducing oxidative stress at the cellular level, thus supporting the hypothesis. The obtained results point to the potential of hemp seed oil for the prevention and treatment of conditions caused by the action of reactive oxygen species

Application of Hempseed (Cannabis sativa L.) Oil in the Treatment of Ear, Nose and Throat (ENT) Disorders
Oleg V. Grigoriev
June 2002 Journal of Industrial Hemp 7(2):5-15
DOI: 10.1300/J237v07n02_02
Hempseed oil was dispensed in an open clinical trial involving in- and out patients, for the treatment of chronic Ear, Nose and Throat disorders (external otitis, laryngitis, pharyngitis, sinusitis, tonsillitis), acute external otitis, and one case of trauma, for a total of 117 cases. The application of hempseed oil made it possible to reduce the period of treatment of all mentioned cases by eight days (9%), compared with standard treatments. The results allowed us to consider hempseed oil as an effective means for treating ENT disorders as well as injuries resulting from traumas or burns.

Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content
G. Lesmaa, R. Consonnib, V. Gambaroc, C. Remuzzia, G. Rodac, A.
Silvania, V. Vecea & G.L. Visconti
Natural Product Research: (2014)
DOI: 10.1080/14786419.2014.926354
Within a project aimed to reintroduce non-drug hemp cultivars in the Italian Po valley, for fibre but also high added-value nutraceutical production, investigation on locally grown plants has been performed, in order to assess their oil and metabolic content. This study provides useful information regarding three different hemp cultivars, from two sites, in view of their potential industrial application. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance ofv-3 and v-6 fatty acids, as requested for healthy foods. The alcoholic extracts, for which a high content of amino acids and phenolic compounds has been highlighted, could provide dietary supplements to help in preventing oxidative stress. By investigating the Carmagnola cultivar, six known and four new lignanamides have been identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants.

Comparing Hemp Seed Yields (Cannabis sativa L.) of an On-Farm Scientific Field Experiment to an On-Farm Agronomic Evaluation Under Organic Growing Conditions in Lower Austria
Christian Reinhard Vogl, Gunilla Lissek-Wxsolf, Andreas Surböck
June 2004 Journal of Industrial Hemp 9(1):37-49
Hemp seed yields of the variety Fedora-19 in an on-farm scientific field experiment on small plots and in an on-farm evaluation in 11 hemp fields under practical organic growing conditions in Lower Austria were compared to give a realistic view of the variability of yields. Dry matter seed yields from the on-farm field experiment ranged from 127 to 143 g m2. Under practical growing conditions, yields ranged from 34 to 151 g m2 in the sample plots. The reported hemp seed yield after combine harvesting, drying, and cleaning was between 324 kg ha1 and 717 kg ha1. The results of the experiment show that harvesting by hand considerably influences yields. Yields of the manual harvest in sample plots indicate a high correlation with yields harvested by the combine harvester (R2 = 0.91). The commercial yield is 71% of the yields recorded in sample plots in the fields. Our data questions the transfer of results and conclusions drawn from the data of scientific field experiments that employ manual harvest to that of practical circumstances, and support the notion of on-farm research.

Cannabinoid Profiling of Hemp Seed Oil by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.
Citti, C., Linciano, P., Panseri, S., Vezzalini, F., Forni, F., Vandelli, M. A., & Cannazza, G.
Frontiers in Plant Science, 10.(2019).*
doi:10.3389/fpls.2019.00120
Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.


Fatty Acid and Tocopherol Profiles of Industrial Hemp Cultivars Grown in the High Latitude Prairie Region of Canada
Stanford F Blade, Kwesi Ampong-Nyarko, Roman Przybylski
January 2005 Journal of Industrial Hemp 10(2):33-43
DOI: 10.1300/J237v10n02_04
Hemp (Cannabis sativa L.) is being promoted as a diversification option with applications in fiber, paper, particleboard, and biodegradable plastics. Hemp seed oil is valued for its nutritional properties
and health benefits. Seed protein content and oil composition show promise for animal feed opportunities. The beneficial effects of hemp oil need to be studied to improve the profitability of industrial hemp seed in Canada. This study examined the influence of cultivar on fatty acid profiles and tocopherol content of hemp grown in Alberta in 1997 and 1998. The average seed yield varied between years (1997, 987 kg ha1; 1998, 1633 kg ha1). Linoleic acid concentrations ranged from 47% (Yugo) to 57% (Felina 34) of total fatty acid. Linolenic acid concentrations ranged from 16% (Irene) to 23% (Yugo) of total fatty acid. The gammalinolenic acid varied between 1.5%-4.0% among cultivars. The hemp cultivars varied greatly in the ratio of omega-6 to omega-3 fatty acids, but certain cultivars produced ratios at the 3:1 level, which has been identified as nutritionally optimal. There were varietal differences in both specific and total tocopherol content. The high tocopherol antioxidant capacity, in addition to linolenic acid, alpha-linolenic acid and gamma-linolenic acid, ultimately adds to the nutritional profile of hemp seed oil. All of the cultivars grown in the trials had levels of delta-9 tetrahydrocannabinol (THC) below the Government of Canada limit of 0.3%.

Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.) seed oil extracted by supercritical carbon dioxide
C. Da Porto, D. Decorti, F. Tubaro
Industrial Crops and Products 36 (2012) 401–404
doi:10.1016/j.indcrop.2011.09.015
Supercritical carbon dioxide (SC-CO2) was employed to extract oil from hemp (Cannabis sativa L.) seeds. For ground seeds, the supercritical extraction was carried out at temperatures of 40, 60 and 80 ?C and pressures of 300 and 400 bar. Different solvent-ratios were applied. Supercritical CO2 extractions were
compared with a conventional technique, n-hexane in Soxhlet. The extraction yields, fatty acid composition of the oil and oxidation stability were determined. The seed samples used in this work contained 81% PUFAs, of which 59.6% was linoleic acid (_-6), 3.4% _-linolenic (_-3), and 18% _-linolenic (_-6). The
highest oil yield from seeds was 22%, corresponding to 72% recovery, at 300 bar and 40 ?C and at 400 bar and 80 ?C. The highest oxidation stability corresponding to 2.16mMEq Vit E was obtained at 300 bar and 80 ?C.

Fatty acid composition of Achene oils from five Moroccan climatic cultivars of Cannabis
Abderrahmane Merzouki, Joaquín Molero Mesa
May 1997 Grasas y Aceites 48(3)
DOI: 10.3989/gya.1997.v48.i3.781
The fatty acid composition of achene oil from five Cannabis climatic cultivars cultivated in the nort of Morrocco is determined. Linoleic acid predominated (40 to 45%), followed by linolenic (12 to 17%) and oleic (7 to 10%) acids. Differences in the fatty acid composition of oils are attributed to environmental factors. Se estudia la composición en ácidos grasos del aceite de los aquenios de cinco variedades climáticas del cáñamo ( Cannabis sativa L) cultivadas en el norte de Marruecos. Predomina el ácido linoleico (40 a 45%) seguido por el linolénico (12 a 17%) y el oleico (7 a 10%). Las diferencias en la composición de los ácidos grasos del aceite se atribuyen a factores ambientales.

Fatty acid composition of hempseed oils from different locations in Turkey
M. Kiralan, V. Gül and S. Metin Kara
Spanish Journal of Agricultural Research 2010 8(2), 385-390
https://www.inia.es/gcontrec/pub/385...5034311203.pdf
Recent interest in hempseed as a source of food has largely focused on its oil content and fatty acid profile. The oil content and fatty acid composition (15 fatty acids) of twenty one different hempseed samples of domestic origin from north-western Turkey were monitored. The samples were obtained from seed wholesalers and local spice shops and are of unknown genetic origin. The oil content of the hempseeds ranged between 29.6 to 36.5%. Out of the 15 detected fatty acids, the omega-6 linoleic acid (18:2n-6) was predominant and fluctuated from 55.4 to 56.9%, while the omega- 3 ?-linolenic (18:3n-3) acid ranged from 16.5 to 20.4% and the omega-9 oleic acid (18:1n-9) ranged from 11.4 to 15.9%. Of the minor fatty acids, the highest concentrations were found for ?-linolenic acid (18:3n-6), range 0.6-1.1%, followed by stearidonic acid (18:4n-3), range 0.3-0.5%. These results show that hempseed grown in north-western Turkey provides a well balanced and rich source of dietary omega-6 and -3 essential fatty acids and appears to be a potentially valuable source of food.

Genome Sequence of Paenibacillus polymyxa Strain HOB6, Isolated from Hemp Seed Oil
Mohannad Mahmoud, Suha Jabajia
Microbiol Resour Announc 10
DOI: 10.1128/MRA.00344-21.
https://www.ncbi.nlm.nih.gov/pmc/art...a.00344-21.pdf
Paenibacillus polymyxa strain HOB6 was isolated from hemp seed oil. The strain displays antimicrobial activity against fungal pathogens and has potential for development as a biopesticide against cannabis diseases. Its genome was sequenced and annotated, uncovering the ability to encode the biosynthetic pathways for antimicrobial lanthipeptides and nonribosomal peptides.

Hemp (Cannabis sativa L.) as a Resource for Green Cosmetics: Yield of Seeds and Fatty Acids Composition of 20 Varieties under the Growing Conditions of Organic Farming in Austria
Christian Reinhard Vogl, J. KOBERT, Helga Mölleken, A. Surböck, G. Lissek-Wolf
January 2004 Journal of Industrial Hemp 9(1):51-68
DOI: 10.1300/J237v09n01_05
Hemp seed yields of the variety Fedora-19 in an on-farm scientific field experiment on small plots and in an on-farm evaluation in 11 hemp fields under practical organic growing conditions in Lower
Austria were compared to give a realistic view of the variability of yields. Dry matter seed yields from the on-farm field experiment ranged from 127 to 143 g m2. Under practical growing conditions, yields ranged from 34 to 151 g m2 in the sample plots. The reported hemp seed yield after combine harvesting, drying, and cleaning was between 324 kg ha1 and 717 kg ha1. The results of the experiment show that harvesting by hand considerably influences yields. Yields of the manual harvest in sample plots indicate a high correlation with yields harvested by the combine harvester (R2 = 0.91). The commercial yield is 71% of the yields recorded in sample plots in the fields. Our data questions the transfer of results and conclusions drawn from the data of scientific field experiments that employ manual harvest to that of practical circumstances, and support the notion of on-farm research.

Hemp (Cannabis sativa L.) seed oil: Analytical and phytochemical characterization of unsaponifiable fraction
Sergio Montserrat-de la Paz, Fabiola Marín-Aguilar, M.
Dolores Garcia Gimenez, and Maria Angeles Fernandez-Arche
J. Agric. Food Chem., J• DOI: 10.1021/jf404278q • Publication Date (Web): 15 Jan 2014
DOI: 10.1021/jf404278q
Non-drug varieties of Cannabis sativa L., collectively namely as “hemp”, have been an interesting source of food, fiber and medicine for thousands of years. The ever-increasing demand for vegetables oils has made it essential to characterize additional vegetable oil through innovative uses of its components. The lipid profile showed that linoleic (55%), ?-linolenic (16%) and oleic (11%) were the most abundance fatty acids. A yield (1.84-1.92%) of unsaponifiable matter was obtained and the most interesting compounds were: ?-sitosterol (1905.00 ± 59.27 mg/ Kg oil), campesterol (505.69 ± 32.04 mg / Kg oil), phytol (167.59 ± 1.81 mg/ Kg oil), cycloartenol (90.55 ± 3.44 mg/ Kg oil) and ?-tocopherol (73.38 ± 2.86 mg/ 100 g oil). This study is an interesting contribution for Cannabis sativa L. consideration as a source of bioactive compounds contributing to research novel applications for hemp seed oil in the pharmaceutical, cosmetic food, and other non-food industries.

Hempseed as a nutritional resource: An overview
Euphytica 140: 65–72, (2004).
J.C. Callaway
DOI: 10.1007/s10681-004-4811-6
The seed of Cannabis sativa L. has been an important source of nutrition for thousands of years in Old World cultures. Non-drug varieties of Cannabis, commonly referred to as hemp, have not been studied extensively for their nutritional potential in recent years, nor has hempseed been utilized to any great extent by the industrial processes and food markets that have developed during the 20th century. Technically a nut, hempseed typically contains over 30% oil and about 25% protein, with considerable amounts of dietary fiber, vitamins and minerals. Hempseed oil is over 80% in polyunsaturated fatty acids (PUFAs), and is an exceptionally rich source of the two essential fatty acids (EFAs) linoleic acid (18:2 omega-6) and alpha-linolenic acid (18:3 omega-3). The omega-6 to omega-3 ratio (n6/n3) in hempseed oil is normally between 2:1 and 3:1, which is considered to be optimal for human health. In addition, the biological metabolites of the two EFAs, gamma-linolenic acid (18:3 omega-6; ‘GLA’) and stearidonic acid (18:4 omega-3; ‘SDA’), are also present in hempseed oil. The two main proteins in hempseed are edestin and albumin. Both of these high-quality storage proteins are easily digested and contain nutritionally significant amounts of all essential amino acids. In addition, hempseed has exceptionally high levels of the amino acid arginine. Hempseed has been used to treat various disorders for thousands of years in traditional oriental medicine. Recent clinical trials have identified hempseed oil as a functional food, and animal feeding studies demonstrate the long-standing utility of hempseed as an important food resource.

Hempseed Oil—Influence of the Genotype on the Composition in a Two-Year Study
Bertrand Matthäus, Erika Schumann, Ludger Brühl, Uta Kriese
January 2006 Journal of Industrial Hemp 10(2):45-65
DOI: 10.1300/J237v10n02_05
Seeds of 51 different hemp genotypes, 25 hemp varieties, 19 hemp accessions as well as 7 hemp samples from a cross-breeding program of a Hungarian Institute (GATE), grown in 2000 and 2001 were investigated regarding the oil content as well as fatty acid and tocopherol composition. The oil content ranged between 26.3 in 2001 and 37.5 g/ 100 g in 2000 with 33.2 g/100 g as mean amount in 2000 and 31.2 g/100 g in 2001. The oil contained high amounts of unsaturated fatty acids like linoleic and ?-linolenic acid, which comprised about 80% of the total fatty acids. Hemp seed oil also consisted of remarkable amounts of ?-linolenic and stearidonic acid. The concentrations varied from 0.67 to 4.08 g/100 g and 0.39 to 1.60 g/100 g, respectively, in dependence of the genotype. The content of ?-linolenic and stearidonic acid were both highly correlated (r = 0.87), while ?-tocopherol was not correlated to the total sum of polyunsaturated fatty acid. Tocopherols mainly consisted of ?-tocopherol (36 to 97 mg/100 g). This made up to about 85% of the total tocopherols. In the oil also small amounts of ?-tocopherol were found (2 to 10 mg/100 g). The effect of the year of cultivation on the composition of the oil was less pronounced than the influence of the genotype. Therefore the seed material, presented in this paper shows interesting possibilities to improve the composition of hemp seed oil

Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes
U. Kriese, E. Schumann, W.E. Weber, Matthäus
Euphytica 137(3):339-351 June 2004
DOI: 10.1023/B:EUPH.0000040473.23941.76
The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25% (w/w) to 37.50%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 1.45% in 2000 and 31.21 0.96% in 2001. The -tocopherol, -tocopherol, -tocopherol, P-8- and -tocopherol contents of the 51 genotypes averaged 21.68 3.19, 1.82 0.49, 1.20 0.40, 0.18 0.07 and 0.16 0.04 mg 100g–1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The -linolenic acid yield of hemp (3–30 kg ha–1) was similar to the -linolenic acid yield of plant species that are currently used as sources of -linolenic acid (borage (19–30 kg ha–1), evening primrose (7–30 kg ha–1)). The linoleic acid yield of hemp (129–326 kg ha–1) was similar to flax (102–250 kg ha–1), but less than in sunflower (868–1320 kg ha–1). Significant positive correlations were detected between some fatty acids and

Preliminary Results of Selection for Seed Oil Content in Hemp (Cannabis sativa L.)
I. Bócsa, Finta-Korpelová, P. Máthé
June 2005 Journal of Industrial Hemp 10(1):5-15
DOI: 10.1300/J237v10n01_02
The ‘Kompolti’ variety and the ‘Fibrimon 21-63’ hybrid parent were selected for high oil content and the resulting lines were named ‘Kolaj’ and ‘Fibrolaj’, respectively. Individual selection was carried
out on the basis of pressed oil content. The selection gains calculated from the pressing data were checked using the extraction method. For this purpose an experiment was set up in a random block design with four replications, taking selection as the treatment. In the case of ‘Kolaj’ five years of selection resulted in a 2.96% increase on the basis of the pressed oil content and in a selection gain of 2.83% according to the data of extraction with hexane. The pressing method could not be applied in ‘Fibrolaj’ in two years due to the small seed yield, so no selection could be carried out in these years. This meant that selection was only carried out three times between 1997 and 2002. Analysis of variance indicated a higher selection gain for the extracted oil content (1.38%) than for the pressed oil content (0.87%) in ‘Fibrolaj’. At present the pressed oil content of ‘Kolaj’ is 29.8% and the extracted oil content 35.4%, while for ‘Fibrolaj’ these figures are 29.7% and 35.9%, respectively

Not Cannabis specific
Preventing seed quality loss during storage. The role of oxygen is underestimated
Steven P.C. Groot
https://www.researchgate.net/publica...underestimated
DOI: 10.13140/2.1.5013.3447
Crop seeds can often survive for a considerable time under dry conditions, but eventually they lose their germination capacity due to accumulating damage. There is a large variation in shelf life between seeds of different crops. Whereas primed seeds deteriorate relatively fast. To reduce the rate of ageing and prolong shelf life of the seeds, it is recommended to store the seeds dry and cool. Most if not all damage is related to oxidation of proteins, membranes, DNA, mRNA and lipids. Although the effect of both low temperatures and low moisture seed moisture content is through a reduction in the rate of oxidation, the role of oxygen in the storage environment is hardly or not considered. The deleterious effects of oxygen during dry seed storage are presented, as well methods to reduce oxygen levels during dry seed storage and prolong the shelf life of seeds.

Not Cannabis specific
[COLOR=“red"]*Prolonging the longevity of ex situ conserved seeds by storage under anoxia.[/COLOR]
Groot, S. P. C., de Groot, L., Kodde, J., & van Treuren, R.
Plant Genetic Resources, 13(01), 18–26.(2014).
doi:10.1017/s1479262114000586
Plant genetic resources are conserved by genebanks mainly in the form of seeds. In most of the cases, the dried seeds can be stored for a considerable period of time, but eventually seed deterioration results in the inability to generate healthy seedlings. Prolonging seed longevity during storage reduces the frequency of regeneration, which is beneficial from a genetic as well as a management point ofview. To reduce the rate ofdeterioration,cool and dry storage conditions are usually practised for long-term seed storage. In spite of the growing body of evidence that seed deterioration is predominantly caused by oxidative processes, the importance of seed storage under anoxic conditions has received little attention from the genebank community. Herein, we report on the effects of anoxia on seed viability, the oxygen uptake by dry seeds in closed containers and the permeability for oxygen of various seed storage containers. Our results confirm that the ageing of dry seeds is accelerated by the presence of oxygen in the storage environment. Therefore, we recommend that genebanks store dry seeds under anoxic conditions to prolong their longevity during ex situ conservation. To reduce the initial rate of viability loss, we further recommend that the period of temporary storage after seed harvest be minimized and also that the seeds are kept during this period under controlled conditions, including anoxia.

Relationship between cannabinoids content and composition of fatty acids in hempseed oils.
Petrovi?, M., Debeljak, Ž., Kezi?, N., & Džidara, P.
Food Chemistry, 170, 218–225. (2015).
doi:10.1016/j.foodchem.2014.08.039
Hempseed oils acquired on the Croatian markets were characterised by cannabinoid content and fatty acid composition. The new method for determination of cannabinoid content was developed and validated in the range of 0.05–60 mg/kg, and the content of tetrahydrocannabinol varied between 3.23 and 69.5 mg/kg. Large differences among the samples were obtained for phenotype ratio suggesting that not all of analysed hempseed oils were produced from industrial hemp. Sample clustering based on cannabinoid content assigned samples to two groups closely related to the phenotype ratios obtained. The results of this study confirm that hempseed oil is a good source of polyunsaturated fatty acids, especially c-linolenic and stearidonic acid, but the content varies a lot more than the omega-6/omega-3 ratio. The grouping of samples on fatty acid content assigned samples to two groups which were consistent with the groups obtained based on cannabinoid content clustering

Not Cannabis specific
Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions.
Groot, S. P. C., Surki, A. A., de Vos, R. C. H., & Kodde, J. Annals of Botany, 110(6), 1149–1159.(2012).
doi:10.1093/aob/mcs198
† Background and Aims Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen. † Methods Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 8C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed. †Key Results The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under longterm dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 8C after equilibration at 85 % RH. †Conclusions Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice

Survey of minor fatty acids in Cannabis sativa L. fruits of various origins
Helga Mölleken and Roland R. Theimer
Journal of the International Hemp Association 4(1): 13-17.
https://www.internationalhempassocia.. ./jiha4107.html
Interest in the fruits of Cannabis sativa has focused on the composition of the fat because of its growing importance for nutritional and pharmaceutical purposes. We have therefore initiated analyses of fatty acids (FAs) in the fruit and its compartments. Cannabis fruits provide an oil with a favorable fraction of certain very desirable FAs, including ?-linolenic acid (GLA; 18:3, cis-6,9,12), stearidonic acid (SDA; 18:4, cis-6,9,12,15), and eicosenoic acid (EA; 20:1, cis-11). Origin of the seed seems to influence FA composition. The FA profile is different in the various compartments of the fruit and points to a possible physiological role for these compounds.

Synthesis and Analytical Study of Fatty Acid Methyl Ester of Cannabis sativa (Hemp) Seed Oil in the Presence of Triglycerides
Shobha S. Borhade
https://www.statperson.com/Journal/S...ctra/PDF/2.pdf
Hemp seed oil contains linoleic acid (LA) and linolenic acid (LNA) as its major omega-6 and omega-3 polyunsaturated fatty acids (PUFA), respectively It is necessary to apply transesterification procedure to convert fatty acids into more volatile compounds such as fatty acid methyl ester (FAME). Fatty acid methyl ester is made virgin or used vegetable oils (both edible and nonedible) and animal fats. Fatty acid methyl ester operates in compression ignition engines like petro-disel. Fatty acid methyl ester can be blended in any ratio with petroleum disel fuels. It can be stored just like the petroleum disel fuel. Petrodisel can be replaced by biodiesel due to its superiority. It has various advantages. The seed of cannabis sativa (Hemp) contain 32.21 % oil. Methyl ester were prepared from seed oil containing a high free fatty acid content by using a two stage process. Sulphuric acid was used as catalyst in an esterification reaction which was then followed by a transesterification reaction using hydroxide as the catalyst. It was found that methanol and sulphuric acid were suitable to perform the transesterificatin reaction. Fatty acid methyl ester was characterized by UV, IR and LCMS.

Targeted mutation of ?12 and ?15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil
Monika Bielecka, Filip Kaminski, Ian Adams, Helen Poulson, Raymond Sloan, Yi Li,
Tony R. Larson, Thilo Winzer, Ian A. Graham
Plant Biotechnology Journal (2014) 12, pp. 613–623
doi: 10.1111/pbi.12167
We used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic?based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven ?12 desaturases and one of three ?15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have ?x+3 activity, while CSFAD3A activity was exclusively at the ?15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non?sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major ?12 and ?15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a?1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1?9) oil at yields similar to wild type. Cold?pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2?6,9 and 18:2?9,15, were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2?9,15 from endogenous 18:1?9 when expressed inS. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop.

Temperature and Moisture Content for Storage Maintenance of Germination Capacity of Seeds of Industrial Hemp, Marijuana, and Ditchweed Forms of Cannabis sativa
Ernest Small & Brenda Brookes
Journal of Natural Fibers, 9:240–255, 2012
DOI: 10.1080/15440478.2012.737179
Cannabis sativa seeds of three industrial hemp cultivars, a medicinal strain of marijuana, and a ruderal strain were subjected to combinations of four temperatures (20?C, 5?C, ?20?C, and ?80?C) and three seed moisture contents (approximately 11%, 6%, and 4%) for 66 months. Storage of seeds with a moisture content of 11% at 20?C reduced the germinability of seeds of all varieties to zero in less than 18 months. Either reducing the temperature to at
least 5?C or reducing the seed moisture content to at least 6% had a huge beneficial effect on maintaining seed viability. Additional reduction of temperature, but not additional reduction of moisture content had a small supplementary beneficial effect. No apparent benefit was noticed from oxygen-free seed storage.

The Composition of Hemp Seed Oil and Its Potential as an Important Source of Nutrition.
Leizer, C., Ribnicky, D., Poulev, A., Dushenkov, S., & Raskin, I.
Journal of Nutraceuticals, Functional & Medical Foods, 2(4), 35–53.(2000).
doi:10.1300/j133v02n04_04
The fatty acid and natural product content of hemp seed oil was analyzed by GC-MS and LC-MS. The presence of linoleic acid (LA) and -linolenic acid (LNA) were confirmed in their previously reported ratio of 3:1 LA:LNA. The presence of -caryophyllene (740 mg/L), myrcene (160 mg/L), -sitosterol (100-148 g/L) and trace amounts of methyl salicylate was observed in the oil which had not been previously reported. Trace amounts of cannabidiol (CBD) were also detected. Bioassays were performed with the oil to determine its effectiveness as an antimicrobial agent. Some bioactivity was observed during the primary screening.

The CPRO Cannabis germplasm collection
E.P.M. de Meijer & L.J.M. van Soest
Euphytica 62: 201-211, 1992.
DOI:10.1007/BF00041754
In an effort to reduce the overproduction of a too limited number of arable food crops, several research programmes dealing with industrial crops
have been initiated in the Netherlands. The 'National Hemp Programme' investigates the feasibility of hemp (Cannabis sativa L.) as a raw material
for paper pulp production. In this context breeding, agronomy, mechanisation and processing of hemp are subject of a comprehensive study. Two previous attempts to introduce fibre hemp in the Netherlands have been reported (de Jonge, 1944; Friederich, 1960). Breeding of fibre hemp in the Netherlands is unprecedented. In the framework of the current programme, improvement of cultivars is considered necessary in order to increase yield, quality, and disease resistance. A collection of Cannabis accessions is therefore being established at CPRO since 1988. In this report, the present state of the collection and its maintenance are described.

The feasibility of converting Cannabis sativa L. oil into biodiesel
Si-Yu Li, James D. Stuart, Yi Li, Richard S. Parnas
Bioresource Technology 101 (2010) 8457–8460
doi:10.1016/j.biortech.2010.05.064
Cannabis sativa Linn, known as industrial hemp, was utilized for biodiesel production in this study. Oil from hemp seed was converted to biodiesel through base-catalyzed transesterification. The conversion is greater than 99.5% while the product yield is 97%. Several ASTM tests for biodiesel quality were implemented on the biodiesel product, including acid number, sulfur content, flash point, kinematic viscosity, and free and total glycerin content. In addition, the biodiesel has a low cloud point (_5 _C) and kinematic viscosity (3.48 mm2/s). This may be attributed to the high content of poly-unsaturated fatty acid of hemp seed oil and its unique 3:1 ratio of linoleic to a-linolenic acid.

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications
Salvador Soler, Dion?s Borras, Santiago Vilanova, Alicia Sifres, Isabel
Andujar, Maria R. Figas, Ernesto R. Llosa, and Jaime Prohens
J Forensic Sci, March 2016, Vol. 61, No. 2
doi: 10.1111/1556-4029.12995
Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis



IC Classification/Taxonomy


A CHEMOTAXONOMIC ANALYSIS OF CANNABINOID VARIATION IN CANNABIS (CANNABACEAE)
Karl Hillig
American Journal of Botany 91(6): 966–975. 2004.
DOI: 10.3732/ajb.91.6.966
Cannabinoids are important chemotaxonomic markers unique to Cannabis. Previous studies show that a plant’s dry-weight ratio of D9-tetrahydrocannabinol (THC) to cannabidiol (CBD) can be assigned to one of three chemotypes and that alleles BD and BT encode alloenzymes that catalyze the conversion of cannabigerol to CBD and THC, respectively. In the present study, the frequencies of BD and BT in sample populations of 157 Cannabis accessions were determined from CBD and THC banding patterns, visualized by starch gel electrophoresis. Gas chromatography was used to quantify cannabinoid levels in 96 of the same accessions. The data were interpreted with respect to previous analyses of genetic and morphological variation in the same germplasm collection. Two biotypes (infraspecific taxa of unassigned rank) of C. sativa and four biotypes of C. indica were recognized. Mean THC levels and the frequency of BT were significantly higher in C. indica than C. sativa. The proportion of high THC/CBD chemotype plants in most accessions assigned to C. sativa was ,25% and in most accessions assigned to C. indica was .25%. Plants with relatively high levels of tetrahydrocannabivarin (THCV) and/or cannabidivarin (CBDV) were common only in C. indica. This study supports a two-species concept of Cannabis.

A chemotaxonomic analysis of terpenoid variation in Cannabis
Karl Hillig
Biochemical Systematics and Ecology 32 (2004) 875–891
doi:10.1016/j.bse.2004.04.004
To determine whether the terpenoid composition of the essential oil of Cannabis is useful for chemotaxonomic discrimination, extracts of pistillate inflorescences of 162 greenhouse grown plants of diverse origin were analyzed by gas chromatography. Peak area ratios of 48 compounds were subjected to multivariate analysis and the results interpreted with respect to geographic origin and taxonomic affiliation. A canonical analysis in which the plants were pre-assigned to C. sativa or C. indica based on previous genetic, morphological, and chemotaxonomic studies resulted in 91% correct assignment of the plants to their pre-assigned species. A scatterplot on the first two principal component axes shows that plants of accessions from Afghanistan assigned to the wide-leaflet drug biotype (an infraspecific taxon of unspecified rank) of C. indica group apart from the other putative taxa. The essential oil of these plants usually had relatively high ratios of guaiol, isomers of eudesmol, and other unidentified compounds. Plants assigned to the narrow-leaflet drug biotype of C. indica tended to have relatively high ratios of trans-b-farnesene. Cultivars of the two drug biotypes may exhibit distinctive medicinal properties due to significant differences in terpenoid composition

A classification of endangered high-THC cannabis (Cannabis sativa subsp. indica) domesticates and their wild relatives
John M. McPartland, Ernest Small
PhytoKeys 144: 81–112 (2020)
doi: 10.3897/phytokeys.144.46700
Two kinds of drug-type Cannabis gained layman’s terms in the 1980s. “Sativa” had origins in South Asia (India), with early historical dissemination to Southeast Asia, Africa, and the Americas. “Indica” had origins in Central Asia (Afghanistan, Pakistan, Turkestan). We have assigned unambiguous taxonomic names to these varieties, after examining morphological characters in 1100 herbarium specimens, and analyzing phytochemical and genetic data from the literature in a meta-analysis. “Sativa” and “Indica” are recognized as C. sativa subsp. indica var. indica and C. sativa subsp. indica var. afghanica, respectively. Their wildgrowing relatives are C. sativa subsp. indica var. himalayensis (in South Asia), and C. sativa subsp. indica var. asperrima (in Central Asia). Natural selection initiated divergence, driven by climatic conditions in South and Central Asia. Subsequent domestication drove further phytochemical divergence. South and Central Asian domesticates can be distinguished by tetrahydrocannabinol and cannabidiol content (THC/CBD ratios, ?7 or <7, respectively), terpenoid profiles (absence or presence of sesquiterpene alcohols), and a suite of morphological characters. The two domesticates have undergone widespread introgressive hybridization in the past 50 years. This has obliterated differences between hybridized “Sativa” and “Indica” currently available. “Strains” alleged to represent “Sativa” and “Indica” are usually based on THC/CBD ratios of plants with undocumented hybrid backgrounds (with so-called “Indicas” often delimited simply on possession of more CBD than “Sativas”). The classification presented here circumscribes and names four taxa of Cannabis that represent critically endangered reservoirs of germplasm from which modern cannabinoid strains originated, and which are in urgent need of conservation.

A Combined Analysis of Agronomic Traits and Allozyme Allele Frequencies for 69 Cannabis Accessions
Karl Hillig
Journal of Industrial Hemp, 10:1, 17-30,
DOI: 10.1300/J237v10n01_03
This study reports on a combined analysis of phenotypic traits and allozyme allele frequencies for 69 accessions from the CPRO Cannabis germplasm collection. Sample populations of each accession
were evaluated for achene traits, cannabinoid content, days to maturity, plant height, leaflet shape, fiber traits, and root-knot nematode resistance. Sample populations were also assayed for 11 enzymes using starch gel electrophoresis and the frequencies of 49 alleles encoded at 17 gene loci were determined. The phenotypic and genetic data sets were combined and analyzed by principal components (PC) analysis. A scatter plot on the first two PC axes shows that southern and eastern Asian hemp (fiber/seed) accessions group apart from hemp accessions from Europe, Asia Minor, and central Asia. On average, the former group matured later and had significantly higher levels of THC and lower levels of CBD, wider leaflets relative to their length, longer xylem fibers, and lower bast fiber content.

A Multivariate Analysis of Allozyme Variation in 93 Cannabis Accessions from the VIR Germplasm Collection
Karl Hillig
Journal of Industrial Hemp, 9:2, 5-22,
DOI: 10.1300/J237v09n02_02
The Vavilov Institute (VIR) in Russia maintains the world’s largest collection of Cannabis sativa hemp germplasm through periodic seed regeneration. Sample populations of 93 accessions from the VIR collection were assayed for allozyme variation at 17 gene loci by starch-gel electrophoresis. The frequencies of 47 alleles were subjected to principal components (PC) analysis. A scatter plot on the first two PC axes showed little tendency for the accessions to cluster according to country of origin or their assignment to eco-geographical groups. Three Chinese accessions previously assigned to the hemp biotype of C. indica clustered separately. Eleven other Chinese accessions clustered with accessions from Europe, Asia Minor, and central Asia, and are likely to be hybridized. Conservation of C. indica hemp landraces is of vital importance to the hemp industry.

A new Cannabis germplasm classification system and research strategies of non-psychoactive medicinal cannabis.
[Article in Chinese]
Li QS1, Meng Y2, Chen SL1.
Zhongguo Zhong Yao Za Zhi. 2019 Oct;44(20):4309-4316.
doi: 10.19540/j.cnki.cjcmm.20190916.101.
At present,China manages cannabis into industrial and medical types. Industrial cannabis( THC<0. 3%) is grown for fiber and seed,while medical cannabis( THC>0. 3%) is prohibited from being planted and used. In recent years,breakthroughs have been made in the applications for a variety of fields of CBD,a non-psychoactive cannabinoid of Cannabis. However,China made a minimal contribution to this international research hotspot,mainly due to the outdated way of classification on cannabis. Here we reviewed the medicinal use history ofcannabis,achievements in the study of Cannabis germplasm,and first proposed the three-level classification of management: psychoactive medicinal cannabis( THC>0. 3%),non-psychoactive medicinal cannabis( THC<0. 3%,high CBD)and industrial cannabis( THC<0. 3%,for fiber and seed,low CBD). Based on summarizing the research progress of the non-psychoactive medicinal cannabis,four key research strategies of breeding,sex control,plant factory,and synthetic biology are proposed.

A Practical and Natural Taxonomy for Cannabis
Ernest Small and Arthur Cronquist
Taxon, Vol. 25, No. 4 (Aug., 1976), pp. 405-435
DOI: 10.2307/1220524
Variation in Cannabis is evaluated in the context of the confusing systematic history of this genus. Aside from some experimentally produced polyploids, all Cannabis is diploid (n = io), and there appear to be no barriers to successful hybridization within the genus. The present pattern of variation is due in large part to the influence of man. Two widespread classes of plant are discernible: a group of generally northern plants of relatively limited intoxicant potential, influenced particularly by selection for fibre and oil agronomic qualities, and a group of generally southern plants of considerable intoxicant potential, influenced particularly by selection for inebriant qualities. These two groups are treated respectively as subsp. sativa and indica, of C. sativa, the only species of the genus Cannabis. Within each subspecies two parallel phases are recognizable. The "wild" (weedy, naturalized or indigenous) phase is more or less distinguishable from the domesticated (cultivated or spontaneous) phase by means of an adaptive syndrome of fruit characteristics. The resulting four discernible groups are recognized as varieties.

A Proposal to Establish a Comprehensive Cannabis Varietal Registry in Washington State
David J. Mighell, Dominic Corva, Michelle Sexton
https://visionwashington.files.wordp.. .r_06162013.pdf
Current analytic methods for identifying and tracking cannabis cultivars (“strains”) currently test for THC, CBD and CBN content, which constitute only three of the approximately 30 principal chemical compounds represented in cannabis varietals in various proportions. In fact, it is the precise combination of the secondary metabolites – terpenes, flavonoids and minor cannabinoids - that are responsible for various qualities of the human/plant biochemical interaction. The authors assert that capturing and understanding a more complex and precise profile of commercial cannabis products is necessary to inform enlightened policy regulation.
At the same time, scientists have now demonstrated the means to reliably capture exhaustive chemical profiles of plant matter, and importantly, have demonstrated methods to use the resulting quantitative data to chemically distinguish cannabis varietals from one another. This means that we now have the capability to classify cannabis strains by common compositional relationships, and identify morphological change in plants over time and responsive to growing conditions. Thus, we can baseline cannabis products made available to Washington consumers, and manage important dimensions of product continuity and quality assurance that are not currently addressed in draft regulations.
We propose to establish a lab in Washington State that can collect comprehensive cultivar profiles and analyze them in support of Washington State policy goals, in particular the need to establish analytic standards that are based on robust scientific data that currently does not exist. The data collection and analysis done by the lab will be used to create a comprehensive Cannabis Varietal Registry based on chemo-taxonomic analytical methods that are far more comprehensive and informative than we expect to be reflected in final rules for recurrent crop testing for safety and potency.

A Systematic Investigation of Cannabis
Thesis for: Karl Hillig Ph.D.Advisor: Paul G. Mahlberg March 2005
DOI: 10.13140/RG.2.1.2648.3680
https://www.researchgate.net/publica...on_of_Cannabis
Botanists disagree whether Cannabis (Cannabaceae) is a monotypic or polytypic genus. A systematic investigation was undertaken to elucidate underlying evolutionary and taxonomic relationships within the genus. Genetic, morphological, and chemotaxonomic analyses were conducted on 157 Cannabis accessions of known geographic origin. Sample populations of each accession were surveyed for allozyme variation at 17 gene loci. Principal component (PC) analysis of the allozyme allele frequencies revealed that most accessions were derived from two major gene pools corresponding to C. sativa L., and C. indica Lam. A third putative gene pool corresponds to C. ruderalis Janisch. Previous taxonomic treatments were tested for goodness of fit to the pattern of genetic variation. Based on these results, a working hypothesis for a taxonomic circumscription of Cannabis was proposed that is a synthesis of previous polytypic concepts. Putative infraspecific taxa were assigned to “biotypes” pending formal taxonomic revision. Genetic variation was highest in the hemp and feral biotypes and least in the drug biotypes. Morphometric traits were analyzed by PC and canonical variates (CV) analysis. PC analysis failed to differentiate the putative species, but provided objective support for recognition of infraspecific taxa of C. sativa and C. indica. CV analysis resulted in a high degree of discrimination of the putative species and infraspecific taxa. Variation in qualitative and quantitative levels of cannabidiol (CBD), tetrahydrocannabinol (THC), and other cannabinoids was determined, as were frequencies of alleles that control CBD and THC biosynthesis. The patterns of variation support a two-species concept, but not recognition of C. ruderalis as a separate species from C. sativa. PC analysis of terpenoid variation showed that the wide-leaflet drug (WLD) biotype of C. indica produced enhanced mean levels of guaiol and isomers of eudesmol, and is distinct from the other putative taxa. In summary, the results of this investigation show that a taxonomic revision of Cannabis is warranted. However, additional studies of putative wild populations are needed to further substantiate the proposed taxonomic treatment.

Cannabis — a polytypic genus
William Allen Emboden
Published in Economic Botany 1974, 28(3), 304–310.
DOI:10.1007/BF02861427
It has been a general assumption by botanists who have not worked taxonomically on the genus that Cannabis is monotypic. The preponderance of literature has treated it as such in the absence of any thorough taxonomic review to establish whether the epithet sativa must be restricted to a single morphologically distinct taxon within a more variable genus than presupposed or whether the specimens and literature concerning Cannabis permit the recognition of more than one specific epithet, in accordance with the most recent appearance of the International Code of Botanical Nomenclature.

Cannabis: An Example of Taxonomic Neglect.
Cannabis and Culture.
RICHARD EVANS SCHULTES, WILLIAM M. KLEIN, TIMOTHY PLOWMAN, and TOM E. LOCKWOOD
Botanical Museum Leaflets, Harvard University
Vol. 23, No. 9 (February 28, 1974), pp. 337-367
doi:10.1515/9783110812060.21
Native apparently somewhere in central Asia, cannabis is at present one of the most widely disseminated cultivated plants. Cannabis as we know it has developed together with man as a multi-purpose economic plant; and, as a result of selection for desirable characteristics, it has become one of the most variable of cultivated plants. Due to the extraordinary plasticity and variability of cultivated cannabis, there can be no progress in unravelling the taxonomic complexities in the genus until the biology of the wild populations is investigated. The genus has been and still is widely considered to be monotypic, especially by botanists who have not studied the classification of the genus in depth: we believe this results from lack of taxonomic investigations of wild cannabis in its native habitat or even of comparative studies of the range of variation in cultivated hemp. A polytypic concept of the genus is not new: in 1783 Lamarck recognized Cannabis imlica as "very distinct" from the species which Linnaeus had named Cannabis sativa. In 1924, Russian botanists, who studied wild populations of cannabis, recognized a third species, Cannabis ruderalis. Their work has not been widely accepted, partly due to conservative attitudes to changing established beliefs in the monotypic nature of the genus. Several British and American taxonomists who have investigated the genus now favor the polytypic concept. There may be significant chemical differences in content of cannabinolic and other constituents among the species. The paper reviews the taxonomic history of the genus and presents data in support of the polytypic concept.

Cannabis chemovar classification: terpenes hyper-classes and targeted genetic markers for accurate discrimination of flavours and effects
Philippe Henry PhD
Doi: 10.7287/peerj.preprints.3307v1
https://peerj.com/preprints/3307v1.pdf
The classification of Cannabis varieties has been increasingly discussed in the past years, particularly in the wake of emerging legal markets, with implications for intellectual property development, marketing and improvement of the scientific understanding of this contentious plant. While the concept of chemovars has been proposed and has gained popularity of late, the lack of guidance in introducing this concept and the fact that chemovars are based on indirectly assessed traits with a heritable basis has likely impeded the implementation of the concept to a broader audience. Here I propose a simplified version of terpene hyper-classes based on three dominant terpenes that is shown to outperformed the classic indica-sativa-hybrid scheme of classification as well as a recently proposed terpene super-class scheme. This information was used to identify the most informative genetic markers for chemovar classification based on the terpene hyperclasses. I demonstrate the ability of clearly clustering accessions based on their dominant terpene and propose to extent this approach as a benchmark for chemovar classification in lieu of previously proposed model

Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples
Ulrich Reimann-Philipp, Mark Speck, Cindy Orser, Steve Johnson, Aaron Hilyard, Helen Turner, Alexander J. Stokes, and Andrea L. Small-Howard
Cannabis and Cannabinoid Research
Volume ?, Number ?, 2019
DOI: 10.1089/can.2018.0063
Introduction: Medical cannabis patients receive clinical benefits from the secondary metabolites of the plant, which contain a variety of cannabinoids and terpenoids in combinations that can be used to classify the chemovars. State-regulated medical cannabis programs rely on breeder-reported ‘‘strain’’ names both within diversion control systems and to describe the medical cannabis products that are sold to patients in medical cannabis dispensaries. In state-regulated medical cannabis programs, there is no conventional nomenclature system that correlates
the breeder-reported names with their profiles of active ingredients, and these ‘‘strain’’ names are invalid as they refer to chemical differences properly referred to as to chemovars.
Materials and Methods: To determine the actual levels of chemical diversity represented in 2662 samples of Cannabis flower collected between January 2016 and June of 2017 in Nevada, chemical profile data were measured from these samples by a state-qualified third-party testing laboratory. Principal component analysis (PCA) was used to define clusters in data sets representing both cannabinoids and terpenoids, cannabinoids only, or terpenoids only.
Results: The PCA of the terpenoid only data set revealed three well-defined clusters. All three terpenoids only data clusters had high tetrahydrocannabinolic acid synthase, but the terpene profiles listed in reverse-order of abundance best defined these chemovars. The three chemovars in Nevada were labeled with 396 breeder reported sample names, which overestimate the diversity and do not inform patients regarding chemical properties. Representative DNA samples were taken from each chemovar to determine whether the genetic diversity was greater than the chemical diversity. The limited genotyping experiment was based on DNA sequence polymorphisms. The genetic analysis revealed twelve distinct genetic clades, which still does not account for the entirety of the 396 reported sample names. The finite genotypes did not correlate with the chemotypes determined for the samples. This suggests that either the DNA-markers used were too narrowly restricted for factual separation or that environmental factors contributed more significantly to the chemical profiles of cannabis than genetics.
Conclusion: The three chemovars and twelve genotypes reflect low medical diversity on the market in Nevada
during its ‘‘medical use only’’ phase. Furthermore, the 396 breeder-reported sample names within this set imply a false sense of diversity of products in Nevada dispensaries.


Cannabis: from cultivar to chemovar II-a metabolomics approach to Cannabis
classification.

Hazekamp A, Tejkalová K, Papadimitriou S (2016)
Cannabis Cannabinoid Res 1: 202–215
DOI: 10.1089/can.2016.0017
https://pdfs.semanticscholar.org/01a...808.1560054937
Introduction: There is a large disparity between the ''cultural'' language used by patients using cannabis for self-medication and the ''chemical'' language applied by scientists to get a deeper understanding of cannabis effects in laboratory and clinical studies. The distinction between Sativa and Indica types of cannabis, and the different biological effects associated with them, is a major example of this. Despite the widespread use of cannabis by self-medicating patients, scientific studies are yet to identify the biochemical markers that can sufficiently explain differences between cannabis varieties.
Methods: A metabolomics approach, combining detailed chemical composition data with cultural information available for a wide range of cannabis samples, can help to bridge the existing gap between scientists and patients. Such an approach could be helpful for decision-making, for example, when identifying which varieties of cannabis should be made legally available under national medicinal cannabis programs. In our study, we analyzed 460 cannabis accessions obtained from multiple sources in The Netherlands, including hemp-and drug-type cannabis.
Results: Based on gas chromatography analysis of 44 major terpenes and cannabinoids present in these samples , followed by Multivariate Data Analysis of the resulting chromatographic data, we were able to identify the cannabis constituents that may act as markers for distinction between Indica and Sativa. This information was subsequently used to map the current chemical diversity of cannabis products available within the Dutch medicinal cannabis program, and to introduce a new variety missing from the existing product range.
Conclusion: This study represents the analysis of the widest range of cannabis constituents published to date. Our results indicate the usefulness of a metabolomics approach for chemotaxonomic mapping of cannabis varieties for medical use.

Cannabis Systematics at the Levels of Family, Genus, and Species
John M. McPartland
Cannabis and Cannabinoid Research
Volume 3.1, 2018
DOI: 10.1089/can.2018.0039
New concepts are reviewed in Cannabis systematics, including phylogenetics and nomenclature. The family Cannabaceae now includes Cannabis, Humulus, and eight genera formerly in the Celtidaceae. Grouping Cannabis, Humulus, and Celtis actually goes back 250 years. Print fossil of the extinct genus Dorofeevia (=Humularia) reveals that Cannabis lost a sibling perhaps 20 million years ago (mya). Cannabis print fossils are rare (n = 3 worldwide), making it difficult to determine when and where she evolved. A molecular clock analysis with chloroplast DNA (cpDNA) suggests Cannabis and Humulus diverged 27.8 mya. Microfossil (fossil pollen) data point to a center of origin in the northeastern Tibetan Plateau. Fossil pollen indicates that Cannabis dispersed to Europe by 1.8– 1.2 mya. Mapping pollen distribution over time suggests that European Cannabis went through repeated genetic bottlenecks, when the population shrank during range contractions. Genetic drift in this population likely initiated allopatric differences between European Cannabis sativa (cannabidiol [CBD] >D9-tetrahydrocannabinol [THC]) and Asian Cannabis indica (THC > CBD). DNA barcode analysis supports the separation of these taxa at a subspecies level, and recognizing the formal nomenclature of C. sativa subsp. sativa and C. sativa subsp. indica. Herbarium specimens reveal that field botanists during the 18th–20th centuries applied these names to their collections rather capriciously. This may have skewed taxonomic determinations by Vavilov and Schultes, ultimately giving rise to today’s vernacular taxonomy of ‘‘Sativa’’ and ‘‘Indica,’’ which totally misaligns with formal C. sativa and C. indica. Ubiquitous interbreeding and hybridization of ‘‘Sativa’’ and ‘‘Indica’’ has rendered their distinctions almost meaningless.

Cannabis Taxonomy: The 'Sativa' versus 'Indica' Debate
By Robert C. Clarke and Mark D. Merlin, PhD
HerbalEGram: Volume 13, Issue 4, April 2016
https://cms.herbalgram.org/herbalgra...63299d67c54e61

Classification of Cannabis
Mark Lewis
https://youtube.com/@medicinal.genomics?si=BOg_Qde9I2E-JdXa

Nice idea for the cultivars classification instead of indica/sativa



Classification of cannabis strains in the Canadian market with discriminant analysis of principal components using genome-wide single nucleotide polymorphisms
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
DOI: 10.1371/journal.pone.0253387
The cannabis community typically uses the terms “Sativa” and “Indica” to characterize drug strains with high tetrahydrocannabinol (THC) levels. Due to large scale, extensive, and unrecorded hybridization in the past 40 years, this vernacular naming convention has become unreliable and inadequate for identifying or selecting strains for clinical research and medicinal production. Additionally, cannabidiol (CBD) dominant strains and balanced strains (or intermediate strains, which have intermediate levels of THC and CBD), are not included in the current classification studies despite the increasing research interest in the therapeutic potential of CBD. This paper is the first in a series of studies proposing that a new classification system be established based on genome-wide variation and supplemented by data on secondary metabolites and morphological characteristics. This study performed a whole-genome sequencing of 23 cannabis strains marketed in Canada, aligned sequences to a reference genome, and, after filtering for minor allele frequency of 10%, identified 137,858 single nucleotide polymorphisms (SNPs). Discriminant analysis of principal components (DAPC) was applied to these SNPs and further identified 344 structural SNPs, which classified individual strains into five chemotype-aligned groups: one CBD dominant, one balanced, and three THC dominant clusters. These structural SNPs were all multiallelic and were predominantly tri-allelic (339/344). The largest portion of these SNPs (37%) occurred on the same chromosome containing genes for CBD acid synthases (CBDAS) and THC acid synthases (THCAS). The remainder (63%) were located on the other nine chromosomes. These results showed that the genetic differences between modern cannabis strains were at a whole-genome level and not limited to THC or CBD production. These SNPs contained enough genetic variation for classifying individual strains into corresponding chemotypes. In an effort to elucidate the confused genetic backgrounds of commercially available cannabis strains, this classification attempt investigated the utility of DAPC for classifying modern cannabis strains and for identifying structural SNPs.

Classification of Various Marijuana Varieties by Raman Microscopy and Chemometrics
Luis Ramos-Guerrero, Gemma Montalvo, Marzia Cosmi, Carmen García-Ruiz and Fernando E. Ortega-Ojeda
Toxics2022, 10, 115.
Doi: 10.3390/ toxics10030115
https://www.mdpi.com/2305-6304/10/3/...m_medium=email
The Raman analysis of marijuana is challenging because of the sample’s easy photo- degradation caused by the laser intensity. In this study, optimization of collection parameters and laser focusing on marijuana trichome heads allowed collecting Raman spectra without damaging the samples. The Raman spectra of ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) standard cannabinoids were compared with Raman spectra of five different types of marijuana: four Sativa varieties (Amnesia Haze, Amnesia Hy-Pro, Original Amnesia, and Y Griega) and one Indica variety (Black Domina). The results verified the presence of several common spectral bands that are useful for marijuana characterization. Results were corroborated by the quantum chemical simulated Raman spectra of their acid-form (tetrahydrocannabinol acid (THCA), cannabidiol acid (CBDA)) and decarboxylated cannabinoids (THC, CBD, and CBN). A chemometrics-assisted method based on Raman microscopy and OPLS-DA offered good classification among the different marijuana varieties allowing identification of the most significant spectral bands. .

Evolution and classification of Cannabis sativa (marijuana, hemp) in relation to human utilization.
Small E (2015)
Bot Rev 81: 189–294
DOI: 10.1007/s12229-015-9157-3
Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wildgrowing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.

Genetic Evidence for Speciation in Cannabis (Cannabaceae)
Karl Hillig
Genetic Resources and Crop Evolution · January 2005
DOI: 10.1007/s10722-003-4452-y
Sample populations of 157 Cannabis accessions of diverse geographic origin were surveyed for allozyme variation at 17 gene loci. The frequencies of 52 alleles were subjected to principal components analysis. A scatter plot revealed two major groups of accessions. The sativa gene pool includes fiber/seed landraces from Europe, Asia Minor, and Central Asia, and ruderal populations from Eastern Europe. The indica gene pool includes fiber/seed landraces from eastern Asia, narrow-leafleted drug strains from southern Asia, Africa, and Latin America, wide-leafleted drug strains from Afghanistan and Pakistan, and feral populations from India and Nepal. A third putative gene pool includes ruderal populations from Central Asia. None of the previous taxonomic concepts that were tested adequately circumscribe the sativa and indica gene pools. A polytypic concept of Cannabis is proposed, which recognizes three species, C. sativa, C. indica and C. ruderalis, and seven putative taxa.

GENOMIC, TERPENE AND CANNABINOID PROFILES OF A PUTATIVELY NOVEL CANNABIS SPECIES
Kevin McKernan, Cameron Miller, Brad Douglass. Jessica Spangler, Braden Doane, Vasisht Tadigotla, Colin Montgomery, Yvonne Helbert1 , Lei Zhang, Douglas Smith, Jeffrey Raber
https://www.medicinalgenomics.com/wp...cies_sbmt3.pdf

An Australian feral male cannabis strain (Australian Bastard Cannabis or ABC) was crossed with Cannabis Sativa L. (Purple skunk Oregon) and presented with atypical leaf patterns and stunted but hardy growth (offspring termed ABCh). Microscopy identified small trichomes and encouraged further evaluation with UV-HPLC (UV confirmed high performance liquid chromatography) that identified THCA and CBCA peaks. This cultivar was further tested for terpene profiles with GC-FID (gas chromatography-flame ionization detector) identifying many common terpenes in Cannabis. Thin layer
chromatography (TLC) was used to confirm the cannabinoid results against Cerilliant standards and the sample was selected for genomic analysis.

Identification of Phenotypic Characteristics in Three Chemotype Categories in the Genus Cannabis
Dan Jin, Philippe Henry, Jacqueline Shan, Jie Chen
HORTSCIENCE
DOI: 10.21273/HORTSCI15607-20
https://journals.ashs.org/hortsci/vi...e=The+Cannabis +%2B+Cannabinoid+Curator&utm_c ampaign=70b2ae2af2-EMAIL_CAMPAIGN_2020_03_13_07_5 9_COPY_01&utm_medium=email&utm _term=0_47f6c7c552-70b2ae2af2-365750754
Modern Cannabis cultivars are morphologically distinguished by their leaflet shapes (wide for “Indica” and narrow for “Sativa”) by users and breeders. However, there are no scientific bases or references for determining the shape of these leaflets. In addition, these two categories contained mostly THC dominant (high THC) cultivars while excluded CBD dominant (high CBD) and intermediate (intermediate level of both THC and CBD) cultivars. This study investigated the phenotypic variation in 21 Cannabis cultivars covering three chemical phenotypes, referred to as chemotypes, grown in a commercial greenhouse. Thirty morphological traits were measured in the vegetative, flowering, and harvest stages on live plants and harvested inflorescences. The collected data were subjected to correlation analysis, hierarchical clustering, principal component analysis, and canonical correlation analysis with preassigned chemotypes. Canonical correlation analysis assigned individual plants to their chemotypes with 92.9% accuracy. Significant morphological differences were identified. Traits usable as phenotype markers for CBD dominant cultivars included light-green and narrow leaflets, a greater number of primary and secondary serrations, loose inflorescences, dense and resinous trichomes, and Botrytis cinerea resistance. Traits for intermediate cultivars included deep-green and medium-wide leaflets, more primary and secondary serrations, medium compact inflorescences, trichomes that are less dense and less resinous, and Botrytis cinerea resistance. Traits for THC dominant cultivars included deep-green and wide leaflets, large and compact inflorescences, dense and resinous trichomes, and Botrytis cinerea susceptibility. The results of this study provide a comprehensive profile of morphological traits of modern Cannabis cultivars and provides the first such profile for CBD dominant and intermediate cultivars. Additionally, this study included the traits of inflorescences, which have not been compared between three chemotypes in the literature. Phenotype markers identified in this study can facilitate preliminary cultivar identification and selection on live plants before or as a supplement to chemical and genetic analysis.

Letter to the Editor: Ordering the chaos in cannabinoid-related research: is it time for a task force on taxonomy?
Schräder NHB, Bolling MC, Wolff AP
Cannabis and Cannabinoid Research X, 1–2, (2020)
DOI: 10.1089/can.2020.0145.
To the editor of Cannabis and Cannabinoid Research, the cannabinoid research associations and institutes, and to the scientific community endeavoring the field of cannabinoid-related research:
An increasing number of countries are loosening restrictions on both recreational and medical cannabinoid use, which has inevitably led to growing scientific interest. In fact, cannabinoid research is one of the fastest growing scientific fields, seeing exponential number of publications per year on topics spanning therapeutic, preventive, and basic research in numerous diseases.1
As a result of expanding research, there is an increasing heterogeneity of terminology in this immense data set. One can get lost in the numerous terms describing cannabinoid-related substances, their chemical constituents, and biological derivatives. We can already see the natural evolution of scientifically appropriated cannabinoid-related terminology moving from ordered to chaotic, where a simple literature search must encompass multiple terms, such as cannabis, cannabinoids, delta-9-tetrahydrocannabinol, THC, cannabidiol, CBD, marijuana, nabilone, dronabinol, and nabiximols, taken from the search query of a recent meta-analysis.
The problem with heterogeneous terminology for cannabinoid-related science is that it facilitates confusion among both new and established scientists. In the absence of a framework, researchers are likely to harness imprecise nomenclature used within their network, leading to the fragmentation of fields of thought. Without uniform communication, authors are obliged to develop their own classifications whereby research studies cannot reliably be compared. This is a significant hurdle to the scientific community who wish to communicate sound and reproducible research, and international organizations such as the United Nations, who are impaired in their decision making “…by a lack of international standards on issues such as terminology,” with respect to cannabinoid-related policy.2 It must go without saying that harnessing the benefits from a terminological consensus will excel the efficiency in cannabinoid-related scientific research (from basic science through clinical trials and meta-analyses), streamline collaboration, and maintain coherent education.


Letter to the Editor: Small, Ernest. 2015. Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization.
Robert C. Clarke, & Mark D. Merlin
Botanical Review 81(3): 189-294. The Botanical Review, 81(4), 295–305. 2015
doi:10.1007/s12229-015-9158-2
Few plant genera have received as much attention concerning their taxonomy and domestication as Cannabis. “Solving the taxonomy puzzle” is important for botanical, agricultural, legal, political and medical reasons (Lausen, 2015). However, for the authors of this rebuttal, resolving the issue of one or more species is not as fascinating as understanding the evolution of Cannabis. It is in this context that we offer our rebuttal.
In his comprehensive literature review Ernest Small covers diverse topics such as hemp fibers and processing, hemp seed nutrition, cannabinoid biosynthesis, cannabinoid-receptor interactions, medical uses and modes of action, among many more. The author’s concise narrative style, and excellent illustrations (including naïve art), add to the educational tone of this long contribution. However, the authors of this rebuttal challenge Small’s use of outdated terminology, question his renaming of the functional evolutionary and taxonomic groupings, support the use of cultivar names, are confused by his conclusions concerning resin gland function, and wish to elaborate on the particulars of drug Cannabis domestication.
Throughout his review Small uses the terms “narcotic” and “intoxicant” to describe drug Cannabis plants and their psychoactive products. The Oxford English Dictionary Online (http://www.oxforddictionaries.com. Accessed September 2, 2015) defines a “narcotic” as a “drug or other substance affecting mood or behavior and sold for nonmedical purposes, especially an illegal one” while the online Merriam-Webster Unabridged Dictionary (http://www.merriam-webster.com. Accessed September 2, 2015) defines a “narcotic” as “a drug (such as cocaine, heroin, or marijuana) that affects the brain and that is usually dangerous and illegal.” And, in a medical context defines a “narcotic” as “a drug (as codeine, methadone, or morphine) that in moderate doses dulls the senses, relieves pain, and induces profound sleep but in excessive doses causes stupor, coma, or convulsions” or “a drug (as marijuana or LSD) subject to restriction similar to that of addictive narcotics whether in fact physiologically addictive and narcotic or not.” According to these definitions drug Cannabis could be termed “narcotic” because it “affects the brain” but there is little if any evidence to support its being “usually dangerous.” And, although Cannabis remains “illegal” in many jurisdictions where it is “subject to restriction,” calling cannabis a “narcotic” is a defamatory artifact of the legal system, rather than a choice backed by scientific observation

Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests.
Kevin McKernan, Jessica Spangler, Yvonne Helbert, Ryan C. Lynch , Adrian Devitt-Lee, Lei Zhang, Wendell Orphe, Jason Warner, Theodore Foss, Christopher J. Hudalla, Matthew Silva, Douglas R. Smith.
DOI: 10.12688/f1000research.9662.1
Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation.
Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media.
Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal
species further influenced the differential growth of species in the microbiome of each sample.
Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

Models of Cannabis Taxonomy, Cultural Bias, and Conflicts between Scientific and Vernacular Names
John M. McPartland & Geoffrey W. Guy
Bot. Rev. 83(4), 327-381
DOI 10.1007/s12229-017-9187-0
Debates over Cannabis sativa L. and C. indica Lam. center on their taxonomic circumscription and rank. This perennial puzzle has been compounded by the viral spread of a vernacular nomenclature, “Sativa” and “Indica,” which does not correlate with C. sativa and C. indica. Ambiguities also envelop the epithets of wildtype Cannabis: the spontanea versus ruderalis debate (i.e., vernacular “Ruderalis”), as well as another pair of Cannabis epithets, afghanica and kafirstanica. To trace the rise of vernacular nomenclature, we begin with the protologues (original descriptions, synonymies, type specimens) of C. sativa and C. indica. Biogeographical evidence (obtained from the literature and herbarium specimens) suggests 18th–19th century botanists were biased in their assignment of these taxa to field specimens. This skewed the perception of Cannabis biodiversity and distribution. The development of vernacular “Sativa,” “Indica,” and “Ruderalis” was abetted by twentieth century botanists, who ignored original protologues and harbored their own cultural biases. Predominant taxonomic models by Vavilov, Small, Schultes, de Meijer, and Hillig are compared and critiqued. Small’s model adheres closest to protologue data (with C. indica treated as a subspecies). “Sativa” and “Indica” are subpopulations of C. sativa subsp. indica; “Ruderalis” represents a protean assortment of plants, including C. sativa subsp. Sativa and recent hybrids.

Response to the Erroneous Critique of my Cannabis Monograph by R. C. Clarke and M.D. Merlin
Ernest Smal
Bot. Rev. 81(4)
DOI: 10.1007/s12229-015-9159-1
I have been involved in debates in scientific journals concerning the classification of Cannabis sativa for almost a half century. Its variation pattern is by no means more complex than posed by thousands of other plant species, which usually receive just a paragraph of mention in the literature. However, because it is the world’s most controversial plant, C. sativa has attracted very widespread interest, not only by scientists but indeed by the public, who regrettably have been confused by its classification and nomenclature. Even in recent scientific publications there is frequent misunderstand about names applied to forms of C. sativa. The issues are not merely academic, since appropriate classification is the means by which certain biotypes are
authorized for industrial and medical purposes, or subject to civil and criminal penalties. Accordingly, the evolutionary classification of C. sativa is very important, and Iam very grateful to the editors of Botanical Review for having permitted me to publish a very extensive article on the subject (Small, 2015). Of course, as with most controversial issues, there is not unanimity of opinion on all aspects, as reflected by the criticism of Clarke and Merlin (2015). While I do not question their expertise, as detailed in the following, every one of their substantive criticisms of my contribution is mistaken. Much of the presentation of Clarke and Merlin quotes or rephrases material I presented in my paper, and of course I have no quarrel with this. My rejoinder is limited to their challenges regarding facts or interpretations in my presentation.

The Cannabis sativa Versus Cannabis indica Debate:
An Interview with Ethan Russo, MD

Daniele Piomelli and Ethan B. Russo
Cannabis and Cannabinoid Research Jan 2016
DOI: 10.1089/can.2015.29003.ebr
Dr. Ethan Russo, MD, is a board-certified neurologist, psychopharmacology researcher, and Medical Director of PHYTECS, a biotechnology company researching and developing innovative approaches targeting the human endocannabinoid system. Previously, from 2003 to 2014, he served as Senior Medical Advisor and study physician to GW Pharmaceuticals for three Phase III clinical trials of Sativex for alleviation of cancer pain unresponsive to optimized opioid treatment and studies of Epidiolex for intractable epilepsy. He has held faculty appointments in Pharmaceutical Sciences at the University of Montana, in Medicine at the University of Washington, and as visiting Professor, Chinese Academy of Sciences. He is a past President of the International Cannabinoid Research
Society and former Chairman of the International Association for Cannabinoid Medicines. He serves on the Scientific Advisory Board for the American Botanical Council. He is the author of numerous books, book chapters, and articles on Cannabis, ethnobotany, and herbal medicine. His research interests have included correlations of historical uses of Cannabis with modern pharmacological mechanisms, phytopharmaceutical treatment of migraine and chronic pain, and phytocannabinoid/terpenoid/serotonergic/vanilloid interactions.

The Case for the Entourage Effect and Conventional Breeding of Clinical Cannabis: No “Strain,” No Gain
Ethan B. Russo
Frontiers in Plant Science 1 January 2019 | Volume 9 | Article 1969
doi: 10.3389/fpls.2018.01969
The topic of Ccannabis curries controversy in every sphere of influence, whether politics, pharmacology, applied therapeutics or even botanical taxonomy. Debate as to the speciation of Cannabis, or a lack thereof, has swirled for more than 250 years. Because all Cannabis types are eminently capable of cross-breeding to produce fertile progeny, it is unlikely that any clear winner will emerge between the “lumpers” vs. “splitters” in this taxonomical debate. This is compounded by the profusion of Cannabis varieties available through the black market and even the developing legal market. While labeled “strains” in common parlance, this term is acceptable with respect to bacteria and viruses, but not among Plantae. Given that such factors as plant height and leaflet width do not distinguish one Cannabis plant from another and similar difficulties in defining terms in Cannabis, the only reasonable solution is to characterize them by their biochemical/pharmacological characteristics. Thus, it is best to refer to cannabisCannabis types as chemical varieties, or “chemovars.” The current wave of excitement in cannabisCannabis commerce has translated into a flurry of research on alternative sources, particularly yeasts, and complex systems for laboratory production have emerged, but these presuppose that single compounds are a desirable goal. Rather, the case for cannabisCannabis synergy via the “entourage effect” is currently sufficiently strong as to suggest that one molecule is unlikely to match the therapeutic and even industrial potential of cannabisCannabis itself as a phytochemical factory. The astounding plasticity of the cannabisCannabis genomeadditionally obviates the need for genetic modification techniques.

The Name of Cannabis: A Short Guide for Nonbotanists
Antonino Pollio
Cannabis and Cannabinoid Research Volume 1.1, 2016
DOI: 10.1089/can.2016.0027
The genus Cannabis (Family Cannabaceae) is probably indigenous to wet habitats of Asiatic continent. The long coexistence between mankind and Cannabis led to an early domestication of the plant, which soon showed an amazing spectrum of possible utilizations, as a source of textile fibers, as well as narcotic and psychoactive compounds.
Nowadays, the specie(s) belonging to the genus Cannabis are represented by myriads of cultivated varieties, often with unstable taxonomic foundations. The nomenclature of Cannabis has been the object of numerous nomenclatural treatments. Linnaeus in Species Plantarum (1753) described a single species of hemp, Cannabis sativa, whereas Lamarck (1785) proposed two species of Cannabis: C. sativa, the species largely cultivated in Western Continent, and Cannabis indica, a wild species growing in India and neighboring countries. The dilemma about the existence of the species C. indica considered distinct from C. sativa continues up to present days. Due to their prevalent economic interest, the nomenclatural treatment is particularly important as far as it concerns the cultivated varieties of Cannabis. In this context, we propose to avoid the distinction between sativa and indica, suggesting a bimodal approach: when a cultivar has been correctly established. It could be advisable to apply a nomenclature system based on the International Code of Nomenclature for Cultivated Plants (ICNCP): it is not necessary to use the species epithets, sativa or indica, and a combination of the genus name and a cultivar epithet in any language and bounded by single quotation marks define an exclusive name for each Cannabis cultivar. In contrast, Cannabis varieties named with vernacular names by medical patients and recreational users, and lacking an adequate description as required by ICNCP, should be named as Cannabis strain, followed by their popularized name and without single quotation marks, having in mind that their names have no taxonomical validity.

What’s in a Shape
Daniela Vergara
The Cannabis Scientist
https://thecannabisscientist.com/res...ats-in-a-shape
Back in the summer of 2015, I was busy helping collect physical trait information from the results of a hemp-BLMT cross – 300 plants in all! In 2021 – on April 20 (4/20!) – we published our results (1), and I hope you’ll agree that all the hard work was well worth it. Before we get to our findings (the paper title is more than a teaser!), a little background reading...
Even though Cannabis is considered as one species (Cannabis sativa L), according to Carl Linnaeus (who classified it in 1753), we know there are at least three groupings within C. sativa (1; see Figure 1). These three groupings include industrial hemp, and two other clusters that are used in medicinal and recreational settings – broad-leaf marijuana type (BLMT) and narrow-leaf marijuana type (NLMT)
 
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"RESIN BREEDER"
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IC DNA

A comprehensive study of thiol reduction gene expression under stress conditions in Arabidopsis thaliana
C. Belin, T. Bashandy, J. Cela, V. Delorme-Hinoux, C. Riondet, & J. P. Reichheld
Plant, Cell and Environment (2014)
doi: 10.1111/pce.12276
Thiol reduction proteins are key regulators of the redox state of the cell, managing development and stress response programs. In plants, thiol reduction proteins, namely thioredoxin (TRX), glutaredoxin (GRX), and their respective reducers glutathione reductase (GR) and thioredoxin reductase (TR), are organized in complex multigene families. In order to decipher the function of the different proteins, it is necessary to have a clear picture of their respective expression profiles. By collecting information from gene expression databases, we have performed a comprehensive in silico study of the expression of all members of different classes of thiol reduction genes (TRX, GRX) in Arabidopsis thaliana. Tissue expression profiles and response to many biotic and abiotic stress conditions have been studied systematically. Altogether, the significance of our data is discussed with respect to published biochemical and genetic studies.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativaL.)
Dowling CA, Shi J, Toth JA, Quade MA, Smart LB, McCabe PF, Melzer R, Schilling S
Hemp ( Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is tightly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive (“autoflower”) cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2 , a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T ( FT ) from Arabidopsis thaliana which we termed CsFT1 . Extensive sequence divergence between alleles of CsFT1 was identified between photoperiod-sensitive and insensitive cultivars of C. sativa , including a duplication of CsFT1 and sequence differences especially in introns. Genotyping of several mapping populations and a diversity panel confirmed a strong correlation between CsFT1 alleles and photoperiod response as well as affirming that at least two independent loci for this agriculturally important trait, Autoflower1 and Autoflower2 , exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.
DOI: 10.1101/2023.04.21.537862


Not Cannabis specific
A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce
Charles J. Underwood, Kitty Vijverberg, Diana Rigola, Shunsuke Okamoto, Carla Oplaat, Rik H. M. Op den Camp, Tatyana Radoeva, Stephen E. Schauer, Joke Fierens, Kim Jansen, Sandra Mansveld, Marco Busscher, Wei Xiong, Erwin Datema, Koen Nijbroek, Evert-Jan Blom, Ross Bicknell, Andrew Catanach, Sylvia Erasmuson, Christopher Winefield, Arjen J. van Tunen, Marcel Prins, M. Eric Schranz & Peter J. van Dijk
Nature Genetics (2022)
https://www.nature.com/articles/s41588-021-00984-y
Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelionPAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.
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A Belated Green Revolution for Cannabis: Virtual Genetic Resources to Fast-Track Cultivar Development
Matthew T. Welling, Tim Shapter, Terry J. Rose, Lei Liu, Rhia Stanger, and Graham J. King,
July 2016 Frontiers in Plant Science 7
DOI: 10.3389/fpls.2016.01113
Cannabis is a predominantly diecious phenotypically diverse domesticated genus with few if any extant natural populations. International narcotics conventions and associated legislation have constrained the establishment, characterization, and use of Cannabis genetic resource collections. This has resulted in the underutilization of genepool variability in cultivar development and has limited the inclusion of secondary genepools associated with genetic improvement strategies of the Green Revolution. The structured screening of ex situ germplasm and the exploitation of locally-adapted intraspecific traits is expected to facilitate the genetic improvement of Cannabis. However, limited attempts have been made to establish the full extent of genetic resources available for pre-breeding. We present a thorough critical review of Cannabis ex situ genetic resources, and discuss recommendations for conservation, pre-breeding characterization, and genetic analysis that will underpin future cultivar development. We consider East Asian germplasm to be a priority for conservation based on the prolonged historical cultivation of Cannabis in this region over a range of latitudes, along with the apparent high levels of genetic diversity and relatively low representation in published genetic resource collections. Seed cryopreservation could improve conservation by reducing hybridization and genetic drift that may occur during Cannabis germplasm regeneration. Given the unique legal status of Cannabis, we propose the establishment of a global virtual core collection based on the collation of consistent and comprehensive provenance meta-data and the adoption of high-throughput DNA sequencing technologies. This would enable representative core collections to be used for systematic phenotyping, and so underpin breeding strategies for the genetic improvement of Cannabis.

A Cannabis sativa STR Genotype Database for Australian Seizures: Forensic Applications and Limitations
Christopher Howard, Simon Gilmore, James Robertson, Rod Peakall
J Forensic Sci, May 2009, Vol. 54, No. 3
doi: 10.1111/j.1556-4029.2009.01014.x
A genetic database was established with the aim of documenting the genetic diversity of Cannabis sativa in Australia for future utilization in forensic investigations. The database consisted of genotypes at 10 validated short tandem repeat loci for 510 plants representing drug seizures from across Australia and 57 fiber samples. A total of 106 alleles and 314 different genotypes were detected. All fiber samples exhibited unique genotypes while 55% of the drug samples shared a genotype with one or more samples. Shared genotypes were mostly found within seizures; however, some genotypes were found among seizures. Statistical analysis indicated that genotype sharing was a consequence of clonal propagation rather than a lack of genetic resolution. Thus, the finding of shared genotypes among seizures is likely due to either a common supplier, or direct links among seizures. Notwithstanding the potential intelligence information provided by genetic analysis of C. sativa, our database analysis also reveals some present limitations.

NOT CANNABIS SPECIFIC
A Century of Sex Determination in Flowering Plants
Alex Harkess and Jim Leebens-Mack
Journal of Heredity, 2017, 69–77
doi:10.1093/jhered/esw060
Plants have evolved a diverse array of strategies for sexual reproduction, particularly through the modification of male and female organs at distinct points in development. The immense variation in sexual systems across the land plants provides a unique opportunity to study the genetic, epigenetic, phylogenetic, and ecological underpinnings of sex determination. Here, we reflect on more than a century of research into flowering plant sex determination, placing a particular focus on the foundational genetic and cytogenetic observations, experiments, and hypotheses. Building on the seminal work on the genetics of plant sex, modern comparative genomic analyses now allow us to address longstanding questions about sex determination and the origins of sex chromosomes.

Not Cannabis specific
A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed Genes
Sheila A. Johnson-Brousseau and Sheila McCormick
The Plant Journal (2004) 39, 761–775
doi: 10.1111/j.1365-313X.2004.02147.x
This article provides detailed protocols for collecting pollen and outlines genetic crosses and phenotypic assays that are useful for characterizing mutants that affect pollen development

A complete Cannabis chromosome assembly and adaptive admixture for elevated cannabidiol (CBD) content
Christopher J. Grassa, Jonathan P. Wenger, Clemon Dabney, Shane
G. Poplawski, S. Timothy Motley, Todd 5 P. Michael, C.J. Schwartz1,
George D. Weiblen
doi: 10.1101/458083
Cannabis has been cultivated for millennia with distinct cultivars providing either 20 fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol /cannabidiol segregating population and a complete 25 chromosome assembly from a high-cannabidiol cultivar together resolve the linkage of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated
with transposable elements. High-cannabidiol cultivars appear to have been
generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests 30 that overall drug potency, however, is associated with other genomic regions needing additional study.

A comprehensive molecular approach to the detection of drug-type versus fiber-type hemp varieties
Giuseppe Forlani, Davide Petrollino
Forensic Sci Int Genet 2021 May;52:
DOI: 10.1016/j.fsigen.2021.102464
The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sativa cultivars would allow fast and cheap analysis of any plant specimen, including seeds and leaves. Several approaches to this issue have been described, mainly using polymorphisms in the genes coding for tetrahydrocannabinol acid synthase or cannabidiolic acid synthase. Some studies reported sequencing of these genes from small groups of hemp varieties belonging to both chemotypes, showing the occurrence of specific DNA signatures. However, the effectiveness of the corresponding primers to discriminate among chemotypes has been validated on a limited number of cultivars, or not tested at all. Here we report a thorough in silico analysis of available gene sequences for both synthases, showing the existence of hypervariable regions at 3’ and 5’ ends. This notwithstanding, some possible signatures were identified, and 12 putatively specific primer pairs were designed and tested on 16 fiber-type and 11 drug-type varieties. In most cases inconsistent results were obtained, further strengthening the high genetic variability of these genes in hemp germplasm, yet some highly informative polymorphisms were identified. Potentiality and perspectives of this approach are discussed.
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Not Cannabis specific
A draft sequence reference of the Psilocybe cubensisgenome [version 1; peer review: awaiting peer review]
Kevin McKernan, Liam T. Kane, Seth Crawford Chen-Shan Chin, Aaron Trippe, Stephen McLaughlin
F1000Research 2021, 10:281 Last updated: 09 APR 2021
https://f1000researchdata.s3.amazonaws .com/manuscripts/54802/de389924-8eb1-4f5b-8107-d3d1bde170f6_51613_-_kevin_mckernan.pdf?doi=10.126 88/f1000research.51613.1&numberOf BrowsableCollections=27&number OfBrowsableInstitutionalCollec tions=4&numberOfBrowsableGatew ays=26
10.12688/f1000research.51613.1
We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.)

Caroline A. Dowling, Jiaqi Shi, Jacob A. Toth, Michael A. Quade, Lawrence B. Smart, Paul F. McCabe, Rainer Melzer,
doi: https://doi.org/10.1101/2023.04.21.537862
Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is tightly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive (“autoflower”) cultivars are available.

Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. Extensive sequence divergence between alleles of CsFT1 was identified between photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences especially in introns. Genotyping of several mapping populations and a diversity panel confirmed a strong correlation between CsFT1 alleles and photoperiod response as well as affirming that at least two independent loci for this agriculturally important trait, Autoflower1 and Autoflower2, exist in the C. sativa gene pool.

This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

A GOLD RUSH FOR CANNABIS
Elie Dolgin
18 OCTOBER 2018 | VOL 562 | NATURE
https://www.nature.com/articles/d415...q84OpP39j2pJoY
Jonathan Page has been around cannabis all his life. Growing up on Canada’s Vancouver Island in the 1970s, he was surrounded by hippie beachcombers and dope smokers. So after earning a PhD in plant biology and phytochemistry, he felt completely at ease working with the plant Cannabis sativa as a postdoc in Germany in the early 2000s.
During that time, Page helped to characterize a pair of genes that some varieties of the plant uses to make fragrant oils responsible for pine- and lemon-like aromas 1. And during an interview for a position with Canada’s National Research Council (NRC), Page proposed simi¬lar projects to reveal how cannabis produces pharmaceutically active compounds known as cannabinoids.

A high-throughput segregation analysis identifies the sex chromosomes of Cannabis sativa
Djivan Prentout, Olga Razumova, Bénédicte Rhoné, Hélène Badouin, Hélène Henri, Cong Feng, Jos Käfer, Gennady Karlov, Gabriel AB Marais,
DOI: 10.1101/721324
https://www.biorxiv.org/content/10.1...324v1.full.pdf
Cannabis sativa-derived tetrahydrocannabinol (THC) production is increasing very fast worldwide. C. sativa is a dioecious plant with XY chromosomes, and only females (XX) are useful for THC production. The C. sativa sex chromosomes sequence would improve early sexing and better management of this crop; however, the C. sativa genome projects failed to identify the sex chromosomes so far. Moreover, dioecy in the Cannabaceae family is ancestral, C. sativa sex chromosomes are potentially old and thus very interesting to study as little is known about the last steps of sex chromosome evolution in plants. Here we RNA-sequenced a C. sativa family (2 parents and 10 male and female offspring) and performed a segregation analysis for all C. sativa genes using the probabilistic method SEX-DETector. We identified >500 sex-linked genes. Mapping of these sex-linked genes to a C. sativa genome assembly identified a single chromosome pair with a large non-recombining region. Further analysis of the >500 sex-linked genes revealed that C. sativa has a strongly degenerated Y chromosome and represents the oldest plant sex chromosome system documented so far. Our study revealed that old plant sex chromosomes can have large non-recombining regions and be very differentiated and still be of similar size (homomorphic).

A high-quality reference genome of wild Cannabis sativa
Shan Gao, Baishi Wang, Shanshan Xie, Xiaoyu Xu, Jin Zhang, Li Pei, Yongyi Yu, Weifei Yang and Ying Zhang
Horticulture Research (2020) 7:73
https://www.nature.com/articles/s41438-020-0295-3.pdf
DOI: 10.1038/s41438-020-0295-3
Cannabis sativa is a well-known plant species that has great economic and ecological significance. An incomplete genome of cloned C. sativa was obtained by using SOAPdenovo software in 2011. To further explore the utilization of this plant resource, we generated an updated draft genome sequence for wild-type varieties of C. sativa in China using PacBio single-molecule sequencing and Hi-C technology. Our assembled genome is approximately 808 Mb, with scaffold and contig N50 sizes of 83.00 Mb and 513.57 kb, respectively. Repetitive elements account for 74.75% of the genome. A total of 38,828 protein-coding genes were annotated, 98.20% of which were functionally annotated. We provide the first comprehensive de novo genome of wild-type varieties of C. sativa distributed in Tibet, China. Due to long-term growth in the wild environment, these varieties exhibit higher heterozygosity and contain more genetic information. This genetic resource is of great value for future investigations of cannabinoid metabolic pathways and will aid in promoting the commercial production of C. sativa and the effective utilization of cannabinoids. The assembled genome is also a valuable resource for intensively and effectively investigating the C. sativa genome further in the future.

A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana
Christopher J. Grassa, George D. Weiblen, Jonathan P. Wenger, Clemon Dabney, Shane G. Poplawski, S. Timothy Motley, Todd P. Michael, C. J. Schwartz
New Phytologist01 February 2021
doi: 10.1111/nph.17243
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Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for CBDRx, a potent CBD-type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed. Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD-type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio. Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

A New Crop of Marijuana Geneticists Sets Out to Build Better Weed
Katie M. Palmer
WIRED 2016 – 04
https://www.wired.com/2016/04/the-science-of-marijuana/
THE MARIJUANA ANALYTICS company Steep Hill doesn’t smell dank, or skunky, or “loud”—unless you happen to arrive when a client is dropping off a sample. No seven-pointed-leaf logos ornament the walls; no Tibetan prayer flags flutter from the doorframe. Inside, a half-dozen young scientists work in a glass-walled lab to the sounds of whirring ventilation and soft jazz. The effect is one of professionalism and scientific objectivity.
Still, this place is all about weed. And Reggie Gaudino, Steep Hill’s burly and dreadlocked 53-year-old vice president of scientific operations, does look the part. Steep Hill is headquartered in famously 420-friendly Berkeley, California, after all. “I’ve been smoking since I was 13 years old,” he says, looking down over a railing at the lab. It’s a world he has long appreciated. Now he’d like to give a little back. “There’s so much good that can be done with cannabis, and so little of it is being done.”

A PCR marker Linked to a THCA synthase Polymorphism is a Reliable Tool to Discriminate Potentially THC-Rich Plants of Cannabis sativa L.
Christina Staginnus Ph.D., Siegfried Zörntlein Ph.D., Etienne de Meijer Ph.D.
3 March 2014 Journal of Forensic Sciences 59(4)
DOI: 10.1111/1556-4029.12448
†The nucleotide sequences reported in this paper are deposited in the NCBI GenBank under accession numbers JQ935235–JQ935244.
Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant's life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (BT and BD). The homozygous BT/BT genotype underlies the THC-predominant phenotype, BD/BD is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (BT/BD). Using PCR-based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC-predominant or the THC-intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species-specific marker system was applied to a large number of casework samples and fiber hemp cultivars.

A physical and genetic map of Cannabis sativa identifies extensive rearrangement at the THC/CBD acid synthase locus.
Laverty KU, Stout JM, Sullivan MJ, Shah H, Gill N, Bolbrook L, Deikus G, Sebra R, Hughes TR, Page JE, van Bakel H (2018)
Genome Res, in press,
doi: 10.1101/gr.242594.118
Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain ‘Purple Kush’ and the hemp variety ‘Finola’. The map reveals that cannabinoid biosynthesis genes are generally unlinked, but that aromatic prenyltransferase (AP) – which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS) – is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. Strikingly, THCAS and CBDAS (which determine the drug vs hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly non-homologous between drug- and hemp-type alleles, and are furthermore embedded within ~40 Mb of non-recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant.

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples
Fidelia Cascini, Simona Martello
Forensic Science International 217 (2012) 134–138
DOI: 10.1016/j.forsciint.2011.10.041
In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and
storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drugtype from the fibre-type varieties.
A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency
plants, we chose the DDCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished.
As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested.

Not Cannabis specific
A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species
PLoS ONE 6(5):e19379 May 2011
DOI: 10.1371/journal.pone.0019379
Robert J Elshire, Jeffrey C Glaubitz, Qi Sun, Sharon Mitchell
Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

A Simple DNA Extraction Method for Marijuana Samples Used in Amplified Fragment Length Polymorphism (AFLP) Analysis*
Heather Miller Coyle, Gary Shutler, Sharon Abrams, Janet Hanniman, Suzanne Neylon, Carll Ladd, Timothy Palmbach, and Henry C. Lee,
J Forensic Sci, Mar. 2003, Vol. 48, No. 2: 343-7
https://pdfs.semanticscholar.org/6c4...9c05f1314a.pdf
DOI: 10.1520/JFS2001207
As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125–500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA.

A Single Nucleotide Polymorphism assay sheds light on the extent and distribution of genetic diversity, population structure and functional basis of key traits in cultivated North American Cannabis
Philippe Henry, Surender Khatodia, Karan Kapoor, Britni Gonzales, Alexis Middleton, Kevin Hong, Aaron Hilyard, Steve Johnson, Davis Allen, Zachary Chester, Dan Jin, José Carlos Rodriguez Jule, Iain Wilson, Manu Gangola, Jason Broome, Deron Caplan, Dinesh Adhikary, Michael K. Deyholos, Michael Morgan, Oliver W.Hall, Brent Guppy, Cindy Orser
bioRxiv 2020
Doi: 10.1101/2020.02.16.951459
Background The taxonomic classification of the Cannabis genus has been delineated through three main types: sativa (long and less branched plant with long and narrow leaves), indica (short but highly branched plant with broader leaves) and ruderalis(wild type with short stature, less branching and small thick leaves). While still under discussion, particularly whether the genus is polytypic or monotypic, this broad classification reflects putative geographical origin of each group and putative chemotypic and pharmacology.
Methods Here we describe a thorough investigation of cannabis accessions using a set of 22 highly informative and polymorphic SNP markers associated with important traits such as cannabinoid and terpenoid expression as well as fibre and resin production. The assay offers insight into cannabis population structure, phylogenetic relationship, population genetics and correlation to secondary metabolite concentrations and demonstrate the utility of this assay for rapid, repeatable and cost-efficient genotyping of commercial and industrial cannabis accessions for use in product traceability, breeding programs, regulatory compliance and consumer education.
Results The main outcomes are the identification of 5 clusters in the sample set available, including industrial hemp, resin hemp which likely underwent a bottleneck to stabilize CBDA accumulation (Type II & III). THC resin (type I) make up the other three clusters with terpinolene (colloquial “sativa” or “NLD”), myrcene/pinene and myrcene/limonene (colloquial “indica”, “BLD”), which also putatively harbour an active CBCAS.
Conclusion The functional basis of key traits is also discussed as recently enabled by the NCBI Cannabis sativa Annotation Release 100, allowing for hypothesis testing with regards to secondary metabolite production as well as other key traits of importance for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

Abnormal Meiosis in Tetraploid (4x) Cannabis sativa (L.) from Lahaul-Spiti (Cold Desert Higher Altitude Himalayas)-A Neglected But Important Herb
J. Biol. Chem. Chron. 2015, 2(1), 38-42
eresearchco.com/jbcc-pdf/6%20VKS%20JBCC%202_1_.pdf
Vivek Sharma, Devendra K. Srivastava, Raghbir C. Gupta, Bikram Singh
Present paper deals with the chromosome numbers, meiotic course and pollen viability of Cannabis sativa L., which was assessed cytologically in 12 populations growing in different localities of Lahaul-Spiti of Himachal Pradesh (H.P.) viz. Trilokinath (3,020m), Kaza (3,820m), Udaipur (2,743m), Losar (4,079m), Keylong (3,350m), Manali (2,050m), Gramphoo (3,060m), Chamba (920m), Kullu (1,362m), Khoksar (3,140m), Batal (3,960m) and Darcha (3,360m). Except, the population growing in Darcha (2n=4x=40), all the other populations were reported with diploid chromosome count of 2n=2x=20. The tetraploid (4x) meiotic chromosome count of 2n=40 in Cannabis sativa L. from cold desert area of Lahul-Spiti is reported here for the first time. Microsporogenesis was normal in most of the populations. It was present with Cytomixis (33.36%), laggards (19.52%), bridges (15.60%), chromosome stickiness (27.17%), diads (07.50%), triads (06.33%), polyads (09.50%), micronuclei and unequal daughter cells in plants of Darcha population (2n=4x=40). The consequences of these meiotic abnormalities were in the form of heterogenous sized pollen grains and low (67.33%) pollen viability.

Not Cannabis specific
Accelerated Yield Technology™ : A Platform for Marker Assisted Selection of Simple and Complex Traits
Scott Sebastian, Lizhi Feng, and Les Kuhlman
Marker assisted selection (MAS) strategies known as ‘forward selection’ have been used effectively in soybean since the mid 1990’s to pre-screen breeding populations for simply-inherited disease resistance traits (Cahill and Schmidt, 2004). But many complex traits have not been amenable to forward selection because quantitative trait loci (QTL) detected within one genetic context have not been sufficiently predictive of other genetic contexts (Bernardo, 2008; Holland, 2004, 2007; Xu and Crouch, 2008). This has prompted us to investigate a ‘Context Specific MAS’ (CSM) approach for MAS of complex traits (Sebastian et al., 2010). For essential complex traits such as high grain yield potential, CSM has already demonstrated both technical feasibility and commercial success. The efficiency gains will only improve with experience and with increasingly affordable genome-wide markers. The combination of forward selection for simple traits followed by CSM for grain yield and other complex traits is now a key product development strategy known commercially as Accelerated Yield Technology™ or AYT™. Since simple trait mapping and forward selection techniques are already covered extensively in the literature, this chapter focuses on CSM for grain yield potential per unit land area - herein referred to as ‘yield’. CSM generates a customized genetic model for yield within each breeding population and geographic zone (the ‘context’) during the first year of yield testing. A genetic prediction of yield potential for each line is then used in combination with the phenotypic score to increase the heritability of yield selections. Efficiency with CSM is gained by focusing expensive multi-environment yield trials on selections that are less likely to be artifacts of Type I and Type II measurement errors and/or genotype by environment (GxE) interactions. A specific example of CSM is shown, but the basic principles can be tailored to accommodate plant breeding programs that vary greatly in scope and budget.

*Accumulation of somatic mutations leads to genetic mosaicism in Cannabis
Kristian Adamek, Davoud Torkamaneh, A. Maxwell P. Jones
DOI: 10.1101/2021.02.11.430823
Feb 2021
Cannabis is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigour and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50x depth of coverage) to compare the variability within an individual Cannabis sativa cv. "Honey Banana" plant sampled at the bottom, middle and top. Overall, we called over 6 million sequence variants based on a published reference genome (SNPs, MNPs, and indels) and found that that the top had the most by a sizable amount. We compared the variants among the samples and uncovered that nearly 600K (34%) were unique to the top while the bottom only contained 148K (12%) and middle with 77K (9%) unique variants. Bioinformatics tools were used to identify high impact mutations in critical cannabinoid/terpene biosynthesis pathways. While none were identified, some contained more than double the average level of nucleotide diversity (π) in or near the gene, including OLS, CBDAS, HMGR2 and CsTPS9FN. The first two genes code for essential enzymes required for the cannabinoid pathway while the other two are involved in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, these data identified a measurable number of intra-plant genetic diversity that could impact the long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.

An extreme phenotype genome? wide association study identifes candidate cannabinoid pathway genes in Cannabis
Matthew T. Welling, Lei Liu, Tobias Kretzschmar, Ramil Mauleon, OmidAnsari, & Graham J. King
Nature Scientifc Reports | (2020) 10:18643`
DOI: 10.1038/s41598-020-75271-7
Cannabis produces a class of isoprenylated resorcinyl polyketides known as cannabinoids, a subset of which are medically important and exclusive to this plant. The cannabinoid alkyl group is a critical structural feature that governs therapeutic activity. Genetic enhancement of the alkyl side-chain could lead to the development of novel chemical phenotypes (chemotypes) for pharmaceutical end-use. However, the genetic determinants underlying in planta variation of cannabinoid alkyl side-chain length remain uncharacterised. Using a diversity panel derived from the Ecofbre Cannabis germplasm collection, an extreme-phenotype genome-wide association study (XP-GWAS) was used to enrich for alkyl cannabinoid polymorphic regions. Resequencing of chemotypically extreme pools revealed a known cannabinoid synthesis pathway locus as well as a series of chemotype-associated genomic regions. One of these regions contained a candidate gene encoding a ?-keto acyl carrier protein (ACP) reductase (BKR) putatively associated with polyketide fatty acid starter unit synthesis and alkyl sidechain length. Association analysis revealed twenty-two polymorphic variants spanning the length of this gene, including two nonsynonymous substitutions. The success of this frst reported application of XP-GWAS for an obligate outcrossing and highly heterozygote plant genus suggests that this approach may have generic application for other plant species

Not Cannabis Specific
An Improved Genotyping by Sequencing (GBS) Approach Offering Increased Versatility and Efficiency of SNP Discovery and Genotyping.
Sonah, H., Bastien, M., Iquira, E., Tardivel, A., Légaré, G., Boyle, B., … Belzile, F. (2013). PLoS ONE, 8(1), e54603.
doi:10.1371/journal.pone.0054603
Highly parallel SNP genotyping platforms have been developed for some important crop species, but these platforms typically carry a high cost per sample for first-time or small-scale users. In contrast, recently developed genotyping by sequencing (GBS) approaches offer a highly cost effective alternative for simultaneous SNP discovery and genotyping. In the present investigation, we have explored the use of GBS in soybean. In addition to developing a novel analysis pipeline to call SNPs and indels from the resulting sequence reads, we have devised a modified library preparation protocol to alter the degree of complexity reduction. We used a set of eight diverse soybean genotypes to conduct a pilot scale test of the protocol and pipeline. Using ApeKI for GBS library preparation and sequencing on an Illumina GAIIx machine, we obtained 5.5 M reads and these were processed using our pipeline. A total of 10,120 high quality SNPs were obtained and the distribution of these SNPs mirrored closely the distribution of gene-rich regions in the soybean genome. A total of 39.5% of the SNPs were present in genic regions and 52.5% of these were located in the coding sequence. Validation of over 400 genotypes at a set of randomly selected SNPs using Sanger sequencing showed a 98% success rate. We then explored the use of selective primers to achieve a greater complexity reduction during GBS library preparation. The number of SNP calls could be increased by almost 40% and their depth of coverage was more than doubled, thus opening the door to an increase in the throughput and a significant decrease in the per sample cost. The approach to obtain high quality SNPs developed here will be helpful for marker assisted genomics as well as assessment of available genetic resources for effective utilisation in a wide number of species.

An overview of DNA methods for the identification and individualization of marijuana
Heather Miller Coyle, Timothy M Palmbach, Henry C Lee, Nicholas Juliano
July 2003 Croatian Medical Journal 44(3):315-21
https://forendex.safs1966.org/upload..._Marijuana.pdf
The purpose of this review is to summarize the status of DNA-based methods for the identification and individualization of marijuana. In forensics, both identification of a substance as marijuana and the subsequent individualization of a sample may be desired for casework. Marijuana identification methods in the United States primarily include biochemical tests and, less frequently, DNA-based tests. Under special circumstances, DNA-based tests can be useful. For example, if the quantity of seized marijuana is extremely small and/or biochemical tests do not detect any D9-tetrahydrocannabinol (THC), DNA identification of plant material as Cannabis is still possible. This circumstance can arise when seeds, trace residue, tiny leaf fragments, or fine roots need to be analyzed. Methods for the individualization of marijuana include amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and short tandem repeat (STR) techniques that link an evidentiary sample to a source. Marijuana growers propagate their plants either by seed or by cloning. Seed-generated marijuana plants are expected to have unique DNA profiles analogous to a human population. Cloned marijuana plants, however, exhibit identical DNA profiles that allow for tracking of plant material derived from a common genetic lineage. The authors have validated the AFLP method for marijuana samples and are constructing a comparative database of marijuana seizure samples to estimate the expected frequency of a DNA profile match between unrelated plants. Continued development of DNA-based methods for plants can be useful for marijuana and other types of plant evidence in forensics

ANALYSIS OF GENES FROM CANNABINOID BIOSYNTHETIC PATHWAY
STIASNA KLARA, PRESINSZKA MARIA, VYHNANEK TOMAS, TROJAN VACLAV, MRKVICOVA EVA, HRIVNA LUDEK, HAVEL LADISLAV
Conference Paper · November 2015 International Ph.D. Students Conference on MendelNet 2015 Fac Agron, Brno, Czech Republic
https://www.researchgate.net/publica...THETIC_PATHWAY
Cannabis, or hemp, (Cannabis sativa L.) has been grown for thousands of years all around the world for its valuable traits in fabric making industry and traditional medicine. Today it is still considered as an important crop and medicinal plant. The most studied cannabinoids, secondary metabolites of genus Cannabis, are ?-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Ratio between THC and CBD content is relevant marker in differentiation of “fiber-type” and “drug-type”. Biosynthesis of THC and CBD is catalyzed by enzymes tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. Sequence heterogeneity of genes encoding these enzymes in six varieties of industrial hemp, namely ‘Finola’, ‘Tiborszálási’, ‘Tisza’, ‘Kompolti’, ‘Kompolti hybrid TC’ and ‘Carmagnola’, was studied. Partial sequences of cannabidiolic acid synthase gene with numerous indels and single nucleotide polymorphisms were detected. Similar situation was observed in full-length tetrahydrocannabinolic acid synthase sequences. According to PCR marker, three tested varieties were indicated as potentially rich in THC content, what will be verified by HPLC in future.

Maybe a paper of the work will be published besides this poster?
Analysis of Published PCR Primers to Determine Sex of Industrial Hemp Seedlings
Allison Nalesnik and Les Erickson
2020 Posters on the Bay
Salisbury University Student Research Luncheon at the Maryland General Assembly
https://future4200.com/uploads/short...6cX23N0RDF.pdf
Industrial hemp refers to non-intoxicating strains of Cannabis sativa that produce high levels of cannabidiolic acid (CBDA), the precursor to cannabidiol (CBD). CBD has known health benefits and is being tested for a wide range of potential medicinal applications. When growing hemp for CBD, unfertilized female plants are desired as they produce high amounts of CBDA in their flower organs. Identification and elimination of male hemp plants is imperative and allows cultivators to spend fewer resources on plants that will not produce flower and could possibly pollinate female plants. It has been reported that male cannabis plants can be identified weeks before any visual sex features using the polymerase chain reaction (PCR), however the results from reported studies have been inconsistent in identifying male plants. Here, genomic DNA from 13 hemp seedlings was analyzed using male-DNA-specific PCR primer pairs described in the research literature. Three different primer pairs (SCAR323, SCAR119, and MADC2) were used in the analyses. SCAR323 primers have been reported to amplify a male-specific amplicon of 323 base pairs (bp); SCAR119 are reported to amplify a male-associated amplicon of 119 bp; and MADC2 primers have been reported to amplify a male-associated amplicon of 300 bp and a female-associated amplicon of 450 bp

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase
Flavia Fulvio, Roberta Paris, Massimo Montanari, Cinzia Citti, Vincenzo Cilento, Laura Bassolino, Anna Moschella, Ilaria Alberti, Nicola Pecchioni, Giuseppe Cannazza and Giuseppe Mandolino
Plants 10(9):1857 September 2021
DOI: 10.3390/plants10091857
https://www.researchgate.net/publica..._acid_synthase
Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids

Analysis of THCA synthase gene expression in cannabis: A preliminary study by real-time quantitative PCR
Fidelia Cascinia, Stella Passerottib, Ilaria Boschia
Forensic Science International Volume 231, Issues 1–3, 10 September 2013, Pages 208–212
doi: 10.1016/j.forsciint.2013.05.019
In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression.
Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.

Analysis of the genetic diversity of Chinese native Cannabis sativa cultivars by using ISSR and chromosome markers
L.G. Zhang, Y. Chang, X.F. Zhang, F.Z. Guan, H.M. Yuan, Y. Yu, L.J. Zhao (2014).
Genet. Mol. Res. 13, 10490–10500.
doi: 10.4238/2014.december.12.10
Hemp (Cannabis sativa) is an important fiber crop, and native cultivars exist widely throughout China. In the present study, we analyzed the genetic diversity of 27 important Chinese native hempcultivars, by using inter-simple sequence repeats (ISSR) and chromosome markers. We determined the following chromosome formulas: 2n = 20 = 14m + 6sm; 2n = 20 = 20m; 2n = 20 = 18m +2sm; 2n = 20 = 16m + 4sm; and 2n = 20 = 12m + 8sm. The results of our ISSR analysis revealed the genetic relationships among the 27 cultivars; these relationships were analyzed by using the unweighted pair-group method based on DNA polymorphism. Our results revealed that all of the native cultivars showed considerable genetic diversity. At a genetic distance of 0.324, the 27 varieties could be classified into five categories; this grouping corresponded well with the chromosome formulas. All of the investigated hemp cultivars represent relatively primitive types; moreover, the genetic distances show a geographical distribution, with a small amount of regional hybridity.

Not Cannabis specific
Application of CRISPR/Cas9 in plant biology
Xuan Liu, SuruiWu, JiaoXu, ChunSuin, JianheWei
Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Doi: 10.1016/j.apsb.2017.01.002
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR- associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation , high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considereda revolutionary technology in plant biology. Here, we introduce the mechanism of the typeII CRISPR/Cas called CRISPR/Cas9, update its recentadvances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.

Not Cannabis specific
Apoptosis gene EI24, compositions, and methods of use
https://patents.google.com/patent/US5843659A/en
https://patentimages.storage.googleapi s.com/72/63/11/675686448e1963/US5843659.pdf
Disclosed is the isolation and characterization of EI24, a novel gene whose 2.4 kb mRNA is induced following etoposide treatment. Induction of EI24 mRNA by etoposide required expression of wild-type p53. Overexpression of functional p53 was sufficient to induce expression of the EI24 mRNA. The EI24 mRNA was also induced in a p53-dependent manner by ionizing irradiation of primary murine thymocytes. The invention is thus directed to an isolated EI24 protein, nucleotide sequences coding for and regulating expression of the protein, antibodies directed against the protein, and recombinant vectors and host cells containing the genetic sequences coding for and regulating the expression of the protein sequence. The invention is also directed to genomic DNA, cDNA, and RNA encoding the EI24 protein sequence and to corresponding antisense RNA sequences. Antibodies can be used to detect EI24 in biological specimens, including, for example, human tissue samples. The present invention is further directed to methods of treating degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death. The present invention is further directed to methods for diagnosing degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death, as well as methods for monitoring the progress of such degenerative disorders.

Assessing Genetic Diversity in Cannabis sativa Using Molecular Approaches.
Punja ZK, Rodriguez G, Chen S (2017)
In S Chandra, H Lata, MA ElSohly, eds, Cannabis sativa L. - Botany and Biotechnology. Springer International Publishing, Cham, pp 395–418
DOI :10.1007/978-3-319-54564-6_19

I liked this one:
Assessment of Genetic Diversity and Population Structure in Iranian Cannabis Germplasm
Aboozar Soorni, Reza Fatahi, David C. Haak, Seyed Alireza Salami & Aureliano Bombarely
Cannabis sativa has a complex history reflected in both selection on naturally occurring compounds and historical trade routes among humans. Iran is a rich resource of natural populations which hold the promise to characterize historical patterns of population structure and genetic diversity within Cannabis. Recent advances in high-throughput DNA sequencing technologies have dramatically increased our ability to produce information to the point that it is now feasible to inexpensively obtain population level genotype information at a large scale. In the present investigation, we have explored the use of Genotyping-By-Sequencing (GBS) in Iranian cannabis. We genotyped 98 cannabis samples 36 from Iranian locations and 26 accessions from two germplasm collections. In total, 24,710 high-quality Single Nucleotide Polymorphisms (SNP) were identified. Clustering analysis by Principal Component Analysis (PCA) identified two genetic clusters among Iranian populations and fineSTRUCTURE analysis identified 19 populations with some geographic partitioning. We defined Iranian cannabis in two main groups using the results of the PCA and discovered some strong signal to define some locations as population according to fineSTRUCTURE analyses. However, single nucleotide variant analysis uncovered a relatively moderate level of variation among Iranian cannabis.

Assessment of the Genetic Stability of Micropropagated Plants of Cannabis sativa by ISSR Markers
Hemant Lata, Suman Chandra, Natascha Techen, Ikhlas A. Khan, Mahmoud A. ElSohly
Planta Med 2010; 76: 97–100
DOI 10.1055/s-0029-1185945
Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic stability of the micropropagated plants of Cannabis sativa over 30 passages in culture and hardening in soil for 8 months. A total of 15 ISSR primers resulted in 115 distinct and reproducible bands. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants,
confirming the genetic stability among clones and mother plants. Chemical analysis of cannabinoids, using gas chromatography/flame ionization detection (GC/FID), was done to further confirm whether the qualitative and quantitative differences in the major secondary metabolites exist between the mother plant and micropropagated plants. Six major cannabinoids – Δ9-THC, THCV, CBD, CBC, CBG, and CBN – were identified and compared with
the mother plant. Our results clearly showed a similar cannabinoid profile and insignificant differences in THC content between the two types of plants. These results suggest that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and applicable for clonal mass propagation of C. sativa.

BLOCKCHAINED CANNABIS DNA
THE INFORMATION CHAIN FOR ADVANCED GROWERS AND REGULATORS

https://cdn2.hubspot.net/hubfs/34029..._hsmi=57554917
This document will provide an overview of how the cannabis industry can use DNA sequencing and blockchain technology to create a transparent supply chain that cultivators, dispensaries, regulators, and consumers can trust.
A blockchain-based genetic catalog of all cannabis varieties in a given market provides transparency to an opaque world of underground names and folklore medicine. We have the unique ability in time to build the most comprehensive cannabis tracking system in the world that will bring patient safety, manufacturer transparency, and regulatory comfort.
Such a system is attractive to regulators, because it delivers emergent incentives against diversion and counterfeiting. Blockchain-linked mobile phone applications could instantly verify legitimate material with QR code links to public strain data on Kannapedia.net.
Cannabis breeders and cultivators can also use genetic sequencing to accurately fingerprint their varieties and publish their genetic data to the Bitcoin blockchain. They can then use the information to defend against any future patents or file for their own patent.

Not Cannabis Specific
Broad-Scale Genetic Diversity of Cannabis for Forensic Applications
Christophe Dufresnes, Catherine Jan, Friederike Bienert, Jérôme Goudet, and Luca Fumagalli
PLoS One. 2017; 12(1): e0170522.
doi: 10.1371/journal.pone.0170522
Cannabis (hemp and marijuana) is an iconic yet controversial crop. On the one hand, it represents a growing market for pharmaceutical and agricultural sectors. On the other hand, plants synthesizing the psychoactive THC produce the most widespread illicit drug in the world. Yet, the difficulty to reliably distinguish between Cannabis varieties based on morphological or biochemical criteria impedes the development of promising industrial programs and hinders the fight against narcotrafficking. Genetics offers an appropriate alternative to characterize drug vs. non-drug Cannabis. However, forensic applications require rapid and affordable genotyping of informative and reliable molecular markers for which a broad-scale reference database, representing both intra- and inter-variety variation, is available. Here we provide such a resource for Cannabis, by genotyping 13 microsatellite loci (STRs) in 1 324 samples selected specifically for fibre (24 hemp varieties) and drug (15 marijuana varieties) production. We showed that these loci are sufficient to capture most of the genome-wide diversity patterns recently revealed by NGS data. We recovered strong genetic structure between marijuana and hemp and demonstrated that anonymous samples can be confidently assigned to either plant types. Fibres appear genetically homogeneous whereas drugs show low (often clonal) diversity within varieties, but very high genetic differentiation between them, likely resulting from breeding practices. Based on an additional test dataset including samples from 41 local police seizures, we showed that the genetic signature of marijuana cultivars could be used to trace crime scene evidence. To date, our study provides the most comprehensive genetic resource for Cannabis forensics worldwide.

Can public online databases serve as a source of phenotypic information for Cannabis genetic association studies?
Aardema ML, DeSalle R
PLOS ONE 16(2): e0247607. (2021)
DOI: 10.1371/journal.pone.0247607
The use of Cannabis is gaining greater social acceptance for its beneficial medicinal and recreational uses. With this acceptance has come new opportunities for crop management, selective breeding, and the potential for targeted genetic manipulation. However, as an agricultural product Cannabis lags far behind other domesticated plants in knowledge of the genes and genetic variation that influence plant traits of interest such as growth form and chemical composition. Despite this lack of information, there are substantial publicly available resources that document phenotypic traits believed to be associated with particular Cannabisvarieties. Such databases could be a valuable resource for developing a greater understanding of genes underlying phenotypic variation if combined with appropriate genetic information. To test this potential, we collated phenotypic data from information available through multiple online databases. We then produced a Cannabis SNP database from 845 strains to examine genome wide associations in conjunction with our assembled phenotypic traits. Our goal was not to locate Cannabis-specific genetic variation that correlates with phenotypic variation as such, but rather to examine the potential utility of these databases more broadly for future, explicit genome wide association studies (GWAS), either in stand-alone analyses or to complement other types of data. For this reason, we examined a very broad array of phenotypic traits. In total, we performed 201 distinct association tests using web-derived phenotype data appended to 290 uniquely named Cannabis strains. Our results indicated that chemical phenotypes, such as tetrahydrocannabinol (THC) and cannabidiol (CBD) content, may have sufficiently high-quality information available through web-based sources to allow for genetic association inferences. In many cases, variation in chemical traits correlated with genetic variation in or near biologically reasonable candidate genes, including several not previously implicated in Cannabis chemical variation. As with chemical phenotypes, we found that publicly available data on growth traits such as height, area of growth, and floral yield may be precise enough for use in future association studies. In contrast, phenotypic information for subjective traits such as taste, physiological affect, neurological affect, and medicinal use appeared less reliable. These results are consistent with the high degree of subjectivity for such trait data found on internet databases, and suggest that future work on these important but less easily quantifiable characteristics of Cannabis may require dedicated, controlled phenotyping.

Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples
Ulrich Reimann-Philipp, Mark Speck, Cindy Orser, Steve Johnson, Aaron Hilyard, Helen Turner, Alexander J. Stokes, and Andrea L. Small-Howard
Cannabis and Cannabinoid Research
Volume ?, Number ?, 2019
DOI: 10.1089/can.2018.0063

Cannabis DNA Typing Methods
H. Miller Coyle
Encyclopedia of Forensic Sciences (Second Edition) 2013, Pages 382–386
DOI: 10.1016/B978-0-12-382165-2.00073-8
This article focuses on Cannabis, primarily, the development of biomarkers to classify and uniquely identify marijuana. DNA methods such as short tandem repeat, amplified fragment length polymorphism, and single-nucleotide polymorphism are discussed with an emphasis on forensic data-basing applications and the benefits and limitations of each method. It also provides an overview of marijuana regarding the history of cultivation in the United States, associative evidence at crime scenes and current law enforcement concerns in the US, and microscopic and chemical methods for classification, geo-sourcing and high-throughput screening.

Cannabis gene variation - comparison of multiple genome assemblies
Oori Weisshaus NRGene Ltd.
Journal of Plant Genetics and Breeding
https://www.omicsonline.org/open-acc...assemblies.pdf
In recent years the Cannabis sativa genome is at the cen¬tre of research efforts due to the species’ large array of secondary metabolism products and their potential to interact with mammalian systems. Removal of legislative barriers has enabled the publication of multiple genome assemblies using various technologies for sequencing and assembly, mostly using highly heterozygous plants. In this work we demonstrate a pan-genome comparison of multiple cannabis genomes of hemp and drug type culti¬vars. We have de-novo assembled two new heterozygous elite-line genomes with fully phased high accuracy assem¬blies and compared them to four public reference-level non-phased assemblies and several commercial clones at WGS-level assembly. This comparison was carried out in a pan-genome structure based on a common coordi¬nate system of the CBDRx reference genome. We have aligned and ordered multiple genome assemblies with phased haplotypes and created uniform chromosome mapping. We have generated a non-redundant dataset of 43,000 transcripts and mapped it to each haplotype. This enabled identification of allelic variation and novel homologues of important genes for cannabinoid biosyn¬thesis, as well as an accurate comparison of copy number, present-absent and structural variations, identification of highly conserved gene region duplications and identifi¬cation of novel candidate genes. We have identified hy¬per-variable regions and massive genome rearrangements that may hold great significance to cannabis and hemp research and breeding.

Cannabis Genomics
Francesco Tonolo, Cannabis biotechnology research, Leiden University
Four classroom meetings: January 18, 21, 25, 28, 2022 Class delayed until mid 2022 I will post dates when I have them.
Hortus Botanicus, Amsterdam
Cost €1200 (exclusive btw)
In cooperation with MCS Medical Cannabis Society
https://medicalcannabisociety.eu/etn/cannabis-genomics/
(I will attend classes)

Cannabis Genomics & Cannabinoid Genes, Cannabis Conversations Dr. Daniela Vergara - YouTube

Overview of Cannabis genes
Nov 3 2021 YouTube

CannabisGDB: a comprehensive genomic database for Cannabis Sativa L.
Sen Cai, Zhiyuan Zhang, Suyun Huang Xu Bai, Ziying Hua Cng, Yiping Jason Zhang,
Likun Huang, Weiqi Tang, George Haughn, Shijun You, Yuanyuan Liu,
Plant Biotechnology Journal 19 January 2021
https://onlinelibrary.wiley.com/doi/10.1111/pbi.13548
DOI: 10.1111/pbi.13548
Being one of the world’s oldest crops with significant economic and medicinal importance, Cannabis Sativa L. (cannabis) has attracted an enormous amount of attention and become one of the most popularly cultivated plants worldwide (Gao et al., 2020). The content of ?9?tetrahydrocannabinol (THC) determines the legal status of the cannabis varieties. Uncontrolled varieties called hemp are defined as those that have 0.3% or less THC, while marijuana is defined as varieties with higher than 3% (4%?35%).

Cannabis Microbiome Sequencing: Implications for Cannabis Safety Testing
Kyle Boyar
DOI: 10.13140/RG.2.2.18308.17288
Conference: Cannabis Science Conference East April 2019
Project: Medicinal Genomics
The cannabis plant and cannabis products are highly varied and complex matrices with each different product and route of administration having its own considerations for microbial testing. In the absence of rigorous study, this immature industry has decided to adopt methods commonly used in food testing to obtain information about the potential microbial hazards present. However, DNA sequencing of both the cannabis microbiome and the conditions before and after culturing tell a tale of inaccurate methodology. Many of the methods that are currently being employed are leading the cannabis industry astray, while blinding them to the real hazards that could be present. This presentation will walk you through the data that shows this and the discoveries we've made along the way that will hopefully open fresh discussions with new perspective on how to tackle microbiological contaminants in cannabis.

Cannabis Phylotree
https://www.kannapedia.net/cannabis-phylotree/
The cannabis phylogenetic tree or evolutionary tree shown below is a branching diagram or “tree” showing the inferred evolutional relationships among various cannabis strains—their phylogeny—based upon similarities and differences in their genetic characteristics. Here the genetic characteristics are the identity and frequency of Single Nucleotide Polymorphism (SNP) in the captured regions of each cannabis straingenome (see description in each Tab under the tree for details). Genetic distance is a summary measure of the genetic divergence between cannabis strains, and the genetic distance of any two strains is approximately proportional to the total length of the radial parts in the path connecting the two ”leafs”, which represent the two strains. SNPs of each strain are identified by DNA sequencing and bioinformaticsanalysis in our StrainSEEK™ Strain Identification and Registration Service.

Cannabis Plant Breeding and Genetics Cannabis Conversations with Mariolla Schrool

Review of Schrool Medical activities
Dec 20 2021 YouTube

CANNABIS RESEARCH GENOMIC INITIATIVE CGRI
https://cannabisgenomics.org/research/
Our Research:
Our work is divided up between the plants, and wetlab where we extract and amplify DNA, and our computers where the analysis happens. Learn more about our research goals below.
1. Build an Ultra-high density genetic map of Cannabis
2. Develop the history, phylogeny and phylogeography of Cannabis
3. Discover the hybrid origins of modern Cannabis cultivars
4. Understand the morphologic differences between Cannabis strains

Cannabis seed identification by chloroplast and nuclear DNA
Tsai, L., Hsieh, H.-M., Huang, L.-H., Wang, J, Linacre, A., & Lee, J. C.
Forensic Science International 158 (2006) 250–251
doi:10.1016/j.forsciint.2005.10.021 (2006).
The Control Substances Hazard Prevention Act classifies Cannabis sativa as a controlled substance under schedule II of the Act in Taiwan. The punishment for trafficking may be a sentence of up to life in prison. Huo Ma Jen is usually imported as a Chinese medicinal remedy and also as bird food. Importers of cannabis material make use of loopholes in national legislation and claim that the botanical material is from the plant known as Huo Ma Jen and as such avoids normal regulations. The term Huo Ma Jen is therefore frequently used as a cover for the importation of cannabis seeds for germination. The identification of C. sativa plant material is conducted using morphological observation based upon the cystolithic hairs on the leaves [1], chemical tests to identify of the presence of cannabinoids [2] and more recently using DNA analysis [3,4]. When seeds suspected of contravening national legislation are seized, the seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. There is a delay in the process while the seeds germinate. In the case outlined it was possible to obtain species identification from the seeds directly without the need for propagation. The methods used analyzed species conserved loci and were used in conjunction with the 2,3,5-triphenyltetrazolium chloride (TTC) method [5] to detect viability. The gene loci used were the intergenic spacer (IGS) of trnL (UAA)–trnF (GAA) in the chloroplast genome and the internal transcribed spacer I (ITS1) of nuclear rDNA.

Cannabis, The Multibillion Dollar Plant That No Genebank Wanted
Davoud Torkamaneh , Andrew Maxwell Phineas Jones
Genome 2021
DOI: 10.1139/gen-2021-0016
Although cannabis is legalized and accepted as an agricultural commodity in many places around the world, a significant lack of public germplasm repositories remains an unresolved problem in the cannabis industry. The acquisition, preservation and evaluation of germplasm including landraces and ancestral populations is key to unleash the full potential of cannabis in the global marketplace. We argue here that accessible germplasm resources are crucial for long-term economic viability, preserving genetic diversity, breeding, innovation, and the long-term sustainability of the crop. We believe that cannabis restrictions require a second look to allow genebanks to play a fuller and more effective role in conservation, sustainable use, and exchange of cannabis genetic resources.
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Characterization; Genome Sizes and Morphology of Sex Chromosomes in Hemp (Cannabis sativa ).
Sakamoto, K., Akiyama, Y., Fukui, K., Kamada, H., & Satoh, S.
CYTOLOGIA, 63(4), 459–464.(1998).
doi:10.1508/cytologia.63.459
A dioecious plant, Cannabis sativa has two sex chromosomes (X and Y). The genome sizes of the diploid female and male plants were determined to be 1636 and 1683 Mbp, respectively, by flow cytometry. By the karyotype analysis, the X and Y chromosomes were found to be submetacentric and subtelocentric, respectively. The Y chromosome had the largest long arm with a satellite in the terminal of its short arm. Conspicuous condensation was specifically observed in the long arm and satellite of the Y chromosome during the prometaphase to metaphase stages. These results indicate that the Y chromosome, especially in its long arm, specifically differentiates in Cannabis sativa and might contribute to the sex determination


Characterization of Cannabinoid Synthase Family members

Anthony Torres, Keith Allen, Christian Cizek, Kim Neubauer, Daniela Vergara, Robert M Givens, Kymron DeCesare, Donald P Land, Reginald J Gaudino
Cannabis sativa, a unique plant has a long history of domestication by humans for thousands of years it’s medicinal properties have been utilized. Find ptt link I have paper .

Characterization of 15 STR cannabis loci: Nomenclature proposal and SNPSTR haplotypes
Laura Valverdea, Christian Lischkaa, Stefanie Scheipera, Johanna Nedelea, Rachel Challisa, Marian M. de Pancorbob, Heidi Pfeiffera, Stephan Köhnemanna,
Forensic Science International: Genetics
Volume 9, March 2014, Pages 61–65
DOI: 10.1016/j.fsigen.2013.11.001
The standardization of methods for individualizing Cannabis sativa plants could offer new possibilities in the investigation of its illegal trade. Here we present the first nomenclature proposal for 15 cannabis STRs, which allows an initial standardization for performing comparisons between laboratories and generating genotype databases. Several alleles of the 15 STR loci have been sequenced. This has revealed that not all the STR loci are equally suitable for the individualization purposes. Moreover, several nucleotide variations have been detected both inside the repeat structure and/or in the flanking region. All the different SNPSTR haplotypes are presented and compared with the previous sequence raw data of the 15 STR loci. The SNPSTR data could considerably increase the informative value of the STRs, which could be very useful in complex cases.

Characterization of the polymorphic repeat sequence within the rDNA IGS of Cannabis sativa
Hsing-Mei Hsieh, Chia-Ling Liu, Li-Chin Tsai, Rur-Jyun Hou,
Kuo-Lan Liu, Adrian Linacre, James Chun-I Lee
Forensic Science International 152 (2005) 23–28
doi:10.1016/j.forsciint.2005.02.022
The rDNA intergenic spacer (IGS) structure of Cannabis sativa contains six variable repeat motifs within a locus spanning 1387 base pairs. The degree of variation of the first three motifs was examined using 77 samples from cannabis samples. The samples originated from five seizures in Taiwan and seed stocks from six different countries. The results showed that there were four types of sequences producing PCR products at either 255, 260, 264 or 265 base pairs. The data obtained indicates that this region of rDNA IGS exhibits a degree of polymorphism that while insufficient by itself can be added to a multiplex with other cannabis STR loci.

Chromothripsis and Epigenetics in the Heritable Genotoxicity, Carcinogenicity and Fetotoxicity of Cannabis
and Other Addictions.
Albert Stuart Reece, Gary Kenneth Hulse
Mutat Res. 2016 Jul;789:15-25.
doi:10.1016/j.mrfmmm.2016.05.002
The recent demonstration that massive scale chromosomal shattering or pulverization can occur abruptly due to errors induced by interference with the microtubule machinery of the mitotic spindle followed by haphazard chromosomal annealing, together with sophisticated insights from epigenetics, provide profound mechanistic insights into some of the most perplexing classical observations of addiction medicine, including cancerogenesis, the younger and aggressive onset of addiction-related carcinogenesis, the heritability of addictive neurocircuitry and cancers, and foetal malformations. Tetrahydrocannabinol (THC) and other addictive agents have been shown to inhibit tubulin polymerization which perturbs the formation and function of the microtubules of the mitotic spindle. This disruption of the mitotic machinery perturbs proper chromosomal segregation during anaphase and causes micronucleus formation which is the primary locus and cause of the chromosomal pulverization of chromothripsis and downstream genotoxic events including oncogene induction and tumour suppressor silencing. Moreover the complementation of multiple positive cannabis-cancer epidemiological studies, and replicated dose-response relationships with established mechanisms fulfils causal criteria. This information is also consistent with data showing acceleration of the aging process by drugs of addiction including alcohol, tobacco, cannabis, stimulants and opioids. THC shows a non-linear sigmoidal dose-response relationship in multiple pertinent in vitro and preclinical genotoxicity assays, and in this respect is similar to the serious major human mutagen thalidomide. Rising community exposure, tissue storage of cannabinoids, and increasingly potent phytocannabinoid sources, suggests that the threshold mutagenic dose for cancerogenesis will increasingly be crossed beyond the developing

Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.
Raharjo, T. J., Chang, W.-T., Verberne, M. C., Peltenburg-Looman, A. M. G., Linthorst, H. J. M., & Verpoorte, R.
Plant Physiology and Biochemistry, 42(4), 291–297 (2004). doi:10.1016/j.plaphy.2004.02.011
A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

Comparative genetic structure of Cannabis sativa including federally produced, wild collected, and cultivated samples.
Anna L. Schwabe, Connor J. Hansen, Richard M. Hyslop and Mitchell E. McGlaughlin
Front. Plant Sci. (2021)
doi: 10.3389/fpls.2021.675770
https://www.frontiersin.org/articles...75770/abstract
Currently in the U.S. the sole licensed facility to cultivate Cannabis sativa L. for research purposes is the University of Mississippi, which is funded by the National Institute on Drug Abuse (NIDA). Studies researching Cannabis flower consumption rely on NIDA-supplied “research grade marijuana”. Previous research found that cannabinoid levels of NIDA supplied Cannabis do not align with commercially available Cannabis. We sought to investigate the genetic identity of Cannabis supplied by NIDA relative to common categories within the species. This is the first genetic study to include “research grade marijuana” from NIDA. Samples (49) were assigned as Wild Hemp (feral;6) and Cultivated Hemp (3), NIDA (2), CBD drug type (3), and high THC drug type subdivided into Sativa (11), Hybrid (14), and Indica (10). Ten microsatellites targeting neutral non-coding regions were used. Clustering and genetic distance analyses support a division between hemp and drug type Cannabis. All hemp samples clustered genetically, but no clear distinction of Sativa, Hybrid, and Indica subcategories within retail marijuana samples was found. Interestingly, the two analyzed “research grade marijuana” samples obtained from NIDA were genetically distinct from most drug type Cannabis available from retail dispensaries. Although the sample size was small, “research grade marijuana” provided for research is genetically distinct from most retail drug type Cannabis that patients and patrons are consuming.
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Comparative RNA?Seq analysis reveals genes associated with masculinization in female Cannabis sativa
Ayelign M. Adal · Ketan Doshi · Larry Holbrook · Soheil S. Mahmoud
Planta (2021) 253:17
doi: 10.1007/s00425-020-03522-y
https://link.springer.com/article/10...25-020-03522-y
Genetically female Cannabis sativa plants normally bear female flowers, but can develop male flowers in response to environmental and developmental cues. In an attempt to elucidate the molecular elements responsible for sex expression in C. sativa plants, we developed genetically female lines producing both female and chemically-induced male flowers. Furthermore,
we carried out RNA-Seq assays aimed at identifying differentially expressed genes responsible for male flower development in female plants. The results revealed over 10,500 differentially expressed genes, of which around 200 potentially control masculinization of female cannabis plants. These genes include transcription factors and other genes involved in male organ (i.e., anther and pollen) development, as well as genes involved in phytohormone signalling and male-biased phenotypes. The expressions of 15 of these genes were further validated by qPCR assay confirming similar expression patterns
to that of RNA-Seq data. These genes would be useful for understanding predisposed plants producing flowers of both sex types in the same plant, and help breeders to regulate the masculinization of female plants through targeted breeding and plant biotechnology.

Comparison of effectiveness of ISSR and RAPD markers in genetic characterization of seized marijuana (Cannabis sativa L.) in Turkey
Ali Seyit, Seyit Ali Kayis, Erdogan E Hakki, Emine Pinarkara
African journal of agricultural research 5(21):2925-2933 December 2010
https://www.researchgate.net/publica...va_L_in_Turkey
This paper presents results from comparison of effectiveness of Inter Simple Sequence Repeats (ISSR), Randomly Amplified Polymorphic DNA (RAPD), and ISSR and RAPD (ISSR-RAPD) markers together in characterization of Cannabis accessions. The plant material used was common accessions of psychoactive Cannabis samples (a total of 17 accessions), which were used in discriminating drug type Cannabis from hemp type Cannabis via ISSR and characterization of Cannabis samples via RAPD. Data were analyzed via cluster and principal coordinate analyses (PCoA). Discriminating powers of ISSR and RAPD markers on the seized Cannabis accessions were evaluated by utilising polymorphism information content, resolving power and marker index (MI). The PCoA of ISSR and ISSR-RAPD markers data produced similar results. Average resolving power and MI values of ISSR assay found to be slightly higher than those of RAPD assay. Consequently, ISSR markers would be a better choice compared to RAPD markers in characterization of Cannabis accessions. Key words: Cannabis sativa L., individualization of seized marijuana, inter simple sequence repeat (ISSR), randomly amplified polymorphic DNA (RAPD), principal coordinate analysis, cluster analysis.

Comparison of Hemp Varieties Using Random Amplified Polymorphic DNA Markers
Silvia Forapani, Andrea Carboni, Claudia Paoletti, V. M. Cristiana Moliterni, Paolo Ranalli, and Giuseppe Mandolino
Crop Science 41(6) November 2001
DOI: 10.2135/cropsci2001.1682
The objective was to study the genetic structure and degree of variability of hemp (Cannabis sativa L.) varieties. Six varieties of hemp were analyzed by random amplified polymorphic DNA (RAPD) analysis, using 10 plants per variety. The varieties were a dioecious landrace, a dioecious selection from it, a cross-bred cultivar, a monoecious variety, a drug strain, and an inbred female line. The genetic complexity of each cultivar was investigated by determining the number of bands produced by the primers used, the number of fixed and polymorphic loci, the average allele frequency, and the heterozygosity. A good correlation was found between these parameters and the genetic origin and breeding strategy of each variety. The average polymorphism over all varieties and loci was 97.1%; the single cultivar polymorphism ranged from 31.1 to 85.5%. Heterozygosity ranged from 0.05 (inbred female line) to 0.26 (cross-bred Fibranova). The average heterozygosity calculated over all 102 loci and all plants studied was 0.29. The Fst (Wright's fixation index) value calculated for all loci was 0.48, and only 33.3% of the scored loci had higher values and can be considered informative for cultivar identification. A Fisher's test based on allele frequencies suggested complete differentiation among all varieties, with the exception of the Italian dioecious varieties Carmagnola and CS, for which no discriminating alleles were found. The correlations among the molecular data and the genetic structure of the different cultivars and the consequences in relation to variety discrimination in hemp are discussed.

Constructing a Draft Map of the Cannabis Proteome
Conor Jenkins, Benjamin C Orsburn
March 2019
DOI: 10.1101/577635
Recently we have seen a relaxing on the historic restrictions on the use and subsequent research on the cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. In this study we present our initial findings consisting of the identification of 17,269 unique proteins identified from Cannabis plant materials, as well as 6,110 post-translational modifications identified on these proteins. The results presented demonstrate the first steps toward constructing a complete draft map of the Cannabis proteome.

Creating the First Cannabis Pangenome
(Original Press Release from NRGene)
NRGENE AND PURE CANNABIS RESEARCH AG TEAM UP FOR NOVEL CANNABIS PRODUCTION
https://www.nrgene.com/PRESS-RELEASE...CH-AG-TEAM-UP/
Complete genomes and pangenomes of multiple varieties to enhance R&D for new and improved cannabis varieties for superior cultivation that are rich in beneficial compounds
The varieties of Pure Cannabis Research will be tailored to its customers’ needs, including healthier, hardier plants that are optimal for production, as well as custom cannabinoid, terpenoid, and flavonoid profiles. These metabolite profiles are key for targeting specific therapeutic uses, such as pain relief and sleep support, as well as providing unique flavor profiles for energy drinks and tobacco-alternative products.
“We’re at the forefront of providing safe, non-addictive, CBD-based products for tobacco alternatives, beverages, cosmetics, and other uses for the Swiss and European markets,” said Stevens Senn, CEO of Pure Holding Group. “NRGene has refined the process for identifying key genetic traits across virtually any species of plants, so we are partnering with them in order to support our breeding program and develop plants with any trait that is biologically possible to best serve the international and national markets with new varieties.”
Pure Cannabis Research is leveraging NRGene’s DeNovoMAGIC™ technology for the assembly of multiple cannabis genomes. NRGene’s DeNovoMAGIC is a powerful platform that was used successfully to assemble the first-ever complete wheat genome. Their success has continued to potato, strawberry, barley, rye, and oat, which all have larger and/or more complex genomes than that of humans.
On behalf of Pure Cannabis Research, NRGene will use its PanMAGIC™ technology to create the world’s first cannabis pangenome. This pangenome project will create a collection of sequences that will provide unique and essential insights into the natural genetic diversity of cannabis. PanMAGIC will compare all cultivars to each other to create the highest resolution of local genomic differences of each cultivar and genomic regions that define traits that give the cultivar its prized characteristics. Then, NRGene’s GenoMAGIC will be used to identify markers for key traits. GenoMAGIC™, NRGene’s cloud-based multi-purpose platform, will couple the genetic diversity of cannabis with Pure Cannabis Research breeding program. GenoMAGIC has already been adopted by some of the world’s leading seed companies, including Monsanto and Syngenta.
“The recognition of the importance of cannabis in specific and hemp in general is growing globally, whether for a plethora of medical problem or as a valuable, renewable resource with many eco-friendly uses,” Dr. Gil Ronen, CEO of NRGene. “Cannabis is being increasingly recognized as the most viable solution for many health problems, and we’re gratified that we can contribute to people’s well-being.”

CRISPR/Cas9: A powerful tool for crop genome editing
Gaoyuan Song, Meiling Jia, Kai Chen, Xingchen Kong, Bushra Khattak, Chuanxiao Xie, Aili Li, Long Mao
Crop Science Society of China and Institute of Crop Science, CAAS 2016
Doi: 10.1016/j.cj.2015.12.002
The CRISPR/Cas9 technology is evolved froma type II bacterial immune systemand represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides) that is complementary to a target gene or locus and is anchored by a protospaceradjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or
multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, webriefly survey the history of the CRISPR/Cas9 systeminvention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications ofmodified Cas9 variants, and discuss the
potential of this systemfor targeted genome editing and modification for crop improvement.

CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops
Suhas G. Karkute1, Achuit K. Singh, Om P. Gupta2 Prabhakar M. Singh and
Bijendra Singh
Front. Plant Sci. 8:1635.
doi: 10.3389/fpls.2017.01635
Horticultural crops are an important part of agriculture for food as well as nutritional security. However, several pests and diseases along with adverse abiotic environmental factors pose a severe threat to these crops by affecting their quality and productivity. This warrants the effective and accelerated breeding programs by utilizing innovative biotechnological tools that can tackle aforementioned issues. The recent technique of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR
associated 9 (CRISPR/Cas9) has greatly advanced the breeding for crop improvement due to its simplicity and high efficiency over other nucleases such as Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases. CRISPR/Cas9 tool contains a non-specific Cas9 nuclease and a single guide RNA that directs Cas9 to the specific genomic location creating double-strand breaks and subsequent repair process creates insertion or deletion mutations. This is currently the widely adopted tool for reverse genetics, and crop improvement in large number of agricultural crops. The use of CRISPR/Cas9 in horticultural crops is limited to few crops due to lack of availability of regeneration protocols and sufficient sequence information in many horticultural crops. In this review, the present status of applicability of CRISPR/Cas9 in horticultural crops was discussed along with the challenges and future potential for possible improvement of these crops for their yield, quality, and resistance to biotic and abiotic stress.

Not Cannabis specific
CRISPR-Cas Methods.
Springer Protocols Handbooks.
Islam, M. T., Bhowmik, P. K., & Molla, K. A. (Eds.). (2020).
doi:10.1007/978-1-0716-0616-2
Sometimes in February 2016, I got in touch with Prof. Tofazzal Islam—an editor of this book—about applying pathogenomics to a disease epidemic that just broke out in his native country of Bangladesh. This story has been told over and over, and our perspective is documented in an article we wrote a few months ago after an emotional visit to affected farms in Bangladesh (Kamoun, S., Talbot, N.J., and Islam, M.T. 2019. PLOS Biology, 17: e3000302). However, what I doubt is that either one of us expected at the time the chain of events that have transformed our career paths and ultimately led to this book. What happened is that the blast fungus—the culprit of that dreadful outbreak—not only infected the wheat crop but also ended up redirecting our research activities towards investigating this formidable foe. That journey took us to view CRISPR-Cas9 gene editing of wheat for blast disease resistance as a particularly promising approach to deliver solutions to this problem. How to manage destructive plant pathogens such as the wheat blast fungus? Genetics has to be part of the answer. Crop germplasm screens and breeding towards disease resistance is valuable but can be slow with limited potential. Biotechnology is a promising alternative that has yet to deliver its full potential to plant health problems like wheat blast. One such technology is gene editing or bioediting. Geneticists have long dreamed about editing their organisms’ genomes as one would edit text on a computer with a word processor. The dream has started to become a reality when CRISPR-Cas9 took the biological world by storm just a few years ago in 2013. Other gene editing methods, notably zinc-finger nucleases and TALENs, were available at the time but CRISPR-Cas9 democratized gene editing in the biological sciences. It has made gene editing relatively easy and accessible to pretty much any laboratory with basic molecular biology skills. The breadth of topics and authors that Prof. Islam and his co-editors Dr. Pankaj K. Bhowmik and Dr. Kutubuddin A. Molla have lined up in this book is a great example of the range of developments and applications of this technology and its potential to impact plant health and many other areas of biology. Why another book on CRISPR? The series of chapters reflect the unique perspective of the authors and their aspiration to see CRISPR-Cas9 bioediting applied to the improvement of crops and the human condition in general. My hope is that the book would guide a new generation of plant gene editors from all over the world. I hope it would inspire early career scientists, particularly from developing countries, to embrace gene editing technology and forge their own path in this rapidly expanding area of genetics.

Cryptocurrencies and Zero Mode Wave guides: An unclouded path to a more contiguous Cannabis sativa L. genome assembly.
Kevin McKernan, Yvonne Helbert, Liam T. Kane, Heather Ebling, Lei Zhang, Biao Liu, Zachary Eaton, Luo Sun, Eileen T Dimalanta, Sarah Kingan, Primo Baybayan, Maximilian Press ,William Barbazuk, Timothy Harkins
DOI 10.17605/OSF.IO/N98GP
We describe the use of a Decentralized Autonomous Organization (DAO) to crypto-fund the single molecule sequencing and publication of a Type II Cannabis plant. This resulted in the construction of the most contiguous Cannabis genome assembly to date. The combined use of the Dash cryptocurrency, DAOs, and Pacific Biosciences sequencing delivered a 1.03 Gb genome with a N50 of 665Kb in 77 days from funding to public upload. This represents a 230 fold improvement in the contiguity of the first cannabis assemblies in 2011 and a 4 fold improvement over all cannabis assemblies to date. 34Gb of additional sequencing pushed the assembly to a N50 of 3.8Mb. Hi-C data from Phase Genomics further scaffolded the assembly to 35 contigs at an N50 of 74Mb but requires additional curation. The genome is partially phased and larger than previously reported (2N = 1.33Gb). The CBCA, THCA and CBDA synthase gene clusters have been phased onto respective contigs demonstrating tandem repeat expansions.

Crypto-Funded Cannabis Genomes
Kevin McKernan | CannMed 2018
YOUTUBE talk on sequencing the Cannabis geneome and the need for longer DNA reads and how to achieve them


Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa
Chiho Taguchi, Futoshi Taura, Taro Tamada, Yoshinari Shoyama, Yukihiro Shoyama, Hiroyuki Tanaka, Ryota Kurokib, and Satoshi Morimoto
Acta Cryst. (2008). F64, 217–220
doi:10.1107/S1744309108003795
Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that
catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa
(Mexican strain). PKS-1 was overproduced in Escherichia coli , purified and
finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w /v )
polyethylene glycol 3350 diffracted to 1.65 A ° resolution and belonged to space group P 1, with unit-cell parameters a = 54.3, b = 59.3, c = 62.6 A ° , _ = 69, _ = 81, _ = 80_ . Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w /v ) polyethylene glycol 3350 diffracted to 1.55 A ° resolution and belonged to space group P 21 21 21 , with unit-cell parameters a = 54.3, b = 110, c = 130 A ° . These data will enable us to determine the crystal structure of PKS-1.

Crystallization of ?1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa
Yoshinari Shoyama, Ayako Takeuchi, Futoshi Taura, Taro Tamada, Motoyasu Adachi, Ryota Kuroki, Yukihiro Shoyamaa and Satoshi Morimotoa
Acta Cryst. (2005). F61, 799–801
DOI: 10.1107/S1744309105023365
_1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A ° resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A ° . The calculated Matthews coefficient was approximately 4.1 or 2.0 A ° 3 Da_1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

CYTOLOGICAL STUDIES OF CANNABIS SATIVA IN SHIMLA HILLS OF HIMACHAL PRADESH
Suman Kausha
Indian Journal of Fundamental and Applied Life Sciences 2012 Vol. 2 (1) January- March, pp.240 -245
https://www.cibtech.org/J-LIFE-SCIEN...l%20Kangra.pdf
Chromosome counts made in Cannabis sativa, clearly depict that the genus is diploid with 2n= 20. On the basis of the position of centromere, they can be represented as 10M + 6SM + 4ST i.e., 10 median, 6
sub-median and 4 sub-terminal. Polyploidy were also observed in some of the tetraploid cells with 4n=40. The root-tips showing polyploidy were thicker in appearance than those showing diploid number.

De novo assembly and annotation of transcriptomes from two cultivars of Cannabis sativa with different cannabinoid profiles.
McGarvey, P., Huang, J., McCoy, M., Orvis, J., Katsir, Y., Lotringer, N., … Madhavan, S.
Gene, 145026.(2020).
doi:10.1016/j.gene.2020.145026
Cannabis has been cultivated for millennia for medicinal, industrial and recreational uses. Our long-term goal is to compare the transcriptomes of cultivars with different cannabinoid profiles for therapeutic purposes. Here we describe the de novo assembly, annotation and initial analysis of two cultivars of Cannabis, a high THC variety and a CBD plus THC variety. Cultivars were grown under different lighting conditions; flower buds were sampled over 71 days. Cannabinoid profiles were determined by ESI-LC/MS. RNA samples were sequenced using the HiSeq4000 platform. Transcriptomes were assembled using the DRAP pipeline and annotated using the BLAST2GO pipeline and other tools. Each transcriptome contained over twenty thousand protein encoding transcripts with ORFs and flanking sequence. Identification of transcripts for cannabinoid pathway and related enzymes showed full-length ORFs that align with the draft genomes of the Purple Kush and Finola cultivars. Two transcripts were found for olivetolic acid cyclase (OAC) that mapped to distinct locations on the Purple Kush genome suggesting multiple genes for OAC are expressed in some cultivars. The ability to make high quality annotated reference transcriptomes in Cannabis or other plants can promote rapid comparative analysis between cultivars and growth conditions in Cannabis and other organisms without annotated genome assemblies.

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L. Running title: High-throughput molecular markers in Cannabis
Jacob A. Toth, George M. Stack, Ali R. Cala, Craig H. Carlson, Rebecca L. Wilk, Jamie L. Crawford, Donald R. Viands, Glenn Philippe, Christine D. Smart, Jocelyn K.C. Rose, and Lawrence B. Smart
GCB Bioenergy January 2020
doi: 10.1111/GCBB.12667
Hemp Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high-throughput PACE (PCR
Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa
Masashi Kitamura, Masako Aragane, Kou Nakamura, Kazuhito Watanabe, and Yohei Sasaki
Biol. Pharm. Bull. 39, Vol. 39, No. 7, 1144–1149 (2016)
DOI: 10.1248/bpb.b16-00090
In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60–66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.

Development of transient expression assay for Cannabis sativa which revealed differential Agrobacterium susceptibility among cannabis cultivars
Alexei Sorokin, Narendra Singh Yadav, Daniel Gaudet, Igor Kovalchuk
bioRxiv preprint (2020)
DOI: 10.1101/2020.03.12.988618
In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest and subsequently leads the path to develop an improved crop variety with better agronomic traits. In this study, we have reported a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated
transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay was used to detect the uidA expression. A 1% hydrogen peroxide (H2O2) solution was used for both seed sterilization and rapid germination steps. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Comparison to Nicotiana tabacum (tobacco) showed that cannabis seedlings were less susceptible to transformation with Agrobacterium tumefaciens. The susceptibility to Agrobacterium infection also varied with the different cannabis cultivars. The method established in this study has potential to be an important tool for gene-function studies and genetic improvement in cannabis.
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The following 8 articles are by Sunrise Genetics or Marigene I did not agree with all that was said, but this is the future of Cannabis breeding
Decoding Cannabis
https://www.marijuanaventure.com/dec...abis-genetics/
Sunrise Genetics CEO C.J. Schwartz
https://www.sunrisegenetics.com/
An Introduction to Cannabis Genetics, Part I
https://cannabisindustryjournal.com/...netics-part-i/
By Dr. CJ Schwartz
Understanding natural genetic variation and family trees is essential to breeding successful cannabis strains.
DNA stores information about how to build an organism. Just as a series of 0’s and 1’s represents digital data, DNA data is represented by four letters (A, C, G and T), which inherently allows DNA to store more information per unit The amount of DNA required to build a human is mind-boggling. The human genome has 3.2 billion A’s, C’s, G’s, or T’s, (called nucleotides). Cannabis has 820 million nucleotides. This is true for every cell in the organism. The DNA from a single human cell when spread out would stretch six feet long. A cell is not visible to the naked eye, yet it contains a microscopic thread of DNA six feet long! If you put all the DNA molecules in your body end to end, the DNA would reach from the Earth to the Sun.

An Introduction to Cannabis Genetics, Part II
https://cannabisindustryjournal.com/...etics-part-ii/
By Dr. CJ Schwartz
The most widely used method for gene discovery is quantitative trait loci mapping (QTL).
Plants and animals have roughly 25,000 to 30,000 genes. The genes provide the information needed to make a protein, and proteins are the building blocks for all biological organisms. An ideal analogy is a blueprint (DNA) for an alternator (the protein) in a car (the plant). Proteins are the ‘parts’ for living things. Some proteins will work better than others, leading to visible differences that we call phenotypes. Many traits, and the genes controlling them, are of interest to the cannabis industry. For hemp seed oil, quality, quantity and content can be manipulated through breeding natural genetic variants. Hemp fibers are already some of the best in nature, due to their length and strength. Finding the genes and proteins responsible for elongating the fibers can allow for the breeding of hemp for even longer fibers. In cannabis, the two most popular genes are THCA and CBDA synthases. There are currently over 100 sequences of the THCAS/CBDAS genes, and many natural DNA variations are known. We can make a family tree using just the THCAS, gene data and identify ‘branches’ that result in high, low or intermediate THCA levels. Generally most of the DNA changes have little to no effect on the gene, but some of the changes can have profound effects.

An Introduction to Cannabis Genetics, Part III
https://cannabisindustryjournal.com/...tics-part-iii/
By Dr. CJ Schwartz
Dr. CJ Schwartz discusses polyploidy, epigenetics, sex determination and GMOs as they relate to Cannabis.
Polyploidy in Cannabis
Polyploidy is defined as containing more than two homologous sets of chromosomes. Most species are diploid (all animals) and chromosomal duplications are usually lethal, even partial duplications have devastating effects (Down’s syndrome). Plants are unique as in being able to somewhat “tolerate” chromosomal duplications. We often observe hybrid vigor in the F1, while the progeny of the F1 (F2) will produce mostly sickly or dead plants, as the chromosomes are unable to cleanly segregate.
Polyploids are generated when chromosomes fail to separate (non-disjunction) during pollen and egg generation. The chromosomes normally exist in pairs, thus having only one, or three, interferes in pairing in subsequent generations. Chromosomal duplications, either one chromosome or the whole genome, happen frequently in nature, and actually serves as a mechanism for evolution. However the vast majority (>99.99%) results in lethality. Thus there is polyploidy in Cannabis, and a few examples are supported by scientific evidence. The initial hybrid may show superior phenotypes and can be propagated through cloning, but there may be little potential for successful breeding with these plants.
Epigenetics and Phenotypic Consistency in Clones
One mechanism of turning off genes is by the DNA becoming physically inaccessible due to a structure resembling a ball. In addition, making molecules similar to DNA (RNA) that prevents expression of a gene can turn off certain genes. Both mechanisms are generally termed epigenetics.
Epigenetic regulation is often dependent on concentrations of certain proteins. Through the repeated process of cloning, it is possible that some of these proteins may be diluted, due to so many total cell divisions and epigenetic control of gene expression can be attenuated and results in phenotypic variability.
Sexual reproduction, and possibly tissue culture propagation, may re-establish complete epigenetic gene regulation, however the science is lacking. Epigenetic gene regulation is one of the hottest scientific topics and is being heavily investigated in many species including humans.
Hermaphrodites and Sex Determination
Cannabis is an extremely interesting genus (species?) for researching sex determination. Plants are usually either monoecious (both male and female organs on a single plant), or dioecious, separate sexes. Sex determination has evolved many times in many species. Comparing the mechanisms of sex determination in different organisms provides valuable opportunities to contrast and compare, thereby developing techniques to control sex determinations.
Cannabis is considered a male if it contains a Y-chromosome. Females have two X chromosomes. Even though female Cannabis plants do not have the “male” chromosome, they are capable of producing viable pollen (hermaphrodite) that is the source of feminized seeds. Therefore, the genes required to make pollen are NOT on the Y-chromosome, but are located throughout the remainder of the Cannabis genome. However, DNA based tests are available to identify Male Associated Sequence (MAS) that can be used as a test for the Y-chromosome in seedlings/plants.
Natural hermaphrodites may have resulted from Polyploidization (XXXY), or spontaneous hermaphrodites could be a result of epigenetic effects, which may be sensitive to the environment and specific chemical treatments.
Feminized seeds will still have genes segregating, thus they are not genetically identical. This shouldn’t lead to a necessary decrease in health, but could. A clone does not have this problem.
The other issue is that “inbreeding depression” is a common biological phenomenon, where if you are too inbred, it is bad…like humans. Feminized seeds are truly inbred. Each generation will decrease Heterozygosity, but some seeds (lines) may be unhealthy and thus are not ideal plants for a grower.

Researching Cannabis Genetics: A Q&A with CJ Schwartz, Ph.D.
https://cannabisindustryjournal.com/...schwartz-ph-d/
CJ Schwartz hopes to use genetic variations and marker-assisted breeding to provide cannabis as a more precise medical treatment.
Studying cannabis genetics is a convoluted issue. Strain classification, medicinal effects and plant breeding are particular areas in the science of cannabis that still require heavy research. Marigene, a company researching cannabis genetics, is currently working with universities and research institutes to help map the cannabis genome and catalog genetic variation.
According to CJ Schwartz, Ph.D., chief executive officer and founder of Marigene, their mission is to “to classify, certify, and improve cannabis.” After studying genetics and cellular biology at the University of Minnesota, Schwartz received his Ph.D. in biochemistry from the University of Wisconsin. Hisresearch in the past decade has focused on genetic variations that control flowering time, discovering the expression of a gene called Flowering Locus T leads to differential flowering time of plants and is dependent on their native locations. We sat down with Schwartz to learn more about his research and collaborative efforts.

MARKER ASSISTED BREEDING, ARTIFICIAL SELECTION, AND THE NEW AGRICULTURAL REVOLUTION
HTTPS://GROWERSNETWORK.ORG/INDUSTRY/...AL-REVOLUTION/
Marie Turner of Marigene Consulting describes advancements in breeding with Marker-Assisted Selection. Find out how you can take advantage of modern genetic technology to speed up your breeding program!
Humans have been breeding plants for almost as long as they have been growing agricultural crops. At first, this ‘breeding’ was mostly unintentional, because people saved seeds from the best plants in order to grow them in the following season (Figure 1). Over time, this meant that crops came to more closely resemble what humans wanted for successful harvests. Traits such as higher yield, pest resistance, environmental resilience, reduced branching habit, uniform maturity, and non-shattering (shattering is the natural dispersal of seed onto the ground) developed. To this day, the non-shattering characteristic remains one of the major traits separating domesticated plants such as corn from their wild relatives (in corn’s case, this is a plant called teosinte). Artificial selection in agriculture is similar to Darwin’s natural selection, in which organisms with beneficial traits are more likely to successfully reproduce. Successful reproduction means their genes become more prevalent in subsequent generations. In artificial selection, humans control the reproduction, and thus select the traits to pass on.
It wasn’t long before humans took a more proactive approach to breeding. The first generally recognized example of deliberate cross-pollination of plants by humans is that of date palms, illustrated in Assyrian reliefs from circa 800 B.C. (Figure 2). Of course, in modern times, hybridization is common and fundamental to many of our major crops, including Cannabis. Hybridization is not only a way of generating high-performing (heterotic) varieties, but also of both generating plant diversity and stabilizing desired traits through introgression. The measurement of plant characteristics and the search for/discovery of good traits and varieties is known as phenotyping.
The job of a plant breeder is twofold:
1. Generate diversity and identify useful phenotypes and varieties.
2. Once desirable phenotypes are found, introgress them into a more desirable and reproductively stable state.
For example, in Cannabis, it may be desirable to introgress good terpene profiles from one strain into another strain that has high yields and good disease resistance. The difficulty with old-fashioned plant breeding, however, is that the plants have to be grown out until maturity to collect this kind of data, substantially delaying the development of commercial varieties. In addition, observation of traits during a growing season is labor intensive and takes additional labor, cost, and time post-harvest. For some major crops, automated systems for High Throughput Phenotyping have been developed

Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics.
Vincent, D., Rochfort, S., & Spangenberg, G.
Molecules, 24(4), 659. (2019).
doi:10.3390/molecules24040659
Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway

WHAT IS DNA? WHAT IS A GENE? WHAT IS PROTEIN?
Drs. CJ Schwartz and Marie Turner
https://growersnetwork.org/cultivati...at-is-protein/
In this contributor article, Drs. CJ Schwartz and Marie Turner of Marigene Consulting explains the basics of genetics when it comes to cannabis cultivation.
Society is currently experiencing a revolution. Like the industrial revolution and the computer revolution, we are now experiencing the DNA revolution. The secrets to the vast and amazing abilities of living things, and human health, lie in the DNA code. So what is DNA?
DNA is a molecule, made up of 4 building blocks. These building blocks, called monomers, are referred to as nucleotides. DNA is typically found as long chains called chromosomes within the nucleus of eukaryotic cells. Despite the fact that cells cannot be observed without a microscope, each individual cell contains several feet of DNA, depending on the species. If you laid out all of the DNA in your body end-to-end, it would extend to the moon and back, multiple times.
DNA serves as the basic form of data storage required to encode life. DNA has the capacity to store an enormous amount of data, similar to a computer hardrive, but orders of magnitude more efficient. To illustrate: Computer code is binary and has two choices (0 or 1) per position, while DNA has 4 choices per position (A, C, G, or T). For example, if there are 5 positions, each with 4 choices, you end up with 4 x4 x4 x4 x4 = 45 = 1024 potential combinations. A binary code, on the other hand, would only have 2 x2 x2 x2 x2 = 25 = 32 potential combinations. As you add positions, the differences become exponentially greater. In the Cannabis genome there are 820 million positions or base-pairs of DNA (humans have 3.2 billion). The number of possible combinations is truly incredible!
DNA sequences encode for the 20 amino acids that are used as the building blocks for proteins found within cells. Proteins can be thought of as the basic working units of an organism, similar to the workers in a construction company. The specific biochemical properties and combinations of the individualamino acids determine the function of a protein, while the proteins themselves can be shaped differently to make different structures. THCAS (THCASynthase) and CBDAS (CBDA Synthase) have very similar amino acid sequences (roughly 90% identical), but the difference in amino acids between the twoproteins result in different end products.
A gene is most commonly defined as a DNA sequence that codes for a specific protein. E. coli has about 5000 genes, while more complex organisms have 25,000-30,000 genes. For any given gene, there exists some natural variation, which can result in a protein with a different function (or even no function). Thus, genetic differences (genotype) can result in physical differences (phenotype). The DNA sequence also determines the timing of gene expression to coordinate developmental processes, such as flowering time. In layman’s terms, the DNA sequence is like a building blueprint.

Next-generation breeding
By C.J. Schwartz
https://www.marijuanaventure.com/nex...tion-breeding/
Traditional breeding in plants and animals depends on the identification of individual subjects with superior performance or traits. These plant qualities are referred to as the organism’s phenotype. Individuals with desired phenotypes are selected for additional cycles of breeding, thereby stabilizing the genetic architecture (inbreeding) and producing consistent offspring with the properties of interest. While traditional breeding relies on phenotype alone, marker-assisted breeding also uses genetic information — the genotype of the plant — to identify the genes that confer the specific phenotype, allowing for a vast acceleration in the development of desirable cultivars.
To perform successful marker-assisted breeding, two strains with differing phenotypes are crossed for the purpose of comparing genetic differences or similarities that allow identification of a correlation between phenotypes and genotype (genes). The progeny of this cross is the F1 generation. F1 plants often display hybrid vigor, which means the progeny’s phenotype is improved compared to either parent.
For some crops, F1 plants are the goal and are propagated in the field. When F1 plants undergo sexual reproduction, the resulting progeny (F2 generation) will display a wide range of phenotypes between those of the original parents. For example, if the mother plant flowered at 45 days and the father plant flowered at 65 days, the F2 plants might flower at 45, 47, 53, 60 or 65 days — a continuous distribution. To identify the gene or genes underlying the flowering time differences, the two extreme classes are genotyped to identify a correlation between the genes and the phenotype. Different genes will likely be identified when using different parents. Additional crosses may be required to assist in gene identification, including a backcross, which is crossing F1 plants back to one of the parents. The progeny from this cross (BC1) will have 75% of its genes from one parent and 25% from the other parent. This substantially decreases the genetic variability of the resulting progeny, thus increasing the probability of identifying the genes of interest.

ARE GENETICS RESHAPING THE MARIJUANA INDUSTRY?
https://static1.squarespace.com/stat...ebMrch2016.pdf
Mapping the cannabis genome with CJ Schwartz of Marigene
We spoke with CJ Schwartz the founder, CEO and CSO of Marigene to try to unravel the sequencing of roughly 30,000 genes that make up the genome of Cannabis. As patients, we marvel at cannabis’ medicinal properties and often wonder how the plant makes THC. With 15 years of plant molecular genetic experience, Schwartz is more than qualifi ed to piece together the puzzle. Schwartz has a BS in Genetics and Cell Biology from the University of Minnesota and a PhD in Biochemistry from the University of Wisconsin. He completed his post-doctoral research at the Salk Institute in San Diego, participating in plant natural genetic variation studies. Schwarz was a Howard Hughes Teaching Fellow and a Scientist at the University of Wisconsin, where he was involved with the development of dedicated bioenergy crops.
Having successfully mapped the cannabis genome, Sunrise Genetics CEO C.J. Schwartz is one step closer to unlocking the plant’s limitless potential
Talk to any cannabis cultivator for more than a couple minutes and the conversation will ultimately drift toward genetics. While some growers and breeders have a deep understanding of their cultivars’ phenotypes, the vast majority of the information about different strains is nothing more than cannabis industry folklore.
Yet, as the business matures, research into the plant has been shifting from secretive, underground operations to universities and well-equipped laboratories overseen by scientists like C.J. Schwartz, who has a Ph.D. in biochemistry and nearly two decades of experience in plant molecular genetics.
More than just a buzzword, genetics are truly the key to unlocking the full potential of cannabis and creating standardized varietals, products designed for a specific purpose, and fine-tuning the cultivation requirements of a crop.
Schwartz is the CEO of Sunrise Genetics, which announced in January that it had mapped the cannabis genome, following a two-year research project in conjunction with the University of Minnesota and CBDRx. Marijuana Venture spoke with Schwartz about what exactly that means for both the short- and long-term future of this fledgling industry.
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Decoding Cannabis
Sunrise Genetics CEO C.J. Schwartz
https://www.marijuanaventure.com/dec...abis-genetics/
Jonathan E. Page and Jake M. Stout have filed Patent WO2015196275 on the nucleotide sequence encoding the enzyme CBCAS based reagents, and methods for producing cannabinoids and/or altering cannabinoid production.
https://patents.google.com/patent/WO2015196275A1/en

Differential expression of genes involved in C1metabolism and lignin biosynthesis in wooden core and bast tissues of fibre hemp (Cannabis sativa L.)
Hetty C. van den Broeck, Chris Maliepaard, Michel J.M. Ebskamp, Marcel A.J. Toonen, Andries J. Koops
Plant Science 174 (2008) 205–220
doi:10.1016/j.plantsci.2007.11.008
Hetty C. van den Broeck, Chris Maliepaard, Michel J.M. Ebskamp, Marcel A.J. Toonen, Andries J. Koops
Plant Science 174 (2008) 205–220
doi:10.1016/j.plantsci.2007.11.008
Plants are the major source of fibres for, e.g., textile and paper applications. Fibre hemp (Cannabis sativa L.) can be grown under a wide variety of agro-ecological conditions, is resistant to weeds and pests and, in general, drought tolerant. Fibre length and content of cellulose and lignin are important quality parameters for raw material used in cordage, textile, paper, and fibreboard industries. To improve currently used hemp varieties, more knowledge about the molecular processes that underlie cell wall metabolism is needed. To study gene expression in hemp, separate cDNA libraries were constructed from developing core fibres and developing and near mature bast fibres. A cDNA microarray was constructed with 3414 randomly selected hemp cDNAs. From these a total number of 1571 sequences was obtained from the 50 ends of the cDNAs. After similarity search, 1036 unique contigs were obtained. To study differences in the genetic background of quality properties of bast and core fibres, bast and core tissues were obtained from middle parts of the stems during various developmental stages and expression of the cDNAs was analyzed. A total number of 178 clones encoding 65 proteins was found to be more highly expressed in core tissue. Most of these 65 proteins are involved in C1 metabolism and lignin biosynthesis. In bast tissue, a total number of 110 clones encoding 44 proteins was more highly expressed. Among these 44 proteins are arabinogalactan proteins, lipid transfer proteins, lipoxygenases, and endoxyloglucan transferases. This paper describes the identification of genes that are differentially expressed in bast fibres and wooden core tissue of hemp, and is the first step in linking the genetic background to quality differences between these two types of hemp tissue. This research may result in biotechnological approaches to increase the value of hemp grown for industrial use.

Differentiation between fiber and drug types of hemp (Cannabis sativa L.) from a collection of wild and domesticated accessions.
Piluzza G, Delogu G, Cabras A, Marceddu S, Bullitta S (2013)
Genet Res Crop Evol 60: 2331–2342
https://www.researchgate.net/publica...ted_accessions
DOI 10.1007/s10722-013-0001-5
Accessions of wild and domesticated hemp (Cannabis sativa L.) originating from Colombia, Mexico, California, Bolivia, Thailand, Afghanistan, Serbia, Hungary, south Africa and different regions of China, were studied by means of DNA polymorphisms in order to discriminate between drug and fiber types.
Analysis of molecular variance (AMOVA) was used to partition the total genetic variance within and among populations. The significance of the variance components was tested by calculating their probabilities based on 999 randompermutations.AMOVA revealed 74 % variation among accessions and 26 % within accessions, all AMOVA variation was highly significant (P\ 0.001). The cluster analysis of molecular data, grouped accessions into eight clusters and gave a matrix correlation value of r = 0.943, indicating a very good fit between the similarity values implied by the phenogram and those of the original similarity matrix. In this study, DNA polymorphisms could discriminate the fiber and drug types, and accessions were grouped in accordance to their classification and uses. In addition, seed size variation and micromorphological characters of seeds were studied by means of a scanning electron microscope (SEM). Seeds varied significantly in size, and were bigger in the fiber types. SEM analysis exhibited variation of micromorphological characters of seeds that could be important for discriminating the fiber or drug types.

Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay
D. Rotherham, S.A. Harbison
Forensic Science International 207 (2011) 193–197
doi:10.1016/j.forsciint.2010.10.006
Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of
tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for
the differentiation of ‘‘drug’’ and ‘‘non-drug’’ Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between ‘‘drug’’ and ‘‘non-drug’’ Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous

Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers
Chunsheng Gao , Pengfei Xin , Chaohua Cheng, Qing Tang, Ping Chen, Changbiao Wang, Gonggu Zang, Lining Zhao
Plos ONE
doi: 10.1371/journal.pone.0110638
Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.

Diversity and evolution of the repetitive genomic content in Cannabis sativa
Rahul Pisupati, Daniela Vergara and Nolan C. Kane
BMC Genomics (2018) 19:156
DOI: 10.1186/s12864-018-4494-3
https://bmcgenomics.biomedcentral.co.. .018-4494-3.pdf
Background: The repetitive content of the genome, once considered to be “junk DNA”, is in fact an essential
component of genomic architecture and evolution. In this study, we used the genomes of three varieties of Cannabis
sativa, three varieties of Humulus lupulus and one genotype of Morus notabilis to explore their repetitive content using
a graph-based clustering method, designed to explore and compare repeat content in genomes that have not been
fully assembled.
Results: The repetitive content in the C. sativa genome is mainly composed of the retrotransposons LTR/Copia and
LTR/Gypsy (14% and 14.8%, respectively), ribosomal DNA (2%), and low-complexity sequences (29%). We observed a
recent copy number expansion in some transposable element families. Simple repeats and low complexity regions of
the genome show higher intra and inter species variation.
Conclusions: As with other sequenced genomes, the repetitive content of C. sativa’s genome exhibits a wide range
of evolutionary patterns. Some repeat types have patterns of diversity consistent with expansions followed by losses
in copy number, while others may have expanded more slowly and reached a steady state. Still, other repetitive
sequences, particularly ribosomal DNA (rDNA), show signs of concerted evolution playing a major role in
homogenizing sequence variation.

Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa
,
Peter V. Apicella et al
Plant Direct (2022).
DOI: 10.1002/pld3.412
Find PDF

DNA Analysis of Natural Fiber Rope
Mignon Dunbar, and Terence M. Murphy
J Forensic Sci, January 2009, Vol. 54, No. 1
doi: 10.1111/j.1556-4029.2008.00906.x
When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis N_e (abaca, ‘‘Manila hemp’’), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase ? oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.

Not Cannabis Specific
DNA Fingerprinting in Plants Principles, Methods, and Applications Second Edition
Kurt Weising, Hilde Nybom, Kirsten Wolff, Gu?nter Kahl
https://www.biocenter.helsinki.fi/bi...erprinting.pdf
The new developments in the decade that has passed since the publication of our
first edition of DNA Fingerprinting in Plants and Fungi have been more impressive than one could ever have imagined at that time. Our first edition encompassed basically all published work that employed DNA fingerprinting in plant or fungal research. In the present edition, we not only had to restrict ourselves to plants, but we also faced the difficult task of extracting a reasonable “core collection” from the tremendous number of scientific articles that had appeared on the topic. We would like to express our apologies to the many authors whose work could not be mentioned because of space limitations, despite the more than 1600 references already listed. Applications of DNA fingerprinting have blossomed in applied as well as in basic plant sciences. In addition, the diversity of techniques has increased as well. In particular, the balance of hybridization- vs. PCR-based methods has completely been reversed during the last 10 years, with the latter now being the mainstay of most molecular laboratories. We have the strong impression that the publications related to the methodology and applications of PCR-based DNA fingerprinting behave like the DNA in a PCR, i.e., they amplify exponentially. Given that a simple update would never have worked, writing the new edition basically meant writing a completely new book. The availability of new techniques and new equipment also indicated that we had to write for an even more diverse audience than before. There are still complete novices around, but the starting level of students has generally improved. Although we have still attempted to present the basic protocols and principles, we have also included some background theory as well as numerous references for and descriptions of more sophisticated methodology. The book is therefore intended to serve as a benchtop manual for the beginner as well as a key reference for a wide variety of DNA profiling techniques and applications. Ten years ago, the average plant molecular marker laboratory employed random amplified polymorphic DNAs (RAPDs) and restriction fragment length polymorphism (RFLP) fingerprinting, whereas the advanced research institutions had already switched to microsatellites and amplified fragment length polymorphisms (AFLPs). Today, the latter two techniques are commonplace in many laboratories, whereas the avant-garde has turned its attention to single-nucleotide polymorphisms (SNPs) and DNA microarrays. It may be assumed that SNPs will become routine markers in the next 10 years or so, but it is quite difficult to predict what kind of exciting novel marker technologies will be on the market in 2014. If the number of techniques and their applications keep increasing as they have done during the last decade, it is also difficult to imagine what a book like the one you have in your hands will look like another 10 years from now. In any case, we hope that the present book will assist in establishing DNA fingerprinting technology in a broad range of laboratories involved in plant research.

DNA Markers to Discriminate Cannabis sativa L. “Cheungsam” with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes.
Doh, E. J., Lee, G., Yun, Y.-J., Kang, L.-W., Kim, E. S., Lee, M. Y., & Oh, S.-E.
Evidence-Based Complementary and Alternative Medicine, 2019, 1–10.
doi:10.1155/2019/8121796
Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. *erefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. *us, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar “Cheungsam” (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars

DNA polymorphism detection of Cannabis using amplified fragment length polymorphism
Jia Guo, Li Pei, Jian-Xiong Peng, Hong Zhai, Gui-Qin Zhang, Qin Jie, Zheng Tu
Fa yi xue za zhi 24(5):330-2 November 2008
OBJECTIVE: To screen the AFLP primers with good diversity to distinguish various species of Cannabis.
METHODS: The AFLP was used to analyze the genetic diversity of 12 species of Cannabis using 55 primer combinations.
RESULTS: A total of 285 AFLP bands were obtained using five primer combinations with better diversity, among which 99 bands were polymorphic and 10 bands were special, with 47-76 bands amplified in each pair of primers.
CONCLUSION: AFLP may has good resolution in the diversity study of Cannabis. It may provide an essential basis for further study of the genetic diversity of Cannabis.

DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in "drug-type" and "fiber-type" Cannabis sativa L.
Kojoma M, Seki H, Yoshida S, Muranaka T (2006)
Forensic Sci Int 159: 132–140
DOI: 10.1016/j.forsciint.2005.07.005
The cannabinoid content of 13 different strains of cannabis plant (Cannabis sativa L.) was analyzed. Six strains fell into the ‘‘drug-type’’ class, with high D-9-tetrahydrocannabinolic acid (THCA) content, and seven strains into the ‘‘fiber-type’’ class, with low THCA using HPLC analysis. Genomic DNA sequence polymorphisms in the THCA synthase gene from each strain were studied. A single PCR fragment of the THCA synthase gene was detected from six strains of ‘‘drug-type’’ plants. We could also detect the fragment from seven strains of ‘‘fiber-type’’ plants, although no or very low content of THCA were detected in these samples. These were 1638 bp from all 13 strains and no intron among the sequences obtained. There were two variants of the THCA synthase gene in the ‘‘drug-type’’ and ‘‘fiber-type’’ cannabis plants, respectively. Thirty-seven major substitutions were detected in the alignment of the deduced amino acid sequences from these variants. Furthermore, we identified a specific PCR marker for the THCA synthase gene for the ‘‘drug-type’’ strains. This PCR marker was not detected in the ‘‘fiber-type’’ strains.

Not directly Cannabis related
Emerging Genome Engineering Tools in Crop Research and Breeding (Chapter 14)
Andriy Bilichak, Daniel Gaudet, and John Laurie
In Vaschetto, L. M. (Ed.). (2020). Cereal Genomics. Methods in Molecular Biology.doi:10.1007/978-1-4939-9865-4
Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant’s genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.

Endogenous synthesis of pyrethrins by cannabis
Adrian Devitt-Lee, Douglas R. Smith, David Chen, Kevin McKernan, Simone
Groves, Ciaran McCarthy
bioRxiv, 2017
doi: 10.1101/169417
Pyrethrins are a class of natural terpenoid pesticides produced by Tanacetum cinerariifolium, commonly known as chrysanthemum. Here we present evidence that cannabis may be able to produce pyrethrins endogenously. Flower from a cannabis plant grown in a closed hydroponic environment contained 2.48 parts per million pyrethrin I by weight. A comparison of the
genetics of T. cinerariifolium and Cannabis demonstrates Cannabis homologues of the genes that contribute to pyrethrins production in T. cinerariifolium. This provides a plausible pathway for the biosynthesis of pyrethrins in cannabis. Although preliminary, these data indicate a potentially significant confounding variable in both cannabis research and regulations on allowable pyrethrins residues in cannabis products.

Engineering Monoterpene Production in Yeast Using a Synthetic
Dominant Negative Geranyl Diphosphate Synthase
Codruta Ignea, Marianna Pontini, Massimo E. Maffei, Antonios M. Makris, and Sotirios C. Kampranis
Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.

Not Cannabis Specific
Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing
Daniel Rodr?´guez-Leal, Zachary H. Lemmon, Jarrett Man, Madelaine E. Bartlett, Zachary B. Lippman
Cell 171, 1–11 October 5, 2017
https://dx.doi.org/10.1016/j.cell.2017.08.030
Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis regulatory
alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans -generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between generegulatory changes and control of quantitative traits.

Epigenetic mechanisms and endocannabinoid signalling
Claudio D'Addario, Andrea Di Francesco, Mariangela Pucci, Alessandro Finazzi Agrò, Mauro Maccarrone FEBS J. 2013 May
doi: 10.1111/febs.12125 https://febs.onlinelibrary.wiley.com.. .111/febs.12125
The endocannabinoid system, composed of endogenous lipids, their target receptors and metabolic enzymes, has been implicated in multiple biological functions in health and disease, both in the central nervous system and in peripheral organs. Despite the exponential growth of experimental evidence on the key role of endocannabinoid signalling in basic cellular processes, and on its potential exploitation for therapeutic interventions, much remains to be clarified about the respective regulatory mechanisms. Epigenetics refers to a set of post-translational modifications that regulate gene expression without causing variation in DNA sequence, endowed with a major impact on signal transduction pathways. The epigenetic machinery includes DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs. Due to the reversibility of epigenetic changes, an emerging field of interest is the possibility of an 'epigenetic therapy' that could possibly be applied also to endocannabinoids. Here, we review current knowledge of epigenetic regulation of endocannabinoid system components under both physiological and pathological conditions, as well as the epigenetic changes induced by endocannabinoid signalling.

Not directly Cannabis related
Eukaryotic Translation Initiation Factors Shape RNA Viruses Resistance in Plants
Jannat Shopan , Xiaolong Lv , Zhongyuan Hu , Mingfang Zhang , Jinghua Yang
Horticultural Plant Journal (2020),
doi: 10.1016/j.hpj.2020.03.001
Viruses are representative of a global threat to agricultural production. Genetic resistance is the preferred strategy for the control of viral infection and against loss of crop yield. Viral protein synthesis requires host cellular factors for translating their viral RNAs, and for regulating their replication and cell to cell systemic movement. Therefore, the viruses are dependent on cellular translation factors. Mutations in the gene encoding eIF4E and eIF4G or their isoforms, eIFiso4E, eIFiso4G and eIF2B? have been mapped as a source of plant potyvirus while other genus of plant virus recessive resistance genes in many species are originated from these loci. Some of other plant translation factors, such as eIF3, eIF4A-like helicases, eEF1A and eEF1B, which are required in interacting with viral RNAs and regulating various aspects of the infection cycle, have also been identified. Here, we summarize the mechanisms utilized by RNA viruses of eukaryotic plants and the essential roles of eIFs in virus infection. Moreover, we discuss the potential of eIFs as a target gene in the development of genetic resistance to viruses for crop improvement. This review highlighted newly revealed examples of abnormal translational strategies and provided insights into natural host resistance mechanisms that have been linked to 3’ cap-independent translational enhancer activity.

Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification
Rachel Houston & Matthew Birck & Sheree Hughes-Stamm & David Gangitano
Int J Legal Med
DOI 10.1007/s00414-015-1296-x
Marijuana (Cannabis sativa) is the most commonly used illicit substance in the USA. The development of a validated method using Cannabis short tandem repeats (STRs) could aid in the individualization of samples as well as serve
as an intelligence tool to link multiple cases. For this purpose, a modified 13-loci STR multiplex method was optimized and evaluated according to ISFG and SWGDAM guidelines. A real-time PCR quantification method for C. sativa was developed and validated, and a sequenced allelic ladder was also designed to accurately genotype 199 C. sativa samples from 11 U.S. Customs and Border Protection seizures. Distinguishable DNA profiles were generated from 127 samples that yielded full STR profiles. Four duplicate genotypes within seizures were found. The combined power of discrimination of this multilocus system is 1 in 70 million. The sensitivity of the multiplex STR system is 0.25 ng of template
DNA. None of the 13 STR markers cross-reacted with any of the studied species, except for Humulus lupulus (hops) which generated unspecific peaks. Phylogenetic analysis and caseto- case pairwise comparison of 11 cases using Fst as genetic distance revealed the genetic association of four groups of cases. Moreover, due to their genetic similarity, a subset of samples (N=97) was found to form a homogeneous population in Hardy-Weinberg and linkage equilibrium. The results of this research demonstrate the applicability of this 13-loci STR system in associating Cannabis cases for intelligence purposes.

Evaluation of the trnK-matK-trnK, ycf3, and accD-psal chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa.
Cheng, YC., Houston, R.
Int J Legal Med (2021).
DOI: 10.1007/s00414-021-0251
Cannabis sativa (marijuana and hemp) is one of the most controversial crops worldwide. In the USA, the state-specific legalization of marijuana and recently legalized hemp pose a problem for law enforcement. This study seeks to utilize chloroplast hSTRs, INDEL, and SNPs markers to develop genotyping methods to aid in the differentiation of legal hemp from illicit marijuana and also for tracking the flow of trafficked marijuana. Three polymorphic regions:trnK-matK-trnK, ycf3, and accD-psal, of the C. sativa chloroplast genome were evaluated in order to distinguish crop type and biogeographic origin. A total of nine polymorphic sites were genotyped from five distinct populations (hemp from the USA and Canada, marijuana from Chile and USA-Mexico, and medical marijuana from Chile) with a custom fragment and SNaPshotTM assay. The study also combined genotype results from the same sample set using 21 additional polymorphic markers from previous studies. The effectiveness of these multi-locus assays to distinguish sample groups was assessed using haplotype analysis, phylogenetic analysis, pairwise comparisons, and principal component analysis. Results indicated a clear separation of Canadian hemp using only the nine polymorphic sites developed in this study. The additional 21 markers were able to separate US hemp from both marijuana groups to a significant level (p < 0.05) when assessing average Fixation Indices (FST). This study demonstrated the applicability of these organelle markers for the determination of crop type and biogeographic origin of C. sativa. However, a more extensive database is needed to evaluate the true discriminatory power of these markers.


Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.
Yang, X., Matsui, T., Mori, T., Taura, F., Noguchi, H., Abe, I., & Morita, H.
Acta Crystallographica Section F Structural Biology Communications, 71(12), 1470–1474. (2015).
doi:10.1107/s2053230x15020385
Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra--ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric + barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 A? resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 A? . Further crystallographic analysis will provide valuable insights into the structure– function relationship and catalytic mechanism of OAC.


Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)
El Alaoui MA, Melloul M, Alaoui Amine S, Stambouli H, El Bouri A, et al. (2013) (PLoS ONE 8(10): e74714.
doi:10.1371/journal.pone.0074714
The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective
of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA
typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis
resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB
protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality
genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol
failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene.
However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different
sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin
derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances

Female-Associated DNA Polymorphisms of Hemp (Cannabis sativa L.)
Hong Shao, Shu-Juan Song, Robert Connell Clarke
January 2003 Journal of Industrial Hemp 8(1):5-9
DOI: 10.1300/J237v08n01_02
The sex identification of pre-floral hemp (Cannabis sativa L.) is problematic for taxonomists and plant breeders, because the distinguishing morphological characteristics of male and female plants do not appear before flowering. Employing the RAPD (Random Amplified Polymorphic DNA) technique with 15 random primers, we have found that the genomes of female individuals bear female-associated polymorphic DNA fragments (870 bp and 1160 bp amplified by primer OPA-04; 1680 bp by primer OPF-05), which are absent in the genomes of male individuals amplified by the same primers. This technique makes it possible to determine the gender of dried herbarium specimens lacking flowers, and to select male or female seedlings for breeding parents and other uses.

Field Testing of Collection Cards for Cannabis sativa Samples With a Single Hexanucleotide DNA Marker
Lindsay Allgeier, John Hemenway, Nicholas Shirley, Tommy LaNier, and Heather Miller Coyle,
J Forensic Sci, September 2011, Vol. 56, No. 5
doi: 10.1111/j.1556-4029.2011.01818.x
The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried ‘‘dispensary’’ samples, U.S. border seizures, and hashish. Using a standardized PCR kit with customlabeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site–seized samples with applications for databasing and market disruption.

Gene copy number is associated with phytochemistry in Cannabis sativa
Daniela Vergara, Ezra L. Huscher, Kyle G. Keepers, Robert M. Givens, Christian G. Cizek, Anthony Torres, Reggie Gaudino and Nolan C. Kane
DOI: 10.1101/736181.
Gene copy number variation is known to be important in nearly every species where it has been examined. Alterations in gene copy number may provide a fast way of acquiring diversity, allowing 15 rapid adaptation under strong selective pressures, and may also be a key component of standing 16 genetic variation within species. Cannabis sativa plants produce a distinguishing set of secondary 17 metabolites, the cannabinoids, many having medicinal utility. Two major cannabinoids --THCA and 18 CBDA -- are products of a three-step biochemical pathway. Using genomic data for 69 Cannabis 19 cultivars from diverse lineages within the species, we found that genes encoding the synthases in this 20 pathway vary in copy number, and that the cannabinoid paralogs may be differentially expressed. We 21 also found that copy number partially explains variation in cannabinoid content levels among 22 Cannabis plants.

Not Cannabis specific
Gene discovered crucial to making crop plants produce clonal seeds
PhysOrg Jan 6, 2022
https://phys.org/pdf560694578.pdf
Researchers from KeyGene and Wageningen University & Research (WUR), in collaboration with colleagues from Japan and New Zealand, have discovered a gene that will make it possible to produce seeds from crops that are genetically identical to the mother plant and that do not need pollination. This phenomenon, called apomixis, enables plants with a desirable combination of traits to produce many offspring with the same desirable combination of genes as the mother plant. Together with researchers from the Japanese breeding company Takii and New Zealand's Plant & Food Research and Lincoln University, the KeyGene and WUR researchers explain in Nature Genetics how the gene works and the way it influenced the work of the 'father of genetics' Gregor Mendel. The discovery is expected to lead to major innovations in plant breeding over the coming years. The gene found has been given the name PAR, shortened from parthenogenesis, the process whereby egg cells grow into plant embryos without fertilization of the egg cells. The discovery marks a definitive breakthrough and crowns the research team's work that started at KeyGene over 15 years ago.

Gene duplication and divergence affecting drug content in Cannabis sativa.
Weiblen, G. D., Wenger, J. P., Craft, K. J., ElSohly, M. A., Mehmedic, Z., Treiber, E. L., & Marks, M. D.
New Phytologist, 208(4), 1241–1250.(2015).
doi:10.1111/nph.13562
Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/ CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency

Not Cannabis specific
WO2020239984A1 Gene for Parthenogensis Patent
https://patentimages.storage.googleapi s.com/d1/99/59/c922ddef2fd1b8/WO2020239984A1.pdf
https://patents.google.com/patent/WO2020239984A1/en
The invention provides the nucleotide sequence and amino acid sequences of the parthenogenesis gene of Taraxacum as well as (functional) homologues, fragments and variants thereof, which provides parthenogenesis as a part of apomixis. Also parthenogenetic plants and methods for making these are provided, as are molecular markers and methods of using these.
Title: Gene for Parthenogenesis
Field of the invention
The present invention relates to the field of biotechnology and in particular to plant biotechnology including plant breeding. The invention relates in particular to the identification and uses of genes relating to and useful e.g. in apomixis and haploid induction. The invention in particular relates to the gene that is associated with parthenogenesis, as well as the encoded protein, and fragments of both. The invention further relates to methods for suppressing and/or inducing parthenogenesis in plants and crops, to the use of the gene and/or the protein or their fragments for apomixis in particular in combination with apomeiotic gene(s), or for the production of haploid plants of which the chromosomes can be doubled to produce doubled haploids.

Gene Networks Underlying Cannabinoid and Terpenoid Accumulation in Cannabis
Jordan Zager, Iris Lange, Narayanan Srividya, Bernd Markus Lange
May 2019 Plant physiology
DOI: 10.1104/pp.18.01506
https://www.plantphysiol.org/content...01506.full.pdf
Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant–environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell and genetic origin. Glandular trichomes were isolated from each of these strains and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from importedprecursors. Terpene synthase genes involved in the biosynthesis of the major mono- and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT ((-) limonene synthase) and CsTPS15CT (?-myrcene synthase) wefunctionally evaluated genes that encode enzymes with activities not previously described incannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases); CsTPS16CC (germacrene B synthase); and CsT

Not Cannabis specific
Gene Specific Molecular Markers for Hop (Humulus lupulus L.)
J. Patzak , J. Matousek
Acta horticulturae 848(848):73-80 December 2009
DOI: 10.17660/ActaHortic.2009.848.7
The development of DNA technology has provided a number of methods, which eliminate any influence of environmental factors, to detect differences at the DNA sequence level for evaluation of hop genotypes. Most of these methods in hops used unknown DNA sequences (e.g., AFLP) or specific DNA sequences (e.g., SSR) in non-coding regions of the genome. A new trend in molecular methods is to develop new molecular markers in specific sequences, near or within structural genes. Recently, markers in intron, 5' and 3'UTR were identified in several hop genes. Increasing sequence information of hop genes in Genebank provides additional opportunities to develop new gene specific molecular markers. In this study, we report new efficient and reliable expressed sequence tag microsatellite repeat (EST-SSR) molecular markers. We used forty primer pairs for twenty-nine gene specific sequences. We evaluated them for identification and clustering in eight hops cultivars. Gene-specific based clustering was compared to clustering based on SSR, AFLP markers and on a combination of molecular markers. These molecular markers are ideal for DNA fingerprinting, for control of authenticity and purity of varieties, for evaluation of genetic diversity in genetic resources, etc. They are readily applicable in breeding, and for genetic linkage mapping and quantitative trait loci (QTL) analysis.

Genetic analysis of cannabis is here
Washington State University Research
Science Daily May 29, 2019
https://www.sciencedaily.com/release...0529145039.htm
Research could provide government regulators with powerful new tools for addressing a bevy of commercial claims and other concerns as non-medical marijuana, hemp and CBD products become more commonplace. The new analysis of the genetic and chemical characteristics of cannabis is believed to be the first thorough examination of its kind.

Genetic and Genomic Tools for Cannabis sativa
Daniela Vergara, Halie Baker, Kayla Clancy, Kyle G. Keepers, J. Paul Mendieta, Christopher S. Pauli, Silas B. Tittes, Kristin H. White & Nolan C. Kane
Critical Reviews in Plant Sciences,
DOI:10.1080/07352689.2016.1267496
The Cannabis industry is currently one of the fastest growing industries in the United States. Given the changing legal status of the plant, and the rapidly advancing research, updated information on the advancement of Cannabis genomics is needed. This versatile plant is used as medicine and for food, fiber, and bioremediation. Insights from modern, high-throughput genomic technology are revolutionizing our understanding of the plant and are providing new tools to further improve our knowledge and utilization of this unique species. This review quantifies and evaluates the currently available genomic resources for Cannabis research, including six whole-genome assemblies, two transcriptomes, and 393 other substantial genomic resources, as well as other smaller publicly available genetic and genomic resources. The open-source approaches followed by many leading scientists in the field promote collaboration and facilitate these rapid advances

(Not directly Cannabis related)
Genetic and Hormonal Regulation of Growth, Flowering, and Sex Expression in Plants.
Chailakhyan, M. K.
American Journal of Botany, 66(6), 717.(1979).*
doi:10.2307/2442417*
The interaction of the genetic and hormonal regulation of growth, flowering, and sex expression in plants is discussed. The genetic control of these processes is characterized, and data on their hormonal regulation are supplied. The interaction of genetic and hormonal regulation is considered with reference to tall-growing and genetic dwarf forms of the pea and wheat plants. It is shown that in the dwarf forms of the pea plant and in many other varieties, growth stimulation in response to treatment with the phytohormone gibberellic acid is clearly manifested and the expression of genetic dwarfism
is eliminated, whereas in dwarf wheats it is expressed only slightly, if at all. At the same time both tall-growing and dwarf forms of both pea and wheat show a clearly defined growth retardation response to treatment with the growth inhibitor, abscisic acid, which causes the expression of physiological
dwarfism. The short- and long-day characteristics of the photoperiodic response of plants are described as genetically controlled features, and data are given on the induction of flowering of a long-day variety coneflower grown under short-day conditions with the aid of gibberellins extracted from leaves of long-day vegetative plants of short-day Mammoth tobacco. Data are also supplied on the induction of flowering of a short-day variety, red-leaved goosefoot, grown under continuous light with the aid of metabolites extracted from leaves of the same Mammoth tobacco plants flowering under short-day conditions. This demonstrates the possibility of hormonal regulation of the genetically controlled long-day and short-day characteristics in photoperiodically sensitive plants. Genetic and hormonal regulation of sex expression in two dioecious plants, hemp and spinach, is discussed. It is
shown that sex expression in these plants is regulated by gibberellins which are synthesized in leaves and cause male sex expression and by cytokinins which are synthesized in the roots and cause female sex expression. These data indicate that sex expression in dioecious plants is the result of interaction
between the genetic apparatus and phytohormones

Genetic Diversity and Population Structure of Cannabis Based on the Genome-Wide Development of Simple Sequence Repeat Markers
Jiangjiang Zhang, Jiangtao Yan, Siqi Huang, Gen Pan, Li Chang, Jianjun Li, Chao Zhang, Huijuan Tang, Anguo Chen, Dingxiang Peng, Ashok Biswas, Cuiping Zhang, Lining Zhao, and Defang Li
Front. Genet. 11:958. (2020)
doi: 10.3389/fgene.2020.00958
Cannabis has been used as a source of nutrition, medicine, and fiber. However, lack of genomic simple sequence repeat (SSR) markers had limited the genetic research on Cannabis species. In the present study, 92,409 motifs were identified, and 63,707 complementary SSR primer pairs were developed. The most abundant SSR motifs had six repeat units (36.60%). The most abundant type of motif was dinucleotides (70.90%), followed by trinucleotides, tetranucleotides, and pentanucleotides. We randomly selected 80 pairs of genomic SSR markers, of which 69 (86.25%) were amplified successfully; 59 (73.75%) of these were polymorphic. Genetic diversity and population structure were estimated using the 59 (72 loci) validated polymorphic SSRs and three phenotypic markers. Three hundred ten alleles were identified, and the major allele frequency ranged from 0.26 to 0.85 (average: 0.56), Nei’s genetic diversity ranged from 0.28 to 0.82 (average: 0.56), and the expected heterozygosity ranged from 0.28 to 0.81 (average: 0.56). The polymorphism information content ranged from 0.25 to 0.79 (average: 0.50), the observed number of alleles ranged from 2 to 8 (average: 4.13), and the effective number of alleles ranged from 0.28 to 0.81 (average: 0.5). The Cannabis population did not show mutation-drift equilibrium following analysis via the infinite allele model. A cluster analysis was performed using the unweighted pair group method using arithmetic means based on genetic distances. Population structure analysis was used to divide the germplasms into two subgroups. These results provide guidance for the molecular breeding and further investigation of Cannabis.

Genetic Diversity Analysis of Wild Cannabis in China Based on Morphological Characters and RAPD Markers
TANG Zhi-cheng, CHEN Xuan, ZHANG Qing-ying, GUO Hong-yan, YANG Ming
J. West China For. Sci. 42, 61–66. (2013)
Wild Cannabis germplasms is not only the natural gene bank of Cannabis research and utilization,but also the important basis of hemp breeding.In this article,genetic diversity of 12 wild Cannabis resources and 4 cultivars were assessed by the methods of phenotypic characters and RAPD markers,and the cluster of phenotypic traits and RAPD markers were constructed by Farthest-neighbor method and UPGMA method respectively.The results showed the genetic diversity in wild cannabis was abundant,and the difference of 11 Morphological characters in all germplasms was significant(P 0.001).The thousand-grain weight had the highest coefficient of variation,while the effective number of branches had the smallest one.After the amplification with 13 RAPD primers,a total of 106 fragments were detected,among which,79 fragments were polymorphic,accounting for 74.52 %.The whole germplasms could be classified into three groups based on morphological cluster.The first group was composed of 12 wild germplasms which further divided into two subgroups based on the degree of latitude,while the other two groups contain only 3 cultivars.RAPD cluster analysis showed that the whole germplasms were also classi-fied into 3 groups clustered with the characteristic of regionalism.Germplasms in Yunnan and Xinjiang were in one group,germplasms in northeast and north China were in the second group,while germplasm in Xizang was alone in another cluster,thewild and cultivars were not distinguished obviously.The results suggest that the genetic diversity of Cannabis in china is complicated.

Genetic diversity of Cannabis sativa germplasm based on RAPD markers
V. FAETI', G. MANDOLINO' and P. RANALLI
Plant Breeding 115, 367—370 (1996)
DOI: 10.1111/j.1439-0523.1996.tb00935.x
Random amplified polymorphic DNA (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Itahan and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variabihty within the two cultivars 'Carmagnola' and 'Fibranova' was also shown, suggesting good possibilities for longterm selection work. RAPD markers provide a powerful tool for the
investigation of genetic variation in cultivars/accessions of hemp.

Genetic identification of Cannabis sativa using chloroplast trnL-F gene
Dias, V. H. G., Ribeiro, A. S. D., Mello, I. C. T., Silva, R., Sabino, B. D., Garrido, R. G. Moura-Neto,
Letter to the Editor/ Forensic Science International: Genetics 14 (2015) 201–202
Doi: 10.1016/j.fsigen.2014.10.003
Techniques using molecular markers have been used to study the differentiation of plant species, including Cannabis sativa. However, it is a field not yet explored in Brazil, especially in forensic routine. Although the use of non-coding region between trnL and trnF genes was described in previous studies [1–3], herein we report a standardized protocol for PCR and DNA sequencing on the use of a different section of the chloroplast genes trnL and trnF, as a potential identifier DNA marker for C. sativa samples, seized by the Pol?´cia Civil do Estado do Rio de Janeiro (PCERJ).

Genetic identification of female Cannabis sativa plants at early developmental stage.
Techen N1, Chandra S, Lata H, Elsohly MA, Khan IA.
Planta Med.
doi: 10.1055/s-0030-1249978
Sequence-characterized amplified region (SCAR) markers were used to identify female plants at an early developmental stage in four different varieties of Cannabis sativa. Using the cetyl trimethylammonium bromide (CTAB) method, DNA was isolated from two-week-old plants of three drug-type varieties (Terbag W1, Terbag K2, and Terbag MX) and one fiber-type variety (Terbag Fedora A7) of C. sativa grown under controlled environmental conditions through seeds. Attempts to use MADC2 (male-associated DNA from Cannabis sativa) primers as a marker to identify the sex of Cannabis sativa plants were successful. Amplification of genomic DNA using MADC2-F and MADC2-R primers produced two distinct fragments, one with a size of approximately 450 bp for female plants and one for male plants with a size of approximately 300 bp. After harvesting the tissues for DNA extraction, plants were subjected to a flowering photoperiod (i.e., 12-h light cycle), and the appearance of flowers was compared with the DNA analysis. The results of the molecular analysis were found to be concordant with the appearance of male or female flowers. The results of this study represent a quick and reliable technique for the identification of sex in Cannabis plants using SCAR markers at a very early developmental stage.

Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system
Maria A. Mendoza, DeEtta K. Mills, Hemant Lata, Suman Chandra, Mahmoud A. ElSohly, Jose R. Almirall
Anal Bioanal Chem (2009) 393:719–726
DOI 10.1007/s00216-008-2500-3
Cannabis sativa is the most frequently used of all illicit drugs in the USA. Cannabis has been used throughout history for its stems in the production of hemp fiber, seed for oil and food, and buds and leaves as a psychoactive
drug. Short tandem repeats (STRs) were chosen as molecular markers owing to their distinct advantages over other genetic methods. STRs are codominant, can be standardized such that reproducibility between laboratories can be easily achieved, have a high discrimination power, and can be multiplexed. In this study, six STR markers previously described for C. sativa were multiplexed into one reaction. The multiplex reaction was able to individualize 98 cannabis samples (14 hemp and 84 marijuana, authenticated as originating from 33 of the 50 states of the USA) and detect 29 alleles averaging 4.8 alleles per loci. The data did not relate the samples from the same state to each other. This is the first study to report a single-reaction
sixplex and apply it to the analysis of almost 100 cannabis samples of known geographic origin.

Not Cannabis specific
Genetic mapping in polyploids Peter M. Bourke Genetic mapping in polyploids
Peter M. Bourke
Propositions
1. Ignoring multivalent pairing during polyploid meiosis simplifies and improves subsequent genetic analyses. (this thesis)
2. Classifying polyploids as either autopolyploid or allopolyploid is both inappropriate and imprecise. (this thesis)
3. The environmental credentials of electric cars are more grey than green.
4. ‘Gene drive’ technologies display once again that humans act more like ecosystem terrorists than ecosystem managers.
5. Heritability is a concept that promises much but delivers little.
6. Awarding patents to cultivars or crop traits is patently wrong.
7. The term “air miles” should refer to one’s lifetime allowance of fossil fuelled air travel.
8. The Netherlands’ most effective educational tool comes on two wheels with a bell.
Molecular markers and Mendel’s missing caveat It is only relatively recently that the terms “marker” and “molecular marker” have been taken to be synonymous. In his pioneering experiments with peas, Mendel was already studying the transmission and inheritance of a now-famous set of genetic “markers” (Mendel, 1866), with the physical expression of seven different traits (what we term “phenotypes”) being the marker set itself. It took almost fifty years before it was realised that these morphological markers also represented specific locations on chromosomes which were either transmitted together (due to genetic linkage) or independently (due to lack of linkage) (Morgan, 1911). Curiously, Mendel had selected only unlinked traits for his study of peas (whether this was by accident or by design we do not know). His second law, the law of independent assortment, states that alleles of one gene sort into gametes independently of the alleles of another gene. However, because chromosomes and their connection to inheritance had not yet been discovered, Mendel missed one vital caveat to this law, namely that independence only occurs if such genes are located on separate chromosomes (or perhaps at opposite ends of a single long chromosome). Most of what follows in this thesis is based on this non-independent segregation of linked loci. One of the major milestones in genetics was the realisation that a collection of linked markers could be arranged in a linear fashion, with distances between their positions estimated from the counts of co-inherited markers (Sturtevant, 1913). In his demonstration of this fact using six linked morphological markers of the common fruit fly Drosophila melanogaster, Sturtevant created the world’s first genetic linkage map (Sturtevant, 1913;Van Ooijen and Jansen, 2013).
Nowadays, we generally rely on DNA markers (a.k.a. molecular markers) to identify positions on chromosomes. In this thesis we exclusively present data on single nucleotide polymorphism (SNP) markers. These are nucleotide positions which generally differ in the individuals being screened and are usually selected to be “bi-allelic” (i.e. alternating between two possible nucleotides in the material under study). However, the methods we develop are general to any bi-allelic marker system for which marker “dosage” counts
can be accurately estimated. The term “dosage” is less commonly used in diploid studies, but is a very important concept in genetic analyses of polyploids. Dosage is generally understood to be the number of copies of the alternative allele carried by an individual at a particular locus. An illustration of the possible marker dosage scores in a tetraploid and hexaploid are shown in Figure 1.

Genetic tools weed out misconceptions of strain reliability in Cannabis sativa: Implications for a budding industry
Anna L. Schwabe1 and Mitchell E. McGlaughlin
bioRxiv preprint
doi: 10.1101/332320
Cannabis sativa is listed as a Schedule I substance by the United States Drug Enforcement Agency and has been federally illegal in the United States since 1937. However, the majority of states in the United States, as well as several countries, now have various levels of legal Cannabis. Products are labeled with identifying strain names but there is no official mechanism to register Cannabis strains, therefore the potential exists for incorrect identification or labeling. This study uses genetic analyses to investigate strain reliability from the consumer point of view. Ten microsatellite regions were used to examine samples from strains obtained from dispensaries in three states. Samples were examined for genetic similarity within strains, and also a possible genetic distinction between Sativa, Indica, or Hybrid types. The analyses revealed genetic inconsistencies within strains. Additionally, although there was strong statistical support dividing the samples into two genetic groups, the groups did not correspond to commonly reported Sativa/Hybrid/Indica types. Genetic differences have the potential to lead to phenotypic differences and unexpected effects, which could be surprising for the recreational user, but have more serious implications for patients relying on strains that alleviate specific medical symptoms.

Genetic Variation in Hemp and Marijuana (Cannabis sativa L.) According to Amplified Fragment Length Polymorphisms
Shannon L. Datwyler, and George D. Weiblen,
Forensic Sci, March 2006, Vol. 51, No. 2
doi:10.1111/j1556-4029.2006.00061.x
Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.

Commentary on: Datwyler SL, Weiblen GD. Genetic Variation
in Hemp and Marijuana (Cannabis sativa L.) According to
Amplified Fragment Length Polymorphisms
. J Forensic Sci 2006; 51:371–5.
John M. McPartland,
J Forensic Sci, November 2006, Vol. 51, No. 6
doi:10.1111/j.1556-4029.2006.00276.x
Sir: The authors are commended for their careful methods, important results, and unique perspectives. I want to extend their results by embedding them in recent Cannabis taxonomic research based on cannabinoid variation (1), terpenoid variation (2), genetic variation (3), morphological traits (4), and host–parasite relationships (5). A taxonomic proposal by Hillig and coworkers is presented in Table 1, alongside previous taxonomic concepts cited
by Datwyler and Weiblen.
The four Cannabis populations studied by Datwyler and Weiblen can be placed in this classification with reasonable certainty, because the works of Datwyler, Weiblen, and Hillig intersect at a character trait: plant production of tetrahydrocannabinol (THC) and cannabidiol (CBD). According to Hillig and de Meijer (6), THC production in Cannabis is controlled by two co-dominant
alleles at a single locus, termed BT (encoding THC-synthase) and BD (encoding CBD-synthase). Cannabis indica biotypes are dominated by the BT allele and plants of this biotype generally produce a high THC: low CBD profile. Datwyler and Weiblen reported that ‘‘Skunk #1’’ produced this profile, consistent with ‘‘Skunk #1’’s pedigree—a hybrid of C. indica narrow-leafed and wide-leafed drug biotypes (7). The C. sativa hemp biotype is
dominated by the BD allele and these plants usually produce a high CBD: low THC profile. Datwyler and Weiblen reported that ‘‘Carmen’’ yielded this profile, which agrees with its origin as a European C. sativa hemp cultivar (Gordon Scheifele, personal communication, 2006).
The lineages of ‘‘Minneapolis’’ and ‘‘Shakopee’’ are unknown, but their high CBD: low THC profile suggests they are likely feral descendents of European C. sativa hemp. In the principal coordinates scatterplot by Datwyler and Weiblen, the three hemp populations and ‘‘Skunk #1’’ are at opposite ends of PC 1. Similarly, C. sativa populations and C. indica populations cluster at opposite ends of PC 1 in a scatterplot based on allozyme frequencies (8).

Genetically Modified Cannabis Sativa Plants And Modified Cannabinoid Compounds For Treatment Of Substance Addiction And Other Disorders
Williams, Jonnie R, Supera Pharmaceuticals, Inc.
https://www.freepatentsonline.com/y2020/0061138.html
A method of increasing a cannabinoid level in a Cannabis sativa plant, comprising: genetically modifying the Cannabis sativa plant with a genetic modification, wherein the genetic modification induces the Cannabis sativa plant to overexpress a gene controlling the expression of a cannabidiolic acid (CBDA) synthase, wherein the CBDA synthase catalyzes an increased synthesis of cannabigerolic acid to cannabidiolic acid, and wherein the cannabidiolic acid is converted into an increased cannabinoid level in the plant, wherein the cannabinoid is a compound having the structure:

Genetics and marker-assisted selection of the chemotype in Cannabis sativa L.
Daniela Pacifico, Francesca Miselli, Mirta Micheler, Andrea Carboni, Paolo Ranalli and Giuseppe Mandolino
Molecular Breeding (2006) 17: 257–268
DOI 10.1007/s11032-005-5681-x
Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp
breeding. In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements. In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible
applications in the forensic and pharmaceutical fields.

Not Cannabis Specific
Genome-Editing Technologies for Enhancing Plant Disease Resistance
Giuseppe Andolfo, Paolo Iovieno, Luigi Frusciante and Maria R. Ercolano
Front. Plant Sci. 7:1813. doi:
10.3389/fpls.2016.01813
One of the greatest challenges for agricultural science in the 21st century is to improve yield stability through the progressive development of superior cultivars. The increasing numbers of infectious plant diseases that are caused by plant??pathogens make it ever more necessary to develop new strategies for plant disease resistance breeding. Targeted genome engineering allows the introduction of precise modifications directly into a commercial variety, offering a viable alternative to traditional breeding methods. Genome editing is a powerful tool for modifying crucial players in the plant immunity system. In this work, we propose and discuss genome-editing strategies and targets for improving resistance to phytopathogens. First of all, we present the opportunities to rewrite the effector-target sequence for avoiding effector-target molecular interaction and also to modify effector-target promoters for increasing the expression of target genes involved in the resistance process. In addition, we describe potential approaches for obtaining synthetic R-genes through genome-editing technologies (GETs). Finally, we illustrate a genome editing flowchart to modify the pathogen recognition sites and engineer an R-gene that mounts resistance to some phylogenetically divergent pathogens. GETs potentially mark the beginning of a new era, in which synthetic biology affords a basis for obtaining a reinforced plant defense system. Nowadays it is conceivable that by modulating the function of the major plant immunity players, we will be able to improve crop performance for a sustainable agriculture.

Genome-Wide Analysis of Alternative Splicing and Non-Coding RNAs Reveal Complicated Transcriptional Regulation in Cannabis sativa L.
Bin Wu, Yanni Li, Jishuang Li, Zhenzhen Xie, Mingbao Luan, Chunsheng Gao, Yuhua Shi and Shilin Chen
Int. J. Mol. Sci. 2021, 22, 11989.
DOI: 10.3390/ijms222111989
https://www.ncbi.nlm.nih.gov/pmc/art...s-22-11989.pdf
It is of significance to mine the structural genes related to the biosynthetic pathway of fatty acid (FA) and cellulose as well as explore the regulatory mechanism of alternative splicing (AS), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the biosynthesis of cannabinoids, FA and cellulose, which would enhance the knowledge of gene expression and regulation at posttranscriptional level in Cannabis sativa L. In this study, transcriptome, small RNA and degradome libraries of hemp ‘Yunma No.1’ were established, and comprehensive analysis was performed. As a result, a total of 154, 32 and 331 transcripts encoding key enzymes involved in the biosynthesis of cannabinoids, FA and cellulose were predicted, respectively, among which AS occurred in 368 transcripts. Moreover, 183 conserved miRNAs, 380 C. sativa-specific miRNAs and 7783 lncRNAs were predicted. Among them, 70 miRNAs and 17 lncRNAs potentially targeted 13 and 17 transcripts, respectively, encoding key enzymes or transporters involved in the biosynthesis of cannabinoids, cellulose or FA. Finally, the crosstalk between AS and miRNAs or lncRNAs involved in cannabinoids and cellulose was also predicted. In summary, all these results provided insights into the complicated network of gene expression and regulation in C. sativa

Genome-wide analysis of LBD(lateral organ boundaries domain) gene family in Cannabis sativa of traditional Chinese medicine hemp seed
Wang Zhen , M I Yao-Lei , Meng Xiang-Xiao , Wan Hui-Hua , J I Ai-Jia , Sun Wei , M A Wei
China Journal of Chinese Materica Medica 2020 Nov;45(22):5477-5486 (in Chinese)
doi: 10.19540/j.cnki.cjcmm.20200818.106
LBD(lateral organ boundaries)transcription factors play an important role in the regulation of plant growth, development and secondary metabolism. In order to explore the function of LBD genes in cannabis, the Cannabis sativa genome and transcriptome were used to identify the C. sativa LBD gene family, and analyzed their expression patterns. Our results showed that the cannabis LBD contains 32 members, which were divided into two major categories, seven sub-families. Class ? was divided into 5 sub-families, named Class ?_a to Class ?_e, while Class ? was divided into 2 sub-families, including Class ?_a and Class ?_b. Analysis showed that the number of amino acids encoded LBDs was between 172 and 356, and the isoelectric point was between 4.92 and 9.43. The mole-cular weight of LBD was between 18 862.92 Da and 40 081.33 Da, and most members are located in the nucleus. Chromosome positioning of LBD showed that 32 members were unevenly distributed on 10 chromosomes of C. sativa LBD transcription factor domain, gene structure and motifs are relatively conservative, and the characteristics of different class members are similar. The upstream promoter region of the gene contains a variety of cis-acting elements related to plant hormones and environmental factors, C. sativa LBD genes have different expression patterns in the stems, leaves, and flowers of ZYS varieties(low tetrahydrocannabinol, high cannabidiol). The members of the LBD gene family are mainly expressed in the flowers and stems of ZYS varieties, while members expressed in the leaves very few; Class ? members CsLBD21 and CsLBD23 are expressed in flowers and stems, and CsLBD8 and CsLBD18 are expressed in flowers, stems and leaves. These genes may participate in the growth and development of cannabis and affect the biosynthesis of cannabinoids. This study laid the foundation for the subsequently functional research of the cannabis LBD gene family

Genome-wide analyses reveal clustering in Cannabis cultivars: the ancient domestication trilogy of a panacea
DOI: 10.7287/PEERJ.PREPRINTS.1553
https://peerj.com/preprints/1553.pdf
In the present research, I used an open access data set (Medicinal Genomics) consisting of nearly 200'000 genome-wide single nucleotide polymorphisms (SNPs) typed in 28 cannabis accessions to shed light on the plant's underlying genetic structure. Genome-wide loadings were used to sequentially cull less informative markers. The process involved reducing the number of SNPs to 100K, 10K, 1K, 100 until I identified a set of 42 highly informative SNPs that I present here. The two first principal components, encompass over 3/4 of the genetic variation present in the dataset (PCA1 = 48.6%, PCA2= 26.3%). This set of diagnostic SNPs is then used to identify clusters into which cannabis accession segregate. I identified three clear and consistent clusters; reflective of the ancient domestication trilogy of the genus Cannabis.

Genome-wide characterization of the MLO gene family in Cannabis sativa reveals two genes as strong candidates for powdery mildew susceptibility
Noémi Pépin, Francois Olivier Hebert, and David L. Joly
Biorxiv 2021
DOI: 10.1101/2021.07.16.452661
https://www.biorxiv.org/content/10.1...661v1.full.pdf
Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of 7 transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Genome-wide development of insertiondeletion (InDel) markers for Cannabis and its uses in genetic structure analysis of Chinese germplasm and sex-linked marker identification
Gen Pan, Zheng Li, Siqi Huang, Jie Tao, Yaliang Shi, Anguo Chen Jianjun L, Huijuan Tang, Li Chang, Yong Deng, Defang Li, and Lining Zhao
BMC Genomics (2021) 22:595
DOI: 10.1186/s12864-021-07883-w
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_7883.pdf
Background: Cannabis sativa L., a dioecious plant derived from China, demonstrates important medicinal properties and economic value worldwide. Cannabis properties have been usually harnessed depending on the sex of the plant. To analyse the genetic structure of Chinese Cannabis and identify sex-linked makers, genome-wide insertion-deletion (InDel) markers were designed and used.
Results: In this study, a genome-wide analysis of insertion-deletion (InDel) polymorphisms was performed based on the recent genome sequences. In total, 47,558 InDels were detected between the two varieties, and the length of InDels ranged from 4 bp to 87 bp. The most common InDels were tetranucleotides, followed by pentanucleotides. Chromosome 5 exhibited the highest number of InDels among the Cannabis chromosomes, while chromosome 10 exhibited the lowest number. Additionally, 31,802 non-redundant InDel markers were designed, and 84 primers evenly distributed in the Cannabis genome were chosen for polymorphism analysis. A total of 38 primers exhibited polymorphisms among three accessions, and of the polymorphism primers, 14 biallelic primers were further used to analyse the genetic structure. A total of 39 fragments were detected, and the PIC value ranged from 0.1209 to 0.6351. According to the InDel markers and the flowering time, the 115 Chinese germplasms were divided into two subgroups, mainly composed of cultivars obtained from the northernmost and southernmost regions, respectively. Additional two markers, “Cs-I1–10” and “Cs-I1–15”, were found to amplify two bands (398 bp and 251 bp; 293 bp and 141 bp) in the male plants, while 389-bp or 293-bp bands were amplified in female plants. Using the two markers, the feminized and dioecious varieties could also be distinguished.

Genome-wide DNA methylation analysis of heavy cannabis exposure in a New Zealand longitudinal cohort.
Osborne, A. J., Pearson, J. F., Noble, A. J., Gemmell, N. J., Horwood, L. J., Boden, J. M., … Kennedy, M. A.
Translational Psychiatry, 10(1). (2020).
doi:10.1038/s41398-020-0800-3
Cannabis use is of increasing public health interest globally. Here we examined the effect of heavy cannabis use, with and without tobacco, on genome-wide DNA methylation in a longitudinal birth cohort (Christchurch Health and Development Study, CHDS). A total of 48 heavy cannabis users were selected from the CHDS cohort, on the basis of their adult exposure to cannabis and tobacco, and DNA methylation assessed from whole blood samples, collected at approximately age 28. Methylation in heavy cannabis users was assessed, relative to non-users (n = 48 controls) via the Illumina Infinium® MethylationEPIC BeadChip. We found the most differentially methylated sites in cannabis with tobacco users were in the AHRR and F2RL3 genes, replicating previous studies on the effects of tobacco. Cannabis-only users had no evidence of differential methylation in these genes, or at any other loci at the epigenome-wide significance level (P < 10?7 ). However, there were 521 sites differentially methylated at P < 0.001 which were enriched for genes involved in neuronal signalling (glutamatergic synapse and long-term potentiation) and cardiomyopathy. Further, the most differentially methylated loci were associated with genes with reported roles in brain function (e.g. TMEM190, MUC3L, CDC20 and SP9). We conclude that the effects of cannabis use on the mature human blood methylome differ from, and are less pronounced than, the effects of tobacco use, and that larger sample sizes are required to investigate this further.

Genome-Wide Expression Profiles of Hemp (Cannabis sativa L.) in Response to Drought Stress
Chunsheng Gao, Chaohua Cheng, Lining Zhao, Yongting Yu, Qing Tang, Pengfei Xin, Touming Liu, Zhun Yan, Yuan Guo, and Gonggu Zang
International Journal of Genomics Volume 2018, Article ID 3057272, 13 pages
DOI: 10.1155/2018/3057272
https://downloads.hindawi.com/journa...18/3057272.pdf
Drought is the main environmental factor impairing hemp growth and yield. In order to decipher the molecular responses of hemp to drought stress, transcriptome changes of drought-stressed hemp (DS1 and DS2), compared to well-watered control hemp (CK1 and CK2), were studied with RNA-Seq technology. RNA-Seq generated 9.83, 11.30, 11.66, and 11.31 M clean reads in the CK1, CK2, DS1, and DS2 libraries, respectively. A total of 1292 differentially expressed genes (DEGs), including 409 (31.66%) upregulated and 883 (68.34%) downregulated genes, were identified. The expression patterns of 12 selected genes were validated by qRT-PCR, and the results were accordant with Illumina analysis. Gene Ontology (GO) and KEGG analysis illuminated particular important biological processes and pathways, which enriched many candidate genes such as NAC, B3, peroxidase, expansin, and inositol oxygenase that may play important roles in hemp tolerance to drought. Eleven KEGG pathways were significantly influenced, the most influenced being the plant hormone signal transduction pathway with 15 differentially expressed genes. A similar expression pattern of genes involved in the abscisic acid (ABA) pathway under drought, and ABA induction, suggested that ABA is important in the drought stress response of hemp. These findings provide useful insights into the drought stress regulatory mechanism in hemp

Genome-Wide Identification and Expression Profiles of bZIP Genes in Cannabis sativa L.
Meng Lu, Xiang-Xiao Meng, Yi-Ming Zhang, Xue-Wen Zhu, Jun Li, Wei-Qiang Chen, Hui-Hua Wan, Si-Fan Wang, Xue Cao, Wei Sun, Yao-Lei Mi, and Jun-Wen Zhai
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0153
Background: The bZIP gene family plays roles in biotic and abiotic stress, secondary metabolism, and other aspects in plants. They have been reported in Arabidopsis thaliana, Oryza sativa, Artemisia annua, and other plants, but their roles in Cannabis sativa have not been determined.
Materials and Methods: In this study, we analyzed the genome-wide identification and expression profile of the bZIP gene family in C. sativa.
Results: A total of 51 members of the bZIP gene family were identified based on the C. sativa genome and numbered in order from CsbZIP1 to CsbZIP51. Their phylogenetic relationships, cis-elements in promoter region, gene structures and motif compositions, physicochemical properties, chromosome locations, and expression profiles, were analyzed. The results showed that the 51 CsbZIPs were unevenly distributed on 10 chromosomes and could be clustered into 11 subfamilies. Furthermore, CsbZIPs located in the same subfamilies presented similar intron/exon organization and motif composition. The expression levels of CsbZIPs in various tissues (flowers, bracts, vegetative leaves, stems, and seeds) were determined using reverse transcription quantitative polymerase chain reaction. The expression levels of CsbZIPs were higher in flowers and bracts. The 51 CsbZIPs were explored, and their structure, evolution, and expression pattern in different tissues of C. sativa were characterized synthetically. The findings indicated that CsbZIPs are essential for the growth and development of C. sativa.
Conclusions: These results provide a theoretical basis for subsequent research on hemp bZIP transcription factors and the cultivation of high-cannabidiol and low-tetrahydrocannabinol high-quality cannabis varieties.
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Genome-wide identification, expression, and sequence analysis of CONSTANS-like gene family in cannabis reveals a potential role in plant flowering time regulation

Gen Pan, Zheng Li, Ming Yin, Siqi Huang, Jie Tao, Anguo Chen, Jianjun Li, Huijuan Tang, Li Chang, Yong Deng, Defang Li. and Lining Zhao
DOI: 10.1186/s12870-021-02913-x
https://bmcplantbiol.biomedcentral.c.. .21-02913-x.pdf
Background Cannabis, an important industrial crop, has a high sensitivity to photoperiods. The flowering time of cannabis is one of its important agronomic traits, and has a significant effect on its yield and quality. The CONSTANS-like (COL) gene plays a key role in the regulation of flowering in this plant. However, the specific roles of the COL gene family in cannabis are still unknown.
Results In this study, 13 CsCOL genes were identified in the cannabis genome. Phylogenetic analysis implied that the CsCOL proteins were divided into three subgroups, and each subgroup included conserved intron/exon structures and motifs. Chromosome distribution analysis showed that 13 CsCOL genes were unevenly distributed on 7 chromosomes, with chromosome 10 having the most CsCOL members. Collinearity analysis showed that two syntenic gene pairs of CsCOL4 and CsCOL11 were found in both rice and Gossypium raimondii. Of the 13CsCOL genes, CsCOL6 and CsCOL12 were a pair of tandem duplicated genes, whereasCsCOL8 and CsCOL11 may have resulted from segmental duplication. Furthermore, tissue-specific expression showed that 10 CsCOL genes were preferentially expressed in the leaves, 1CsCOL in the stem, and 2 CsCOL in the female flower. Most CsCOL exhibited a diurnal oscillation pattern under different light treatment. Additionally, sequence analysis showed that CsCOL3 and CsCOL7 exhibited amino acid differences among the early-flowering and late flowering cultivars.
Conclusion This study provided insight into the potential functions of CsCOL genes, and highlighted their roles in the regulation of flowering time in cannabis. Our results laid a foundation for the further elucidation of the functions of COL genes in cannabis.

Genomic and Chemical Diversity in Cannabis
Ryan C Lynch, Daniela Vergara, Silas Tittes, Kristin White, C.J. Schwartz, Matthew J Gibbs, Travis C Ruthenburg, Kymron deCesare, Donald P Land, Nolan C Kane
Critical Reviews in Plant Sciences Pages 1-15 Published online: 22 Feb 2017
Doi: 10.1080/07352689.2016.1265363
Plants of the Cannabis genus are the only producers of phytocannabinoids, terpenoid compounds that strongly interact with evolutionarily ancient endocannabinoid receptors shared by most bilaterian taxa. For millennia, the plant has been cultivated for these compounds, but also for food, rope, paper, and clothing. Today, specialized varieties yielding high-quality textile fibers, nutritional seed oil or high cannabinoid content are cultivated across the globe. However, the genetic identities and histories of these diverse populations remain largely obscured. We analyzed the nuclear genomic diversity among 339 Cannabis varieties, and demonstrate the existence of at least three major groups of diversity. As well as being genetically distinct, each group produces unique cannabinoid and terpenoid content profiles. This combined analysis of population genomic and trait variation informs our understanding of the potential uses of different genetic variants for medicine and agriculture, providing valuable insights and tools for a rapidly emerging, valuable legal industry. Marijuana legalization continues apace around the globe with governments the world over now recognizing some medical use for cannabis consumption. But that increasing acceptance belies a hidden truth: Researchers still don’t really understand the genetic roots of the plant’s biochemical bounty. Yet, over the past few months, large-scale DNAsequencing efforts have started to chart the genes responsible for the rich spectrum of phytochemicals produced by both drug and hemp varieties of theCannabis sativa plant, offering key insights for research, industry, and policy. “The genome map is a very powerful step forward,” says Jonathan Page, chief scientific officer of Aurora Cannabis, one of the largest cannabis companies in the world. It brings the plant into the modern agricultural era, he adds, noting that “the cutting edge is waiting for cannabis.”

Genomic Evidence That Governmentally Produced Cannabis sativa Poorly Represents Genetic Variation Available in State Markets
Daniela Vergara, Ezra L. Huscher, Kyle G. Keepers, Rahul Pisupati, Anna L. Schwabe, Mitchell E. McGlaughlin and Nolan C. Kane
Front. Plant Sci. 12:668315. (2021)
doi: 10.3389/fpls.2021.668315
https://www.frontiersin.org/articles...21.668315/full
The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genomes of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA’s varieties differ from widely available medical, recreational, or industrial Cannabis . Furthermore, our results suggest that NIDA’s varieties lack diversity in the single-copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome.
Therefore, results based on NIDA’s varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.

GENOMIC, TERPENE AND CANNABINOID PROFILES OF A PUTATIVELY NOVEL CANNABIS SPECIES
Kevin McKernan, Cameron Miller, Brad Douglass. Jessica Spangler, Braden Doane, Vasisht Tadigotla, Colin Montgomery, Yvonne Helbert, Lei Zhang, Douglas Smith, Jeffrey Raber
Paper
https://www.medicinalgenomics.com/wp...cies_sbmt3.pdf
YOUTUBE

An Australian feral male cannabis strain (Australian Bastard Cannabis or ABC) was crossed with Cannabis Sativa L. (Purple skunk Oregon) and presented with atypical leaf patterns and stunted but hardy growth (offspring termed ABCh). Microscopy identified small trichomes and encouraged further evaluation with UV-HPLC (UV confirmed high performance liquid chromatography) that identified THCA and CBCA peaks. This cultivar was further tested for terpene profiles with GC-FID (gas chromatography-flame ionization detector) identifying many common terpenes in Cannabis. Thin layer chromatography (TLC) was used to confirm the cannabinoid results against Cerilliant standards and the sample was selected for genomic analysis.

Genomics blazes a trail to improved cannabis cultivation
Elie Dolgin
PNAS | April 30, 2019 | vol. 116 | no. 18
DOI: 10.1073/pnas.1904094116
Marijuana legalization continues apace around the globe with governments the world over now recognizing some medical use for cannabis consumption. But that increasing acceptance belies a hidden truth: Researchers still don’t really understand the genetic roots of the plant’s biochemical bounty. Yet, over the past few months, large-scale DNAsequencing efforts have started to chart the genes responsible for the rich spectrum of phytochemicals produced by both drug and hemp varieties of theCannabis sativa plant, offering key insights for research, industry, and policy. “The genome map is a very powerful step forward,” says Jonathan Page, chief scientific officer of Aurora Cannabis, one of the largest cannabis companies in the world. It brings the plant into the modern agricultural era, he adds, noting that “the cutting edge is waiting for cannabis.”

NOT CANNABIS SPECIFIC
Haploid plants produced by centromere-mediated genome elimination.
Ravi, M., & Chan, S. W. L.
Nature, 464(7288), 615–618. (2010).
doi:10.1038/nature08842
Production of haploid plants that inherit chromosomes from only one parent can greatly accelerate plant breeding1–3. Haploids generated from a heterozygous individual and converted to diploid create instant homozygous lines, bypassing generations of inbreeding. Two methods are generally used to produce haploids. First, cultured gametophyte cells may be regenerated into haploid plants4 , but many species and genotypes are recalcitrant to this process2,5. Second, haploids can be induced from rare interspecific crosses, in which one parental genome is eliminated after fertilization6–11. The molecular basis for genome elimination is not understood, but one theory posits that centromeres from the two parent species interact unequally with the mitotic spindle, causing selective chromosome loss12–14. Here we show that haploid Arabidopsis thaliana plants can be easily generated through seeds by manipulating a single centromere protein, the centromere-specific histone CENH3 (called CENP-A in human). When cenh3 null mutants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Haploids are spontaneously converted into fertile diploids through meiotic nonreduction, allowing their genotype to be perpetuated. Maternal and paternal haploids can be generated through reciprocal crosses. We have also exploited centromere-mediated genome elimination to convert a natural tetraploid Arabidopsis into a diploid, reducing its ploidy to simplify breeding. As CENH3is universalin eukaryotes, our method may be extended to produce haploids in any plant species

Hemp in ancient rope and fabric from the Christmas Cave in Israel: Talmudic background and DNA sequence identification
Terence M. Murphy, Nahum Ben-Yehuda , R.E. Taylor, John R. Southon
Journal of Archaeological Science 38 (2011) 2579e2588
doi:10.1016/j.jas.2011.05.004
The “Christmas Cave”, a cave in the Qidron Valley near the Dead Sea and Qumran, has yielded a complex collection of plant-derived rope and fabric artifacts. Using polymerase chain reaction (PCR) to amplify DNA of the samples, we estimated the sizes and determined restriction patterns and base sequences of chloroplast genes, primarily rbcL (gene for the large subunit of ribulose bisphosphate carboxylase). DNA was successfully extracted from all samples, but was limited to sizes of approximately 200e300 base pairs. As expected, the DNA extracted from the samples was identified as coming primarily from flax (Linum usitatissamum L.), but two samples had a significant fraction, and all samples had at least a trace, of hemp (Cannabis sativa L.) DNA. Artifacts from the Christmas Cave were thought to date from Roman times, but it was thought possible that some could be much older. Accelerator mass spectrometry (AMS)-based 14C dating confirmed that the samples contained representatives from both the Roman and Chalcolithic periods. This paper provides a synthesis of DNA, isotope, and literary analysis to illuminate
textile history at the Christmas Cave site.

How hemp got high
Naomi Attar
Genome Biology 2011, 12:409
doi:10.1186/gb-2011-12-10-409
The development of RNA-seq, which applies high throughput next-generation sequencing technology to cDNA generated from RNA samples, has resulted in an explosion of transcriptome sequences. Not only does RNA-seq benefit from higher sensitivity than microarrays, it also does not require the a priori knowledge needed for constructing chips; this flexibility has infused the transcriptome explosion with a biologically diverse character and encompassed many species not well covered by commercially available microarrays.
Species such as Cannabis sativa , a plant with a ‘split personality’, whose Dr Jekyll, hemp, is an innocent source of textiles, but whose Mr Hyde, marijuana, is chiefly used to alter the mind. Until now, Cannabis sativa was not one of the many species whose genome had been published during Genome Biology’s lifetime. In common with many plants, genome assembly of cannabis DNA sequence is technically challenging, and so the publication a 534 Mbp draft genome in this month’s issue is in itself a landmark achievement.

Genome-wide development of insertiondeletion (InDel) markers for Cannabis and its uses in genetic structure analysis of Chinese germplasm and sex-linked marker identification
Gen Pan, Zheng Li, Siqi Huang, Jie Tao, Yaliang Shi, Anguo Chen Jianjun L, Huijuan Tang, Li Chang, Yong Deng, Defang Li, and Lining Zhao
BMC Genomics (2021) 22:595
DOI: 10.1186/s12864-021-07883-w
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_7883.pdf
Background: Cannabis sativa L., a dioecious plant derived from China, demonstrates important medicinal properties and economic value worldwide. Cannabis properties have been usually harnessed depending on the sex of the plant. To analyse the genetic structure of Chinese Cannabis and identify sex-linked makers, genome-wide insertion-deletion (InDel) markers were designed and used.
Results: In this study, a genome-wide analysis of insertion-deletion (InDel) polymorphisms was performed based on the recent genome sequences. In total, 47,558 InDels were detected between the two varieties, and the length of InDels ranged from 4 bp to 87 bp. The most common InDels were tetranucleotides, followed by pentanucleotides. Chromosome 5 exhibited the highest number of InDels among the Cannabis chromosomes, while chromosome 10 exhibited the lowest number. Additionally, 31,802 non-redundant InDel markers were designed, and 84 primers evenly distributed in the Cannabis genome were chosen for polymorphism analysis. A total of 38 primers exhibited polymorphisms among three accessions, and of the polymorphism primers, 14 biallelic primers were further used to analyse the genetic structure. A total of 39 fragments were detected, and the PIC value ranged from 0.1209 to 0.6351. According to the InDel markers and the flowering time, the 115 Chinese germplasms were divided into two subgroups, mainly composed of cultivars obtained from the northernmost and southernmost regions, respectively. Additional two markers, “Cs-I1–10” and “Cs-I1–15”, were found to amplify two bands (398 bp and 251 bp; 293 bp and 141 bp) in the male plants, while 389-bp or 293-bp bands were amplified in female plants. Using the two markers, the feminized and dioecious varieties could also be distinguished.

Identification of candidate genes affecting ?9-tetrahydrocannabinol biosynthesis in Cannabis sativa.
Marks, M. D., Tian, L., Wenger, J. P., Omburo, S. N., Soto-Fuentes, W., He, J., … Dixon, R. A.
Journal of Experimental Botany, 60(13), 3715–3726.(2009).
doi:10.1093/jxb/erp210
RNA isolated from the glands of a D9 -tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands



Identification and mapping of major-effect flowering time loci Autoflower1 and Early1 in Cannabis sativa L.
Jacob A. Toth, George M. Stack, Craig H. Carlson† and Lawrence B. Smart
Flowering time is an important trait for all major market classes of hemp (Cannabis sativa), affecting yields and quality of grain, fiber, and cannabinoids. C. sativa is usually considered a short-day plant, flowering once night length reaches a critical threshold. Variations in flowering time within and across cultivars in outdoor grown populations have been previously identified, likely corresponding to genetic differences in this critical night length. Further, some C. sativa are photoperiod insensitive, colloquially referred to as “autoflowering.” This trait has anecdotally been described as a simple recessive trait with major impacts on phenology and yield. In this work, the locus responsible for the “autoflower” trait (Autoflower1), as well as a major-effect flowering time locus, Early1, were mapped using bulked segregant analysis. Breeder-friendly high-throughput molecular marker assays were subsequently developed for both loci. Also detailed are the flowering responses of diverse cultivars grown in continuous light and the result of crossing two photoperiod insensitive cultivars of differing pedigree

Identification of Chemotypic Markers in Three Chemotype Categories of Cannabis Using Secondary Metabolites Profiled in Inflorescences, Leaves, Stem Bark, and Roots
Dan Jin, Philippe Henry, Jacqueline Shan and Jie Chen
Front. Plant Sci. 12:699530.
doi: 10.3389/fpls.2021.699530
https://www.ncbi.nlm.nih.gov/pmc/art...-12-699530.pdf
Previous chemotaxonomic studies of cannabis only focused on tetrahydrocannabinol (THC) dominant strains while excluded the cannabidiol (CBD) dominant strains and intermediate strains (THC ≈ CBD). This study investigated the utility of the full spectrum of secondary metabolites in different plant parts in three cannabis chemotypes (THC dominant, intermediate, and CBD dominant) for chemotaxonomic discrimination. Hierarchical clustering, principal component analysis (PCA), and canonical correlation analysis assigned 21 cannabis varieties into three chemotypes using the content and ratio of cannabinoids, terpenoids, flavonoids, sterols, and triterpenoids across inflorescences, leaves, stem bark, and roots. The same clustering results were obtained using secondary metabolites, omitting THC and CBD. Significant chemical differences were identified in these three chemotypes. Cannabinoids, terpenoids, flavonoids had differentiation power while sterols and triterpenoids had none. CBD dominant strains had higher amounts of total CBD, cannabidivarin (CBDV), cannabichromene (CBC), α-pinene, β-myrcene, (−)-guaiol, β-eudesmol, α-eudesmol, α-bisabolol, orientin, vitexin, and isovitexin, while THC dominant strains had higher total THC, total tetrahydrocannabivarin (THCV), total cannabigerol (CBG), camphene, limonene, ocimene, sabinene hydrate, terpinolene, linalool, fenchol, α-terpineol, β-caryophyllene, trans-β-farnesene, α-humulene, trans-nerolidol, quercetin, and kaempferol. Compound levels in intermediate strains were generally equal to or in between those in CBD dominant and THC dominant strains. Overall, with higher amounts of β-myrcene, (−)- guaiol, β-eudesmol, α-eudesmol, and α-bisabolol, intermediate strains more resemble CBD dominant strains than THC dominant strains. The results of this study provide a comprehensive profile of bioactive compounds in three chemotypes for medical purposes. The simultaneous presence of a predominant number of identified chemotype markers (with or without THC and CBD) could be used as chemical fingerprints for quality standardization or strain identification for research, clinical studies, and cannabis product manufacturing.

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)
G. Mandolino, A. Carboni, S. Forapani V. Faeti, P. Ranalli
Theor Appl Genet (1999) 98: 86‹92
DOI:10.1007/s001220051043
A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8400, present in all male plants and absent in female plants. A screening of 167 plants belonging to di.erent genotypes for the association of the OPA8400 marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-speci?c association, the sequences corresponding to OPA8400 were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and speci?c primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-speci?c. This SCAR is suitable for a precise, early and rapid identi?cation of male plants during breeding programs of dioecious and monoecious hemp

Identification of fasciclin-like arabinogalactan proteins in textile hemp (Cannabis sativa L.): in silico analyses and gene expression patterns in different tissues.
Guerriero, Gea; Mangeot-Peter, Lauralie; Legay, Sylvain; Behr, Marc; Lutts, Stanley; Siddiqui, Khawar Sohail; Hausman, Jean-Francois
BMC Genomics, 18(1), 741–. (2017).
doi:10.1186/s12864-017-3970-5
Background: The fasciclin-like arabinogalactan proteins (FLAs) belong to the arabinogalactan protein (AGP) superfamily and are known to play different physiological roles in plants. This class of proteins was shown to participate in plant growth, development, defense against abiotic stresses and, notably, cell wall biosynthesis. Although some studies are available on the characterization of FLA genes from different species, both woody and herbaceous, no detailed information is available on the FLA family of textile hemp (Cannabis sativa L.), an economically important fibre crop. Results: By searching the Cannabis genome and EST databases, 23 CsaFLAs have been here identified which are divided into four phylogenetic groups. A real-time qPCR analysis performed on stem tissues (isolated bast fibres and shivs sampled at three heights), hypocotyls (6-9-12-15-17-20 days-old), whole seedlings, roots, leaves and female/male flowers of the monoecious fibre variety Santhica 27, indicates that the identified FLA genes are differentially expressed. Interestingly, some hemp FLAs are expressed during early phases of fibre growth (elongation), while others are more expressed in the middle and base of the stem and thus potentially involved in secondary cell wall formation (fibre thickening). The bioinformatic analysis of the promoter regions shows that the FLAs upregulated in the younger regions of the stem share a conserved motif related to flowering control and regulation of photoperiod perception. The promoters of the FLA genes expressed at higher levels in the older stem regions, instead, share a motif putatively recognized by MYB3, a transcriptional repressor belonging to the MYB family subgroup S4. Conclusions: These results point to the existence of a transcriptional network fine-tuning the expression of FLA genes in the older and younger regions of the stem, as well as in the bast fibres/shivs of textile hemp. In summary, our study paves the way for future analyses on the biological functions of FLAs in an industrially relevant fibre crop.

Not Cannabis Specific
Identification of genes required for de novo DNA methylation in Arabidopsis.
Greenberg, M. V. C., Ausin, I., Chan, S. W. L., Cokus, S. J., Cuperus, J. T., Feng, S., Jacobsen, S. E.
Epigenetics, 6(3), 344–354(2011).
doi:10.4161/epi.6.3.14242
De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata.
Hussain, T., Plunkett, B., Ejaz, M., Espley, R. V., & Kayser, O.
Frontiers in Plant Science, 9.(2018).
doi:10.3389/fpls.2018.00537
The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

Identification of QTLs for sex expression in dioecious and monoecious hemp (Cannabis sativa L.).
Faux, A.-M., Draye, X., Flamand, M.-C., Occre, A., & Bertin, P.
Euphytica, 209(2), 357–376. (2016).
doi:10.1007/s10681-016-1641-2
Hemp (Cannabis sativa L.) is a diploid species including both dioecious and monoecious cultivars with hetero- and homomorphic sex chromosomes,
respectively. It displays a high plasticity of sex expression, i.e., the ratio of female and male flowers.
In this study, we investigated the role of sex chromosomes in the genetic determinism of sex expression in dioecious and monoecious hemp. The experimental materials were three F1 segregating populations, two dioecious (C1 and C2: ‘Carmagnola’ $ 9 ‘Carmagnola’# ), and one monoecious (UF: ‘Uso 31’ 9 ‘Fedora 17’). A ‘sex’ phenotypic marker was mapped in C1 and C2. In total, 23, 42, and 26 AFLP markers (71 markers in total) were mapped to three, nine, and three co-segregation groups putatively located on sex chromosomes in C1, C2, and UF, respectively. Recombination rates with sex ranged from 0 to 0.5. Five sexlinked markers were detected in UF, revealing homologies between the X chromosomes of monoecious hemp and the X and Y chromosomes of dioecious hemp. Five QTLs associated with quantitative variations in sex expression were identified in each map. Four markers associated with variations in sex expression in UF segregated with sex or accounted for a putative QTL in C1 or C2. Two QTLs and three of these markers were mapped in UF in a region homologous to the sex-locus region of the dioecious maps. Given these results, conducting further research on the genetic determinism of sex expression in hemp using a quantitative approach appears relevant.

Identification of the aquaporin gene family in Cannabis sativa and evidence for the accumulation of silicon in its tissues.
.Guerriero, G., Deshmukh, R., Sonah, H., Sergeant, K., Hausman, J.-F., Lentzen, E., … Exley, C.
Plant Science, 110167.(2019).
doi:10.1016/j.plantsci.2019.110167
Cannabis sativa is an economically important crop providing bast fibres for the textile and biocomposite sector. Length is a fundamental characteristic determining the properties of bast fibres. Aquaporins, channel-forming proteins facilitating the passage of water, urea, as well as elements such as boron and silicon, are known to play a role in the control of fibre length in other species, like cotton. By mining the available genome, we here identify, for the first time, the aquaporin gene family of C. sativa. The analysis of published RNA-Seq data and targeted qPCR on a textile variety reveal an organ-specific expression of aquaporin genes. Computational analyses, including homology-based search, phylogeny and protein modelling, identify two NOD26-like intrinsic proteins harbouring the Gly-Ser-Gly-Arg (GSGR) aromatic/Arg selectivity filter and 108 amino acid NPA (Asn-Pro-Ala) spacing, features reported to be associated with silicon permeability. SIMS nano-analysis and silica extraction coupled to fluorescence microscopy performed on hemp plantlets reveal the presence of silicon in the bast fibres of the hypocotyl and in leaves. The accumulation of silica in the distal cell walls of bast fibres and in the basal cells of leaf trichomes is indicative of a mechanical role



Identification of the Histone Deacetylases Gene Family in Hemp Reveals Genes Regulating Cannabinoids Synthesis

Liu Yang, Xiangxiao Meng, Shilin Che, Jun , Wei Sun , Weiqiang Che, Sifan Wang , Huihua Wa, Guangtao Qian , Xiaozhe Yi, Juncan Li , Yaqin Zheng , Ming Lu, Shanshan Che, Xia Li and Yaolei M
Frontiers in Plant Science
DOI 103899/fpls.2021.755494
Histone deacetylases (HDACs) play crucial roles nearly in all aspects of plant biology, including stress responses, development and growth, and regulation of secondary metabolite biosynthesis. The molecular functions of HDACs have been explored in depth in Arabidopsis thaliana, while little research has been reported in the medicinal plant Cannabis sativa L. Here, we excavated 14 CsHDAC genes of C. sativa L that were divided into three relatively conserved subfamilies, including RPD3/HDA1 (10 genes), SIR2 (2 genes), and HD2 (2 genes). Genes associated with the biosynthesis of bioactive constituents were identified by combining the distribution of cannabinoids with the expression pattern of HDAC genes in various organs. Using qRT-PCR and transcription group analysis, we verified the expression of candidate genes in different tissues. We found that the histone inhibitor Trichostatin A (TSA) affected the expression of key genes in the cannabinoid metabolism pathway and the accumulation of synthetic precursors, which indirectly indicates that histone inhibitor may regulate the synthesis of active substances in C. sativa L.


Not Cannabis Specific
In a battle between parental chromosomes, a failure to reload.
Chan, S. W. L.
Proceedings of the National Academy of Sciences, 108(33), 13361–13362. (2011).
doi:10.1073/pnas.1110320108
What can go wrong after an egg is fertilized by sperm from a different species? One of the most intriguing outcomes to the chromosome biologist is uniparental genome elimination. In this phenomenon, one of the parental chromosome sets is completely lost during embryonic cell divisions, creating sterile haploid offspring (1). Uniparental genome elimination occurs in diverse taxa, including several distantly related plant species and even in fish (1, 2). The mechanism by which chromosomes from one parent are selectively discarded has been studied cytologically in crosses between different grass species. However, these experiments have not revealed what is defective about the eliminated genome in a hybrid. In PNAS, Sanei et al. (3) make a major breakthrough by showing that missegregated chromosomes in a classic barley interspecies cross (Hordeum vulgare × Hordeum bulbosum) fail to assemble kinetochores, the microtubule attachment sites that mediate chromosome inheritance. The chromosomal location of the kinetochore, termed the centromere, is marked by incorporation of a histone H3 variant named CENH3. CENH3-containing nucleosomes are found at centromeres in all eukaryotes and are essential for recruiting other kinetochore proteins. Sanei et al. (3) used an antibody raised against rice CENH3 to stain embryos created by pollinating cultivated barley (H. vulgare) with its wild relative H. bulbosum—the antibody recognizes CENH3 from both parents. A fascinating observation about this cross is that elimination of the paternal H. bulbosum chromosomes is temperature-dependent. At low temperature, CENH3 was found at the predicted foci on all chromosomes, which segregated accurately. In embryos created by fertilization at higher temperature, chromosomes that lagged behind during mitosis and were eventually discarded showed a lack of CENH3 antibody staining. Fluorescence in situ hybridization with labeled genomic DNA (combined with a wealth of previous genetic data) indicates that lagging chromosomes without functional kinetochores were derived from the H. bulbosum parent.

In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L.
Bassolino, L., Buti, M., Fulvio, F., Pennesi, A., Mandolino, G., Milc, J., … Paris, R.
Plants, 9(11), 1540. (2020).
doi:10.3390/plants9111540
Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids’ metabolic pathways, very little is known about their regulation, while the literature on flavonoids’ metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs.

Induction of Polyploidy and Its Effect on Cannabis sativa L. Cannabis Sativa L.
Mansouri, H., & Bagheri, M.
Botany and Biotechnology, 365–383.(2017).
doi:10.1007/978-3-319-54564-6_17
Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants, and is an important mechanism of speciation. Polyploids can be formed in various ways. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits. We reported the production of putative tetraploid plants of Cannabis sativa L., with the ultimate aim of improving the medicinal and physiological traits of this widely distributed cultivated plant. The production of tetraploid plant was improved with colchicine at different concentrations and time through dropping method. Flow cytometry analysis was used to confirm the ploidy level. Morphologic, anatomic and biochemical characteristics were compared between tetraploid and diploid control plants. The results showed that 0.2% colchicine for 24 h was the most efficient for production of polyploid plants. The percentage of tetraploid plants and the survival rate were lowered by the increasing the treatment time. In addition, the leaf index and height of tetraploid plants exhibited a significant decrease compared to the diploid plants. The size of stomata on epidermis of leaves were larger in tetraploid plant compared to the diploid ones, in spite of the tetraploid plants have less stomata density. However, the amount of total chlorophyll and carotenoids were almost the same in both tetraploid and diploid plants. In addition, some differences were also observed in the cross section of stem of these plants from a descriptive structural point of view. Overall, the results introduced usage of the stomata parameters as an effective, fast and convenient method for detecting the tetraploid plants. We also investigated polyploidy effects on some primary and secondary metabolites. The results of biochemical analyzes showed that soluble sugars and total protein content increased significantly into mixoploid plants compared to tetraploid and diploid plants. Tetraploid plants had higher amount of total proteins compared with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabionol only in mixoploid plants but tetraploid plants had lower amounts of this substance in comparison with diploids. Our results suggest that tetraploidization was not useful for production of tetrahydrocannabinol for commercial use but mixoploids were found suitable

Inner Workings: Genomics blazes a trail to improved cannabis cultivation.
Dolgin, E.
Proceedings of the National Academy of Sciences, 116(18), 8638–8640. (2019).
doi:10.1073/pnas.1904094116
Marijuana legalization continues apace around the globe with governments the world over now recognizing some medical use for cannabis consumption. But that increasing acceptance belies a hidden truth: Researchers still don’t really understand the genetic roots of the plant’s biochemical bounty. Yet, over the past few months, large-scale DNAsequencing efforts have started to chart the genes responsible for the rich spectrum of phytochemicals produced by both drug and hemp varieties of theCannabis sativa plant, offering key insights for research, industry, and policy. “The genome map is a very powerful step forward,” says Jonathan Page, chief scientific officer of Aurora Cannabis, one of the largest cannabis companies in the world. It brings the plant into the modern agricultural era, he adds, noting that “the cutting edge is waiting for cannabis.”

Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa*L.)
Erdogan E Hakki, Seyit Ali Kayis, Emine Pinarkara, Ayla Sag
Electronic Journal of Biotechnology*10(4)
DOI:10.2225/vol10-issue4-fulltext-4
Cannabis sativa*L. is a multiple-use plant that provides raw material for the production of seed oil, natural fiber for textiles, automotive and pulp industries. It has also been used in insulating boards, ropes, varnishes, animal feed, and as medicinal agents.*Cannabis*has potential to be used for phytoremediation: however, its cultivation is strictly controlled due to its psychoactive nature and usage in producing drugs such as marijuana, and hashish.*In this study, psychoactive type*Cannabis*samples, which were seized from 23 different locations of Turkey, and nine hemp type*Cannabis*accessions, as well as an unknown accession*were used. Our interest was to identify*the genetic relatedness of the seized samples and to separate drug and hemp type plants. Inter Simple Sequence Repeats (ISSRs) were employed for analysis based on single plant material (SET1) and bulked samples of them (SET2). Data was analysed*via*cluster analysis and principal coordinate analysis (PCoA). PCoA analyses, by using SET1 and SET2, were able to efficiently discriminate the seized samples from the fiber type accessions. However, separation*of the plants was not clear*via*unweighted pair-group method using arithmetic average (UPGMA) dendogram in SET1, while they were clearly separated in SET2. Hemp type accessions showed high levels of variation compared to drug typeCannabis*both in SET1 and SET2.

Intraspecific variation in Cannabis sativa L. based on intergenic spacer region of chloroplast DNA.
Mareshige Kohjyouma, In Jung Lee, Motoyoshi Satake
Published in Biological & pharmaceutical bulletin 2000
DOI:10.1248/bpb.23.727
We analyzed the nucleotide sequences of the non-coding region of chloroplast DNA: the intergenic spacer between trnL (UAA) 3'exon and trnF (GAA). Two kinds of sequence, "type-1" and "type-2," were detected in 33 populations of Cannabis sativa. The length of the "type-1" fragment was 354 bp. In contrast, the "type-2" fragment from 3 populations was 353 bp long, with only one base deletion compared to "type-1." The fragment length from Humulus lupulus was 353 bp with a 1-bp deletion, and ten 1-bp substitutions compared to the sequences from C. sativa "type-1." Furthermore, we could clearly identify differences between C. sativa and H. lupulus using single-strand conformation polymorphism of PCR products (PCR-SSCP) analysis.

Investigations into the Hypothesis of Transgenic Cannabis
Fidelia Cascini,
J Forensic Sci, May 2012, Vol. 57, No. 3
doi: 10.1111/j.1556-4029.2011.02021.x
The unusual concentration of cannabinoids recently found in marijuana samples submitted to the forensic laboratory for chemical analysis prompted an investigation into whether genetic modifications have been made to the DNA of Cannabis sativa L. to increase its potency. Traditional methods for the detection of genetically modified organisms (GMO) were used to analyze herbal cannabis preparations. Our analyses support the hypothesis that marijuana samples submitted to forensic laboratories and characterized by an abnormal level of D9-THC are the product of breeding selection rather than of transgenic modifications. Further, this research has shown a risk of false positive results associated with the poor quality of the seized samples and probably due to the contamination by other transgenic vegetable products. On the other hand, based on these data, a conclusive distinction between the hypothesis of GMO plant contamination and the other of genetic modification of cannabis cannot be made requiring further studies on comparative chemical and genetic analyses to find out an explanation for the recently detected increased potency of cannabis.

Is Cannabis Testing Now for Growers Too?
Leo Bear-McGuinness
https://www.analyticalcannabis.com/a...ers-too-311485
As marijuana legalization has swept the states, many farmers have seen their once stigmatized hustle grow into a profitable profession. And business is blooming. The ‘green rush’ has been so explosive that in Oregon, zealous growers now have enough cannabis to meet consumer demand for the next six and a half years.
But an expansive, regulated market comes with expansive regulations. In California, for example, all regulated cannabis products must be screened for a multitude of factors before they can grace the shelves. Pesticides, microbes, heavy metals, cannabinoids, and terpenoids, the list goes on, and is extensive enough to keep growers waiting anxiously for their results.
And if it’s a fail, the batch is discarded and the whole process starts again.
But things are changing. Tired of being on the backfoot of regulations, cannabis growers are taking testing into their own hands.
New, affordable technologies, like Medicinal Genomics’ youPCRTM, can screen cannabis plants for cannabinoids, pathogens, and pests, right on the farm itself. For the first time, growers are a step ahead.

Isolation of microsatellite markers in Cannabis sativa L. (marijuana).
Gilmore, S., & Peakall, R.
Molecular Ecology Notes, 3(1), 105–107.(2003).
doi:10.1046/j.1471-8286.2003.00367.x*
We have identified 15 variable microsatellite loci in Cannabis sativa. In 48 samples from five fibre crop seed accessions, we detected an average of 10 alleles per locus (range 2–28) with mean heterozygosity of 0.68 (range 0.28–0.94). Significant genetic differentiation was found between accessions (FST = 0.12, P < 0.001). These markers have utility for characterizing genetic diversity in cultivated and naturalized Cannabis populations.

Karyological Studies in Root-Tip Cells of Cannabis sativa var. indica
P. Srivastava 1, S. Srivastava 2, M. K. Verma3 and S. K. Mishra
January 1999 Cytologia 64:435-440
DOI: 10.1508/cytologia.64.435
Chromosomal study was carried in the dividing root-tip cells of Cannabis sativa (Family: Cannabinaceae). The diploid chromosome number of this species is 2n=20. Karyotype analysis reveals that all the 9 chromosomes in female somatic cells are metacentric and 1 chromosome is submetacentric whereas the X and Y chromosome in male cells is sub-metacentric. The Y chromosome is longer than X chromosome. The third pair bears the satellite.

Not Cannabis Specific
Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants. Hofberger, J. A., Ramirez, A. M., Bergh, E. van den, Zhu, X., Bouwmeester, H. J., Schuurink, R. C., & Schranz, M. E. PLOS ONE, 10(6), e0128808.(2015).
doi:10.1371/journal.pone.0128808
An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineagespecific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant specie

Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa
Guangpeng Ren, Xu Zhang, Ying Li, Kate Ridout, Martha L. Serrano-Serrano, Yongzhi Yang, Ai Liu, Gudasalamani Ravikanth, Muhammad Ali Nawaz, Abdul Samad Mumtaz, Nicolas Salamin8 and Luca Fumagalli
Science Advances 16 Jul 2021:Vol. 7, no. 29, eabg2286
DOI: 10.1126/sciadv.abg2286
https://advances.sciencemag.org/cont...g2286.full.pdf
Cannabis sativa has long been an important source of fiber extracted from hemp and both medicinal and recreational drugs based on cannabinoid compounds. Here, we investigated its poorly known domestication history using whole-genome resequencing of 110 accessions from worldwide origins. We show that C. sativa was first domesticated in early Neolithic times in East Asia and that all current hemp and drug cultivars diverged from an ancestral gene pool currently represented by feral plants and landraces in China. We identified candidate genes associated with traits differentiating hemp and drug cultivars, including branching pattern and cellulose/lignin biosynthesis. We also found evidence for loss of function of genes involved in the synthesis of the two major biochemically competing cannabinoids during selection for increased fiber production or psychoactive properties. Our results provide a unique global view of the domestication of C. sativa and offer valuable genomic resources for ongoing functional and molecular breeding research.

Latitudinal Adaptation and Genetic Insights Into the Origins of Cannabis sativa L.
Qingying Zhang, Xuan Chen, Hongyan Guo, Luisa M. Trindade, Elma M. J. Salentijn, Rong Guo, Mengbi Guo, Yanping Xu and Ming Yang
Frontiers In Plant Science December 2018 | Volume 9 | Article 1876
doi: 10.3389/fpls.2018.01876
https://www.frontiersin.org/articles...018.01876/full
Cannabis is one of the most important industrial crops distributed worldwide. However, the phylogeographic structure and domestication knowledge of this crop remains poorly understood. In this study, sequence variations of five chloroplast DNA (cpDNA) regions were investigated to address these questions. For the 645 individuals from 52 Cannabis accessions sampled (25 wild populations and 27 domesticated populations or cultivars), three haplogroups (Haplogroup H, M, L) were identified and these lineages exhibited distinct high-middle-low latitudinal gradients distribution pattern. This pattern can most likely be explained as a consequence of climatic heterogeneity and geographical isolation. Therefore, we examined the correlations between genetic distances and geographical distances, and tested whether the climatic factors are correlated with the cpDNA haplogroup frequencies of populations. The “isolation-by-distance” models were detected for the phylogeographic structure, and the day-length was found to be the most important factor (among 20 BioClim factors) that influenced the population structures. Considering the distinctive phylogeographic structures and no reproductive isolation among members of these lineages, we recommend that Cannabis be recognized as a monotypic genus typified by Cannabis sativa L., containing three subspecies: subsp. sativa, subsp. Indica, and subsp. ruderalis. Within each haplogroup which possesses a relatively independent distribution region, the wild and domesticated populations shared the most common haplotypes, indicating that there are multiregional origins for the domesticated crop. Contrast to the prevalent Central-Asia-Origin hypothesis of C. saltiva, molecular evidence reveals for the first time that the low latitude haplogroup (Haplogroup L) is the earliest divergent lineage, implying that Cannabis is probably originated in low latitude region

License heritable gene editing like medical cannabis.
Julian H.
Nature. 2019 Jun;570(7762):446.
DOI:10.1038/d41586-019-01971-4
https://www.nature.com/articles/d41586-019-01971-4
In my view, a moratorium on the technology would be regulatory theatre (see E. Lander et al. Nature 567, 165–168; 2019). CRISPR gene editing is cheap and easily accessible, and its practice and products are hard to detect. Like cannabis prohibition, a moratorium risks fostering a black market in unregulated and potentially harmful ‘products’, in this case heritable gene variants.

Long-read nanopore cDNA sequencing and direct DNA methylation detection resolves copy number debate in Cannabis
Todd Michael
Video of talk LC 2020 London Calling
https://londoncallingconf.co.uk/reso...ion-resolves-0
DNA methylation plays an essential role in governing the architecture of gene expression across eukaryotes. In plants, DNA methylation not only protects the genome from transposable element (TE) jumping by repressing their expression, it also mediates environmental-specific expression responses.
Here Todd presents how he used direct DNA methylation detection coupled to full-length cDNA sequencing across several Cannabis chemotypes (high and low THC/CBD) to understand the final step in the cannabinoid pathway. The final step of the cannabinoid pathway is the Tetrahydrocannabinol acid synthase (THCAS) and/or Cannabidiol acid synthase (CBDAS), which are intron-less genes that are nested in 50-80 kb TE units tandemly duplicated at several loci in the genome.
Due to the complexity of the THCAS/CBDAS loci and the fact that they are highly similar, full-length cDNA sequencing was required to resolve which synthases were expressed in different chemotype genomes.
Furthermore, short-read based bisulfite sequencing fails to distinguish the DNA methylation patterns at these loci. However, direct DNA methylation enables the characterization of DNA patterns across these complex loci, providing evidence that DNA methylation in part explains the observed expression patterns.

Loss of pollen?specific phospholipase NOT LIKE DAD triggers gynogenesis in maize LaurineM Gilles, Abdelsabour Khaled, Jean?Baptiste Laffaire, Sandrine Chaignon,Ghislaine Gendrot, Jérôme Laplaige, Hélène Bergès, Genséric Beydon, Vincent Bayle,Pierre Barret, Jordi Comadran, Jean?Pierre Martinant, PeterM Rogowsky, Thomas Widiez
Published online 22.02.2017 The EMBO Journal (2017)
DOI 10.15252/embj.201796603
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow

MAKING CANNABIS HISTORY IN 2020
Tiffanie Yael Maoz, PhD
https://www.nrgene.com/BLOG/MAKING-C...STORY-IN-2020/
2020 IS expected to be an exceptional year for the expansion of work done in Cannabis genomics. At the annual PAG meeting held this January in San Diego, academic experts and industry leaders met to share insights on the work they’ve done with this plant. While there were several scientific presentations made during the conference, the highlight of the event came the last day of the conference when an international group of industry and academic volunteers came together to form the International Cannabis Research Consortium (ICRC).
The ICRC is tasked with creating a unified approach to curating and documenting work done in Cannabis Genomics. As Cannabis sativa includes both hemp and marijuana types, leaders such as Larry Smart from Cornell and Graham King from Southern Cross University as well as industry representatives from the US, Canada, Europe and Israel were all able to contribute to the discussion.
The first order of business was to agree on a ‘reference genome’ and chromosome id’s for Cannabis. This was a bit of a challenge as there are several reference genome assemblies already available in the public space to be considered for use as the de facto reference. Consensus from the committee was that currently, the CBDRX CS10 assembly was the best quality assembly with a supporting transcript dataset and it was proposed that this should be declared the Cannabis reference for future work. However, even with choosing a reference, there remains the challenge of reconciling datasets across all the genomes already out in the public domain. This is because the chromosome assignments for each assembly can vary widely.
As NRGene has recently generated it’s own Cannabis Genomic Database (CannaGene), we’ve already done the analysis on the chromosomal mapping across 4 of the reference genomes in the public space (see the table below). We’re providing this to the ICRC as well as information on the chromosome lengths and N-content for the 4 genomes we’ve looked at. This will be used by the consortium to check chromosome order and length.

Manipulation of Cannabinoid Biosynthesis via Transient RNAi Expression
Lennon Matchett-Oates, German C. Spangenberg and Noel O. I. Cogan Front. Plant Sci., 10 December 2021 |
DOI: 10.3389/fpls.2021.773474 https://www.frontiersin.org/articles...21.773474/fullxnpe_tifc=xDbl4kPshIs_h.hLh.bX h9pZhfEWVjQsVuU_O.VjOIolhkUstI YDhIbA4FP_hNl8bdhNtIHpbDPNxfb7 bDHdb9XJOI_D4fnD4IsN4IoXxFxD&u tm_source=eNews&utm_campaign=T CS%20Newsletter%20week%2003%20 2022&utm_medium=email
Cannabis sativa L. produces unique phytocannabinoids, which are used for their pharmaceutical benefits. To date, there are no reports of in vivo engineering targeting the cannabinoid biosynthesis genes to greater elucidate the role each of these genes play in synthesis of these medically important compounds. Reported here is the first modulation of cannabinoid biosynthesis genes using RNAi via agroinfiltration. Vacuum infiltrated leaf segments of the Cannbio-2 C. sativa strain, transfected with different RNAi constructs corresponding to THCAS, CBDAS, and CBCAS gene sequences, showed significant downregulation of all cannabinoid biosynthesis genes using real-time quantitative PCR. Using RNAi, significant off-targeting occurs resulting in the downregulation of highly homologous transcripts. Significant (p < 0.05) downregulation was observed for THCAS (92%), CBDAS (97%), and CBCAS (70%) using pRNAi-GG-CBDAS-UNIVERSAL. Significant (p < 0.05) upregulation of CBCAS (76%) and non-significant upregulation of THCAS (13%) were observed when transfected with pRNAi-GG-CBCAS, suggesting the related gene’s ability to synthesize multiple cannabinoids. Using this approach, increased understanding of the relationship between cannabinoid biosynthesis genes can be further elucidated. This RNAi approach enables functional genomics screens for further reverse genetic studies as well as the development of designer cannabis strains with over-expression and/or downregulation of targeted cannabinoid biosynthesis genes. Functional genomics screens, such as these, will further provide insights into gene regulation of cannabinoid biosynthesis in Cannabis.

Medicinal Genomics announces Cannabis Pan-Genome project, a major advance in genetics for cannabis breeding
https://www.prnewswire.com/news-rele...300834647.html
Pacific Biosciences Sequel II System genetic analysis platform to catalog structural variants—the genetic hotspots—governing cannabis chemotype
Medicinal Genomics Corp. (MGC), a pioneer in using genomics to improve the quality, quantity and safety of cannabis, announced the initiation of a cannabis pan-genome project, a comparative genetic analysis of several different cannabis cultivars to identify genomic structural variations.
Building upon a recently updated reference genome of the Jamaican Lion cultivar, MGC has identified the genetic variations that cause a plant to produce the important cannabinoids of THC, CBD, or a mixture of the two, referred to as chemotypes (I-IV). This information provides a "cookbook" of genetic recipes for different types and cultivars, and is a key to breeding for cannabis yield, potency and a host of other traits.
"The pan-genome focuses on whole genome sequencing and de novo assembly using the Sequel II System by Pacific Biosciences (PacBio) to catalog structural variation inheritance patterns in closely and distantly related cultivars. This family of genomes will form the foundation for cannabis breeding programs across the industry," said Medicinal Genomics Chief Scientific Officer, Kevin McKernan. "The economic value of the genetics that govern cannabinoid expression, seed development, and fiber yield is one of the most promising opportunities of the decade. And now, every breeder and grower can use this tool, retaining complete ownership of the results for their own cultivars."

Medicinal Genomics Releases Industry's First Comprehensive Cannabis Reference Genome
Genetic milestone in mapping the cannabis genome to be made public to accelerate research and commercialization
https://www.prnewswire.com/news-rele...300781669.html
Medicinal Genomics Corporation (MGC), a pioneer in using genomics to improve the transparency, safety and yield of cannabis has today announced the industry's most comprehensive Cannabis reference genome, a high quality de novo genome assembly of the Jamaican Lion cannabis strain. As the first reference genome to break "usability barrier," the significance of this development cannot be overstated. Having a comprehensive cannabis genome opens the door to a host of industry innovations, including enabling cannabis research in states and countries where cannabis is still illegal, marker-assisted selection (MAS) for genetically-based strain identification, accelerated breeding to improve production yields, reliable seed-to-sale tracking systems, and pathogen identification to ensure cannabis purity and safety.
"For the first time, growers and breeders will have a complete and reliable genome from which to create new strains for increased yields and productivity, while documenting them to protect their work. Cultivators will also be able to embark on a brand new era of intelligent breeding destined to change the way cannabis is grown, sold and consumed," said Kevin McKernan, chief science officer for Medicinal Genomics. "And this is just the beginning of enhancing cannabis yield with genomics. This plant that has the potential to be more valuable than wheat and corn combined, but with far more varied and diverse uses, including everything from CO2 sequestration to pain relief and medical therapies to complete vegetable proteins, clothing and industrial materials."

Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges
Michihito Deguchi, Shriya Kane, Shobha Potlakayala, Hannah George, Renata Proano, Vijay Sheri, Wayne R. Curtis and Sairam Rudrabhatla
Front. Plant Sci. 11:580621. (2020)
doi: 10.3389/fpls.2020.580621
Industrial hemp (Cannabis sativa L.) is a diploid (2n = 20), dioecious plant that is grown for fiber, seed, and oil. Recently, there has been a renewed interest in this crop because of its panoply of cannabinoids, terpenes, and other phenolic compounds. Specifically, hemp contains terpenophenolic compounds such as cannabidiol (CBD) and cannabigerol (CBG), which act on cannabinoid receptors and positively regulate various human metabolic, immunological, and physiological functions. CBD and CBG have an effect on the cytokine metabolism, which has led to the examination of cannabinoids on the treatment of viral diseases, including COVID-19. Based on genomic, transcriptomic, and metabolomic studies, several synthetic pathways of hemp secondary metabolite production have been elucidated. Nevertheless, there are few reports on hemp metabolic engineering despite obvious impact on scientific and industrial sectors. In this article, recent status and current perspectives on hemp metabolic engineering are reviewed. Three distinct approaches to expedite phytochemical yield are discussed. Special emphasis has been placed on transgenic and transient gene delivery systems, which are critical for successful metabolic engineering of hemp. The advent of new tools in synthetic biology, particularly the CRISPR/Cas systems, enables environment-friendly metabolic engineering to increase the production of desirable hemp phytochemicals while eliminating the psychoactive compounds, such as tetrahydrocannabinol (THC).

METHOD FOR DIFFERENTIATING CANNABIS PLANT CULTIVARS BASED ON CANNABINOID SYNTHASE PARALOGS.

US 2020/0270623 A1
University of Colorado & Steep Hill inc.
Download PDF here: https://patents.google.com/patent/WO2019070876A2/en

Missense mutations in THCAS are associated with Cannabigerol expression in Cannabis sativa L.
Kevin McKernan, Lei Zhang, Yvonne Helbert, Liam T. Kane, Stephen McLaughlin
https://zenodo.org/record/5903712#.YfUFYaDMKqC
The chemical expression of THCA and CBDA has been previously attributed to copy number gains and losses in THCAS and CBDAS. Here we describe an alternative mechanism to ablate THCAS activity in 5 CBG dominant cannabis varietals. Five cannabis cultivars from three unique cultivators surprisingly contain a THCAS gene but also share homozygous Pro333Arg and Ser355Asn mutations in THCAS. Pro333Arg is commonly found in THCAS dominant varietals but is uniquely found in a homozygous state in CBG varietals in conjunction with homozygous Ser355Asn missense mutations. These results hint at convergent evolution in cannabinoid synthesis where selection for and against THCA synthesis has been applied.

Mitochondrial genomes do not appear to regulate flowering pattern / reproductive strategy in Cannabis sativa

Ziv Attia, Cloe Pogoda, Daniela Vergara, Nolan C. Kane
https://academic.oup.com/aobpla/advance/doi/10.1093/aobpla/plab068/6414685
Currently, the amount of genetic data for Cannabis is lacking due to the illegal nature of the plant. Our study used 73 Cannabis sativa whole genome shotgun libraries to reveal eight different mtDNA haplotypes. The most common haplotype contained 60 of the 73 samples studied and was composed of only dioecious individuals. However, other haplotypes contained a mix of both mating strategies (i.e., monecious and dioecious). From these haplotype groupings we further examined the fully annotated mitochondrial genomes of four hemp individuals with different mt haplotypes and recorded gene content, copy number variation, and synteny. Our results revealed highly syntenic mitochondrial genomes that contained approximately 60 identifiable sequences for protein coding genes, tRNAs and rRNAs and no obvious rearrangements or chimeric genes. We found no clear evidence that modern reproductive patterns are due to simple CMS mutations. It is likely the interaction between nuclear genetic components and the X/Y sex chromosomes that determines reproductive strategy. Additionally, we added 50% more mitochondrial genomes to the publicly available repository.
Check link or DOI

Not Cannabis Specific
Molecular approaches for the fixation of plant hybrid vigor.
Siddiqi, I., Marimuthu, M. P. A., & Ravi, M.
Biotechnology Journal, 4(3), 342–347.(2009).
doi:10.1002/biot.200900018
Research in the plant sciences is inextricably linked to our dependence on plants as a source of food. With the growing population comes the challenge of increasing food supply and hence the yield of food crops. This issue is of particular relevance for developing countries. The green revolution beginning in the late 1960s, enabled countries such as India to achieve self-sufficiency in food production; however, the increases in yield have plateaued and the world is faced with having to meet the increasing demand for food with far greater constraints in the availability of the two main resources: land and water.The use of hybrids in agriculture is well known to lead to large yield increases of upto 20% for important crops such as maize and rice, a phenomenon known as hybrid vigor. However, hybrid vigor is not stable and declines in each subsequent generation due to segregation of alleles at the time of sexual reproduction. The instability of hybrids requires that seeds of any hybrid have to be synthesized anew at each generation by crossing of the two parental lines. From the perspective of agriculture, the interest in apomixis derives from the possibility of fixing hybrid vigor by the formation of asexual seeds through apomixis, bypassing the normal processes of meiosis and fertilization. The application of apomixis to plant breeding and seed production would have a major impact on crop production by allowing the fixation and propagation of any elite genotype however complex it may be. Apomixis does not occur naturally in the major food crops and would have to be introduced into food crops using biotechnological approaches

Molecular, biochemical and genetic aspects of cannabinoids
Eric Murillo-Rodriguez
June 2015
DOI: 10.13140/RG.2.1.1863.9523
Cannabinoids are a group of terpenophenolic compounds present in Cannabis (Cannabis sativa L). The broader definition of cannabinoids refers to a group of substances that are structurally related to delta-9-tetrahydrocannabinol (?9-THC) or that bind to cannabinoid receptors. Before the 1980’s, it was often speculated that cannabinoids produced their physiological and behavioral effects via nonspecific interaction with cell membranes, instead of interacting with specific membrane-bound receptors. The discovery of the first cannabinoid receptors in the 1980s helped to resolve this debate. These receptors are common in animals, and have been found in mammals, birds, fish, and reptiles. There are currently two known types of cannabinoid receptors, termed CB1 and CB2. The cannabinoid system has been around for over 600 million years…even before the dinosaurs!! The cannabinoid system is present in species such as hydra, mollusks, and insects, leading to speculation on the physiological importance of such a system preserved throughout evolution. To date, the presence in the central nervous system of specific lipids that bind naturally to the CB1/CB2 cannabinoid receptors has been documented. Pharmacological experiments have shown that injection of those compounds induces cannabimimetic effects. The family of endogenous cannabinoids or endocannabinoids comprises Arachidonoylethanolamine, 2-araquidonylglycerol, Virodhamine, noladinether and N-arachidonyldopamine. The endocannabinoids have an active role in modulating diverse neurobiological functions, such as learning and memory, feeding, pain perception and sleep generation. The system of endogenous cannabinoids is present in several species, including humans, leading to speculation regarding the neurobiological role of the endocannabinoid system in diverse functions. Hence, I thought it was time to bring out an editorial book on the subject containing advanced and up-to-date scientific information on this special and exclusive topic. I expect that such a book is likely to attain global circulation among students, teachers and researchers alike. Fortunately, in response to our appeal, a number of leading scientists in the field across the globe agreed to contribute to the book. Thus, this book deals with various aspects of the cannabinoid and endocannabinoid system, from phenomena to molecular processes. I am sincerely grateful to all the contributors for keeping. The purpose of the book is to provide a comprehensive understanding of the endocannabinoid system with adequate emphasis on the pharmacological and molecular foundation and is directed at all who are interested in cannabinoid research.

Molecular characterization of edestin gene family in Cannabis sativa L.
Teresa Docimo, Immacolata Caruso, Elena Ponzoni, Monica Mattana,
Incoronata Galasso
Plant Physiology and Biochemistry 84 (2014) 142e148
DOI: 10.1016/j.plaphy.2014.09.011
Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this
work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98e99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11e12%), but CsEde2 resulted particularly rich in methionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition
determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs.

Molecular characterization, vector identification and sources of phytoplasmas associated with brinjal little leaf disease in India
Manish Kumar, Madhupriya, Govind Pratap Rao,
3 Biotech (2017) 7:7
DOI 10.1007/s13205-017-0616-x

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF 1-TETRAHYDROCANNABINOLIC ACIDSYNTHASE FROM CANNABIS SATIVA L.
Vol. 279, No. 38, Issue of September 17, pp. 39767–39774, 2004
DOI: 10.1074/jbc.M403693200
Supaart Sirikantaramas, Satoshi Morimoto, Yoshinari Shoyama, Yu Ishikawa, Yoshiko Wada, Yukihiro Shoyama, and Futoshi Taura
1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of _1-tetrahydrocannabinol. We cloned a novel cDNA (GenBankTM accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

Molecular cytogenetic analysis of monoecious hemp (Cannabis sativa L.) cultivars reveals its karyotype variations and sex chromosomes constitution
Olga V. Razumova & Oleg S. Alexandrov & Mikhail G. Divashuk & Tatiana I. Sukhorada & Gennady I. Karlov
Protoplasma, 2015
DOI 10.1007/s00709-015-0851-0
Hemp (Cannabis sativa L., 2n?=?20) is a dioecious plant. Sex expression is controlled by an X-to-autosome balance system consisting of the heteromorphic sex chromosomes XY for males and XX for females. Genetically monoecious hemp offers several agronomic advantages compared to the dioecious cultivars that are widely used in hemp cultivation. The male or female origin of monoecious maternal plants is unknown. Additionally, the sex chromosome composition of monoecious hemp forms remains unknown. In this study, we examine the sex chromosome makeup in monoecious hemp using a cytogenetic approach. Eight monoecious and two dioecious cultivars were used. The DNA of 210 monoecious plants was used for PCR analysis with the male-associated markers MADC2 and SCAR323. All monoecious plants showed female amplification patterns. Fluorescence in situ hybridization (FISH) with the subtelomeric CS-1 probe to chromosomes plates and karyotyping revealed a lack of Y chromosome and presence of XX sex chromosomes in monoecious cultivars with the chromosome number 2n?=?20. There was a high level of intra- and intercultivar karyotype variation detected. The results of this study can be used for further analysis of the genetic basis of sex expression in plants.

Molecular Cytogenetic Characterization of the Dioecious Cannabis sativa with an XY Chromosome Sex Determination System
Mikhail G. Divashuk, Oleg S. Alexandrov, Olga V. Razumova, Ilya V. Kirov, Gennady I. Karlov
Plos ONE
doi: 10.1371/journal.pone.0085118
Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

Molecular docking analysis of phyto-constituents from Cannabis sativa with pfDHFR
Temitope . David, Niyi S. Adelakun, Olaposi . Omotuyi, Damilohun S. Metibemu,Oluwafemi E. Ekun Gabriel O. eniafe, Olumide K. Inyang, Bamidele Adewumi, Ojochenemi A. Enejoh, Raymond T. Owolabi, Eunice I. Oribamise
Bioinformation 14(9): 574-579 (2018)
doi: 10.6026/97320630014574.
Available antimalarial drugs have been associated with numerous side effects, which include skin rashes and myelo-suppression. Therefore, it is of interest to explore compounds from natural source having drug-like properties without side effect. This study focuses on the screening of compounds from Cannabis sativa against malaria Plasmodium falciparum dihydrofolate reductase for antimalarial properties using Glide (Schrodinger maestro 2018-1). The result showed that phytochemicals from Cannabis sativa binds with a higher affinity and lower free energy than the standard ligand with isovitexin and vitexin having a glide score of -11.485 and -10.601 respectively, sophoroside has a glide score of -9.711 which is lower than the cycloguanil (co-crystallized ligand) having a glide score of -6.908. This result gives new perception to the use of Cannabis sativa as antimicrobial agent.

Not Cannabis Specific
Molecular Marker Based Genetic Diversity Analysis in Rice Genotypes (Oryza sativa L.) using SSR Markers
Krupa, K. N.* , Shashidhar, H. E., Ningaraj Dalawai, Mahendra Reddy and Vijaykumara Swamy, H. V
Int. J. Pure App. Biosci. 5 (2): 668-674 (2017)
DOI: 10.18782/2320-7051.2892
Assessment of genetic diversity is important in plant breeding if there is to be improvement by selection. The role of a broad genetic base and systematically characterized germplasm in the crop improvement of cultivated plants has been well recognized. Genetic variability studies are important in selection of parents for hybridization as sound crop improvement depends upon the
magnitude of variability in the base population. In the present investigation five rice genotypes were evaluated for genetic diversity. Upon PCR amplification the alleles were separated on Agarose Gel Electrophoresis system. Initial polymorphism detection was conducted using twenty primer pairs distributed on five rice chromosomes. A total of 65 alleles were detected with an average of 3.25 alleles per locus. The polymorphism information content (PIC) reflections of alleles diversity frequency among the varieties, which is ranged from 0.215 to 0.791, with an average of 0.493. RM 260 was found as the best marker for identification of genotypes as revealed by PIC values. The highly informative markers identified in this study could be utilized in further studies for comparative mapping and marker assisted selection for drought tolerance.

Molecular phylogenetics and character evolution of Cannabaceae
Mei-Qing Yang, Robin van Velzen, Freek T. Bakker, Ali Sattarian, De-Zhu Li, Ting-Shuang Yi1
TAXON 62 (3) • June 2013: 473–485
Cannabaceae includes ten genera that are widely distributed in tropical to temperate regions of the world. Because of limited taxon and character sampling in previous studies, intergeneric phylogenetic relationships within this family have been poorly resolved. We conducted a molecular phylogenetic study based on four plastid loci (atpB-rbcL, rbcL, rps16, trnL-trnF) from 36 ingroup taxa, representing all ten recognized Cannabaceae genera, and six related taxa as outgroups. The molecular results strongly supported this expanded family to be a monophyletic group. All genera were monophyletic except for Trema, which was paraphyletic with respect to Parasponia. The Aphananthe clade was sister to all other Cannabaceae, and the other genera formed a strongly supported clade further resolved into a Lozanella clade, a Gironniera clade, and a trichotomy formed by the remaining genera. Morphological ancestral state reconstructions indicated the complex evolution pattern of most analyzed morphological characters, and it is difficult to identify morphological synapomorphies for most clades within Cannabaceae.

Not Cannabis specific
Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement
Stephen P. Moose, Rita H. Mumm
Plant Physiology, July 2008, Vol. 147, pp. 969–977
doi/10.1104/pp.108.118232
The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices has created the foundation for molecular plant breeding, an interdisciplinary science that is revolutionizing 21st century crop improvement. Though the methods of molecular plant breeding continue to evolve and are a topic of intense interest among plant breeders and crop scientists (for review, see Cooper et al., 2004; Nelson et al., 2004; Lo¨rz and Wenzel, 2005; Varshney et al., 2006; Eathington et al., 2007; Mumm, 2007), they have received relatively little attention from the majority of plant biologists engaged in basic scientific research. The objective of this article for an Editor’s Choice series on future advances in crop biotechnology is to briefly review important historical developments in molecular plant breeding, key principles influencing the current practice of molecular plant breeding, and factors that influence the adoption of molecular plant breeding in crop improvement programs. Furthermore, we emphasize how the application of molecular plant breeding is now contributing to discoveries of genes and their functions that open new avenues for basic plant biology research

Multiplex qPCR and Cannabis Microbiome sequencing reveals several Bacteria and Fungi Native to Cannabis flowers
Kevin McKernan, Jessica Spangler, Lei Zhang, Vasisht Tadigotla, Yvonne Helbert, Douglas Smith
Color Poster:
https://system.na3.netsuite.com/core...7c1f3&_xt=.pdf
PDF:
https://www.medicinalgenomics.com/wp...owers_sbmt.pdf

Novel D9-tetrahydrocannabinol formulation Namisol® has beneficial pharmacokinetics and promising pharmacodynamic effects
Linda E. Klumpers, Tim L. Beumer, Johan G. C. van Hasselt, Astrid Lipplaa, Lennard B. Karger, H. Daniël Kleinloog, Jan I. Freijer, Marieke L. de Kam & Joop M. A. van Gerven
Br J Clin Pharmacol / 74:1 / 42–53
DOI:10.1111/j.1365-2125.2012.04164.x
AIMS Among the main disadvantages of currently available D9-tetrahydrocannabinol (THC) formulations are dosing difficulties due to poor pharmacokinetic characteristics. Namisol® is a novel THC formulation, designed to improve THC absorption. The study objectives were to investigate the optimal administration route, pharmacokinetics (PK), pharmacodynamics (PD) and tolerability of Namisol®.
METHODS This first in human study consisted of two parts. Panel I included healthy males and females (n = 6/6) in a double-blind, double-dummy, randomized, crossover study with sublingual (crushed tablet) and oral administration of Namisol® (5 mg THC). Based on these results, male and female (n = 4/5) participants from panel I received oral THC 6.5 and 8.0 mg or matching placebo in a randomized, crossover, rising dose study during panel II. PD measurements were body sway; visual analogue scales (VAS)
mood, psychedelic and heart rate.THC and 11-OH-THC population PK analysis was performed.
RESULTS Sublingual administration showed a flat concentration profile compared with oral administration. Oral THC apparent t1/2 was 72–80 min, tmax was 39–56 min and Cmax 2.92–4.69 ng ml-1.THC affected body sway (60.8%, 95% CI 29.5, 99.8), external perception (0.078 log mm, 95% CI 0.019, 0.137), alertness (-2.7 mm, 95% CI -4.5, -0.9) feeling high (0.256 log mm, 95% CI 0.093, 0.418) and heart rate (5.6 beats min–1, 95% CI 2.7, 6.5). Namisol® was well tolerated.
CONCLUSIONS Oral Namisol® showed promising PK and PD characteristics. Variability and tmax of THC plasma concentrations were smaller for Namisol® than reported for studies using oral dronabinol and nabilone. This study was performed in a limited number of healthy
volunteers. Therefore, future research on Namisol® should study clinical effects in patient populations.

Novel male-specific molecular markers (MADC5, MADC6) in hemp
Otto Törjek, Nandor Bucherna, Erzsebet Kiss, Hajnalka Homoki, Zsuzsanna Finta-Korpelov, Ivan Bocsa, Istvan Nagy & Laszlo E. Heszky,
Euphytica 127(2): 209–218, 2002.
DOI: 10.1023/A:1020204729122
Decamer RAPD primers were tested on dioecious and monoecious hemp cultivars to identify sex-specific molecular markers. Two primers (OPD05 and UBC354) generated specific bands in male plants. These two DNA fragments were isolated, cloned and sequenced. Both markers proved to be unique, since no sequence with significant homology to OPD05961 and UBC354151 markers were found in databases. These markers were named MADC3 (OPD05961) and MADC4 (UBC354151) (Male-Associated DNA from Cannabis sativa). The markers were converted into sequence-characterized amplified region (SCAR) markers. The SCAR markers correlated with the sex of the segregating F2 population and proved the tight linkage to the male phenotype. Results of F2 plant
population analysis suggest these markers are to be linked to the Y chromosome.

Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics.
Vincent, D., Rochfort, S., & Spangenberg, G.
Molecules, 24(4), 659. (2019).
doi:10.3390/molecules24040659
Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway

Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa
November 2007 Forensic science international 172(2-3):179-90
DOI: 10.1016/j.forsciint.2006.10.025
Simon Gilmore, Rod Peakall, James Robertson,
Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.

Phased Diploid Genome Assembly with Single Molecule Real-Time Sequencing
Chen-Shan Chin, Paul Peluso1, Fritz J. Sedlazeck, Maria Nattestad, Gregory T. Concepcion, Alicia Clum, Christopher Dunn, Ronan O’Malley, Rosa Figueroa-Balderas, Abraham Morales-Cruz, Grant R. Cramer, Massimo Delledonne, Chongyuan Luo, Joseph R. Ecker,
Dario Cantu, David R. Rank, Michael C. Schatz.
DOI: 10.1038/nmeth.4035
While genome assembly projects have been successful in a number of haploid or inbred species, one of the current main challenges is assembling non-inbred or rearranged heterozygous genomes. To address this critical need, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble Single Molecule Real-Time (SMRT®) Sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We demonstrate the quality of this approach by assembling new reference sequences for three heterozygous samples, including an F1 hybrid of the model species Arabidopsis thaliana, the widely cultivated V. vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata that have challenged short-read assembly approaches. The FALCON-based assemblies were substantially more contiguous and complete than alternate short or long-read approaches. The phased diploid assembly enabled the study of haplotype structures and heterozygosities between thehomologous chromosomes, including identifying widespread heterozygous structural variations within the coding sequences.

Phytochemical and genetic analyses of ancient cannabis from Central Asia.
Russo EB, Jiang H-E, Li X, Sutton A, Carboni A, del Bianco F, Mandolino G, Potter DJ, Zhao Y-X, Bera S, et al (2008)
J Exp Bot 59: 4171–4182
DOI: 10.1093/jxb/ern260
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2639026/
The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.

Plant genera Cannabis and Humulus share the same pair of well‐differentiated sex chromosomes
Djivan Prentout, Theo Tricou, Celine Brochier-Armanet, Jernej Jakse, Jos Kafer, Gabriel Marais, Andreja Cerenak, Natasa Stajner
New Phytol. 2021 May 12.
DOI: 10.1101/2021.03.11.434957
https://www.researchgate.net/publica...ex_chromosomes
We recently described, in Cannabis sativa , the oldest sex chromosome system documented so far in plants. Based on our estimate of its age, we predicted that it should be shared by its sister genus Humulus , which is known to also possess XY sex chromosomes. Here, we used transcriptome sequencing of a F1 family of Humulus lupulus to identify and study the sex chromosomes in this species using the probabilistic method SEX-DETector. We identified 265 sex-linked genes in H. lupulus , located on the chromosome that is also the C. sativa sex chromosome pair. Using phylogenies of sex-linked genes, we show that a region of these chromosomes had already stopped recombining in the common ancestor of the two species. Furthermore, as in C. sativa , Y gene expression was reduced in correlation to the position on the X chromosome, and strongly Y degenerated genes showed dosage compensation. Here we report, for the first time in the Angiosperms, a sex chromosome system that is shared by two different genera. Recombination suppression started at least 21-25 My ago, and then (either gradually or step-wise) spread to a large part of the sex chromosomes, leading to a strongly degenerated Y chromosome.

Not Cannabis specific
Plant pan-genomes are the new reference
Philipp E. Bayer?, Agnieszka A. Golicz, Armin Scheben?, Jacqueline Batley? and David Edwards
Nature Plants 6(8):1-7 July 2020
DOI: 10.1038/s41477-020-0733-0
Recent years have seen a surge in plant genome sequencing projects and the comparison of multiple related individuals. The high degree of genomic variation observed led to the realization that single reference genomes do not represent the diversity within a species, and led to the expansion of the pan-genome concept. Pan-genomes represent the genomic diversity of a species and includes core genes, found in all individuals, as well as variable genes, which are absent in some individuals. Variable gene annotations often show similarities across plant species, with genes for biotic and abiotic stress commonly enriched within variable gene groups. Here we review the growth of pan-genomics in plants, explore the origins of gene presence and absence variation, and show how pan-genomes can support plant breeding and evolution studies.

Potential of marker-assisted selection in hemp genetic improvement
G. Mandolino & A. Carboni
Euphytica 140: 107–120, 2004.
DOI: 10.1007/s10681-004-4759-6
The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars.
Barcaccia, G., Palumbo, F., Scariolo, F., Vannozzi, A., Borin, M., & Bona, S.
Frontiers in Plant Science, 11.(2020).
doi:10.3389/fpls.2020.573299
Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges—along with potential solutions—behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F1 hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs

Not Cannabis Specific
Powdery Mildew Resistance in Tomato by Impairment of SlPMR4 and SlDMR1
Robin P. Huibers, Annelies E. H. M. Loonen, Dongli Gao, Guido Van den Ackerveken, Richard G. F. Visser, Yuling Bai
PLoS ONE 8(6): e67467.
doi:10.1371/journal.pone.0067467
Genetic dissection of disease susceptibility in Arabidopsis to powdery and downy mildew has identified multiple susceptibility (S) genes whose impairment results in disease resistance. Although several of these S-genes have been cloned and characterized in more detail it is unknown to which degree their function in disease susceptibility is conserved among
different plant species. Moreover, it is unclear whether impairment of such genes has potential in disease resistance breeding due to possible fitness costs associated with impaired alleles. Here we show that the Arabidopsis PMR4 and DMR1, genes encoding a callose synthase and homoserine kinase respectively, have functional orthologs in tomato with respect to their S-gene function. Silencing of both genes using RNAi resulted in resistance to the tomato powdery mildew fungus Oidium neolycopersici. Resistance to O. neolycopersici by SlDMR1 silencing was associated with severely reduced plant growth whereas SlPMR4 silencing was not. SlPMR4 is therefore a suitable candidate gene as target for mutagenesis to obtain alleles that can be deployed in disease resistance breeding of tomato.

Production of Tetraploid and Triploid Hemp
Lauren E. Kurtz, Mark H. Brand, and Jessica D. Lubell-Brand
HortScience Sept 2020
https://journals.ashs.org/hortsci/vi...CI15303-20.xml
To maximize yield, cannabidiol (CBD) hemp producers prefer female plants, and this is accomplished by using expensive feminized seed, vegetatively propagated female clones, or by removing male plants from dioecious seed lots. Hemp pollen drifts long distances on wind, and pollination of females reduces CBD content. Induction of triploidy is a common strategy used by plant breeders to produce sterile cultivars of agricultural crops. Triploid (3n) hemp, with three sets of chromosomes, was developed by crossing naturally diploid (2n) hemp with tetraploid (4n) hemp. Tetraploid plants used to create triploids were produced using pregerminated seeds and the mitotic spindle inhibitor colchicine. Seedlings from seeds of ‘Abacas’ × [(‘Otto2’ × ‘BaOx’) × (‘BaOx’ × ‘Colorado Cherry’)] treated with 0.05% colchicine or 0.02% colchicine for 12 hours and longer were significantly shorter than controls and ?1 cm tall at 10 days after sowing. Surviving seedlings exhibited thickened cotyledons and hypocotyls, which indicated a potential change in ploidy. Tetraploid induction ranged from 26% to 64% for pregerminated seeds of five different hemp cultivars (Abacus × Wife, Cherry Wine, Mountain Mango, Wife, and Youngsim10) treated with 0.05% colchicine for 12 hours. Tetraploids had nearly twice the DNA content as diploids according to flow cytometric analysis. Tetraploid ‘Wife’ had larger stomates and reduced stomatal density compared with diploid ‘Wife’. Four triploid ‘Wife’ genotypes produced from crossing tetraploid ‘Wife’ with diploid ‘Wife’ were acclimated to greenhouse conditions after embryo rescue. DNA content and stomate size of triploid ‘Wife’ was intermediate between the parents. This is the first report of triploid plants of hemp. Future research will evaluate the sterility of triploid hemp.

Proficiency Testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories
Katherine A. Lau, Anders Gonçalves da Silva, Torsten Theis, Joanna Gray, Susan A Ballard, and William D. Rawlinson
Biorxiv (2020)
https://www.biorxiv.org/content/10.1...519v1.full.pdf
DOI: 10.1101/2020.09.18.304519
The adoption of whole genome sequencing (WGS) data over the past decade for pathogen surveillance, and decision-making for infectious diseases has rapidly transformed the landscape of clinical microbiology and public health. However, for successful transition to routine use of these techniques, it is crucial to ensure the WGS data generated meet defined quality standards for pathogen identification, typing, antimicrobial resistance detection and surveillance. Further, the ongoing development of these standards will ensure that the bioinformatic processes are capable of accurately identifying and characterising organisms of interest, and thereby facilitate the integration of WGS into routine clinical and public health laboratory setting. A pilot proficiency testing (PT) program for WGS of infectious agents was developed to facilitate widely applicable standardisation and benchmarking standards for WGS across a range of laboratories. The PT participating laboratories were required to generate WGS data from two bacterial isolates, and submit the raw data for independent bioinformatics analysis, as well as analyse the data with their own processes and answer relevant questions about the data. Overall, laboratories used a diverse range of bioinformatics tools and could generate and analyse high-quality data, either meeting or exceeding the minimum requirements. This pilot has provided valuable insight into the current state of genomics in clinical microbiology and public health laboratories across Australia. It will provide a baseline guide for the standardisation of WGS and enable the development of a PT program that allows an ongoing performance benchmark for accreditation of WGS-based test processes.

PROGRESS IN EARLY SEX DETERMINATION OF CANNABIS PLANT BY DNA MARKERS
Conference Paper · November 2016 MENDELNET 2016, ; 731-735
Peter Mendel, Ajinkya Lalge, Tomáš Vyhnánek, Ladislav Havel, Václav Trojan, Petr Kalousek, Hugo Maassen
https://mendelnet.cz/pdfs/mnt/2016/01/130.pdf
The cannabis plant is a tall annual crop of economic importance. It is mostly dioecious, but fiber hemp varieties have been bred to be monoecious. Separating male and female plants at early developmental stage is useful due to the influence of gender at agriculturally significant traits. Several experiments have been focused on developing a reliable molecular marker for sex determination in cannabis plants. Our study compares three DNA markers for the detection of male genotype in totally twelve samples of industrial hemp and medicinal cannabis plants. Genotype scoring of SCAR119 marker appeared to be the most reliable, followed by MADC2 and SCAR323, when compared to the observed phenotype of plants. The results confirmed the insights given in previous studies. Research and Polymerase Chain Reaction (PCR) analysis should continue in order to find more advanced DNA markers for sex determination of cannabis plants. INTRODUCTION Cannabis (Cannabis sativa L.) is a tall upright annual herb. It is generally dioecious i.e. producing separate male and female plants but fiber hemp varieties have been specifically bred to be monoecious (hermaphrodite) (Debruyne et al. 1994, Srivastava and Yadav 2013). The sex of most dioecious plants can only (reliably) be determined at the time of flowering. The significance of separating male and female plants at seedling stage lies in the fact that in many dioecious plants gender influences the economic value, breeding schemes and opportunities for commercial use of genetically modified materials (Parker and Clark 1991). The discussed demand for tools supporting sex determination in plants gave rise to a series of molecular studies investigating DNA markers that could be used for that purpose. A molecular marker (DNA marker) is a DNA sequence observed in at least two versions that are easy to distinguish (Brown 2002), which reveals individual polymorphisms. The preferred marker should demonstrate the widest possible range of variation in the analyzed trait, and it should not be affected by environmental factors. An effective marker should guarantee reproducibility, and it should be easy to detect. Molecular markers facilitate analyzes of variations between individuals, regardless of their development stage (Sztuba-Soli?ska 2005), which is particularly useful in sex determination studies of plants. Some researchers have suggested that effective markers for plants should be relatively short to support sex determinations in herbarium specimens with damaged DNA. Shorter sequences increase the probability of successful amplification (Korpelainen et al. 2008). This study is focused on testing the reliability and reproducibility of three developed molecular markers linked to male sex in the cannabis plant (Cannabis sativa L.). Standard technical hemp varieties, as well as medicinal ones were included.

Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid
Futoshi Taura, S Morimoto, Yukihiro Shoyama
Journal of Biological Chemistry August 1996
August 1996 271(29):17411-6
DOI: 10.1074/jbc.271.29.17411
We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.

Quantitative trait loci controlling agronomic and biochemical traits in Cannabis sativa
Patrick Woods, Brian J. Campbell, Timothy J. Nicodemus, Edgar B. Cahoon, Jack L. Mullen, and John K. McKay
GENETICS, 2021, 219(2)
DOI: 10.1093/genetics/iyab099
https://www.semanticscholar.org/pape...9129166d4f1b0c
Understanding the genetic basis of complex traits is a fundamental goal of evolutionary genetics. Yet, the genetics controlling complex traits in many important species such as hemp (Cannabis sativa) remain poorly investigated. Because hemp’s change in legal status with the 2014 and 2018 U.S. Federal Farm Bills, interest in the genetics controlling its numerous agriculturally important traits has steadily increased. To better understand the genetics of agriculturally important traits in hemp, we developed an F2 population by crossing two phenotypically distinct hemp cultivars (Carmagnola and USO31). Using whole-genome sequencing, we mapped quantitative trait loci (QTL) associated with variation in numerous agronomic and biochemical traits. A total of 69 loci associated with agronomic (34) and biochemical (35) trait variation were identified. We found that most QTL co-localized, suggesting that the phenotypic distinctions between Carmagnola and USO31 are largely controlled by a small number of loci. We identified TINY and olivetol synthase as candidate genes underlying colocalized QTL clusters for agronomic and biochemical traits, respectively. We functionally validated the olivetol synthase candidate by expressing the alleles in yeast. Gas chromatography-mass spectrometry assays of extracts from these yeast colonies suggest that the USO31 olivetol synthase is functionally less active and potentially explains why USO31 produces lower cannabinoids compared to Carmagnola. Overall, our results help modernize the genomic understanding of complex traits in hemp.

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.
Koichi Sakamoto, Tomoko Abe, Tomoki Matsuyama, Shigeo Yoshida,
Nobuko Ohmido, Kiichi Fukui, and Shinobu Satoh
Genome 48: 931–936 (2005)
doi: 10.1139/G05-056
Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC (male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.

Not Cannabis Specific
Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion
Vladimir Nekrasov, Congmao Wang, Joe Win, Christa Lanz, Detlef Weigel & Sophien Kamoun
Nature Scientific Reports | 7: 482
DOI:10.1038/s41598-017-00578-x
Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non-transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology. We used whole genome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/Cas9 being a highly precise tool, as we did not detect off-target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.

Rapid identification of drug-type and fiber-type cannabis by allele specific duplex PCR.
Yamamuro, T., Segawa, H., Kuwayama, K., Tsujikawa, K., Kanamori, T., & Iwata, Y. T.
Forensic Science International, 110634.(2020).
doi:10.1016/j.forsciint.2020.110634
Cannabis is classified into two types: drug-type cannabis, which is abused worldwide, and fiber-type cannabis, which is used for industrial purposes. The two types are a result of differences in the sequences of tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes. In the present study, we aimed to establish a PCR-based method to distinguish between drug-type and fiber-type cannabis by detecting the differences in the sequences of THCAS and CBDAS. We constructed a single-plex PCR targeting active THCAS, and observed drug-type cannabisspecific amplification when using 10 pg to 1 ng of DNA; however, amplification was also observed in fiber-type cannabis when the DNA content reached 10 ng. Similarly, singleplex PCR targeting active CBDAS showed fiber-type cannabis-specific amplification in 100 pg of DNA, as well as in >1 ng of drug-type cannabis DNA. Therefore, when an allele-specific duplex PCR system was constructed, in which both primer sets were mixed at an appropriate ratio, unintended nonspecific amplification was suppressed and amplicons of different sizes were observed between the drug-type and fiber-type cannabis, using DNA samples in the range of 1 pg to 10 ng. When the constructed duplex PCR was performed on DNA extracted from various cannabis seed samples, it was possible to distinguish between the drug-type and the fiber-type as well as detect a hybrid-type with both active THCAS and active CBDAS and a special type with neither. The identification method developed in the present study can quickly and accurately distinguish between drug-type and fiber-type cannabis, and is expected to be used for various purposes such as the detection of genetic contamination of industrial hemp as well as forensic examination of cannabis-related cases.
? We developed a new method to rapidly distinguish drug-type from fiber-type
cannabis.
? Active THCAS and CBDAS are characteristic of drug-type and fiber-type,
respectively.
? Primer sets targeting active THCAS and active CBDAS were designed.
? Allele-specific duplex PCR identified cannabis type in 1 pg to 10 ng DNA sample.
? We identified a hybrid-type with both active genes and a special type with neither.

Recent advances in cannabis biotechnology.
Hesami, M., Pepe, M., Alizadeh, M., Rakei, A., Baiton, A., & Phineas Jones, A. M.
Industrial Crops and Products, 158, 113026. (2020)
doi:10.1016/j.indcrop.2020.113026
Cannabis sativa L. is a high-value crop with a multi-billion dollar international market, yet due to the long history of prohibition, there is a significant lack of research on the plant and biotechnological techniques are in their infancy. Developing and applying modern techniques to Cannabis will help overcome some species-specific challenges to increase productivity and improve our knowledge about this plant. With regulatory environments relaxing in many parts of the world, there has been a significant increase in biotechnological research with this species. The current manuscript reviews the advances in Cannabis biotechnology, including molecular markers, microRNA, omics-based methods, and functional genes related to the terpene and cannabinoid biosynthesis as well as fiber quality. The foremost aim of this study is to a comprehensive review of the available literature to guide future cannabis studies in the field of genetic engineering and biotechnology.

Recent advances in Cannabis sativa genomics research.
Hurgobin, B., Tamiru‐Oli, M., Welling, M. T., Doblin, M. S., Bacic, A., Whelan, J., & Lewsey, M. G.
New Phytologist. (2020)
doi:10.1111/nph.17140
Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically-useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis

Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology
Supaart Sirikantaramas, Futoshi Taura, Satoshi Morimoto and Yukihiro Shoyama
Current Pharmaceutical Biotechnology, 2007, 8, 237-243
DOI: 10.2174/138920107781387456
Cannabinoids, consiting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate: olivetolate geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These
results open the way for biotechnological production of cannabinoids in the future.

Not Cannabis specific
Regenerant Arabidopsis Lineages Display a Distinct Genome-Wide Spectrum of Mutations Conferring Variant Phenotypes
Caifu Jiang, Aziz Mithani, Xiangchao Gan, Eric J. Belfield, John P. Klingler, Jian-Kang Zhu, Jiannis Ragoussis, Richard Mott, Nicholas P. Harberd
DOI: https://dx.doi.org/10.1016/j.cub.2011.07.002
Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [ 1–3 ]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [ 2 ], and clonal regenerant plants exhibit poorly understood heritable phenotypic (“somaclonal”) variation [ 4–7 ]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutationrate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation.

Research grade marijuana supplied by the National Institute on Drug Abuse is genetically divergent from commercially available Cannabis
Anna L. Schwabe, Connor J. Hansen, Richard M. Hyslop, Mitchell E. McGlaughlin
https://www.biorxiv.org/content/10.1...725v1.full.pdf
DOI: 10.1101/592725
Public comfort with Cannabis (marijuana and hemp) has recently increased, resulting in previously strict Cannabis regulations now allowing hemp cultivation, medical use, and in some states, recreational consumption. There is a growing interest in the potential medical benefits of the various chemical constituents produced by the Cannabis plant. Currently, the University of Mississippi, funded through the National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA), is the sole Drug Enforcement Agency (DEA) licensed facility to cultivate Cannabis for research purposes. Hence, most federally funded research where participants consume Cannabis for medicinal purposes relies on NIDA-supplied product. Previous research found that cannabinoid levels in research grade marijuana supplied by NIDA did not align with commercially available Cannabis from Colorado, Washington and California. Given NIDA chemotypes were misaligned with commercial Cannabis, we sought to investigate where NIDA’s research grade marijuana falls on the genetic spectrum of Cannabis groups. NIDA research grade marijuana was found to genetically group with Hemp samples along with a small subset of commercial drug-type Cannabis. A majority of commercially available drug-type Cannabis was genetically very distinct from NIDA samples. These results suggest that subjects consuming NIDA research grade marijuana may experience different effects than average consumers.

*Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes
Kevin J. McKernan, Yvonne Helbert, Liam T. Kane, Heather Ebling, Lei Zhang, Biao Liu, Zachary Eaton, Stephen McLaughlin, Sarah Kingan, Primo Baybayan,Gregory Concepcion, Mark Jordan, Alberto Riva, William Barbazuk,Timothy Harkins
https://www.biorxiv.org/content/10.1...428v1.full.pdf
Doi: 10.1101/2020.01.03.894428.
Cannabis is a diverse and polymorphic species. To better understand cannabinoid synthesis inheritance and its impact on pathogen resistance, we shotgun sequenced and assembled a Cannabis trio (sibling pair and their offspring) utilizing long read single molecule sequencing. This resulted in the most contiguous Cannabis sativa assemblies to date. These reference assemblies were further annotated with full-length male and female mRNA sequencing (Iso-Seq) to help inform isoform complexity, gene model predictions and identification of the Y chromosome. To further annotate the genetic diversity in the species, 40 male, female, and monoecious cannabis and hemp varietals were evaluated for copy number variation (CNV) and RNA expression. This identified multiple CNVs governing cannabinoid expression and 82 genes associated with resistance to Golovinomyces chicoracearum, the causal agent of powdery mildew in cannabis. Results indicated that breeding for plants with low tetrahydrocannabinolic acid (THCA) concentrations may result in deletion of pathogen resistance genes. Low THCA cultivars also have a polymorphism every 51 bases while dispensary grade high THCA cannabis exhibited a variant every 73 bases. A refined genetic map of the variation in cannabis can guide more stable and directed breeding efforts for desired chemotypes and pathogen-resistant cultivars.

Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype
Chiara Onofri a, Etienne P.M. de Meijer , Giuseppe Mandolino
Phytochemistry 2015 Aug 9;116:57-68
Doi: 10.1016/j.phytochem.2015.03.006
Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases

SEQUENCING OF THREE MALE CANNABIS GENOMES AND DEVELOPMENT OF MULTIPLEX QPCR ASSAYS FOR RAPID MALE SEX DETERMINATION

Kevin McKernan, Vasisht Tadigotla, Yvonne Helbert, Jessica Spangler, Lei Zhang, Douglas Smith
https://www.medicinalgenomics.com/wp...ation_sbmt.pdf
Male associated DNA Cannabis (MADC2) markers have previously been described by Mandolino et al1 . These markers were reported to target the MADC2 repeat and generate many bands with gel electrophoresis of which the male cannabis plant delivered a unique band. While these markers are reported to accurately detect male plants, the use of gels and visual inspection of banding patterns could be improved with more scalable quantitative PCR (qPCR) methods. Towards this end, we cloned and sequenced these bands to over 1000x coverage using next generation sequencing. We discovered highly variable MADC2 sequences complicating qPCR assay design

Sex chromosomes and quantitative sex expression in monoecious hemp (Cannabis sativa L.)
Anne-Michelle Faux, Alice Berhin, Nicolas Dauguet, P. Bertin
Euphytica
DOI: 10.1007/s10681-013-1023-y
Hemp (Cannabis sativa) has a highly variable sexual phenotype. In dioecious hemp, the sex is controlled by heteromorphic sex chromosomes according to an X-to-autosomes equilibrium. However, in monoecious hemp, the sex determinism remains widely unknown and has never been related to a quantitative approach of sex expression. The present paper aims to contribute to the comprehension of the sex determinism in monoecious hemp by assessing the genotypic variability of its sex expression and establishing its sex chromosomes. Five monoecious and one dioecious cultivars were grown in controlled conditions under several photoperiods. The monoecy degree of 194 monoecious plants was recorded at each node by a figure ranging from 0 (male flowers only) to 6 (female flowers only). The genome size of 55 plants was determined by flow cytometry. The DNA of 115 monoecious plants was screened with the male-associated marker MADC2. The monoecy degree varied significantly among monoecious cultivars from 3.36 ± 2.28 in ‘Uso 31’ to 5.70 ± 0.81 in the most feminised ‘Epsilon 68’. The variation of monoecy degree among cultivars remained consistent across trials despite a significant “cultivar × trial” interaction and partly agreed with their earliness. The genome size of monoecious plants (1.791 ± 0.017 pg) was not different from that of females (1.789 ± 0.019 pg) but significantly lower than that of males (1.835 ± 0.019 pg). MADC2 was absent from all monoecious plants. These results strongly support that cultivars of monoecious hemp have the XX constitution and that their sex expression has a genetic basis.

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations
Simon Gilmorea, Rod Peakallb, James Robertson
Forensic Science International 131 (2003) 65–74
DOI: 10.1016/S0379-0738(02)00397-3
Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations.
Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among
accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the
geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.

Not Cannabis Specific
Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.
Sung, Y.-C., Lin, C.-P., Hsu, H.-J., Chen, Y.-L., & Chen, J.-C.
Frontiers in Plant Science, 10. (2019).
doi:10.3389/fpls.2019.01183
Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

Work by Medicinal Genomics in progress
Single molecule sequencing reveals the structure of the Bt:Bd allele in Cannabis
AKA Single molecule sequencing of THCA synthase reveals copy number variation in modern drug-type Cannabis sativa L
https://www.biorxiv.org/content/bior...28654.full.pdf
doi: 10.1101/028654
• Cannabinoid expression is an important genetically determined feature of cannabis that presents clinical and legal implications for patients seeking
Cannabinoid specific therapies like Cannabidiol (CBD).
• Cannabinoid, terpenoid, and flavonoid marker assisted selection can accelerate breeding efforts by offering genetic tools to select for desired traits at an early stage in growth. To this end, multiple models for chemotype inheritance have been described suggesting a complex picture for chemical phenotype determination.
• Here we explore the potential role of copy number variation of THCA Synthase
using phased single molecule sequencing and demonstrate that copy number and
sequence variation of this gene is common and suggests a morenuanced view of
chemotype prediction.

SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L.
Andrea R. Garfinkel, Matthew Otten, Seth Crawford
Genes 2021, 12(2), 228;
DOI: 10.3390/genes12020228
The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant’s legal cultivation. Of the major cannabinoids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The “null” THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa.

Spatio-Temporal and Cultivar-Dependent Variations in the Cannabis Microbiome
Comeau D, Novinscak A, Joly DL and Filion M
Front. Microbiol. 11:491. (2020)
doi: 10.3389/fmicb.2020.00491
The incipient legalization and commercialization of Cannabis sativa in Canada have promulgated research into characterizing the plant’s microbiome as it promotes many facets of plant growth and health. The emblematic production of commercially important secondary metabolites, namely tetrahydrocannabinol (THC), cannabidiol (CBD) and terpenes, has warranted investigating the modulating capacity of these molecules on the plant microbiome. C. sativa cultivars can be classified into chemotypes depending on the relative levels of THC and CBD they produce; their biosynthesis also varies spatially and temporally during the life cycle of the plant. To study the differential microbiome structure and diversity between cultivars in a spatio-temporal manner, we extracted microbial DNA from the rhizosphere, endorhizosphere, and phyllosphere during the entire life cycle of three different chemotypes; CBD Yummy (< 1% CBD). Illumina marker gene sequencing of bacterial (16S) and fungal (ITS) communities were coupled to the QIIME2, PICRUSt, and LEfSe pipelines for analysis. Our study describes spatiotemporal and cultivar-dependent variations in the fungal and bacterial microbiome of C. sativa, and details strong cultivar-dependent variance in the belowground microbiome. Furthermore, the predicted pathway abundance of the bacterial microbiome is concomitantly subject to spatio-temporal variations; pathways related to lipid, amino acid, glucose and pentose metabolism were noteworthy. These results describe, for the first time, spatio-temporal and cultivar-dependent variations in the microbiome of C. sativa produced under strict commercial settings. Describing the microbiome is the first step in discoveries that could help in engineering a plant growth and health promoting microbiome in future works.

Species Identification of Cannabis sativa Using Real-Time Quantitative PCR (qPCR)
Christopher E. Johnson, Amritha Premasuthan, Jessica Satkoski Trask,
and Sree Kanthaswamy
J Forensic Sci, March 2013, Vol. 58, No. 2
doi: 10.1111/1556-4029.12055
Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3? exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa

SSR loci survey of technical hemp cultivars: The optimization of a cost-effective analyses to study genetic variability.
Vyhnánek, T., Nevrtalova, E., Bjelková, M., & Balgová, B.
Plant Science, 110551.(2020).
doi:10.1016/j.plantsci.2020.110551
Our study aimed to optimize a selection of a suitable combination of SSRs (Simple Sequence Repeats) for determination of technical Cannabis cultivars and genetic variability level. We used sequences of 23 published SSR families (107 alleles) and amplified them in 28 cultivars. One of the alleles possesses no selective information (SSR family CAN1660) due to its presence in every single tested cultivar. We excluded it, together with another 11 least informative alleles. After data filtration, we used 96 alleles to do recursive sub-sampling of random alleles' sets. We found a minimal set of 8 alleles (in three different combinations) to distinguish 28 analyzed cultivars from each other. Our results contribute to saving resources and to reduce the performance time of the molecular analysis

Statistical genetic considerations for maintaining germ plasm collections
J. Crossa, C. M. Hernandez, P. Bretting, S. A. Eberhart, S. Taba
Theor Appl Genet (1993) 86:673-678
DOI: 10.1007/BF00222655
One objective of the regeneration of genetic populations is to maintain at least one copy of each allele present in the original population. Genetic diversity within populations depends on the number and frequency of alleles across all loci. The objectives of this study on outbreeding crops are: (1) to use probability models to determine optimal sample sizes for the regeneration for a number of alleles at independent loci; and (2) to examine theoretical considerations in choosing core subsets of a collection. If we assume that k- 1 alleles occur at an identical low frequency of Po and that the k th allele occurs at a frequency of 1 - [(k- 1)po], for loci with two, three, or four alleles, each with a Po of 0.05, 89-t10 additional individuals are required if at least one allele at each of 10 loci is to be retained with a 90% probability; if 100 loci are involved, 134-155 individuals are required. For two, three, or four alleles, when Po is 0.03 at each of 10 loci, the sample size required to include at least one of the alleles from each class in each locus is 150-186 individuals; if 100 loci are involved, 75 additional individuals are required. Sample sizes of 160-210 plants are required to capture alleles at frequencies of 0.05 or higher in each of 150 loci, with a 90-95% probability. For rare alleles widespread throughout the collection, most alleles with frequencies of 0.03 and 0.05 per locus will be included in a core subset of 25-100 accessions.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases
Lewis J. Kearsey, Nicole Prandi, Vijaykumar Karuppiah, Cunyu Yan, David Leys, Helen Toogood, Eriko Takano, and Nigel S. Scrutton
The FEBS Journal 287 (2020) 1511–1524
doi:10.1111/febs.15089
https://www.ncbi.nlm.nih.gov/labs/pm...S-287-1511.pdf
In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 ? C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 ? C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes

6GW3
Structure of TKS from Cannabis sativa in complex with CoA

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa

Xinmei Yang, Takashi Matsui, Takeshi Kodama, Takahiro Mori, Xiaoxi Zhou, Futoshi Taura, Hiroshi Noguchi, Ikuro Abe and Hiroyuki Morita
The FEBS Journal 283 (2016) 1088–1106 Federation of European Biochemical Societies
doi:10.1111/febs.13654
https://asset-pdf.scinapse.io/prod/2...2325675648.pdf
In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant a+b barrel (DABB) protein that catalyzes the C2–C7 aldol cyclization of the linear pentyl tetra-b-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC–OA complex binary crystal structures at 1.32 and 1.70 _A resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future.

Full wwPDB X-ray Structure Validation Report
Worldwide Protein Bank WWPDB Jan 25, 2022 – 06:12 PM JST
PDB ID : 7W6G
Title : TKS-L190G mutant from Cannabis sativa in complex with lauroyl-CoA
Authors : Nakashima, Y.; Lee, Y.E.; Morita, H.
Deposited on : 2021-12-01
Resolution : 2.10 Å(reported)

Studies on tetrahydrocannabinolic acid synthase that produces the acidic precursor of tetrahydrocannabinol, the pharmacologically active cannabinoid in marijuana
Futoshi Taura
Drug Discov Ther. 2009; 3(3):83-87.
https://pdfs.semanticscholar.org/c36...808.1560054937
Tetrahydrocannabinol (THC), the psychoactive component of marijuana, is now regarded as a promising medicine because this cannabinoid has been shown to exert a variety of therapeutic activities. It has been demonstrated that THC is generated from the acidic precursor, tetrahydrocannabinolic acid (THCA) by nonenzymatic decarboxylation, and that THCA is biosynthesized by THCA synthase, which catalyzes a unique biosynthetic reaction, the stereospecific oxidative cyclization of the geranyl group of the substrate cannabigerolic acid. Molecular characterization of THCA synthase has revealed its structural characteristics and reaction mechanism. THCA synthase is the first cannabinoid synthase to be studied and is potentially attractive target for various biotechnological applications as it produces the direct precursor of THC. This review describes the research history of this enzyme, i.e., purification, molecular cloning, biochemical characterization, and possible biotechnological application of THCA synthase.

Subcellular localization defines modification and production of ?9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.
Geissler M, Volk J, Stehle F, Kayser O, Warzecha H.
Biotechnol Lett. 2018 Jun;40(6):981-987.
doi: 10.1007/s10529-018-2545-0. Epub 2018 Apr 4.

Survey on the Germplasm Resources of Cannabis sativa L.
WANG Hua-dong, WEI Ying-fang. (2012)
Cannabis sativa L.is the main original plant of medicinal material FRUCTUS CANNABIS that has a long cultivation history in China.Although numerous rare varieties have been formed,the planting area of C.sativa is sharply reduced due to the adjustment of industrial structure in recent years,which results in the great changes of resources distribution.We find,most of the wild resources in the main producing areas have been consumed,only a small part of which are restricted to Sinkiang Province,Yunnan Province and Shandong Province.Currently,C.sativa is cultivated extensively in Anhui, Yunnan, Gansu, Heilongjiang, Inner Mongolia, Guangxi, Shanxi, Sichuan, Henan and other provinces of China,sporadically cultivated in Ningxia, Shaanxi, Hebei, Hubei, Hunan and Shanxi,and seldom cultivated in the east coastal regions.Therefore,it is imperative to establish the germplasm resource library and nursery garden for special purpose in order to realize the protective development and utilization of C.sativa Furthermore,seeds of native varieties should be screened based upon the drug properties to ensure the effectiveness and safety of its medicinal material FRUCTUS CANNABIS and to guide its rational use.

Testing of DNA isolation for the identification of hemp
Tomáš Vyhnánek, Václav Trojan, Klára Štiasna, Mária Presinszká, Lud?k H?ivna, Eva Mrkvicová, Ladislav Havel
Potravinarstvo, vol. 9, 2015, no. 1, p. 393-397
https://www.researchgate.net/publica...cation_of_hemp
Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb) with nine pairs of autosomes plus XX (?) or XY (?) chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar) of hemp with low ? 9 -tetrahydrocannabinol content are used for industrial applications. Varieties with high ? 9 -tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg) of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic). The DNeasy® Plant Mini Kit (Qiagen, GE) was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 – 38.90 ng.µL-1 ), but with better purity than samples of higher weight (ratio A260nm/A280nm for low-weight samples was near 1.80). To test the applicability of DNA analysis, we used two SSR markers (CAN1347 and CAN2913). PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg). In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.

Tetrahydrocannabinol Levels in Hemp (Cannabis sativa) Germplasm Resources
November 2003 Economic Botany 57(4):545-558
Ernest Small David Marcus
DOI: 10.1663/0013-0001 (2003)057[0545:TLIHCS]2.0.CO;2

The Applications of Molecular Markers in Genetics and Breeding of Hemp
Giuseppe Mandolino, Paolo Ranalli
Journal of Industrial Hemp 2002 7:1, 7-23
Doi: 10.1300/J237v
BACKGROUND:
Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.
RESULTS:
We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of ?9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why thepsychoactive cannabinoid ?9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers ofcannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp.
CONCLUSIONS:
The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.

The bioengineering of cannabis
Elie Dolgin
NATURE OUTLOOK 28 August 2019
https://www.nature.com/articles/d41586-019-02525-4
Genetic modification could enable industrial-scale production of cannabinoids that have pharmaceutical potential.

Not Cannabis specific
The chloroplast genome hidden in plain sight, open access publishing and anti-fragile distributed data sources
Kevin Judd McKernan
Mitochondrial DNA Part A, 27(6), 4518–4519.
DOI:10.3109/19401736.2015.1101541
We sequenced several cannabis genomes in 2011 of June and the first and the longest contigs to emerge were the chloroplast and mitochondrial genomes. Having been a contributor to the Human Genome Project and an eye-witness to the real benefits of immediate data release, I have first hand experience with the potential mal-investment of millions of dollars of tax payer money narrowly averted due to the adopted global rapid data release policy. The policy was vital in reducing duplication of effort and economic waste. As a result, we felt obligated to publish the Cannabis genome data in a similar spirit and placed them immediately on a cloud based Amazon server in August of 2011. While these rapid data release practices were heralded by many in the media, we still find some authors fail to find or reference said work and hope to compel the readership that this omission has more pervasive repercussions than bruised egos
and is a regression for our community

The complete chloroplast genomes of Cannabis sativa and Humulus lupulus.
Vergara, D., White, K. H., Keepers, K. G., & Kane, N. C.
Mitochondrial DNA Part A, 27(5), 3793–3794.(2015).
doi:10.3109/19401736.2015.1079905
Cannabis and Humulus are sister genera comprising the entirety of the Cannabaceae sensu stricto, including C. sativa L. (marijuana, hemp), and H. lupulus L. (hops) as two economically important crops. These two plants have been used by humans for many purposes including as a fiber, food, medicine, or inebriant in the case of C. sativa, and as a flavoring component in beer brewing in the case of H. lupulus. In this study, we report the complete chloroplast genomes for two distinct hemp varieties of C. sativa, Italian ‘‘Carmagnola’’ and Russian ‘‘Dagestani’’, and one Czech variety of H. lupulus ‘‘Saazer’’. Both C. sativa genomes are 153 871 bp in length, while the H. lupulus genome is 153 751 bp. The genomes from the two C. sativa varieties differ in 16 single nucleotide polymorphisms (SNPs), while the H. lupulus genome differs in 1722 SNPs from both C. sativa cultivars

The complete mitochondrial genome for Cannabis Sativa, Mitochondrial DNA Part B
Kristin H. White, Daniela Vergara, Kyle G. Keepers & Nolan C. Kane
DOI: 10.1080/23802359.2016.1155083 To link to this article: https://doi.org/10.1080/23802359.2016.1155083 The following report details the first annotated mitochondrial genome for the Carmagnola variety of Cannabis sativa, the first reference genome for the Cannabaceae family. The total length is 415,499 bp and contains 54 genes, which sub-divide into 38 protein-coding genes, 15 tRNA genes, and 3 rRNA genes

The draft genome and transcriptome of Cannabis sativa
Harm van Bakel, Jake M Stout, Atina G Cote, Carling M Tallon, Andrew G Sharpe, Timothy R Hughes, and Jonathan E Page
Genome Biol. 2011; 12(10): R102.
doi: 10.1186/gb-2011-12-10-r102
Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.
RESULTS:
We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of ?9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why thepsychoactive cannabinoid ?9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers ofcannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp.
CONCLUSIONS:
The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.

The Future Of Cannabis Genetics
Jonathan Page, Anandia Labs
https://www.slideshare.net/Adrienna/...nabis-genetics
Slides Presentation
The Gene Controlling Marijuana Psychoactivity

The Gene Controlling Marijuana Psychoactivity
MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF _1 TETRAHYDROCANNABINOLIC ACID SYNTHASE FROM CANNABIS SATIVA L.*
Supaart Sirikantaramas, Satoshi Morimoto, Yoshinari Shoyama, Yu Ishikawa, Yoshiko Wada,Yukihiro Shoyama, and Futoshi Taura
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 39767–39774, 2004
DOI 10.1074/jbc.M403693200
1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of _1-tetrahydrocannabinol. We cloned a novel cDNA (GenBankTM accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists
of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved
in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis

The Genetic Structure of Marijuana and Hemp
Jason Sawler, Jake M. Stout, Kyle M. Gardner, Darryl Hudson, John Vidmar, Laura Butler, Jonathan E. Page, Sean Myles
Plos ONE
DOI: 10.1371/journal.pone.0133292
Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid ?9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.

The genetics of Cannabis – genomic variations of key synthases and their effect on cannabinoids content
Dr. Aparna Singh, Dr. Andriy Bilichak, and Dr. Igor Kovalchuk
Genome (2020)
DOI: 10.1139/gen-2020-0087
Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalisation in Canada, it is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allows for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single nucleotide polymorphisms in the coding regions of cannabinoid synthases play important role in determining plant chemotype. Deep understanding of how these variants affect enzymes activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profile. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.

The Genomics of Cannabis and Its Close Relatives.
Kovalchuk, I., Pellino, M., Rigault, P., van Velzen, R., Ebersbach, J., R. Ashnest, J., … Sharbel, T. F.
Annual Review of Plant Biology, 71(1). (2020).
doi:10.1146/annurev-arplant-081519-040203*
https://www.annualreviews.org/doi/pd...-081519-040203
https://www.researchgate.net/publica...lose_Relatives
Cannabis sativa L. is an important yet controversial plant with a long history of recreational, medicinal, industrial, and agricultural use, and together with its sister genus Humulus, it represents a group of plants with a myriad of academic, agricultural, pharmaceutical, industrial, and social interests. We have performed a meta-analysis of pooled published genomics data, and we present a comprehensive literature review on the evolutionary history of Cannabis and Humulus, including medicinal and industrial applications.We demonstrate that current Cannabis genome assemblies are incomplete, with ?10% missing, 10–25% unmapped, and 45S and 5S ribosomal DNA clusters as well as centromeres/satellite sequences not represented. These assemblies are also ordered at a low resolution, and their consensus quality clouds the accurate annotation of complete, partial, and pseudogenized gene copies. Considering the importance of genomics in the development of any crop, this analysis underlines the need for a coordinated effort to quantify the genetic and biochemical diversity of this species.

The results of an experimental indoor hydroponic Cannabis growing study, using the ‘Screen of Green’ (ScrOG) method—Yield, tetrahydrocannabinol (THC) and DNA analysis
Glenys Knight, Sean Hansen, Mark Connor, Helen Poulsen, Catherine McGovern, Janet Stacey
Forensic Science International 202 (2010) 36–44
doi:10.1016/j.forsciint.2010.04.022
The results of an indoor hydroponic Cannabis growth study are presented. It is intended that this work will be of assistance to those with an interest in determining an estimation of yield and value of Cannabis crops. Three cycles of six plants were grown over a period of 1 year in order to ascertain the potential
yield of female flowering head material from such an operation. The cultivation methods used were selected to replicate typical indoor hydroponic Cannabis growing operations, such as are commonly encountered by the New Zealand Police. The plants were also tested to ascertain the percentage of the psychoactive chemical D-9 tetrahydrocannabinol (THC) present in the flowering head material, and were genetically profiled by STR analysis. Phenotypic observations are related to the data collected. The inexperience of the growers was evidenced by different problems encountered in each of the three cycles, each of which would be expected to negatively impact the yield and THC data obtained. These data are therefore considered to be conservative. The most successful cycle yielded an average of 881 g (31.1 oz) of dry, groomed female flowering head per plant, and over the whole study the 18 plants yielded a total of 12,360 g (436.0 oz), or an average of 687 g (24.2 oz) of dry head per plant. THC data shows significant intra-plant variation and also demonstrates inter-varietal variation. THC values for individual plants ranged from 4.3 to 25.2%. The findings of this study and a separate ESR research project illustrate that the potency of Cannabis grown in New Zealand has dramatically increased in recent years. DNA analysis distinguished distinct groups in general agreement with the phenotypic variation observed. One plant however, exhibiting a unique triallelic pattern at two of the five loci tested, while remaining phenotypically indistinguishable from three other plants within the same grow.

The sexual differentiation of Cannabis sativa L.: A morphological and molecular study
V. M. Cristiana Moliterni, Luigi Cattivelli, P. Ranalli, Giuseppe Mandolino
Euphytica January 2004, Volume 140, Issue 1-2, pp 95-106
DOI: 10.1007/s10681-004-4758-7
Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova.
Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment.
Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, an SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.

The validation of a 15 STR multiplex PCR for Cannabis species
Stephan Köhnemann & Johanna Nedele &
Daniela Schwotzer & Julia Morzfeld & Heidi Pfeiffer
Int J Legal Med (2012) 126:601–606
DOI 10.1007/s00414-012-0706-6
Trade and acquisition of Cannabis drugs are illegal in many countries worldwide; nevertheless, crimes related with these drugs are a major problem for the investigative authorities. With this manuscript, we want to introduce a 15
short tandem repeat (STR) Cannabis marker set that can be amplified in one PCR reaction. This multiplex PCR is specific to Cannabis species and combines highly informative STR markers. The 15 STR multiplex is easy to use and was
validated according to common laboratory quality standards. Due to the fact that a lot of Cannabis plants are cultivated by clonal propagation and may show aneuploidy, polyploidy or multiple gene loci, it is not possible to apply biostatistics that follow the Hardy–Weinberg law. However, this multiplex will help the police to trace back trade routes of drug syndicates or dealers and it can help to link Cannabis plants to a crime scene.

Not directly Cannabis related
The X chromosome is necessary for ovule production in Silene latifolia.
Veltsos, P., & Delph, L. F. (2019).
PLOS ONE, 14(5), e0217558.
doi:10.1371/journal.pone.0217558
Sex chromosomes stop recombining and accumulate differences over time. In particular, genes on the chromosome restricted to the heterogametic sex degenerate and become non-functional. Here, we investigated whether or not the degeneration of a plant Y chromosome was sufficient to cause ovules containing a Y to fail to develop, thereby eliminating the possibility of YY individuals. We used two genotypic assays to determine the genotype— XX, XY, or YY—of offspring from a single fruit of an otherwise normal male XY plant of Silene latifolia. The fruit contained fewer ovules than normal pistillate flowers, produced an equal offspring sex ratio, and generated no YY offspring. The results indicate that ovaries must contain an X chromosome to develop properly. While haploid selection has slowed down Y-chromosome degeneration in S. latifolia, we find that it has progressed sufficiently to prevent the proper development of ovules, and hence prevent the presence of YY individuals.

Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.
Yuanyuan Liu · Panpan Zhu · Sen Cai · George Haughn · Jonathan E. Page
Plant Molecular Biology Feb 2021
DOI: 10.1007/s11103-021-01129-9
Key message Three novel transcription factors were successfully identifed and shown to interact with the trichomespecifc THCAS promoter regulatory region. Abstract Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving efects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female fowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specifc expression. Using this promoter fragment in a yeast-one-hybrid screen, we identifed 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specifc expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specifc gene expression profles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.

Not Cannabis specific
Time for a paradigm shift in the use of plant genetic resources.
Belzile, F., Abed, A., & Torkamaneh, D.
Genome.(2019).
doi:10.1139/gen-2019-0141
For all major crops, sizeable genebanks are maintained across the world and serve as repositories of genetic diversity and key sources of novel traits used in breeding. Although molecular markers have been used to characterize diversity in a broad sense, the most common approach to exploring these resources has been through phenotypic characterization of subsets of these large collections. With the advent of affordable large-scale genotyping technologies and the increasing body of candidate genes for traits of interest, we argue here that it is time for a paradigm shift in the way that explore and exploit these considerable and highly useful resources. By combining dense genotypic information in and around candidate genes, it is possible to classify accessions based on their haplotype, something approximating the actual alleles at these genes of interest.


TRACKING CLONAL MARIJUANA USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) ANALYSIS: AN OVERVIEW
Heather Miller Coyle, Joselle Germano-Presby, Carll Ladd, Timothy Palmbach and Henry C. Lee.
Forensic Botany. (2004)
DOI: 10.1201/9780203484593.ch12
Cannabis sativa (marijuana) is one of the oldest cultivated crops in the world. As a plant, it is valued for both its hallucinogenic and medicinal properties. Marijuana has been used to treat a variety of ailments including pain, glaucoma, nausea, asthma, depression, insomnia and neuralgia (1). Like many cultivated crops (e.g. wheat, corn), it has been domesticated from a naturally occurring weed species and propagated to yield increasing amounts of ?-9-tetrahydrocannabinol (THC) and for other desirable smoking traits (e.g. flavor, smoothness).
Marijuana is a large cash crop in the United States and marijuana cases account for enormous asset forfeitures. These forfeited assets, in turn, are used to fund law enforcement budgets for drug eradication efforts, community-based treatment and drug prevention programs. Marijuana is
a primary focus for drug intervention programs in schools and is considered by many as a “gateway” drug to introduce young users to other addictive illicit drugs such as heroin and
cocaine. Most drug arrests are for crimes involving marijuana. In 1998, eighty-eight percent of seven hundred thousand drug-related cases were for marijuana possession in the United States

Not Cannabis specific
Transcriptome Analysis of Arabidopsis Wild-Type and gl3–sst sim Trichomes Identifies Four Additional Genes Required for Trichome Development
M. David Marksa, Jonathan P. Wengera, Edward Gildinga, Ross Jilkb and Richard A. Dixon
Molecular Plant • Volume 2 • Number 4 • Pages 803–822 • July 2009
doi: 10.1093/mp/ssp037
https://www.cell.com/action/showPdf?...2814%2960760-7
Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3–sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3–sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3–sst sim trichomes. The analysis of genes preferentially expressed in gl3–sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD–ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

Transcriptome differences between fiber-type and seed-type Cannabis sativa variety exposed to salinity
Jiajia Liu, Qin Qiao, Xia Cheng, Guanghui Du, Gang Deng, Mingzhi Zhao & Feihu Liu
Physiology and Molecular Biology of Plants volume 22, 429–443 (2016)
doi: 10.1007/s12298-016-0381-z
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120038/
The industrial hemp varieties ‘Yunma 5’ and ‘Bamahuoma,’ which demonstrate growth vigor and environmental adaptability, have been primarily cultivated in Yunnan and Guangxi, China, respectively, for fiber and seeds. The results of physiological measurements showed the phenotypic differences between the two varieties in response to salt stress. RNA-Seq analysis was first performed on leaves of both varieties sampled at four time intervals (0, 2, 4, 6 days) after treatment with salt (500 mM NaCl) We identified 220 co-up-regulated differentially expressed genes (DEGs) in the two varieties, while 26 up-regulated DEGs and 24 down-regulated DEGs were identified exclusively in the single varieties after 2 days of salt stress. Among the 220 DEGs, we identified 22 transcription factors, including key transcription factors involved in salt stress, such as MYB, NAC, GATA, and HSF. We applied gene expression profile analysis and found that ‘Yunma 5’ and ‘Bamahuoma’ have variety-specific pathways for resisting salt stress. The DEGs of ‘Yunma 5’ were enriched in spliceosome and amino acid metabolism genes, while the DEGs of ‘Bamahuoma’ were enriched in fatty acid metabolism, amino acid metabolism, and endoplasmic reticulum protein processing pathway. Although there were common DEGs, such as genes encoding cysteine protease and alpha/beta-hydrolase superfamily, the two varieties’ responses to salt stress impacted different metabolic pathways. The DEGs that were co-expressed in both varieties under stress may provide useful insights into the tolerance of cultivated hemp and other bast fiber crops to saline soil conditions. These transcriptomes also represent reference sequences for industrial hemp.


Transcriptomic and reverse genetic analyses of branched-chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana.
Slocombe SP, Schauvinhold I, McQuinn RP, Besser K, Welsby NA, Harper A, Aziz N, Li Y, Larson TR, Giovannoni J, Dixon RA, Broun P.
Plant Physiol. 2008 Dec;148(4):1830-46.
doi: 10.1104/pp.108.129510.
Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) alpha-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with alpha-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance.


Not Cannabis specific
Transgressive segregation in mating traits drives hybrid speciation
Kotaro Kagawa, Gaku Takimoto , Ole Seehausen
Evolution, 2023, 77(7), 1622–1633
doi.org/10.1093/evolut/qpad072
Hybridization can rapidly generate novel genetic variation, which can promote ecological speciation by creating novel adaptive phenotypes. However, it remains unclear how hybridization, creating novel mating phenotypes (e.g., mating season, genitalia shapes, sexual displays, mate preferences), affects speciation especially when the phenotypes do not confer adaptive advantages. Here, based on individual-based evolutionary simulations, we propose that transgressive segregation of mating traits can drive incipient hybrid speciation. Simulations demonstrated that incipient hybrid speciation occurred most frequently when the hybrid population received moderate continued immigration from parental lineages causing recurrent episodes of hybridization. Recurrent hybridization constantly generated genetic variation, which promoted the rapid stochastic evolution of mating phenotypes in a hybrid population. The stochastic evolution continued until a novel mating phenotype came to dominate the hybrid population, which reproductively isolates the hybrid population from parental lineages. However, too frequent hybridization rather hindered the evolution of reproductive isolation by inflating the variation of mating phenotypes to produce phenotypes allowing mating with parental lineages. Simulations also revealed conditions for the long-term persistence of hybrid species after their incipient emergence. Our results suggest that recurrent transgressive segregation of mating phenotypes can offer a plausible explanation for hybrid speciation and radiations that involved little adaptive ecological divergence
Two complete chloroplast genome sequences of Cannabis sativa varieties
Hyehyun Oha, Boyoung Seoa, Seunghwan Leea, Dong-Ha Ahnb, Euna Job, Jin-Kyoung Parkb & Gi-Sik Min
Mitochondrial DNA, 1–3.
Doi: 10.3109/19401736.2015.1053117
In this study, we determined the complete chloroplast (cp) genomes from two varieties of Cannabis sativa. The genome sizes were 153,848 bp (the Korean non-drug variety, Cheungsam) and 153,854 bp (the African variety, Yoruba Nigeria). The genome structures were identical with 131 individual genes [86 protein-coding genes (PCGs), eight rRNA, and 37 tRNA genes]. Further, except for the presence of an intron in the rps3 genes of two C. sativa varieties, the cp genomes of C. sativa had conservative features similar to that of all known species in the order Rosales. To verify the position of C. sativa within the order Rosales, we conducted phylogenetic analysis by using concatenated sequences of all PCGs from 17 complete cp genomes. The resulting tree strongly supported monophyly of Rosales. Further, the family Cannabaceae, represented by C. sativa, showed close relationship with the family Moraceae. The phylogenetic relationship outlined in our study is well congruent with those previously shown for the order Rosales.

Unravelling the Genetic Map of Cannabis: The Road to Designer Strains
Leo Bear-McGuinness
Nov 27, 2018
Analytical Cannabis Extraction, Science, Testing
https://www.analyticalcannabis.com/a...strains-311354
A new ‘genetic map of cannabis’ could help create tailor-made cannabis plants that only express desired genes.
The first-of-its-kind map is made up of the nearly 25,000 genes found in cannabis’ 10 chromosomes from three different cultivars: hemp, a high-CBDA strain and a high-THCA strain (these are the genes which code for CBD and THC respectively).
As a powerful new resource for cannabis companies, the hope is that the map will go on to inform growers of the locations of desired genes and, therefore, help then in breeding bespoke varieties.
Despite several other attempts to sequence the cannabis genome, the map is also the first of its kind to show the lost ancestral links between different cannabis cultivars.
In a paper published in BioRxiv – that is still in pre-print and awaiting peer-review – the map’s researchers reveal that high-CBD cannabis plants appear to have been created when hemp-like and marijuana-type cannabis cultivars were hybridized.
The research was conducted by some of the leading genetic institutions of America, including the Craig Venter Institute, Sunrise Genetics Inc. and the universities of Harvard and Minnesota.

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications
Salvador Soler, Dion?s Borras, Santiago Vilanova, Alicia Sifres, Isabel
Andujar, Maria R. Figas, Ernesto R. Llosa, and Jaime Prohens
J Forensic Sci, March 2016, Vol. 61, No. 2
doi: 10.1111/1556-4029.12995
Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis

Variation in mtDNA haplotypes suggests a complex history of reproductive strategy in Cannabis sativa
Ziv Attia, Cloe S. Pogoda , Daniela Vergara , Nolan C. Kane
https://www.biorxiv.org/content/10.1...591v1.full.pdf
DOI: 10.1101/2020.12.28.424591
Cannabis is one example in angiosperms that appears to have a recent origin of dioecy and X/Y sex chromosomes. Several evolutionary explanations for this transition have been proposed, with the most parsimonious beginning with a mitochondrial mutation leading to cytoplasmic male sterility (CMS). Our study utilized 73 Cannabis sativa whole genome shotgun libraries to reveal eight different mtDNA haplotypes. The most common haplotype contained 60 of the 73 individuals studied and was composed of only dioecious individuals. However, other haplotypes contained a mix of dioecious and monoecious individuals, so haplotype alone does not predict dioecy. From these haplotype groupings we further examined the fully annotated mitochondrial genomes of four hemp individuals and looked for genetic variation affecting reproductive strategy (e.g., monoecious vs. dioecious strategies). Specifically, we searched for markers associated with CMS and for gene rearrangements within these mitochondrial genomes. Our results revealed highly syntenic mitochondrial genomes that contained approximately 60 identifiable sequences for protein coding genes, tRNAs and rRNAs and no obvious rearrangements or chimeric genes. We find no clear evidence that the different reproductive patterns are due to easily identifiable CMS mutations. Our results refute the simplest hypothesis that there was a single recent origin of dioecy in a monoecious ancestor. Instead, the story of the evolution of dioecy is likely much more complex. Further exploration of the nuclear and mitochondrial genomes and their interaction is required to fully understand Cannabis’ mating strategies and the possible existence of CMS in this species.

Not Cannabis Specific
Variation in shade-induced flowering in Arabidopsis thaliana results from FLOWERING LOCUS T allelic variation
C. J. Schwartz, Joohyun Lee, Richard Amasino
PLoS ONE 12(11): e0187768.
Doi: 10.1371/journal.pone.0187768
Plants have evolved developmental mechanisms to ensure reproduction when in sub-optimal local environments. The shade-avoidance syndrome is one such mechanism that causes plants to elongate and accelerate flowering. Plants sense shade via the decreased red:far-red (R:FR) ratio that occurs in shade. We explored natural variation in flowering behavior caused by a decrease in the R:FR ratio of Arabidopsis thaliana accessions. A survey of accessions revealed that most exhibit a vigorous rapid-flowering response in a FR enriched environment. However, a subset of accessions appeared to be compromised in the accelerated-flowering component of the shade-avoidance response. The genetic basis of the muted response to FR enrichment was studied in three accessions (Fl-1, Hau-0, and Mir-0). For all three accessions, the reduced FR flowering-time effect mapped to the FLOWERING LOCUS T (FT) region, and the FT alleles from these accessions are expressed at a lower level in FR-enriched light compared to alleles from accessions that respond robustly to FR enrichment. In the Mir-0 accession, a second genomic region, which includes CONSTANTS (CO), also influenced flowering in FR-enriched conditions. We have demonstrated that variation in the degree of precocious flowering in shaded conditions (low R:FR ratio) results from allelic variation at FT.

[FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp][COLOR=red]Whole genome sequencing of colonies derived from [/COLOR][/B][/FONT][/FONT][FONT=Cambria][B][FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp][COLOR=red]cannabis flowers and the impact of media selection on [/COLOR][/B][/FONT][/FONT][FONT=Cambria][B][FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp][COLOR=red]benchmarking total yeast and mold detection tools [version 2; peer review: 2 approved][/COLOR][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT]
[FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp]Kevin McKernan , Yvonne Helbert, Liam Kane , Nathan Houde, Lei Zhang, [/FONT][/FONT][FONT=Cambria][FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp]Stephen McLaughlin[/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT]
[FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT="PÊˇø◊e\00278D5\0027C0¥*† °&amp]F1000Research 2021, 10:624 Last updated: 10 MAR 2022[/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT][/B][/FONT]
[FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][FONT=Cambria][B][URL="https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8383127/pdf/f1000research-10-70512.pdf"]https://www.ncbi.nlm.nih.gov/labs/pm...h-10-70512.pdf[/URL]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Background:
[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Cannabis products are subjected to microbial testing for human [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]pathogenic fungi and bacteria. These testing requirements often rely [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]on non-specific colony forming unit (CFU/g) specifications without [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]clarity on which medium, selection or growth times are required. We [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]performed whole genome sequencing to assess the specificity of [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]colony forming units (CFU) derived from three different plating media: [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Potato Dextrose Agar (PDA), PDA with chloramphenicol and Dichloran [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Rose Bengal with chloramphenicol (DRBC).[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Methods:[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Colonies were isolated from each medium type and their whole [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]genomes sequenced to identify the diversity of microbes present on [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]each medium selection. Fungal Internal Transcribed Spacer (ITS3) and [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Bacterial 16S RNA(16S) quantitative polymerase chain reactions (qPCR) [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]were performed, to correlate these CFUs with fungi- and bacterialspecific [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]qPCR.[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Results:[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Each plating medium displayed a ten-fold difference in CFU counts. [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]PDA with chloramphenicol showed the highest diversity and the [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]highest concordance with whole genome sequencing. According to [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]ITS3 and 16S qPCR confirmed with whole genome sequencing, DRBC [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]under counted yeast and mold while PDA without chloramphenicol [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]over counted CFUs due to bacterial growth without selection.[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Conclusions:[/FONT]
[FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]Colony Forming Unit regulations lack specificity. Each medium. [FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]produces significant differences in CFU counts. These are further [/FONT][/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]dependent on subjective interpretation, failure to culture most [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]microbes, and poor selection between bacteria and fungi. Given the [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]most human pathogenic microbes found on cannabis are endophytes [/FONT][FONT="PÊˇø◊e\00278D5\0027C0¥*†°&amp]which culture fails to detect, molecular methods offer a solution to this long-standing quantification problem in the cannabis testing field[/FONT]
 
Last edited:

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Drug Cannabis


A preliminary investigation of lung availability of cannabinoids by smoking marijuana or dabbing BHO and decarboxylation rate of THC-and CBD-acids
Marianne Hädenera, Sina Vietenb, Wolfgang Weinmanna, Hellmut Mahler
Forensic Science International 2018
doi:10.1016/j.forsciint.2018.12.021
Highly potent cannabis concentrates obtained by butane or by supercritical carbon dioxide-extraction are gaining popularity. These extracts called butane hash oil (BHO) with D9-tetrahydrocannabinolic acid A (THCA) contents above 60% are consumed by flash vaporization on a glowing titanium nail, followed by inhalation of the resulting vapor through a water pipe in a single puff — _a technique referred to as “dabbing”. We herein investigated the decarboxylation rate of THCA during artificial smoking of cannabis plant material and simulated dabbing, and the lung availability of D9-tetrahydrocannabinol (THC) which we define as the recovery of THC in the smoke and vapor condensates. Preliminary smoking and dabbing experiments were performed using an apparatus built in-house. Due to availability of cannabidiol (CBD)- rich hemp in Switzerland, we included a sample of CBD flowers in our experiments and investigated the decarboxylation and recovery of cannabidiolic acid (CBDA) and CBD, respectively. Decarboxylation of THCA and CBDA during combustion of the plant material and vaporization of the BHO, respectively, was complete. The high recovery of total THC (75.5%) by dabbing cannot be achieved by smoking marijuana. Lung availability ranged from 12% for mixed cannabis material with a rather low THC content, to approximately 19–27% for marijuana flowers, similar for THC in marijuana as for CBD in CBD-rich marijuana. In reality, when smoking a joint, further losses in recovery must be assumed by additional sidestream smoke. The rather high lung availability of THC via dabbing can explain the increased psychoactive and adverse effects associated with this new trend of cannabis consumption

AKT1 genotype moderates the acute psychotomimetic effects of naturalistically smoked cannabis in young cannabis smokers
DOI: 10.1038/tp.2015.219
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872423/

Cannabis Use in Turkey
Serap Annette Akgür, Melike Aydo?du
January 2019
https://www.researchgate.net/publica..._Use_in_Turkey
In Anatolian, psychoactive substances containing cannabis have been cultivated and consumed for various purposes in areas such as health, food and textile due to its special fibrous structure. Marijuana is the name of the public among these plant parts that are intoxicating and pleasant when used. With the recognition of the negative aspects of cannabis use, it has been viewed as a social problem and has been forbidden. Today, cannabis is on the list of prohibited drugs to be trained, consumed and sold under the Turkish Penal Code. In our country, the fight against drugs system established directly under the Prime Ministry is being carried out in a multidisciplinary way. Within the scope of these studies, comparable, reliable and objective scientific data such as the distribution of cannabis amounts seized between years 2002 and 2016, the number of incidents related to cannabis, and the distribution of cannabis related incidents to crime types were obtained. In 2016, 49.1% of illegal substance-related incidents were reported to becaused by cannabis. The prevalence of lifetime cannabis use in our country has been determined as 0.7%. In this research, the prevalence of cannabis use during adolescence which a risky period for substance use, cannabis use in migrants and refugees were discussed in detail. Attention has been drawn to the ambition and sales policies of products with hemp visual. In this context, with senior drug fighting council established in our country, demand reduction in Turkey, awareness, protection and prevention approaches, treatment and precaution implementations, law enforcement precautions, substance trafficking, narcotic operations with national and local projects, activities and education.

Characteristics that infuence purchase choice for cannabis products: a systematic review
Jennifer Donnan, Omar Shogan, Lisa Bishop, Michelle Swab and Maisam Najafzada
Journal of Cannabis Research (2022) 4:9
DOI: 10.1186/s42238-022-00117-0
https://jcannabisresearch.biomedcentra l.com/track/pdf/10.1186/s42238-022-00117-0.pdf
Introduction: When non-medical cannabis use became legal, government regulators implemented policies to encourage safer consumption through access to a regulated market. While this market is growing, sales still occur through unregulated channels. This systematic review identifes factors infuencing cannabis purchasing to help policymakers understand why consumers still purchase illicit market cannabis (registered with PROSPERO CRD42020176079). Methods: A comprehensive search strategy included databases in health, business, and social science felds (inception to June 2020). Studies were eligible for inclusion if they were conducted with persons who purchase cannabis and examine at least one attribute that would infuence purchase choice and were published in the English language. Studies could be of any methodological design. Two independent reviewers completed two levels of screening, and all extraction was verifed by a second reviewer. A qualitative synthesis of the fndings was completed. The quality of the included studies was assessed using the Mixed Methods Appraisal Tool. Results: Of the 4839 citations screened, 96 were eligible for full-text review and 35 were included in the fnal synthesis. Aspects of price were the most common factors (27 studies). Twenty studies measured price elasticity; most studies found that demand was price inelastic. Many other attributes were identifed (e.g., product quality, route of administration, product recommendations, packaging), but none were explored in depth. Eleven studies addressed aspects of product quality including demand elasticity based on quality, potency, and aroma. Studies also explored consumer-perceived “quality” but provided no defnition; diferences in quality appeared to impact consumer choice. Smoking cannabis appeared to be the preferred route of administration but was only examined in three studies. There was insufcient data to understand in the impact of other attributes on choice. There appeared to be preference heterogeneity for diferent attributes based on the consumer’s experience, reason for use, and gender. Conclusion: While price infuences choices, demand is relatively inelastic. This suggests that consumers may be seeking lowest-cost, unregulated cannabis to avoid reducing consumption. Beyond price, there is a signifcant gap in our understanding of consumer choices. Perceived quality does appear to impact choice; however, more research is needed due to the lack of a recognized defnition for cannabis quality

Cooking with cannabis: The rapid spread of (mis)information on YouTube
Lindsey Ouellette, Mary Cearley, Bryan Judge, Brad Riley, Jeffrey Jones
doi: 10.1016/j.ajem.2017.10.060
According to the 2015 National Survey on DrugUse and Health,marijuana
is the most commonly used illicit drug in the United States. Cannabis is traditionally consumed by smoke inhalation, however as marijuana legalization becomes more prominent, oral consumption of products containing cannabis (edibles) has become more prevalent. When consumed in edible form the effects of the primary psychoactive ingredient, delta-9-tetrahydrocannabinol (THC), differ notably from smoking cannabis. Psychoactive effects have a substantially delayed onset (30–60 min vs. 7–8 min), are described as being more intense, and may last over 6 h Because cooking methods and strengths of cannabis can vary, there is often great variation in potency of edibles from one batch to the next. These factors contribute to a risk of accidental overdose, especially by inexperienced users. Both accurate and inaccurate information regarding edibles and cooking with cannabis is freely available to any person with internet access. Video-sharing websites are a popular method of broadcasting that can be viewed by individuals on many platforms. This study aimed to characterize the content and popularity of dangerous trends involved in edible marijuana consumption by teens and young adults on the video-sharing Web site YouTube.

Expansion of female sex organs in response to prolonged virginity in Cannabis sativa (marijuana)
Ernest Small . Steve G. U. Naraine
Genet Resour Crop Evol
doi: 10.1007/s10722-015-0253-3
Female flowers of Cannabis sativa in wild-growing populations and in hemp plantations are almost always well supplied with pollen. The style-stigma portion of the pistils of such plants was found to average only about 3 mm in length and to invariably be two-branched. By contrast, “buds” (congested female inflorescences), the standard form of marijuana now produced in the illicit and medicinal marijuana sectors, are protected against pollen. This report documents that in the absence of pollen, the style-stigma parts of virgin pistils expand notably, average over 8 mm in length, and tend to develop more than two branches and to increase in girth. From an evolutionary viewpoint, this expansion of pollen-receptive tissue is an apparent adaptation for increasing the probability of fertilizing the females when males are extremely scarce. From a practical viewpoint, the expanded presence of stigma tissues may be both advantageous and disadvantageous. The high-THCsecretory gland heads of Cannabis tend to fall away from marijuana buds, significantly decreasing pharmacological potency, but many gland heads become stuck to the receptive papillae of the stigmas, reducing the loss. Although stigmas constitute a small proportion of marijuana, their distinctive chemistry could have health effects.

False hashish without cannabis resin
Lumír O. Hanuš, Dafna De La Vega, Michael Roman & Pavel Tomí?ek (2015)
Israel Journal of Plant Sciences, 62:4, 277-282,
DOI: 10.1080/07929978.2015.1053202

Folk Methodology of Charas (Hashish) Production and Its Marketing at Afridi Tirah, Federally Administered Tribal Areas (FATA), Pakistan
Muhammad Hamayun, Zabta Khan Shinwari
December 2004 Journal of Industrial Hemp 9(2):41-50
DOI: 10.1300/J237v09n02_04

Long term stability of cannabis resin and cannabis extracts
Christian Lindholst
Australian Journal of Forensic Sciences, 42:3, 181-190
Doi: 10.1080/00450610903258144
The aim of the present study was to investigate the stability of cannabinoids in
cannabis resin slabs and cannabis extracts upon long-term storage. The levels of tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and cannabigerol (CBG) on both neutral and acidic form were measured at room temperature, 48C and 7208C for up to 4 years. Acidic THC degrades exponentially via decarboxylation with concentration halve-lives of approximately 330 and 462 days in daylight and darkness, respectively. The degradation of neutral THC seems to occur somewhat slower. When cannabinoids were stored in extracted form at room temperature the degradation rate of acidic THC increased significantly relative to resin material with concentration halve-lives of 35 and 91 days in daylight and
darkness, respectively. Once cannabis material is extracted into organic solvents, care should be taken to avoid the influence of sunlight.

Marijuana
NIH, National Institute on Drug Abuse
Research Report Series
Last Updated July 2019
https://www.drugabuse.gov/node/pdf/1380/marijuana
from the director:
Changes in marijuana policies across states legalizing marijuana for medical and/or recreational use suggest that marijuana is gaining greater acceptance in our society. Thus, it is particularly important for people to understand what is known about both the adverse health effects and the potential therapeutic benefits linked to marijuana Because marijuana impairs short-term memory and judgment and distorts perception, it can impair performance in school or at work and make it dangerous to drive an automobile. It also affects brain systems that are still maturing through young adulthood, so regular use by teens may have a negative and long-lasting effect on their cognitive development, putting them at a competitive disadvantage and possibly interfering with their well-being in other ways. Also, contrary to popular belief, marijuana can be addictive, and its use during adolescence may make other forms of drug abuse or addiction more likely. Whether smoking or otherwise consuming marijuana has therapeutic benefits that outweigh its health risks is still an open question that science has not resolved. Although many states now permit dispensing marijuana for medicinal purposes and there is mounting anecdotal evidence for the efficacy of marijuana-derived compounds, there are currently no FDA-approved indications for “medical marijuana.” However, safe medicines based on cannabinoid chemicals derived from the marijuana plant have been available for decades and more are being developed. This Research Report is intended as a useful summary of what the most up-to-date science has to say about marijuana and its effects on those who use it — both young and old.
Nora D. Volkow, M.D. Director National Institute on Drug Abuse

New trends in cannabis potency in USA and Europe during the last decade (2008–2017)
Suman Chandra · Mohamed M. Radwan · Chandrani G. Majumdar · James C. Church · Tom P. Freeman, Mahmoud A. ElSohly
European Archives of Psychiatry and Clinical Neuroscience
Doi:10.1007/s00406-019-00983-5
Through the potency monitoring program at the University of Mississippi supported by National Institute on Drug Abuse (NIDA), a total of 18108 samples of cannabis preparations have been analyzed over the last decade, using a validated GC/ FID method. The samples are classified as sinsemilla, marijuana, ditchweed, hashish, and hash oil (now referred to as cannabis
concentrate). The number of samples received over the last 5 years has decreased dramatically due to the legalization of marijuana either for medical or for recreational purposes in many US states. The results showed that the mean ?9 –THC concentration has increased dramatically over the last 10 years, from 8.9% in 2008 to 17.1% in 2017. The mean ?9 -THC:CBD
ratio also rose substantially from 23 in 2008 to 104 in 2017. There was also marked increase in the proportion of hash oil samples (concentrates) seized (0.5–4.7%) and their mean ?9 -THC concentration (6.7–55.7%) from 2008 to 2017. Other potency monitoring programs are also present in several European countries such as The Netherlands, United Kingdom, France, and Italy. These programs have also documented increases in ?9 –THC concentrations and ?9 -THC:CBD ratios in cannabis. These trends in the last decade suggest that cannabis is becoming an increasingly harmful product in the USA and Europe.

The origins of cannabis smoking: Chemical residue evidence from the first millennium BCE in the Pamirs
Meng Ren, Zihua Tang, Xinhua Wu, Robert Spengle, Hongen Jiang, Yimin Yang and Nicole Boivin
Science Advances 12 Jun 2019:
Vol. 5, no. 6, eaaw1391
DOI: 10.1126/sciadv.aaw1391

Traditional Cannabis Cultivation in Darchula District, Nepal—Seed, Resin and Textiles
Robert Connell Clarke
November 2007Journal of Industrial Hemp 12(2):19-42
DOI: 10.1300/J237v12n02_03

Vaporized cannabis extracts have reinforcing properties and support conditioned drug-seeking behavior in rats.
Freels TG, Baxter-Potter LN, Lugo JM, Glodosky NC, Wright HR, Baglot SL, Petrie GN, Zhihao Y, Clowers BH, Cuttler C, Fuchs RA, Hill MN, McLaughlin RJ
J Neurosci. 2020 Jan 16. pii: 2416-19.
doi: 10.1523/JNEUROSCI.2416-19.2020.
Recent trends in cannabis legalization have increased the necessity to better understand the effects of cannabis use. Animal models involving traditional cannabinoid self-administration approaches have been notoriously difficult to establish and differences in the drug employed and its route of administration have limited the translational value of preclinical studies. To address this challenge in the field, we have developed a novel method of cannabis self-administration using response-contingent delivery of vaporized ?9-tetrahydrocannabinol-rich (CANTHC) or cannabidiol-rich (CANCBD) whole-plant cannabis extracts. Male Sprague Dawley rats were trained to nosepoke for discrete puffs of CANTHC, CANCBD, or vehicle (VEH) in daily one-hour sessions. Cannabis vapor reinforcement resulted in strong discrimination between active and inactive operanda. CANTHC maintained higher response rates under fixed ratio schedules and higher break points under progressive ratio schedules compared to CANCBD or VEH, and the number of vapor deliveries positively correlated with plasma THC concentrations. Moreover, metabolic phenotyping studies revealed alterations in locomotor activity, energy expenditure, and daily food intake that are consistent with effects in human cannabis users. Furthermore, both cannabis regimens produced ecologically relevant brain concentrations of THC and CBD and CANTHC administration decreased hippocampal CB1 receptor binding. Removal of CANTHCreinforcement (but not CANCBD) resulted in a robust extinction burst and an increase in cue-induced cannabis-seeking behavior relative to VEH. These data indicate that volitional exposure to THC-rich cannabis vapor has bona fide reinforcing properties and collectively support the utility of the vapor self-administration model for the preclinical assessment of volitional cannabis intake and cannabis-seeking behaviors.
SIGNIFICANCE STATEMENT The evolving legal landscape concerning recreational cannabis use has increased urgency to better understand its effects on the brain and behavior. Animal models are advantageous in this respect; however, current approaches typically employ forced injections of synthetic cannabinoids or isolated cannabis constituents that may not capture the complex effects of volitionalcannabis consumption. We have developed a novel model of cannabis self-administration using response-contingent delivery of vaporizedcannabis extracts containing high concentrations of ?9 tetrahydrocannabinol (THC) or cannabidiol (CBD). Our data indicate that THC-richcannabis vapor has reinforcing properties that support stable rates of responding and conditioned drug-seeking behavior. This approach will be valuable for interrogating effects

IC Ethnobotany

A narrative review of the ethnomedicinal usage of Cannabis sativa Linnaeus as traditional phytomedicine by folk medicine practitioners of Bangladesh
Shahriar S. M. Shakil, Matt Gowan, Kerry Hughes, Md. Nur Kabidul Azam, Md. Nasir Ahmed
Journal of Cannabis Research 3(1) December 2021
DOI: 10.1186/s42238-021-00063-3
Background There is a worldwide interest in the use of Cannabis sativa for biomedicine purposes. Cannabis has ethnomedicinal usage as a natural medicine in Bangladesh and cultivated during the British Empire period for revenues. Objective Folk medicine practitioners (FMPs) from different districts of Bangladesh have been using Cannabis sativa , but until now there have not been any compiled studies particularly regarding this practice. Hence, this review is an effort to retrieve the traditional usage of Cannabis sativa as a phytomedicine from published ethnomedicinal studies. Methods and materials Information was searched by using the search terms “ethnomedicinal Cannabis sativa and Bangladesh”; “Bangladesh cannabaceae and ethnomedicinal survey”; “ganja, bhang and folk medicine Bangladesh”; “tetrahydrocannabinol (THC), cannabinoid and therapeutic, clinical trial”; and “cannabis and pharmacological/biological” and retrieved from ethnobotanical articles available on PubMed, Scopus, Science Direct, and Google Scholar databases. A search of the relevant scientific literature also was conducted to assess the efficacy of the ethnomedicinal usage of Cannabis sativa. Results While reviewing over 200 ethnomedicinal plants’ survey articles, we found that FMPs of Bangladesh from 12 different districts used Cannabis sativa to treat cited ailments like sleep-associated problems ( n =5), neuropsychiatric and CNS problems ( n =5), and infections and respiratory problems ( n =5) followed by rheumatism, gastrointestinal, gynecological ( n =4 each), cancer, sexual, and other ailments including hypertension, headache, itch, increases bile secretion, abortifacient, dandruff, fever, and urinary problems ( n =1 each). There are a total of 15 formulations identified from the 11 out of 18 ethnomedicinal plant survey reports. The leaf was the main plant part used (53.8%), followed by root (23%), seed (7.7%) and flower, inflorescence, resin, and all parts 3.8% respectively. Conclusions Sales and cultivation of Cannabis are illegal at present in Bangladesh, but the use of Cannabis sativa as a natural phytomedicine has been practiced traditionally by folk medicine practitioners of Bangladesh for many years and validated through relevant pharmacological justification. Although Cannabis sativa possesses ethnomedicinal properties in the folk medicine of Bangladesh, it is, furthermore, needed to conduct biological research to consolidate pharmacological justification about the prospects and challenges of Cannabis and cannabinoids’ use in Bangladesh as safer biomedicine in the future.

Cannabis Ethnomedicine
Stephen Dahmer, Michael Balick
In book: Understanding Medical Cannabis November 2020
DOI: 10.4324/9780429343803-3
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Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history
Sara Anne Bonini, Marika Premoli, Simone Tambaro. Andrea Mastino
Journal of Ethnopharmacology 227 September 2018
DOI: 10.1016/j.jep.2018.09.004
Ethnopharmacological relevance: Cannabis sativa L. (C. sativa) is an annual dioecious plant, which shares its origins with the inception of the first agricultural human societies in Asia. Over the course of time different parts of the plant have been utilized for therapeutic and recreational purposes, for instance, extraction of healing oils from seed, or the use of inflorescences for their psychoactive effects. The key psychoactive constituent in C. sativa is called Δ-9-tetrahydrocannabinol (D9-THC). The endocannabinoid system seems to be phylogenetically ancient, as it was present in the most primitive vertebrates with a neuronal network. N-arachidonoylethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are the main endocannabinoids ligands present in the animal kingdom, and the main endocannabinoid receptors are cannabinoid type-1 (CB1) receptor and cannabinoid type-2 (CB2) receptor. Aim of the study: The review aims to provide a critical and comprehensive evaluation, from the ancient times to our days, of the ethnological, botanical, chemical and pharmacological aspects of C. sativa, with a vision for promoting further pharmaceutical research to explore its complete potential as a therapeutic agent. Materials and methods: This study was performed by reviewing in extensive details the studies on historical significance and ethnopharmacological applications of C. sativa by using international scientific databases, books, Master's and Ph.D. dissertations and government reports. In addition, we also try to gather relevant information from large regional as well as global unpublished resources. In addition, the plant taxonomy was validated using certified databases such as Medicinal Plant Names Services (MPNS) and The Plant List. Results and conclusions: A detailed comparative analysis of the available resources for C. sativa confirmed its origin and traditional spiritual, household and therapeutic uses and most importantly its popularity as a recreational drug. The result of several studies suggested a deeper involvement of phytocannabinoids (the key compounds in C. sativa) in several others central and peripheral pathophysiological mechanisms such as food intake, inflammation, pain, colitis, sleep disorders, neurological and psychiatric illness. However, despite their numerous medicinal benefits, they are still considered as a menace to the society and banned throughout the world, except for few countries. We believe that this review will help lay the foundation for promoting exhaustive pharmacological and pharmaceutical studies in order to better understand the clinical relevance and applications of non-psychoactive cannabinoids in the prevention and treatment of life-threatening diseases and help to improve the legal status of C. sativa.

Cannflavins – From plant to patient: A scoping review
Simon Erridgea, Nagina Mangala, Oliver Salazara, Barbara Pacchettib, Mikael H. Sodergren
Fitoterapia 146 (2020)
DOI:10.1016/j.fitote.2020.104712 .
Introduction: Cannflavins are a group of prenylflavonoids derived from Cannabis sativa L.. Cannflavin A (CFL-A), B (CFL-B) and C (CFL-C) have been heralded for their anti-inflammatory properties in pre-clinical evaluations. This scoping review aims to synthesise the evidence base on cannflavins to provide an overview of the current research landscape to inform research strategies to aid clinical translation.
Methods: A scoping review was conducted of EMBASE, MEDLINE, Pubmed, CENTRAL and Google Scholar databases up to 26th February 2020. All studies describing original research on cannflavins and their isomers were
included for review.
Results: 26 full text articles were included. CFL-A and CFL-B demonstrated potent anti-inflammatory activity via inhibition of 12-o-tetradecanoylphorbol 13-acetate induced PGE2 release (CFL-A half maximal inhibitory concentration (IC50): 0.7 μM; CFL-B IC50: 0.7 μM) and microsomal prostaglandin E synthase-1 (CFL-A IC50: 1.8 μM; CFL-B IC50: 3.7 μM). Outcomes were also described in preclinical models of anti-oxidation (CFL-A), antiparasitic activity (CFL-A, CFL-C), neuroprotection (CFL-A) and cancer (Isocannflavin B, a CFL-B isomer). In-silico screening identified that CFL-A has binding affinity with viral proteins that warrant further investigation.
Conclusions: Cannflavins demonstrate a number of promising therapeutic properties, most notably as an anti-inflammatory agent. Low yields of extraction however have previously limited research to small pre-clinical
investigations. Identification of cannflavin-rich chemovars, novel extraction techniques and recent identification of a biosynthetic pathway will hopefully allow research to be scaled appropriately. In order to fully evaluate the therapeutic properties of cannflavins focused research now needs to be embedded within institutions with a track-record of clinical translation.

Not Cannabis Specific
Ethnomedicinal ....antiinflammatory and analgesics
Anil Kumar
In book: Ethnomedicine: A Source of Complementary Therapeutics March 2021
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IC Flavonoids


Amorfrutin-type phytocannabinoids from Helichrysum umbraculigerum.
Pollastro, F., De Petrocellis, L., Schiano-Moriello, A., Chianese, G., Heyman, H., Appendino, G., & Taglialatela-Scafati, O.
Fitoterapia, 123, 13–17. (2017).
doi:10.1016/j.fitote.2017.09.010
Helichrysum umbraculigerum Less. has been reported to be a prolific producer of phytocannabinoids from the alkyl-, aralkyl-, normal-, and abnormal types. Investigation of an acetone extract from the aerial parts of the plant afforded two novel amorfrutin-type phytocannabinoids (3b, 4) and the new geranylated phloroglucinol 5a. The presence of cannabigerol (CBG, 1a) and its acidic precursor (pre-CBG, CBGA, 1b), previously reported from this plant, could not be confirmed, but the phenethyl analogue of CBG (Heli-CBG, 2a) and the methyl ester of its carboxylated version (2b) were isolated. Heli-CBG (2a) was assayed against a series of metabotropic (CB1 and CB2)- and ionotropic (thermo-TRPs) targets of phytocannabinoids, comparing its profile with the one of cannabigerol (CBG). A decreased affinity for cannabinoid receptor was observed, along with substantial retention of the thermo-TRP profile. The biogenetic relationships between the isoprenylated phenolics from H. umbraculigerum

Antinociceptive and Anti-Inflammatory Properties of Cannabidiol Alone and in Combination with Standardized Bioflavonoid Composition
Mesfin Yimam, Alexandria O'neal, Teresa Horm, Ping Jiao, Mei Hong, Shayna Rossiter, Lidia Brownell, Qi Jia
J Med Food 2021 Feb 10.
DOI: 10.1089/jmf.2020.0178
Symptom-alleviating therapies for osteoarthritis (OA) management are inadequate. Long-term application of first-line treatments, such as nonsteroidal anti-inflammatory drugs, is limited due to associated side effects. We believe that a combination of traditionally used botanical extracts, which have diverse active components that target multiple inflammatory pathways, may provide a safe and efficacious alternative to address the multifactorial nature of OA. Recently, cannabidiol (CBD), the major nonpsychoactive component of the hemp plant, has gained renewed global attention for its pharmacological actions. It has shown promise in reducing pain and inflammation in preclinical models of arthritis. In this study, widely employed inflammatory and noninflammatory animal pain models, such as the hot plate test, visceral pain model (writhing test), and carrageenan-induced rat paw edema model, were utilized to evaluate the antinociceptive and anti-inflammatory activity of CBD alone and in combination with standardized bioflavonoid compositions. CBD was tested at 5, 10, 20, and 40 mg/kg orally and at 5% topically. Administered alone, CBD produced dose-correlated, statistically significant pain inhibition in all the models. Enhanced performance in pain and inflammation reduction was observed when CBD was orally administered in complex with the bioflavonoid compositions. Data from this study show that for clinically meaningful efficacy against OA, CBD may have to be delivered in higher dosage or formulated with other medicinal plants with similar activities.

Flavonoid Derivative of Cannabis Demonstrates Therapeutic Potential in Preclinical Models of Metastatic Pancreatic Cancer.
Moreau, M., Ibeh, U., Decosmo, K., Bih, N., Yasmin-Karim, S., Toyang, N., … Ngwa, W.
Frontiers in Oncology, 9.(2019).
doi:10.3389/fonc.2019.00660
Pancreatic cancer is particularly refractory to modern therapies, with a 5-year survival rate for patients at a dismal 8%. One of the significant barriers to effective treatment is the immunosuppressive pancreatic tumor microenvironment and development of resistance to treatment. New treatment options to increase both the survival and quality of life of patients are urgently needed. This study reports on a new non-cannabinoid, non-psychoactive derivative of cannabis, termed FBL-03G, with the potential to treat pancreatic cancer. In vitro results show major increase in apoptosis and consequential decrease in survival for two pancreatic cancer modelsPanc-02 and KPC pancreatic cancer cells treated with varying concentrations of FBL-03G and radiotherapy. Meanwhile, in vivo results demonstrate therapeutic efficacy in delaying both local and metastatic tumor progression in animal models with pancreatic cancer when using FBL-03G sustainably delivered from smart radiotherapy biomaterials. Repeated experiments also showed significant (P < 0.0001) increase in survival for animals with pancreatic cancer compared to control cohorts. The findings demonstrate the potential for this new cannabis derivative in the treatment of both localized and advanced pancreatic cancer, providing impetus for further studies toward clinical translation.

Flavonoid variation in Cannabis L.
M. N. CLARK, B. A. BOHM
Botanical Journal of the Linnean Society, Vol. 79, Issue 3, Oct 1979, Pages 249-257
DOI: 10.1111/j.1095-8339.1979.tb01517.x
Fifty- three individual plants of Cannabis sativa L., grown from seed obtained from nine countries, were analyzed for their flavonoid constituents. Compounds present were the C-glycoflavones vitexin, orientin, cytisoside, and an 0"-glucoside of each. A C-glycuronide of luteolin was detected in several plants. Karmpferol and quercetin-3-0-diglucosides were also seen in many specimens. The distribution of compounds supported the subspecific treatment of the genus suggested by Small & Crouquist i 1976). The data were less helpful in resolving trends amongst the varieties recognized by those authors. A considerable amount of plant to plant variation was observed.

ISOLATION FROM CANNABIS SATIVA L. OF CANNFLAVIN-A NOVEL INHIBITOR OF PROSTAGLANDIN PRODUCTION
M. L. BARRETT, D. GORDON and F. J. Evmst
Biochemical Pharmacology. Vol. 34. No. 11. pp. 2019-2024. 1985
DOI: 10.1016/0006-2952(85)90325-9
The isolation from Cannabis sativa L. of an inhibitor of prostaglandin (PG) E2 production by cultured rheumatoid synovial cells is described. This agent, for which the name Cannflavin has been coined, is distinct from cannabinoids on the basis of isolation procedure, preliminary structural analysis and biological properties. The activity of Cannflavin has been compared with several established antiinflammatory drugs and the major cannabinoids.
 
Last edited:

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Hemp/Cannabis Cultivation

A call for weed research in industrial hemp ( Cannabis sativa L).
Sandler, L. N., & Gibson, K. A.
Weed Research.(2019).*
doi:10.1111/wre.12368
Industrial hemp (Cannabis sativa L.) is grown in more than 30 countries for fibre, seed and flowers, and acreage of cultivation is increasing globally. Hemp has long been promoted as a crop that competes well with weeds and requires little intervention to prevent yield losses. We conducted a literature review and found little peer-reviewed research to support this claim. We identified only three articles that specifically addressed weed management under field conditions and none provided information on hemp yield losses from weeds. These findings highlight a clear need for research-based information on interactions between weeds and hemp to address potential yield losses under various production conditions and provide a research-based framework for weed management in industrial hemp.

A comprehensive study of planting density and nitrogen fertilization effect on dual-purpose hemp (Cannabis sativa L.) cultivation
K.Tang, P.C. Struik, X. Yin, D. Calzolari, S. Musio, C. Thouminot,
M. Bjelková, V. Stramkale, G. Magagnini, S. Amaducci
Industrial Crops and Products Volume 107, 15 Nov 2017, Pages 427-438
doi:10.1016/j.indcrop.2017.06.033
Harvesting hemp (Cannabis sativa L.) for both stems and seeds is now a common practice in Europe while crop management strategies for dual-purpose hemp cultivation have not been properly addressed so far. In the present study, the effects of planting density and nitrogen fertilization on hemp stem and seed yields were tested with the cultivars Futura 75 and/or Bialobrzeskie in eight contrasting environments (Italy in 2013; Italy and Latvia in 2014; Italy (two sites), Latvia, the Czech Republic, and France in 2015). Stem yield ranged between 1.3 and 22.3 Mg ha?1 . The effects of planting density and nitrogen fertilization on stem yield did not interact significantly with each other, or with cultivar and harvest time. Increasing planting density from 30 to 120 plants m?2 and increasing nitrogen fertilization rate from 0 to 60 kg N ha?1 increased stem yield by 29% and 32%, respectively. Further increase in planting density and nitrogen fertilization did not result in a significant increase in stem yield. Seed yield ranged from 0.3 to 2.1 Mg ha?1 . The seed yield was not affected significantly by planting density between 30 and 240 plants m?2 . Although the seed yield showed an increasing trend with increasing nitrogen fertilization, the effects of nitrogen fertilization on seed yield were not statistically significant. To grow hemp as a dual-purpose crop it is recommended to plant 90–150 plants m?2 across all tested environments. Nitrogen fertilization rate at 60 kg N ha?1 was generally sufficient in the tested environments whereas further optimization of nitrogen fertilization requires accurate assessment of plant nitrogen status. To facilitate assessing plant nutritional status, a critical nitrogen dilution curve was determined for hemp and a practical method to determine nitrogen nutritional status was discussed.

A Dynamical Phyllotaxis Model to Determine Floral Organ Number
Miho S. Kitazawa and Koichi Fujimoto
PLoS Comput Biol. 2015 May; 11(5): e1004145.
doi: 10.1371/journal.pcbi.1004145
How organisms determine particular organ numbers is a fundamental key to the development of precise body structures; however, the developmental mechanisms underlying organ-number determination are unclear. In many eudicot plants, the primordia of sepals and petals (the floral organs) first arise sequentially at the edge of a circular, undifferentiated region called the floral meristem, and later transition into a concentric arrangement called a whorl, which includes four or five organs. The properties controlling the transition to whorls comprising particular numbers of organs is little explored. We propose a development-based model of floral organ-number determination, improving upon earlier models of plant phyllotaxis that assumed two developmental processes: the sequential initiation of primordia in the least crowded space around the meristem and the constant growth of the tip of the stem. By introducing mutual repulsion among primordia into the growth process, we numerically and analytically show that the whorled arrangement emerges spontaneously from the sequential initiation of primordia. Moreover, by allowing the strength of the inhibition exerted by each primordium to decrease as the primordium ages, we show that pentamerous whorls, in which the angular and radial positions of the primordia are consistent with those observed in sepal and petal primordia in Silene coeli-rosa, Caryophyllaceae, become the dominant arrangement. The organ number within the outmost whorl, corresponding to the sepals, takes a value of four or five in a much wider parameter space than that in which it takes a value of six or seven. These results suggest that mutual repulsion among primordia during growth and a temporal decrease in the strength of the inhibition during initiation are required for the development of the tetramerous and pentamerous whorls common in eudicots

A Preliminary Study of Pollen Dispersal in Cannabis sativa in Relation to Wind Direction
Ernest Small, Tanya Antle
March 2003 Journal of Industrial Hemp 8(2):37-50
DOI: 10.1300/J237v08n02_03
Pollen of Cannabis sativa is disseminated by wind in large amounts and for long distances, and regulations concerning the production of pedigreed seed of industrial hemp, therefore, often call for extremely large isolation distances to prevent unwanted pollination. In Europe and Canada, a standard distance of 5 km is required for the highest classes of hemp seed. This study examines the relative distribution of pollen from an isolated field over the 3-week maximum flowering period, with particular reference to wind direction. The amount of pollen distributed downwind was about six times the amount distributed upwind. In effect, this means that an isolation distance of 5 km on the downwind side is about equivalent to an isolation distance of 0.9 km on the upwind side. In theory, at the experimental site examined, the required isolation area could be reduced by about 58% while still achieving the equivalent of 5 km isolation in all directions. Given that weedy and illegally cultivated plants are widespread, making it difficult to ensure their absence over a distance of 5 km, it seems advisable, when possible, to take advantage of the considerably reduced isolation distance that is necessary on the upwind side. Pollen distribution appeared to follow the expected leptokurtic curve, reducing rapidly with initial distance from the source, but much more slowly with increasing distance. This makes it impossible to guarantee complete absence of potentially contaminating pollen in the field and, for practical purposes, a very low amount of undesired gene flow needs to be tolerated.

A Review on the Current State of Knowledge of Growing Conditions, Agronomic Soil Health Practices and Utilities of Hemp in the United States
Ifeoluwa Adesina, Arnab Bhowmik * , Harmandeep Sharma and Abolghasem Shahbazi
Agriculture 2020, 10, 129
doi: 10.3390/agriculture10040129
https://www.researchgate.net/publica..._United_States
Hemp (Cannabis sativa L.) is an emerging high-value specialty crop that can be cultivated for either fiber, seed, or cannabidiol (CBD). The demand for hemp and its products has been consistently on the rise in the 21st century. The United States of America (USA) has reintroduced hemp and legalized its production as an agricultural commodity through the 2018 Federal Farm Bill. Although there is a renewed interest in the adoption of hemp due to the emerging market, its production in the United States (US) remains limited partly because of unclear agronomic guidance and fertilization recommendations. This review article provides information on the current agronomic management practices that are available in the literature and identifies the future research needs for cultivating this multipurpose crop to address the growing market demands. Hemp production could be beneficial if managed properly. Hemp fertilizer requirements vary in accordance with the type of hemp grown (seed, fiber, or CBD), soil, environmental conditions and requires a wide range of macro- and micronutrients. Integrating management practices in hemp cultivation intended to build soil health is promising since the hemp cropping system is suitable for crop rotation, cover cropping, and livestock integration through animal waste applications. Hemp also has significant environmental benefits since it has the potential to remediate contaminated soils through phytoremediation, convert high amounts of atmospheric CO2 to biomass through bio-sequestration, and hemp biomass for bioenergy production. This review identifies that most of the agronomic research in the past has been limited to hemp fiber and, to some extent, hemp seed but not CBD hemp. With the increase in the global markets for hemp products, more research needs to be conducted to provide agronomic guidelines for sustainable hemp production.

A Study of Cotyledon Asymmetry in Cannabis sativa L.
Ernest Small, Tanya Antle
May 2007Journal of Industrial Hemp 12(1):3-14
DOI: 10.1300/J237v12n01_02
Although it has been known that the two cotyledons of Cannabis sativa L. seedlings tend to differ in size, the development and magnitude of this phenomenon have not previously been described. In
the dry seeds, the “outer cotyledon” (remote from the radicle) is about 50% heavier than the “inner cotyledon” (adjacent to the radicle). Two days after germination, the difference in fresh weight has been reduced considerably, but thereafter the outer cotyledon remains slightly more than 20% heavier than the inner. In about one in four seedlings, the tip of the outer (normally the larger) cotyledon is persistently covered by remnants of the seed coat, including a characteristic dark area which enclosed the tips of the cotyledons in the seed. In about one in ten seedlings, the achene shell (pericarp) remains up to several days on the tip of the outer cotyledon. These features serve to confirm the identity of very young seedlings of C. sativa, for which there has not previously been a reliable means of identification. Seeds from several sources did not differ significantly from each other with respect to weight ratio of the cotyledons, nor were there significant differences in cotyledon ratio found when seedlings were grown in darkness, a 16-hour light regime, and in continuous light. Microscopic connections of unknown significance were observed between the adpressed adaxial surfaces of the cotyledons in the seed.


Agronomy and photosynthesis physiology of hemp (Cannabis sativa L.)
Kailei Tang Thesis 174 pages 2018
DOI: /10.18174/434837
Hemp (Cannabis sativa L.) is a sustainable high-yielding crop that delivers valuable fibres, seeds and psychoactive substances. However, there is a lack of field experimental data on the cultivation of hemp because its production was largely abandoned in the last century. Hemp is now considered as an ideal crop to produce innovative biomaterials, and in particular, the dual-purpose hemp production (fibre + seed) is now the norm in European countries, driven by the shift of a rapidly expanding market for hemp seeds coupled with lower quality fibre requirements for innovative biomaterials. This study brought new information on the agronomy and photosynthesis physiology for the resurging production of hemp, particularly for dual-purpose production in Europe.
The effects of important agronomic factors, i.e. cultivar, planting density, and nitrogen fertilization, on the performance of the hemp crop were investigated under contrasting European environments. Based on the experimental data, for dual-purpose hemp production, a planting density of 90–150 plants m-2 is recommended for a monoecious cultivar that gives a long vegetative phase while leaving enough time for seed growth. A nitrogen fertilization rate of 60 kg N ha-1 was generally sufficient in the tested environments whereas further optimization of nitrogen fertilization requires accurate and precise assessment of plant nutritional status. To facilitate assessing plant nutritional status, a critical nitrogen dilution curve was determined for hemp.
The responses of leaf photosynthesis to nitrogen content and temperature were quantified using a biochemical model of C3 leaf photosynthesis, based on a complete set of photosynthetic measurements for hemp leaves. Then, by combining measurements and modelling, an upscaling was made from the leaf to the canopy level to analyse hemp’s photosynthetic nitrogen-use efficiency (NUE) and water-use efficiency (WUE) in response to water and nitrogen supply. The effect of nitrogen supply level on hemp’s NUE and WUE was largely determined by its effect on canopy size or leaf area index (LAI). The effect of short-term water stress on WUE and NUE was reflected in the stomatal regulation, whereas long-term water stress enhanced leaf senescence, reduced LAI but retained total canopy nitrogen content, and thus resulted in a further increase in WUE.
Findings in this thesis provided an improved understanding of the agronomy and photosynthesis physiology of hemp, particularly in relation to the dual-purpose production of hemp in Europe. Such understanding not only provides additional evidence that hemp can be grown as a sustainable crop over a wide range of climatic and agronomic conditions, but also provides essential information for parameterizing crop growth models. Prospects for further research were discussed in view of using the findings in this thesis in combination with a crop growth model to develop strategies for optimization of hemp cultivation and breeding.

AGRONOMY and CROP PHYSIOLOGY of FIBRE HEMP, A LITERATURE REVIEW
H.M.G. van der Werf
Center for Agrobiological Research (CABO-DLO)
Objectives of this literature review.
https://edepot.wur.nl/346939
Within the framework of the National Hemp Research Programme, research
is conducted to investigate the potential of hemp as a source of fibre for the paper industry in the Netherlands. This programme started on January 1, 1990 and should reach a conclusion on the economic feasability of hemp for fibre by the end of 1992. Major subjects of research in the program are:
-Breeding
-Soil-borne diseases
-Crop physiology and agronomy
-Harvesting and conservation
-Pulp and paper technology
-Economy and logistics
Research on crop physiology is being carried out within a research project at CABO, research on agronomy within a joint LUW-PAGV project. The objective of the research projects concerning crop physiology and agronomy of fibre hemp is to answer the question: How much and what kind of fibre can hemp yield in the Netherlands? This literature review will focus on the literature results most relevant with respect to that question. The objectives of the literature review are:
1. The identification of research objectives in crop physiology and agronomy of fibre hemp, resulting in a coherent program of experiments serving the objectives.
2. Relevant sections of this literature review will be used in an article presenting the results of the experiments conducted by Meijer and Matthijssen in 1988 and 1989.
3. The literature review may be useful to researchers involved in other parts of the hemp research programme.
While conducting the literature research and writing the review,
literature on crop physiology of hemp turned out to be rare.
Consequently this review mainly covers the agronomy of hemp production

Agronomy of fibre hemp (Cannabis sati6a L.) in Europe
P.C. Struik, S. Amaducci, M.J. Bullard, N.C. Stutterheim, G. Venturi, H.T.H. Cromack
Industrial Crops and Products 11 (2000) 107–118
Doi: 10.1016/S0926-6690(99)00048-5
Fibre hemp may yield up to 25 t above ground dry matter per hectare (20 t stem dry matter ha?1) which may contain as much as 12 t ha?1 cellulose, depending on environmental conditions and agronomy. Its performance is affected by the onset of flowering and seed development. Effects of cultivar and management on yield and quality were tested at three contrasting sites in Italy, the Netherlands and the UK in three years, making use of standardized protocols for experimental design and research methodology. Highest yields (up to 22.5 t dry matter ha?1) were
obtained in Italy when later cultivars were used. Attainable yields proved slightly lower in the Netherlands and much lower in the UK. The quality of the cellulose was relatively stable over the growing season, but lignification may proceed rapidly some time after flowering. Crop development was very rapid and crops maintained green leaf area for a long time, thus radiation interception was considerable. The radiation use efficiency changed during development. It was lower after flowering (about 1.0 g MJ?1 PAR) than before (about 2.2 g MJ?1 PAR). Growing earlier cultivars to obtain some seed set advanced the reduction in radiation use efficiency. Nitrogen proved to affect yield only slightly. A relatively small amount of fertiliser will be adequate to cover the crop’s needs. Plant density declined during growth in a site-specific manner when it was high initially. Very low plant densities may not show this
self-thinning but reduced yield and (especially) quality. Final plant densities were proven to depend more on initial plant stands than expected from literature. This was true at all three contrasting sites and in the different years. Nitrogen and plant density hardly interacted within one site. Results suggest that hemp can yield large quantities of useful cellulose when ecologically adapted cultivars are sown in proper plant densities. The cultivation is environmentally friendly with little harmful accumulation or emission of chemical inputs. More research on ideotyping is required and breeding efforts should be broadened.

*AHP Cannabis Inflorescence Quality Control Monograph
Roy Upton RH DAyu, Lyle Craker, Mahmoud ElSohly, Aviva Romm , Ethan Russo, Michelle Sexton, Jahan Marcu, Diana Swisher
American Herbal Pharmacopoeia Cannabis Inflorescence and Leaf 2013 264 pages
https://american-safe-access.s3.amaz...ph_Preview.pd f
https://herbal-ahp.org/online-orderi...-qc-monograph/
This AHP monograph is historic in that it is the first formal pharmacopoeial monograph on cannabis (Cannabis spp.) developed in the US in more than 70 years. The first monograph was introduced into the United States Pharmacopoeia in 1850 and was removed from the twelfth edition in 1942. Considering the widespread use of cannabis, it is important for there to be quality control guidance, whether used for medicinal or non-medicinal purposes. While identification of the species is typically not in question, quantitative testing of cannabinoids is in question. With a variety of analytical platforms being used, including TLC, HPLC, and GC, all with varying levels of accuracy, this an issue addressed in the monograph.

An Introduction to Industrial Hemp and Hemp Agronomy
D.W. Williams, Rich Mundell
https://www2.ca.uky.edu/agcomm/pubs/ID/ID250/ID250.pdf
I ndustrial hemp (Cannabis sativa L.) was at one time a major agronomic crop in Kentucky. In A History of the Hemp Industry in Kentucky (1951), author James F. Hopkins reported that it was a common crop on central Kentucky farms from the late eighteenth and nineteenth centuries. Nearly all farms growing hemp at that time were mostly interested in the fiber it produced. Farms with limited labor pools grew less or no hemp. It was a very labor-intensive crop both at harvest and during processing. Hemp became impractical as a crop in 1937 with the passage of the Marihuana Tax Act, which was an attempt by the U.S. federal government to reduce the production of Cannabis. The attempt to reduce production was largely due to the fact it was also being consumed as an intoxicant in addition to being utilized as a fiber crop. The new tax and licensing requirements made Cannabis production less profitable and much more bureaucratic, essentially ending production on large scales. There was a brief resurrection of hemp production during World War II, namely the Hemp for Victory campaign. During the campaign, U.S. farmers were asked to grow hemp for fiber to be used in the war effort. In Kentucky, most hemp grown during the campaign was for seed to be shipped to northern states where it was planted as a fiber crop. All Cannabis became fully illegal to produce or even possess following the passage of the Controlled Substance Act (often abbreviated CSA) in 1978. Under the CSA, all forms of Cannabis were classified as Schedule 1 controlled substances and remain in that classification today. Other substances under Schedule 1 classification include the drugs heroin and cocaine. The Agricultural Act of 2014, also known as the Farm Bill, provided for legal pilot research programs with industrial hemp to be administered by either state departments of agriculture or universities. In Kentucky, the General Assembly had passed Senate Bill 50 in 2013 in anticipation of the 2014 Farm Bill. Under those parameters, the Kentucky Department of Agriculture (KDA) began administering the statewide pilot research program in spring 2014. The pilot research program in Kentucky has grown each year since and continues to grow today under the management of the KDA


An Update on Plant Photobiology and Implications for Cannabis Production
Mark Lefsrud, Samuel Eichhorn Bilodeau, Bo-Sen Wu, Anne-Sophie Rufyikiri, Sarah MacPherson
Plant Sci. 10:296.
DOI: 10.3389/fpls.2019.00296
This review presents recent developments in plant photobiology and lighting systems for horticultural crops, as well as potential applications for cannabis (Cannabis sativa and C. indica) plant production. The legal and commercial production of the cannabis plant is a relatively new, rapidly growing, and highly profitable industry in Europe and North America. However, more knowledge transfer from plant studies and horticultural communities to commercial cannabis plant growers is needed. Plant photosynthesis and photomorphogenesis are influenced by light wavelength, intensity, and photoperiod via plant photoreceptors that sense light and control plant growth. Further, light properties play a critical role in plant vegetative growth and reproductive (flowering) developmental stages, as well as in biomass secondary metabolite synthesis and accumulation. Advantages and disadvantages of widespread greenhouse lighting systems that use high pressure sodium lamps or light emitting diode (LED) lighting are known. Some artificial plant lighting practices will require improvements for cannabis production. By manipulating LED light spectra and stimulating specific plant photoreceptors, it may be possible to minimize operation costs while maximizing cannabis biomass and cannabinoid yield, including tetrahydrocannabinol (or ? 9 -tetrahydrocannabinol) and cannabidiol for medicinal and recreational purposes. The basics of plant photobiology (photosynthesis and photomorphogenesis) and electrical lighting systems are discussed, with an emphasis on how the light spectrum and lighting strategies could influence cannabis production and secondary compound accumulation.

Apparent Increase in Biomass and Seed Productivity in Hemp (Cannabis sativa) Resulting from Branch Proliferation Caused by the European Corn Borer (Ostrinia nubilalis)
Ernest Small, David Marcus, Gail Butler, A. R. McElroy
May 2007 Journal of Industrial Hemp 12(1):15-26
DOI: 10.1300/J237v12n01_03
The European corn borer (Ostrinia nubilalis Hübner), or ECB, is a major pest of hemp (Cannabis sativa L.). During the course of a study of hemp germplasm and cultivar accessions, the ECB attacked
the vertical stem leader of hundreds of plants. At the site of invasion the main stem was typically destroyed, and the plant became strongly branched. Although the damaged plants were an average 9% shorter, mean shoot weight was 20% heavier. Seed productivity was also greater, based on a visual scale. The practical significance of such an “overcompensation” response to insect damage, with damaged plants growing more robustly and productively than their undamaged counterparts, is controversial. Certainly, some aspects of productivity, such as fibre quality, are detrimentally affected by the ECB. The insect showed a preference for larger plants, but was indifferent to the level of tetrahydrocannabinol, the chief intoxicant of C. sativa.

Architecture and Florogenesis in Female Cannabis sativa Plants
April 2019Frontiers in Plant Science 10
Ben Spitzer-RimonShai DuchinNirit BernsteinRina Kamenetsky
DOI: 10.3389/fpls.2019.00350
The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be “non-inductive” or “vegetative”, but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture, and to maximize plant productivity and uniformity in medical Cannabis.

ATMOSPHERIC TRANSPORTATION OF MARIHUANA POLLEN FROM NORTH AFRICA TO THE SOUTHWEST OF EUROPE
BALTASAR CABEZUDO, MARTA RECIO, JOSt MARIA SANCHEZ-LAULHI, MARIA DEL MAR TRIGO, FRANCISCO JAVIER TORO and FAUSTO POLVORINOS
Atmospheric Environment Vol. 31, No. 20, pp. 3323 3328, 1997
DOI: 10.1016/S1352-2310(97)00161-1
As a result of aerobiological samples taken on the Costa del Sol (S. Spain), Cannabis sativa L. (marihuana) pollen was detected from May to September 1991-1996, always sporadically and usually during the afternoons. Sampling was by two volumetric spore traps set up in Malaga and Estepona, two coastal towns approximately 90 km apart. A study of the days when this pollen was recorded points to the movement of air masses from North Africa to southern Spain. Furthermore, the isentropic air trajectories calculated for these days reinforce the possibility of the pollen originating in marihuana plantations in northern Morocco (Rif). This study demonstrates the application of aerobiology to the control of the source, quantity and phenology of the crop.

Auxin and Sexuality in Cannabis sativa.
Heslop-Harrison, J. (1956).
Physiologia Plantarum, 9(4), 588–597.
doi:10.1111/j.1399-3054.1956.tb07821.x
It is now well established that sex expression in various monoecious Cucurbitaceae may be modified by auxin treatment. Laibach and Kribben (l'.loOa, 1950 b. 1950 c and 1950 d) bave shown that the proportion of female flowers produced by the cucumber (C'j/c///ni.v .satiinis) may be sub.slantially increased by treatment during early growtb witb [3-indoIeacetic acid flAA) and a-naplitbaleaeacotic acid (NAA). applied eilber in liinolin paste medium or by spraying' in aqueous solution. Nilsch. Kurtz. Liverman and Went il952) bave reporled a similar result witb squash (Cucurbita pepo var. Table Queen), in which spraying with NAA in aqueous solution al 100 p.p.m. at the two leaved stage induced the formation of the first temiilc flower bud al about Ibe ninth node, the first female flowers in control plants not being formed until the twentieth node or later. Corresponding results have been obtained hy Wittwcr and Hillycr (1954) with the cucumber varieties National Pickling.; aud Burpee Hybrid as well as with squash.
Arguing from their findings witb cucumber, Laibach and Kribben (1950) bave suggested tbal tbe sexuality of flowers is dependent upon the conctentration of native auxin available in the leaf axil during the period of flower formation. Were this to be eslablisbed as generally true for the flowering plants, it would constitute an important step forward in our understanding of the control of flowering aud flower morphogenesis. Whilst the results so far reported for the monoecious cucurbits are themselves unequivocal it would obviously be valuable to have a demonstration that auxiu levels govern flower sexuality in dioecious plants also. The present paper provides evidence that tbis may indeed he .so in the dioecious species Cannabis sativa (hemp).

?-Cyclocitral is a conserved root growth regulator
Alexandra J. Dickinsona, Kevin Lehnera, Jianing Mid, Kun-Peng Jiad, Medhavinee Mijara, José Dinnenyc, Salim Al-Babilid, and Philip N. Benfeya
PNAS May 21, 2019 116 (21) 10563-10567; first published May 8, 2019
Doi: 10.1073/pnas.1821445116
Natural compounds capable of increasing root depth and branch- ing are desirable tools for enhancing stress tolerance in crops. We devised a sensitized screen to identify natural metabolites capable of regulating root traits in Arabidopsis. ?-Cyclocitral, an endoge- nous root compound, was found to promote cell divisions in root meristems and stimulate lateral root branching. ?-Cyclocitral res- cued meristematic cell divisions in ccd1ccd4 biosynthesis mutants, and ?-cyclocitral–driven root growth was found to be independent of auxin, brassinosteroid, and reactive oxygen species signaling pathways. ?-Cyclocitral had a conserved effect on root growth in tomato and rice and generated significantly more compact crown root systems in rice. Moreover, ?-cyclocitral treatment enhanced plant vigor in rice plants exposed to salt-contaminated soil. These results indicate that ?-cyclocitral is a broadly effective root growth promoter in both monocots and eudicots and could be a valuable tool to enhance crop vigor under environmental stress.

Bee diversity and abundance on flowers of industrial hemp (Cannabis sativa L.)
C. O'Brien, H.S. Arathi
Biomass and Bioenergy 122 (2019) 331–335
doi: 10.1016/j.biombioe.2019.01.015
Industrial hemp, (Cannabis sativa L.), one of the earliest crops spun for fiber, is now used for a variety of commercial products including paper, textiles, clothing, biodegradable plastics, biofuel, food, animal feed etc., all of which are derived from hemp fiber or seeds. Being wind pollinated, dioecious and staminate hemp plants produce large amounts of pollen that are attractive to bees. Hemp flowering in northern Colorado, where this study was conducted, occurs between the end of July and the end of September. This time period coincides with a dearth of pollinator-friendly crop plants in the region, making hemp flowers a potentially valuable source of pollen for foraging bees. Here we present the diversity and abundance of bees collected in the fields of flowering hemp. A total of 23 different genera of bees were collected of which the European honeybee, Apis mellifera at 38% of the total abundance was the most dominant followed by Melissodes bimaculata at 25% and Peponapis pruinosa at 16%. These three genera made up nearly 80% of the total abundance. While hemp does not produce any nectar, the pollen rich nature of the flowers can make hemp an ecologically valuable crop. As cultivation of hemp continues to expand, we expect insect pests on hemp to also become prevalent. Our results documenting bee diversity in flowering hemp provides the impetus for the development of integrated pest management plans that protect pollinators while controlling pests

Best Management Practices
Northern California Farmers Guide

Hezekih Allen & Kristin Nevedal
https://static1.squarespace.com/stat...sGuide_HHC.pdf
Introduction
Water
Erosion and sediment
Amendments and fertilizers
Disease and pest control
Rats, Mice, and other critters
Recommended sources
Conclusion
The Northern California Farmers Guide is a community-based collaborative project that was written for the benefit of our communities and the watersheds we thrive in. We would like to acknowledge and thank the many community members, farmers, environmentalists, advocates, and sponsors who have contributed to the writing, designing, and funding of this still evolving project. Enjoy!

Botany of Cannabis sativa L.: Identification, Cultivation and Processing
Suman Chandra, Hemant Lata, A. M. Galal, MA ElSohly
March 2011 Planta Medica 77(05)
DOI: 10.1055/s-0031-1273533
Cannabis sativa L. is among the oldest known cultivated plants, with a long history of medical use. Cannabis produces a unique class of terpenophenolic compounds called cannabinoids, 104 of which have been isolated, the major biologically active one being ?9-tetrahydrocannabinol. Cannabidiol, an antiepileptic, is also important. Cannabis is an annual, normally dioecious and occasionally monoecious, wind-pollinated species and is highly allogamous (cross-fertilization) in nature. Therefore, maintaining the efficacy of selected high ?9-tetrahydrocannabinol-yielding elite varieties grown from seeds under field or greenhouse conditions is very difficult. Thus, a careful screening of elite mother clones using GC-FID and their propagation using vegetative cuttings or advanced biotechnological approaches, including micropropagation, is the most suitable way to maintain quality. This chapter describes the botany, species debate, phenotype, screening of high-yielding clones using GC-FID, indoor and outdoor cultivation, micropropagation, quality assurance of propagated plants, harvesting, processing, and storage

Byssinosis in Hemp Mill Workers
John McPartland
January 2003 Journal of Industrial Hemp 8(1):33-44
DOI: 10.1300/J237v08n01_04
This review article concerns byssinosis, a respiratory disease that affects workers in textile mills. According to experts in the field of occupational medicine, hemp mill workers suffer worse than workers in flax, cotton, jute, and sisal mills. The causative factor in hemp dust has not been determined with certainty. However, this review assembles evidence that implicates hemp dust contaminated by bacterial endotoxins, rather than fungal toxins or constituents in hemp itself. Endotoxins are expressed by Gram negative bacteria, and Enterobacter cloacae is a prime suspect. It is proposed that endotoxin contamination occurs during the biological retting process, and not before (in living hemp plants) or after (within the textile mill). Methods of preventing and treating byssinosis are assessed, including some new proposals for management.

*California Agriculture Journal (special issue Cannabis)
JULY–DECEMBER 2019 • VOLUME 73, NUMBER 3–4
https://ucanr.edu/repository/fileAcc...l_attachment=N
In 1953, amid reports that cannabis was growing around San Mateo County, the local sheriff’s office and the UC Agricultural Extension Service in Half Moon Bay issued a booklet entitled Identify and Report Marihuana. The booklet envisioned “total eradication” of cannabis. The authors couldn’t have imagined that, in 2017, the San Mateo County Board of Supervisors would pass an ordinance allowing greenhouse cultivation of cannabis in the county’s unincorporated areas. A lot can happen in 60-plus years — such as voter approval of Proposition 64, the 2016 ballot measure that altered California law to allow the recreational use of cannabis by adults. The measure’s passage presented policymakers with the challenge of regulating, licensing and taxing a large, complex and fast-changing recreational cannabis industry — a challenge made more acute because scientific research on many aspects of cannabis in California had never been conducted at scale. UC is now working to fill that research gap. At least nine UC research centers, most of them new, now focus entirely or in part on cannabis (page 106). A sense of momentum has begun to suffuse cannabis research.
NEWS AND OPINIONS
100 Special issue: Cannabis
101 California cannabis regulation: An overview
, Goldstein and Sumner, In 2016, Proposition 64 decriminalized the possession and use of cannabis by anyone in California aged 21 or over. But the 2015 Medical Marijuana Regulation and Safety Act had begun the process of regulating cannabis in the state.
103 Sidebar: A concise cannabis guide: History, laws and regulations, Crowder
RESEARCH NEWS
104 Restrictions and opportunities for UC cannabis research
, Crowder Cannabis is legal in California but illegal in the United States. The plant’s ambiguous status cuts off many avenues of cannabis research — but leaves other approaches wide open.
106 The rapid evolution of UC cannabis research, Crowder, At campuses across the UC system, cannabis researchers are grappling with questions that have accompanied legalization.
CONVERSATIONS
113
Richard Parrott, CalCannabis
115 Scott Bauer, California Department of Fish and Wildlife
117 Amanda Reiman, Flow Kana
RESEARCH AND REVIEW ARTICLES
119 First known survey of cannabis production practices in California
Wilson et al. Most growers in this survey produced their crop outdoors or in greenhouses, relied primarily on groundwater, used biologically based inputs for pest management and employed seasonal workers paid at fixed piece rates. 128 Characteristics of farms applying for cannabis cultivation permits Schwab et al. In Humboldt County, larger and faster-growing cannabis farms apply for permits at higher rates than do smaller or slowergrowing farms.
136 Retail cannabis prices in California through legalization, regulation and taxation Goldstein et al. A study investigates price patterns at California cannabis retailers during a period of major regulatory changes.
146 Watering the Emerald Triangle: Irrigation sources used by cannabis cultivators in Northern California Dillis et al. Reported subsurface water use among North Coast cannabis cultivators is widespread and may become increasingly common.
154 Costs of mandatory cannabis testing in California Valdes-Donoso et al. California’s safety standards for cannabis — compared to standards in other states and to standards for food products in California — are tight.
161 Perceptions of cannabis among Humboldt County timberland and ranchland owners Valachovic et al. A Humboldt County survey investigates traditional agriculturalists’ views on cannabis cultivation.
169 “We can’t just be a county that supports inebriants”: Voices of the noncannabis agricultural community LaChance Interviews with noncannabis producers in Northern California revealed a variety of concerns about legal cannabis production, from access to land and crop shifts to outsider investments.
177 Growers say cannabis legalization excludes small growers, supports illicit markets, undermines local economies Bodwitch et al. The survey sample was small, but results suggest regulations may need to be modified to incentivize grower participation in state licensing programs.
185 Cannabis farmers or criminals? Enforcement-first approaches fuel disparity and hinder regulation Polson and Petersen-Rockney Siskiyou County, and many other counties, chose not to recognize cannabis cultivation as agriculture. This ethnographic study reveals the effects on parity in farmer rights and access to resources.

Canada’s ‘croptober’ surge pushes cannabis inventory over 1 million kilograms
https://mjbizdaily.com/canadas-cropt...lion-kilograms

Cannabis cultivation in the world: heritages, trends and challenges
Pierre-Arnaud Chouvy
EchoGeo 48 | 2019 : avril / juin 2019
https://journals.openedition.org/echogeo/17591
Despite cannabis being the most common illegal drug crop in the world and its worldwide presence, very little is known about its production, trade, and consumption at the global scale. This is due mostly to over a century of global prohibition and the dangers associated to researching illegal drug crop production. Worse, the limited data available about cannabis cultivation is most often inaccurate, unreliable, and highly controversial. While this has always been problematic, in terms of sheer knowledge and informed policy-making, it has now become even more acute of an issue as global trends towards decriminalisation and legalisation are already provoking negative unintended consequences in poor producing countries. This article is an effort to present the state of the current knowledge and the present and

Cannabis cultivation: Methodological issues for obtaining medical-grade product.
Chandra, S., Lata, H., ElSohly, M. A., Walker, L. A., & Potter, D.
Epilepsy & Behavior, 70, 302–312. (2017).
doi:10.1016/j.yebeh.2016.11.029
As studies continue to reveal favorable findings for the use of cannabidiol in the management of childhood epilepsy syndromes and other disorders, best practices for the large-scale production of Cannabis are needed for timely product development and research purposes. The processes of two institutions with extensive experience in producing large-scale cannabidiol chemotype Cannabis crops—GW Pharmaceuticals and the University of Mississippi—are described, including breeding, indoor and outdoor growing, harvesting, and extraction methods. Such practices have yielded desirable outcomes in Cannabis breeding and production: GW Pharmaceuticals has a collection of chemotypes dominant in any one of eight cannabinoids, two of which—cannabidiol and cannabidivarin—are supporting epilepsy clinical trial research, whereas in addition to a germplasm bank of high-THC, high-CBD, and intermediate type cannabis varieties, the team at University of Mississippi has established an in vitro propagation protocol for cannabis with no detectable variations in morphologic, physiologic, biochemical, and genetic profiles as compared to the mother plants. Improvements in phytocannabinoid yields and growing efficiency are expected as research continues at these institutions

Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.
Livingston, S. J., Quilichini, T. D., Booth, J. K., Wong, D. C. J., Rensing, K. H., Laflamme?Yonkman, J., … Samuels, A. L.
The Plant Journal. (2019).
doi:10.1111/tpj.14516
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile-like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two-photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12–16 secretory disc cells and strongly monoterpene-dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed redshifted autofluorescence, eight secretory disc cells and less monoterpene-dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co-expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.

Cannabis (hemp) positive skin tests and respiratory symptoms
Jeffrey R Stokes, MD; Rita Hartel, RN; Linda B Ford, MD; and Thomas B Casale, MD
Cannabis (hemp) positive skin tests and respiratory symptoms.
Annals of Allergy, Asthma & Immunology, 85(3), 238–240. (2000)
doi:10.1016/s1081-1206(10)62473-8*
Background: We have noted several patients who had rhinitis and/or asthma
symptoms when exposed to Cannabis plants in the summer months. Cannabis plants
are common in the Midwest.
Objectives: To examine whether Cannabis might be a clinically important allergen, we determined Cannabis pollination patterns in the Omaha area for 5 years, the prevalence of skin test positivity, and the association with respiratory symptoms.
Methods: Airborne Cannabis (and other weed) pollens were collected using a Rotorod air impactor, and pollen counts were done using a standardized protocol.
Results: Measurable Cannabis pollen count was not recorded until the last 2 weeks of July. Peak pollination typically occurred during mid- to late-August, and comprised up to 36% of the total pollen counts. Cannabis pollen was not observed after mid-September. To determine the prevalence of skin test positivity, we added Cannabis to the multi-test routine skin test battery. Seventy-eight of 127 patients tested (61%) were skin test positive. Thirty of the 78 patients were randomly selected to determine if they had allergic rhinitis and/or asthma symptoms during the Cannabis pollination period. By history, 22 (73%) claimed respiratory symptoms in the July through September period. All 22 of these subjects were also skin test positive to weeds pollinating during the same period as Cannabis (ragweed, pigweed, cocklebur, Russian thistle, marsh elder, or kochia).
Conclusions: The strong association between skin test reactivity, respiratory symptoms, and pollination period suggests that Cannabis could be a clinically important aeroallergen for certain patients and should be further studied.

UCDAVIS
Cannabis & Hemp Research Center
https://cannabis.ucdavis.edu/
Advancing Knowledge
Unlocking new insights through research to guide production, policy, use and testing

Cannabis sativa L.,
Heslop-Harrison J. and Heslop-Harrison Y.
In :Evans L. T. (Ed.) The induction of flowering. (1969).
Ithaca, New York: Cornell University Press, pp. 206-226. Macmillan
FIND DOI or LINK

CBD HEMP PRODUCTION COSTS & RETURNS FOR CONNECTICUT FARMERS IN 2020
Jeremy Jelliffe, Rigoberto A. Lopez, & Shuresh Ghimire
https://are.uconn.edu/wp-content/upl...OR66_Feb25.pdf
WHAT IS THE ISSUE?
Since its approval in the 2014 Federal Farm Bill under a pilot program, production of industrial hemp, particularly for cannabinoid (CBD) products, has skyrocketed in the United States. This trend has been largely driven by the potential for large profits from a burgeoning consumer demand for CBD products. In Connecticut, hemp cultivation became legal for growers licensed by the Connecticut Department of Agriculture in May 2019. However, in
spite of the excitement, there is lack of information on potential revenues, costs, and risks of producing CBD hemp to guide investment decisions. This report aims to provide such information for the first time to current and potential Connecticutfarmers for 2020, the second year of hemp cultivation in the state.
WHAT DID THIS STUDY FIND?
For a representative farm of 10 acres, as shown in Table 1 (back cover), this study finds that for the 2020 growing season:
• The total cost per acre is estimated at $19,189 or $9.59 per pound of dried hemp flower.
• About two-thirds of the total cost per acre ($12,619) is variable, meaning that it changes with the level of production, and one-third of it ($6,570) is fixed.
• At the prevailing local price of $1.25, yield of 2,000 lbs/acre, and 9% CBD, total revenues are $22,500 per acre, leading to $3,311 in profits per acre, or $9,881 per acre net return over variable costs.
• Break-even (zero profit) combinations of dry flower yield, CBD content, and prices are shown in Figure 1. Points above the curves indicate positive
profits and points below them indicate losses.
• The ultimate profitability of this enterprise will depend on technical abilities of individual farmers (yield and CBD content) as well as external
market forces, predominately CBD prices. Because CBD hemp prices continue to decline as many states are rapidly expanding production and there is a possible further threat from CBD imports, policy changes challenges remain to ensure the long-term profitability and economic sustainability of CBD hemp production in Connecticut. Challenges also remain about finding a suitable buyer and controlling THC content in this nascent industry.

Challenges towards Revitalizing Hemp: A Multifaceted Crop
Craig Schluttenhofer, and Ling Yuan
Trends in Plant Science, November 2017, Vol. 22, No. 11
DOI: 10.1016/j.tplants.2017.08.004
https://www.cell.com/action/showPdf?...2817%2930177-2
Hemp has been an important crop throughout human history for food, fiber, and medicine. Despite significant progress made by the international research community, the basic biology of hemp plants remains insufficiently understood. Clear objectives are needed to guide future research. As a semi-domesticated plant, hemp has many desirable traits that require improvement, including eliminating seed shattering, enhancing the quantity and quality of stem fiber, and increasing the accumulation of phytocannabinoids. Methods to manipulate the sex of hemp plants will also be important for optimizing yields of seed, fiber, and cannabinoids. Currently, research into trait improvement is hindered by the lack of molecular techniques adapted to hemp. Here we review how addressing these limitations will help advance our knowledge of plant biology and enable us to fully domesticate and maximize the agronomic potential of this promising crop.

Characterisation of Cannabis accessions with regard to cannabinoid content in relation to other plant characters
E.P.M. de Meijer, H.J. van der Kamp & F.A. van Eeuwijk
Euphytica 62: 187-200, 1992.
DOI:10.1007/BF00041753
Ninety seven Cannabis accessions were evaluated for cannabinoid content and non-chemical plant characters. Variation within populations for cannabinoid content, and consistency of chemical characters at the population level were investigated. The relationship between chemical and other plant characters was very limited. Leaflet width and phenological data can be used for a rough prediction of the chemical phenotype on a population level. Various combinations of cannabinoid content and other economic plant characters were observed, thus a breeding programme will not be hampered by strict linkage. For a selection programme a direct analysis of cannabinoids will be inevitable

Closing the yield gap for cannabis: a meta-analysis of factors determining cannabis yield
Backer RG, Rosenbaum P, Mccarty V, Eichhorn_bilodeau S, Lyu D, Ahmed M, Robinson W, Lefsrud M, Wilkins O and Smith DL
Front. Plant Sci. 10:495. (2018) .
doi:10.3389/fpls.2019.00495
Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square metre. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square metre and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis and biocontrol.

COLLECTION OF CANNABIS AT INF
Przemys?aw Baraniecki and Gra?yna Ma?kowska
The INF Cannabis collection
https://archive-ecpgr.cgiar.org/file...nnabis_INF.pdf
Polish Cannbis Collection kept at the Institute of Natural Fibres has been formally founded in 1998. Together with the Linum collection it is a part of the Polish Gene Bank supervised and partially funded by the Ministry of Agriculture. The scientific supervision and coordination of plant genetic resources in Poland is the Plant Breeding and Acclimatization Institute (IHAR) in Radzików. The Collection is held at the Department of Department of Bast Fibre Plants Breeding and Agronomy of the Institute. The collection holds 131 Cannabis accessions, including 24 breeding lines. Mrs. Gra?yna Ma?kowska is in charge of the collection. Poland to ratify the Convention on Biological Diversity in 1995
• Foundation of National Program of Cultivated Plant Genetic Resources Protection with Coordination Center at Plant Breeding and Acclimatization Institute in Radzików
• Foundation of Linum Gene Collection at the INF - 1982
• Foundation of Cannabis Gene Collection at the INF – 1998

Combined HPLC Analysis of Organic Acids and Furans Formed During Organosolv Pulping of Fiber Hemp.
Gosselink, R. J. A., van Dam, J. E. G., & Zomers, F. H. A.
Journal of Wood Chemistry and Technology, 15(1), 1–25.(1995).
doi:10.1080/02773819508009497
During organosolv pulping of fiber hemp (Cannabis sativa L) with a
mixture of ethanollwater, delignification is catalyzed by released acetic acid and formic acid in the effluent. The major sources of acetic acid are the acetyl groups, as determined by means of the acetyl balance, whereas formic acid is mainly formed as degradation product of polysaccharides. Trace amounts of other short chain organic acids and furans, formed from cellulose and hemicellulose, are also present in the effluent. A relatively simple and reliable HPLC method is described to analyze simultaneously the carboxylic acids and furans quantitatively. Acetyl and formyl contents of hemp core (4.3% and 0.2% respectively) and hemp bast (1.3% and 0.2%) are also analyzed with HPLC after an alkaline saponification.

Comparative Study of Three Varieties of Cannabis sativa L. Cultivate in Different Region of Morocco
Bouayoun Taoufik, Stambouli Hamid, El Bouri Aziz, Farah Abdellah, Saidi Seddik, Ez zoubi Yassine, Fadil Mouhcine, Charrouf Zoubida, Tabyaoui Mohamed
International Journal of Pharmacognosy and Phytochemical Research Vol 9, No 5 (2017)
DOI: 10.25258/phyto.v9i5.8142
In this study, an experiment of hemp crops was conducted between in four different regions of the country, and concerned three varieties Santhica 27, Epsilon 68 and Futura 75. The tests were conducted in areas with geographical, climatic and soil characteristics different than European cultures, thats why it was necessary to verify the behavior of these plants by controlling their ?-9-THC content. Sampling, drying and ?-9-THC evaluation in these crops were performed according to procedure B of European Regulation, and using a simplified method based on the determination of the ratio ? = ?-9- THC/CBD. The best dry matter yields, plant height and density are obtained with the variety Futura 75 in the four sites, followed by Epsilon 68 and Santhica 27. The average contents of these crops in ?-9-THC were ranged from 0.013% to 0.027% for the variety Epsilon 68, between 0.023% and 0.035% for the variety Futura 75. Analysis of this latter variety by GC / MS SIM mode and LC/MS/MS allowed the identification of traces of ?-9-THC. In the other hand, the ratio ? has been evaluated to 0.042 and 0.047 respectively for Epsilon 68 and Futura 75 varieties which is similar to those usually obtained for hemp fiber crops and stays much lower than 0.2. The Principal Component Analysis confirms the existence of possible correlations for each variety separately. Overall, the results reveal the existence of correlations between different parameters including the one between the dry matter yield, plant height and density

Comparing hemp (Cannabis sativa L.) cultivars for dual-purpose production under contrasting environments
K.Tang, P.C.Struik, X.Yin, C.Thouminot, M.Bjelková, V.Stramkale, S.Amaducci
Industrial Crops and Products Volume 87, Sept 2016, Pages 33-44
doi: 10.1016/j.indcrop.2016.04.026
Interest in hemp as a multi-purpose crop is growing worldwide and for the first time in 2015 it was cultivated in Europe on more than 20.000 ha as a dual-purpose crop, for the seeds and for the fibre. In the present study, fibre and seed productivity of 14 commercial cultivars were tested in four contrasting European environments (Latvia,the Czech Republic, France, Italy). Atfull flowering,the stem yield ranged from 3.7 Mg ha?1 to 22.7 Mg ha?1, the bast fibre content ranged from 21% to 43%, and the bast fibre yield ranged from 1.3 Mg ha?1 to 7.4 Mg ha?1. When harvesting was postponed from full flowering until seed maturity, the stem yield of monoecious cultivars significantly increased but in dioecious cultivars it decreased at all tested sites, except for Italy. Only the early cultivars Fedora 17 and Markant produced seed in the most northern location Latvia. The seed yield ranged from 0.3 Mg ha?1 to 2.4 Mg ha?1 in Italy, France and the Czech Republic. The cultivar effect on stem and seed yield was mainly determined by the genetic variation in time of flowering. Stem yield at full flowering was strictly related to the duration of the vegetative phase while seed yield was lowest in the late flowering cultivar. The late cultivar CS is suitable for stem and fibre production as it had the highest stem yield at full flowering in all locations. Both Fedora 17 and Futura 75 are candidate cultivars for dual-purpose production in Italy, France and the Czech Republic, with Fedora 17 being more suitable for seed production and Futura 75 for fibre production. The application of modelling to design production strategies for dual-purpose hemp is promising. However, accurate parameterisation is needed based on large data sets and diverse genetic background.

Constraints to dry matter production in fibre hemp (Cannabis sativa L.).
Meijer, W. J. M., van der Werf, H. M. G., Mathijssen, E. W. J. M., & van den Brink, P. W. M.
European Journal of Agronomy, 4(1), 109–117.1995).
doi:10.1016/s1161-0301(14)80022-1
Fibre hemp (Cannabis sativa L.) may be an alternative to wood as a r aw material for pulp and paper production. Research was done to assess the potential productivity of fibre hemp and to identify constraints to that productivity. Growth analyses were done on hemp crops in three consecutive years, using several cultivars and seeding rates. In 1987 the crops suffered severely from fungal diseases;
stem dry matter yields were 9.4 t h a-
In 1988 and 1989 fungicides were applied and stem dry matter yields were respectively 11.9 t ha- 1 and 13.6 t ha- 1
The number of living plants m-2 ranged from 86 to 823 at emergence, depending on treatment, and from 38 to 102 at final harvest. Increased seeding rates led to earlier canopy closure and higher initial biomass production, but more plants died during the growing season and the stem yield at final harvest was not affected by seeding rate. Average radiation-use efficiency (RUE; above-ground accumulated dry matter divided by intercepted photosynthetically active radiation) for the entire growing season under favourable growing conditions was 1.9 g Mrl, which is low compared to other C3 crops. Leaf photosynthesis rates at saturating light
intensities, however, were high: 30 kg C02ha-1 h- 1
The light extinction coefficient of hemp canopies was high (0.96), and may have reduced canopy photosynthesis rate and RUE. The high lignin content of the stems, and the high fat and protein content in the seed, also reduced RUE. Furthermore, dry matter invested in the tap root and in shed dead leaves was ignored. The RUE was also underestimated because an appreciable proportion of the biomass of plants that died during the growing season could not be collected. More research is needed to optimize cultural practices in fibre hemp.

COUNTRY REPORT FOR SLOVAK REPUBLIC
https://archive-ecpgr.cgiar.org/file...ollections.pdf


Crop physiology of fiber hemp (Cannabis sativa L,)
Hayo van der Werf
Thesis 1994
https://edepot.wur.nl/202103
Fibre hemp (Cannabis sativa L.) may be an alternative to wood as a raw material for the production of paper pulp. The effects of environmental factors and cultural measures on the functioning, yield and quality of fibre hemp crops in the Netherlands were investigated. Until flowering (generally in August), the radiation use efficiency (RUE, above-ground dry matter accumulated per unit of photosynthetically active radiation intercepted) of hemp was 2.2 g MJ"1 , after flowering it dropped to 1.1 g MJ"1 . This reduction was mainly caused by senescence, resulting in a lower rate of canopy photosynthesis. When flowering was prevented by artificial daylength extension the RUE remained high throughout September, and stem dry matter yield was increased by 2.7 t ha"1 Breeding late-flowering hemp seems a promising stategy to improve the stem yield potential of hemp. Hemp grows at low temperatures, its base temperature is 1 °C for leaf appearance, and 2.5 °C for canopy establishment. Thermal time is a simple and accurate tool to describe leaf appearance and light interception in field-grown fibre hemp. Hemp is grown at high plant densities to improve stem quality and to increase the allocation of above-ground dry matter to the stem. However, in dense hemp crops self-thinning (densityinduced mortality) may occur, causing losses of dry matter. It was established that in fibre hemp optimum plant density is approximated by the highest density possible without inducing self-thinning. During self-thinning an increase in biomass is accompanied by a reduction in plant density, consequently, in fibre hemp optimum plant density is a function of yield. N fertilization affected self-thinning: at similar above-ground dry matter, a higher plant density was sustained at 80 than at 200 kg N ha"1 . The reduced mortality at the low nitrogen level resulted from a shift from mainly competition for light, which enhances differences in plant size, to competition for nutrients, which does not give large plants a disproportionate advantage over small plants. Variability of both weight and height of hemp plants was higher at 200 than at 80 kg ha"1 N, and more suppressed plants were present at 200 than at 80 kg ha"1 N. Sexual dimorphism contributed to variability of height and weight, but the effects were smaller than those of nitrogen fertilization. It is concluded that fibre hemp may be an interesting 'new' crop for arable farmers in the Netherlands: hemp may be profitable, its stems potentially have a large non-food market, the crop requires little or no biocide and suppresses weeds and some major soil-borne diseases

Not Cannabis specific
Cryopreservation of Pollen and Pollen Embryos, and the Establishment of Pollen Banks
Y.P.S.Bajaj
International Review of Cytology Volume 107, 1987, Pages 397-420
doi: 10.1016/S0074-7696(08)61083-9
The cryopreservation of pollen plays an important role in hybridization programs and in the conservation of genetic resources in agricultural and forest biotechnology. In addition to the several potentials outlined, the most important aspect is the storage of “recalcitrant” pollen of important fruit and forest trees and agricultural crops for overcoming geographic, seasonal, and physiological barriers in hybridization. Moreover, in some plant species the flowering period is very short and the pollen grains are very short-lived; thus the plant breeder is not able to handle it. In many fruit crops where yield is erratic, controlled pollination with stored pollen would help to stabilize production. It also avoids the interference of foreign or illegitimate pollen. The establishment of “pollen banks,” in addition to fulfilling various other needs, considerably reduces maintenance of orchards and nurseries for the regular supply of pollen.

Cultivation of low tetrahydrocannabinol (THC) Cannabis sativa L. cultivation in Victoria,
Australia: Do we know enough?
Talia Humphries, Singarayer Florentine
AJCS 13(06):911-919 (2019)
doi: 10.21475/ajcs.19.13.06.p1669
Late 2017, the ban on the cultivation and consumption of low tetrahydrocannabinol (THC) Cannabis sativa L. in Victoria, was lifted by the Federal Government of Australia. Its legalization presents the opportunity for Victoria to become a leading producer and distributer of these economically valuable hemp products. However, as a novel crop to Victoria, there is little information available for obtaining economically viable yields. Therefore, the objectives of this review were to firstly, develop an understanding of the environmental requirements shared by C. sativa cultivars, and what conditions promote fibre and grain yields. Secondly, it seeks to identify what farming practices have been conducted throughout Europe, Canada and China, and to explore whether these practices could be adapted to Victoria. Thirdly, the review will assist in making recommendations regarding which cultivars would be ‘potential’ candidates for commencing trials under Victorian climates so to find out the varieties that can provide high yields for fibre, grain and dual-purpose production. This review notes that Victoria shares a similar climate to central Europe, and has an ideal climate for the development of a successful hemp industry, as it has suitable lengths of daylight throughout spring and summer months and meets the precipitation requirements. This review has thus strongly suggested that the properties and attributes of European varieties of C. sativa should be further researched for site-specific cultivation in Victoria for fibre, grain and dual-purpose production in order to maximise harvest yields.

Current status of the Dutch Hemp collection
Noor Bas Marcel Toonen and Luisa Trindade
https://archive-ecpgr.cgiar.org/file...Collection.pdf
A collection of Cannabis accessions has been established since 1988 at the Centre for Plant Breeding and Reproduction Research (CPRO-DLO), currently Plant Research International B.V. (PRI), a private organization. Seed samples were acquired from genebanks, breeding
and research institutes, seed companies and botanical gardens.
Material of the collection has been used in the ‘National Hemp Programme’ to investigate the feasibility of hemp as a raw material for paper pulp production and to improve cultivars in order to increase yield, quality and disease resistance.

Czech Hemp and National Industry Program
Václav Sladký, Marie Široká
May 2007 Journal of Industrial Hemp 12(1):81-85
DOI: 10.1300/J237v12n01_09
The Czech Hemp Association (CHA) was founded to reintroduce industrial hemp to Czech fields as well as to processing plants, bringing it to various industries. The CHA aims for every region to establish small hemp industrial centers able to process 7400 to 10000 tons of hemp fiber annually. The basic prerequisite for a successful regional development is a sufficient growing area within a distance of 70 to 100 km from the functional scutching line, together with mechanisms for harvesting. In several Czech regions pilot projects of this type have been started.

Defining Hemp: A Fact Sheet
Renée Johnson
Congressional Research Service
https://www.everycrsreport.com/files...47c1ee833d.pdf
Botanically, hemp and marijuana are from the same species of plant, Cannabis sativa, 1 but from different varieties or cultivars.2 However, hemp and marijuana are genetically distinct forms of cannabis that are distinguished by their use and chemical composition as well as by differing cultivation practices in their production. While marijuana generally refers to the cultivated plant used as a psychotropic drug (whether used for medicinal or recreational purposes), hemp is cultivated for use in the production of a wide range of products, including foods and beverages, personal care products, nutritional supplements, fabrics and textiles, paper, construction materials, and other manufactured and industrial goods. Hemp and marijuana also have separate statutory definitions in U.S. law. Despite these differences, growing hemp has been restricted in the United States until recently, and the U.S. market has been largely dependent on imports for finished products and as an ingredient for use in further processing. Hemp’s association with marijuana placed its production under U.S. drug laws wherein all cannabis varieties, including hemp, were considered Schedule I controlled substances under the Controlled Substances Act (CSA).3 Since the late 1950s, the U.S. Drug Enforcement Administration (DEA) has strictly controlled and regulated hemp production. Prior to the late 1950s, hemp in the United States was considered an agricultural commodity, and the U.S. Department of Agriculture (USDA) supported its production. 4 Restrictions on U.S. hemp production and marketing were relaxed by changes enacted in the 2014 farm bill (Agricultural Act of 2014, P.L. 113-79) and were further relaxed in the 2018 farm bill (Agriculture Improvement Act of 2018, P.L. 115-334). These changes provide further differentiation between hemp and marijuana in terms of farm policy and federal regulatory oversight. The Food and Drug Administration (FDA) maintains oversight of hemp-derived consumer products under the Federal Food, Drug, and Cosmetic Act (21 U.S.C. §§ 301 et seq.). FDA’s jurisdiction includes hemp and hemp-derived products as a food and food ingredient, as well as an ingredient for use in body products, cosmetics, dietary supplements, and therapeutic products. Hemp and marijuana are distinct in several key ways: (1) statutory definitions and regulatory oversight, (2) chemical and genetic compositions, and (3) production practices and use. This fact sheet describes these differences, which are summarized in Figure 1.


IN DUTCH
De geschiktheid van de bodem van Nederland voor de teelt van Cannabis sativa (hennep) en Miscanthus sinensis 'Giganteus'
G.A. van Soesbergen
H.A.J. van Lanen
https://edepot.wur.nl/303291

IN DUTCH
De invloed van de temperatuur op de groei en ontwikkeling van vezelhennep (Cannabis sativa)
M. Wijlhuizen
https://edepot.wur.nl/333922
Temperatuur is een belangrijke factor die de plantengroei beïnvloedt. De invloed van de temperatuur op de groei van hennep is nog nooit gekwantificeerd. In dit verslag is getracht de invloed van de temperatuur op vezelhennep te kwantificeren. Hiervoor zijn fytotron- en veldproeven
uitgevoerd. Bij de fytotronproeven werden de planten bij verschillende temperaturen tussen 10 en 28 °C geteeld. Bij de veldproeven werd in 1990, 1991 en 1992 op verschillende tijdstippen van maart tot en met mei gezaaid. Met de gegevens van zowel de fytotron- als de veldproeven zijn basistemperaturen voor groei en ontwikkeling berekend. Een basistemperatuur (Tb) van 0 à 1 °C is berekend voor de periode van zaai tot 50 % opkomst in het veld. Een temperatuursom (Tb = 0) van 88,3 (68 -109,5) graaddagen was berekend van zaai tot 50 % opkomst. Bij de veldproeven werd een Tb van 1 °C voor bladverschijning en een Tb van
2,5 °C voor de toename van het percentage onderschepte PAR berekend. Bij de fytotronproeven namen de bladverschijningssnelheid en de lengtegroeisnelheid lineair toe tussen 10 en 28 °C. Voor de bladverschijning werd een Tb van 5,7 °C en voor de lengtegroei een Tb van 2,5 °C berekend. Bij de veldproeven verklaarde de tijd uitgedrukt in de temperatuursom vanaf opkomst, met de juiste T^ 97,8 % van de variantie van het bladstadium en 98,6 % van de variantie van het percentage onderschepte PAR. De tijd uitgedrukt in dagen na opkomst verklaarden veel minder. De temperatuursom is een eenvoudige en nauwkeurige methode om zowel de bladverschijningssnelheid als de toename van de onderschepte PAR te beschrijven. Uit de gegevens van de fytotronproeven bleek dat de temperatuur geen invloed had op de
drogestofverdeling tussen blad en stengel, noch op de SLA (specifiek bladgewicht). Ook het bastaandeel in de stengel werd niet beïnvloed door de temperatuur. De planten die bij de lagere temperaturen groeiden, waren korter bij eenzelfde plantgewicht dan de planten die bij de hogere temperaturen groeiden. De extinctiecoëfficiënt, berekend uit de gegevens van de veldproeven, was ongeveer 1.

Diamidoximated cellulosic bioadsorbents from hemp stalks for elimination of uranium (VI) and textile waste in aqueous systems
Manpreet Kaura Preeti Tewatiaa Gaurav Rattanb Sonal Singhalc Anupama Kaushik
Journal of Hazardous Materials Available online 11 May 2021
https://www.sciencedirect.com/scienc...7zsza4CYo2X3bU
Selective abolition of hazardous U(VI) ions from nuclear power plants and removal of toxic colorants from textile industries pose great challenge. The work aims to develop cellulosic bioadsorbents from waste stalks of local weed,Cannabis sativa, commonly known as hemp. Cellulose nanofibers (PCFs) were chosen as substrates owing to their unique characteristics like surface hydroxyl groups, large surface to volume ratio and excellent mechanical properties. PCFs were isolated from hemp stalks and their structural characterization using FTIR, TGA and XRD ensured retrieval of pure crystalline cellulose. PCFs were modified via copolymerization to obtain diaminomaleonitrile adorned cellulose grafts (DAMNC) and further converted to get diamidoxime functionalized cellulose (DAOC). DAOC exhibited exceptional affinity with uranium (VI) ions, safranin-o and methylene blue dyes due to presence of two amidoxime groups. Sorption capability was ascertained for optimization of parameters like contact time, pH selectivity, adsorbent dosage and concentration. Sorption followed Pseudo second-order kinetic model with maximum sorption of 220 mg/g, 19.01 mg/g and 46.4 mg/g for U(VI) ions, SO and MB, respectively. EDX mapping revealed uniform adsorption of all the three pollutants on DAOC while XPS ascertained that the sorption originated from multiple interactions between the adsorbent and the pollutants.
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Diketopiperazine Indole alkaloids from hemp seed
Phytochemistry Letters
Volume 18, December 2016, Pages 77-82
doi: 10.1016/j.phytol.2016.09.001
Hemp seeds from non-drug varieties of Cannabis sativa L. are an important source of food and medicine. In continuation of our ongoing study on hemp seed,two pairs of stereoisomers of diketopiperazine indole alkaloid (12S, 22R)-Dihydroxyisoechinulin A (1), (12S, 22S)-Dihydroxyisoechinulin A (2) and (12R/S)- Neoechinulin A (3) were isolated. Their structures were elucidated with UV, IR, NMR, MS, CD spectra, ECD and chiral HPLC analysis techniques. This type of alkaloid is more often reported from fungi, such as Aspergillus and Eurotium, than from plant sources. Since common molds can contaminate herbal medicines, various hemp seed samples and the metabolites of the main fungi isolated from hemp seed were analyzed using HPLC. The data suggested thatthe isolated compounds are rare constituents of hemp seed, rather than fungal contamination. Thus, in this study, these compounds were isolated from hemp seed for the first time, and a previous study’s prediction of endogenous indole alkaloids in hemp was confirmed. Meanwhile, Neoechinulin A could promote SIRT1 expression in HEK293 cell lines. SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases.

Dioecious hemp (Cannabis sativa L.) plants do not express significant sexually dimorphic morphology in the seedling stage
Lesley G. Campbell, Kristen Peach & Sydney B. Wizenberg
Nature Scientific Reports 2021 11:16825
https://www.ncbi.nlm.nih.gov/pmc/art..._96311.pdf.pdf
DOI: 10.1038/s41598-021-96311-w
Some economically important crop species are dioecious, producing pollen and ovules on distinct, unisexual, individuals. On-the-spot diagnosis of sex is important to breeders and farmers for crop improvement and maximizing yield, yet diagnostic tools at the seedling stage are understudied and lack a scientific basis. Understanding sexual dimorphism in juvenile plants may provide key ecological, evolutionary and economic insights into dioecious plant species in addition to improving the process of crop cultivation. To address this gap in the literature, we asked: can we reliably differentiate males, females, and co-sexual individuals based on seedling morphology in Cannabis sativa, and do the traits used to distinguish sex at this stage vary between genotypes? To answer these questions, we collected data on phenotypic traits of 112 C. sativa plants (50 female, 52 male, 10 co-sexuals) from two hemp
cultivars (CFX-1, CFX-2) during the second week of vegetative growth and used ANOVAs to compare morphology among sexes. We found males grew significantly longer hypocotyls than females by week 2, but this difference depended on the cultivar investigated. Preliminary evidence suggests that co-sexual plants may be distinguished from male and female plants using short hypocotyl length and seedling height, although this relationship requires more study since sample sizes of co-sexual plants were small. In one of the cultivars, two-week old male plants tend to produce longer hypocotyls than other plants, which may help to identify these plants prior to anthesis. We call for increased research effort on co-sexual plants, given their heavy economic cost in industrial contexts and rare mention in the literature. Our preliminary data suggests that short hypocotyl length may be an indicator of co-sexuality. These results are the first steps towards developing diagnostic tools for predicting sex using vegetative morphology in dioecious species and understanding how sexual dimorphism influences phenotype preceding sexual maturity.


DIVERSITY IN CANNABIS
ETIENNE DE MEIJER
https://edepot.wur.nl/207065

Economic Viability of Industrial Hemp in the United States: A Review of State Pilot Programs
Tyler Mark, Jonathan Shepherd, David Olson, William Snell, Susan Proper, and Suzanne Thornsbury
USDA
Economic Research Service, Economic Information Bulletin Number 217 February 2020
https://www.ers.usda.gov/webdocs/pub...30/eib-217.pdf
After a hiatus of almost 45 years, the Agricultural Act of 2014, Public Law 113-79 (the 2014 Farm Bill) reintroduced industrial hemp production in the United States through State pilot programs. U.S. industrial hemp acreage reported by State pilot programs increased from 0 in 2013 to over 90,000 acres in 2018, the largest U.S. hemp acreage since the 146,200 acres grown in 1943. While the U.S. hemp industry grew rapidly and commercial hemp production was legalized again by the 2018 Farm Bill, the industry’s long-term economic viability is uncertain. This study documents outcomes and lessons learned from the State pilot programs and examines legal, agronomic, and economic challenges that may affect the transition from the pilot programs to economically viable commercial production. Competition with alternative crops for acreage, global competitiveness, market transparency, and the ability to manage regulatory and market risks will determine patterns of development in the emerging U.S. hemp industry.

(not in English)
Effect of drip irrigation on yield and evapotranspiration of fibre hemp (Cannabis sativa L.)
Borivoj Peji?, Stanko Mili?, Vladimir Sikora, Ivana Baji?
January 2018
Ratarstvo i povrtarstvo 55(3):130-134
https://scindeks.ceon.rs/Article.asp...1-39441803130P
DOI: 10.5937/RatPov1803130P
The experiments showing the effect of drip irrigation on yield and evapotranspiration of fibre hemp (Cannabis sativa L.) were conducted at the experimental field of the Alternative Crops Department, Institute of Field and Vegetable Crops, Novi Sad, Serbia. Irrigation was based on the water balance method. Daily evapotranspiration (ETd) was computed from the reference evapotranspiration (ETo) and crop coefficient (kc) 0.5, 0.9 and 1.1 from sowing to 3-4 pair of leaves, from 3-4 pair of leaves to appearance of male flowers and from appearance of male flowers to the end of the season, respectively. ETo was calculated using Hargreaves equation. The irrigation depth was restricted to the soil depth of 0.4 m. In other words, irrigation started when readily available water in the soil layer of 0.4 m was completely depleted by plants. The irrigation rate was 30 mm at the beginning of the season, 40 mm in the middle of the season, and the amount of water added by irrigation was 320 mm during the entire season. Irrigation significantly affected the yield of fresh stems, fresh leaves, flowers and plant height, but not stem diameter and fibre content. Water used on evapotranspiration in irrigation conditions (ETm) was 470 mm, while in non-irrigated control variant it amounted to 129 mm (ETa). These preliminary results could be used as a good platform for hemp growers in the region, in terms of optimizing the use of irrigation water.

Effect of industrial hemp (Cannabis sativa L) planting density on weed suppression, crop growth, physiological responses, and fibre yield in the subtropics
Jack Hall, Surya P. Bhattarai* and David J. Midmore
Renewable Bioresources 2014; 2:1.
https://www.hoajonline.com/journals/...2-6237-2-1.pdf
doi: 10.7243/2052-6237-2-1
Population density of industrial hemp (Cannabis sativa L.) in the field influences crop growth habit, fibre yield and quality. Therefore, optimization of plant population density is required to control growth and secure fibre yield and quality. Initial hand-thinned plant populations of 100, 200, 300 and 400 plants m-2 were established in a replicated field trial. Density of planting significantly influenced weed suppression and a number of phenological characters, and yield of industrial hemp. Weed suppression increased with increasing plant population. An increase from 100 to 200 plants m-2 markedly reduced weed weight from 23.2 to 6.5 g m-2. Further reductions in weed weights were observed at 300 plants m-2 (2.6 g m-2) and 400 plants m-2 (1.5 g m-2). Weekly height data showed that the high-density plantings resulted in shorter plants at harvest due to a more rapid decline in growth rate than for the low density planting (100 plants m-2). Stem thickness was inversely related to plant population density as low density produced thicker stems compared to that of high density planting. Leaf chlorophyll content and root mass m-2 were not significantly affected by differences in planting density. Raw fibre yields were greatest at 300 plants m-2, which was significantly higher in comparison to 100 plants m-2 (128.4 vs 102.8 g dry weight m-2). Yields were very poor overall with a maximum of 1.28 t ha-1 of raw bast compared to European yields of 2–3 t ha-1. Low yields were attributed to the unsuitable short photoperiods that caused early flowering and therefore shorter stem length in the current variety under trial. New varieties or crop management practices that delay flowering are necessary for regions of short day duration to produce economically viable fibre yields for the industry in subtropical Queensland, Australia.

Effect of Nitrogen Fertilization on Growth and Yield of Industrial Hemp
PAPASTYLIANOU, P., KAKABOUKI, I., & TRAVLOS, I.
(Cannabis sativa L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 46(1), 197.(2017).
doi:10.15835/nbha46110862
Fibre hemp is grown for a multitude of end products derived from its cannabinoids, seed, fibre and wooden core. A key factor that influences the quantity and quality of the production of hemp is nitrogen fertilization. The aim of this study was to determine the response of five well-adapted industrial hemp cultivars to different nitrogen fertilization rates during the 2016 growing season. The experiment was laid out in a split-plot design with two replicates, five main plots (hemp cultivars: ‘Bialobrzeskie’, ‘Tygra’, ‘Felina 32’, ‘Sanhtica 27’, ‘Futura 75’) and sub-plots [fertilization treatments: control (N0), fertilizer 46-0-0 at 120 kg ha-1 (N1), 180 kg ha-1 (N2), 240 kg ha-1 (N3)]. For the computation of height, biomass yield, stem dry weight, length and weight of the inflorescences and mean seed weight, 10 plants were randomly selected in each plot. In general, increasing N fertilization rate positively impacts hemp biomass yield, stem dry weight, plant height, and inflorescence indices. Biomass yield, stem dry weight and inflorescence weight increased by 37.3%, 48.2% and 16%, respectively, with the application of 240 kg N ha-1 when compared with the unfertilized control. Plant height and inflorescence length increased from 1.66 to 1.76 m and from 66.2 to 82.9 cm, respectively, with the application of the higher N rate compared with the control, while there were no significant differences between the fertilization treatments for mean seed weight. The varieties ‘Tygra’ and ‘Futura 75’ showed the highest values for all the measurement characters. Our results indicate that hemp responded well to the addition of N fertilizer.


Effects of Cold Temperature and Acclimation on Cold Tolerance and Cannabinoid Profiles of Cannabis sativa L. (Hemp)

Andrei Galic,Heather Grab, Nicholas Kaczmar Kady MaserWilliam B. Miller, And Lawrence B. Smart
https://www.mdpi.com/2311-7524/8/6/...=TCS Newsletter Week 24 2022&utm_medium=email
Hemp (Cannabis sativa) is a multi-use crop garnering newfound attention from researchers and consumers. While interest has emerged, a lack of substantiated research still exists regarding effects of adverse weather events on physiological health and secondary metabolite production of hemp. The aim of this experiment was to assess cold tolerance of hemp using the cultivars ‘FINOLA’ and ‘AutoCBD’. Effects of cultivar, plant age, cold acclimation, frequency of cold treatments, and intensity of cold treatments were all considered in regard to their influence on physiological stress, biomass, and cannabinoid profile. Few effects of sequential cold treatments were noted, and they were not moderated by cold acclimation, which tended to have negative effects across many responses. This detrimental effect of cold acclimation conditions was further observed in decreased total CBD % and total THC % compared to non-acclimated plants. These findings bear consideration when assessing the unpredictability of a changing climate’s effects on the heath and cannabinoid profile of hemp. .

Effects of Drought and Heat on Photosynthetic Performance, Water Use and Yield of Two Selected Fiber Hemp Cultivars at a Poor-Soil Site in Brandenburg (Germany).
Herppich, W. B., Gusovius, H.-J., Flemming, I., & Drastig, K.
Agronomy, 10(9), 1361.(2020).
doi:10.3390/agronomy10091361
Hemp currently regains certain importance as fiber, oil and medical crop not least because of its modest requirements of biocides, fertilizer and water. During recent years, crops were exposed to a combination of drought and heat, even in northern Central-Europe. Dynamic responses of photosynthesis and stomatal conductance to these stresses and their persistent effects had been studied, if at all, in controlled environment experiments. Comprehensive field studies on diurnal and long-term net photosynthesis and gas exchange, and yield properties of hemp during a drought prone, high-temperature season in northern Central-Europe are obviously missing. Thus, in whole season field trails, the essential actual physiological (rates of net photosynthesis and transpiration, stomatal conductance, water use efficiencies, ambient and internal CO2 concentrations) and the yield performance of modern high-yielding multi-purpose hemp cultivars, ‘Ivory’ and ‘Santhica 27’, were evaluated under extreme environmental conditions and highly limited soil water supply. This provides comprehensive information on the usability of these cultivars under potential future harsh production conditions. Plants of both cultivars differentially cope with the prevailing climatic and soil water conditions. While ‘Ivory’ plants developed high rates of CO2 gain and established large leaf area per plant in the mid-season, those of ‘Santhica 27’ utilized lower CO2 uptake rates at lower leaf area per plant most time. This and the higher germination success of ‘Santhica 27’ resulted in nearly twice the yield compared to ‘Ivory’. Although stomatal control of CO2 gain was pronounced in both cultivars, higher stomatal limitations in ‘Ivory’ plants resulted in higher overall intrinsic water use efficiency. Cultivation of both hemp cultivars with only basic irrigation during seed germination was successful and without large effects on yield and quality. This was valid even under extremely hot and dry climatic conditions in northern Central Europe.


Effects of Light Spectra on Morphology, Gaseous Exchange, and Antioxidant Capacity of Industrial Hemp

Xia Cheng, Rong Wang, Xingzhu Liu, Lijuan Zhou, Minghua Dong, Muzammal Rehman, Shah Fahad, Lijun Liu and Gang Deng
Front. Plant Sci., 02 June 2022 |
DOI: 10.3389/fpls.2022.937436
https://www.frontiersin.org/article...=TCS Newsletter Week 26 2022&utm_medium=email
One of the most important growth factors in cannabis cultivation is light which plays a big role in its successful growth. However, understanding that how light controls the industrial hemp growth and development is poor and needs advanced research. Therefore, a pot study was conducted to investigate the effects of different colors of light, that is, white light (WL), blue light (BL), red light (RL), and 50% red with 50% blue mix light (RBL) on morphology, gaseous exchange and antioxidant capacity of industrial hemp. Compared with WL, BL significantly increase hemp growth in terms of shoot fresh biomass (15.1%), shoot dry biomass (27.0%), number of leaves per plant (13.7%), stem diameter (10.2%), root length (6.8%) and chlorophyll content (7.4%). In addition, BL promoted net photosynthesis, stomatal conductance, and transpiration, while reduces the lipid peroxidation and superoxide dismutase and peroxidase activities. However, RL and RBL significantly reduced the plant biomass, gas exchange parameters with enhanced antioxidant enzymes activities. Thus, blue light is useful for large-scale sustainable production of industrial hemp .

Effects of ?-ray treatment on Cannabis saliva pollen viability
Michela Zottini, Giuseppe Mandolino & Paolo Ranalli
Plant Cell, Tissue and Organ Culture volume 47, pages189–194(1997)
DOI: 10.1007/BF02318957
The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with ?-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased ?-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum ?-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.

Efficiency of the photosynthetic apparatus in Cannabis sativa L. fertilized with sludge froma wastewater treatment plant and with phosphogypsum
Dariusz Zielonka, Mariusz Nierebi?ski, Hazem M. Kalaji, Janusz Augustynowicz, Anna Pr?decka, Stefan Russel
DOI: 10.12775/EQ.2017.039
Sewage sludge and phosphogypsum are by-products of wastewater treatment and phosphorus fertiliser production, respectively. Considering all known methods of their management, it seems rational to use these waste products in agriculture. While assimilating nutrients contained in sewage sludge or phosphogypsum, agricultural crops contribute to the recycling of these otherwise noxious materials. The objective of this study was to identify the effect of fertilization with sewage sludge and with phosphogypsum on selected physiological parameters that determine the efficiency of the photosynthetic apparatus in Cannabis sativa L. Field tests were conducted on three varieties of Cannabis sativa: Bia?obrzeskie, Tygra and Beniko. Plots were fertilized with sewage sludge as an equivalent of nitrogen nutrition in a dose of 170 kg N.ha-1 and with phosphogypsum applied in three doses: 100, 500 and 1000 kg.ha-1. The plants were subjected to physiological assays (relative content of chlorophyll and leaf area index) on three dates in 2014: 29 June (early development stage), 26 July (full development) and 20 September (final development stage). The study discusses the effect of fertilization with sewage sludge and phosphogypsum on the efficiency of the photosynthetic apparatus . inCannabis sativa.

In Turkish but can be easily translated with Google translate.
Endüstriyel Kenevir (Cannabis sativa L.) Gerçe?i
Derya Aksoy, R?za Pasl?, Selim Aytaç
ULUSLARARASI 19 MAYIS YEN?L?KÇ? B?L?MSEL YAKLA?IMLAR KONGRES? Uygulamal? Bilimle
https://www.researchgate.net/publica...egi/references
Cannabis sativa L. is a cannabis plant; It is a woody and one-year plant from the cannabinaceae family close to nettle. Hemp (Cannabis sativa L.) is a plant of 2n = 20 chromosomes, one-year, C3 group, cultivated for long and strong fibers and seeds, which the human being has cultivated. Hemp is called hemp, gang, hemp and twine in our country. The main homeland of cannabis is Central Asia and it spreads from temperate to subtropical. Hemp; China, Manchuria, Japan, North India, Turkey, Russia, Australia, Hungary, Italy, France, Belgium, Germany, Chile and the United States has grown for
fiber production in US. It is also produced in China and Manchuria to obtain oil from seeds. Hemp is a plant grown for the fibers obtained from the stem, seed and oil obtained from the seed. Fibers of sails, sacks and wicker cloths, cars, steamers, ball covers, rope, twine, textile and so on. productions are made. Fiber plants are the raw material source of the textile industry. Fiber plants are also an important source of vegetable oil, animal feed raw material and cellulose. In our country, cotton is produced as the main fiber plant.
Cannabis is one of the most important fiber plants that can be grown in areas where cotton is not grown. Cannabis can be grown successfully for fiber purposes in the Black Sea Region ecology; With its long quality and durable fiber, it has the potential to form an alternative fiber source to the textile industry. Hemp, on the other hand, has begun to find modern uses in biopolymers, construction and cosmetics and pharmaceuticals in many countries of the world. In the future, cannabis agriculture and industry are expected to find more shares in the economy. In this review, properties and superior aspects of industrial cannabis will be discussed.

Engineering Perspectives of the Hemp Plant, Harvesting and Processing
Ying Chen, Jude Liu, Jean-Louis Gratton
December 2004 Journal of Industrial Hemp 9(2):23-39
DOI: 10.1300/J237v09n02_03
The special characteristics of the hemp (Cannabis sativa L.) plant make it one of the most challenging crops to handle. Several studies, both in the laboratory and the field, have been conducted at the University of Manitoba, Canada, on the engineering perspectives of hemp production, including the physical and mechanical properties of the hemp plant, hemp harvesting and processing. Physical properties of the hemp plant, such as plant height, seed-head length, stem diameter and stem specific mass, vary highly within a field and across fields. The force and energy required for cutting a hemp stem are much greater than those required for cutting maize stalk and forage crops. The two-windrow harvesting concept has been demonstrated to be feasible and can be implemented into a commercial windrower for harvesting dual-purpose hemp. Conditioned hemp dries significantly faster than unconditioned hemp. However, conditioning hemp requires more power than conditioning a forage crop. The basic machine functions required for hemp fibre processing are separating the fibre from the core and cleaning the fibre. A field-going processing unit can be formed by combining a modified forage harvester and a straw walker from a grain combine. However, the effectiveness of such a unit is limited, and the design of new separating and cleaning devices may be required for higher fibre yield and purity

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.
Husain, R., Weeden, H., Bogush, D., Deguchi, M., Soliman, M., Potlakayala, S., … Rudrabhatla, S.
PLOS ONE, 14(8), (2019).
doi:10.1371/journal.pone.0221570
Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.

Ethephon application stimulats cannabinoids and plastidic terpenoids production in Cannabis sativa at flowering stage.
Mansouri, H., Salari, F., & Asrar, Z.
Industrial Crops and Products, 46, 269–273. (2013).
doi:10.1016/j.indcrop.2013.01.025
We studied the effect of ethephon on levels of the major cannabinoids (tetrahydrocannabinol and cannabidiol) and chlorophyll, carotenoids and -tocopherol in Cannabis sativa at productive stage. Results revealed that ethephon increased THC content of leaf in male and female plants and of male flowers. However, ethephon unable to enhancing THC content in female flowers. Treatment with etheohon increased CBD content in male and female leaf and female flowers. The treatment of male flowers with low ethephon concentration caused an increase, and those treated with high ethephon concentration resulted in a decrease in CBD content. The lowest level of ethephon (1 M) enhanced chlorophyll a, b and total chlorophyll in male and female plants. Both sexes treated with ethephon showed an increase in carotenoids content, but 1 M ethephon had the stronger effect in this regards. Male and female plants had a higher content of -tocopherol when treated with ethephon. These results showed ethephon is a suitable treatment for increasing cannabinoids and -tocopherol in productive stage of cannabis and there was not a relation between primary and secondary terpenoids.

Evaluation of a phenological model for strategic decisions for hemp (Cannabis Sativa L.) biomass production across European sites
Stefano Amaducci, Michele Colauzzi, Gianni Bellocchi, Salvatore Luciano Cosentino, Katri Pahkala, Tjeerd Jan Stomphf, Wim Westerhuis, Alessandro Zatta, Gianpietro Venturi
Industrial Crops and Products 37 (2012) 100–110
doi: 10.1016/j.indcrop.2011.11.012
The optimal combination of yield and quality of hemp fibres from field grown crops is around flowering. Therefore prediction of flowering time would support in planning production and optimization of the cultivar choice for different agro-ecological zones. In the current paper the validation of a recently published model (Amaducci et al., 2008a) is carried out for four varieties across a wide range of sites and thus of air temperature–photoperiod combinations. The model was evaluated by comparing its output to field observations of the duration between emergence and 50% flowering. The model output and observed times from emergence to 50% of flowering generally corresponded well, but some discrepancies were apparent. The biggest discrepancies between estimates and actual data were observed at extreme latitudes. The level of accuracy ofthe model predictions is satisfactory for strategic decision regarding sowing and harvesting time and cultivar choice, but tactical decisions (e.g. time of harvest based on flowering time) cannot be accurately supported

Evaluation of microbial inoculants pretreatment in straw and manure co-composting process enhancement
Yupeng Wu, Yanxin Chen, Muhammad Shaaban , Duanwei Zhu , Chenxiao Hu, Zhongbing Chen, Yan Wang,
CHEMICAL ENGINEERING TRANSACTIONS VOL. 56, 2017
doi: 10.1016/j.jclepro.2019.118078
Commercial microbial inoculants (MI) are usually inoculated during the co-composting of straw and manure to improve the compost process. In the present study, MI was prior used to pretreat straw before co-composting (MI pretreated) and compared with a treatment with inoculated MI during the co-composting (MI inoculated) as well as a treatment without MI utilization (CK). Compared with the CK, MI utilization (MI inoculated and MI pretreated) significantly improved the compost maturity index of C/N (Sig. values 0.016), Cwater-soluble organic/Norganic (Sig. values 0.000), EC (Sig. values 0.043), and GI (Sig. values 0.009), as well as increased the final products’ total nutrient content by 4–13%. The MI pretreatment disrupted straw structure and enhanced lignocellulose degradation before co-composting as shown by a decreased straw cellulose content by 8–18% and hemicellulose content by 20–23%. However, no noticeable difference was found in the maturity index between MI pretreated and MI inoculated treatments, although the microbial community of compost differed between them during the thermophilic stage. In conclusion, MI utilization is proposed during the composting process for enhancing compost quality. However, straw pretreatment by MI before co-composting is not recommended, as this practice showed limited effects on compost quality improvement.

Evaluation of substrate composition and exogenous hormone application on vegetative propagule rooting success of essential oil hemp (Cannabis sativa L.)
Sean M. Campbell, Steven L. Anderson, Zachary T. Brym, Brian J. Pearson
PLoS ONE 16(7): e0249160.
DOI: 10.1371/journal.pone.0249160
https://journals.plos.org/plosone/ar...l.pone.0249160
To support the rapidly expanding industrial hemp industry, a commercial supply of highquality starter plants with low genetic variability from nurseries will be key to consistent and efficient cultivation efforts. Rooting success was evaluated across four propagation medias, five rooting hormones, and eight commercially available high-cannabidiol (CBD) essential oil hemp cultivars. Cuttings were placed in a climate-controlled room and assessed for rooting success 12 days after cloning. Rooting success was determined by quantifying total root number, cumulative total root length, and total root mass. Propagation media had the greatest effect on rooting success (13–80%). Rockwool had the highest rooting success resulting in 10-fold increases in rooting traits over the next highest scoring medium (Berger BM6). Hormone applications significantly improved (15- to 18-fold) rooting success compared to no hormone application, while non-statistical differences were observed across auxin hormone concentrations and application methods. Genetic variation in rooting response was observed between cultivars with ‘Cherry Wine’ outperforming all other cultivars with an approximate 20% increase in rooting success over the next highest rooting cultivar, ‘Wife’. Although the ideal combination was not specifically identified in this study, findings provide insight into how rooting hormone application and medium selection impact vegetative propagule rooting success of essential oil hemp

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes
Oier Aizpurua-Olaizola, Umut Soydaner, Ekin O?ztu?rk, Daniele Schibano, Yilmaz Simsir, Patricia Navarro, Nestor Etxebarria, and Aresatz Usobiaga
Journal of Natural Products 79(2) February 2016
DOI: 10.1021/acs.jnatprod.5b00949
The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and fl owers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantifi ed using GC-FID and verifi ed via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/ cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and IIIneeded more time to reach peak production of THCA, CBDA, and monoterpenes. Diff erences in the cannabigerolic acid
development among the diff erent chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of diff erent chemotypes were clearly diff erentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

Experimental Endozoochory of Cannabis sativa Achenes
John M. McPartland, Steve G. Naraine
Med Cannabis Cannabinoids Published online: October 8, 2018
DOI: 10.1159/000492971
The mechanism by which Cannabis sativa dispersed from its center of origin remains an open question. The literature provides many hypotheses, which we review for the first time, but experiments are few. Darwin was interested in zoochory – the transport of plants by animals. He demonstrated endozoochory (transport of seeds via animal digestive systems) of C. sativa achenes (seeds) by carrier pigeons, but he did not quantify achene survival rates. We assessed mammalian endozoochory in a triplicate experiment: feeding C. sativa achenes into a simulated gastrointestinal system, a dog, and a human. The in vitro system subjected achenes to sequential digestive enzymes. Achenes were planted in potting soil and monitored for emergence under growroom conditions. The in vivo experiments added achenes to a normal morning meal (dog food or granola). Feces were collected for daily instillation into an outdoor garden and monitored for seedling emergence for 16 days. Control achenes were planted directly into soil without ingestion. In the in vitro study, 34.7% of the digested achenes emerged as seedlings. The in vivo emergence rates were 10.3, 1.3, and 76.0% for the dog, human, and control conditions. The three groups differed significantly (?2 = 1,264.93, p < 0.0001). Achene survival was greatest under in vitro conditions, which lacked a mastication step, compared to dog (minimal chewing) and human (maximal chewing) conditions. Although C. sativa lacks evolutionary traits for classic endozoochory (i.e., a fleshy fruit), it seems well adapted to this manner of seed dispersal.

First known survey of cannabis production practices in California
Houston Wilson, Hekia Bodwitch, Jennifer Carah, Kent Daane, Christy Getz, Theodore E. Grantham and Van Butsic
CALIFORNIA AGRICULTURE • VOLUME 73, NUMBER 3-4
DOI: 10.3733/ca.2019a0015
Legalization of cannabis production has daylighted a unique and highly valuable crop in California agriculture. State and regulatory agencies must now address the ecological, social and agricultural effects of cannabis production, but little is known about how growers produce this crop. Using an online survey, we gathered information from growers in July 2018 on their production practices. According to responses from about 100 growers, most cannabis was produced outdoors or in greenhouses, relied primarily on groundwater and used biologically based inputs for pest management. Many farms employed seasonal workers paid at fixed piece rates. Regulatory compliance varied according to farm size. Beginning to document growing practices will help scientists formulate key environmental, social and agronomic questions and develop relevant research and extension programs to promote best management practices and minimize negative environmental impacts of production.

Flowering Dynamics in Monoecious and Dioecious Hemp Genotypes
Stefano Amaducci, Michele Colauzzi, Alessandro Zatta, Gianpietro Venturi
April 2008 Journal of Industrial Hemp 13(1):5-19
DOI: 10.1080/15377880801898691
Flowering is a crucial phase in hemp cultivation. It influences both stem and seed yield. A uniform and short flowering duration in hemp is desirable, because it favors uniform crop development. Moreover, flowering is often taken as a reference point for harvesting, and very long durations of flowering might mislead the operator in judging the proper time for mowing. In this article, a large dataset of flowering time and duration for different monoecious and dioecious varieties was studied and the effect of sowing time and genotype on flowering duration is discussed. Minimal flowering duration was observed when the time from emergence to flowering was short. This, was related however, to low yields. Dynamics of flowering was accurately described by a bi-logistic curve that indicates the presence of two underlying logistic processes.

Foliar Sprays of Silver Thiosulfate Produce Male Flowers on Female Hemp Plants
Mark H Brand, Jessica D Lubell
HortTechnology 28(6):743-747 December 2018
DOI: 10.21273/HORTTECH04188-18
Interest in hemp ( Cannabis sativa ) for its medicinal compounds, cannabidiol (CBD), and ?-9-tetrahydrocannabidiol (THC), continues to increase. Maximizing yield of CBD and/or THC requires female plants because female inflorescences accumulate significantly greater concentrations of these compounds than male inflorescences. Production of all female seed requires induction of female plants to develop male flowers that produce genetically female pollen. Growers would like access to feminized seed to produce all-female crops. We evaluated the efficacy of 0, 0.3, and 3 m m silver thiosulfate (STS) applied as a foliar spray (on three occasions 7 days apart) to produce male flowers on four strains of female hemp (having a THC concentration of ?0.3%), designated CBD hemp A, CBD hemp B, CBD hemp C, and industrial hemp. Silver thiosulfate at 3 m m was the most efficacious treatment for all strains. The majority of inflorescences had 100% male flowers at 3 m m STS, and terminal inflorescences had ?95% conversion to male flowers. Silver thiosulfate at 0.3 m m produced partial conversion to male flowers, whereas most inflorescences had around 50% male flowers, except for CBD hemp A, which demonstrated greater levels of masculinization. At 0.3 m m STS, terminal inflorescences of CBD hemp A had 91% conversion to male flowers. This study demonstrates that male flowers can be produced easily and consistently on female plants through application of foliar sprays of STS under short-day conditions.

Genetic Variability of Morphological, Flowering, and Biomass Quality Traits in Hemp (Cannabis sativa L.)
Jordi Petit, Elma M. J. Salentijn, Maria-João Paulo, Claire Thouminot, Bert Jan van Dinter, Gianmaria Magagnini, Hans-Jörg Gusovius, Kailei Tang, Stefano Amaducci, Shaoliang Wang, Birgit Uhrlaub, Jörg Müssig and Luisa M. Trindad
Front. Plant Sci. 11:102.
doi: 10.3389/fpls.2020.00102
Hemp (Cannabis sativa L.) is a bast-fiber crop well-known for the great potential to produce sustainable fibers. Nevertheless, hemp fiber quality is a complextrait, and little is known about the phenotypic variability and heritability of fiber qualitytraits in hemp. The aim ofthis study isto gain insights into the variability in fiber quality within the hemp germplasm and to estimate the genetic components, environmental components, and genotype-by-environment (G×E) interactions on fiber quality traits in hemp. To investigate these parameters, a panel of 123 hemp accessions was phenotyped for 28 traits relevant to fiber quality at three locations in Europe, corresponding to climates of northern, central, and southern Europe. In general, hemp cultivated in northern latitudes showed a larger plant vigor while earlier flowering was characteristic of plants cultivated in southern latitudes. Extensive variability between accessions was observed for all traits. Most cell wall components (contents of monosaccharides derived from cellulose and hemicellulose; and lignin content), bast fiber content, and flowering traits revealed large genetic components with low G×E interactions and high broad-sense heritability values, making these traits suitable to maximize the genetic gains of fiber quality. In contrast, contents of pectin-related monosaccharides, most agronomic traits, and several fiber traits (fineness and decortication efficiency) showed low genetic components with large G×E interactions affecting the rankings across locations. These results suggest that pectin, agronomic traits, and fiber traits are unsuitable targets in breeding programs of hemp, as their large G×E interactions might lead to unexpected phenotypes in untested locations. Furthermore, all environmental effects on the 28 traits were statistically significant, suggesting a strong adaptive behavior of fiber quality in hemp to specific environments. The high variability in fiber quality observed in the hemp panel, the broad range in heritability, and adaptability among alltraits prescribe positive prospects forthe development of new hemp cultivars of excellent fiber quality.

Genome-Wide Expression Profiles of Hemp (Cannabis sativa L.) in Response to Drought Stress
Chunsheng Gao, Chaohua Cheng, Lining Zhao, Yongting Yu, Qing Tang, Pengfei Xin, Touming Liu, Zhun Yan, Yuan Guo, and Gonggu Zang
International Journal of Genomics Volume 2018, Article ID 3057272, 13 pages
DOI: 10.1155/2018/3057272
https://downloads.hindawi.com/journa...18/3057272.pdf
Drought is the main environmental factor impairing hemp growth and yield. In order to decipher the molecular responses of hemp to drought stress, transcriptome changes of drought-stressed hemp (DS1 and DS2), compared to well-watered control hemp (CK1 and CK2), were studied with RNA-Seq technology. RNA-Seq generated 9.83, 11.30, 11.66, and 11.31 M clean reads in the CK1, CK2, DS1, and DS2 libraries, respectively. A total of 1292 differentially expressed genes (DEGs), including 409 (31.66%) upregulated and 883 (68.34%) downregulated genes, were identified. The expression patterns of 12 selected genes were validated by qRT-PCR, and the results were accordant with Illumina analysis. Gene Ontology (GO) and KEGG analysis illuminated particular important biological processes and pathways, which enriched many candidate genes such as NAC, B3, peroxidase, expansin, and inositol oxygenase that may play important roles in hemp tolerance to drought. Eleven KEGG pathways were significantly influenced, the most influenced being the plant hormone signal transduction pathway with 15 differentially expressed genes. A similar expression pattern of genes involved in the abscisic acid (ABA) pathway under drought, and ABA induction, suggested that ABA is important in the drought stress response of hemp. These findings provide useful insights into the drought stress regulatory mechanism in hemp

Genotype × Environment Interactions of Industrial Hemp Cultivars Highlight Diverse Responses to Environmental Factors.
Campbell, B. J., Berrada, A. F., Hudalla, C., Amaducci, S., & McKay, J. K.
Age, 2(1), 0.(2019).
doi:10.2134/age2018.11.0057
Starting with the 2014 Farm Bill, hemp (Cannabis sativa L.) is being re-introduced as an industrial crop in the United States. Since the crop has been absent for over 70 yr, little is known regarding the genetic mechanisms controlling economically relevant traits. Particularly, with federal legality of the crop hinging on a stringent tetrahydrocannabinol (THC) content of 0.3% or less, it is necessary to assess variance in this trait due to environmental effects and genotype × environment interactions (GEI) to avoid running afoul of federal law. Understanding how physical and biochemical traits respond to the environment also plays a strong role in selecting and developing appropriate cultivars for production in diverse growing regions. In 2016 we performed cultivar trials in multiple environments in Colorado to assess performance characteristics of a diverse set of germplasm from breeding programs across Europe and Asia. From these data, we were able to identify traits nearly entirely controlled by genetic factors, like days to maturity and THC and cannabidiol (CBD) production. We also identified traits strongly influenced by the environment and GEI, like grain yield, plant height, and water use. Individual cultivars also exhibited widely varying degrees of sensitivity to the environment. This underscores the importance of continued work to characterize genetic control of hemp traits to expedite breeding of cultivars that are well-adapted to target growing regions.

Greenhouse propagation of Cannabis Sativa L. by vegetative cuttings.
Coffman, C. B., & Gentner, W. A. (1979).
Economic Botany, 33(2), 124–127.
doi:10.1007/bf02858280
Previous work revealed significant variations in cannabinoid profiles of Cannabis sativa L. derived from a single seed source (P.I. 378939) and subjected to the same growth environment. Studies were conducted to evaluate the efficacy of propagation of C. sativa by vegetative cuttings in order to increase uniformity of cannabinoid concentrations within a given plant population. C. sativa was successfully propagated by vegetative cuttings. However, there were both morphological and biochemical differences between seed-derived plants and their vegetative propagules. Delta-9-tetrahydrocannabinol concentrations were 4.1 times higher in vegetative propagules than in seed propagules. Vegetative cuttings also generally developed more profuse lateral branch growth; hence, foliage increased relative to their parent plants. Cannabinoid levels within the population of vegetative cuttings remained highly variable.

Hemp: A New Crop with New Uses for North America
Ernest Small and David Marcus
Trends in new crops and new uses. 2002. J. Janick and A. Whipkey (eds.).
https://www.hort.purdue.edu/newcrop/ncnu02/v5-284.html
“Hemp” refers primarily to Cannabis sativa L. (Cannabaceae), although the term has been applied to dozens of species representing at least 22 genera, often prominent fiber crops. For examples, Manila hemp (abaca) is Musa textilis Née, sisal hemp is Agave sisalina Perrine, and sunn hemp is Crotolaria juncea L. Especially confusing is the phrase “Indian hemp,” which has been used both for narcotic Asian land races of C. sativa (so-called C. indica Lamarck of India) and Apocynum cannabinum L., which was used by North American Indians as a fiber plant. Cannabis sativa is a multi-purpose plant that has been domesticated for bast (phloem) fiber in the stem, a multi-purpose fixed oil in the “seeds” (achenes), and an intoxicating resin secreted by epidermal glands. The common names hemp and marijuana (much less frequently spelled marihuana) have been applied loosely to all three forms, although historically hemp has been used primarily for the fiber cultigen and its fiber preparations, and marijuana for the drug cultigen and its drug preparations. The current hemp industry is making great efforts to point out that “hemp is not marijuana.” Italicized, Cannabis refers to the biological name of the plant (only one species of this genus is commonly recognized, C. sativa L.). Non-italicized, “cannabis” is a generic abstraction, widely used as a noun and adjective, and commonly (often loosely) used both for cannabis plants and/or any or all of the intoxicant preparations made from them

Hemp and flax germplasm collections at CRA-ISCI
G. Mandolino, A. Carboni, F. Fontana, C. Maestrini and G. Fila
The hemp collection at CRA-ISCI
https://archive-ecpgr.cgiar.org/file...y%20report.pdf
Hemp seed was collected and stored at ISCi since the Sixties, when hemp was a major crop for the economy of the plains of Northern Italy. At ISCI (in its denomination before 1967) several highfibre quality and content varieties were bred, such as CS and Fibranova, starting from traditional, high-yield and well-adapted italian ecotypes such as Carmagnola, Bolognase, Napoletana, etc. ISCI (now CRA-ISCI was the main centre of collection, characterization andbreeding of hemp germplasm, until the Seventies only from the point of view of the fibre content and quality, and more recently also as the institute officially in charge to determine the cannabinoids content of hemp stands, for making the european subsidies accessible to growers. The italian hemp
germplasm, along with experimental hybrids and material from other germplasm banks (e.g. Gatersleben), was the starting material for CRA-ISCI research work on the sexual determination and differentiation, for molecular mapping and for the development of DNA markers for the marker-assisted selection for sex, chemotype, and for germplasm fingerprinting studies.

Hemp as Food at High Latitudes
J. C. Callaway PhD
Journal of Industrial 2002 Hemp 7(1):105-117
DOI: 10.1300/J237v07n01_09
Hempseed offers a unique nutritional package, in terms of dietary oil, protein, vitamins and minerals, which can be produced at high latitudes ( > 50° latitude). Hempseed oil is highly unsaturated and contains both essential fatty acids (linoleic acid and alpha-linolenic acid) in a nutritionally balanced ratio, in addition to considerable amounts of biochemically important gamma-linolenic acid (GLA) and stearidonic acid (SDA). The protein in hempseed is complete, in that it contains all of the essential amino acids in nutritionally significant amounts, and lacks the nutritional inhibiting factors found in soya. Hempseed could become a viable replacement for imported soya in Northern Europe, particularly as feed stock for animals.

Hemp-Based Phytoaccumulation of Heavy Metals from Municipal Sewage Sludge and Phosphogypsum Under Field Conditions.
Zielonka, D., Szulc, W., Skowro?ska, M., Rutkowska, B., & Russel, S.
Agronomy, 10(6), 907.(2020).
doi:10.3390/agronomy10060907
The aim of this study was to evaluate the ability of three hemp cultivars to accumulate heavy metals under sewage sludge (SS) and phosphogypsum (PG) application. The field study was carried out from 2014 to 2016 on Luvisol (loamy sand) in Poland. The experiment scheme included five treatments—T0: the control without fertilization, T1: 170 kg N (nitrogen) ha?1 from sewage sludge, T2: 170 kg N ha?1 from sewage sludge and 100 kg ha?1 of phosphogypsum, T3: 170 kg N ha?1 from sewage sludge and 500 kg ha?1 of phosphogypsum, and T4: 170 kg N ha?1 from sewage sludge and 1000 kg ha?1 of phosphogypsum. It was found that the application of municipal sewage sludge enriched the soil with the bioavailable forms of heavy metals to the greatest extent and contributed to the highest increase in their contents in vegetative and generative organs of hemp plants. These parameters showed a phosphogypsum dose-dependent decline, which could hinder the phytoextraction process. The greatest extractions of heavy metal(loid)s (HMs) from the soil treated with SS and PG were achieved by the Tygra variety, which had the highest bioconcentration factor (BCF) and biomass yield.

Hemp (Cannabis) Cultivation and Use in the Republic of Korea
Robert Connell Clarke
April 2006 Journal of Industrial Hemp 11(1):51-86
DOI: 10.1300/J237v11n01_07
This paper summarizes the history of hemp (Cannabis) cultivation and traditional use in the Republic of Korea and investigates the cultivation and processing techniques currently being employed to produce hemp ribbon and seed and weave hemp textiles. Recent production levels and market conditions are reviewed. Hemp is cultivated for bast fiber and seed in several of the numerous fertile inland valleys of the Republic of Korea. Present distribution and level of production are much reduced in comparison with the 20th century. There is no local tradition of its use as either medicine or inebriant. Comparisons with European hemp cultivation and processing, (representative of commercial Western hemp production), Chinese hemp cultivation and production in Shandong Province, (representative of modern Chinese commercial production), and Vietnamese Hmong-Miao cultivation, processing, and weaving, (representative of traditional non-commercial Asian hemp production), are provided where appropriate. Comparisons to hemp growing in the Republic of Korea during the 1950s and early 1960s are made.

Hemp (Cannabis sativa L.)
Mistianne Feeney and Zamir K. Punja
Methods in Molecular Biology, vol. 344: Agrobacterium Protocols, 2/e, volume 2
DOI:10.1385/1-59745-131-2:373
Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent, mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection were screened for PMI expression using a
chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines and the presence of the PMI gene was confirmed using polymerase chain reaction and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1 to 55.3%.

Hemp (Cannabis sativa L.) Cultivation in North-Central Turkey
Justin S. Tiret
June 2002Journal of Industrial Hemp 7(2):73-81
DOI: 10.1300/J237v07n02_07
This paper summarizes the history of hemp (Cannabis sativa L.) cultivation and traditional use in the north-central region of Turkey, and investigates the cultivation and processing techniques currently being used to produce hemp bast fiber and hemp seed. Hemp bast fibers are used by the Turks for making paper, hand-made rope, and machine processed twine and rope. The hurd is used locally for kindling. The flowers are used for compost and the seeds are used for human food and bird feed. The use of hemp in recent years has declined significantly. The cultural and economic reasons for the reduced cultivation and use of hemp are discussed.

Hemp (Cannabis sativa L.) Cultivation in the Tai'an District of Shandong Province, Peoples Republic of China
https://www.votehemp.com/wp-content/...ha_vol2no2.pdf
Robert C. Clarke
Journal of the International Hemp Association Vol. 2 No. 2 December 1995
This paper summarizes the history of hemp (Cannabis sativa L.) cultivation and traditional use in the Tai'an District of Shandong Province in the People's Republic of China, and investigates the cultivation and processing techniques currently being employed to produce hemp ribbon and hemp seed. Recent production levels and market conditions are reviewed. Comparisons with Hungarian hemp cultivation and processing, being representative of Western hemp production, are provided where appropriate. Wild types and escaped plants are also described.

Hemp (Cannabis sativa L.) leaf photosynthesis in relation to nitrogen content and temperature: implications for hemp as a bio-economically substainable crop.
Kailei Tang , Paul C. Struik, Stefano Amaducci, Tjeerd?Jan Stomph, Xinyou Yin
GCB Bioenergy (2017) 9, 1573–1587,
doi: 10.1111/gcbb.12451
Hemp (Cannabis sativa L.) may be a suitable crop for the bio-economy as it requires low inputs while producing a high and valuable biomass yield. With the aim of understanding the physiological basis of hemp’s high resource use efficiency and yield potential, photosynthesis was analysed on leaves exposed to a range of nitrogen and temperature levels. Light-saturated net photosynthesis rate (Amax) increased with an increase in leaf nitrogen up to 31.2 ± 1.9 ?mol m-2 s -1 at 25 °C. The Amax initially increased with an increase in leaf temperature (TL), levelled off at 25-35°C and decreased when TL became higher than 35 °C. Based on a C3 leaf photosynthesis model, we estimated mesophyll conductance (gm), efficiency of converting incident irradiance into linear electron transport under limiting light (?2LL), linear electron transport capacity (Jmax), Rubisco carboxylation capacity (Vcmax), triose phosphate utilization capacity (Tp) and day respiration (Rd), using data obtained from gas exchange and chlorophyll fluorescence measurements at different leaf positions and various levels of incident irradiance, CO2 and O2. The effects of leaf nitrogen and temperature on photosynthesis parameters were consistent at different leaf positions and among different growth environments except for ?2LL, which was higher for plants grown in the greenhouse than for those grown outdoors. Model analysis showed that compared with cotton and kenaf, hemp has higher photosynthetic capacity when leaf nitrogen is less than 2.0 g N m-2. The high photosynthetic capacity measured in this study, especially at low nitrogen level, provides additional evidence that hemp can be grown as a sustainable bio-energy crop over a wide range of climatic and agronomic conditions.

Hemp (Cannabis sativa L.) Trials in Southern Sweden 1999-2001
B. Svennerstedt, G. Sevenson
April 2006 Journal of Industrial Hemp 11(1):17-25
DOI: 10.1300/J237v11n01_03
Results from hemp experiments in European countries indicate that long day conditions may be beneficial for the fibre content and quality of hemp. The long day conditions during summer in Sweden are therefore of interest. This paper presents results from the first fibre hemp experiments carried out in Sweden since the 1960s. Field trials were performed in southern Sweden, during the years 1999-2001, testing three monoecious varieties (Felina, Fedora and Futura) at seed rates of 30 and 60 kg/ha. Total biomass yield in autumn varied from 7.8 to 14.5 ton DM/ha and fibre yield from 1.9 to 3.3 ton DM/ha. The highest fibre yields per hectare were produced by the variety Futura, the lowest by Fedora. The seed rates 30 and 60 kg/ha gave similar biomass and fibre yields, but the plant density was lower and the stem diameter was larger at 30 kg/ha. Results of chemical analyses of stem and leaves are presented

Hemp Farming Systems
https://www.hempfarmingsystems.com/
Hemp Farming Systems (HFS) is a consultancy partnership. It is based in Australia, but will work with farmers, anywhere, to help them grow industrial hemp as a component of sustainable and productive farming systems.
Industrial hemp has been grown by Eurasian farmers for more than 10,000 years. Some communities saw it as a source of fibre, others stayed healthy by eating the grain. Early entrepreneurs turned it into paper – as soon as writing was invented.
But now, new industries are developing around the unique properties of this plant – plastics, construction materials, components of motor vehicles, and many more. The HFS team can demonstrate cutting edge national and international agricultural experience, and has worked intensively with farmers growing hemp for biomass (fibre, etc.), seed, and grain (food and oil) over the past 8 years

Hemp Fiber as a Sustainable Raw Material Source for Textile Industry: Can We Use Its Potential for More Eco-Friendly Production?
Gedik, G., & Avinc, O.
Sustainability in the Textile and Apparel Industries, 87–109. (2020).
doi:10.1007/978-3-030-38541-5_4
Sustainable production defines an environmental friendly production that we produce without changing the balance of the nature. Processes and the utilized materials should be renewable, and our whole production should be harmless so that nature can recover itself in an indigenous way. All natural fibers are biodegradable and sustainable, and consequently, they are commonly called as biofibers. Providing a sustainable production chain for textile processes requires individual attention for each input in the first place. One of the most important parts of these inputs is raw material selection and therefore fiber supply. Right at this point, hemp fiber step forwards and shines out with its huge sustainable production potential for textile industry. In this chapter, sustainable and biodegradable hemp fiber, which is an alternative to cotton and petroleum-based synthetic fibers, for textile raw material sourcing is reviewed in detail. The parameters that make this fiber sustainable are also investigated. Present common and special uses and possible future innovative alternatives of hemp fibers for technical textiles production are also stated. Mainly, composite material production with this sustainable fiber is reviewed for a replacement of nonsustainable synthetic competitors. When sustainable composite materials are produced not only ecofriendly textile production is carried out but also other materials can be produced with an ecofriendly path leading to more sustainable world.

Hemp for textiles: plant size matters
Willem Westerhuis Thesis 2016
.doi: 10.18174/378698
Fibre hemp (Cannabis sativa L.) may be an alternative to cotton and synthetic fibres as a raw material for textile yarn production in the European Union. The agronomic options to manipulate plant development and crop growth with the aim to optimise hemp long fibre production were investigated. Field trials with factors sowing density, sowing date, harvest time and variety were conducted. Stems were traditionally processed by retting, drying, breaking, and scutching. Following standard protocols, almost 1500 hemp stem samples were analysed. Varieties differ widely in their fibre content, but this thesis shows that when variety and plant size are known, the amounts of fibres, wood, and retting losses are known. The dry weight of the stems at harvest, not the factors underlying this weight, are determinant. In retted stems the dry matter is split–up into fibres and wood in a fixed way. The options to manipulate this ratio by crop management, given variety, are very small and for practical reasons they can be neglected. In fibre hemp two bast fibre types occur. Primary or long fibres are valuable for yarn spinning. Secondary fibres are too short and their presence hampers the production of fine yarns. This thesis shows that the secondary fibre front height increases with plant weight. Although a causal relationship between secondary fibre formation and flowering does not exist, the secondary fibre front is found higher in flowering plants when compared to non–flowering plants of the same height. This is likely to be caused by the higher weight or momentum of flowering plants as compared with non–flowering plants of the same height. Consequently, a harvest before flowering is preferable. This was shown in a greenhouse experiment, in which the short–day response of hemp was used to create size ranges of flowering and non–flowering plants. To produce high–quality raw materials for textile production, short crops should be grown. The options to produce plants with the desired size are manifold. Since sowing density, sowing date, and harvest time do not have an additional effect on the primary fibre content besides the indirect effect through stem weight, any combination of these factors could be chosen to optimize plant size.

Chapter 6: Hemp Genetics and Genomics
Brian Campbell, Dong Zhang, and John K. McKay
DOI: 10.2134/industrialhemp.c6
Cannabis sativa L. is an economically important crop that has been surrounded by controversy over the last 100 years. Despite its widespread use as an intoxicant and an industrial crop, governments worldwide have struggled to appropriately regulate Cannabis use and production. The lack of uniformity in Cannabis law, both spatially and temporally, has made research on this plant difficult through traditional channels. With increasing public support for medical marijuana and
growing interest in applications for industrial hemp, laws are changing and doors are opening providing for long-overdue research. The purpose of this chapter is to review the current body of knowledge relative to Cannabis genetics, specifically regarding speciation and classification, evolutionary origin, genetics of industrial hemp breeding, genetic diversity and population structure of the species, and Cannabis genomics, as well as a look at important areas of genetics and genomics research for the future of industrial hemp. The origin and the taxonomy of Cannabis is surrounded by uncertainty; academics debate questions of both where its evolutionary roots lie, as well as whether or not the diversity observed in Cannabis warrants distinguishing common types as separate species or subspecies. Eurasia has been proposed as its evolutionary center of origin, with more specific recommendations of central Asia, in the Himalayas, or possibly two distinct centers of Hindustani and European-Siberian origin (Clarke and Merlin, 2016; Hillig, 2005; Andre et al., 2016).
As defined in previous chapters, Cannabis has been utilized since ancient times. Archeological findings date the use of hemp rope to 10,000 years ago in Taiwan (Laursen, 2015). This early human use led to vectoring of seed thousands of years ago (Hillig, 2005) that has made clearly distinguishing evolutionary origins by genetic analyses difficult, if not practically impossible. The diverse morphology of C. sativa, combined with a multitude of uses, has caused no shortage of confusion over the classification of this species. The debate of whether or not Cannabis is a single species began long ago. The species was first labeled Cannabis sativa by Carl Linnaeus in 1753 (Watts, 2006). This monospecific
viewpoint was challenged in 1785 when Jean-Baptiste Lamarck found that some Cannabis specimens from India exhibited distinctly different morphology from those described by Linnaeus and created a new classification, Cannabis indica (Watts, 2006). Sativa samples were tall, with long intermodal spacing, and narrow leaflets, while indica-types are shorter and bushier plants with wide leaflets. Although several other classifications have been proposed, the only generally accepted third possible species was proposed in 1924 by Dmitri Janischevsky as Cannabis ruderalis (Hillig and Mahlberg, 2004). This newest classification of Cannabis was created to describe Russian samples that did not exhibit the same “domestication syndrome” traits as indica and sativa samples and were essentially small, wild (ruderal; or generally occurring in regularly disturbed ecosystems) plants (Small and Cronquist, 1976). Since there are no reproductive barriers between these three types of Cannabis, we consider this as a single species, but the debate between the “splitters” and the “lumpers” remains very active (Watts, 2006)

Hemp in the British Isles
Kenyon Gibson
October 2006 Journal of Industrial Hemp 11(2):57-67
DOI: 10.1300/J237v11n02_04
Hemp has been traced back to 343 BC in the UK, with record of its use in Roman times as well, and on into the Middle Ages. Cultivation was widespread, with record of hemp in Scotland, England, Wales and Ireland. In each of these countries there is record of it being grown in many locations, with place names reflecting this fact to this very day. The availability of hemp made it possible for the English to greatly increase their naval powers, but ultimately hemp had to be imported to maintain their military and economic primacy. By the nineteenth century, most of the hemp used by the United Kingdom was imported from Russia, with Italy as a supplier of the highest quality grades. The dependence of the nation on such a product was of concern to many legislators, who argued for an increase in domestic cultivation for reasons of national security. Ultimately, metal ships and steam power reduced the need for hemp in the British Navy, and production virtually ceased. In the 1980s the ecological movement took an interest in hemp, and hemp was re-introduced. It is now legal to grow hemp with a licence from the Home Office. Acreage has been increasing since the mid-1990s, when a few thousand acres were being grown. Most of the hemp grown in the UK is for seed and oil production. The industry is growing dramatically in the UK, where locally grown hemp provides most of the hemp based food products sold. Other applications of hemp, such as clothing, are also available to the public, but are imported, as in the case of Braintree Hemp Clothing (UK), which used Chinese hemp. This shop and other high profile shops have caught the public imagination in the UK, with a little help from such well-known fig
ures as Anita Roddick and Woody Harrelson. Outlets from Tony’s Hemp Cornerin London to The Hemp Shop in Brighton are now doing well with a range of products including paper, body products, clothing and food.

Hemp Museum Tour of Nagano Prefecture, Japan
Robert Connell Clarke
April 2006 Journal of Industrial Hemp 11(1):95-101
DOI: 10.1300/J237v11n01_09
Industrial hemp has a long and fascinating history in Japan and was grown in many regions. History and culture museums in small towns occasionally host small displays of a few hemp artifacts. This article highlights three local museums in northern Nagano Prefecture that feature comprehensive displays of hemp cultivation and processing. They are located near enough to each other to facilitate a short visit of high educational value

Hemp of Russian Northern Regions as a Source of Spinning Fibers
Sergey Grigoryev
January 2006 Journal of Industrial Hemp 10(2):105-114
DOI: 10.1300/J237v10n02_09
The present paper deals with several aspects of hemp studies, for instance, earliness as a factor for increasing hemp yield potential and extending the crop northwards. Another aspect of interest– the possibility of hemp fiber cottonization–will permit producing mixed hemp-cotton yarns which are in demand with the Russian textile industry. The latter may be the key precondition for expanding areas under hemp in appropriate regions of Russia, including its northern agricultural regions. Consequently, there is a n eed for genetic material with the desired characteristics for breeding cultivars that would meet demands of the textile industry.

Hemp Production in Aotearoa
John McPartland, Steve Cutler, Donald J. McIntosh
June 2004 Journal of Industrial Hemp 9(1):105-115
DOI: 10.1300/J237v09n01_12
Hemp is new to Aotearoa, the indigenous name of New Zealand (NZ). The NZ government approved the experimental cultivation of hemp in 2001. Eleven cultivars have been cultivated to date, ‘Anka’, ‘Carmen’, ‘Fasamo’, ‘Felina’, ‘Finola’, ‘Futura 77’, ‘Kompolti’, ‘Uniko B’, ‘USO 14’, ‘USO 31’, and ‘Zola’. Crops have been planted at 19 sites the past two seasons, in a wide range of latitudes, climates and soil types. NZ’s fragile soil necessitates careful management of its fertility. Hemp fits into the paradigm of sustainable stewardship, organic soil fertilization, and responsible crop rotation. It can be rotated with existing fodder crops and vegetable crops. Hemp’s well-known ability to suppress weeds makes its rotation with pasture an attractive way to clean soil banks of weed seeds. Hemp cultivated for seed produced maximal yields of 2800 kg ha1; and fiber crops yielded stalk biomass (dry matter) as high as 13,900 kg ha1. These yields are consistent with or greater than reports from the European literature. Several pests new to hemp were discovered in NZ, but none required pesticides. Birds caused problems in seed crops, requiring control with repellents and bird netting. Future prospects look promising for this new crop.

Hemp Production in Italy
Stefano Amaducci
June 2005 Journal of Industrial Hemp 10(1):109-115
DOI: 10.1300/J237v10n01_09
After a short history of hemp in Italy, this article lists the events that have brought back the cultivation of this fibre crop in Italy in recent years. The cultivation technique used for baby hemp is briefly described and the preliminary results of its processing are given. Baby hemp is a hemp crop grown at high plant densities (400-500 plants m_2) that is chemically desiccated when the height of 120-140 cm is reached and that is harvested with the machines used for flax. Advantages, problems and possible solutions for this technique are presented.

Hemp Production in Western Canada
Cecil L. Vera, Arthur Hanks
December 2004 Journal of Industrial Hemp 9(2):79-86
DOI: 10.1300/J237v09n02_08
Hemp production has been successfully adapted to meet local conditions in western Canada. Production has been driven by commercial interest in this species as an oilseed crop. As hemp is a photosensitive plant, the higher latitudes of western Canada appear to contribute to increased levels of essential fatty acids in the seed. Cultivars of shorter stature and early maturity have been imported, or locally bred, for the specific purpose of oilseed production or dual purpose (seed and fiber). Over 2,700 ha were seeded in western Canada in 2003. Research continues in order to improve production practices and optimize seed yield of the crop, under conventional and Certified Organic systems

Hemp Program
Summary of Varieties: Including Varieties of Concern and Prohibited Varieties

Kentucky Department of Agriculture
Ryan F. Quarles Commissioner
https://www.kyagr.com/marketing/docu..._List_2019.pdf
All license holders should review this document before purchasing seed or plant propagules for the Hemp Program in Kentucky. This document provides important guidance for growers based on past THC test results from crops grown in Kentucky. Provided here is a list of Prohibited Varieties and Varieties of Concern, as well as all other varieties that have been grown and tested in Kentucky in the past 2 years.
KRS 260.850(5) define industrial hemp as “the plant Cannabis sativa L. and any part of such plant, whether growing or not, with a delta-9 tetrahydrocannabinol [THC] concentration of not more than 0.3 percent on a dry weight basis.”
302 KAR 50:020 provides the following definitions:
(27) “Prohibited Variety” means a variety or strain of cannabis excluded from the Department’s Program.
(39) “Variety of Concern” means any variety of hemp in the Department’s Program that tests above 3000 ppm or 0.3000% delta-9-THC in one (1) or more Pre-Harvest Samples. A hemp variety designated as a “Variety of Concern” may be subject to restrictions and additional testing.

Hemp Yields and Its Rotation Effects on Wheat under Rainfed Mediterranean Conditions
Gorchs, G., Lloveras, J., Serrano, L., & Cela, S.
Agronomy Journal, 109(4), 1551.(2017).
doi:10.2134/agronj2016.11.0676
Hemp (Cannabis sativa L.) has a low impact on the environment requiring few added resources, and has multiple downstream applications. Th ere is little information on hemp biomass, seed yields, and response to NPK fertilization under humid rainfed Mediterranean conditions. Moreover, the eff ects of hemp on subsequent wheat (Triticum aestivum L.) crops have not been determined. To address these issues, we performed a fi eld study for 6 yr in Catalonia (northeastern Spain). Hemp treatments included: hemp monoculture, unfertilized hemp succeeding wheat, and NPK-fertilized hemp succeeding wheat. In turn, wheat treatments included: wheat monoculture, fi rst-, second-, and third-year wheat succeeding unfertilized hemp, and fi rst-, second-, and third-year wheat succeeding NPK-fertilized hemp. Th e hemp biomass yields (5340–10,090 kg ha–1) were similar to or lower than those achieved in other European regions whereas the hemp seed yields were relatively high (604–1434 kg ha–1). Both the biomass yield and seed yield greatly increased with NPK fertilization. Th e rotation eff ects of hemp on the subsequent wheat crops increased the wheat yield by 1368 and 155 kg ha–1 in the fi rst and second years, respectively, but in the third year the yield was similar to the wheat monoculture. Th e benefi cial eff ects of hemp on wheat therefore appear to last for 2 yr. We conclude that dual-purpose hemp (harvested for fi ber and seed) is an excellent predecessor for wheat, improving the sustainability of cereal-based cropping systems under humid rainfed Mediterranean conditions.

How to Increase Cannabinoids and Terpenoids in Cannabis Through Horticultural Management
Deron Caplan
Poster
https://www.canadiangreenhouseconfer.. .20Cannabis.pdf

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells
Satoshi Morimoto, Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama, Futoshi Taura
Journal of Biological Chemistry 282(28):20739-51 Aug 2007
DOI: 10.1074/jbc.M700133200
Cannabinoids are secondary metabolites stored in capitatesessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and 1 -tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2 and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT,indicating that this reaction was notinduced via a caspasedependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

IMPACT OF PHYSICAL AND CHEMICAL MUTAGENS ON SEX EXPRESSION IN CANNABIS SATIVA
Suman Kaushal Department of Botany, Govt. College Shahpur, H.P (India)
Indian Journal of Fundamental and Applied Life Sciences 2012 Vol. 2 (4) October-December, pp.97-103/ Kaushal Research Article 97
https://www.cibtech.org/J-LIFE-SCIEN...t...Sativa.pdf
The phenomenon of sexual dimorphism was observed in all the plants growing under wild condition as well as in the cultivated fields of cannabis. But in the present study late-flowering plants (Winter condition) and the plants raised from the seeds treated with physical (gamma-rays with 1.0 KR dose) and physical and chemical mutagens in combination (pre-treatment with sodium azide and then with gamma rays) showed changed sex- expression under glass house condition. The female plants showed the presence of bisexual flowers, reduced female flowers and male flowers. Probably the changed conditions outside and inside the plant might have disturbed the balance of hormones to such an extent that in the female plants, male hormones also made their existence. Therefore, it may be inferred that like all morphogenetic processes, the development of the male and female flowers in the same or different plants is under the influence of hormones. The plants showing change in sex-expression showed bends in the stems. The thickened growth observed at these regions of bends is probably due to the accumulation of growth hormones at these regions.


Impact of varying plant densities on two industrial hemp cultivars grown in the Manawatu

Q.M. Swanepoel, R. Barge, E. Kawana-Brown3and L.H.J Kerckhoffs
Agronomy New Zealand 48, 2018
https://www.agronomysociety.org.nz/f...ty_effects.pdf
Industrial hemp (Cannabis sativa L) is a fast-growing annual multi-purpose crop grown for its fibres, hurd, seeds and pharmaceuticals. The commercial production of industrial hemp is strictly legislated and only approved cultivars with delta-9-tetrahydrocannibinol (THC) levels under 0.35% are permitted in New Zealand. The effect of varying plant densities was studied for two industrial hemp cultivars (Fasamo and Ferimon 12) in the Manawatu region. Four planting densities (40, 80, 120 and 160 plants/m2 ) at two row spacings (15 cm and 30 cm) were evaluated. Ferimon 12 produced the greatest average biomass of 34.6 t/ha with 160 plants/m2 at a 30 cm row spacing. Cultivar not plant density or row spacing had the most effect on agronomic measurements. Ferimon 12 produced longer, heavier stems with higher overall biomass compared to Fasamo regardless of plant density or row spacing. Individual stem weight decreased as plant population increased for both cultivars. Higher planting density and closer row spacing improved weed suppression. .

Impacts of Surface Water Diversions for Marijuana Cultivation on Aquatic Habitat in Four Northwestern California Watersheds
Scott Bauer , Jennifer Olson , Adam Cockrill, Michael van Hattem, Linda Miller, Margaret Tauzer, Gordon Leppig
Plos ONE
doi: 10.1371/journal.pone.0120016
Marijuana (Cannabis sativa L.) cultivation has proliferated in northwestern California since at least the mid-1990s. The environmental impacts associated with marijuana cultivation appear substantial, yet have been difficult to quantify, in part because cultivation is clandestine and often occurs on private property. To evaluate the impacts of water diversions at a watershed scale, we interpreted high-resolution aerial imagery to estimate the number of marijuana plants being cultivated in four watersheds in northwestern California, USA. Low-altitude aircraft flights and search warrants executed with law enforcement at cultivation sites in the region helped to validate assumptions used in aerial imagery interpretation. We estimated the water demand of marijuana irrigation and the potential effects water diversions could have on stream flow in the study watersheds. Our results indicate that water demand for marijuana cultivation has the potential to divert substantial portions of streamflow in the study watersheds, with an estimated flow reduction of up to 23% of the annual seven-day low flow in the least impacted of the study watersheds. Estimates from the other study watersheds indicate that water demand for marijuana cultivation exceeds streamflow during the low-flow period. In the most impacted study watersheds, diminished streamflow is likely to have lethal or sub-lethal effects on state-and federally-listed salmon and steelhead trout and to cause further decline of sensitive amphibian species.

Increasing Inflorescence Dry Weight and Cannabinoid Content in Medical Cannabis Using Controlled Drought Stress
Deron Caplan, Mike Dixon, and Youbin Zheng
HORTSCIENCE 54(5):964–969. 2019.
doi: 10.21273/HORTSCI13510-18
Controlled application of drought can increase secondary metabolite concentrations in some essential oil-producing crops. To evaluate the effects of drought on cannabis (Cannabis sativa L.) inflorescence dry weight and cannabinoid content, drought stress was applied to container-grown cannabis plants through gradual growing substrate drying under controlled environment. Fertigation was withheld during week 7 in the flowering stage until midday plant water potential (WP) was approximately L1.5 MPa (drought stress threshold). This occurred after 11 days without fertigation. A well-irrigated control was used for comparison. Leaf net photosynthetic rate (Pn), plant WP, wilting (leaf angle), and volumetric moisture content (VMC) were monitored throughout the drying period until the day after the drought group was fertigated. At the drought stress threshold, Pn was 42% lower and plant WP was 50% lower in the drought group than the control. Upon harvest, drought-stressed plants had increased concentrations of major cannabinoids tetrahydrocannabinol acid (THCA) and cannabidiolic acid (CBDA) by 12% and 13%, respectively, compared with the control. Further, yield per unit growing area of THCA was 43% higher than the control, CBDA yield was 47% higher, ?9 -tetrahydrocannabinol (THC) yield was 50% higher, and cannabidiol (CBD) yield was 67% higher. Controlled drought stress may therefore be an effective horticultural management technique to maximize both inflorescence dry weight and cannabinoid yield in cannabis, although results may differ by cannabis cultivar or chemotype

Indigenous Uses and Ethnobotany of Cannabis sativa L. (Hemp) in Uttaranchal (India)
N. C. Shah
June 2004 Journal of Industrial Hemp 9(1):69-77
DOI: 10.1300/J237v09n01_07
Cannabis sativa L. is one of the oldest food, fibre, medicinal, psychoactive and oil plants known. It has been used by innumerable ethnic societies in Asia. Uttaranchal (India) is an ethnic region where the plant is a part of the local culture. In this paper the indigenous uses and ethnobotany of its seed, seed oil, stems, fibre, leaves, inflorescences and resin along with various recipes of seeds are described. A theory of its introduction to Uttaranchal by ethnic races is also given. It is concluded that in the light of the present commercial and industrial uses of Cannabis its cultivation should be promoted in Uttaranchal and other parts of the Himalayan regions of India, where it grows naturally and is cultivated for folk uses.

Induction of male flowers on female plants of Cannabis sativa by gibberellins and its inhibition by abscisic acid
H. Y. Mohan Ram, V. S. Jaiswal
September 1972, Planta Volume 105, Issue 3, pp 263–266|
https://www.researchgate.net/publica..._abscisic_acid
DOI: 10.1007/BF00385397
Gibberellins (GA3, GA4+7, GA7 and GA9) induce male flowers on female plants of Cannabis sativa. This is, depending on concentration, partially or fully inhibited by abscisic acid (ABA). The ABA effect can in turn be partially overcome by increasing the concentration of GA3.

Industrial Hemp as a Modern U.S. Commodity Crop
D W Williams
University of Kentucky (2019)
https://manitowoc.extension.wisc.edu.. .PP-Handout.pdf
Preface https://onlinelibrary.wiley.com/doi/...mp.frontmatter
Chapter 1: The History of Hemp John Fike, Ph.D.* DOI: 10.2134/industrialhemp.c1
Chapter 2: Hemp Grain D.W. Williams, Ph.D.*
DOI: 10.2134/industrialhemp.c2
Chapter 3: Hemp Fibers Trey Riddle,* Jared Nelson, and Patrick Flaherty
DOI: 10.2134/industrialhemp.c3
Chapter 4: Hemp AgronomyGrain and Fiber Production Jeff Kostuik and D.W Williams*
DOI: 10.2134/industrialhemp.c4
Chapter 5: Cannabinoids-Human Physiology and Agronomic Principles for Production R.A. Williams, M.D. and D.W. Williams, Ph.D.*
DOI: 10.2134/industrialhemp.c5
Chapter 6: Hemp Genetics and Genomics
Brian Campbell, Dong Zhang, and John K. McKay
DOI: 10.2134/industrialhemp.c6
Chapter 7: Economic Issues and Perspectives for Industrial Hemp Tyler B. Mark* and Will Snell
DOI: 10.2134/industrialhemp.c7
Epilogue D.W. Williams, PhD
DOI: 10.2134/industrialhemp.epilogue

Industrial Hemp as a Raw Material for Energy Production
Henryk Burczyk, Lidia Grabowska, Jacek Ko?odziej, Ma?gorzata Strybe
April 2008 Journal of Industrial Hemp 13(1):37-48
DOI: 10.1080/15377880801898717
According lo the "strategy lor the development of a renewable energy sector" the ,shyre ol renewable energy in the primarily energy balance in Poland should grow to l.yVr by 2010, The main souree of renewable energy will be biomass from annual and perennial energy erops. Sinec January I. 2(K)7 Pnland has been included in a European Union system of subsidies to energy crop prinluction and hemp will be included in the grttup of energy ert)ps. In Poland, climate and soil eonditions are suitable for hemp euhivation. Currently, there arc four eultivars of moiUMieious hemp regislered (Beniko, liialobr/.eskie. Silesia, and Tygra). yielding 1()-15 tons of bminass (x:r heetare. The technologies for hemp eultivation and harvesting are developed. Research conducted at the Institute of Natural Fibres has shown that hemp is a valuable raw material for energy production (its heat of combustion is about 18 MJ/kg). The source of energy is either the whole plant or its shives (75% of yield mass). Trials on producing briquettes from shives, conducted at the INF Experimental Farm at Steszew, were successful and a pilot line for the prtxluction of briquettes was installed.

Industrial Hemp (Cannabis sativa subsp. sativa) as an Emerging Source for Value-Added Functional Food Ingredients and Nutraceuticals
H. P. Vasantha Rupasinghe, Amy Davis, Shanthanu K. Kumar, Beth Murray and Valtcho D. Zheljazkov
Molecules 2020, 25(18), 4078;
DOI: 10.3390/molecules25184078
https://www.ncbi.nlm.nih.gov/pmc/art...s-25-04078.pdf
Industrial hemp (Cannabis sativa L., Cannabaceae) is an ancient cultivated plant originating from Central Asia and historically has been a multi-use crop valued for its fiber, food, and medicinal uses. Various oriental and Asian cultures kept records of its production and numerous uses. Due to the similarities between industrial hemp (fiber and grain) and the narcotic/medical type of Cannabis, the production of industrial hemp was prohibited in most countries, wiping out centuries of learning and genetic resources. In the past two decades, most countries have legalized industrial hemp production, prompting a significant amount of research on the health benefits of hemp and hemp products. Current research is yet to verify the various health claims of the numerous commercially available hemp products. Hence, this review aims to compile recent advances in the science of industrial hemp, with respect to its use as value-added functional food ingredients/nutraceuticals and health benefits, while also highlighting gaps in our current knowledge and avenues of future research on this high-value multi-use plant for the global food chain

Industrial Hemp (Cannabis sativa) for Phytoremediation of Uranium Contaminated Soil

Hanah Rheay
Catherine Brewer
FIND PDF.

INDUSTRIAL HEMP (CANNABIS SATIVA L.) PRODUCTIVITY AND RISK ASSESSMENT IN HEMP PRODUCTION
Aleksandrs ADAMOVIČS, Sandija ZĒVERTE-RIVŽA
NJF 25th Congress June 16-18, 2015
https://llufb.llu.lv/conference/NJF/...ia-242-248.pdf
The sector of hemp growing and processing is subjected to significant changes through the past decades, historically hemp growing was widespread and in Latvia hemp was broadly used in everyday life – in the local cuisine, for feeding animals and also as a building and textile material. It had lost its popularity but following the global tendencies in agriculture, growing and processing of hemp (cannabis sativa L.) is slowly regaining its positions mainly due to the versatile possibilities of using hemp. Though the total number of hemp growers is significantly smaller nowadays as it has been historically, the use of hemp is expanding and during recent years several enterprises have introduced new products that includes using hemp in food production, production of ecological construction materials, paper, production of textiles, biodegradable plastics and as mentioned, renewable energy production. Agriculture, including hemp production and processing, is one of the industries subject to risks due to changing weather conditions, diseases and pests and volatile market prices on inputs and products produced. This article aims to reflect the results of the risk evaluation in hemp production
and processing, for the evaluation purposes the hemp production is divided in five stages – preparation of soil and sowing of hemp; growing of hemp; hemp harvesting; hemp processing; and realisation of the produced
output. The evaluation of risks was made within a system of 18 risks, divided in 6 main groups – agrometeorological; technological and production; personnel; environment; legislative; economical and market risks. The results reviles, that the highest risk level in the entire hemp production and processing process was specific to the group of personnel risks, whereas the lowest – to the group of environmental risks. However, some risks were assessed as the highest for the group of technological and production risks, for instance, the unavailability of machinery during hemp harvesting and processing.

INDUSTRIAL HEMP PERFORMANCE IN ILLINOIS TRIALS - 2020
Crop Sciences Special Report 2020-04, October 2020
Darin K. Joos and Allen Parrish
http://vt.cropsci.illinois.edu/hemp/...ages%201-3.pdf
The first industrial hemp trials were conducted in 2020 at the University of Illinois Research and Education Center located in Urbana, IL. The trials were conducted to evaluate commercial varieties for fiber and grain yields.
Materials and Methods
Each variety had a 1000 seed weights measured (Table 1) in order to fill packets with the proper quantity of seed. The fiber trial had a target seeding rates of 26, 35, and 44 PLS per ft2. The grain trials had a target seeding rate of 18 PLS per ft2. Trials were fertilized with urea prior to planting at 50 lbs of nitrogen per acre for fiber and 100 lbs of nitrogen per acre for grain. Our industrial hemp variety trials consisted of six, 7.5-inch rows planted to a depth range of .5-.75 inches and trimmed to about 19 feet in length before harvest. Stand counts were taken two weeks after emergence to sure adequate plant populations for trial evaluations (Table 2 and Table 3). Flowering started in mid-July and ran through the middle of August (Table 1). After flowering, samples were taken to evaluate THC content along with uniformity ratings. Uniformity ratings are a subjective note taken for variety homogeneity (Table 1). All varieties fell below the legal limit of .3%. A period of hot, dry weather accelerated the industrial hemp crop. The fiber trial was harvested on August 31st with a Wintersteiger Cibus S and grain trial was harvested with an Almaco SPC 20 on September 17th. A subsample was collected for the fiber trials and allowed to remain in a drying oven for seven days at 140oF till no moisture was present. Whole plots were collected for grain trials. Each plot sample was placed in dryer for 18 hours at 90oF. The whole sample was passed through an aspirator to remove foreign material and reweighed to determine grain yields. Additional subsamples were collected immediately after cleaning, weighed, and placed back in the dryer for five days till 0% moisture was achieved. Yields were corrected to 0% moisture for both grain and fiber. AGROBASE Generation II by Agronomix Software, Inc statistical software was used to create each randomized complete block trial for grain and fiber and perform statistical analysis. No seed treatments were used during this trial.
Results and Discussion
The 2020 industrial hemp growing season in Illinois had a slow start. Early spring rains prevented a May planting and a flooding event on June 3rd resulted in both trials being replanted on June 15th. After replanting weather conditions improved and were favorable the rest of the growing season. Hemp was able to outcompete the majority of weeds observed and relatively low abundance in the plots used for compiling yield data. Pest pressure and no major diseases were observed throughout the growing season.
Jin Ma was dropped from the fiber trial due to poor germination results and not enough seed as available for replanting. Notes were taken from an observational plot (Table 1) for Jin Ma. No entry achieved the target population two weeks after planting for grain and fiber. No final stand counts were taken to identify if dormant seeds emerged later in the season. Fiber and grain yields, grain moisture and dry matter content, and stand counts (Tables 2 and 3) are provided with CV, LSD, and Test means.

INDUSTRIAL HEMP TRIALS WITH A NOVEL SOIL AMENDMENT: SOIL BALANCE PRO
Robert I. Bruck,
https://l.facebook.com/l.php?u=https://lookasid e.fbsbx.com%2Ffile%2F2019-Kind_Roots-Soil_Balance_Pro-Hemp_White_Paper.pdf%3Ftoken%3 DAWwSTLf0ZXIviYh_49cdutMxbT19z shvrru86IxvNEpffBAA16r6SBDMLgc wD0pGxFiCUrZCq4To3EvjyDCv0OfH8 7SkB7OKXETfSTeZWI18cZoVBDLU_pA RueU9hoMeWYKVc8zVMXng8HK4eIyIx z9vkTcxzVic28XpUM6_m5siplwApeh 7peyfEXeJsgF4L5fEpiV4EXccr1tgl oo9Q8lX-5lzMNThYy9F3ls9NFkSzgazVM18_aW GdB1cRHw_MyT3T3c0wgbOk1aO7gjLQ 7A4G1r_D6jmGMM1-lLTJnyRb5xTRb9Q5n9vcisfB5GSDYN 3KV6_buG1jtjYWFzsqdCv3Nj92rpqz 7FvAWvdFPZukjrJ5PbbOsG6iWM962O yl15pFvIoE-l1SWN7xL9DHBBLtWiiYwy-a6uxgrBRF5tYlwpr4u3gBKG8-zyng53qBlGeUvvoMxi7NOwkawV9aDJ hjNlXAqJGdFHn038vLhg8ZI1613aQ5 0vaQ4mb_4PA4y8KO47U3YvVgxxOtH2 eoCRXhqKitVofb-xg52OcUtGg41g9-nxUjUadpNKdYA-Y9TM85dG8-ytUOL--Ap_o3-BfiMG9UOjodW39h9X0LUTw5w&h=AT3 RnX3DvI_32ybLf55o8e0nPgdi2EzYi aIkO-haHeMbFg9kvl-qW7h2US9nriqN-CJWYkhqbmnPV1C0wSimwJi7H0Cl0TJ h0yOcfxqcYujZlDWf1eO84XWVC0deK LxED5B8EATqYVzow-DgWw&__tn__=H-R&c[0]=AT1x0JwfFddn5n-hNb9D_UEK1l2cjaLQ5zGurNigwWnXk 2c2J7pYWOoB_65wjPiaYh4oYWZKqUD fHH-N0sPRqhqxoBVhdrNIZcSEKBh62bLRq SISmW6_oIpXEio-FUwkljP2WMk7lZzccxZZqtG53Tivof eDHn8q4UXlOm4Id6-mn6tILrsn1C30dWPthSJ5Fq6fAG2Pm pJ-ybudiD14QLRWXsEl5ew
During the 2019 growing season in Franklin County, NC, research was conducted to evaluate the efficacy of Soil Balance Pro (SBP) on the phenological development of industrial hemp cultivar, T1. Data was
meticulously gathered on the growth, concentration, quality, and yield of the hemp to elucidate cannabinoid and terpene content. Throughout the growing process, up to one, two, or three applications of SBP were applied depending on the growth stage. Data collected at harvest (October 7) indicated significant improvement in the overall health and vitality of the hemp plant’s root, shoot, and flower (colas) tissues, increased the number of leaf bearing trichomes, increased the number and weight of colas, and doubled, or tripled the CBD, CBD-A, CBG, CBG-A cannabinoid and aromatic terpene content, respectively.

Influence of agronomic factors on yield and quality of hemp ( Cannabis sativa L.) fibre and implication for an innovative production system
Stefano Amaducci, Alessandro Zatta,Federica Pelatti, Gianpietro Venturi
May 2008 Field Crops Research 107(2):161-169DOI: 10.1016/j.fcr.2008.02.002
Research and development of an innovative production system for hemp (Cannabis sativa L.) fibre for textile use requires the integration of multidisciplinary knowledge from cultivation technique to realization of end products. Research was carried out to study the effect of the agronomic factors cultivation year (2003–2004), genotype (Futura 75 and Tiborszallasi), plant population (120, 240 and 360 plants m2 ) and harvesting time (beginning and full flowering) on fibre yield and quality in the whole hemp stem, and in the basal and apical stem portions separately. The study of separate stem portions was done to determine the effect on fibre quality of an innovative harvesting and processing system in which hemp stems are cut in two portions of approximately 1 m at harvest to enable processing on modern flax scutching lines. Stem and fibre yield were affected by most of the agronomic factors. The extreme drought experienced in the first year reduced stem and fibre yield, but stems had higher percentage of fibre (16.5%), that were finer (22.9 mm) and with a higher degree of maturity (73.6%) in 2003 than in 2004 (respectively 16.0%; 24.5 mm; 55.8%). Between the two genotypes under trial the monoecious Futura 75 largely out yielded the dioecious Tiborszallasi in both years. The latter however had finer primary fibres and less secondary ones. In both genotypes primary fibres maturity and quantity of secondary fibres increased at later harvest. Plant population affected stem biometrics and fibre characteristics, with finer fibres and less secondary growth at higher stands. It can be concluded that cultivation technique can be exploited in order to maximize the quality and yield of stems destined for the innovative harvesting and processing system herein described.

Influence of Temperature on the Water Retting Process of Hemp (Cannabis sativa L.) Cultivated Under Swedish Climate Conditions
Karin Magnusson, Bengt Svennerstedt
November 2007Journal of Industrial Hemp 12(2):3-17
DOI: 10.1300/J237v12n02_02
Hemp has recently received increased interest as a sustainable crop for a multitude of possible uses. This paper focuses on the retting process, which is an important part in the processing of high quality fibre for, e.g., textile products. Water retting process trials were performed on stem plant material from Futura 75 grown at the Lönnstorp Farm research station in the south of Sweden in 2004. Three retting treatments at water temperature 30, 37.5 and 45°C were performed on dried hemp material. Hemp samples were taken from each of the three treat ments at 48, 96, 144 and 196 hours. The retting process was investigated and documented by using the change in pectin content and the change in pH value as measures of the rettability. It was not possible to determine an optimal temperature for the retting process by these trials. However it could be concluded that 45°C was too high a water temperature.

Introduction to emerging industrial applications of cannabis (Cannabis sativa L.)
Giuseppe Sorrentino
Rendiconti Lincei. Scienze Fisiche e Naturali 2021
DOI: 10.1007/s12210-021-00979-1
https://www.researchgate.net/publica...nabis_sativa_L
The Italian Law of 22 November 2016 has legalized the cultivation of hemp, which drives the development of sustainable agriculture by generating new products with high added value in the new context of circular economy. Hemp cultivation is known for its low environmental impact, as hemp grows fast, suppresses weeds and does not need pesticides. It has no specialized parasites, favors pollination and improves the physical and chemical soil fertility. Recently, many countries have increased their interest in hemp ( Cannabis Sativa L.), considering it as a climate-friendly crop that can mitigate climate change and desertification. For these reasons, hemp can be a new protagonist of Italian agriculture already oriented towards the objectives of EU 2030 which predicts 40% decrease in greenhouse gas emissions compared to 1990. The hemp cultivation can activate a new supply chain by allowing using different parts of the plant, benefiting farmers, environment, and human health. Indeed, although a very old plant, hemp will be one of the main protagonists of the green economy in the near future. Its seeds can be used by agri-food industry to produce flour, pasta, pastry and oil, while the stem through canapulo (woody part of stem) in green building sector. Its fiber (external part of stem) will find new applications in textile industry. As for its inflorescences and roots, thanks to the extraction of bioactive molecules, they will play an important role in the pharmaceutical and parapharmaceutical industry. Finally, only the medical sector with Δ ⁹ ‐tetrahydrocannabinol (THC) extraction from inflorescence is not yet regulated by the aforementioned Italian Law.


Intrusive growth of primary and secondary phloem fibres in hemp stem determines fibre-bundle formation and structure.
Anastasia Snegireva, Tatyana Chernova, Marina Ageeva, Tatyana Gorshkova
May 2015 AoB PLANTS 7(1)
DOI: 10.1093/aobpla/plv061
Plant fibres -cells with important mechanical functions and a widely used raw material - are usually identified in microscopic sections only after reaching a significant length or after developing a thickened cell wall. We characterized the early developmental stages of hemp (Cannabis sativa) stem phloem fibres, both primary (originating from the procambium) and secondary (originating in the cambium), when they still had only a primary cell wall. We gave a major emphasis to the role of intrusive elongation, the specific type of plant cell growth, by which fibres commonly attain large cell length. We could identify primary phloem fibres at a distance of only 1.2-1.5 mm from the shoot apical meristem when they grew symplastically with the surrounding tissues. 0.5 mm more basal, fibres began their intrusive elongation, which led to a sharp increase in fibre numbers visible within the stem cross-sections. The intrusive elongation of primary phloem fibres was completed within the several distal centimeters of the growing stem, before the onset of their secondary cell wall formation. The formation of secondary phloem fibres started long after the beginning of secondary xylem formation. Our data indicate that only a small portion of the fusiform cambial initials (<10%) give rise directly or via their derivatives to secondary phloem fibres. The key determinant of final bundle structure, both for primary and secondary phloem fibres, is intrusive growth. Through bi-directional elongation, fibres join other fibres initiated individually in other stem levels, thus forming the bundles. Our results provide the specific developmental basis for further biochemical and molecular-genetic studies of phloem fibre development in hemp, but may be applied to many other species.

Irrigation Management Strategies for Medical Cannabis in Controlled Environments
Jonathan Stemeroff
A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science (2017)
https://atrium.lib.uoguelph.ca/xmlui...=8&isAllowed=y
Medical cannabis production is a new industry in Canada and represents a challenge for the production of a repeatable and standardized product for medical use. A reliable and reproducible environmental control strategy can contribute significantly to meeting this challenge. Irrigation management and control of plant water status is one of the key environmental control elements. To assess the effects of various irrigation management strategies this study deployed in situ stem psychrometers to measure the water status of plants. As a routine feedback device for irrigation control these devices are not ideal for large-scale production so correlation with the key environment variable representing the aerial demand for moisture (vapour pressure deficit) was assessed. By establishing a relationship between cumulative water potential (cWP) and cumulative vapour pressure deficit (cVPD) an irrigation management strategy that predicted plant water status based on
measurements of cVPD could be employed. Three treatments; control (irrigation events every 1-2 days), mild-stress (irrigation events every 2 days), and moderate-stress (irrigation events every 3 days) were tested. The effects of flushing were also investigated to determine whether it had the intended effect of reducing nutrient concentrations within the dried bud. Through the use of psychrometers, water status (cWP) thresholds were correlated with humidity (cVPD) thresholds and reduced irrigation frequency resulting in water use reductions up to 45.7% which had negligible impacts on yield and cannabinoid profile. Flushing was found to be ineffective in removing any significant amount of nutrient from the bud.

Karyological Studies in Root-Tip Cells of Cannabis sativa var. indica
P. Srivastava 1, S. Srivastava 2, M. K. Verma3 and S. K. Mishra
January 1999 Cytologia 64:435-440
DOI: 10.1508/cytologia.64.435
Chromosomal study was carried in the dividing root-tip cells of Cannabis sativa (Family: Cannabinaceae). The diploid chromosome number of this species is 2n=20. Karyotype analysis reveals that all the 9 chromosomes in female somatic cells are metacentric and 1 chromosome is submetacentric whereas the X and Y chromosome in male cells is sub-metacentric. The Y chromosome is longer than X chromosome. The third pair bears the satellite.

Key cultivation techniques for hemp in Europe and China
July 2014 Industrial Crops and Products 68
Stefano Amaducci, Danilo Scordia, Fei-Hu Liu, Salvatore Luciano Cosentino
DOI: 10.1016/j.indcrop.2014.06.041
Project: Fibre crops as feedstock for biobased products and bioenergy "FIBRA"
Hemp (Cannabis sativa L.) is a multiuse, multifunctional crop that provides raw material to a large number of traditional and innovative industrial applications. A relatively simple, low input cultivation technique and the sustainability of its products are the main drivers for a future expansion of the hemp crop. In Europe, the large political support of bioenergy in recent years has fuelled numerous studies on the potential cultivation of hemp for bioenergy production. In China the main drivers for a renewed interest in hemp are its traditional applications. For any given destination, the main target of hemp cultivation is the maximization of biomass production, but each end-use destination has specific quality requirements in terms of properties of the bast fibre, characteristics of the oil and proteins in the seeds, or profile of secondary metabolites in the inflorescence.
In this paper, traditional and innovative end use destinations and cultivation systems for hemp are introduced, together with some notes on hemp botany, biology, and resource use efficiency. This information, together with a review of the practical experience of hemp cultivation in Europe and China and knowledge gathered form scientific literature, highlights the effect of agronomic factors in determining the yield potential and quality level of hemp for specific end use destinations. To conclude, future perspectives and recommendations for hemp cultivation and research are discussed.

Light dependence of photosynthesis and water vapor exchange characteristics in different high _9-THC yielding varieties of Cannabis sativa L.
Suman Chandraa, Hemant Lataa, Zlatko Mehmedica, Ikhlas A. Khana, Mahmoud A. ElSohly
• Journal of Applied Research on Medicinal and Aromatic Plants 2(2) March 2015
Doi: 10.1016/j.jarmap.2015.03.002
The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600and 2000 _mol m?2s?1) on gas and water vapor characteristics of four high _9-tetrahydrocannabinol (_9-THC) yielding drug type varieties (HPM, K2, MX and W1) ofCannabis sativa was studied. Plants of each variety were grown from seeds. On flowering,male plants were removed and vegetatively propagated clones of selected female plantswere used for gas and water vapor studies at different photosynthetic photon flux densi-ties (PPFDs). Our data show an increasing trend in photosynthesis (PN), transpiration (E)and stomatal conductance (gCO2) with increase in PPFD up to 2000 _mol m?2s?1in allvarieties at optimum growth temperature (25 ± _3?C). However, the magnitude of increaseand maximum rate of PN(PN max) varied considerably with the varieties. Highest PNwasobserved in W1 followed by MX, K2 and HPM. Water use efficiency (WUE) in W1, MX andHPM increased with PPFDs up to the highest level tested, whereas, in K2 the highest WUEwas observed at 1600 _mol m?2s?1. Our results suggest that this species is able to use high level of PPFDs for its PNand therefore, may be cultivated in sun exposed areas in the field orunder high PPFDs using indoor grow lights for the optimum growth. Strict control of otherenvironmental factors, however, needs to be maintained while growing the plants indoor.

List of Approved Cultivars for the 2019 Growing Season: Industrial Hemp Varieties Approved for Commercial Production
https://www.canada.ca/en/health-cana...is-sativa.html

Medical cannabis production: Starting from the root zone
Poster
DOI: 10.13140/RG.2.2.33760.58888
https://www.researchgate.net/publica..._the_root_zone

Microscopic Study on Hemp Bast Fibre Formation.
Hernandez, A., Westerhuis, W., & van Dam, J. E. G.
Journal of Natural Fibers, 3(4), 1–12. (2007).
doi:10.1300/j395v03n04_01
Commonly, fibre hemp (Cannabis sativa) is not considered for the production of soft and easy-care textiles. However, initial tests in the textile industry have shown that certain processes allow improvement in the everyday-wear characteristics of hemp textiles. In the framework of the EU R&D project Hemp-sys [QLRT-01-01363] the potential of hemp as a feedstock in textile processing is investigated. Therefore, a detailed study on the effects of agronomy and fibre extraction processing on fibre properties for textile processing has been carried out. To relate the crop growing conditions with fibre quality and productivity it is required that information is obtained on hemp bast fibre formation. At various stages of the plant development the fibre development has been monitored by microscopic techniques. The two distinct bast fibre types occurring in hemp–the so called primary and secondary fibres–show different and independent development. The primary fibres appear earlier in the development in the rapid growth stage and are formed from the primary meristem in the outer layers (phloem). Primary fibres are thick, extremelyong and with compact cell walls and small lumen and are present from bottom to top of the plant. In contrast, the secondary fibres are formed in later stages during thickness growth from the secondary meristem. Secondary fibres are thinner, shorter and with higher lignin content than the primary fibres. In early developmental stages secondary fibres only occur at the bottom of the stem. Substantial secondary fibre formation is observed at the start of flowering or when the plants grow taller than 2.25 m and/or thicker than approximately 1 cm. Based upon these observations a selection of conditions can be made for growing and harvesting of hemp to obtain the best quality fibre for textile processing. To produce a crop with maximum amount of primary fibres free from secondary fibres, the recommended practice is to grow hemp at high seed density to avoid thickness growth and to harvest the hemp stems around flowering of the plants

Minnesota wild hemp: a crucial botanical source in early cannabinoid discovery
Crist N. Filer
Journal of Cannabis Research (2020) 2:25
doi 10.1186/s42238-020-00031-3
Renewed and sustained Cannabis chemistry exploration was initiated by Roger Adams at the University of Illinois Chemistry Department with cooperation from the Treasury Department Narcotics Laboratory in the early 1940’s. This partnership and time investment by both parties made practical sense. Adams was able to explore natural products chemistry and the Narcotics Laboratory began to clarify the chemistry mysteries of Cannabis. Minnesota wild hemp, often viewed as just a roadside weed, was employed as the critical botanical source. Based on its widespread cultivation during World War II, this was also a very pragmatic decision. Although the unique Illinois – Washington D. C. collaboration lasted only a few short years (1939–1942), the stunning results included the isolation and extensive characterization of cannabidiol, the structure elucidation and total synthesis of cannabinol as well as the identification of the tetrahydrocannabinol structure as an intoxicating pharmacophore. Furthermore, this research well prepared many junior chemists for prolific careers in both academia as well as industry, inspired the discoveries of later Cannabis investigators and also provided a successful model of a productive academic-government partnership.

Morpho-Physiological and Metal Accumulation Responses of Hemp Plants (Cannabis Sativa L.) Grown on Soil from an Agro-Industrial Contaminated Area
Fabrizio Pietrini, Laura Passatore, Valerio Patti, Fedra Francocci, Alessandro Giovannozzi and Massimo Zacchini
Water (2019) 11, 808;
doi:10.3390/w11040808
Hemp is a promising plant for phytomanagement. The possibility to couple soil restoration to industrial crop cultivation makes this plant attractive for the management of contaminated sites. In this trial, Cannabis sativa L. plants were grown in a greenhouse on soils from two sites of “Valle del Sacco” (Lazio Region, Italy), a wide area contaminated by agro-industrial activities.
One site was representative of moderate and diff use metal(loid) multi-contamination, above the Italian concentration limit for agriculture (MC—moderately contaminated). The second site showed a metal(loid) content below the aforementioned limit, as a typical back ground level of the district (C—control). After 90 days, biometric and physiological parameters revealed satisfactory growth in both soil types. MC-grown plants showed a slight, but significant reduction in leaf area, root, and leaf biomass compared with C-grown plants. Chlorophyll content and chlorophyll fluorescence parameters, namely the quantum yield of primary photochemistry (Fv/ Fm ) and the Performance Index (PIABS ), confirmed the good physiological status of plants in both soils. Metal(loid) analyses revealed that As, V, and Pb accumulated only in the roots with significant differences in MC- and C-grown plants, while Zn was found in all organs. Overall, preliminary results showed a satisfactorily growth coupled with the restriction of toxic metal translocation in MC-grown hemp plants, opening perspectives for the phytomanagement of moderately contaminated areas.

New developments in fiber hemp (Cannabis sativaL.) breeding
Elma M.J.Salentijn, Qingying Zhang, Stefano Amaducci, Ming Yang, Luisa M.Trindade
Industrial Crops and Products
Volume 68, June 2015, Pages 32-41
doi: 10.1016/j.indcrop.2014.08.011
Fiber hemp (Cannabis sativa L.) is a sustainable and high yielding industrial crop that can help to meet the high global demand for fibers. Hemp can be grown for fiber, seeds, and/or for dual purpose in a wide range of geographic zones and climates. Currently the main hemp producing regions in the world are China, Europe, and Canada. The number of new cultivars developed for each of these regions has gradually increased, with each region producing its own typical hemp cultivars for different purposes. In this article, the state of the art of fiber hemp breeding programs in Europe, China, and Canada are reviewed. The breeding strategies and tools used in the breeding of hemp cultivars are discussed. We also provide an overview of genetic diversity in hemp for different traits. In addition, the current knowledge of the main breeding goals for fiber hemp, which are an improvement of fiber quality and fiber yield, breeding for specific cannabinoid profiles, control of flowering behavior, male flowering control, and breeding of cultivars for specific environments are evaluated. Lastly, we discuss the inestimable value of next generation technologies to breed new hemp cultivars that are suitable for a biobased economy.

New evidence on the introduction, cultivation and processing of hemp (Cannabis sativaL.) in southern Sweden.
Larsson, M., & Lagerås, P.
Environmental Archaeology, 20(2), 111–119.(2014).
doi:10.1179/1749631414y.0000000029
Subfossil remains of Cannabis sativa L. (hemp) have been found at Lindängelund in the region of Malmö, southern Sweden. These represent the earliest robust evidence so far for hemp retting in Scandinavia. Finds of seeds, stems and pollen of C. sativa from a waterlogged context on a settlement dating to the Roman Iron Age demonstrate that the plant was locally cultivated and processed during the 1st–2nd centuries AD. An introductory phase in Scandinavia is proposed (c. AD 1–400) during which the cultivation of hemp was apparently small scale and processing was probably carried out within settlements. In the succeeding centuries, c. AD 400–550 (the Migration Period), remains of hemp are mostly found in pollen records from lake sediments, and less frequently in the archaeological record. This could indicate that the process of hemp retting relocated from settlements to lakes shores where activity became larger in scale and more integrated with the prevailing agricultural system.

New Technology of Harvesting Hemp Grown for Seed
Henryk Burczyk, Ryszard Kaniewski
June 2005 Journal of Industrial Hemp 10(1):49-60
DOI: 10.1300/J237v10n01_05
The expected growth of hemp cultivation world wide for industrial purposes will clearly cause a growing demand for sowing material. Hence, there is a need for the development of a new, more efficient method for harvesting hemp seed. The technology described in this paper, elaborated at the Institute of Natural Fibres (INF), presents the new concept of a panicle cutting device that involves mowing and cutting stems into smaller sections. The cut-off panicles are collected on a trailer hooked behind the mower. The panicles harvested during the flowering phase are used for the production of essential oils, while those harvested in full maturity are the source for sowing seed. In the latter case, the panicles–before ginning–must be initially dried in conditions allowing for preservation of good germination capacity. The seed must be cleaned immediately and dried further to 12% moisture content and protected with fungicidal treatments. After mowing and cutting stems are swathed in the field and left for retting or collected–after drying–using agricultural presses and then decorticated.

New Tropical Industrial Hemp
T. Jobling, P. Warner
https://www.agronomyaustraliaproceed.. ./c/jobling.pdf
Industrial hemp trials in Australia have previously been limited to using European certified cultivars that are specifically adapted to long summer daylengths for production. In Australia, these varieties flower prematurely thereby limiting yield. Australian Hemp Resource and Manufacture (AHRM) has developed subtropical cultivars and in the summer 1999 – 2000 conducted a comprehensive variety trial near Toowoomba. The subtropical variety INSX achieved yields over 10 t/ha DM harvested stalk, with all subtropical variety yields higher than those of European cultivars. The trial used staggered sowing dates to coordinate maturity of early flowering and late flowering varieties. The results are discussed with respect to sowing dates, THC concentrations, harvesting times and maturation.

Nitrogen fertilization and row width affect self-thinning and productivity of fibre hemp (Cannabis sativa L.).
Van der Werf, H. M. G., van Geel, W. C. A., van Gils, L. J. C., & Haverkort, A. J.
Field Crops Research, 42(1), 27–37. (1995).
doi:10.1016/0378-4290(95)00017-k
In fibre hemp (Cannabis sativa L.) a high plant density is desirable, but inter-plant competition may cause self-thinning, which reduces stem yield and quality. We investigated whether agronomic factors could reduce self-thinning in hemp. The effects of soil nitrogen level (80 and 200 kg ha- ~ ), row width ( 12.5, 25 and 50 cm), type of sowing implement, and thinning method on self-thinning, growth, yield and quality of hemp were determined in field experiments in 1991 and 1992. Soil nitrogen level affected plant morphology before self-thinning occurred. Due to enhanced competition for light more plants died from self thinning at 200 than at 80 kg N ha 1. In August, stem yield of living plants was similar at the two nitrogen levels, but 5% of the
plants had died at 80 kg N ha ~ and 25% at 200 kg N ha- 1. Although dry matter losses resulting from self-thinning were greater at 200 than at 80 kg N ha- ~, crop growth rate was greater at 200 than at 80 kg N ha- i. Apparently, the crop growth at 80 kg N ha- ~ was affected by a lack of nitrogen. At final harvest in September stem yield of living plants was 10.4 t ha- ~ at 80 and 11.3 t ha ~ at 200 kg N ha- ~, bark content in the stem was 35.6% at 80 and 34,0% at 200 kg N ha- ~. The effect of row width on self-thinning was small relative to that of nitrogen level. More self-thinning took place at 50 cm row width than at 12.5 and 25 cm. During early growth and also in August stem yield was smaller when row width was larger; in September row width did not
affect stem yield or quality. Type of sowing implement and thinning method did not affect self-thinning or stem yield.

Nitrogen fertilization and sex expression affect size variability of fibre hemp (Cannabis sativa L.).
Van der Werf, H. M. G., & van den Berg, W.
Oecologia, 103(4), 462–470.)1995).
doi:10.1007/bf00328684
Mechanical harvesting and industrial processing of fibre hemp (Cannabis' sativa L.) require uniformity of stem length and weight. In 1991 and 1992 we carried out field experiments to investigate the effects of soil nitrogen level (80 and 200 kg ha 1 N) and row width (12.5, 25 and 50 cm) on the variability of weight and height in hemp plants. The crops were sampled 5 times between early June and early September. Row width did not affect size variability. At final harvest coefficients of variation (CV) of both weight and height were about 1.5 times higher at 200 than at 80 kg ha-1 N. Distributions of dry weight were positively skewed at all sampling dates except the first, with skewness larger at 200 than at 80 kg ha-1 N. Distributions of height were negatively skewed at all sampling dates except the first at 80 kg ha- 1 N. At 200 kg ha- 1 N they changed from negative skewness during the first part of the growing season to negative kurtosis in the second part of the growing season. More suppressed plants were present at 200 than at 80 kgha-lN. Contrary to most published results, we did not find a reduction of CV of weight nor of CV of height at the onset of self-thinning. Suppressed hemp plants can survive
relatively well in the low-light environment under the canopy. Sexual dimorphism contributed to variability of height and weight, but the effects were smaller than those of nitrogen fertilization. The ratio of female to male plants was higher at 200 than at 80 kg ha- ~ N, as a result of a shift in sex-ratio within the population of suppressed plants. As suppressed plants were much more slender than dominating plants, self-thinning eliminated the most slender individuals in a hemp crop. However, the presence of many more heavy individuals of low slenderness at 200 than at 80 kg ha- 1 N was probably the major cause of the difference in slenderness between 200 and 80 kg ha-1 N.

Northern Limits to Fiber Hemp Production in Europe
Katri Pahkala, Heidi Syrjälä
October 2008 Journal of Industrial Hemp 13(2):104-116
DOI: 10.1080/15377880802391084
During the 1900’s fiber hemp was commercially grown in Scandinavia at latitudes as far north as 65°N and 66°N. Hemp is sensitive to temperature and day length with a quantitative short-day requirement, though long days can be beneficial for fiber production in late varieties, provided that other growing conditions are optimal. The response of fiber hemp varieties to photo period and low temperatures at northern latitudes was studied in Finland in 2003 and 2004. The article also reports on risks and prerequisites of hemp growing at high latitudes showing that if a suitable variety is used, stem yields of more than 10 t ha?1 are possible. If a variety adapted to southern European climates should be grown, however, the short growing season and the frosts in the beginning and at the end of the summer can restrict the plant growth. The northern limit for hemp production in Europe is currently at 64 to 65°N.

Nutrient concentrations, digestibility, and cannabinoid concentrations of industrial hemp plant components.
Kleinhenz, M. D., Magnin, G., Ensley, S. M., Griffin, J. J., Goeser, J., Lynch, E., & Coetzee, J. F.
Applied Animal Science, 36(4), 489–494.(2020).
doi:10.15232/aas.2020-02018
Objective: The cultivation and production of industrial hemp [Cannabis sativa containing <0.3% tetrahydrocannabinol (THC)] has increased. Information regarding the nutrient composition and cannabinoid concentration of different plant parts are deficient. Materials and Methods: Single lots of industrial hemp plants and by-products of cannabinoid production were obtained from a licensed research station located in south-central Kansas. Samples obtained were (1) whole industrial hemp plants (no roots), (2) stalks remaining after seed harvesting, (3) unprocessed female flowers intended for cannabinoid extraction, (4) whole seed heads for seed production, (5) leaves obtained from the drying process, (6) chaff obtained after seed harvesting and cleaning, and (7) processed female flowers after cannabinoid extraction. Plant materials were submitted for nutrient concentration and digestibility analysis, and for cannabinoid concentration determination, to a commercial nutrition laboratory. Results and Discussion: Dry matter of the plant material ranged from 65 to 96.6%. Crude protein ranged from 5.3 to 24.5%. Calcium concentration was from 1.0 to 5.7% DM. The plants tested had high fiber concentrations, with NDF ranging from 28 to 80% and ADF ranging from 18 to 65% DM. Total digestible nutrients was 19.8 to 61.5. Six of the 10 cannabinoids tested were detected in all samples. Cannabidiolic acid, cannabidiol, and ?9-tetrahydrocannabinolic acid A were detected in all samples at the highest concentrations. Implications and Applications: These findings will assist livestock producers in using industrial hemp in animal feeds through consideration of both the nutritional and cannabinoid concentrations in the ration.

Oliehennep
Cannabis sativa
https://edepot.wur.nl/221807 IN DUTCH

Optimal Rate of Organic Fertilizer during the Flowering Stage for Cannabis Grown in Two Coir-based Substrates
Deron Caplan, Mike Dixon, and Youbin Zheng
HORTSCIENCE 52(12):1796–1803. 2017.
doi: 10.21273/HORTSCI12401-17
In the expanding North American medical cannabis industry, growers lack reliable and systematically investigated information on the horticultural management of their crops, especially with regard to nutrient management and growing substrates. To evaluate organic substrates and their optimal nutrient management, five rates that supplied 57, 113, 170, 226, and 283 mg N/L of a liquid organic fertilizer (2.00N–0.87P– 3.32K) were applied to container-grown plants [Cannabis sativa L. ‘WP:Med (Wappa)’] in two coir-based organic substrates. The trial was conducted in a walk-in growth chamber and the two substrates used were ABcann UNIMIX 2-HP (U2-HP) with lower container capacity (CC) and ABcann UNIMIX 2 (U2) with higher CC. U2-HP produced 11% higher floral dry weight (yield), 13% higher growth index (GI), 20% higher ?9 - tetrahydrocannabinol (THC) concentration, 57% higher THC yield (per plant), 22% higher D9 -tetrahydrocannabidiolic acid (THCA) yield, and 20% higher cannabigerolic acid (CBGA) yield than U2. Increasing fertilizer rate led to increased growth and yield but also to a dilution of THC, THCA, and CBGA. In U2-HP, to maximize both yield and cannabinoid yield, the optimal organic fertilizer rates were those which supplied 212– 261 mg N/L. For U2, the highest applied rate, that supplied 283 mg N/L, maximized yield; although lower rates delivered higher cannabinoid concentrations in dry floral material. The results on these substrates and recommended fertilizer rates can serve as a guide when using other organic fertilizers and substrates; although results may differ with cannabis variety.

Optimal Rate of Organic Fertilizer during the Vegetative-stage for Cannabis Grown in Two Coir-based Substrates
Deron Caplan, Mike Dixon, and Youbin Zheng
HORTSCIENCE 52(9):1307–1312. 2017.
doi: 10.21273/HORTSCI11903-17
Cannabis producers, especially those with organic operations, lack reliable
information on the fertilization requirements for their crops. To determine the optimal organic fertilizer rate for vegetative-stage cannabis (Cannabis sativa L.), five rates that supplied 117, 234, 351, 468, and 585 mg N/L of a liquid organic fertilizer (4.0N–1.3P–1.7K) were applied to container-grown plants with one of two coir-based organic substrates. The trial was conducted in a walk-in growth chamber and the two substrates used were ABcann UNIMIX 1-HP with lower water-holding capacity (WHC) and ABcann UNIMIX 1 with higher WHC. No differences in growth or floral dry weight (yield) were found between the two substrates. Pooled data from both substrates showed that the highest yield was achieved at a rate that supplied 389 mg N/L (interpolated from yield-fertilizer responses) which was 1.8 times higher than that of the lowest fertilizer rate. The concentration of ?9-tetrahydrocannabinol (THC) in dry floral material was maximized at a rate that supplied 418 mg N/L, and no fertilizer rate effects were observed on D9-tetrahydrocannabidiolic acid (THCA) or cannabinol (CBN). The highest yield, cannabinoid content, and plant growth were achieved around an organic fertilizer rate that supplied 389 mg N/L during the vegetative growth stage when using the two coirbased organic substrates.

Overview on Italian hemp production chain, related productive and commercial activities and legislative framework
Luca Giupponi, Valeria Leoni, Matteo Carrer, Giulia Ceciliani, Stefano Sala, Sara Panseri, Radmila Pavlovic, Annamaria Giorgi
Italian Journal of Agronomy (2020)
DOI: 10.4081/ija.2020.1552
Once a very important crop, the cultivation of hemp has seen a significant reduction in Europe for a long time, but it is now subject to direct payments and promotion initiatives. Italy used to be an important producer and exporter of hemp textiles until the Second World War but currently information is lacking regarding the hemp production chain and legislation on the issue is often misunderstood by producers. Moreover, there has been an important development of economic activities connected with hemp, such as the so-called “hemp shops” or “grow shops” and the market of a product called “cannabis light”, the dried inflorescences of industrial hemp. The aim of the study was to investigate 30 Italian hemp farms in order to identify the characteristics of the production chain and the uses of the crop. Some considerations on this sector in the Italian economic and legislative contest are made and an anonymous web survey on the commercial activities associated with hemp (grow shops) was conducted. Most farms are multifunctional, 83% have been set up recently (in the last ten years) and directly as hemp producers. They are run by young entrepreneurs (57% holders under 35) and allow the use of marginal abandoned territories (43% of the farms). The 30 farms cultivate 460 ha of hemp and the extension of the field crops is very variable, from small patches in the mountains of 0,001 ha to more than 100 ha farms in the plains (in particular in Campania region). Almost all the farms use the crop to produce more than one end-product (seeds, flour, decorticated seeds, hemp-beer, seeds for animal nutrition and food oil from seeds, seeds and inflorescences harvested for cosmetic use, herbal use and extraction of active ingredients). In some cases, “technical use” linked to selling of the dry top inflorescences of industrial hemp for smoking was declared and it was found that there has been a significant increase in grow shops in Italy, from 4 in 2002 to more than 700 in 2018. As emerges from the analyses of European and Italian legislation, there is a need for clear regulations and a system of control by regulatory organizations considering the actual criticalities. At the same time, the renewed appeal of this crop derives from the multiple possibilities of use of the plant and from growing consumer demand for eco-compatible and sustainable products

Phenological growth stages of hemp (Cannabis sativa L.): codification and description according to the BBCH scale
Sergey Mishchenko, Jurij Mokher, Iryna Laiko, Natalija Burbulis, Hanna Kyrychenko, Svetlana Dudukova
https://www.lmaleidykla.lt/ojs/index...view/3496/2295
10.6001/zemesukiomokslai.v24i2.3496
The current paper discusses the characteristics of principal growth stages of hemp (Canabis sativa), provides a brief description of these stages and of the coding system according to the BBCH scale. The growing season of hemp is divided into nine principal growth stages: germination and sprouting (0), leaf development (1), formation of lateral shoots (2), stem elongation (3), inflorescence & emergence (5), flowering (6), development of fruit (7), ripening of fruit (8) and senescence (9). The principal growth stages are subdivided into secondary stages based on their main distinctive features. The classification system of hemp growth stages presented in the paper is designed for hemp growers and researchers, including plant breeders, physiologists, entomologists and other agricultural producers. Using the uniform coding system of the phenological stages of hemp is a valuable tool to ensure a more efficient planning and implementation of crop and soil management practices and techniques

Phenotypic Variation of Cell Wall Composition and Stem Morphology in Hemp (Cannabis sativa L.): Optimization of Methods.
Petit, J., Gulisano, A., Dechesne, A., & Trindade, L. M.
Frontiers in Plant Science, (2019).
10.doi:10.3389/fpls.2019.00959
The growing demands for sustainable fibers have stimulated the study of genetic diversity in the quality of hemp fiber (Cannabis sativa L.). Nevertheless, the lack of highthroughput phenotyping methods that are suited for the analysis of hemp fiber, hampers the analysis of many accessions, and consequently the breeding for this complex trait. In the present report, we developed and optimized the throughput of five methods to study the diversity in hemp fiber quality including cell wall extraction, biochemical composition of cell wall polysaccharides, quantification of lignin, quantification of crystalline polysaccharides and morphology of the stems. Six hemp accessions contrasting for cell wall properties were used to assess the throughput and suitability of these methods for genetic studies. The methods presented revealed to be highly repeatable, with low coefficients of variation between technical replicates. With these methods we were able to detect significant phenotypic variation in cell wall composition and stem morphology between the six accessions. In addition, the throughput of the methods has been upgraded to a level that enables their use for phenotyping cell wall traits in breeding programs. The cell wall extraction was optimized to extract enough material for the complete characterization of the cell wall of hemp while reducing the time for the entire analysis. The throughput of the stem morphological analysis was improved by decreasing the timing of fixation, infiltration, and embedding of mature and dry hemp stems. Notwithstanding, our methods already have the potential to phenotype large number of accessions in a relatively short period of time. Our methods will enable exploration of genetic diversity of fiber quality and will contribute to the development of new hemp varieties with advanced quality of fibers

Photosynthetic response of Cannabis sativa L. to variations in photosynthetic photon flux densities, temperature and CO2 conditions
Suman Chandra, Hemant Lata, Ikhlas A. Khan, and Mahmoud A. Elsohly
Physiol. Mol. Biol. Plants, 14(4)–October, 2008
doi:10.1007/s12298-011-0066-6
The effect of elevated CO2 concentrations (545 and 700 ?mol mol(-1)) on gas exchange and stomatal response of four high ?(9)-THC yielding varieties of Cannabis sativa (HPM, K2, MX and W1) was studied to assess their response to the rising atmospheric CO2 concentration. In general, elevated CO2 concentration (700 ?mol mol(-1)) significantly (p?<?0.05) stimulated net photosynthesis (P N), water use efficiency (WUE) and internal CO2 concentration (C i), and suppressed transpiration (E) and stomatal conductance (g s) as compared to the ambient CO2 concentration (390 ?mol mol(-1)) in all the varieties whereas, the effect of 545 ?mol mol(-1) CO2 concentration was found insignificant (p?<?0.05) on these parameters in most of the cases. No significant changes (p?<?0.05) in the ratio of internal to the ambient CO2 concentration (C i/C a) was observed in these varieties under both the elevated CO2 concentrations (545 and 700 ?mol mol(-1)). An average increase of about 48 %, 45 %, 44 % and 38 % in P N and, about 177 %, 157 %, 191 % and 182 % in WUE was observed due to elevated CO2 (700 ?mol mol(-1)) as compared to ambient CO2 concentration in HPM, K2, MX and W1 varieties, respectively. The higher WUE under elevated CO2 conditions in Cannabis sativa, primarily because of decreased stomatal conductance and subsequently the transpiration rate, may enable this species to survive under expected harsh greenhouse effects including elevated CO2 concentration and drought conditions. The higher P N, WUE and nearly constant C i/C a ratio under elevated CO2 concentrations in this species reflect a close coordination between its stomatal and mesophyll functions.

Photosynthetic response of Cannabis sativa L. to variations in photosynthetic photon flux densities, temperature and CO2 conditions
Suman Chandra, Hemant Lata, Ikhlas A. Khan, and Mahmoud A. Elsohly
Physiol. Mol. Biol. Plants, 14(4)–October, 2008 ), 299–306.
doi:10.1007/s12298-008-0027-x
Effect of different photosynthetic photon flux densities (0, 500, 1000, 1500 and 2000 ?mol m-2s-1), temperatures (20, 25, 30, 35 and 40 oC) and CO2 concentrations (250, 350, 450, 550, 650 and 750 ?mol mol-1) on gas and water vapour exchange characteristics of Cannabis sativa L. were studied to determine the suitable and efficient environmental conditions for its indoor mass cultivation for pharmaceutical uses. The rate of photosynthesis (PN) and water use efficiency (WUE) of Cannabis sativa increased with photosynthetic photon flux densities (PPFD) at the lower temperatures (20-25 oC). At 30 oC, PN and WUE increased only up to 1500 ?mol m-2s-1 PPFD and decreased at higher light levels. The maximum rate of photosynthesis (PN max) was observed at 30 oC and under 1500 ?mol m-2s-1 PPFD. The rate of transpiration (E) responded positively to increased PPFD and temperature up to the highest levels tested (2000 ?mol m-2s-1 and 40 0C). Similar to E, leaf stomatal conductance (gs) also increased with PPFD irrespective of temperature. However, gs increased with temperature up to 30 oC only. Temperature above 30 oC had an adverse effect on gs in this species. Overall, high temperature and high PPFD showed an adverse effect on PN and WUE. A continuous decrease in intercellular CO2 concentration (Ci) and therefore, in the ratio of intercellular CO2 to ambient CO2 concentration (Ci/Ca) was observed with the increase in temperature and PPFD. However, the decrease was less pronounced at light intensities above 1500 ?mol m-2s-1. In view of these results, temperature and light optima for photosynthesis was concluded to be at 25-30 oC and ~1500 ?mol m-2s-1 respectively. Furthermore, plants were also exposed to different concentrations of CO2 (250, 350, 450, 550, 650 and 750 ?mol mol-1) under optimum PPFD and temperature conditions to assess their photosynthetic response. Rate of photosynthesis, WUE and Ci decreased by 50 %, 53 % and 10 % respectively, and Ci/Ca, E and gs increased by 25 %, 7 % and 3 % respectively when measurements were made at 250 ?mol mol-1 as compared to ambient CO2 (350 ?mol mol-1) level. Elevated CO2 concentration (750 ?mol mol- 1) suppressed E and gs ~ 29% and 42% respectively, and stimulated PN, WUE and Ci by 50 %, 111 % and 115 % respectively as compared to ambient CO2 concentration. The study reveals that this species can be efficiently cultivated in the range of 25 to 30 oC and ~1500 ?mol m-2s-1 PPFD. Furthermore, higher PN, WUE and nearly constant Ci/Ca ratio under elevated CO2 concentrations in C. sativa, reflects its potential for better survival, growth and productivity in drier and CO2 rich environment.

Physical Properties of Traditional Thai Hemp Fiber (Cannabis sativa L.)
Thiprada Poonsawat, Lily Kaveeta, Jörg Müssig
April 2008 Journal of Industrial Hemp 13(1):20-36
DOI: 10.1080/15377880801898709
Hemp (Cannabis sativa L.) from a tradiiional Thai variety was used as a source of fiber in this research. The variety has been grown for a long time by the Hmong, a hill tribe, for the purpose of textiles. Hemp was grown in Chiang Mai, in the North of Thailand. Fiber fineness, fiber strength, fiber length, and lignin content of bast fibers from the lop, middle, and bottom part of hemp stems aged 65 and 120 days were measured after chemical separation. The results showed that ihe finest fiber was from the top of the stem. The fiber fineness determined from tbe airflow method varied from 14.2 to 27.0 FBAI200, and according to tbe OFDA melhtid it varied from 14.1 to 19.1 ^m. The highest strength fiber was from the middle part of the stem; fiber strength (Stelomeler ~ collective test) varied from 10.9 to 27.4 cN/tex. Fiber length varied from 22.0 to 28.4 mm. At the early stages

Plant density and nitrogen fertilization affect agronomic performance of industrial hemp ( Cannabis sativa L.) in Mediterranean environment.
Campiglia, E., Radicetti, E., & Mancinelli, R.
Industrial Crops and Products, 100, 246–254.(2017).
doi:10.1016/j.indcrop.2017.02.022
The renewed interest in industrial hemp (Cannabis sativa L.) is due to its large number of applications and for the wide range of agro-environmental conditions under which it can be cultivated. Two-year field experiments (2007 and 2008 growing seasons) were carried out in a Mediterranean environment of central Italy with the aim of assessing the impact of genotype, plant density and N fertilization on hemp yield, in terms of stems, inflorescences and seeds. The treatments consisted in: (a) seven genotypes (Epsilon68, Fedora17, Felina32, Ferimon, Futura75, Santhica27, and Uso31); three plant density (40, 80, and 120 plants m?2); two N fertilization levels (50 and 100 kg of N ha?1). Physiological parameters, plant height, stem weight and diameter, inflorescence yield, seed yield and the characteristics of hemp and weed aboveground biomass were recorded. High plant density resulted in shorter plant height compared with low plant density (?41%) as the hemp plants tended to reach the reproductive stage early at high density. At full flowering, stem yield ranged from 3.4 to 8.0 t ha?1 of dry matter and was positively correlated with the duration of vegetative phase, which tended to be high in the intermediate flowering genotypes (Epsilon68, Futura75 and Santhica27). Stem diameter was inversely correlated with plant density (6.7, 5.8 and 5.2 mm at 40, 80 and 120 plants m?2, respectively). Conversely to stem yield, inflorescence and seed production proved to be higher in the early flowering genotypes (Fedora17, Felina32, Ferimon and Uso31) and increased as plant density increased. High N fertilization level had a positive impact on stems rather than inflorescence and seed yields (on average +28%, +17% and +4% in 100 kg of N ha?1 compared with 50 kg of N ha?1 fertilization level, respectively). Farmers should consider making a dual-purpose production of stems and inflorescences or stems and seeds, even if it is clear that yield is related to the choice of genotype. Further research should be carried out to find various genotypes as well as flexible agronomical practices that are able to improve both traditional (stems) and innovative (inflorescences and seeds) hemp yields under Mediterranean conditions

Plant Population Effects on Fibre Hemp Morphology and Production
Stefano Amaducci, Marco Errani, Gianpietro Venturi
June 2002Journal of Industrial Hemp 7(2):33-60
DOI: 10.1300/J237v07n02_04
This paper reports on a 4-year study into the effect of plant density on the morphology and production of fibre hemp. At high density (180-270 plants m2), strong competition for light early in the growing season promoted internode extension (primary growth) and inhibited increase of stem diameter (secondary growth). In relating plant density to fibre hemp production it can therefore be said that a high plant population favours a high yield of long bast fibres for the textile industry. Demands only for high total yield can however be satisfied by targeting 30-45 plants m2. This is further supported by results of self-thinning, showing plant loss in 1999 was negligible at low density (30-90 plants m2), while at high density (180 and 270 plants m2) 50% and 60% of the initial stand was lost, respectively. In view of this result, and because of the fact that no significant morphological difference was found between 180 and 270 plants m2, it is also worth to note that when growing for long bast fibres 180 plants m2 should be chosen instead of 270 in order to save seed costs

Plant weight determines secondary fibre development in fibre hemp (Cannabis sativa L.).
Westerhuis, W., van Delden, S. H., van Dam, J. E. G., Pereira Marinho, J. P., Struik, P. C., & Stomph, T. J.
Industrial Crops and Products, 139, 111493.(2019).
doi:10.1016/j.indcrop.2019.111493
In fibre hemp (Cannabis sativa L.) grown for the production of high?quality textile yarns the presence of secondary fibres is unwanted. These fibres are too short for spinning and their presence hampers the production of fine and homogeneous yarns from the primary or long fibres. Primary fibres are present along the stem from bottom to top and hemp for fibres is traditionally harvested around the time of flowering, when the cell walls of these fibres are sufficiently thickened with cellulose to be extracted. In literature indications are found that the height up to which secondary fibres are present, moves upwards along the stem during the growing season, and that this process accelerates around flowering. To optimise the length of the stem part with primary fibres, but without secondary fibres, the background of secondary fibre development should be elucidated. It can be hypothesised that either flowering or the increasing plant size accelerates the formation of secondary fibres. To investigate this, an indoor experiment was conducted in greenhouses with mobile covers in which the day–- length sensitivity of hemp was used to create size ranges of flowering and non–flowering plants for a single cultivar, Futura 75. Secondary fibre formation was recorded using microscopic techniques. The height up to which secondary fibres were present, depended on plant weight. The higher secondary fibre front in flowering plants was most likely caused by the higher weight of these plants as compared with non–flowering plants of the same height. As seed carrying inflorescences contribute to plant weight, dual use of fibre hemp for seed and high–quality textile fibres is not an option. Results from a field experiment confirmed the correlation between plant size and the height of the secondary fibre front. Therefore, to optimise the length of the stem part with primary fibres, but without secondary fibres above stubble height, for Futura 75 a relatively short crop of around 1.3–1.4 m should be harvested before flowering. This ideal crop height is likely to differ between varieties.

Pollen Appearance and In Vitro Germination Varies for Five Strains of Female Hemp Masculinized Using Silver Thiosulfate
Jessica DiMatteo, Lauren Kurtz, Jessica D. Lubell-Brand
HortScience 2020
DOI: 10.21273/HORTSCI14842-20
Genetically female seed is sought for cannabidiol (CBD) hemp production because CBD is extracted from the flowers of female hemp plants. The production of all female seed requires masculinization of female plants to produce genetically female pollen that reliably generates female seed. Of the five female hemp genotypes that we masculinized using foliar sprays of silver thiosulfate (Abacus, Cherry Wine, Mountain Mango, Youngsim10, Wife), all genotypes produced fewer large and more irregular or misshapen pollen grains than genetically male plants. The masculinized female genotypes Wife and Cherry Wine produced pollen with germination rates similar to those of the male genotype Kentucky Sunshine. Female hemp genotypes vary in their ability to produce usable pollen that disperses well, is easily collected, and germinates as well as pollen from genetically male hemp plants.

Postponed sowing does not alter the fibre/wood ratio or fibre extractability of fibre hemp (Cannabis sativa).
Westerhuis, W., Struik, P. C., van Dam, J. E. G., & Stomph, T. J.
Annals of Applied Biology, 155(3), 333–348.(2009).
doi:10.1111/j.1744-7348.2009.00342.x
Because hemp is a short-day plant, postponing the sowing date might be a suitable strategy to obtain shorter and smaller plants around flowering, when primary fibres are ‘ripe’ enough to be harvested. Smaller plants can be processed on existing flax scutching and hackling lines and might have fibre characteristics that are desirable for producing high-quality ’long fibres’ for yarn spinning. It was investigated whether sowing beyond the normal sowing period in the Netherlands affects the ratio in which fibres and wood are produced, and what proportion of these fibres are long fibres, suitable for long fibre spinning. About 400 stem samples were fractioned into retting losses, wood, tow, and long fibre, and the ratios between fractions were analysed using multiple linear regression analyses. A normal sowing date at the end of April was compared with a postponed sowing date at the end of May. The total fibre/wood ratio was not affected. More than 95% of the variance in total fibre was accounted for by the wood weight per stem (55.5%), the variety (+33.3%) and the stem part (+6.5%). The amount of long fibre per stem mainly depended on the amount of the total fibre per stem (95.4% variance was accounted for) and the stem part (+2.0%). For economic reasons, it could be interesting to grow two successive highquality hemp crops in one growing season. Therefore, in an additional experiment with one variety, the effect of sowing fibre hemp up to 12 weeks later than normal on the quantity and quality of the fibres was studied. Postponing the sowing date up to 12 weeks had no important effects on retting losses, the total fibre/wood ratio, and the long fibre/total fibre ratio. It is therefore technically possible to grow two successive hemp crops. Whether this fits well in farming systems and a hemp production chain remains to be studied.

Potential of bioenergy production from industrial hemp (Cannabis sativa): Pakistan perspective
Muhammad Saif Ur Rehman, Naim Rashid, Ameena Saif, Tariq Mahmood, Jong-In Han
2013 Renewable and Sustainable Energy Reviews 18(154):164
doi: 10.1016/j.rser.2012.10.019
Pakistan is facing severe economical crunch due to continuously growing gap between energy demand and supply. The shortage in power and gas supply has already halted many industrial sectors such as textile, small and medium enterprises and local transportation. The government has spent US $ 9 billion on energy import during 2008–2009 to fulfill current energy requirements. Indigenous energy resources, mainly fossil fuels, are already being exploited at their maximum. Besides these short term steps, energy demand is expected to double during next decade. Thus, renewable and sustainable energy resources, such as biomass, needs to be exploited so that a sustainable energy mix could be employed to ensure energy security. Industrial hemp (Cannabis sativa) has been successfully investigated for its potential to be used as a renewable feedstock for the production of biofuels. Hemp is an environmental friendly and low cost feedstock which grows wildly in most parts of Pakistan. Thus, hemp can be grown as a potential energy crop in Pakistan to meet its energy requirements by producing various kinds of biofuels. This sustainable feedstock will help the country to reduce its energy import bills, and ensure sustainable energy supply.

Predicting the chemical composition of fibre and core fraction of hemp (Cannabis sativa L.).
Toonen, M. A. J., Maliepaard, C., Reijmers, T. H., van der Voet, H., Mastebroek, H. D., van den Broeck, H. C., … Kessler, R. W.
Euphytica, 140(1-2), 39–45.(2004).
doi:10.1007/s10681-004-4753-z
Fibre formation in hemp (Cannabis sativa L.) is a dynamic process. In order to follow this process, the chemical composition of the hemp stem was studied during plant development using the acid and neutral detergent fibre and lignin methods. Additionally, near infra-red spectroscopy was carried out. To predict the chemical composition of the stem samples partial least square (PLS) analysis was carried out. The developed PLS models can predict the level of %ADF and %NDF in the hemp bast and core samples. For %ADL, the models are only suited for either core or bast tissues and can not be used for both tissues simultaneously.

Propagation and Root Zone Management for Controlled Environment Cannabis Production
Deron M. Caplan
A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy
https://pdfs.semanticscholar.org/0ec...8e971977ff.pdf
Cannabis producers lack reliable information on the horticultural management of their crops. This thesis research was designed to improve horticultural practices for controlled environment cannabis production; topics included propagation, growing substrates, fertilization, and irrigation. To optimize the procedures for taking vegetative stem cuttings in cannabis, several factors were evaluated on how they affect rooting success and quality (Chapter Two). These included number of leaves, leaf tip removal, basal/apical position of cutting on the stock plant, and type of rooting hormone. Removing leaf tips reduced rooting success and cuttings with three fully-expanded leaves had higher rooting success and quality than those with two. Also, a 0.2% indole-3-butyric gel was more effective than a 0.2% willow extract gel to stimulate rooting and cutting position had no effect on rooting. Coir-based substrates with different physical properties were evaluated during the vegetative and flowering stage of cannabis production; optimal organic fertilizer rates were established for each substrate (Chapters Three and Four). During the vegetative stage, cannabis performed well in both tested substrates despite the ?11% difference in container capacity (CC) between them. During the flowering stage, the substrate with lower CC increased floral dry weight (yield) and the concentration and/or yield of some cannabinoids, including THC, compared to the substrate with higher CC. The optimal organic fertilizer rate varied by substrate during the flowering stage but not during the vegetative stage; higher fertilizer rate during the flowering stage increased growth and yield but diluted some cannabinoids. Finally, the effects of controlled drought stress timing and frequency during the flowering stage were explored on floral dry weight and secondary metabolism (Chapters Five and Six). When drought was applied during week seven of the flowering stage, through gradual substrate drying over eleven days, floral concentration and content per unit growing area of major cannabinoids were increased. When drought was applied over a period of ?8 days during week seven, cannabinoid content was similar to a well-watered control; though, dependent on drought timing, the content of some terpenoids varied. This research provided evidence-based
information that can help growers improve the quality and yield of their cannabis crops.

Quality Aspects in Hemp Fibre Production—Influence of Cultivation, Harvesting and Retting
Jörg Müssig, Reent Martens
January 2003 Journal of Industrial Hemp 8(1):11-32
DOI: 10.1300/J237v08n01_03
For hemp cultivar Fedrina 74 we investigated the influence of different sowing dates in 1997 and 1998. The highest yields occurred with early sowing dates, at the middle of April. The pure fibre content of the plants of the last sowing date increased rapidly, reaching the same amounts as earlier sown plants. We measured the influence of harvesting technique and retting duration on strength and fineness. Comparison of the results of the purely visual assessment of the degree of retting with the Near Infrared Spectroscopy (NIRS) results shows a good correlation. The NIRS method can supply a reproducible method of analysis for retting. In 1998 we examined damage to the fibres due to the wet conditions during retting. In 1997 an increase in retting time led to a decrease of fibre bundle width. In the North German region, with high hu midity in autumn, early sowing is advantageous. For both years early sowing in combination with early harvesting proved the optimal way to get a good yield and a secure collection of the stems after retting.

Quality of hemp (Cannabis sativa L.) stems as a raw material for paper.
Van der Werf, H. M. G., Harsveld van der Veen, J. E., Bouma, A. T. M., & ten Cate, M.
Industrial Crops and Products, 2(3), 219–227. (1994).
doi:10.1016/0926-6690(94)90039-6
Fibre hemp (Cannabis sativu L.) stems consist of high-cellulose low-lignin bark containing long fibres and lowcellulose high-lignin core containing short fibres. The bark, which contains a variable proportion of less valuable secondary bast fibre, is more valuable as a raw material for paper than the core. A study of the factors affecting the quality of hemp grown in The Netherlands for paper production is described. The bark content in the stem was determined by mechanically separating bark and core. The chemical composition of bark and core was determined according to procedures defined by the Technical Association of the Pulp and Paper Industry. The secondary bast fibre content was determined by manually separating primary and secondary phloem tissue after boiling in a 2% NaOH solution. Bark content in the stem decreased during the growing season; at harvest in September it ranged from 30 to 35% depending on cultivar and plant density. The proportion of secondary bast fibre in the bast fibre fraction increased with stem weight, from 10 to 45%. Measurement of secondary bast fibre in a representative stem section allowed the proportion of this fibre in the bast fibre fraction of the entire stem to be estimated accurately. Differences in chemical composition within sets of samples of bark or core were small compared with the difference between bark and core. The bark of the French cultivars tested contained less cellulose than that of the Hungarian cultivars. Bark quality for paper making improved during the growing season because the cellulose content in the bark increased, whereas the content of lignin and extractives decreased

*Regeneration of shoots from immature and mature inflorescences of Cannabis sativa
Kevin F. Piunno, Gregory Golenia, Ekaterina A. Boudko, Cassandra Downey, A. Maxwell P. Jones
Canadian Journal of Plant Science Can. J. Plant Sci April 2019
DOI: 10.1139/CJPS-2018-0308
Cannabis sativa is usually clonally propagated from plants in the vegetative phase. However, phenotypic traits such as yield and chemical composition can only be assessed in unfertilized plants reaching the end of their life cycle and there are no peer reviewed methods to propagate flowering plants. In this study, immature (3 cultivars) and mature (1 cultivar) floral explants were cultured on thidiazuron and shoot development was observed from both immature and mature explants. This provides the first report of micropropagation from floral tissues in C. sativa and will enable plants to be clonally propagated up to the date of harvest

Regulatory sampling of industrial hemp plant samples (Cannabis sativa L.) using UPLC-MS/MS method for detection and quantification of twelve cannabinoids
Erin C. Berthold, Rui Yang, Abhisheak Sharma, Shyam H. Kamble, Siva R. Kanumuri, Tamara I. King, Raluca Popa, Joshua H. Freeman, Zachary T. Brym, Bonnie A. Avery, and Christopher R. McCurdy
Journal of Cannabis Research (2020) 2:42
DOI: 10.1186/s42238-020-00050-0
https://jcannabisresearch.biomedcentra l.com/track/pdf/10.1186/s42238-020-00050-0.pdf
Background: In 2018, the Farm Bill mandated the United States Department of Agriculture to develop regulations governing the cultivation, processing, and marketing of industrial hemp. Industrial hemp is defined as Cannabis sativa L. with a total Δ-9-tetrahydrocannabinol (Δ-9-THC) content ≤0.3%. Therefore, for hemp to become an agricultural commodity, it is important to regulate production by developing standard methods for sampling and testing of the plant material.
Methods: An ultra-performance liquid chromatography-tandem mass spectrometry analytical method for the quantification of twelve cannabinoids was developed. The method was applied to a regulatory sampling trial of three hemp varieties cultivated for cannabidiol (CBD) production. Two samples were taken from 28 plants with one sample being flower only while the other was a composite sample that included flowers, leaves, and stems.
Results: The assay method was validated for specificity, range, repeatability, reproducibility, and recovery in accordance with all applicable standards for analytical methods. The results of the regulatory study indicated a significant decrease in the concentration of total Δ-9-THC and total CBD of 0.09% and 1.32%, respectively, between a flower only and a composite sample.
Conclusions: There are many factors that may influence reported total Δ-9-THC content in industrial hemp. A robust analytical method was developed to analyze hemp samples in a trial regulatory study. The results indicate that the way hemp is sampled and analyzed may influence the legality of a crop, which could have negative economic and legal consequences.

Response of essential oil hemp (Cannabis sativa L.) growth, biomass, and cannabinoid profiles to varying fertigation rates
Steven L. Anderson II, Brian Pearson , Roger Kjelgren, Zachary Brym
PLoS ONE Published: July 29, 2021
DOI: 10.1371/journal.pone.0252985
https://journals.plos.org/plosone/ar...l.pone.0252985
Five essential oil hemp (Cannabis sativa L.) cultivars (Cherry Blossom, Cherry Blossom (Tuan), Berry Blossom, Cherry Wine, and Cherry Blossom × Trump) were treated with six fertigation treatments to quantify the effects of synthetic fertilizer rates and irrigation electrical conductivity on plant growth, biomass accumulation, and cannabinoid profiles. Irrigation water was injected with a commercial 20-20-20 fertilizer at rates of 0, 50, 150, 300, 450, and 600 ppm nitrogen equating to 0.33 (control), 0.54, 0.96, 1.59, 2.22, and 2.85 dS m−1, respectively. Plants were grown under artificial lighting (18 hr) to maintain vegetative growth for eight weeks, followed by an eight-week flowering period. High linear relationship between chlorophyll concentrations and SPAD-502 measurements validated the utilization of SPAD meters to rapidly identify nutrient deficiency in essential oil hemp. Cultivars expressed significant variation in plant height and cannabinoid profiles (% dry mass), in concurrence with limited biomass and cannabinoid (g per plant) yield variation. Cherry Blossom was the best performing cultivar and Cherry Wine was the least productive. Variation in plant growth, biomass, and cannabinoid concentrations were affected to a greater extent by fertilizer rates. Optimal fertilizer rates were observed at 50 ppm N, while increased fertilizer rates significantly reduced plant growth, biomass accumulation, and cannabinoid concentrations. Increased fertilizer rates (> 300 ppm N) resulted in compliant THC levels (< 0.3%), although when coupled with biomass reductions resulted in minimal cannabinoid yields. Additionally, CBD concentration demonstrated higher sensitivity to increased fertilizer rates (> 300 ppm N) compared to THC and CBG (> 450 ppm N). The results of this study can serve as a guide when using fertigation methods on essential oil hemp cultivars; although results may differ with cultivar selection, environmental conditions, and management practices.

Root Comparison: Seedlings vs Clones
Mick Donahue
North Carolina Farms
https://ncfarmsinc.com/articles/root...q_y6S13xrc_ssE
There’s an ongoing debate about which is superior: hemp clones or hemp seedlings. You’ll hear arguments that go something like this: clones are more uniform in the field, more true to type, and 100% female, but seeds are more easily stored, free from pests from the beginning, and–this is the big one–produce a taproot.
We decided to put that last one to the test. How would seedlings and clones compare in a side-by-side root test?

Ruderalis
By DMT on July 22, 2003
https://www.cannabisculture.com/cont...03/07/22/3033/
Deep in the North American woods lurks a recent addition to the marijuana gene pool: Ruderalis hybrids! The forests and fields are coming alive with resinated plants, blasting their way to maturity under the intense light of the summer sun, long before the buzzing of choppers or moldy autumn weather.
Until recently, Ruderalis had been almost unanimously given a bad name by cultivators and breeders alike. Early Dutch seed pioneers like Super Sativa Seed Club voiced their concerns about Ruderalis early in the homegrown revolution. This was all with understandable reason, as pure Ruderalis varieties are almost completely devoid of THC and come with a host of other problems for the grower or breeder.
Breeding programs between Ruderalis and drug type strains can and have produced plants of notable quality. The aim of this article is to shine some light on this recent advance in marijuana breeding and pave the way to what could be the future of outdoor marijuana cultivation for many areas of the world.

S1084: Industrial Hemp Production, Processing, and Marketing in the U.S.
https://www.nimss.org/projects/view/mrp/outline/17716
Statement of Issues and Justification
Industrial hemp (Cannabis sativa L.) was at one time a major agronomic crop in the U.S. An excellent history of the scope and legality of industrial hemp production can be found in the UK extension publication Economic Considerations for Growing Industrial Hemp: Implications for Kentucky’s Farmers and Agricultural Economy (https://www.uky.edu/Ag/AgEcon/pubs/r...mpfarmer28.pdf). Since that publication was released in 2013, Section 7606 of the 2014 Farm Bill has provided for pilot research projects in states where supporting legislation has been established.
Hemp is broadly adapted, having essentially a global distribution (Johnson, 1999), but historically production has been concentrated in more northern temperate regions of the globe. The plant grows best at temperatures between about 60 and 80°F, but its tolerance to quite low temperatures makes it suitable to plant before corn (Ehrensing, 1998). This tolerance to low temperatures allows early plantings to reach a closed canopy early in the season, supporting rapid growth and minimizing weed competition. Hemp is sensitive to day length, setting seeds as day length shortens in the fall (USDA, 2000). Industrial hemp is produced for one or both of two, main harvestable components: 1) Stalks, 2) Seed/grain. The stalk is largely used for fiber, although the core or hurd of the stalk also has commercial uses including bedding, insulation and building materials. Fiber from the plant can be utilized in numerous ways ranging from yarn and fabric to electrical super-capacitors manufactured from carbon nanosheets. Hemp hurds and fibers also can be used as an alternative to wood in construction materials (chip board and particle board), for batts or blown-in insulation, as insulative fill in lightweight concrete applications (hempcrete), and for structural reinforcement in molded plastic composites (replacing synthetic fibers). Today, applications using hemp fibers are different and much broader than when industrial hemp was last grown extensively in the U.S. in the 1940s. Although the historic uses for hemp (e.g., in rope and canvas for ship sails) have declined, new opportunities for the crop are growing with the realization that hemp fibers possess several very positive attributes that make them useful in modern industrial applications. The seed is sold as hemp grain and also has several valuable markets. Hemp grain is relatively high in oil content; generally containing 30% or more by weight. This oil is very healthful as a dietary constituent or supplement for humans. It is rich in omega-3 fatty acids and has a very favorable omega 3 to omega 6 ratio of about 3:1. This is much higher than that found in many other oil seeds. The grain is also high in protein and contains all 20 amino acids (Russo and Reggiani 2015).

Sequenced Anaerobic-Aerobic Treatment of Hemp Pulping Wastewaters
Sjon Kortekaas
https://edepot.wur.nl/193312
In the 1980's, increasing problems with soil-borne pathogens due to intensive cultivation of a limited number of crops in short rotations forced Dutch arable farming to look for new profitable crops intended for large-scale non-food markets. Hemp was considered, due to the outstanding quality of hemp bark fibres for papermaking. The potential of fibre hemp as a raw material for the pulp and paper industry was subject of a comprehensive 4 year study, the Hemp Research Programme, which took place from 1990 to 1994. The aim of the programme was the development of economically feasible and environmentally safe pulping processes for hemp fibres grown in The Netherlands. Environmental demands were addressed by avoiding sulphur-based cooking chemicals and chlorine-based bleaching processes as well as research on wastewater treatment to minimize the environmental impact of discharged pulping effluents. The task of the Department of Environmental Technology was the characterization of hemp pulping wastewaters, evaluation of biological methods for the treatment of these wastewaters and development of adequate technology to overcome constraints for treatment.
In this chapter, the importance of hemp in the history of papermaking will be traced. Subsequently, the chemical composition of hemp feedstocks in relation to wastewater characteristics will be discussed. Thereafter, the perspectives of anaerobic and aerobic biological treatment methods will be evaluated, with a focus on the toxicity and
biodegradability of pulp and paper industry wastewater constituents.

Shifting the Paradigm: An Ecological Systems Approach to Weed Management
Karla L. Gage, Lauren M. Schwartz-Lazaro
Agriculture 2019, 9(8), 179;
doi: 10.3390/agriculture9080179
Weeds have been historically, and are still today, the primary and most economically important pest in agriculture. Several selection pressures associated with weed management, such as an overreliance on herbicides, have promoted the rapid evolution of herbicide-resistant weeds. Integrated Weed Management (IWM) is promoted as an ecological systems approach, through the combination of biological, chemical, cultural, ecological, and mechanical control methods. The concept of a systems approach is defined as managing weeds by combining practice and knowledge with the goals of increasing yield and minimizing economic loss, minimizing risks to human health and the environment, and reducing energy requirements and off-target impacts. The reliance on herbicides in modern cropping systems has shifted the management focus from requiring intimate knowledge of biology, ecology, and ecological systems to herbicide chemistry, mixes, and rotations, application technology, and herbicide-tolerant crop traits. Here, an ecological systems approach is considered, examining new trends and technologies in relation to IWM and weed ecology. Prevention of spread, seedbank management, crop rotations, tillage, cover crops, competitive cultivars, biological weed control, and future solutions in concept-only are presented, and knowledge gaps are identified where research advancements may be possible. An ecological systems approach will provide improved stewardship of new herbicide technologies and reduce herbicide resistance evolution through diversification of selection pressures. Agroecological interactions should be studied in light of new, developing weed control technologies. The science of weed management needs to refocus on the foundations of weed biology and ecology to enable an ecological systems approach and promote agricultural sustainability

SILVANA (LV. 200) - A NEW DIOIC HEMP CULTIVAR DEVELOPED AT THE SCDA LOVRIN (TIMIS COUNTY)
V. TAB?R?; CARMELLA FLORICA DUMA, CAMELIA MOLDOVAN
This paper can be downloaded by typing in the title into a browser and the Pdf is the first link.
The Silvana dioic hemp cultivar has been developed at the Agricultural Research and Development Station in Lovrin (Timis County), as a result of a series of hybridisations followed by repeated individual selection. It is characterised by a very good yielding capacity (11,360 kg/ha pf stems in the STAS network, 775 kg/ha seed, and over 31% fiber content in the stems. The hemp cultivar has a good adaptability to environmental conditions, and a vegetation period of 135 days in seed crops and 83-108 days in fibber crops

Sowing density and harvest time affect fibre content in hemp (Cannabis sativa) through their effects on stem weight
W. Westerhuis, S. Amaducci, P.C. Struik, A. Zatta, J.E.G. van Dam, & T.J. Stomph
Annals of Applied Biology 155(2), 225-244
doi: 10.1111/j.1744-7348.2009.00334.x
Sowing density and harvest time are considered important crop management factors influencing fibre quantity and quality in hemp (Cannabis sativa). We investigated whether the effects of these factors are essentially different or that both factors affect stem weight and thereby total and long-fibre content. The effects of all combinations of three sowing densities and three harvest times were studied for six different stem parts. Almost 500 samples consisting of stem parts from 50 plants and with a length of 50 cm were tested. Fibres were extracted by a controlled warm-water retting procedure, followed by breaking and scutching. The initial sample weight was fractionated into retting losses, wood, tow and long fibre. In both Italy and the Netherlands, crops were successfully established with different stem densities (99–283 m22 ), plant heights (146–211 cm) and stem diameters (4.5–8.4 mm) at harvest. Stem dry matter yields (6.8–11.7 Mg ha21 ) increased with a delay in harvest time but were not affected by sowing density. Retting loss percentages were lower in lower stem parts and decreased with later harvest because maturation was associated with increasing amounts of fibre and wood. Within a certain stem part, however, the absolute retting losses were constant with harvest time. Multiple linear regression analyses showed that the amount of fibre in a hemp stem is almost completely determined by the weight and the position of that stem part. When the plant grows, the increase in dry matter is split up into fibres and wood in a fixed way. This total fibre/ wood ratio was highest in the middle part of the stem and lower towards both bottom and top. Sowing density and harvest time effects were indirect through stem weight. The long-fibre weight per stem increased with the total fibre weight and hence with stem weight. Stem weight increased with harvest time; as harvest time did not affect plant density, the highest long-fibre yields were obtained at the last harvest time. The long fibre/total fibre ratio was lowest in the bottom 5 cm of the stems but similar for all other parts. Sowing density and harvest time effects again were indirect. Fibre percentages in retted hemp decreased with increasing stem weights towards a level that is presumably a variety characteristic. The dry matter increase between harvests, however, is much more important with respect to total and long-fibre yield.

Sowing time and prediction of flowering of different hemp (Cannabis sativa L.) genotypes in southern Europe
Salvatore Luciano Cosentino?, Giorgio Testa, Danilo Scordia, Venera Copani
Industrial Crops and Products 37 (2012) 20– 33
doi:10.1016/j.indcrop.2011.11.017
Fibre hemp can be grown under a wide range of agro-ecological conditions, but it requires special attention for several physiological features and crop management. A management strategy in order to reduce inputs and thus achieve acceptable yield could be achieved optimizing sowing time. With this respect, the effects of sowing date on hemp biology and yield was studied, using two monoecious and two dioecious genotypes. Field experiments were carried out in two subsequent years (2003–2004) in South of Italy, using drip irrigation system. Sowing time, in the two year period, ranged between March 10th and July 22nd. Optimal sowing time was observed between the end of April and the first three weeks of May; in that range, the dioecious Fibranova yielded the most in terms of aboveground biomass and stem dry yield, followed by Tiborszallasi, while the two monoecious showed the lowest yield. On the contrary, before and after that period, the shorter day length caused an early floral induction that strongly reduced stem and fibre elongation, and thus aboveground dry biomass and consequently stem yields. Based on this study
a simulation model focusing on flowering prediction in Mediterranean environment was developed.

STATUS OF INDUSTRIAL HEMP IN TENNESSEE
Eric Walker
https://extension.tennessee.edu/Chea...emp%20W777.pdf
Since 2015, Tennessee participants of the Industrial Hemp Agricultural Pilot Program have produced industrial hemp (Cannabis sativa L.) for fiber, grain and phytocannabinoids, or cannabinoids that occur naturally in the Cannabis spp. Production has been of relatively small scale compared to other crops, and there have been successes and failures in the pilot program. Participants, Tennessee Department of Agriculture (TDA), Tennessee Crop Improvement Association, Tennessee Hemp Industries Association (TN HIA), lawmakers, and university scientists have collaborated on the pilot project to: • Develop, improve, and abide by federal and state laws, regulations, and guidelines. • Identify suitable varieties, production practices, and pest management strategies to consistently produce high-quality industrial hemp. • Identify existing markets, develop new markets, and improve crop profitability.

Status report on the German collections of flax and hemp
Frank Höppner and Helmut Knu?pffer
https://archive-ecpgr.cgiar.org/file...rt%20final.pdf
For hemp, only a few descriptors are being observed and recorded during routine seed multiplication, namely, Date of sowing, Date of emergence, Date of flowering, Flower colour, Plant height, Date of harvest, Yield, Thousand-seed-weight. These data are also computerised for the last ca. 10 years.
All above characterisation and evaluation results will be made available through the new Genebank Information System, GBIS/I.
Since 2001, the subsidy is lower than in the years before and has been divided into two parts: area subsidies for cultivation for farmers and fibre subsidies for fibre extracting factories. A new requirement is that fibres have to be extracted from stems in order to receive a subsidy. These new guidelines and the lack of fibre extracting industries initially led to a decrease in hemp cultivation (Table 5). Today, hemp cultivation has stabilized at a level about 2000 ha with an increase in the area per farm. In 2005, the following 10 cultivars were grown: ‘Beniko’, ‘Epsilon 68’, ‘Fedora 17’, ‘Felina 32’, ‘Felina 34’, ‘Futura 75’, ‘Red Petiole’, ‘Uso 31’, ‘Bialobrzeskie’ and ‘Chamaeleon’. Currently eight licensed primary processors exist in Germany and cultivation is concentrated near them (Figure 1). The greatest hemp cultivation area is found in the south of Germany around Malsch. A specialty market exists for peeled hemp seeds and hemp oil as a raw material for the food, pharmaceutical, and cosmetic industries. Under the modified framework it could be more economically attractive for farmers if both hemp stems and hemp seeds were to be harvested (Höppner et al. 2004). In Southern Germany the climate promotes the production of sufficient seeds for a profitable harvest. The Gatersleben hemp collection (with 22 accessions) was included in a larger study of hemp germplasm by Small and Marcus (2000, 2002).

XXI.—Studies on Flowering-Plant Growth and Organogenesis. I. Morphogenetic Effects of 2, 3, 5-Triiodobenzoic Acid on Cannabis sativa
January 1957 Volume 66, Issue 41957 , pp. 409-423
Proceedings of the Royal Society of Edinburgh (1844 - 1940),
J. Heslop-Harrison, Y. Heslop-Harrison
DOI: 10.1017/S0080455X0000059X
Not with standing its reported effect upon the levels of native auxin in plant tissues, 2, 3, 5~triiodobenzoic acid (TIBA) does not influence sex expression in Cannabis sativa, a dioecious species in -uhich there is evidence that flower sexuality may be regulated by auxin levels in the neighbourhood of developing primordia. The total production of flowers is, however, increased in both sexes by TIBA, an effect possibly resulting from an alleviation of the correlative inhibition of lateral buds. Whilst hardly influencing morphogenetic activity at a cellular level at all, TIBA treatment leads to gross ontogenetic abnormalities, of which the common feature is a tendency for the elimination of localized growth and the substitution instead of generalized growth fields, resulting in the formation of infundibuliform and cylindrical foliar and floral structures. It seems probable that the bulk of the effects of TIBA in the plant may be due to an influence on auxin distribu- tion, perhaps through interference with the mechanism of polar transport.

XXII.—Studies on Flowering-Plant Growth and Organogenesis. II. The Modification of Sex Expression in Cannabis sativa by Carbon Monoxide
J. Heslop-Harrison, Y. Heslop-Harrison
January 1957
DOI: 10.1017/S0080455X00000606
Brief exposure in the young stage to carbon monoxide in concentration c. 1 per cent modifies subsequent sex expression in male plants of the dioecious Cannabis sativa (hemp), inducing the formation of intersexual or even functionally female flowers. The effect is registered in flower primordia of a particular age; older ones are already sexually committed, and younger ones apparently recover from treatment to pursue the genetically established path of ontogeny. Grafting experiments show that the stimulus is retained in the apex even when it is transferred to an untreated plant before the flower primordia have begun differentiation. The effect of carbon monoxide in modifying sex expression is similar to that induced by auxin administration, and it is possible that the basic function of the gas at the effective concentrations is to upset auxin metabolism in treated tissues, perhaps by inhibiting enzyme systems normally responsible for regulating endogenous auxin levels.

XXII--Studies on flowering-plant growth and organogenesis - III. Leaf shape changes associated with flowering and sex differentiation in Cannabis sativa.
Heslop-Harrison J, Heslop-Harrison Y.
Proc.Roy.Irish Acad. 59: 257-283. 1958.
https://sci-hub.tw/10.2307/20494799
https://www.jstor.org/stable/pdf/204...o_tab_contents
IN short-day races of the dioecious Cannmabis sativ'a, the two sexes cannot be distinguished morphologically whilst out of flower, but major secondary sexual differences develop when flowering begins in short days. In both sexes the digitate leaves undergo a diminution in area from node to node during flowering accompanied by a reduction in the numbers of lobes and marginal serrations, but the trends are much more pronounced in the male. These differences appear concurrently with the differentiation of flowers, and are probably to be regarded as by-products of the primary sex determming mechanism. Treatment with the auxin, a-naphthaleneacetie acid, which under some circumstances can cause sex reversal in male plants, also affects the trend of hetero blastic leaf development, reducing the d-egree to which leaf-lobing and marginal serration declin-e following brief photoperiodic induction, and so causing male plants to behave vegetatively more in the manner of females. In each sex, both the type of leaf primordium formed and the (tegree of expansion of the lamina depend upon prevailing day l4ngth; primordia initiated in one day length can be caused to develop in another, with a resultant production of leaves of anomalous types. Some implications of these findings are discussed.

XXII--Studies on flowering-plant growth and organogenesis. IV Effects of gibberellic acid on flowering and the secondary sexual difference in stature in Cannabis sativa.
Proc. Roy. Irish Acad., Sect. B 61, 219-232 (1961)
https://www.jstor.org/stable/2049482...n_tab_contents
Gibberellic acid does not bring about flowering in the short-day dioecious species Cannabis sativa (hemp) under non-inductive photoperiodic conditions. The two sexes do, however, respond to gibberellic acid treatment in long days by a substantial increase in stem growth, and leaf shape changes ensue similar to those which follow short-day induction. When gibberellic acid is supplied during short-day induction, flowering is somewhat delayed, and in genetically male plants the total numbers of flowers produced may be reduced. Treatment after photoperiodic induction may, however, increase total flower production in both sexes. In stem extension growth, female plants respond more to gibberellic acid under short-day conditions than males, and as a consequence, the secondary sexual difference in stature which normally results from the greater growth of male plants during entry into a flowering period is abolished or reversed. These results can be interpreted on the assumption that stem extension growth in hemp is governed by native gibberellin-like substances, and that, while the sexes show no difference in gibberellin metabolism under long days, a difference develops during the early phase of induction in short days such that gibberellin production in the male plants rises while that of the females falls. It remains to be shown whether or not gibberellins are concerned in primary sex differentiation in hemp.

Not directly Cannabis related
Super Moisture Absorbent Gels for Sustainable Agriculture via Atmospheric Water Irrigation.
Zhou, X., Zhang, P., Zhao, F., & Yu, G.
ACS Materials Letters, 1419–1422.
doi:10.1021/acsmaterialslett.0c00439 (2020).
An atmospheric water irrigation system based on super moisture absorbent gels (SMAG) is developed for sustainable agriculture technology. The SMAG-soil can harvest water from the air and provide water to the plants upon solar heating regardless of the local accessibility to liquid water resources. As a benefit of this solar-powered atmospheric water irrigation system, agriculture can become geographically and hydrologically independent. Thus, crop planting in underdeveloped and drought areas can be liberated from the long-distance water and power supplies.

Sustainable Agriculture Reviews 42 Hemp Production and Applications
Crini, G., & Lichtfouse, E. (Eds.).
Sustainable Agriculture Reviews. (2020).
doi:10.1007/978-3-030-41384-2
1 Hemp Production .....1. Krystyna ?uk-Go?aszewska and Janusz Go?aszewski
2 Traditional and New Applications of Hemp . . 37. Grégorio Crini, Eric Lichtfouse, Gilles Chanet, and Nadia Morin-Crini
3 Industrial Hemp in the USA: A Brief Synopsis . . . 89. John H. Fike, Heather Darby, Burton L. Johnson, Larry Smart, and David W. Williams
4 Hemp Fibers in Serbia: Cultivation, Processing and Applications . . 111. Biljana Peji?, Marija Vuk?evi?, and Mirjana Kosti?
5 Physico-chemical Characterization and Development of Hemp Aggregates for Highly Insulating Construction Building Materials . 147. Yunhong Jiang, Atif Hussain, Davoud M. Heidari, Michael Lawrence, and Martin Ansell
6 Modelling the Hygrothermal Behaviour of Hemp Concrete: From Material to Building . . 171. Yacine Aït Oumeziane, Florence Collet, Christophe Lanos, and Bassam Moujalled
7 Hempseed Protein: Processing and Functional Properties . . 223. Anne Pihlanto, Markus Nurmi, and Sari Mäkinen
8 Functional and Bioactive Properties of Hemp Proteins . . 239. Tamara Dap?evi?-Hadna?ev, Miroslav Hadna?ev, Manda Dizdar, and Nataša Jovanovi? Lješkovi?
9 Hemp Seed as a Source of Food Proteins . . 265. François Potin and Rémi Saurel
10 Hemp Fibers for Wastewater Treatment . . . 295 Lavinia Tofan, Carmen Paduraru, and Carmen Teodosiu

Tasmania Hemp (Cannabis) Fiber and Seed Cultivar Field Trials—2018-2019
Robert C. Clarke
International Journal of Food Science and Agriculture, 2020, 4(4), 470-481
DOI: 10.26855/ijfsa.2020.12.016
http://www.hillpublisher.com/journals/jsfa/
Tasmania has a temperate climate well-suited for hemp production, is located nearer to the south pole than other regions of Australia, and therefore presents an important location for hemp variety trials. The primary aim of this research is to evaluate the performance of hemp (Cannabis) fiber and seed cultivars from var- ious geographical origins and latitudes when grown in southern Tasmania. Se- venteen registered hemp cultivars were sown on three dates in large replicate trial blocks to allow combine harvesting and collection of accurate yield data. Results are discussed with respect to site selection, crop performance, sowing dates, sowing rates, crop development, pest infestation, THC levels, seed yields, crop management and cultivar selection. Canadian cultivars performed well for grain seed production with the highest yields and their short crop height accom- modates mechanical harvesting. French cultivars yielded much less seed than Canadian cultivars and grew to heights nearly beyond the reach of a standard grain header. Chinese cultivars flowered too late to produce viable seeds and are not suitable for hemp seed grain production in Tasmania, although their vigorous growth and late flowering makes them good candidates for biomass production. THC levels in the sampled Chinese cultivars were too high for seed production, but those that do not flower at Tasmanian latitudes are suitable for fiber and biomass production.
TETRAHYDROCANNABINOL LEVELS IN HEMP (CANNABIS SATIVA) GERMPLASM RESOURCES
ERNEST SMALL AND DAVID MARCUS
Economic Botany 57(4) pp. 545-558. 2003
10.1663/0013-0001(2003)057[0545:TLIHCS]2.0.CO;2
In most of the western worm where industrial hemp, Cannabis sativa, is licensed for cultivation, the plants must not exceed a level of 0.3% tetrahydrocannabinol (THC), the principal intoxicating constituent of the species. Because there are no publicly available germplasm hemp collections in North America and only a very few, recent North American cultivars have been bred, the future breeding of cultivars suitable for North America is heavily dependent on European cultivars and European germplasm collections. Based mostly on material from Europe, this study surveyed THC levels of 167 accessions grown in southern Ontario, making this the largest survey to date of germplasm intended for breeding in North America. Forty-three percent of these had THC levels >-0.3% and, therefore, are unsuitable for hemp development in North America. Discrepancies were found between THC levels reported for some germplasm holdings in Europe when they were grown in Canada and, accordingly, verification of THC

The 5 biggest challenges in the growing of Medicinal Cannabis and how to solve them
Delphy
FIND DOI or Link

The Ancient Cultivation of Hemp
H. Godwin
Antiquity Volume 41, Issue 161 March 1967 , pp. 42-49
doi:10.1017/S0003598X00038928
The discovery in a long pollen diagram from East Anglia of a substantial curve for a pollen-grain referable to Cannabis sativa, L.,
the Indian hemp, raised the hope that we might, through palynology, have the means of tracing the history of cultivation of this important and sinister economic plant in England and in Western Europe. It was clearly essential that pollen-analytic evidence should be related fully
to existing historical and archaeological knowledge, and aided by a notice in this journal (ANTIQUITY, 1964, 287), and by the notable
kindness of a great many academic colleagues, I have put together a condensed historical account of the plant in antiquity as preface to a description of the pollen-analytic data.

The Applications of Molecular Markers in Genetics and Breeding of Hemp
Giuseppe Mandolino, Paolo Ranalli
Journal of Industrial Hemp 2002 7:1, 7-23
Doi: 10.1300/J237v07n01_03
Molecular markers were employed to the characterization and analysis of hemp genetic structure by using RAPD technique. The results are presented about the statistical treatment of the molecular data. In addition, markers tightly linked to the male sex and their applications are discussed, and a short protocol for direct amplification of such markers from hemp tissue is provided. A molecular map of hemp, including a number of RAPD markers obtained from a progeny of a cross between a female Carmagnola plant and a monoecious accession is also presented. Finally, the state of the art of sex genetics in hemp and the possibilities of developing molecular markers linked to different hemp chemotypes are discussed.

The Botany of Cannabis sativa L
Andreu FV
https://www.academia.edu/25681288/Th...nabis_sativa_L
Cannabis sativa L. is a widespread species in nature It is found invarious habitats ranging from sea level to the temperate and alpine foothills of the Himalayas, from where it was probably spread over the last 10,000 years. The age-old cultivation makes its original distribu-tion difficult to pinpoint. Cannabis has a long history of medicinaluse in the Middle East and Asia, with references as far back as the 6thcentury BCE, and it was introduced in Western Europe as a medicine in the early 19th century to treat epilepsy, tetanus, rhe rumatism, migraine, asthma, trigeminal neuralgia, fatigue, and insomnia. As a plant, it is valued for its hallucinogenic and medicinal prop-erties, more recently being used for pain, glaucoma, nausea, asthma, depression, insomnia, and neuralgia.
Derivatives are used inHIV/AIDS and multiple sclerosis. The pharmacology and therapeutic efficacy of cannabis preparations and its main activeconstituent
?9-tetrahydrocannabinol (?9-THC) have been extensively reviewed. The other important cannabinoid constituent of current interest is cannabidiol (CBD). There has been a significantinterest in CBD over the last few years because of its reportedactivity as an antiepileptic agent, particularly its promise for thetreatment of intractable pediatric epilepsy.
Other than ?9 –THC and CBD, tetrahydrocannabivarin (THCV), cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC) are major isolates.

The Cannabis Conundrum
David Kuack
https://gpnmag.com/wp-content/upload...sConundrum.pdf
If you’re considering growing cannabis, don’t expect to easily find a lot of credible information on how to produce and process the crop. If you are a grower of ornamental crops interested in producing cannabis, be aware there are a lot of unknowns about producing, harvesting and processing this crop. “For ornamental crops, the research literature provides a wealth of information related to cultural practices including fertility, light intensity and other factors,” says Allison Justice, vice president of cultivation at OutCo in El Cajon, California. Justice, who has been with the cannabis production and wholesale/retail company for two years, received her PhD in plant and environmental science from Clemson University in 2014. OutCo has been operating since 2015 and produces cannabis for both medicinal and recreational sales. “The biggest problem I experience with producing cannabis, even after working with the crop for two years, is the lack of a baseline,” Justice says. “If I was growing poinsettias, there is a baseline of nutrient concentrations that are used. If a tweak needs to be made in the nutrients, it’s easily made.”

The cannabis conundrum
Richard J. Miller
Proc Natl Acad Sci U S A. 2013 Oct 22; 110(43): 17165.
doi: 10.1073/pnas.1317663110
The debate about cannabis (also known as marijuana or Indian hemp) continues. Is it “dangerous”? Is it “addictive”? Does it make you “crazy”? Everybody has an opinion, but what is the truth of the matter and how should this influence the legal status of the drug? At the present time the cannabis laws in the United States are very confusing. Consider this: the federal government has classified cannabis as a Schedule 1 drug, the same status accorded heroin. Schedule 1 drugs are supposed to have no medical applications or therapeutic uses whatsoever and are also supposed to be the most easily abused and the most addictive. It is difficult to obtain such drugs, even for research purposes. On the other hand, many individual states have moved to decriminalize the possession and use of small amounts of cannabis. The major psychotropic molecule in cannabis, ?9–tetrahydrocannabinol (?9-THC), is actually available by prescription throughout the United States. The drug, known as dronabinol and sold under the trade name Marinol, is a Schedule 3 drug and thus is considered to be much less dangerous than the crude material from which it was originally isolated.

The complex interactions between flowering behaviour and fibre quality in hemp
https://sci-hub.tw/10.3389/fpls.2019.00614
doi: 10.3389/fpls.2019.00614
Hemp, Cannabis sativa L., is a sustainable multipurpose fibre crop with high nutrient and water use efficiency and with biomass of excellent quality for textile fibres and construction materials. The yield and quality of hemp biomass are largely determined by the genetic background of the hemp cultivar but are also strongly affected by environmental factors, such as temperature and photoperiod. Hemp is a facultative short-day plant, characterized by a strong adaptation to photoperiod and a great influence of environmental factors on important agronomic traits such as “flowering-time” and “sex determination”. This sensitivity ofhemp can cause a considerable degree of heterogeneity, leading to unforeseen yield reductions. Fibre quality for instance is influenced by the developmental stage of hemp at harvest. Also, male and female plants are differing in stature and produce fibres with different properties and quality. Next to these causes, there is evidence for specific genotypic variation in fibre quality among hemp accessions. Before improvedhemp cultivars can be developed, with specific flowering-times and fibre qualities, and adapted to different geographical regions, a better understanding of the molecular mechanisms controlling important phenological traits such as “flowering-time” and “sex determination” in relation to fibre quality in hemp is required. It is well known that genetic factors play a major role in the outcome of both phenological traits but, the major molecularfactors involved in this mechanism are not characterized in hemp. Genome sequences and transcriptome data are available but their analysis mainly focussed on the cannabinoid pathway for medical purposes. Herein, we review the current knowledge of phenotypic- and genetic data vailable for “flowering-time”, “sex determination” and “fibre quality” in short-day and dioecious crops, respectively, and compare them with the situation in hemp. A picture emerges for several controlling key genes, for which natural genetic variation may lead to desired flowering behaviour, including examples of pleiotropic effects on yield quality and on carbon partitioning. Finally we discuss the prospects for using this knowledge for the molecular breeding of this sustainable crop via a candidate gene approach.

The Constituents of Cannabis sativa Pollen
M. Paris, F. Boucher, L. Cosson
Economic Botany, Vol. 29, No. 3 (Jul. - Sep., 1975), pp. 245-253 : https://www.jstor.org/stable/4253616
https://www.jstor.org/stable/4253616
This research concerns the study of various constituents, chiefly the cannabinoids and nitrogenous substances of the pollen of Cannabis sativa that had been cultivated under artificial climatic conditions in the C. N. R. S. phytotron at 91 Gif-sur-Yvette, France. It covers principally an examination with qualitative and quantitative analyses of the main cannabinoids found as well as observations on the alkaloids and flavonoids present. The conditioned rooms of the phytotron enabled enough pollen to be produced to carry out these tests. The pollen of Cannabis sativa L. was rich in cannabinoids and particularly in THC and THCA, the latter being able to be transformed into physiologically active THC. Climatic factors and particularly temperature played an important role, since the THC content at 24° C 16 h was 30 times as great as at 22° C 12° C 16h. Determinations of the phenol compounds in the corresponding flowering heads had not been completed, but those that had been finished showed that the optimum content was given by plants cultivated at 24° C 16 h. The highest concentration of alkaloid type substances was also given under this climatic regime: these substances were different from choline and trigonelline and studies were under way to identify them. With regard to the flavonoids that were examined by two-dimensional paper chromatography, two principal spots were detected corresponding to two glycosides. After hydrochloric acid hydrolysis two different genins were identified as apigenin and luteolin respectively. These flavone glycosides were also found in the leaves together with several others, and a further study is being made of them the results of which will be published shortly.

The CPRO Cannabis germplasm collection
E.P.M. de Meijer & L.J.M. van Soest
Euphytica 62: 201-211, 1992.
DOI:10.1007/BF00041754
In an effort to reduce the overproduction of a too limited number of arable food crops, several research programmes dealing with industrial crops
have been initiated in the Netherlands. The 'National Hemp Programme' investigates the feasibility of hemp (Cannabis sativa L.) as a raw material
for paper pulp production. In this context breeding, agronomy, mechanisation and processing of hemp are subject of a comprehensive study. Two previous attempts to introduce fibre hemp in the Netherlands have been reported (de Jonge, 1944; Friederich, 1960). Breeding of fibre hemp in the Netherlands is unprecedented. In the framework of the current programme, improvement of cultivars is considered necessary in order to increase yield, quality, and disease resistance. A collection of Cannabis accessions is therefore being established at CPRO since 1988. In this report, the present state of the collection and its maintenance are described.

The effect of daylength on yield and quality of fibre hemp (Cannabis sativa L.).
H. M. G. van der Werf, H. J. Haasken , M. Wijlhuizen
Eur. J. Agron., 1994, 3(2), 117-123
Doi: 10.1016/S1161-0301(14)80117-2
Stem growth of the short-day plant fibre hemp (Cannabis sativa L.) decreases after flowering. In the Netherlands, the hemp cultivars currently available flower in August. In 1990 and 1991 the ambient daylength was compared with a 24-hour daylength in field experiments on two cultivars. Crop development, interception of photosynthetically active radiation, dry matter accumulation, stem yield and stem composition were recorded. The 24-hour daylength did not totally prevent flowering, but did greatly reduce the allocation of dry matter to floral parts. It enhanced the efficiency of post-flowering radiation use, and increased stem dry matter yield by 2.7 t ha- 1 • The continued stem growth resulted in higher yields, which in one cultivar were accompanied by a lower bark content of the stem. At final harvest, the 1 per cent NaOH solubility indicated a lower fibre content in the bark of plants from the 24-hour daylength. Breeding late-flowering hemp may be a promising strategy to improve the potential stem yield of hemp in the Netherlands, but the stem quality of such cultivars may be slightly poorer

The effect of N and P fertilization on growth, seed yield and quality of industrial hemp in the Parkland region of Saskatchewan
C. L. Vera, S. S. Malhi, J. P. Raney, and Z. H. Wang
Can. J. Plant Sci. 84: 939–947
https://cdnsciencepub.com/doi/pdf/10.4141/P04-022
Industrial hemp (Cannabis sativa L.) has sparked renewed interest in western Canada in recent years, and there is very little research information available on its fertilizer requirements. The objective of this study was to determine the effect of surface-broadcast ammonium nitrate and seedrow placed monoammonium phosphate fertilizers on the production and seed quality attributes of industrial hemp (cv. Fasamo and Finola). Field experiments were conducted on a Black Chernozem silty loam soil at Melfort, Saskatchewan, Canada, in 2000, 2001 and 2002. Increasing N rates significantly increased plant height, biomass, seed yield and seed protein content of hemp in all years. Seed-applied P fertilizer increased plant height in all years, and biomass in 2000, but reduced plant density, biomass and seed yield in 2001 and 2002. Finola consistently had lower plant height, earlier maturity, heavier seeds, and higher seed yield, seed protein content and seed oil content than Fasamo. The average amount of nitrate-N in the 0–60 cm soil was 40 kg N ha–1. Seed yield kg–1 of N was 9.4, 5.9, 4.5 and 3.7 kg ha–1 for Fasamo, and 10.6, 7.7, 6.0 and 4.5 kg ha–1 for Finola, respectively, at 40, 80, 120 and 160 kg ha–1 of soil plus fertilizer N.

The effect of temperature on leaf appearance and canopy establishment in fibre hemp (Cannabis sativa L.).
WERF, H. M. G. V. D., BROUWER, K., WIJLHUIZEN, M., & WITHAGEN, J. C. M.
Annals of Applied Biology, 126(3), 551–561.(1995).
doi:10.1111/j.1744-7348.1995.tb05389.x
The effects of temperature on the development and growth of hemp (Cannabis satiua L.) have never been quantified. Therefore, to establish the effect of temperature on leaf appearance and canopy establishment of fibre hemp under
controlled and field conditions, plants were grown in growth chambers at 11 regimes with average temperatures between 10°C and 28"C, and three cultivars were sown in the field in March, April and May in 1990, 1991 and 1992. In the field, thermal time (base 0°C) between sowing and emergence ranged from 68"Cd to 109.5"Cd (average 88.3"Cd). Rates of leaf appearance and stem elongation increased linearly with temperature between 10°C and 28°C. The base temperature for leaf appearance was 5.7"C from the growth chamber experiments and 1°C from the field experiments. In the field, the base temperature for the relationship between light interception by the canopy and thermal time was 2.5"C, and thermal time, calculated at the appropriate base temperature, accounted for about 98% of the variance in the number of leaves and for 98.6% of the variance in the proportion of light intercepted by the canopy. Days from emergence accounted for less of the variance in both parameters than thermal time. Interception of 90% of light was attained on average at 465"Cd (base 0°C) after emergence. It is concluded that thermal time is a simple and accurate tool to describe leaf appearance and light interception in fibre hemp.

The Effects of Organic and Inorganic Nitrogen Fertilizer on the Morphology and Anatomy of Cannabis sativa “Fédrina” (Industrial Fibre Hemp) Grown in Northern British Columbia, Canada
MSc Charlene Forrest BSc, Jane P. Young BSc, MSc, PhD
October 2006 Journal of Industrial Hemp 11(2):3-24
DOI: 10.1300/J237v11n02_02
The effects of organic and inorganic nitrogen fertilizer on the morphology and anatomy of Cannabis sativa “Fédrina” (industrial fibre hemp) was investigated in both a greenhouse and field setting in Northern British Columbia. Plots (90 stems/m2) treated with 0, 75, 150 or 300 kg N/ha of inorganic nitrogen, or fish meal, blood meal or sea star organic fertilizers were also replicated with 90 kg inorganic P2O5/ha application. The application of 150 and/or 300 kg N/ha of any nitrogen fertilizer type benefited field-grown plant morphology, secondary phloem fibre and xylem development, while greenhouse-grown plant morpology, secondary phloem fibre and xylem were positively influenced by 90 kg P2O5/ha. Primary phloem fibre characteristics of both greenhouse and field-grown plants were benefited by the absence of either nitrogen or phosphorus fertilizer. This study determined that the response of C. sativa “Fédrina” fibre production to organic nitrogen application was comparable to that of inorganic nitrogen application

The emergence of cannabis agriculture frontiers as environmental threats.
Butsic, V., Carah, J., Baumann, M., Stephens, C., & Brenner, J. C.
Environmental Research Letters.(2018).
doi:10.1088/1748-9326/aaeade
On agricultural frontiers, minimal regulation and potential windfall profits drive opportunistic land use that often results in environmental damage. Cannabis, an increasingly decriminalized agricultural commodity in many places throughout the world, may now be creating new agricultural frontiers. We examined how cannabis frontiers have boomed in northern California, one of the United States’ leading production areas. From 2012–2016 cannabis farms increased in number by 58%, cannabis plants increased by 183%, and the total area under cultivation increased by 91%. Growth in number of sites(80%), as well as in site size (56% per site) contributed to the observed expansion. Cannabis expansion took place in areas of high environmental sensitivity, including 80%–116% increases in cultivation sites near high-quality habitat for threatened and endangered salmonid fish species. Production increased by 40% on steep slopes, sites more than doubled near public lands, and increased by 44% in remote locations far from paved roads. Cannabis farm abandonment was modest, and driven primarily by farm size, not location within sensitive environments. To address policy and institutions for environmental protection, we examined state budget allocations for cannabis regulatory programs. These increased six-fold between 2012–2016 but remained very low relative to other regulatory programs. Production may expand on frontiers elsewhere in the world, and our results warn that without careful policy and institutional development these frontiers may pose environmental threats, even in locations with otherwise robust environmental laws and regulatory institutions.

THE EVALUATION OF HEMP {Cannabis sativa L.) AS A NON-WOOD SOURCE OF PAPER PULP
E.P.M, de Meiier
https://edepot.wur.nl/412584
A study of non-wood paper pulp production in the Netherlands by means of hemp (Cannabis sativa L.) is presently in its final stage. In the context of this comprehensive study and prior to the breeding of improved cultivars, a germplasm collection (de Meijer & van Soest, 1992), covering the variation within the genus Cannabis has been evaluated in various experimental set-ups. This paper reports on the observed variation for, and relations between the most relevant agronomic traits. Conclusions will be presented with reference to breeding prospects. Conclusions will be presented with reference to breeding prospects.

The Fluctuation Curve of Sex Reversal in Staminate Hemp Plants Induced by Photoperiodicity
John H. Schaffner
American Journal of Botany, Vol. 18, No. 6 (Jun., 1931), pp. 424-430
DOI:10.1002/j.1537-2197.1931.tb09602.x
In past experiments on sex reversal in the hemp, Cannabis sativa L., the writer did not succeed in producing ioo percent reversal of the individuals of any given population grown in the greenhouse during the short day period of winter (Schaffner 9, io). The highest percentage of reversal obtained was 88+ percent for carpellate plants and 93+ percent for staminate plants. When these results were interpreted by some as indicating a multiple factor constitution of sex determination, with females, female intersexes, male intersexes, males, and supermales as the possible Mendelian combinations, further experiments became necessary to obtain definite information on the subject (see Hirata 3, 4). An experiment was devised through which "pure females" were rejuvenated after the usual blooming period and then changed to male expression in the second or rejuvenated ontogenetic cycle (Schaffner iI). It is evidently desirable to learn how to set the ecological stage so that IOO percent of sex reversal would appear in the staminate plants in the first or usual ontogenetic cycle. A record of sex reversal in relation to photoperiodicity throughout the changing daily light period of the different seasons also seemed desirable. The aim, then, in the present experiments was to obtain, if possible, IOO percent of sex reversal of the staminate plants, including the "supermales," of a given plot; to find out how perfect the curve of reversal might be, and also to discover how definitely the per-centage of reversal for any seasonal planting might be predicted. The staminate plants only were studied because of a lack of proper greenhouse space and because the carpellate plants take a much longer period to reach maturity. The carpellate plants were mostly removed as soon as their sex reaction was definitely developed. The plants were grown on greenhouse benches with about 8 in. (20 cm.) of good, rich soil and were well watered. The temperature was kept at about 55-65 degrees Fahrenheit (I3-I8o C.) but the fluctuation was often considerably beyond these points. The plants were not disturbed in any way by removing flowers, branches, or leaves so whatever unusual effects were obtained were due entirely to difference of daily illumination.

The Future of Hemp Fibres Under Changing Climate Conditions
Fieke Dhondt
https://www.researchgate.net/publica...ate_Conditions
Climate change is posing a significant threat to textile fibres. Research has shown how increased temperatures, drought and extreme weather influences the availability of raw materials for fashion. Climate change does affect textile fibres, but on the other hand, the fashion industry increases the prospects of global warming. The industry contaminates freshwater and uses an extensive amount of chemicals, pesticides and insecticides. This study aims to determine how hemp as a textile fibre can persevere under a changing climate and become a less polluting textile for the future apparel industry. By analysing and combining existing studies targeted on the cultivation and processing of hemp crops to textile fibres, the study answers: What are the impacts, challenges and opportunities of climate change on hemp fibres? Derived from a review of the literature on hemp's preferred climate and climate change data gathered from the Intergovernmental Panel on Climate Change (IPCC) reports and the Köppen Climate Classification, a predictive analysis was formed. Next to that, possible implications and opportunities and challenges hemp will face as a textile of the future are discussed. The results of this study emphasise how hemp is not only a sustainable alternative for the current most used fibres but also exceeds those fibres in climate change resistance. Given this, it is recommended that fashion brands use hemp as a key material in the future. Further research is needed to identify other factors that could secure hemp against climate change, such as seawater cultivation, hydroponic farms and the research into climate-resistant hemp strains. Next to that, the thesis can be enriched by fashion-related business cases and local supply chain case studies.

The future of industrial hemp in the Netherlands
E. van Vliet
https://dspace.library.uu.nl/handle/1874/295451
Master thesis Environmental Biology July, 2014
Allocation of the Common Agricultural Policy (CAP) subsidies from fiber crops including industrial hemp to biofuel and bio-energy crops, induces competition for cropland and jeopardizes the Dutch hemp cultivation. By means of scientific and professional literature and questionnaires, future prospects were drawn for industrial hemp in the Netherlands. In addition, the role of the Dutch hemp industry in the European and global market was investigated. Future prospects were drawn based on the expectations expressed by HempFlax and Dun Agro, the only Dutch hemp processors, and dr. ir. Trindade, hemp breeding expert at Wageningen University. It was assessed that although hemp is receiving more attention, especially in the automotive industry, hemp processors will have difficulty with finding sufficient cropland to grow raw material. It is expected that with ongoing competition for cropland, industrial hemp will disappear from the Netherlands. However, contradicting this, the industrial hemp acreage is expected to grow as a result of the many applications and beneficial properties of hemp. Hemp is well adapted to Dutch climate, grows well on poor Dutch soils without chemical additives, provides a sustainable alternative for cotton and synthetic fibers and may be used as bioremediation, making hemp a very interesting crop for the Netherlands. However, under the current circumstances, the Dutch hemp industry has difficulty to overcome the startup phase and therefore it is recommended that the Dutch and European policies are adapted to create a more supporting environment for industrial hemp.

The Hemp collections in Italy
Dr. Giuseppe MANDOLINO
Dr. Mario DI CANDILO
https://archive-ecpgr.cgiar.org/file...ollections.pdf
Italian hemp germplasm at CRA-ISCI
•Different seed lots of the same variety (e.g. Carmagnola);
•Rare traditional italian ecotypes (e.g. Carmagnola gigante,
Eletta Campana, Napoletana, Pinnatofida);
•New varieties (Yellow apex, Red petiole, monoecious
varieties);
•Breeding lines;
•Experimental hybrids between fibre and drug strains
(to study heterosis and cannabinoid segregation).

The History of Hemp in Norway
Jan Bojer Vindheim
January 2002Journal of Industrial Hemp 7(1):89-103
DOI: 10.1300/J237v07n01_08
Around the year 1000 we may assume that hemp was grown in several places in Norway, but at all times the importation has been greater than local production. This article discusses the history of hemp in Norway and the many ways the plant has been used, including both ritual and common purposes.

The Influenec of Relative Daylight On The Reversal Of Sex In Hemp
John H. Schaffner
Ecology Vol. 4:323-334 Oct 1923
DOI: 10.2307/1929179
Since investigating, by observation and experiment, the fundamental nature of sex in organisms, especially plants, the writer has been impressed by the
great influence of the environment in determining the characteristics of the individual. The inevitable conclusion has been reached that ecological factors
are of prime importance, in many species, in determining directly the actual sexual nature of the individual or in inducing actual reversals of the sexual
state in case the sex had already been determined. The results on which these conclusions have hitherto rested have been published in various papers,
the most important of which from the ecological point of view are those on Arisaema triphyllum (L.) Torr. (Schaffner, '22), where it was shown that
the sexual expression of the perennial individual could be changed absolutely from year to year through control of the nutrition and water supply, and on
Cannabis sativa L. ('21), in which it was shown that ordinary hemp produces pure diecious (two sexes separate) individuals when planted in the field in
spring, but that about 90 per cent of both male and female plants reversed their sexual expression, the carpellate individual giving rise to male structures
and the staminate to female, when planted in the greenhouse during the winter period.

The long history of Cannabis and its cultivation by the Romans in central Italy, shown by pollen records from Lago Albano and Lago di Nemi
Anna Maria Mercuri, Carla Alberta Accorsi, Marta Bandini Mazzanti
Vegetation History and Archaeobotany December 2002, Volume 11, Issue 4, pp 263–276
DOI:10.1007/s003340200039
The cores from the Albano and Nemi lakes, near Rome, were studied within the European Union funded PALICLAS project and provided high resolution records of the Late-glacial and Holocene. Pollen evidence of increasing human influence on vegetation was recorded in the Holocene parts of both diagrams, and the Cannabis (hemp) curve was one of the major signs. In this paper we present unambiguous pollen evidence from the Cannabaceae records for the cultivation of hemp in central Italy by the Romans. The oldest records of Cannabis andHumulus (hop) date from to the Late-glacial. Hop pollen values rise during the mid Holocene, while hemp pollen becomes more abundant from ca. 3000 cal B.P. onwards. The highest earliest hemp peak (21%) is dated to the 1st century A.D. This ‘Cannabis phase’, with the abrupt rise of hemp pollen soon after the rise of cultivated trees (Castanea, Juglans and Olea) is associated with the increase in cereals and ruderal plants. This unambiguous proof of cultivation by Romans around 2000 B.P. occurs as well as a long lasting pre-Roman presence of hemp in the area, which is natural and possibly also anthropogenic. Subsequent clear episodes of cultivation in the medieval period were found.

The National Cannabis Collection in Hungary
Attila Simon
https://archive-ecpgr.cgiar.org/file...collection.pdf
Hungarian genebank activity
Long history and rich tradition
Several reorganizations during 50 years
Last: December 31st 2005
Institute for Agrobotany as an independent institute directly reporting to the
Ministry of Agriculture was dissolved
Successor: The Research Centre for Agrobotany as a department of National Institute for Agricultural Quality Control (NIAQC)
Heritage: entire gene bank activity
Exploration and collection if PGR
Multiplication and regeneration
Isoclimatic regeneration
Characterisation and evaluation
Documentation
Medium and long term conservation
Participation in the IPGRI ECP/GR program
Long term conservation of Hungarian National Base Collection
Distribution of seed samples
Nation-wide responsibility for the coordination of PGR activities

The National Cannabis Collection in Hungary
Attila Simon
https://archive-ecpgr.cgiar.org/file...y%20report.pdf
The national gene bank activity has a long history and a rich tradition in Hungary. The most outstanding result of the last half a century is that the collection includes more than 71 500 accessions, which is available for Hungarian and foreign breeders and other users as well. The Research Centre for Agrobotany (RCA) - as its predecessor the Institute for Agrobotany - as a
department of the National Institute for Agricultural Quality Control (NIAQC) is in charge of the Hungarian national gene bank activities and performs entire gene bank activity. As a national designated authority, the NIAQC is responsible for the state registration of plant varieties and animal breeds, with the examination, quality control, certification and official supervision of seed and reproductive materials, and with activities connected with animal breeding and feed quality.

The Next Evolution in Horticulture Lighting
Adding value & differentiation to your Horticulture products through use of UV LEDs
Dr. Peter Barber
Director of NA UV Sales
SETi/ SeoulViosys
https://issuu.com/amsterdamrai/docs/peter_barber.pptx
Walkthrough history
?Where are we now—key factors
?Where can we go
?UV 101 & Solar Spectrum
?Plant response mechanism to UVB
?Secondary plant metabolites (cannabinoids/terpenoids)
?Post-harvest

The Performance and Potentiality of Monoecious Hemp (Cannabis sativa L.) Cultivars as a Multipurpose Crop.
Baldini, M., Ferfuia, C., Piani, B., Sepulcri, A., Dorigo, G., Zuliani, F., … Cattivello, C.
Agronomy, 8(9), 162. (2018).
doi:10.3390/agronomy8090162
Given the growing interest in multipurpose hemp crop, eight monoecious cultivars were compared in a two-year trial for quantitative and qualitative yield in a Mediterranean environment characterized by a temperate and humid climate with hot summers. All hemp cultivars were evaluated for yield potential of (i) seed plus stem at seed maturity, and (ii) essential oil yield from inflorescences harvested at full flowering. The second goal was set to test the ability of cultivars to supply new seeds after the removal of inflorescence at full flowering. Among the cultivars, Fedora obtained the best results for seed (0.79 and 0.52 t ha?1 ) and vegetable oil yield (0.17 and 0.09 t ha?1 ) normally and with inflorescence removed plants, respectively. Futura, conversely, showed the best results for inflorescence (3.0 t ha?1 ), essential oil (9 L ha?1 ), and stem yield at seed maturity (8.34 t ha?1 ), as means across the two years. The cultivars studied generally reached the grain-filling stage during a period that was drier and warmer than the average of the same multi-year period, and this negatively affected seed quality. The oil fatty acid composition was mainly composed of polyunsaturated fatty acids (75% on average) and not affected by the cultivar. In conclusion, although the hemp grower should always clearly know the main production objective of the crop, the monoecious cultivars available today allow a multipurpose use of hemp crop, improving the sustainability of the cultivation activity

The potential of hemp (Cannabis sativa L.) for sustainable fibre production: a crop physiological appraisal.
WERF, H. V. D., MATHUSSEN, E. W. J. M., & HAVERKORT, A. J.
Annals of Applied Biology, 129(1), 109–123. (1996).
doi:10.1111/j.1744-7348.1996.tb05736.x
Hemp (Cannabis sativa L.) fibre can be used as a raw material for paper and
textile production. A comprehensive research programme in the Netherlands has concluded that fibre hemp is a potentially profitable crop, having the right profile to fit into sustainable farming systems. This paper presents an appraisal of the crop physiological characteristics and the agronomic potential of hemp. Parameter values of basic crop physiological characteristics such as light interception potential, radiation use efficiency and dry matter partitioning coefficients are given. The effect of crop management decisions such as cultivar choice, sowing date, plant density, and harvest date on the value of these parameters is discussed. A simple crop growth model was used to assess the yield potential of hemp for the climate of the Netherlands. Calculations made for a non-stressed late-flowering hemp crop sown on 15 April and harvested on 15 September give a stem dry matter yield of 17.1 t ha-'. The effects of advancing or delaying sowing or harvest date on stem yield were calculated. Crop physiological characteristics of hemp are compared to those of kenaf (Hibiscus cannabinus L.). Radiation use efficiency and dry matter partitioning coefficients of the two crops are similar. Base temperatures for development and growth are lower in hemp than in kenaf. In a temperate
climate with cool springs, canopy establishment will be more rapid in hemp than in kenaf. Hemp seems an excellent candidate to fill the niche for an annual fibre crop in a temperate climate.

THE PROPAGATION, CHARACTERISATION AND OPTIMISATION OF CANNABIS SATIVA L AS A PHYTOPHARMACEUTICAL
David Potter
Phd Thesis
https://www.scribd.com/document/2140...pharmaceutical
https://www.yumpu.com/en/document/re...thesisdjpotter
https://docslide.net/download/link/potter-thesis-2009
In response to known pharmacology, and an increasing weight of anecdotal evidence of efficacy clinical trials have been preformed to support the licensing of Cannabis based botanical medicines. The initial applications envisaged were the treatment of cancer pain, neuropathic pain, and various symtoms associated with multiple sclerosis. With effective alternatives often unavailable, otherwise law-abiding UK patients have regularly turned to illicit Cannabis for medicinal relief.
The main axtive ingredients in this are the Cannabinoids THC and CBD, but other pharmacologically active cannabinoids are present. One study reported here quantifies these cannabinoids and assesses the likely implicarions for efficacy. Using light microscopy studies are performed to expand current knowledge of the forms and function of trichomes in
Cannabis sativa L. Supporting chemical analyses ascertain what secondary metabolites are biosynthesized within these trichomes and determines where and when this occurs. To comply with the demands of the pharmaceutical industry, and in marked contrast to illicit Cannabis, a phytopharmaceutical feedstock must meet high expectations regarding the minimum and maximum content of a range of compounds. Specific studies are performed to ascertain how growing methods affect the secondary metabolite content. They also aim to find out how a tight specification can be met while satisfying commercial and environmental expectations. This involves studing plant development and secondary metabolite are biosynthesis in both indoor and outdoor conditions. The first approved cannabis based botanical medicine supported by this research is Sativex®. This became available in Canada in 2005 for the treatment of central neuropathic pain in multiple sclerosis and in 2007 for intractable cancer pain. The medicine is also available in the UK and many other countries on a ‘named patient basis’ this thesis has also supported the production of a range of other cannabinoids which are undergoing in-vitro and vito testing. This could lead to the commercial production of an increasing rage of phytopharmaceuticals.

The sexual differentiation of Cannabis sativa L.: A morphological
and molecular study

V.M. Cristiana Moliterni, Luigi Cattivelli, P. Ranalli & Giuseppe Mandolino
Euphytica 140: 95–106, 2004.
DOI: 10.1007/s10681-004-4758-7
Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova. Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment. Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.

The state of the apex and the response to induction in Cannabis sativa.
Heslop-Harrison J, Heslop-Harrison Y.
1970 In: Bernier G, ed. Proceedings of the Symposium on Cellular and Molecular Aspects of Floral Induction, Liege, Belgium. Longmans Green, 3-26.
FIND DOI or LINK

The Terroir of Cannabis: Terpene Metabolomics as a Tool to Understand Cannabis sativa Selections.
Mudge, E., Brown, P., & Murch, S. (2019).
Planta Medica.
doi:10.1055/a-0915-2550
The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, ?-myrcene, and ?-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

Time Dependent Metabolomics and Transcriptional Analysis of Cannabinoid Biosynthesis in Cannabis sativa var. Bedrobinol and Bediol Grown under Standardized Condition and with Genetic Homogeneity
Remco Muntendam, Nizar Happyana, Tjalling Erkelens, Freerk Bruining, Oliver Kayser
Volume 1, Issue 2, pp. 31-40; December 2012 International Journal of Medicinal Plants Research
DOI: 10.13140/RG.2.1.2263.5360
Cannabis sativa L. is mostly grown under non standardized conditions resulting in high fluctuations of cannabinoid contents. The exact measure of these metabolites is hampered as most of the research has been conducted on seized, extracted or non-standardized cultivated C. sativa L. material. In this
report legal standardized cultivation of two C. sativa L. chemotypes is investigated by HPLC-UV, qPCR and 1H-NMR, respectively during two cultivation phases of four (4) weeks each. Based on HPLC the cannabinoid spectrum over 4 week cultivation period is monitored under specific light conditions and in correlation with the expression profile of thca and cbda genes in both chemotypes. Cannabinoid accumulation is observed specifically after shortening of the light period. Gene transcript based chemotyping was only possible comparing the thca and cbda transcription level ratio at the middle of the cultivation period, while at the end differences in gene transcription ratio diminished. 1H-NMR results indicate a clear difference between both chemotypes and cultivation phases by applying pattern
recognition software. The differences detected by using simple and non-destructive 1H-NMR were used to build a model, which can be applied to chemotyping and cultivation phase separation using selective 1H-NMR signals.

Traditional Cannabis Cultivation in Darchula District, Nepal—Seed, Resin and Textiles
Robert Connell Clarke
November 2007Journal of Industrial Hemp 12(2):19-42
DOI: 10.1300/J237v12n02_03
The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, ?-myrcene, and ?-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

Traditional ecological knowledge and skills: hemp plants cultivation and use by a local community (Vel?ice village case)
Pavol Elias sen
December 2016
https://www.researchgate.net/publica...e_village_case
Hemp (Cannabis sativa L. subsp. sativa) was an important and beneficial crop which provided textile raw material – fibre and food – seeds and seed oil for local communities in southern Slovakia for centuries. Cumulative community knowledge, practice and belief of the industrial hemp were transfered from one generation to next ones orally and by sharing practical experiences, being integral part of every day work of poeple, including folk saying. The poeple recognized and established areas/sites most suitable for the hemp cultivation which were used as crops for many years (in Slovak language the site is called „konopisko“). Sowing in high density by hands from a sheet resulted in tall hemp plants and controled weeds in the crops. The crop was harvested in two different periods - male (first harvesting) and female (second harvesting) plants separately. Water retting by submerging bundles of hemp stalks in meandred brook ?ereš?ový potok. In the paper individual elements of TEK of hemp growing/cultivation, primary processing and usage by a local community in Vel?ice village (district Zlaté Moravce, southwest Slovakia, Central Europe) are described and discussed in details. Key words: industrial hemp, Cannabis sativa, cultivation, primary processing, treatment, use, local community, Vel?ice village, Slovakia

Traditional Nepali Hemp Textiles.
Clarke, R. C.
Journal of Industrial Hemp, 12(2), 97–113.(2007).
doi:10.1300/j237v12n02_07
Farmers living in the western districts of the Nepal Himalaya grow Cannabis and use the fibrous bark to make various textile products. Nepali hemp cordage crafts and woven fabrics are becoming increasingly known amongst textile collectors. This paper will introduce some of the collectible hemp textiles of Nepal.

Not Cannabis Specific
Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening.
Mou, W., Li, D., Luo, Z., Mao, L., & Ying, T. (2015).
PLOS ONE, 10(6), e0129598.
doi:10.1371/journal.pone.0129598
Abscisic acid (ABA) has been proven to be involved in the regulation of climacteric fruit ripening, but a comprehensive investigation of its influence on ripening related processes is still lacking. By applying the next generation sequencing technology, we conducted a comparative analysis of the effects of exogenous ABA and NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) on tomato fruit ripening. The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples, 10388 were identified as significantly differently expressed genes. Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these processes. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis. Our results highlighted the significance of ABA in regulating tomato ripening, which provided insight into the regulatory mechanism of fruit maturation and senescence process.

Understanding Cultivar-Specificity and Soil Determinants of the Cannabis Microbiome
Max E. Winston , Jarrad Hampton-Marcell, Iratxe Zarraonaindia, Sarah M. Owens, Corrie S. Moreau, Jack A. Gilbert, Josh Hartsel, Suzanne J. Kennedy, S. M. Gibbons
Plos ONE
doi: 10.1371/journal.pone.0099641
Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

KeyGene works with Cannabis
UniSNP: unique partnership between KeyGene and INCOTEC for variety tracing and seed purity testing
Press Release September 10, 2014
https://news.agropages.com/News/News...---13115-e.htm
KeyGene and INCOTEC have intensified their collaboration in order to develop SNP assays in various crops. These assays, UniSNP sets, are used for quality control testing in commercial seed production, such as variety tracing and (hybrid to open pollinated) seed purity testing. INCOTEC is the world’s largest independent seed enhancement company and KeyGene is one of the world’s leading Agro Biotechnology companies specialized in molecular plant genetics. The two companies teamed up in 2012 to work on this venture.
The UniSNP sets are unique SNP assays which have been developed through the united effort of INCOTEC, KeyGene and their customers. In the last 2 years UniSNP sets have been developed and commercialized worldwide for tomato, pepper, cucumber, melon, watermelon, lettuce and sunflower. These assays have proven to be highly predictive and effective. A unique feature for the proprietary SNPs involved is that they have been developed from broad crop germplasm collections obtained from seed companies all over the world. Together, INCOTEC and KeyGene have designed broadly applicable SNP assays for the crops mentioned above and they have been made available to all seed companies. Crops which are currently under assay development are: eggplant, several brassicas, onion, corn and squash with rice and cotton to follow soon. All UniSNP sets will be made commercially available shortly. INCOTEC and KeyGene provide customers the opportunity to send samples for testing. As the demands of the market will determine which crops will be added to the list, customers are able to influence the choice of crops for which assays will be developed

USDA National Hemp Report 2022, Feb 17
2021 Industrial Hemp Value $824 Million
2021 Industrial Hemp in the Open Value $712 Million
2021 Industrial Hemp Under Protection Value $112 Million
All industrial hemp: In 2021, the value of hemp production in the open and under protection for the United States totaled $824 million.

Industrial hemp in the open: Planted area for the Nation in 2021 for all utilizations totaled 54.2 thousand acres. The value of hemp production in the open for the United States totaled $712 million. Area harvested for all purposes in the open totaled 33.5 thousand acres.
Floral hemp in the open: United States floral hemp production grown in the open for 2021 was estimated at 19.7 million pounds. Area harvested for floral hemp in the open in the United States was estimated at 16.0 thousand acres. The average yield for 2021 floral hemp in the open was estimated at 1,235 pounds per acre. The value of floral hemp grown in the open totaled $623 million.
Grain hemp in the open: National production of hemp grown in the open for grain in 2021 totaled 4.37 million pounds. Area harvested for hemp grown in the open for grain in the United States was estimated at 8,255 acres. The average yield for 2021 hemp grown in the open for grain was estimated at 530 pounds per acre. The value of hemp grown in the open for grain totaled $5.99 million.
Fiber hemp in the open: In 2021, production of hemp grown in the open for fiber was estimated at 33.2 million pounds. Area harvested for hemp grown in the open for fiber in the United States was estimated at 12.7 thousand acres. The average yield for 2021 hemp grown in the open for fiber was estimated at 2,620 pounds per acre. The value of hemp grown in the open for fiber totaled $41.4 million.
Seed hemp in the open: Production of hemp grown in the open for seed in 2021 was estimated at 1.86 million pounds. Area harvested for hemp grown in the open for seed in the United States was estimated at 3,515 acres. The average yield for 2021 hemp grown in the open for seed was estimated at 530 pounds per acre. The value of hemp grown in the open for seed totaled $41.5 million.
Hemp under protection: In 2021, hemp growers used 15.6 million square feet under protection for production. The 2021 value of hemp production under protection in the United States totaled $112 million.
Hemp clones and transplants grown under protection in the Nation for 2021 totaled 20.2 million plants. The value of hemp clones and transplants grown under protection totaled $23.8 million. United States production of floral hemp grown under protection was estimated at 310 thousand pounds. The value of floral hemp grown under protection totaled $64.4 million. Hemp grown under protection for seed totaled 4,059 pounds. The value of hemp grown under protection for seed totaled $23.7 million.
https://downloads.usda.library.cornell .edu/usda-esmis/files/gf06h2430/xd07hw825/v692v917t/hempan22.pdf

UT-Arlington and UT-El Paso to Evaluate Phytochrome Manipulation in Hemp
https://cannabisindustryjournal-com....fb00-365750754
The Collaborative Laboratories for Environmental Analysis and Remediation (CLEAR) at the University of Texas at Arlington (UT-Arlington) and the University of Texas at El Paso (UT-El Paso) has begun collaborating with Curtis Mathes Grow Lights (CMGL), a subsidiary of the Curtis Mathes Corporation, and the hemp genetics company ZED Therapeutics. The research will involve characterizing the phytochemical effects of phytochrome manipulation using various LED horticultural lights of differing light spectrum, and novel high-yielding varietals of hemp. All of the hemp plants will be grown by renowned geneticists Adam Jacques, Christian West, and Oriah Love of ZED Therapeutics under the CMGL Harvester LED lights at their Oregon facility. Drs. Kevin Schug and Zacariah Hildenbrand will oversee the analysis of the corresponding samples for the expression of terpenes, flavonoids, and other classes of therapeutic compounds. The expression of 15 primary cannabinoid species will be performed concurrently by Matthew Spurlock of ZED Therapeutics.

Variations and origin of the atmospheric pollen of Cannabis detected in the province of Tetouan (NW Morocco): 2008–2010
Nadia Aboulaich, M. Mar Trigo, Hassan Bouziane, Baltasar Cabezudo, Marta Recio, Mohamed El Kadiri, Mohammed Ater
Science of the Total Environment 443 (2013) 413–419
DOI: 10.1016/j.scitotenv.2012.10.075
Cannabis, also called marihuana or hemp, is a wind-pollinated plant that produces hundreds of flowers on large inflorescences. It is also one of the oldest psychoactive plants known to humanity. Morocco has become one of the main producers of Cannabis resin (hashish), primarily supplying the European market. The aim of this paper is to ascertain whether the atmospheric monitoring of Cannabis pollen can play a role, from a criminological point of view, in the surveillance of Cannabis cultivation in the area of Tetouan (NW Morocco) as well as to estimate pollen emission so that the sensitive population can be warned about the allergic diseases that its pollen can cause. Aerobiological samplings were made with the aid of a Hirst type volumetric trap (Hirst, 1952), which worked uninterruptedly during a 3-year period (2008–2010) according to the methodology proposed by the Spanish Aerobiology Network, the REA. Cannabis pollen was present in the atmosphere of Tetouan mainly from early April to late August, a period in which about 95% of the annual counts were registered. The highest levels were detected in June and July, with concentrations more or less evenly distributed throughout the day with slight increases of 5% between 12:00 and 16:00 h. The strong association between skin test reactivity, respiratory symptoms, and pollination period found by other authors, together with the levels registered, suggests that Cannabis pollen could be a clinically important aeroallergen for sensitive patients. On the other hand, the data obtained could serve as an indicator of the cultivation activity of this species and should be taken into account by the state authorities since they provide strong evidence of the existence of Cannabis crops in the region of Tetouan.

Variations of 9-THC content in single plants of hemp varieties
Klemens Mechtler, Josef Bailer, Karl de Hueber
Industrial Crops and Products. 19(1), 19-24
DOI: 10.1016/S0926-6690(03)00077-3
Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. To test the distribution of 9-tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of five well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30–61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribution of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single-plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC values far outside this bandwidth.

Vegetative propagation of cannabis by stem cuttings: effects of leaf number, cutting position, rooting hormone and leaf tip removal
Deron Caplan, Jonathan Stemeroff, Mike Dixon and Youbin Zheng
Canadian Journal of Plant Science
https://tspace.library.utoronto.ca/b...-2018-0038.pdf
This study evaluated the influence of the several factors and their interactive effects on propagation success using stem cuttings of cannabis (Cannabis sativa L.). Factors included: (i) Leaf number (two or three) (ii) leaf tip removal (1/3 of leaf tips removed) (iii) basal/apical position of stem cutting on the stock plant, and (iv) rooting hormone (0.2% indole-3-butyric (IBA) acid gel or 0.2% willow (Salix alba L.) extract gel). Cuttings were placed in a growth chamber for twelve days then assessed on their rooting success rate and root quality using a relative root quality scale. The IBA gel delivered a 2.1 times higher rooting success rate and 1.6 times higher root quality than the willow extract. Removing leaf tips reduced rooting success rate from 71% to 53% without influencing root quality. Cuttings with three leaves had 15% higher root quality compared to those with two, but leaf number did not influence rooting success rate. Position of cutting had little effect on rooting success or quality. To achieve maximum rooting success and root quality, cuttings from either apical or basal positions should have at least three fully expanded, uncut leaves and the tested IBA rooting hormone is preferential to the willow based product.

Water- and Nitrogen-Use Efficiencies of Hemp (Cannabis sativa L.) Based on Whole-Canopy Measurements and Modeling
Kailei Tang, Alessandra Fracasso, Paul C. Struik, Xinyou Yin* and Stefano Amaducci
Front. Plant Sci., 16 July 2018
doi: 10.3389/fpls.2018.00951
Interest in hemp (Cannabis sativa L.) as a crop for the biobased economy is growing worldwide because hemp produces a high and valuable biomass while requiring low inputs. To understand the physiological basis of hemp’s resource-use efficiency, canopy gas exchange was assessed using a chamber technique on canopies exposed to a range of nitrogen (N) and water levels. Since canopy transpiration and carbon assimilation were very sensitive to variations in microclimate among canopy chambers, observations were adjusted for microclimatic differences using a physiological canopy model, with leaf-level parameters estimated for hemp from our previous study. Canopy photosynthetic water-use efficiency (PWUEc), defined as the ratio of gross canopy photosynthesis to canopy transpiration, ranged from 4.0 mmol CO2 (mol H2O)?1 to 7.5 mmol CO2 (mol H2O)?1 . Canopy photosynthetic nitrogen-use efficiency (PNUEc), the ratio of the gross canopy photosynthesis to canopy leaf-N content, ranged from 0.3 mol CO2 d ?1 (g N)?1 to 0.7 mol CO2 d ?1 (g N)?1 . The effect of N-input levels on PWUEc and PNUEc was largely determined by the N effect on canopy size or leaf area index (LAI), whereas the effect of water-input levels differed between short- and long-term stresses. The effect of short-term water stress was reflected by stomatal regulation. The long-term stress increased leaf senescence, decreased LAI but retained total canopy N content; however, the increased average leaf-N could not compensate for the lost LAI, leading to a decreased PNUEc. Although hemp is known as a resource-use efficient crop, its final biomass yield and nitrogen use efficiency may be restricted by water limitation during growth. Our results also suggest that crop models should take stress-induced senescence into account in addition to stomatal effects if crops experience a prolonged water stress during growth.

Weighing the risk of cannabis cross-pollination
James DeDecker
Michigan State University Extension (2019)
https://www.canr.msu.edu/news/weighi...ss-pollination
Cannabis is what’s known as a dioecious species, meaning that male and female flowers are borne on separate plants. There are some monoecious varieties of cannabis with male and female flowers on the same plant, and stress can also induce the production of male flowers on female plants, but these are exceptions to the plant’s normally dioecious nature. Flowering is induced when day and night lengths become equal. Male cannabis plants flower for a period of two to four weeks, and a single male flower can produce 350,000 pollen grains. Pollen is carried to female plants on the wind and can travel great distances when conditions are favorable. Bees will collect cannabis pollen but are generally not attracted to the female flowers to contribute to pollination.
In the 1970s, marijuana growers found that preventing pollination by rogueing out male plants or producing only females (through clonal propagation or sowing of feminized seed) could greatly increase the yield and potency of their crop. This works because cannabis is one of the few plant species that can actively increase the number and size of its female sex organs in response to prolonged virginity, according to Small and Naraine, 2016. The longer female plants go unpollinated, the more flowers are produced and the larger they get.

What is Industrial Hemp?
V.D. Jeliazkov, J. Noller, S. Angima, S. Rondon, R. Roseberg, S. Summers, G. Jones, V. Sikora
OREGON STATE UNIVERSITY EXTENSION SERVICE
https://www.researchgate.net/publica...ndustrial_Hemp
ntroduction Industrial hemp (Cannabis sativa L.) was grown as a commodity fiber crop in the United States from the mid-18th century until the mid-1930s. As in many other countries, C. sativa was banned and was considered an illegal crop in the U.S. for several decades. In 2014, Section 7606 of the federal Agricultural Act of 2014, commonly called the Farm Bill, allowed the cultivation of industrial hemp within authorized pilot programs for “the plant Cannabis sativa L. and any part of such plant, whether growing or not, with a delta-9 tetrahydrocannabinol [THC] concentration of not more than 0.3 percent on a dry weight basis.”
 
Last edited:

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Hemp Processing


A new technology for predicting the fiber content in hemp bast

Deng Gang, Ding Ming liang, Cheng Xia, Liu Fei Hu
PeerJ Preprints (2017)
doi.: 10.7287/peerj.preprints.3458v1
Hemp (Cannabis sativa L.) is a traditional fiber crop, which is becoming one of the most important industrial fibers, with a promising future in many fields. To accelerate the breeding of hemp cultivars with increased fiber content, it is important to establish a preflowering method that can be used to predict the potential fiber content in hemp bast. This study investigated the correlation between fiber content in the stem bast portion and in the entire stem. In addition, the variation in the bast fiber content during the sampling period was studied in three hemp cultivars. It was clear that the bast fiber content in hemp stems was determined up to 40 d before emergence of the staminate buds. The fiber content of the bast (in a sample piece 30cm long and covering one-quarter of the stem girth, sampled at two-fifths of the plant height from the soil, 20 d before the staminate buds emerged) was shown to be representative of the fiber content of the entire stem. In conclusion, this new method would allow breeders to select the hemp plants for high bast fiber content during the early to middle growth periods, before the male buds emerged, potentially accelerating the genetic improvement of fiber content in industrial hemp

A review of the properties of hemp concrete for green building applications.
Jami, T., Karade, S. R., & Singh, L. P.
Journal of Cleaner Production, (2019).
doi:10.1016/j.jclepro.2019.117852
The growing concerns surrounding the rising carbon emissions have impelled the leaders around the world to make efforts to prevent catastrophic manifestations of climate change and global warming. This has led to the resurrection of vegetal concrete building materials using biomass, which have the added benefits of carbon sequestration apart from low embodied energy and renewability. Vegetal concretes are made up of an organic or inorganic binder, and biomass originating from agro-forestry industries such as rice husk, straw bale, hemp, kenaf, cork, and so on. Hemp concrete, a variety of vegetal concrete has been widely researched and is arguably one of the most researched building materials in current times. This paper presents a review of the state-of-the-art of hemp concrete research, with a view to identifying research gaps that shall guide future research for its implementation in the fast-growing green buildings industry. The reviewed aspects of hemp concrete include properties of hemp relevant to construction, binder characteristics, mechanical properties, durability, hygric and thermal properties, environmental credentials, manufacturing processes, and current applications. Several research gaps with regards to the hydraulicity of the binder, strength and durability, and fire resistance of hemp concrete were identified. It was also established that hemp concrete has very low embodied carbon and embodied energy, making it ideal for green building applications. The paper ends with a discussion outlining the need and direction for future research on improving the manufacturing processes and mechanical performance of hemp concrete for wider adoption by the construction industry

Assessing the Mechanical Performance Cannabis Sativa Composites– Reinforced with Long Time Dried fibre
Nadendla Srinivasababu
Procedia Engineering 97 ( 2014 ) 986 – 993
doi: 10.1016/j.proeng.2014.12.375
An increase in environmental concern invites the development new eco friendly materials having light weight and superior mechanical performance. In this connection an attempt is made to manufacture composites reinforced with long time exposed dora hemp fibre at ambient conditions into the unsaturated polyester resin. The fabricated composite specimens are processed at P1, P2 and P3 conditions to assess their mechanical performance as per ASTM procedures. Out of all the composites investigated in the present work the dora hemp composites at P2 have shown good tensile and flexural properties, whereas the highest impact strength is achieved for the composites at P1. Hence this class of natural fibre composites is suitable for making automobile components after thorough investigation of necessary parameters.

A Technical and Economic Feasibility Study of Green Decortication of Hemp Fibre for Textile Uses
Sue Riddlestone, Emily Stott, K. Blackburn, James Brighton
October 2006 Journal of Industrial Hemp 11(2):25-55
DOI: 10.1300/J237v11n02_03
In 2003-4 BioRegional Development Group carried out a technical and economic feasibility study of a method of green decortication of hemp and degumming for the production of textile fibre. The study was published in December 2004. Two field trials of the green decortication technology took place in the UK and Australia, Cranfield University evaluated the process in both instances. Some of the fibre produced was subsequently processed in preparation for spinning. This paper contains the findings of these trials.

Application of cryogenic and mechanical treatment in degumming of hemp stems.
Liu, J., Guan, Z., & Li, Z.
Biosystems Engineering, 174, 144–152.(2018).
doi:10.1016/j.biosystemseng.2018.07.007
Natural fibre reinforced composites are lightweight materials widely applied to automotive interior parts. However, there are some problems such as volatile organic compound (VOC) emissions exceeding standard levels and weak interfacial bonding, which limits their use in industrial applications. In order to overcome the existing problems, a new degumming method (cryogenic and mechanical treatment) is proposed in this paper. Firstly, hemp fibre bundles became loose after cryogenic treatment, and there was some micropore creation or microcracking. Secondly, mechanical treatment separated hemp fibres from the fibre bundles. Finally, hemp fibres were cleaned with alkaline liquid. The influences of cryogenic and mechanical treatment on the fibres were investigated by using scanning electron microscope (SEM), Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The results revealed that cellulose content was increased from 66.25% to 78.93%, and hemicellulose and lignin were reduced to 7.16% and 2.82%, respectively. Decreases in the diameter and tensile properties of the treated hemp fibres were also observed. In addition, in differential thermogravimetric analysis (DTG), the untreated hemp fibres had a significant peak at about 336 C, while the significant peaks of treated hemp fibres were approximately at 360 C. The significant peaks at 336 C and 360 C represent the maximum oxidative decomposition rate, and indicate that the treated fibres had a higher thermal stability than the untreated fibres. It was found that cryogenic and mechanical treatment is feasible for the degumming of hemp fibres.

not Cannabis specific
Bioprocessing of bast fibers.
Yu, Y., Wang, Q., & Wang, P.
Advances in Textile Biotechnology, 1–19.(2019).
doi:10.1016/b978-0-08-102632-8.00001-3
Bast fiber, also called phloem fiber, is a type of plant fiber that can be collected from the phloem or bast surrounding the stem of certain dicotyledonous plants. Bast fibers can be obtained either from cultivated herbs, such as flax, hemp, and ramie, or wild plants, such as linden, wisteria, and mulberry. The strands of bast fibers are usually released from the cellular and woody tissue of the stem by mechanical, biological, or chemical methods. Bast fibers have higher tensile strength than other natural fibers, thereby are usually used in the production of high-quality textiles (Faruk et al., 2012; Summerscales et al., 2010). Bast fibers are processed and utilized in many industries, such as textiles, ropes and nets, carpets and mats, brushes, and mattresses industries, in addition to paper and board materials industries (Paridah et al., 2011). In recent years, bast fibers, such as flax, kenaf, and hemp, have received attention from researchers and industries for their use as reinforcement in polymerematrix composites because of environmental awareness of consumers and government regulation in some countries (Anuar and Zuraida, 2011; Bos et al., 2002; Saba et al., 2015; Stuart et al., 2006). Biotechnological process has been used in textile processing of bast fibers, such as microbial retting of bast fibers, which took place during BC periods. During the past decades, textile biotechnology has been an important research area, and thus several enzyme-based processes have now been well-established and are available for use in bast fiber processing, such as retting, scouring, bleaching, and functionalization (Table 1.1) (Kozlowski et al., 2006; Shahid et al., 2016).

Cell Wall Composition of Hemp Shiv Determined by Physical and Chemical Approaches
Maya-Sétan Diakité, Hélène Lenormand, Vincent Lequart, Santiago Arufe, Patrick Martin, and Nathalie Leblanc
Molecules. 2021 Nov; 26(21): 6334. 2021 Oct 20.
doi: 10.3390/molecules26216334
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8587414/
The use of agricultural by-products in the building engineering realm has led to an increase in insulation characteristics of biobased materials and a decrease in environmental impact. The understanding of cell wall structure is possible by the study of interactions of chemical compounds, themselves determined by common techniques like Van Soest (VS). In this study, a global method is investigated to characterise the cell wall of hemp shiv. The cell wall molecules were, at first, isolated by fractionation of biomass and then analysed by physical and chemical analysis (Thermal Gravimetric Analysis, Elementary Analysis, Dynamic Sorption Vapor and Infra-Red). This global method is an experimental way to characterise plant cell wall molecules of fractions by Thermal Gravimetric Analysis following by a mathematical method to have a detailed estimation of the cell wall composition and the interactions between plant macromolecules. The analyzed hemp shiv presents proportions of 2.5 ± 0.6% of water, 4.4 ± 0.2% of pectins, 42.6 ± 1.0% (Hemicellulose–Cellulose), 18.4 ± 1.6% (Cellulose–Hemicellulose), 29.0 ± 0.8% (Lignin–Cellulose) and 2.0 ± 0.4% of linked lignin.

Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity
Jae Kwon Kim, Hee-Young Heo, SeonJu Park, Haheon Kim, Jeong Ju Oh, Eun-Hwa Sohn, Se-Hui Jung, and Kooyeon Lee
ACS Omega 2021, 6, 47, 31945–31954 Publication Date:November 18, 2021
DOI: 10.1021/acsomega.1c04727
https://pubs.acs.org/doi/pdf/10.1021/acsomega.1c04727
Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α- MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 μM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-transcaffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skinwhitening agent

Dry Grinding in the cannabis industry
IKA
http://www.ikausa.com/wp-content/upl...bis_CONIKA.pdf
An essential step for cannabis and hemp production Milling is an essential step in the CBD oil extraction. With several standard sieve sizes available, the IKA CONIKA provides a simple yet reliable ability to target sizing, which is critical to the extraction process. The arrangement of the mill provides a continuous processing method for rapid dry grinding of stalk, trim, or flower.

Effect of aluminium sulphate-catalysed hydrolysis process on furfural yield and cellulose degradation of Cannabis sativa L. shives
Prans Brazdausks, Aigars Paze, Janis Rizhikovs, Maris Puke, Kristine Meile,
Nikolajs Vedernikovs, Ramunas Tupciauskas, Martins Andzs
Biomass and Bioenergy 89 February 2016
doi.org/10.1016/j.biombioe.2016.01.016
Commercially, furfural is produced from pentosan-rich biomass using mineral acids as homogeneous catalysts. This study investigated a novel hydrolysis method that allows to obtain furfural from hemp shives with high yield and also to preserve the cellulose in the remaining biomass for other bioconversion processes. To date, hemp shives have not been investigated for furfural production. Cannabis sativa L. (“Bialobrzeskie” variety) shives were used as a feedstock due to the high content of pentosan (17.6% of oven-dried biomass). It means that the theoretically possible amount of furfural was 12.8% of oven-dried hemp shives. The effect of temperature (140e180 _C), the amount of catalyst (3e7% of ovendried biomass) and the treatment time (10e90 min) on the furfural formation were studied. Whereas, the effect of the same temperature and the amount of catalyst on the changes of lignocellulose were studied after 90 min treatment time. Al2(SO4)3*18H2O was used as a catalyst for the conversion of C5- sugars to furfural. To show the catalytic properties of Al2(SO4)3*18H2O, autocatalysis was performed as a reference process using the same parameters. The highest yield of furfural, 73.7% of the theoretical yield, was obtained at 180 _C, 5% Al2(SO4)3*18H2O of oven-dried mass and 90 min. From the biorefinery perspective, the optimal hydrolysis parameters were 160 _C, 5% Al2(SO4)3*18H2O of oven-dried mass and 90 min. With these parameters, the yield of furfural was 62.7% of the theoretical yield, 99.2% of hemicelluloses were removed and 95.8% of cellulose was preserved and slightly depolymerized.

Effect of steam treatment on the chemical composition of hemp (Cannabis sativa L.) and identification of the extracted carbohydrates and other compounds
Taneli Väisänena, Petri Kilpeläinenb, Veikko Kitunenb, Reijo Lappalainena, Laura Tomppo
Industrial Crops & Products 131 (2019) 224–233
Doi: 10.1016/j.indcrop.2019.01.055
The overarching aim of this study was to investigate the possibility of extracting carbohydrates and other compounds from hemp (Cannabis sativa L.) using a continuous steam treatment that would also separate the fibres from the hurd. Different parts of hemp, such as stalk, leaves, and decorticated hemp with hurd, were extracted by a steam treatment. After processing, the fibers in the stalk could be easily separated. The products obtained at different extraction temperatures were characterized using multiple techniques. The hemicellulose content of untreated dry hemp was reduced from 275 mg/g to 237 mg/g at 160 °C treatment while the corresponding cellulose content increased from 376 mg/g to 418 mg/g. For example, the iron (Fe) content of the extracts increased with elevated treatment temperatures; for dry hemp at 100 °C, the Fe content in extracts was 1.33 mg/kg, whereas at 160 °C, it was much higher, 16.58 mg/kg. The results demonstrate that the extraction temperature influences the composition of the extracts. Compounds with potentially useful applications, such as in medicine and in the chemical industry, were also identified in the extracts. However, more research will be needed to optimize the treatment and the further processing of the products in order to estimate the commercial potential of this technique.

Engineering Perspectives of the Hemp Plant, Harvesting and Processing
Ying Chen, Jude Liu, Jean-Louis Gratton
December 2004 Journal of Industrial Hemp 9(2):23-39
DOI: 10.1300/J237v09n02_03
The special characteristics of the hemp (Cannabis sativa L.) plant make it one of the most challenging crops to handle. Several studies, both in the laboratory and the field, have been conducted at the University of Manitoba, Canada, on the engineering perspectives of hemp production, including the physical and mechanical properties of the hemp plant, hemp harvesting and processing. Physical properties of the hemp plant, such as plant height, seed-head length, stem diameter and stem specific mass, vary highly within a field and across fields. The force and energy required for cutting a hemp stem are much greater than those required for cutting maize stalk and forage crops. The two-windrow harvesting concept has been demonstrated to be feasible and can be implemented into a commercial windrower for harvesting dual-purpose hemp. Conditioned hemp dries significantly faster than unconditioned hemp. However, conditioning hemp requires more power than conditioning a forage crop. The basic machine functions required for hemp fibre processing are separating the fibre from the core and cleaning the fibre. A field-going processing unit can be formed by combining a modified forage harvester and a straw walker from a grain combine. However, the effectiveness of such a unit is limited, and the design of new separating and cleaning devices may be required for higher fibre yield and purity

not Cannabis specific
Environmental impact of textile fibres – what we know and what we don’t know. Fiber Bible part 2.
Report number: Mistra Future Fashion report number 2019:03
Affiliation: RISE Research Institutes of Sweden
Project: Mistra Future Fashion
Gustav Sandin, Sandra Roos, Malin Johansson
DOI: 10.13140/RG.2.2.23295.05280
Production of cotton and synthetic fibres are known to cause negative environmental effects. For cotton, pesticide use and irrigation during cultivation contributes to emissions of toxic substances that cause damage to both human health and the ecosystem. Irrigation of cotton fields cause water stress due to large water needs. Synthetic fibres are questionable due to their (mostly) fossil resource origin and the release of microplastics. To mitigate the environmental effects of fibre production, there is an urgent need to improve the production of many of the established fibres and to find new, better fibre alternatives. For the first time ever, this reports compiles all currently publicly available data on the environmental impact of fibre production. By doing this, the report illuminates two things: • There is a glaring lack of data on the environmental impact of fibres – for several fibres just a few studies were found, and often only one or a few environmental impacts are covered. For new fibres associated with sustainability claims there is often no data available to support such claims. • There are no ”sustainable” or ”unsustainable” fibre types, it is the suppliers that differ. The span within each fibre type (different suppliers) is often too large, in relation to differences between fibre types, to draw strong conclusions about differences between fibre types. Further, it is essential to use the life cycle perspective when comparing, promoting or selecting (e.g. by designers or buyers) fibres. To achieve best environmental practice, apart from considering the impact of fibre production, one must consider the functional properties of a fibre and how it fits into an environmentally appropriate product life cycle, including the entire production chain, the use phase and the end-of-life management. Selecting the right fibre for the right application is key for optimising the environmental performance of the product life

Enzymatic Degumming of Hemp.
Jörg Müssig, Holger Fischer, Cornelia Bluhm
Conference: COST Action 847 „Textile Quality and Biotechnology“ WG2 & WG3 Meetings., Maribor, Slovenia: Institute of Textilies, University of Maribor, Maribor, 2004-02-26 till 2004-02-27.
University of Maribor, Maribor, Slovenia February 2004 Volume: 25
https://www.researchgate.net/publica...umming_of_Hemp
Hemp (Cannabis sativa L.) as renewable resource is a good candidate for usage in high-performance composite materials as well as in yarns for textile production. Since the cultivation of hemp was permitted in 1996, a lot of research work has been done to establish cultivation, harvesting and processing techniques. Up to now, the mechanical separation of hemp fibres is realised in industrial scale. The actual fibre qualities are good enough for usage in e.g. needle felts and low-price composites, but the fineness is not sufficient for e.g. yarn production. Hemp fibres for the purpose of textile production are nowadays imported from eastern Europe and China and are mostly produced by chemical separation in NaOH. In view of sustainable production this process is not feasible in Germany due to its high consumption of water, chemicals and energy. Alternatives are for example the steam explosion process or enzymatic separation, where the enzymatic process tends to be more sustainable due to its lower energy consumption. Enzymatic separation of hemp has been carried out successfully in laboratory scale. Now the results have to be transferred to an industrial scale. Main topics are: - selection of appropriate fibre qualities - quality management from the plant to the final product - optimization of the fibre pretreatment processes (harvesting, mechanical separation etc.) - selection or development of especially adapted enzymes - planning and realization of a process integrated waste-water treatment to ensure nearly complete recycling - production of yarns, textiles and composites from the enzymatic modified fibres To ensure a sustainable process this pilot plant operates with a water circuit to minimise water consumption and wastewater effluent by a special process design. To establish the new system on market as soon as possible the project team consists of five research institutes, specialised for the research on botany, biotechnology, environmental process engineering and fibre quality and seven partners from industry for hemp treatment, enzyme production, membrane design, system engineering, waste management, yarn production and marketing studies.

ENZYMATIC MODIFICATION OF HEMP FIBRES FOR SUSTAINABLE PRODUCTION OF HIGH QUALITY MATERIALS:COMPARING DIFFERENT COMMERCIAL ENZYMES
Holger Fischer, Jörg Mu?ssig, Cornelia Bluhm, Jan Marek, Viktor Antonov
https://www.researchgate.net/publica...ercial_Enzymes
Main focus of this paper is the influence of different commercial enzymes in enzymatic hemp separation on the fibre quality. The influence of fibre pre-treatment has to be considered. The results show, that processing parameters from e.g. flax treatment can not easily be transferred to hemp, since there is a different pectin structure. For hemp a fibre pre-treatment by soda improves the efficiency of the subsequent enzymatic step substantially. Tested enzymes comprise pectate lyases like BioPrep™ 3000L, Baylase™ EVO, pectinases like Lyvelin™, Texazym™ BFE and hemicellulases like Texazym™ DLG. Best results in fibre fine separation were obtained by the pure pectinases. The pectate lyases were less efficient in fibre separation and the hemicellulase tends to cause loss of fibre tenacity as undesirable side effect

Enzymatic Modification of Hemp Fibres for Sustainable Production of High Quality Materials: Effects on Chemical Fibre Composition.
Holger Fischer, Jörg Müssig, Dinora Abdulajeva
Conference: Proceedings of The 4th International Conference on Textile Biotechnology, June 4–7, 2006, COEX, Seoul, Korea.
At: COEX, Seoul, Korea. Volume: 4
https://www.researchgate.net/publica...re_Composition
Hemp (Cannabis sativa L.) as a renewable resource is a good candidate for usage in high-performance composite materials as well as in yarns for textile production. Since the cultivation of hemp in Germany was permitted in 1996, a lot of research work has been done to establish cultivation, harvesting and processing techniques. Up to now, the mechanical separation of hemp fibres is realised in industrial scale. The actual fibre qualities are good enough for usage in e.g. needle felts and low-price composites, but the fineness is not sufficient for e.g. yarn production. Hemp fibres for the purpose of textile production in Europe are nowadays imported e.g. from China and are mostly produced by chemical separation in NaOH. In view of sustainable production this process is not feasible due to its high consumption of water, chemicals and energy. Alternatives are for example the steam explosion process, microbiological separation or enzymatic separation, where the micobiological and enzymatic processes tend to be more sustainable due to their lower energy consumption. Different commercial enzymes have been used in enzymatic hemp separation with specific influence on the fibre quality. It is known, that processing parameters from e.g. flax treatment can not easily be transferred to hemp, since there is a different pectin structure. Best results in hemp fibre separation were obtained by pure pectinases like Lyvelin™ and Texazym™ BFE. Main focus of this presentation is the influence of enzymatic hemp fibre treatment on the chemical composition of the fibres. Results are presented for lignin and hemicellulose content of different raw materials. These are compared to influence of chemical fibre separation. The results show clearly, that enzymatic and chemical treatment cause a similar reduction of lignin content, but that reduction of hemicellulose content is much bigger by chemical separation than by enzymatic separation.

Enzymatic Modification of Hemp Fibres for Sustainable Production of High Quality Materials: Influence of Processing Parameters.
Conference: INTB 04 — 3rd International Conference on Textile Biotechnology. (Graz, Austria, 2004-06-13 till 2004-06-16). Graz, Austria: Graz University of TechnologyVolume: 3
Holger Fischer, Jörg Müssig, Cornelia Bluhm
https://www.researchgate.net/publica...ing_Parameters
Hemp (Cannabis sativa L.) as renewable resource is a good candidate for usage in high-performance composite materials as well as in yarns for textile production. Since the cultivation of hemp was permitted in 1996, a lot of research work has been done to establish cultivation, harvesting and processing techniques. Up to now, the mechanical separation of hemp fibres is realised in industrial scale. The actual fibre qualities are good enough for usage in e.g. needle felts and low-price composites, but the fineness is not sufficient for e.g. yarn production. Hemp fibres for the purpose of textile production are nowadays imported from eastern Europe and China and are mostly produced by chemical separation in NaOH. In view of sustainable production this process is not feasible in Germany due to its high consumption of water, chemicals and energy. Alternatives are for example the steam explosion process or enzymatic separation, where the enzymatic process tends to be more sustainable due to its lower energy consumption. Enzymatic separation of hemp has been carried out successfully in laboratory scale. Now the results have to be transferred to an industrial scale. Main topics are: selection of appropriate fibre qualities - quality management from the plant to the final product optimization of the fibre pretreatment processes (harvesting, mechanical separation etc.) selection or development of especially adapted enzymes construction of a pilot plant planning and realization of a process integrated waste-water treatment to ensure nearly complete recycling production of yarns, fabrics, felts and composites from the enzymatic modified fibres, and last but not least optimization of the enzymatic separation process to adapt the fibre bundles to the needs of the product. Outlook: To establish the new system on market as soon as possible the project team consists of five research institutes, specialised on botany, biotechnology, environmental process engineering and fibre quality and seven partners from industry for hemp treatment, enzyme production, membrane design, system engineering, waste management, yarn production and marketing studies.

Enzymatic separation of hemp fibres — aspects of quality control along the value-added chain.
Holger Fischer, Jörg Müssig, Cornelia Bluhm
Conference: COST Action 847 „Textile Quality and Biotechnology“ the joint meeting of WG/1 and WG/2. WG/1 and WG/2, Budapest, Hungary 2004-09-23 till 2004-09-24.
Budapest, Hungary: Budapest University of Technology and Economics, Dept. Plastics and Rubber Technology
https://www.researchgate.net/publica...ue-added_chain
Main focus of this presentation is the importance of a good quality control along the value-added chain for hemp products in order to achieve constantly high product qualities. Hemp (Cannabis sativa L.) as renewable resource is a good candidate for usage in high-performance composite materials as well as in yarns for textile production. Since the cultivation of hemp in Germany was permitted in 1996, a lot of research work has been done to establish cultivation, harvesting and processing techniques. Quality control has to start with the selection of the appropriate cultivar for the location depending on climatic conditions, soil quality etc. The fibre quality is strongly dependent on the type and time of harvesting and the intensity of retting. Thus the final fibre quality after mechanical coarse separation is mainly a result of these processing steps. Consequently fibre properties should be examined before harvesting and several times during the retting process to ensure an optimal result of retting. The set-up of a quality control system is exemplarily presented for the control of different growth locations and selection of the optimal lots for enzymatic retting and subsequent production, depending on the desired product quality. In addition it is discussed, which fibre characteristics should be controlled at which process step.

European hemp industry: Cultivation, processing and product lines
Michael Karus, Dominik Vogt
Euphytica 140(1):7-12 January 2004
DOI: 10.1007/s10681-004-4810-7
Two fibre plants are under cultivation in the European Union – flax on approximately 125,000 ha and hemp on approximately 15,500 ha in the year 2004. Seeds, hurds and especially fibres of hemp are used for further processing. The most important markets for hemp fibres produced in the EU are pulp and paper and the automotive industry. Just under 5% of the EU hemp fibres were used in the construction sector. Approximately 95% the whole production of 40,000 t of the hemp hurds are used as animal bedding and 95% of the estimated 6000 t per year hemp seeds are sold for animal feed, mainly as bird feed

Evaluation of hemp (Cannabis sativa L.) as an industrial crop: a review
Muzammal Rehman , Shah Fahad , Guanghui Du , Xia Cheng , Yang Yang , Kailei Tang , Lijun Liu , Fei-Hu Liu , Gang Deng
Environ Sci Pollut Res Int. 2021 Sep 2.
doi: 10.1007/s11356-021-16264-5
Rising human population has increased the utilization of available resources for food, clothes, medicine, and living space, thus menacing natural environment and mounting the gap between available resources, and the skills to meet human desires is necessary. Humans are satisfying their desires by depleting available natural resources. Therefore, multifunctional plants can contribute towards the livelihoods of people, to execute their life requirements without degrading natural resources. Thus, research on multipurpose industrial crops should be of high interest among scientists. Hemp, or industrial hemp, is gaining research interest because of its fastest growth and utilization in commercial products including textile, paper, medicine, food, animal feed, paint, biofuel, biodegradable plastic, and construction material. High biomass production and ability to grow under versatile conditions make hemp, a good candidate species for remediation of polluted soils also. Present review highlights the morphology, adaptability, nutritional constituents, textile use, and medicinal significance of industrial hemp. Moreover, its usage in environmental conservation, building material, and biofuel production has also been discussed.
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First experimental evidence of hop fibres in historical textiles
Git Skoglund & Bodil Holst & Hana Lukešová
Archaeological and Anthropological Sciences (2020) 12:214
DOI: 10.1007/s12520-020-01171-6
Hop (Humulus lupulus) has been used in Scandinavia since at least the ninth century AD, as documented through archaeological findings and written, historical records. The written records are mainly focused on the use of cone-shaped flowers for beer brewing and medical purposes, but there are also records, for example, from the famous Swedish botanist Carl von Linne, who mentions the use of hop fibres for textile production. However, until now no experimental investigations have been published on the use of hop fibres in cultural heritage objects. A major reason for this has been the lack of a suitable characterization method. Hop is a bast fibre, just as flax and hemp and bast fibres cannot be distinguished from each other by simple optical inspection. Recently a new identification method for hop fibres was published by the authors of this article. Here we apply the new method in an investigation of two Swedish cultural heritage objects: (i) a woman’s garment from the nineteenth century, which was labelled as having an upper section made from coarse linen and a bottom section made of hemp and hop and (ii) a textile fragment from an eighteenth-century textile sample book, which was labelled as being made from hop. We show that the woman’s garment is made with hop and hemp fibres and the textile fragment from the textile sample book is made with hop. Our work provides the first direct proof that hop fibres were used for textiles in the past.

Hemp Fiber as a Sustainable Raw Material Source for Textile Industry: Can We Use Its Potential for More Eco-Friendly Production?
Gedik, G., & Avinc, O.
Sustainability in the Textile and Apparel Industries, 87–109. (2020). doi:10.1007/978-3-030-38541-5_4
Sustainable production defines an environmental friendly production that we produce without changing the balance of the nature. Processes and the utilized materials should be renewable, and our whole production should be harmless so that nature can recover itself in an indigenous way. All natural fibers are biodegradable and sustainable, and consequently, they are commonly called as biofibers. Providing a sustainable production chain for textile processes requires individual attention for each input in the first place. One of the most important parts of these inputs is raw material selection and therefore fiber supply. Right at this point, hemp fiber step forwards and shines out with its huge sustainable production potential for textile industry. In this chapter, sustainable and biodegradable hemp fiber, which is an alternative to cotton and petroleum-based synthetic fibers, for textile raw material sourcing is reviewed in detail. The parameters that make this fiber sustainable are also investigated. Present common and special uses and possible future innovative alternatives of hemp fibers for technical textiles production are also stated. Mainly, composite material production with this sustainable fiber is reviewed for a replacement of nonsustainable synthetic competitors. When sustainable composite materials are produced not only ecofriendly textile production is carried out but also other materials can be produced with an ecofriendly path leading to more sustainable world.


Identification of histone acetyltransferase genes responsible for cannabinoid synthesis in hemp Yufei Cheng1 Kang Ning, Yongzhong Chen, Cong Hou, Haibin Yu, Huatao Yu, Shilin Chen, Xiaotong Guo and Linlin Dong
Chinese Medicine (2023) 18:16
Identification of histone acetyltransferase genes responsible for cannabinoid synthesis in hemp
Identification of histone acetyltransferase genes responsible for cannabi
Background Histone acetyltransferases (HATs) play an important role in plant growth and development, stress response, and regulation of secondary metabolite biosynthesis. Hemp (Cannabis sativa L.) is famous for its high indus- trial, nutritional, and medicinal value. It contains non-psychoactive cannabinoid cannabidiol (CBD) and cannabinol (CBG), which play important roles as anti-inflammatory and anti-anxiety. At present, the involvement of HATs in the regulation of cannabinoid CBD and CBG synthesis has not been clarified.

Methods The members of HAT genes family in hemp were systematically analyzed by bioinformatics analysis. In addition, the expression level of HATs and the level of histone acetylation modification were analyzed based on transcriptome data and protein modification data. Real-time quantitative PCR was used to verify the changes in gene expression levels after inhibitor treatment. The changes of CBD and CBG contents after inhibitor treatment were veri- fied by HPLC-MS analysis.

Results Here, 11 HAT genes were identified in the hemp genome. Phylogenetic analysis showed that hemp HAT family genes can be divided into six groups. Cannabinoid synthesis genes exhibited spatiotemporal specificity, and histones were acetylated in different inflorescence developmental stages. The expression of cannabinoid synthesis genes was inhibited and the content of CBD and CBG declined by 10% to 55% in the samples treated by HAT inhibitor (PU139). Results indicated that CsHAT genes may regulate cannabinoid synthesis through altering histone acetylation.

Conclusions Our study provides genetic information of HATs responsible for cannabinoid synthesis, and offers a new approach for increasing the content of cannabinoid in hemp.

Keywords Histone acetyltransferase, Hemp, Acetylation, Cannabinoid, Abiotic stress





Background Histone acetyltransferases (HATs) play an important role in plant growth and development, stress response, and regulation of secondary metabolite biosynthesis. Hemp (Cannabis sativa L.) is famous for its high indus- trial, nutritional, and medicinal value. It contains non-psychoactive cannabinoid cannabidiol (CBD) and cannabinol (CBG), which play important roles as anti-inflammatory and anti-anxiety. At present, the involvement of HATs in the regulation of cannabinoid CBD and CBG synthesis has not been clarified.

Methods The members of HAT genes family in hemp were systematically analyzed by bioinformatics analysis. In addition, the expression level of HATs and the level of histone acetylation modification were analyzed based on transcriptome data and protein modification data. Real-time quantitative PCR was used to verify the changes in gene expression levels after inhibitor treatment. The changes of CBD and CBG contents after inhibitor treatment were veri- fied by HPLC-MS analysis.

Results Here, 11 HAT genes were identified in the hemp genome. Phylogenetic analysis showed that hemp HAT family genes can be divided into six groups. Cannabinoid synthesis genes exhibited spatiotemporal specificity, and histones were acetylated in different inflorescence developmental stages. The expression of cannabinoid synthesis genes was inhibited and the content of CBD and CBG declined by 10% to 55% in the samples treated by HAT inhibitor (PU139). Results indicated that CsHAT genes may regulate cannabinoid synthesis through altering histone acetylation.

Conclusions Our study provides genetic information of HATs responsible for cannabinoid synthesis, and offers a new approach for increasing the content of cannabinoid in hemp.

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Influence of field retting duration on the biochemical, microstructural, thermal and mechanical properties of hemp fibres harvested at the beginning of flowering.
Mazian, B., Bergeret, A., Benezet, J.-C., & Malhautier, L.
Industrial Crops and Products, 116, 170–181.(2018).
doi:10.1016/j.indcrop.2018.02.062
The use of hemp fibres as reinforcements in polymer composites requires a thorough understanding of the hemp fibres transformation processes to obtain a constant quality. In this context, the upstream processing termed field retting is considered. Retting allows a subsequent fibre separation from the plant stems by degradation of cementing compounds by microorganisms. This operation depends on weather conditions and is currently empirically carried out in fields, so that a large variability in the hemp fibres quality (color, morphology, biochemical composition, thermal properties and mechanical properties) is resulting. Therefore, the present study aims to investigate the influence of different retting durations (up to 9 weeks) on hemp fibres properties when harvested at the beginning of flowering growth stage to survey their temporal dynamic. Various assessments were applied on fibres: color observations, morphological (optical microscope), surface (ESEM) and biochemical (gravimetry) analyses, spectrocolorimetric measurements (pectins content), thermogravimetric (TGA) analysis, and mechanical in tensile mode testings. The results reveal that increasing the field retting duration leads to a change of color characteristics from light green to grey due to the development of microbial communities (most probably fungal and bacteria) at the stem surface. A separation of the fibres bundle to elementary fibres occurs with the degradation of pectins during retting. An increase of thermal stability of the fibres is also observed. Both increase of cellulose fraction and crystallinity induce an enhancement in tensile properties.

Influence of Temperature on the Water Retting Process of Hemp (Cannabis sativa L.) Cultivated Under Swedish Climate Conditions
Karin Magnusson, Bengt Svennerstedt
November 2007Journal of Industrial Hemp 12(2):3-17
DOI: 10.1300/J237v12n02_02
Hemp has recently received increased interest as a sustainable crop for a multitude of possible uses. This paper focuses on the retting process, which is an important part in the processing of high quality fibre for, e.g., textile products. Water retting process trials were performed on stem plant material from Futura 75 grown at the Lönnstorp Farm research station in the south of Sweden in 2004. Three retting treatments at water temperature 30, 37.5 and 45°C were performed on dried hemp material. Hemp samples were taken from each of the three treat ments at 48, 96, 144 and 196 hours. The retting process was investigated and documented by using the change in pectin content and the change in pH value as measures of the rettability. It was not possible to determine an optimal temperature for the retting process by these trials. However it could be concluded that 45°C was too high a water temperature

Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept
Mariusz Kuglarz, Merlin Alvarado-Morales, Dimitar Karakashev, Irini
Angelidaki
Bioresource Technology (2015),
doi: 10.1016/j.biortech.2015.10.081
The aim of this study was to develop integrated biofuel (cellulosic bioethanol) and biochemical (succinic acid) production from industrial hemp (Cannabis sativa L.) in a biorefinery concept. Two types of pretreatments were studied (dilute-acid and alkaline oxidative method). High cellulose recovery (> 95%) as well as significant hemicelluloses solubilization (49-59%) after acid-based method and lignin solubilization (35-41%) after alkaline H2O2 method were registered. Alkaline pretreatment showed to be superior over the acid-based method with respect to the rate of enzymatic hydrolysis and ethanol productivity. With respect to succinic acid production, the highest productivity was obtained after liquid fraction fermentation originated from steam treatment with 1.5% of acid. The mass balance calculations
clearly showed that 149 kg of EtOH and 115 kg of succinic acid can be obtained per 1 ton of dry hemp. Results obtained in this study clearly document the potential of industrial hemp for a biorefinery.

Interconnected Carbon Nanosheets Derived from Hemp for Ultrafast Supercapacitors with High Energy
Huanlei Wang, Zhanwei Xu, Alireza Kohandehghan, Zhi Li, Kai Cui, Xuehai Tan, Tyler James Stephenson, Cecil K. King’ondu, Chris M. B. Holt, Brian C. Olsen, Jin Kwon Tak, Don Harfield, Anthony O. Anyia, and David Mitlin
ACS Nano 7(6) :5131-41 June 2013
DOI: 10.1021/nn400731g
We created unique interconnected partially graphitic carbon nanosheets (10_30 nm in thickness) with high specific surface area (up to 2287 m2 g_1), significant volume fraction of mesoporosity (up to 58%), and good electrical conductivity (211_226 S m_1) from hemp bast fiber. The nanosheets are ideally suited for low (down to 0 _C) through high (100 _C) temperature ionicliquid-based supercapacitor applications: At 0 _C and a current density of 10 A g_1, the electrode maintains a remarkable capacitance of 106 F g_1. At 20, 60, and 100 _C and an extreme current density of 100 A g_1, there is excellent capacitance retention (72_92%) with the specific capacitances being 113, 144, and 142 F g_1, respectively. These characteristics favorably place the materials on a Ragone chart providing among the best power_energy characteristics (on an active mass normalized basis) ever reported for an electrochemical capacitor: At a very high power density of 20 kW kg_1 and 20, 60, and 100 _C, the energy densities are 19, 34, and 40 Wh kg_1, respectively. Moreover the assembled supercapacitor device yields a maximum energy density of 12 Wh kg_1, which is higher than that of commercially available supercapacitors. By taking advantage of the complex multilayered structure of a hemp bast fiber precursor, such exquisite carbons were able to be achieved by simple hydrothermal carbonization combined with activation. This novel precursor-synthesis route presents a great potential for facile large-scale production of high performance carbons for a variety of diverse applications including energy storage

Isolation and screening of basidiomycetes with high peroxidative activity.
De Jong, E., De Vries, F. P., Field, J. A., van der Zwan, R. P., & de Bont, J. A. M.
Mycological Research, 96(12), 1098–1104. (1992).
doi:10.1016/s0953-7562(09)80121-4
Sixty-seven Poly R-478 decolorizing basidiomycetes were isolated with a selective medium (containing hemp (Cannabis sativa) stem wood, guaiacol and benomyl). Several of the new isolates seemed to be promising manganese peroxidase-containing white-rot fungi. Enzyme assays indicated that either glyoxal or veratryl alcohol oxidase were present in the culture fluids of peroxidative strains. In contrast, lignin peroxidase was only detected in Phanerochaete chrysosporium, despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from a-keto-y-methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R. Three of the new isolates had Significantly higher Poly R decolorizing activities compared to P. chrysosporium. The Poly R decolorization rate is a good assay when trying to optimize culture conditions for peroxidase/H20 2 production.

Microbial diversity observed during hemp retting
Alexandra Ribeiro & Philippe Pochart & Arnaud Day & Sarah Mennuni & Pierre Bono & Jean-Luc Baret & Jean-Louis Spadoni & Irène Mangin
Applied Microbiology and Biotechnology 99(10) January 2015
DOI 10.1007/s00253-014-6356-5
Historically used in textile and paper industry, hemp fibres have started to find new applications in composite materials with important economic and ecological advantages. However, their applications are limited since manufacturers have some difficulties to standardise fabrication processes.
This study is a first step before selection and isolation of strains that could later be used to optimise microbial retting efficiency and hence fibre quality. We studied six samples harvested on different ground types, at different dates and with different retting durations on field to obtain an exhaustive representation of the process. After DNA extraction, total bacteria and fungi associated with stems during retting were specifically quantified using real-time PCR. Then, using sequence analysis of randomly cloned 16S and 18S ribosomal RNA (rRNA) genes, a phylogenetic characterisation of the dominant microorganisms was carried out. Quantitatively, we showed that there were 8.1–9.5 log10 16S rRNA gene copies per gram of hemp straw for bacteria and 8.6–9.6 log10 18S rRNA gene copies per gram for fungi. Qualitatively, we noticed a higher bacterial diversity in comparison to fungi.
This work showed that in the different samples, the same species were present but in significantly different proportions according to ground type, harvest dates and retting durations on field. The most frequent bacterial sequences were affiliated to species Escherichia coli, Pantoea agglomerans, Pseudomonas rhizosphaerae, Rhodobacter sp., Pseudomonas fulva, Rhizobium huautlense and Massilia timonae, whereas fungal sequences were principally related to the genera Cladosporium and Cryptococcus.

MICROWAVE-ASSISTED EXTRACTION OF CANNABINOIDS AND ANTIOXIDANTS FROM CANNABIS SATIVA AERIAL PARTS AND PROCESS MODELING
Zorica Drini, Senka Vidovi, Jelena Vladi, Anamarija Petrovi, Biljana Kiprovski, Vladimir Sikora
• Journal of Chemical Technology & Biotechnology November 2019
• DOI: 10.1002/jctb.6273
24th International Symposium on Analytical and Environmental Problems, Szeged, Hungary, October 2018., Book of abstracts, p 118.
Cannabis (Cannabis sativa L.) is one of the most controversial plants. So far, its multiple application in the industry for the production of fabrics, paper and construction materials is known. Today, there is an increasing interest in its application for medical purposes, as opposed to its opiate activity. In addition to well?known cannabinoids, the presence of polyphenolic compounds and their antioxidative and reductive abilities are of great importance. Therefore, the extraction of both constituents, polyphenolic and cannabinoids (?9?tetrahydrocannabinol (THC) and cannabidiol (CBD)), of industrial hemp was carried out using microwave extraction. The effects of different extraction parameters (ethanol concentration (30?70%), extraction time (10?30 min) and solid/liquid ratio (1:5, 1:10, 1:20) on the extraction yield, total phenol, total flavonoid content, antioxidant activity, reductive capacity, CBD content and TCH content, were investigated using a response surface method. For experimental design was chosen Box?Behnken's design. In obtained extracts following ranges of targeted compounds were detected: total phenols from 0.8499 to 2.7060 mg GAE/mL, total flavonoids from 0.4707 to 1.4246 mg CE/mL, ?9?tetrahydrocannabinol from range 0.0339 mg/mL to 0.0637 mg/mL, cannabidiol from 0.2243 mg/mL to 1.8415 mg/mL. Antioxidant activity was from 0.0009 to 0.2079 mL/mL, while the reductive capacity was from 0.0021 to 0.0066 mL/mL. Microwave?assisted extraction proved to be a simple, efficient, fast and low environmental impact method for obtaining polyphenols and cannabinoids from C. sativa L.. Cannabis herb, which presents a by?product of fiber and cannabis product, showed to be a promising source of bioactive compounds.

Performance of lightweight hemp concrete with alkali-activated cenosphere binders exposed to elevated temperature.
Kristombu Baduge, S., Mendis, P., San Nicolas, R., Nguyen, K., & Hajimohammadi, A.
Construction and Building Materials, 224, 158–172.(2019).
doi:10.1016/j.conbuildmat.2019.07.069
This study investigates the performance of three different types of cenosphere as a lightweight supplementary cementitious material for alkali-activated binder for lightweight carbon-negative hempconcrete for non-load bearing applications. Mechanical performance of hemp concrete exposed to three temperatures, room temperature (RT), 300 C and 600 C are studied using mechanical testing, thermogravimetric analysis (TGA) and Fourier-transform Infrared Spectroscopy (FTIR). Hemp concrete with cenosphere binder remained its integrity and showed a lower load carrying capacity even after exposure to elevated temperatures. Compressive strength capacity and elastic modulus of the samples reduced with the increase of temperature and shows the composite material is more suitable for non-load bearing application considering its mechanical behavior and fire requirements. The density, age, and type of cenosphere showed effects on mechanical properties at room temperature and elevated temperatures. The study shows that alkali activated cenosphere binders can potentially be a sustainable alternative to the lime binder.

Processing and extraction methods of medicinal cannabis: a narrative review
Masoumeh Pourseyed Lazarjani, Owen Young, Lidya Kebede and Ali Seyfoddin
J Cannabis Res (2021) 3:32
DOI: 10.1186/s42238-021-00087-9
https://www.researchgate.net/publica...rrative_review
Introduction: As the cannabis industry transitions from a black market to a legal market, product development, and methods of extraction have become a focal point. To date, more than thousands of chemical constituents have been identified from the cannabis plant, all of which possess different chemical properties that require different conditions for preservation during drying and extraction. However, scientific publications that explore these areas for the cannabis plant are currently lacking.
Method: This is a narrative review paper which focuses on critiquing drying and extraction methods of Cannabis sativa L. plant. Relevant keywords such as medicinal cannabis, extraction, solvent, cannabinoids, and terpenes have been searched in PubMed, EMBASE, MEDLINE, Google Scholar, and Cochrane Library (Wiley) databases.
Result: To find relevant papers for this narrative review, 93 papers have been reviewed. Among them, 12 irrelevant papers were discarded. The excluded papers were either about hemp seed oil or hemp fiber and protein. Based on this review, solvent extraction is the most common method for cannabis plants. Although solventless and hydrodynamic extraction are known for their high yield and feasibility, more investigation is needed in these areas. Regarding the drying process, hang-drying is the most convenient method; however, it may be substituted by freeze-drying in the near future.
Discussion: This review analyses various drying and extraction processes to guide the selection of suitable methods for various types of cannabis products and applications. This is done by outlining traditional and modern methods of drying techniques, exploring the importance of solvents for extraction, visiting solventless extraction procedures, and finally comparing conventional and alternative methods of extraction.
Conclusion: In conclusion, based on the current knowledge, using organic solvents is the most convenient method for medicinal cannabis extraction. However, more research is needed for some of the drying and extraction methods. Also, developing a green and sustainable cannabis extraction method should be considered for future studies.

Quality Aspects in Hemp Fibre Production—Influence of Cultivation, Harvesting and Retting
Jörg Müssig, Reent Martens
January 2003 Journal of Industrial Hemp 8(1):11-32
DOI: 10.1300/J237v08n01_03
.For hemp cultivar Fedrina 74 we investigated the influence of different sowing dates in 1997 and 1998. The highest yields occurred with early sowing dates, at the middle of April. The pure fibre content of the plants of the last sowing date increased rapidly, reaching the same amounts as earlier sown plants. We measured the influence of harvesting technique and retting duration on strength and fineness. Comparison of the results of the purely visual assessment of the degree of retting with the Near Infrared Spectroscopy (NIRS) results shows a good correlation. The NIRS method can supply a reproducible method of analysis for retting. In 1998 we examined damage to the fibres due to the wet conditions during retting. In 1997 an increase in retting time led to a decrease of fibre bundle width. In the North German region, with high humidity in autumn, early sowing is advantageous. For both years early sowing in combination with early harvesting proved the optimal way to get a good yield and a secure collection of the stems after retting.

Reinforcing mechanisms of natural fibers in green composites: Role of fibers morphology in a PLA/hemp model system.
Mazzanti, V., Pariante, R., Bonanno, A., Ruiz de Ballesteros, O., Mollica, F., & Filippone, G.
Composites Science and Technology.(2019).
doi:10.1016/j.compscitech.2019.05.015
Green composites, i.e. biodegradable polymers reinforced with natural fibers, are attracting interest as potential substitutes for conventional composites based on petroleum derived plastics. The role of the inherently complex morphology of natural fibers in their reinforcing mechanisms is not completely understood and this is the topic of the present study. The selected system was poly-(lactic acid) filled with 3 and 6 wt% of short hemp fibers. Such a low fiber amount was chosen to help visualization of the fiber – matrix interface at the scanning electron microscope. Remarkable differences in the mechanical behavior were found between composites containing fibers that were alkali treated with respect to untreated fiber filled materials, but unexpectedly it was found that the quality of the fiber – matrix interface was only marginally influenced by the alkaline treatment. Interface properties were thus not exhaustive in explaining the observed differences. On the other hand, the main difference between treated and untreated fibers was the presence, in the untreated fibers population, of a volumetrically relevant sub-population of thick fiber bundles. It was further argued that this fraction did not carry the loads transferred across the fiber-matrix interface uniformly in its cross section, thus determining a reduction in the effective fiber volume fraction. In contrast, the combined action of alkalization and the mechanical stresses during melt mixing resulted in a narrow distribution of isolated elementary fibers, which were more effective in providing higher mechanical properties, in agreement with theoretical predictions. The key message for the scientific community interested in maximizing the mechanical performances of green composites is that, besides trying to improve the quality of the fiber-matrix interface, one should also aim at minimizing the amount of fiber bundles.

Safety assessment of the innovative functional food ingredient from Cannabis sativa L. wastes
Fatmanur Gönce, Elmas Ersöz, Meryem Kara, Gökhan Kars, Saliha Dinç, Serpil Edebali, Manuel Roman, and Meltem D. Kars
The EuroBiotech Journal Volume 4: Issue 3
DOI: 10.2478/ebtj-2020-0015
Xylooligosaccharides (XOS) are the oligomers of ?-1,4 linked xylose monomers and they have health promoting effect by modulating the beneficial microorganisms in intestine. In this study, hydrolysate obtained from hemp (Cannabis sativa) shives was investigated in terms of its in vitro toxicological impacts at cellular and genetic levels and antioxidant activity. The hydrolysate was found to contain 0.264 mg mL-1 of xylose, 0.789 mg mL-1 of xylobiose and 0.171 mg mL-1 of xylotriose in addition to hydroxymethlyfurfural (HMF) and furfural (F) at concentrations of 0.545 mg mL-1 and 0.107 mg mL-1, respectively. The cells, colon epithelial cells (CoN) and colon cancer cells (Caco-2), exposed to 5.00 mg mL-1 or lower XOS hydrolysate showed very similar growth profiles to the untreated control cells. At the genetic level, the oxidative responses of the cell types to XOS hydrolysate were different as measured by NFE2L2 (Nuclear factor, erythroid-derived 2-like 2) gene expression. Regarding antioxidant activity, the amount of XOS hydrolysate (IC50) that cleared 50 % of the 2,2-diphenyl-l-picrylhydrazyl (DPPH) in the medium was calculated as 0.12 mg mL-1. To conclude, based on in vitro studies, XOS hydrolysate obtained from lignocellulosic hemp shives emerges as an innovative, alternative and safe functional food candidate.


SURVEY OF ETHNOMEDICINAL PLANTS USED BY MIGRATORY SHEPHERDS IN SHIMLA DISTRICT OF HIMACHAL PRADESH
Radha and Sunil Puri
Plant Archives Vol. 19, Supplement 1, 2019 pp. 477-482
https://www.plantarchives.org/PDF%20...__477-482_.pdf
In Himachal Pradesh tribal migratory shepherds have rich traditional knowledge about ethnomedicinal plants and its uses, in this respect, an ethnobotanical survey was carried out in district Shimla of Himachal Pradesh from 2017 to 2018. The required information on ethnomedicines used by tribal migratory shepherds was collected through personal field visits, interview method and by using a pretested questionnaire. It was observed that in all 32medicinal plant species were reported viz. Bergenia ciliata, Berberis lyceum, Cannabis sativa, Dioscrea deltoidea, Rhododendron arboretum, Solanum nigrum, Zanthoxylem armatum and Picrorhiza kurroa was recorded that herb species were markedly high (13) followed by shrub (9), tree (8), climber (1), and fern (1). This study shows that shepherds in tribal areas are highly dependent on ethnobotanical medicines, which evolved over
generations of experience, for the healthcare. This survey can help as baseline data on ethnomedicinal plants used in Shimla district and could be helpful in conservation of traditional knowledge as well as medicinal plants.

Sustainable Production of High Quality Hemp Fibres by Enzymatic Modification.
Michael Schlueter, M Meyer, S Risse, C Bluhm
Conference: 27th International Exhibition-Congress on Chemical Engineering, Environmental Protection and Biotechnology (Frankfurt am Main 2003-05-19 – 2003-05-23) — Abstracts.
At: DECHEMA, Frankfurt/Main, Germany Volume: 27
https://www.researchgate.net/publica...c_Modification
Hemp (Cannabis sativa L.) as renewable resource is a good candidate for usage in high-performance composite materials as well as in yarns for textile production. Since the cultivation of hemp was permitted in 1996, a lot of research work has been done to establish cultivation, harvesting and processing techniques. Up to now, the mechanical separation of hemp fibres is realised in industrial scale. The actual fibre qualities are good enough for usage in e.g. needle felts and low-price composites, but the fineness is not sufficient for e.g. yarn production. Hemp fibres for the purpose of textile production are nowadays imported from eastern Europe and China and are mostly produced by chemical separation in NaOH. In view of sustainable production this process is not feasible in Germany due to its high consumption of water, chemicals and energy. Alternatives are for example the steam explosion process or enzymatic separation, where the enzymatic process tends to be more sustainable due to its lower energy consumption. Enzymatic separation of hemp has been carried out successfully in laboratory scale and with satisfying results in first experiments in industrial scale. Additional work is done in developing adapted enzymes and a tailored pilot plant for enzymatic hemp separation. To ensure a sustainable process this pilot plant operates with a water circuit to minimise water consumption and wastewater effluent by a special process design. A bioreactor with integrated membranes allows high enzyme and biomass concentrations for heavy loads of hemp and a short residence time. To establish the new system on market as soon as possible the project team consists of five research institutes, specialised on botany, biotechnology, environmental process engineering and fibre quality and seven partners from industry for hemp treatment, enzyme production, membrane design, system engineering, waste management, yarn production and marketing studies. For more detailed information please visit our booth on the ACHEMA 2003 or the homepage https://www.naturfaserprojekt.de . Acknowledgement This work was supported by the BMBF (project no. 0312640C, D)

The complex interactions between flowering behaviour and fibre quality in hemp
https://sci-hub.tw/10.3389/fpls.2019.00614
Hemp, Cannabis sativa L., is a sustainable multipurpose fibre crop with high nutrient and water use efficiency and with biomass of excellent quality for textile fibres and construction materials. The yield and quality of hemp biomass are largely determined by the genetic background of the hemp cultivar but are also strongly affected by environmental factors, such as temperature and photoperiod. Hemp is a facultative short-day plant, characterized by a strong adaptation to photoperiod and a great influence of environmental factors on important agronomic traits such as “flowering-time” and “sex determination”. This sensitivity ofhemp can cause a considerable degree of heterogeneity, leading to unforeseen yield reductions. Fibre quality for instance is influenced by the developmental stage of hemp at harvest. Also, male and female plants are differing in stature and produce fibres with different properties and quality. Next to these causes, there is evidence for specific genotypic variation in fibre quality among hemp accessions. Before improvedhemp cultivars can be developed, with specific flowering-times and fibre qualities, and adapted to different geographical regions, a better understanding of the molecular mechanisms controlling important phenological traits such as “flowering-time” and “sex determination” in relation to fibre quality in hemp is required. It is well known that genetic factors play a major role in the outcome of both phenological traits but, the major molecularfactors involved in this mechanism are not characterized in hemp. Genome sequences and transcriptome data are available but their analysis mainly focussed on the cannabinoid pathway for medical purposes. Herein, we review the current knowledge of phenotypic- and genetic data vailable for “flowering-time”, “sex determination” and “fibre quality” in short-day and dioecious crops, respectively, and compare them with the situation in hemp. A picture emerges for several controlling key genes, for which natural genetic variation may lead to desired flowering behaviour, including examples of pleiotropic effects on yield quality and on carbon partitioning. Finally we discuss the prospects for using this knowledge for the molecular breeding of this sustainable crop via a candidate gene approach.

The influence of unintended field retting on the physicochemical and mechanical properties of industrial hemp bast fibres.
Placet, V., Day, A., & Beaugrand, J.
Journal of Materials Science, 52(10), 5759–5777.(2017).
doi:10.1007/s10853-017-0811-5
Developing hemp fibre composites for structural applications requires both reconsideration and optimisation of the transformation processes to obtain stable, high-quality fibre reinforcements. In this context, field retting remains an important issue because it is weather dependent and has not been completely mastered by the hemp industries. Retting can be achieved voluntarily to facilitate fibre separation and extraction from the stalks prior to mechanical decortication. However, retting can also be involuntary and result from climatic misfortune and unforeseeable events at the time of harvest. Therefore, this study aimed to quantify the influence of involuntary and non-controlled field retting on the physicochemical and mechanical properties of industrial hemp bast fibres. A wide spectrum of analytical techniques was applied, including colour spectrophotometry; morphological, microscopic (SEM), surface (EDX, roughness), biochemical (HPLC and pXRD) and thermogravimetric (TGA) analyses; dynamic vapour sorption; and tensile characterisation. The results indicate that retting induced a decrease in the average width of fibre elements after mechanical processing and a loss of pectic substances. We also observed a change in colour from yellow to dark grey, an increase in surface roughness and an increase in the decomposition temperature for the third mass loss region. A decrease in tensile properties at the scales of both single and technical fibres was also observed. Since no significant decrease in cellulose content was measured, this decay in mechanical performance was connected with both the significant degradation of hemicelluloses and a decrease in the fraction of crystalline cellulose that was quantified in this work.

Tracking the dynamics of hemp dew retting under controlled environmental conditions.
Bleuze, L., Lashermes, G., Alavoine, G., Recous, S., & Chabbert, B.
Industrial Crops and Products, 123, 55–63.(2018).
doi:10.1016/j.indcrop.2018.06.054
The use of natural fibers such as hemp (Cannabis sativa L.) as substitutes for nonrenewable fibers increases the life cycle performance of composite materials. The management of retting in fields as a natural pretreatment prior to fiber extraction remains challenging due to a lack of knowledge about the relative importance of environmental and biotic factors, which continually interact under field conditions. Here, we studied the dynamics of hemp retting under controlled air temperature and humidity conditions and with simulated rain. We tracked the color and infrared spectral absorbance of the stem surface, the chemical composition and microbial enzyme activities of the bast tissues, and the stem architecture over 42 days at 15 °C. Color changes on the stem surfaces were the first indicators of retting progress, with a significant decrease in the L*, a* and b* values (CIELAB) from day 14 onward. These findings were closely correlated with the surface colonization progress as revealed by scanning electron microscopy, the changes in enzymatic activities and the decohesion of the bast tissues. Additional investigations are needed to study other environmental scenarios to provide an accurate assessment of the retting process over time.

Traditional Fiber Hemp (Cannabis) Production, Processing, Yarn Making, and Weaving Strategies—Functional Constraints and Regional Responses. Part 1
Robert Connell Clarke
Journal of Natural Fibers 7(2):118-153 April 2010
DOI: 10.1080/15440478.2010.482324
Traditional (preindustrial) hemp (Cannabis) production and processing methods once used in Europe are compared with those of Asia. The commonality of climate throughout the hemp-growing regions of Eurasia, in league with the essential characteristics of all bast fibers and the utilization of hemp in particular, impose functional constraints on hemp cultivation and processing, and as a result many production protocols are shared among Eurasian hemp-fiber-growing cultures. Despite the commonalities imposed by environment and biology, fundamentally different methods have evolved within Asian and European hemp processing traditions. Two processing strategies may have arisen because of ancient relationships involving the earliest fibers adopted for weaving by different cultures—the hair of animals (e.g., sheep) being an early fiber source that still characterizes European weaving and bast fibers derived from annual plants (e.g., hemp, nettle, and ramie) being the ancient fibers of choice in East Asia. Here we explore the general commonalities and essential regional differences of traditional Cannabis hemp processing from harvest through fiber extraction and processing to making yarn and weaving cloth. Commonalities in Eurasian hemp processing methods have arisen largely from the essential protocols associated with the processing of hemp and other bast fibers (e.g., extracting the fibers from the stalk, softening and whitening the fibers, and constructing yarn), while differences in processing strategies have arisen as localized regional responses to these common challenges.

Traditional Fiber Hemp (Cannabis) Production, Processing, Yarn Making, and Weaving Strategies—Functional Constraints and Regional Responses. Part 2
Robert Connell Clarke
July 2010 Journal of Natural Fibers 7(3):229-250
DOI: 10.1080/15440478.2010.504043
Traditional (pre-industrial) hemp (Cannabis) production and processing methods once used in Europe are compared with those of Asia. The commonality of climate throughout the hemp-growing regions of Eurasia, in league with the essential characteristics of all bast fibers and the utilization of hemp in particular, imposes functional constraints on hemp cultivation and processing, and as a result many production protocols are shared amongst Eurasian hemp fiber-growing cultures. Despite the commonalities imposed by environment and biology, fundamentally different methods have evolved within Asian and European hemp-processing traditions. Two processing strategies may have arisen because of ancient relationships involving the earliest fibers adopted for weaving by different cultures—the hair of animals (e.g., sheep) being an early fiber source which still characterizes European weaving; and bast fibers derived from annual plants (e.g., hemp, nettle, and ramie) being the ancient fibers of choice in East Asia. Here we explore the general commonalities and essential regional differences of traditional Cannabis hemp processing from harvest through fiber extraction and processing, to making yarn and weaving cloth. Commonalities in Eurasian hemp-processing methods arise largely from the essential protocols associated with the processing of hemp and other bast fibers (e.g., extracting the fibers from the stalk, softening and whitening the fibers and constructing yarn) while differences in processing strategies have arisen as localized regional responses to these common challenges.

Traditionally used common fibre plants in outer siraj area, Himachal Pradesh
Arvind Bhardwaj, Savita Rani, J C Rana
Indian Journal of Natural Products and Resources Vol. 5(2), June 2014 pp. 190-194
https://www.researchgate.net/publica...machal_Pradesh
An ethonobotanical study was conducted to document the traditional use of fibre yielding plants used by the indigenous people of Outer Siraj area of district Kullu of Himachal Pradesh. Personal interviews and group discussions were conducted to gather information on the indigenous knowledge. A checklist of 'categories of plant use' was developed and used to identify, categorize and document fibre plant species in the area. The ethnobotanical fabrics/ articles, viz. Pula, Mandri of this region are rich in traditional practices and people living in the Outr Siraj area of Himachal Pradesh, India use natural fibre plants in various ways for their subsistence. Fifteen plants were identified as most commonly used fibre plants belonging to 10 families of which family Poaceae and Urticaceae are most important. 67.00 % of fibre is obtained from stem and bark. Cannabis sativa L. is used by 92.19 % people followed by Grewia optiva Drumm. (76.56 %). Euphorbia royleana Boiss. is the least used fibre plant (1.56 %). New ethnobotanical uses of Agave cantula Roxb. and Urtica dioica L. are reported for the first time.

Valorizing industrial hemp (Cannabis sativa L.) by-products: Cannabidiol enrichment in the inflorescence essential oil optimizing sample pre-treatment prior to distillation.
Fiorini, Dennis; Molle, Arnaud; Nabissi, Massimo; Santini, Giuseppe; Benelli, Giovanni; Maggi, Filippo
Industrial Crops and Products, 128(), 581–589. (2019).
doi:10.1016/j.indcrop.2018.10.045
By products of industrial hemp (Cannabis sativa L.), including inflorescences, represent an exploitable material to produce niche products for the pharmaceutical, nutraceutical, cosmetic and pesticide industry. One of them is the essential oil, whose composition can be properly modulated on an industrial level by optimizing the extractive conditions and sample pretreatment. This allows to achieve high concentrations of bioactive compounds, such as cannabidiol (CBD) and sesquiterpenes [e.g. (E)-caryophyllene]. In the present work, we evaluated the effects of type of distillation apparatus, status of conservation of the plant material, grinding and sample pretreatment with microwave and heat, on the hemp essential oil chemical profile obtained from the monoecious cultivar Felina 32. Seven marker compounds, including the monoterpenes α-pinene, myrcene and terpinolene, the sesquiterpenes (E)-caryophyllene, α-humulene and caryophyllene oxide, and the cannabinoid CBD were quantified in the different hemp essential oil samples by gas chromatography-flame ionization detection (GC-FID) analysis, whereas the overall chemical profiles were achieved by gas chromatography-mass spectrometry (GC–MS) analysis. Results showed that hydrodistillation (HD) in comparison with steam distillation (SD) gave a higher content of cannabinoids. Drying was fundamental to induce decarboxylation of cannabinoid acids to the relative alcoholic forms, coupled with an increase of the sesquiterpene fraction. The optimization of sample pretreatments pointed out that the exposure of dry inflorescences to microwave heating at 900 W power for 1 min was the best method to increase the abundance of bioactive compounds in the essential oil, with special reference to CBD, (E)-caryophyllene and caryophyllene oxide. Overall, these results give new insights into the exploitation of hemp byproducts in different fields such as pharmaceuticals, nutraceuticals and eco-friendly insecticides.

Valorization of Byproducts of Hemp Multipurpose Crop: Short Non-Aligned Bast Fibers as a Source of Nanocellulose
Sara Dalle Vacche, Vijayaletchumy Karunakaran, Alessia Patrucco, Marina Zoccola, Loreleï Douard, Silvia Ronchetti Marta Gallo,
Aigoul Schreier, Yves Leterrier, Julien Bras, Davide Beneventi
Molecules 2021, 26, 4723.
DOI: 10.3390/molecules26164723
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-04723.pdf
Nanocellulose was extracted from short bast fibers, from hemp (Cannabis sativa L.) plants harvested at seed maturity, non-retted, and mechanically decorticated in a defibering apparatus, giving non-aligned fibers. A chemical pretreatment with NaOH and HCl allowed the removal of most of the non-cellulosic components of the fibers. No bleaching was performed. The chemically pretreated fibers were then refined in a beater and treated with a cellulase enzyme, followed by mechanical defibrillation in an ultrafine friction grinder. The fibers were characterized by microscopy, infrared spectroscopy, thermogravimetric analysis and X-ray diffraction after each step of the process to understand the evolution of their morphology and composition. The obtained nanocellulose suspension was composed of short nanofibrils with widths of 5–12 nm, stacks of nanofibrils with widths of 20–200 nm, and some larger fibers. The crystallinity index was found to increase from 74% for the raw fibers to 80% for the nanocellulose. The nanocellulose retained a yellowish color, indicating the presence of some residual lignin. The properties of the nanopaper prepared with the hemp nanocellulose were similar to those of nanopapers prepared with wood pulp-derived rod-like nanofibrils
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Valorization of CBD‑hemp through distillation to provide essential oil and improved cannabinoids profile
Valtcho D. Zheljazkov, Filippo Maggi
Nature, scientific reports (2021)
DOI: 10.1038/s41598-021-99335-4
https://www.researchgate.net/publica...inoids_profile
Hemp (Cannabis sativa L.) synthesizes and accumulates a number of secondary metabolites such as terpenes and cannabinoids. They are mostly deposited as resin into the glandular trichomes occurring on the leaves and, to a major extent, on the flower bracts. In the last few years, hemp for production of high-value chemicals became a major commodity in the U.S. and across the world. The hypothesis was that hemp biomass valorization can be achieved through distillation and procurement of two high-value products: the essential oil (EO) and cannabinoids. Furthermore, the secondary hypothesis was that the distillation process will decarboxylate cannabinoids hence improving cannabinoid composition of extracted hemp biomass. Therefore, this study elucidated
the effect of steam distillation on changes in the content and compositional profile of cannabinoids in the extracted biomass. Certified organic CBD-hemp strains (chemovars, varieties) Red Bordeaux, Cherry Wine and Umpqua (flowers and some upper leaves) and a T&H strain that included chopped whole-plant biomass, were subjected to steam distillation, and the EO and cannabinoids profile were analyzed by gas chromatography-mass spectrometry (GC–MS) and HPLC, respectively. The distillation of hemp resulted in apparent decarboxylation and conversion of cannabinoids in the distilled biomass. The study demonstrated a simple method for valorization of CBD-hemp through the production of two high-value chemicals, i.e. EO and cannabinoids with improved profile through the conversion of cannabidiolic acid (CBD-A) into cannabidiol (CBD), cannabichromenic acid (CBC-A) into cannabichromene (CBC), cannabidivarinic acid (CBDV-A) into cannabidivarin (CBDV), cannabigerolic acid (CBG-A) into cannabigerol (CBG), and δ-9-tetrahydrocannabinolic acid (THC-A) into δ-9-tetrahydrocannabinol (THC). In addition, the distilled biomass contained CBN while the non-distilled did not. Distillation improved the cannabinoids profile; e.g. the distilled hemp biomass had 3.4 times higher CBD in variety Red Bordeaux, 5.6 times in Cherry Wine, 9 times in variety Umpqua, and 6 times in T&H compared to the original non-distilled samples, respectively. Most of the cannabinoids remained in the distilled biomass and small amounts of CBD were transferred to the EO. The CBD concentration in the EO was as follows: 5.3% in the EO of Umpqua, 0.15% in the EO of Cherry Wine and Red Bordeaux and 0.06% in the EO of T&H. The main 3 EO constituents were similar but in different ratio; myrcene (23.2%), (E)-caryophyllene (16.7%) and selina-3,7(11)-diene (9.6%) in Cherry Wine; (E)-caryophyllene (~ 20%), myrcene (16.6%), selina-3,7(11)-diene (9.6%), α-humulene (8.0%) in Red Bordeaux; (E)-caryophyllene (18.2%) guaiol (7.0%), 10-epi-γ-eudesmol (6.9%) in Umpqua; and (E)-caryophyllene (30.5%), α-humulene (9.1%), and (E)-α-bisabolene (6.5%) in T&H. In addition, distillation reduced total THC in the distilled biomass. Scanning electron microscopy (SEM) analyses revealed that most of the glandular trichomes in the distilled biomass were not disturbed (remained
intact); that suggest a possibility for terpenes evaporation through the epidermal membrane covering the glandular trichomes leaving the cannabinoids in the trichomes. This explained the fact that distillation resulted in terpene extraction while the cannabinoids remained in the distilled material.

Variation of Cannabis with reference to stem quality for paper pulp production
EPM. de Meijer
Industrial Crops and Products 3 (1994) 201-211
doi:10.1016/0926-6690(94)90067-1
Ca. 160 Cannabis accessions were evaluated for stem quality characteristics. Stems from field trials in two years were analysed. The mass fractions of woody core, bark and primary and secondary bark fibres in the stem dry weight, and the dimensions of the wood fibres were considered most relevant for the evaluation of Cannabis germplasm as a raw material for paper pulp.
The accession means for the woody core fraction ranged from 50% in fibre cultivars to 78% in wild populations, drug strains and fibre landraces. Consequently, the bark tissue, being the complementary stem fraction, ranged from 50% down to 22%. The ranges of the accession means were 8 to 27% and 0 to 14% for the fractions of primary and secondary bark fibres, respectively. The total bark fibre fraction ranged from 9 to 34%. Fibre cultivars had strongly increased fractions of primary and secondary bark fibres in comparison to wild populations, drug strains and fibre landraces. The levels of the assessed stem fractions were stable over years. Significant differences in wood fibre length among accessions were only detected in one of the two trials. The accession means in this trial ranged from 433 to 613 pm. For wood fibre width the accession means ranged from 24 to 37 km and from 25 to 41 ,um in the two trials, respectively. The stability of wood fibre dimensions over years was low. Within accessions, the wood fibres of male plants were usually somewhat shorter and wider than those of females. Apart from this difference between sexes there was little variation for wood fibre dimensions among individual plants within accessions. Breeding for improved woody core quality is considered not very promising. The best way for genetic improvement of hemp pulping quality is continuous selection for increased bark fibre content, which implies a reduction of the woody core fraction.
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
I fixed the interviews now the links work.
-SamS

IC Interviews Cannabis People

https://druglibrary.net/olsen/HEMP/IHA/iha01113.html
Interview: Professor Dr. Raphael Mechoulam, (his photo in my greenhouse)
Interview: Professor Dr. Raphael Mechoulam
internationalhempassociation.o rg/jiha/iha01113.html delete the empty space in org and add after http : // www. (with the empty spaces removed)

https://druglibrary.net/olsen/HEMP/IHA/iha01215.html
Interview: Professor Dr. Iván Bócsa, the breeder of Kompolti hemp
Vol. 1 No. 2 December 1994 Journal International Hemp Association
add internationalhempassociation.o rg/jiha/iha01215.html

https://druglibrary.net/olsen/HEMP/IHA/iha02114.html
Interview: Dr. Paul G. Mahlberg American Cannabis researcher
Vol. 2 No. 1 June 1995 Journal International Hemp Association
add org/jiha/iha02114.html

https://druglibrary.net/olsen/HEMP/IHA/iha02213.html
Interview: Prof. Ryszard Kozlowski, Director of the Institute of Natural Fibres (INF) in Poznan, Poland
Vol. 2 No. 2 December 1995 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/iha03112.html
Interview: Dr. Mahmoud A. ElSohly, University of Mississippi,Head of Research Institute of Pharmaceutical Sciences (RIPS), is famous for hosting the NIDA Marijuana Project, which grows the Cannabis it supplies to researchers.
Vol. 3 No. 1 June 1996 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/iha03211.html
Interview: Dr. Janos Berenji Institute of Field and Vegetable Crops in Novi Sad, Yugoslavia (Serbia) and directs the hemp research.
Vol. 3 No. 2 December 1996 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha4109.html
Interview: Dr. Rudolf Brenneisen Professor of Phytochemistry and Pharmacognosy in the Department of Clinical Research at the University of Bern, Switzerland
Vol. 4 No. 1 June 1997 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha4211.html
Interview: Dr. Yukihiro Shoyama is a pharmacognosist at Kyushu University in Japan. His specialty interest, and the subject of several fundamental publications, is the biogenesis of cannabinoids in Cannabis
Vol. 4 No. 2 December 1997 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha5111.html
Interview: Dr. V. G. Virovets, head of the Hemp Breeding Department at the Institute of Bast Crops in Glukhov, the Ukraine.
Vol. 5 No. 1 June 1998 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha5209.html
Interview: Michael Karus, co-founder of the nova-Institute, based in Hürth, Germany
Vol. 5 No. 2 December 1998 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha6108.html
Interview: Mr. Francois Desanlis vice-chairman of the board of directors at La Chanvrière de l'Aube (LCDA) a French hemp cooperative and research institute.
Vol. 6 No. 1 June 1999 Journal International Hemp Association

https://druglibrary.net/olsen/HEMP/IHA/jiha6208.html
Interview: Dr. Ernest Small, Canadian Cannabis researcher
Vol. 6 No. 2 December 1999 Journal International Hemp Association

Roger Pertwee Interview
The Cannabis Scientist
https://thecannabisscientist.com/res...m_medium=email

A passion for plant life
H Y MOHAN RAM
Journal of Biosciences 27(7):651-64 Dec 2002 651–664
DOI: 10.1007/BF02708373
Chemical manipulation of sex expression was investigated by V S Jaiswal and Rina Sett using the dioecious in female Cannabis plants by the application of gibberellins (GAs) and antiethylene agents such as silver nitrate (AgNO3 ), silver thiosulphate anionic complex (STS), aminoethoxyvinyl glycine (AVG) and cobalt chloride (CoCl2 ). Fertile female flowers can be induced in male
plants by ethephon (2-chloroethanephosphonic acid) and NIA 10637 (ethylhydrogen-l-propylphosphonate). Interestingly, stamens could be seen arising even from fruits. Stopping the application of growth regulators caused the plants to revert to their original sex. We hypothesized that in Cannabis , GA and ethylene act as male and female hormones respectively, and that the expression of sex is controlled by a balance between their endogenous levels. Abscisic acid (ABA) is able to overcome the GA induced male flower formation (Mohan Ram and Jaiswal 1973; Mohan Ram and Sett 1985).

Cannabis Breeding: Biotech, Traits, Terpenes & Taste with Mark Lewis Emerald Cup 2018
https://www.youtube.com/watch?fbclid=IwAR06W20_Xmx2M4I KebP3R_hHYuZ8BWEKT3aJ8ZYAWKagU UvyiGfSWenHWfc&v=6GIY9d_cm_s&f eature=youtu.be
This is the second part in the three part cannabis breeding speaker series I recorded for you at The Emerald Cup 2018. The link to part one is below. Mark always packs the house with people there for his unique perspective and insights. Mark's business partner Ryan Lee joined Mark from off-stage during the Q&A which totally didn't work for the camera but you can hear him just fine. The first panel is available here:

Classification of Cannabis Cultivars - Dr. Mark Lewis
https://www.youtube.com/watch?fbclid=IwAR3PG9CI2ZRAY0r CveMAW9dDjapOYics2mClJaVmAea3v FIxU6ERCDVIYZo&v=SWgMrJD4sjI&f eature=youtu.be

Conversation with Raphael Mechoulam
Addiction, 102, 887–893
doi:10.1111/j.1360-0443.2007.01795.x
In this occasional series we record the views and personal experience of people who have specially contributed to the evolution of ideas in the Journal’s field of interest. Entering what was, at the start, a neglected
field, Raphael Mechoulam went on to pursue, over the next 40 years, a highly productive career in cannabis research. This interview charts a sequence of
discoveries and reveals something of the dedication and intellectual daring which has characterized the enterprise throughout.

The Cannabis sativa Versus Cannabis indica Debate: An Interview with Ethan Russo, MD
Daniele Piomelli and Ethan B. Russo
Cannabis and Cannabinoid Research Jan 2016
DOI: 10.1089/can.2015.29003.ebr
Dr. Ethan Russo, MD, is a board-certified neurologist, psychopharmacology researcher, and Medical Director of PHYTECS, a biotechnology company researching and developing innovative approaches targeting the human endocannabinoid system. Previously, from 2003 to 2014, he served as Senior Medical Advisor and study physician to GW Pharmaceuticals for three Phase III clinical trials of Sativex for alleviation of cancer pain unresponsive to optimized opioid treatment and studies of Epidiolex for intractable epilepsy. He has held faculty appointments in Pharmaceutical Sciences at the University of Montana, in Medicine at the University of Washington, and as visiting Professor, Chinese Academy of Sciences. He is a past President of the International Cannabinoid Research
Society and former Chairman of the International Association for Cannabinoid Medicines. He serves on the Scientific Advisory Board for the American Botanical Council. He is the author of numerous books, book chapters, and articles on Cannabis, ethnobotany, and herbal medicine. His research interests have included correlations of historical uses of Cannabis with modern pharmacological mechanisms, phytopharmaceutical treatment of migraine and chronic pain, and phytocannabinoid/terpenoid/serotonergic/vanilloid interactions.

Tatyana Yakovlevna Serebryakova: a forgotten hemp expert
J. M. McPartland PROCEEDINGS ON APPLIED BOTANY GENETICS AND BREEDING 182(4):166-171 December 2021 DOI: 10.30901/2227-8834-2021-4-166-171 https://www.researchgate.net/publica...a_Yakovlevna_S erebryakova_a_forgotten_hemp_e xpert https://elpub.vir.nw.ru/jour/article...0?locale=en_US
Tatyana Yakovlevna Serebryakova (1893-?) conducted taxonomic research on Cannabis sativa L. for 20 years. She published important papers and a book on the subject. Next to every great man stands a great woman, and Serebryakova was Vavilov’s hemp expert. Yet her biography has never been written, and many details are lost. We have reconstructed her history based on historical archives, her publications, and her-barium specimen labels. Highlights of her scientific contributions are presented.

Using Genetics to Differentiate Cannabis Cultivars: A Conversation with Philippe Henry of Egret Bioscience
https://terpenesandtesting.com/using...et-bioscience/

Conversation with Raphael Mechoulam
Addiction, 102, 887–893
doi:10.1111/j.1360-0443.2007.01795.x
In this occasional series we record the views and personal experience of people who have specially contributed to the evolution of ideas in the Journal’s field of interest. Entering what was, at the start, a neglected
field, Raphael Mechoulam went on to pursue, over the next 40 years, a highly productive career in cannabis research. This interview charts a sequence of
discoveries and reveals something of the dedication and intellectual daring which has characterized the enterprise throughout.

Shaping Fire Ep. 64 - Feminized Seeds and Female-Only Breeding with guest Ryan Lee

Feminized seeds have a reputation based on devotion, hyperbole, and myth. Cultivators have strong feelings about these particular seeds, no matter if they love or hate them. On this episode of Shaping Fire, host Shango Los talks with biologist and cannabis geneticist Ryan Lee about the science behind feminized seeds, how to make them, and why they used to have a bad reputation. The third set is dedicated to understanding "female-only" breeding.

Shaping Fire Ep. 74 - Tissue Culture and Micropropagation with guest Ryan Lee

The phrase "tissue culture" can get cannabis folks excited and yet not many have a good idea of what it is or how the cannabis plant can benefit from its opportunities. In this episode of Shaping Fire, host Shango Los welcomes back cannabis geneticist Ryan Lee to discuss what tissue culture is, the opportunities found in micropropagation, how to remove viruses and other impediments from important cannabis cultivars, and the wide range of other scientific possibilities that tissue culture holds

IC In Vitro


A bio-inspired cell-free system for cannabinoid production from inexpensive inputs

Meaghan A. Valliere, Tyler P. Korman, Mark A. Arbing & James U. Bowie
Nature Chemical Biology (2020)
DOI: 10.1038/s41589-020-0631-9
Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5?g?l?1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

A boosted crop
ELIE DOLGIN
NATURE 29 VOL 572 AUGUST 2019
https://media.nature.com/original/ma...19-02525-4.pdf
annabis is the only plant known to produce tetrahydrocannabinol (THC), but it remains an imperfect vessel for producing the chemical on an industrial scale. The psychoactive substance is normally found only in small outgrowths from the plant known as trichomes, which means that its stalk, stems and leaves are wasted biomass. Genetic engineering could provide more efficient alternatives. Some researchers and biotechnology companies are aspiring to replace cannabis plants with microorganisms that have been genetically enhanced to spit out THC, the non–psychoactive compound cannabidiol (CBD) and myriad other cannabinoids of pharmaceutical interest. Others are aiming to modify chemical synthesis in the cannabis plant by genetically altering its cells to make the desired molecules from shoot to tip, thereby boosting yield.

A cell-free platform for the prenylation of natural products and application to cannabinoid production
Meaghan A. Valliere, Tyler P. Korman, Nicholas B. Woodall, Gregory A. Khitrov, Robert E. Taylor, David Baker & James U. Bowie
NATURE COMMUNICATIONS | (2019) 10:565 |
doi:10.1038/s41467-019-08448-y
Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and
stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.

Anther culture and callus induction in Cannabis sativa L.
Luwanska, K. Wielgus
Institute of Natural Fibres and Medicinal Plants, Poznan, Poland
doi:10.1016/j.nbt.2009.06.960
Anther cultures and regeneration of haploids plants are important breeding methods in the field of research on the improvement of applied traits. Presented study provides a basis for future work on androgenesis and obtaining haploid Cannabis sativa plants. The aim of this study was to initiate sterile anther cultures of hemp and to determine optimal condition for induction of callus from the anthers grown in vitro. Plant material included anthers collected from seven hemp cultivars (Bialobrzeskie, Finola, Jermakowskie, Juso11, Silistrenskie, W1, Zenit). The anthers were placed on PYL medium, a modified Murashige Skoog medium, supplemented with plant growth regulators: BAP (1 mg/l) and NAA (0.5 mg/l). After cultivation in dark for two weeks cultures were transferred to a growing chamber (temperature 23?C, photoperiod 16/8). The cultivar Jermakowskie showed the maximum (42%) callus induction rate. In Finola, Juso11 and Silistenskie callus production was not observed.

Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis
Andrea Kodym, Christian J. Leeb
Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 138:399–402
DOI: 10.1007/s11240-019-01635-1
The aim of this protocol was to develop an alternative in vitro propagation system for Cannabis sativa L. by mimicking nursery-based vegetative propagation. Photoautotrophic micropropagation (PAM) was achieved on rockwool blocks as substrate combined with commercially available fertilizer suitable for cannabis cultivation. Stock plants were initiated after sterilisation in forced-ventilated glass jars which then provided a continuous supply of shoot tip and nodal cuttings. A 97.5% rooting rate of in vitro shoot tip cuttings and successful acclimatisation were achieved within 3 weeks in glass vessels with passive ventilation.

Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast.
Thomas, F., Schmidt, C., & Kayser, O.
Applied Microbiology and Biotechnology. (2020).
doi:10.1007/s00253-020-10798-3
Heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa.

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR
Kostas Ioannidis, Evangelos Dadiotis , Vangelis Mitsis, Eleni Melliou and Prokopios Magiatis
Molecules 2020, 25, 5928;
DOI: 10.3390/molecules25245928
High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or ?-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ? 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

Biotransformation of Tetrahydrocannabinol
Muhammad T. Akhtar. Khozirah Shaari. Robert Verpoorte
Phytochem Rev October 2016, Volume 15, Issue 5, pp 921–934
DOI 10.1007/s11101-015-9438-9
Cannabinoids are terpenophenolic compounds consisting of an aromatic polyketide and derived from the geranyl diphosphate C10 terpenoid unit. They are the active constituents in Cannabis sativa and have been utilized in a number of cannabisbased medicines. Biotransformation of cannabinoids
is an important field of xenobiochemistry and toxicology and the study of the metabolism of these compounds can lead to the discovery of new compounds,
unknown metabolites with unique structures and new therapeutic entities. Different fungi, bacteria, plants and animal cells have been used for the regioand stereoselective transformation of cannabinoids. All of the above mentioned organisms have distinct enzymes which catalyze the conversion of a specific cannabinoid at different positions and thus provide a variety of derivatives. All organisms are able to transform the alkyl side chain where as mammalians are unique in the formation of the carboxy derivatives. This review article assesses the current knowledge on the biotransformation of tetrahydrocannabinol and with particular focus on D9 -THC.

Cannabidiol as the Substrate in Acid-Catalyzed Intramolecular Cyclization.
Marzullo, P., Foschi, F., Coppini, D. A., Fanchini, F., Magnani, L., Rusconi, S., … Passarella, D.
Journal of Natural Products.(2020).
doi:10.1021/acs.jnatprod.0c00436
The chemical reactivity of cannabidiol is based on its ability to undergo intramolecular cyclization driven by the addition of a phenolic group to one of its two double bonds. The main products of this cyclization are ?9 -THC (trans-?-9-tetrahydrocannabinol) and ?8 -THC (trans-?-8- tetrahydrocannabinol). These two cannabinoids are isomers, and the first one is a frequently investigated psychoactive compound and pharmaceutical agent. The isomers ?8 -iso-THC (trans-?-8-iso-tetrahydrocannabinol) and ?4(8)-iso-THC (trans-?-4,8-iso-tetrahydrocannabinol) have been identified as additional products of intramolecular cyclization. The use of Lewis and protic acids in different solvents has been studied to investigate the possible modulation of the reactivity of CBD (cannabidiol). The complete NMR spectroscopic characterizations of the four isomers are reported. High-performance liquid chromatography analysis and 1 H NMR spectra of the reaction mixture were used to assess the percentage ratio of the compounds formed.

Cannabinoids production in Cannabis sativa L.: An in vitro approach
Sayed Hussein Farag Hussein
DOI:10.17877/DE290R-16424
Cannabis sativa L. (Cannabaceae) is the oldest known medicinal plant. For millennia, the plant has also been used for fibre and oil production.The most prominent feature of C. sativa is the psychoactive effect ascribed to its secondary metabolites, cannabinoids (mainly to tetrahydrocannabinol, THC). However, many other pharmacological properties of the aforementioned specialized compounds have been described. Currently, the demand for THC for various medical applications is substantial, while cultivation and breeding of Cannabis in most countries is strictly regulated and limited to serving research purposes and meeting therapeutic needs. Therefore, the hereby proposed and discussed production of THC using in vitro cultures could be a viable alternative. In the work presented here, in vitro organogenesis from callus cultures (undifferentiated plant cell masses grown on solid media) was successfully established, ultimately resulting in regeneration of the complete C. sativa plant. Further, production of THC as well as other important cannabinoids was achieved in cell suspension, hairy root and trichome cultures of Cannabis. The optimal combination of phytohormones, as applied to the B5 growth medium, fostering the development of meristemoids from callus cultures was: 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and adenine hemisulfate salt (AS) in respective concentrations of 0.5, 5 and 40 mg/l. Concurrently, the most favourable augmentation protocols of the B5 medium for the induction and differentiation of shootlets (small plants with leaves but without roots) were: 0.5 mg/l of gibberelic acid (GA3) or 0.25 mg/l of thidiazouron (TDZ) and 3 mg/l of GA3 (8.5 ± 1.73 and 7.25 ±1.03 shootlets/callus, respectively). The subsequent root formation of shootlets was most prominent after supplementation with 1.5 mg/l of indole-3-acetic acid (IAA). In vitro acclimatized plants growing in Erlenmeyer flasks formed tetrahydrocannabinolic acid (THCA), cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA), retrieved at respective concentrations of about 0.33, 0.45 and 157.1 mg/g fresh weight. In contrast, ex vitro acclimatized plants (grown hydroponically for 8 weeks) synthesized THCA, THC, CBGA, cannabigerol (CBG) and CBDA at corresponding concentrations of 1.54, 28.30, 6.0, 0.125 and 1121.4 mg/g fresh weight. The obtained results confirmed the generation of pharmacologically important cannabinoids; however, the biosynthetic abilities of the investigated cell and hairy root cultures did not provide sufficient levels of the valuable metabolites to warrant scaling-up of the proposed in vitro production platform.

Cannabis control in Europe

Danilo Ballotta, Henri Bergeron and Brendan Hughes
2008, EMCDDA MONOGRAPHS
https://www.academia.edu/3453338/Cannabis_control_in_Europe
Smoked, eaten, imbibed — or just talked about — it seems the world has a strong appetite for cannabis. An estimated one in five European adults have tried it. Thirteen million Europeans have consumed it in the past month. Globally, nearly 50 000 tonnes of cannabis herb or resin is produced for consumption each year. Little wonder, then, that cannabis has become a controversial cultural and commercial phenomenon. Today, cannabis has a unique ability to divide opinion among policymakers, scientists, law enforcers, drugs professionals and consumers.
This EMCDDA cannabis monograph addresses one basic question. How can I find quality information on cannabis, amid all the bias and opinion? During the editing of this monograph it soon became clear that the EMCDDA was entering an area crowded with general guides, even competing cannabis monographs. This is where the idea of a cannabis ‘reader’ emerged. Our audience — researchers, parliamentarians, drugs professionals, students, European citizens — is currently faced with an overload of professional publications. Added to this is the daily flood of information on the Internet, often crusading in nature, and sometimes misleading. This threatens to obscure the genuine progress made in cannabis research during the past two decades.
The EMCDDA cannabis reader underlines the point that cannabis is not just a static, unchanging plant, but a dynamic product that is subject to gradual evolution in potency, prevalence, cultivation, legislative and public health concerns. In this monograph, leading experts provide short, sharp insights on a range of cannabis topics while offering advice on further reading for each topic. Brief editorial notes provide concise introductions to each topic, occasionally drawing attention to political sensitivities and the need for a ‘critical eye’. So this cannabis reader has a value, both as a shortcut to researchers entering the area and a synthesis for experts.
You will find a wide range of views expressed in the chapters in this monograph, not all of them in agreement. The arguments, tone and conclusion of each chapter is the responsibility of the author alone, and is not necessarily endorsed or supported by the EMCDDA. This reflects the wider discourse on cannabis where different positions and perspectives often lead to different conclusions being drawn from the same evidence. We believe each chapter represents a useful contribution to the overall debate, even if their individual perspectives differ. .

Cannabis 25 , biotransformation of cannabidiol and cannabidiolic acid by Pinellia ternata tissue segments
Hiroyuki Tanaka, Ryuji Takahashi, Satoshi Morimoto, and Yukihiro Shoyama
Plant Cell Reports (1996) 15:819- 823
doi: 10.1007/BF00233147.
Two marihuana constituents, cannahidiol (1) and eannabidiolic acid (4) were each converted mainly to two metabolites using tissue segments of Pinellia ternata tuber. The structures of the metabolites formed from 1 were determined to be 1-O- [3-D-glucopyranoside and 1-O- 13-D-diglueopyranoside by 1H umr, 33C nmr and fabms. Those from 4 were determined as 4-O- [3-D-glucopyranoside and 10'- hydroxyl 4-0- [3-D-glucopyranoside. In time course experiments, 1 was absorbed rapidly by the tissues and glueosylated. Hydroxylation subsequent to the glucosylation occurred at the pentyl group in 4.

Cannabis without the plant - history in the makin

https://bioharvest.com

Cell Suspensions of Cannabis sativa (var. Futura): Effect of Elicitation on Metabolite Content and Antioxidant Activity
Damiano Gabotti, Franca Locatelli, Erica Cusano, Elena Baldoni, Annamaria Genga,Laura Pucci, Roberto Consonni and Monica Mattana
Molecules 2019, 24, 4056;
doi: 10.3390/molecules24224056
https://www.researchgate.net/publica...idant_Activity
Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp ( C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts
under the effect of specific elicitors

Coming soon to a lab near you? Genetically modified cannabis
Narure 559, 162 (2018)
doi: 10.1038/d41586-018-05659-z
Legal hurdles to exploring marijuana’s medicinal properties might soon fall in the wake of the US Food and Drug Administration’s (FDA) first approval of a cannabis-derived drug. On 25 June, the FDA announced its approval of Epidiolex — a treatment for epileptic seizures that is based on a cannabis compound called cannabidiol (CBD). The US Drug Enforcement Administration (DEA) has until 24 September to re-classify Epidiolex so that it’s legal for doctors across
the country to prescribe it. Many researchers hope that the agency will re-classify CBD itself, instead of just Epidiolex, so that they can more
easily study this non-psychedelic component of marijuana.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast
Xiaozhou Luo, Michael A. Reiter, Leo d’Espaux, Jeff Wong, Charles M. Denby, Anna Lechner, Yunfeng Zhang, Adrian T. Grzybowski, Simon Harth, Weiyin Lin, Hyunsu Lee, Changhua Yu, John Shin, Kai Deng, Veronica T. Benites, George Wang, Edward E. K. Baidoo, Yan Chen, Ishaan Dev, Christopher J. Petzold & Jay D. Keasling1
Nature Letter
doi: 10.1038/s41586-019-0978-9
Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, ?9-tetrahydrocannabinolic acid, cannabidiolic acid, 9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway. We also introduced the Cannabis genes that encodethe enzymes involved in the biosynthesis of olivetolic acid6,
as well as the gene for a previously undiscovered enzyme with
geranylpyrophosphate:eek:livetola te geranyltransferase activity and the genes for corresponding cannabinoid synthases. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural
and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification
Esther Uchendu, Hemant Lata, Suman Chandra, Ikhlas A. Khan, Mahmoud A. ElSohly
Med Cannabis Cannabinoids
Accepted: January 8, 2019
DOI: 10.1159/000496869
Cannabis sativa L. (marijuana or hemp) is recognized worldwide for its psychoactive properties as well as for fiber production. This study focused on the evaluation of 3 droplet vitrification protocols for long-term conservation of shoot tips in liquid nitrogen (LN). Shoot tips (?0.5 mm) were excised from 3- to 4-week-old in vitro-grown shoots of 3 cultivars (MX, VI-20, and B-5: high tetrahydrocannabinol [THC], high cannabidiol [CBD], and intermediate THC?CBD, respectively) and pretreated on 5% dimethyl sulfoxide agar plates for 48 h. The shoot tips were then vitrified in LN using 3 separate cryoprotectant (plant vitrification solutions [PVS] #2, #3, and #4) droplets on an aluminum cryoplate. There was no significant difference between the regrowth of cryopreserved shoot tips exposed to PVS2 for 15 and 20 min, but regrowth of all 3 cultivars significantly declined after 20 min of exposure. Exposure duration of 15 min was adapted for subsequent experiments. Regrowth of cryopreserved MX was significantly higher with PVS2 (63%) than with PVS3 and PVS4 (?5%). Regrowth of cryopreserved VI-20 was highest with PVS2 (57%) and significantly higher than with PVS3 and PVS4 (?25%). The regrowth of cryopreserved shoot tips of B-5 was significantly different between all 3 protocols with PVS2 > PVS4 > PVS3. Both PVS2 and PVS4 produced regrowth above 55%, while regrowth with PVS3 was significantly lower (31%). These results indicate that 15–20 min of exposure to PVS2 are most suitable for cryopreservation of these varieties.
This is the first report on protocol development for the cryopreservation of organized tissues of C. sativa L. for germplasm conservation

Crystallization of ?1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa
Yoshinari Shoyama, Ayako Takeuchi, Futoshi Taura, Taro Tamada, Motoyasu Adachi, Ryota Kuroki, Yukihiro Shoyamaa and Satoshi Morimotoa
Acta Cryst. (2005). F61, 799–801
DOI: 10.1107/S1744309105023365
_1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase
that catalyzes the biosynthesis of the psychoactive compound THCA in
Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A ° resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A ° . The calculated Matthews coefficient was approximately 4.1 or 2.0 A ° 3 Da_1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

Current Perspectives on Biotechnological Cannabinoid Production in Plants.
Schachtsiek, J., Warzecha, H., Kayser, O., & Stehle, F.
Planta Medica, 84(04), 214–220. (2018).
doi:10.1055/s-0043-125087
The plant Cannabis sativa contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of Cannabis for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other Cannabis-derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic Cannabis plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on Cannabis cell culture systems, in vitro propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated.

Delta9-Tetrahydrocannabinolic acid synthase: the application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis
Kerstin Lange, Andreas Schmid, Mattijs K. Julsing
J Biotechnol. 2016 Sep 10;233:42-8.
doi:10.1016/j.jbiotec.2016.06.022
?(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize ?(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of ?(9)-tetrahydrocannabinol (?(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids.

Designing microorganisms for heterologous biosynthesis of cannabinoids.
Carvalho, Â., Hansen, E. H., Kayser, O., Carlsen, S., & Stehle, F.
FEMS Yeast Research, 17(4).(2017).
doi:10.1093/femsyr/fox037
During the last decade the use of medical Cannabis has expanded globally and legislation is getting more liberal in many countries, facilitating the research on cannabinoids. The unique interaction of cannabinoids with the human endocannabinoid system make these compounds an interesting target to be studied as a therapeutic agent for the treatment of several medical conditions. However, currently there are important limitations in the study, production and use of cannabinoids as pharmaceutical drugs. Besides the main constituent tetrahydrocannabinolic acid, the structurally related compound cannabidiol is of high interest as drug candidate. From the more than 100 known cannabinoids most of them are mostly unknown with regard to their pharmacological profile and in very low amounts extractable. Today, cannabinoids are isolated from the strictly regulated Cannabis plant and the supply of compounds with sufficient quality is a major problem. Biotechnological production could be an attractive alternative mode of production. Herein, we explore the potential use of synthetic biology as an alternative strategy for synthesis of cannabinoids 2 in heterologous hosts. We summarize the current knowledge surrounding cannabinoids biosynthesis and present a comprehensive description of the key steps of the genuine and artificial pathway, systems biotechnology needs and platform optimization.

Development of a Direct in vitro Plant Regeneration Protocol From Cannabis sativa L. Seedling Explants: Developmental Morphology of Shoot Regeneration and Ploidy Level of Regenerated Plants
Alberto Galán-Ávila, Edgar García-Fortea, Jaime Prohens and
Francisco Javier Herraiz
Front. Plant Sci., 27 May 2020 |
DOI: 10.3389/fpls.2020.00645
https://internal-journal.frontiersin...020.0064 5/full
In vitro shoot regeneration can efficiently contribute to the improvement of recalcitrant Cannabis sativa L. We aimed at developing a highly efficient protocol for in vitro direct regeneration of C. sativa plants from different explants (cotyledon, hypocotyl, and true leaf) from seedlings of monoecious C. sativa short-day varieties Ferimon, Felina32, Fedora17, and USO31, together with dioecious neutral-day variety Finola. Ten regeneration media, including already published protocols, and self-designed combinations of plant growth regulators were tested. The developmental morphology since germination of seeds to the development of rooted plantlets was followed. Additionally, the ploidy level of explants and in vitro regenerants was analyzed. We concluded that hypocotyl is the best explant for in vitro direct regeneration of C. sativa plants with 49.45% of responding explants, while cotyledon and true leaf had a poor response with, respectively, 4.70 and 0.42% of explants developing plantlets. In terms of shoot regeneration, we found significant differences among the culture media evaluated and the varieties studied. Overall, the best regeneration media were ZEARIB 2.0 (mg/L) and ZEARIB 1.0 (mg/L) + NAA 0.02 (mg/L) with 66.67% of responding hypocotyls. Amazingly, hypocotyls cultured in medium without plant growth regulators showed an excellent response (61.54% of responding hypocotyls) and spontaneous rooting of regenerants (17.94%).In vitro regenerated plants were acclimatized just 6 weeks after culture initiation. The developmental morphology study suggests that regenerated shoots originate from pericycle cells adjacent to xylem poles. Polysomaty was detected in hypocotyls and cotyledons of all varieties studied, and diploid (>80%) and mixoploid (with diploid and tetraploid cells) plants were regenerated. Our protocol allows a high shoot organogenesis efficiency in different C. sativa varieties. The fact that a significant percentage of plants are mixoploid may provide an alternative way to develop polyploids in C. sativa. Our results show that direct in vitro regeneration may make a significant contribution to the development of improved C. sativa materials for medical applications.

Does recreational cannabis legalization change cannabis use patterns? Evidence from secondary school students in Uruguay

Ariadne Rivera-Aguirre,Alvaro Castillo-Carniglia,Hannah S. Laqueur,Kara E. Rudolph,Silva S. Martins,Jessica Ramírez,Rosario Queirolo,Magdalena Cerdá
First published: 02 May 2022
https://doi.org/10.1111/add.15913
https://onlinelibrary.wiley.com/doi/pdf/10.1111/add.15913

Background and Aims​

In 2013, Uruguay became the first country to legalize and regulate the production and distribution of cannabis for recreational use. We measured whether Uruguay’s non-commercial model of recreational cannabis legalization was associated with changes in the prevalence of risky and frequent cannabis use among secondary school students.

Design​

We used data from repeated cross-sectional surveys of secondary students in Uruguay and Chile (2007-2018). Using a difference-in-difference approach, we evaluated changes in the prevalence of past-year, past-month, any risky and frequent cannabis use following enactment (2014) and implementation (2016) of cannabis legalization among the full sample of secondary students and among students who reported past-year/month use. We examined changes separately for students aged 12-17, and students for whom cannabis became legally accessible, ages 18-21.

Setting​

Uruguay and Chile (2007-2018).

Participants​

Secondary school students in 8th, 10th, and 12th grade (n=204,730).

Measurements​

Past-year and past-month cannabis use; any risky cannabis use measured with the Cannabis Abuse Screening Test (CAST); and frequent cannabis use (10+ days in the past-month).

Findings​

We found a decrease in past-year and past-month use following enactment or implementation. Among students ages 18-21, post-enactment, we observed a transitory increase in 2014 that decreased thereafter for: any risky use among those who reported past-year use (prevalence difference [PD]=13.5%; 95% confidence interval [CI]: 2.0, 24.9), frequent use in the full sample (PD=4.5%; 95%CI: 1.0, 8.1), and frequent use among those who reported past-month use (PD=16.8%; 95%CI: 1.9, 31.8).

Conclusion​

The legalization of recreational cannabis in Uruguay was not associated with overall increases in either past-year/past-month cannabis use or with multi-year changes in any risky and frequent cannabis use among young people.
FIND PDF .

Effect of different cryoprotectants and transfer temperatures on the survival rate of hemp (Cannabis sativa L.) cell suspension in deep freezing.
Jekkel Z, Heszky LE, Ali AH
Department of Genetics and Plant Breeding, University of Agricultural Sciences, Gödölló, Hungary.
Find DOI or Link
Acta Biologica Hungarica [1989, 40(1-2):127-136]
Adequate cell dehydration is the precipitating element in the successful cryopreservation of plant cells and organs. This could be achieved by using different cooling rates, transfer temperatures and cryoprotectants. Experiments were performed to determine these critical points in the freeze preservation procedure of Cannabis sativa (L.) suspension cultures. The explants were frozen at a cooling rate of 2 degrees C/min, while the transfer temperatures were -10 degrees C, -20 degrees C, -30 degrees C, -40 degrees C and -50 degrees C. The applied cryoprotectants were the DMSO, glycerol, proline and PEG in different concentration. The highest viability (58%) was obtained by using 10% DMSO and at -10 degrees C transfer temperature. The optimum transfer temperature varied remarkably by different cryoprotectant concentrations indicating the importance of their interactions

Elicitation studies in cell suspension cultures of Cannabis sativa L.
Flores-Sanchez, I. J., Pe?, J., Fei, J., Choi, Y. H., Dušek, J., & Verpoorte, R.
Journal of Biotechnology, 143(2), 157–168.(2009).
doi:10.1016/j.jbiotec.2009.05.006
Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by 1H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathwa

Engineering Monoterpene Production in Yeast Using a Synthetic Dominant Negative Geranyl Diphosphate Synthase
Codruta Ignea, Marianna Pontini, Massimo E. Maffei, Antonios M. Makris, and Sotirios C. Kampranis
May 2014 ACS Synthetic Biology 3:298-306
DOI: 10.1021/sb400115e
Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.

Engineering yeasts as platform organisms for cannabinoid biosynthesis.
Zirpel, B., Degenhardt, F., Martin, C., Kayser, O., & Stehle, F.
Journal of Biotechnology, 259, 204–212 (2017).
doi:10.1016/j.jbiotec.2017.07.008
? 9 -tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa L. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme’s coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and 3 geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.

Estimation of Cannabis sativa L. Tissue Culture Conditions Essential for Callus Induction and Plant Regeneration
K. Wielgus, A. Luwanska, W. Lassocinski & Z. Kaczmarek
Journal of Natural Fibers, 5:3, 199-207
Doi: 10.1080/15440470801976045
The effects of different combinations of plant growth regulators on callus induction and plant regeneration were observed in three Polish monoecious hemp cultivars (Bialobrzeskie, Beniko, Silesia).
Callus was induced from different explant sources (fragment of cotyledons, stems, roots) on Daria ind+ medium. There was no considerable difference noticed in callus induction between tested cultivars and explants. However, callus obtained from different genotypes had different ability for organogenesis and plant regeneration. Interaction between tested explant and cultivar has significant effect on the efficiency of plant regeneration: the highest regeneration was observed for cotyledon explant (Beniko), the lowest for stem explants (Silesia).

Genetic Transformation of Cannabis sativa Linn: A Multi Purpose Fibre Crop
Lucy MacKinnon (Thesis)
University of Dundee March 2003
https://ethos.bl.uk/OrderDetails.do?...l.ethos.680 201 (Requires free registration on EThOS to download)
Cannabis sativa L. (Hemp) has been cultivated since 2700 BC and has a wide range of applications from cosmetics to paper. It was prohibited in Britain in the early 1900's due to misuse. However it has made a comeback in the past decade due to the breeding of low THC (Tetrahydrocannabinolic acid) cultivars and the demand for novel non-food crops. This project aimed to develop efficient tissue culture and transformation systems for hemp with an objective to incorporate industrially or environmentally relevant genes. It was found that Cannabis does not respond well to tissue or explant culture, thus a non de-differentiation system utilising the shoot apex was investigated. The addition of the antibiotic cefotaxime combined with a reduced light treatment allowed the efficient regeneration of shoot tip cultures. In addition a method of acclimation allowed tissue cultures to be transferred to soil where they were grown to full maturity. This method allowed Agrobacterium-mediated transformation of Cannabis via the shoot apex to be undertaken where it was found that transformation efficiencies of up to 17% (by PCR) were achieved. Transformation was confirmed by Southern analysis (66% of those plants tested showed integration of the BAR gene). Genes encoding polygalacturonase-inhibiting proteins (PGIPs), which convey resistance to Botrytis cinerea were introduced to Cannabis via the above method. Innoculation of these transformants with Botrytis cinerea 0510 showed that plants transformed with the raspberry fruit PGIP gene incurred a mean reduction in lesion formation of 39% compared to plants transformed with an empty vector control. Western analysis confirmed the use of PGIPs for protection against Botrytis infection. This is the first report of successful transformation in Cannabis.

Hemp (Cannabis sativa L.)
Feeney, M., & Punja, Z. K.
Agrobacterium Protocols, 319–329. (2014).
doi:10.1007/978-1-4939-1658-0_25
Hemp ( Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confi rmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23 % ± 0.14 was obtained for all transformation experiments, with a range of 15.1–55.3 %.

HIGH LEVEL IN VIVO BIOSYNTHESIS AND ISOLATION OF WATER – SOLUBLE CANNABINOIDS IN PLANT SYSTEMS
Pub . No . : US 2019 / 0085347 A1
United States Patent Application
Applicant : Trait Biosciences , Inc .
Inventors : Richard T . Sayre, Elton Carvalho Gonçalves , Tawanda Zidenga ,
https://patents.google.com/patent/WO2018176055A3/en
The inventive technology relates to systems and methods for enhanced in vivo production , accumulation and modification of cannabinoids . In one embodiment , the invention may include systems and methods for enhanced in vivo biosyn thesis of chemically - modified water - soluble cannabinoids in a whole plant , or a cell suspension culture system .

INFLUENCE OF CULTIVAR, EXPLANT SOURCE AND PLANT GROWTH REGULATOR ON CALLUS INDUCTION AND PLANT REGENERATION OF CANNABIS SATIVA L.
AURELIA S´ LUSARKIEWICZ-JARZINA, ALEKSANDRA PONITKA, AND ZYGMUNT KACZMAREK
ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 145–151, 2005
https://citeseerx.ist.psu.edu/viewdo...=rep1&type=pdf
The effects of different combinations of plant growth regulators (PGRs) on callus induction and plant regeneration were investigated in five cultivars of Cannabis sativa L. Callus was induced from different explant sources (young leaves, petioles, internodes, axillary buds) on MS basal medium with various concentrations of PGRs (2,4-D,DICAMBA, KIN, NAA). The highest frequency of callus induction (avg. 82.7% of eight medium combinations) was exhibited by petiole explants of cv. Fibrimon-24. Plant regeneration was obtained from all studied cultivars. The highest number of plants was regenerated from callus tissue of petiole explants on MS medium containing DICAMBA. A total of 46 plants (1.35% of callus) were regenerated: 16 (0.47%) from cv. Silesia petioles, 7 (0.20%) from cv. Novosadska petioles, 6 (0.18%) from cv. Fedrina-74 petioles, 12 (0.35%) from cv. Fibrimon-24 axillary buds, and 5 (0.15%) from cv. Juso 15 internodes. Significant improvement of hemp plant regeneration in vitro was achieved

Investigations into the Hypothesis of Transgenic Cannabis
Fidelia Cascini,
J Forensic Sci, May 2012, Vol. 57, No. 3
doi: 10.1111/j.1556-4029.2011.02021.x
The unusual concentration of cannabinoids recently found in marijuana samples submitted to the forensic laboratory for chemical analysis prompted an investigation into whether genetic modifications have been made to the DNA of Cannabis sativa L. to increase its potency. Traditional methods for the detection of genetically modified organisms (GMO) were used to analyze herbal cannabis preparations. Our analyses support the hypothesis that marijuana samples submitted to forensic laboratories and characterized by an abnormal level of D9-THC are the product of breeding selection rather than of transgenic modifications. Further, this research has shown a risk of false positive results associated with the poor quality of the seized samples and probably due to the contamination by other transgenic vegetable products. On the other hand, based on these data, a conclusive distinction between the hypothesis of GMO plant contamination and the other of genetic modification of cannabis cannot be made requiring further studies on comparative chemical and genetic analyses to find out an explanation for the recently detected increased potency of cannabis.

In vitro germplasm conservation of high ?9 -tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions
Hemant Lata, Suman Chandra, Zlatko Mehmedic, Ikhlas A. Khan, Mahmoud A. ElSohly
Acta Physiologiae Plantarum
DOI:10.1007/s11738-011-0874-x
Germplasm conservation of a high ?9-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl2. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 ?M). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, ?9-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.

In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants
Hemant Lata, Suman Chandra, Natascha Techen, Ikhlas A. Khan, Mahmoud A. ElSohly
Journal of Applied Research on Medicinal and Aromatic Plants
DOI: 10.1016/j.jarmap.2015.12.001
The present study describes a simple, efficient and one step regeneration system for rapid shoot proliferation and in vitro rooting of Cannabis sativa nodal explants using meta-topolin (mT), an aromatic natural cytokinin. The best response in terms of explants producing maximum number of shoots with maximum shoot length and percent explants producing shoots was recorded on Murashige and Skoog (MS) medium supplemented with 2 ?M mT. Shoots multiplied on the same medium for two sub-cultures were able to induce healthy roots within 4–6 weeks. A separate medium containing auxin was not required for root induction. Regenerated plantlets were successfully acclimatized and hardened off in the climatic controlled grow room with 100% survival rate. Genetic fidelity of in vitro propagated plants was tested using inter simple sequence repeat (ISSR) markers. Our results show that all the ISSR profiles from in vitro propagated plants were monomorphic and comparable to that of the mother plant, thereby confirming the genetic fidelity. Qualitatively and quantitatively, cannabinoid profiles and the content, using gas chromatography-flame ionization detector (GC–FID), in mother plant and in vitro propagated plants were found to be similar to each other. Furthermore, regenerated plants were eco-ph

Not Cannabis specific
In vitro pollen germination and transient transformation of Zea mays and other plant species
Daniela Nicole Schreiber, Thomas Dresselhaus
Plant Molecular Biology Reporter 21(1):31-41March 2012
DOI: 10.1007/BF02773394
To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed.

In Vitro Propagation of Cannabis sativa L. and Evaluation of Regenerated Plants for Genetic Fidelity and Cannabinoids Content for Quality Assurance
Hemant Lata , Suman Chandra , Ikhlas A. Khan , and Mahmoud A. ElSohly
Methods in molecular biology (Clifton, N.J.) 1391:275-288 April 2016
DOI: 10.1007/978-1-4939-3332-7_19
Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has been used throughout history for fi ber, food, as well as for its psychoactive properties. The dioecious and allogamous nature of C. sativa is the major constraint to maintain the consistency in chemical profi le and overall efficacy if grown from seed. Therefore, the present optimized in vitro propagation protocol of the selected elite germplasm via direct organogenesis and quality assurance protocols using genetic and chemical profiling provide an ideal pathway for ensuring the efficacy of micropropagated Cannabis sativa germplasm.
A high frequency shoot organogenesis of C. sativa was obtained from nodal segments in 0.5 ?M thidiazuron medium and 95 % in vitro rhizogenesis is obtained on half-strength MS medium supplemented with 500 mg/L activated charcoal and 2.5 ?M indole-3-butyric acid. Inter Simple Sequence Repeats (ISSR) and Gas Chromatography-Flame Ionization Detection (GC-FID) are successfully used to monitor the genetic stability in micropropagated plants up to 30 passages in culture and hardened in soil for 8 months.

In vitro propagation of hemp : application to selected clones of Cannabis sativa L. for preservation of plants. (FRENCH)
Christine RICHEZ-DUMANOIS, Françoise BRAUT-BOUCHER, Louis COSSON, Michel PARIS
Agronomie, EDP Sciences, 1986, 6 (5), pp.487-495.
https://hal.archives-ouvertes.fr/hal-00884901/document
Much work has been done on the breeding of hemp for agronomic and chemical criteria. In order to keep plants available for the long period required for chemical analysis, we have propagated clones as horticultural cuttings, under controlled conditions, with or without micro-propagation. Different temperature and photoperiod conditions were used to obtain cuttings of several clones. Under these conditions, growth could be controlled : morphological and chemical development decreased at low temperature and were promoted by a regime of 22 °C (daily temperature) and 17 °C (night temperature) under 24 h illumination and 70 070 relative humidity. In the in vitro experiment, shoot proliferation was obtained three weeks later on the U MRASHIGE and K SOOG medium with added cytokinin (BAP 5.10-! 7 Mil) and auxin (AIB 1 70- M/1). The axillary shoots which developed were used as mother-plants in vitro ; they provided numerous cuttings after repeated sub-culturing on the same medium. A long thinning stage was necessary for rooting the microcuttings in the presence of charcoal (2 g/1) and AIB ( 510- M). The best method for rooting in vivo shoots involved non aseptic conditions (3 to 4 weeks). The further growth of plants at 22 °C/17 °C was comparable to that of corresponding horticultural cuttings and the cannabinoid pattern was similar to that of mother-plants.

In Vitro Screening of Three Commercial Cannabis-Based Products on ATP-Binding Cassette and Solute-Carrier Transporter Function.
Anderson, L. L., Etchart, M. G., MacNair, L., Land, M. H., Mosesova, I. A., Bonn-Miller, M. O., & Arnold, J. C.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2020.0053
Introduction: Legalization of medicinal cannabis around the world has led to an increase in the use of commercial cannabis-based products in the community. These cannabis-based products are being used in combination with conventional drugs to treat a variety of health conditions. Moreover, recreational cannabis-based products may be used in combination with other drugs. In this setting, there is increased potential for drug–drug interactions (DDIs) involving commercial cannabis-based products. Since DDIs can lead to serious adverse events, drug regulatory bodies require that every investigational drug be evaluated for DDI potential at metabolic enzymes and transporters. However, this seldom occurs for cannabis-based products due to legislation in many jurisdictions allowing a direct pathway to market. This study aimed to examine the inhibitory potential of three commercially available cannabis-based products at human ATP-binding cassette (ABC) and solute-carrier (SLC) transporters.
Materials and Methods: Three commercial cannabis-based products (Spectrum Yellow, Tweed Argyle, and Spectrum Red) that contain differing concentrations of cannabidiol (CBD) and D9 -tetrahydrocannabinol (D9 -THC) were evaluated for DDI potential at 12 drug transporters. HEK293 cells or vesicles expressing human ABC transporters (ABCB1, ABCC2, ABCG2, or ABCB11) and SLC transporters (SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLCO1B1, SLCO1B3, SLC47A1, and SLC47A2) were used to measure transporter function.
Results: Spectrum Yellow and Tweed Argyle inhibited ABCG2 transporter function. The IC50 value of Spectrum Yellow based on CBD and D9 -THC content was 4.5 lM for CBD and 0.20 lM for D9 -THC, and the IC50 value of Tweed Argyle was 9.3 lM for CBD and 6.0 lM for D9 -THC. Tweed Argyle also inhibited ABCB11 transporter function with an IC50 value of 11.9 lM for CBD and 7.7 lM for D9 -THC. SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 transporter functions were modestly inhibited by high concentrations of the cannabis-based products. The three cannabis-based products did not inhibit ABCB1, ABCC2, SLC47A1, SLC47A2, or SLC22A8 transporters.
Discussion: Novel findings were that the cannabis-based products inhibited ABCB11, SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 (although modestly in most instances). Spectrum Yellow and Tweed Argyle potently inhibited ABCG2, and future in vivo DDI studies could be conducted to assess whether cannabis products affect the pharmacokinetics of medications that are ABCG2 substrates.

In vitro TISSUE CULTURE AND GENETIC ANALYSIS OF TWO HIGH-CBD MEDICAL CANNABIS (Cannabis sativa L.) BREEDING LINES
Špela MESTINŠEK MUBI1, Sinja SVETIK1, Marko FLAJŠMAN1, Jana MUROVEC
Genetika, Vol 52, No.3, 925-941.
DOI: 10.2298/GENSR2003925M
http://www.dgsgenetika.org.rs/abstra...2_no3_rad8.pdf
The species Cannabis sativa L. has recently witnessed a resurgence of interest all over the world due to its multipurpose applications and the scientific confirmation of its pharmacological properties. Genotypes with high cannabinoid content are appreciated in the pharmaceutical and cosmetic industries due to their therapeutic potential. These genotypes, with predominantly high cannabidiol (CBD) content, are the subject of research and breeding in several programs, but to date, little data is published on the in vitro tissue culture of cannabis. Our study focused on the establishment of an efficient micropropagation method for two high-CBD breeding lines (MX-CBD-11 and MXCBD-707) as the basis for advanced biotechnological breeding approaches. The results demonstrated that the in vitro culture of medical cannabis can be initiated on different culture media, that cultured plants can be successfully acclimatized, and that nodal position, and especially the genotype, have a significant influence on the success of shoot culture establishment. They showed that the published tissue culture media optimized for one high-THC strain of Mexican cannabis are not as efficient for other genotypes of (medical) cannabis. We complemented this research with a genetic study of 95 plants of the two breeding lines with 16 microsatellite (SSR) markers which clustered the plants based on breeding line. The results demonstrated that only 8 markers are needed for discrimination of all analyzed plants and their usefulness for clonal identification

Marijuana compounds brewed using yeast by Canadian biotech firms
https://www.cbc.ca/news/technology/m...east-1.3527950
New medical marijuana products produced by yeast could soon be on the market, the co-founder of a biotech company says. That could potentially lead to a wider range of cannabinoid-based drugs that proponents say could be more effective for treating certain medical conditions than medical marijuana itself.
Cannabinoids are chemical compounds found in marijuana. Kevin Chen's Montreal-based company, Hyasynth, has successfully made the cannabinoid cannabigerol (CBG) from genetically engineered yeast, and he says this could be a precursor to other major and minor cannabinoids.
The appropriate use of medical marijuana has been a controversial topic, with many arguing that further research is needed to evaluate its efficacy as a treatment for a variety of ailments

Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential
Dinesh Adhikary, Manoj Kulkarni, Aliaa El-Mezawy, Saied Mobini, Mohamed Elhiti,
Rale Gjuric, Anamika Ray, Patricia Polowick, Jan J. Slaski, Maxwell P. Jones and
Pankaj Bhowmik
Front. Plant Sci. 12:627240.
doi: 10.3389/fpls.2021.627240
https://www.frontiersin.org/articles...21.627240/full
In recent years high-THC (psychoactive) and low-THC (industrial hemp) type cannabis (Cannabis sativa L.) have gained immense attention in medical, food, and a plethora of other consumer product markets. Among the planting materials used for cultivation, tissue culture clones provide various advantages such as economies of scale, production of disease-free and true-to-type plants for reducing the risk of GMP-EuGMP level medical cannabis production, as well as the development and application of various technologies for genetic improvement. Various tissue culture methods have the potential application with cannabis for research, breeding, and novel trait development, as well as commercial mass propagation. Although tissue culture techniques for plant regeneration and micropropagation have been reported for different cannabis genotypes and explant sources, there are significant variations in the response of cultures and the morphogenic pathway. Methods for many high-yielding elite strains are still rudimentary, and protocols are not established. With a recent focus on sequencing and genomics in cannabis, genetic transformation systems are applied to medical cannabis and hemp for functional gene annotation via traditional and transient transformation methods to create novel phenotypes by gene expression modulation and to validate gene function. This review presents the current status of research focusing on different aspects of tissue culture, including micropropagation, transformation, and the regeneration of medicinal cannabis and industrial hemp transformants. Potential future tissue culture research strategies helping elite cannabis breeding and propagation are also presented.

METHOD FOR DIFFERENTIATING CANNABIS PLANT CULTIVARS BASED ON CANNABINOID SYNTHASE PARALOGS.
US 2020/0270623 A1. (PATENT)
University of Colorado & Steep Hill inc.
Find PDF Link

Not Cannabis specific:
Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system.
Tuttle, J., Haigler, C. H., & Robertson, D.
Plant Methods, 8(1), 27. (2012).
doi:10.1186/1746-4811-8-27
Background: We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium(Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results: The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions: These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage
Hemant Lata, Suman Chandra , Natascha Techen, Ikhlas A. Khan, Mahmoud A. ElSohly
Biotechnology Letters December 2011, Volume 33, Issue 12, pp 2503-2508
DOI: 10.1007/s10529-011-0712-7
The increasing utilization of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. We have assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for 6 months at different growth conditions using inter simple sequence repeat (ISSR) DNA fingerprinting. Molecular analysis of randomly selected plants from each batch was conducted using 14 ISSR markers. Of the 14 primers tested, nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro stored plants were monomorphic and comparable to the mother plant which confirms the genetic stability among the clones. GC analysis of six major cannabinoids [?9-tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, cannabichromene, cannabigerol and cannabinol] showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following 6 months storage.

Not Cannabis specific
Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement
Stephen P. Moose, Rita H. Mumm
Plant Physiology, July 2008, Vol. 147, pp. 969–977
doi/10.1104/pp.108.118232
The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices has created the foundation for molecular plant breeding, an interdisciplinary
science that is revolutionizing 21st century crop improvement. Though the methods of molecular plant breeding continue to evolve and are a topic of intense interest among plant breeders and crop scientists (for review, see Cooper et al., 2004; Nelson et al., 2004; Lo¨rz and Wenzel, 2005; Varshney et al., 2006; Eathington et al., 2007; Mumm, 2007), they have received relatively little attention from the majority of plant biologists engaged in basic scientific research. The objective of this article for an Editor’s Choice series on future advances in crop biotechnology is to briefly review important historical developments in molecular plant breeding, key principles influencing the current practice of molecular plant breeding, and factors that influence the adoption of molecular plant breeding in crop improvement programs. Furthermore, we emphasize how the application of molecular plant breeding is now contributing to discoveries of genes and their functions that open new avenues for basic plant biology research

Optimization of Δ9-tetrahydrocannabinolic acid synthase production in Komagataella phaffii via post-translational bottleneck identification
Bastian Zirpela, Friederike Degenhardta, Chantale Zammarellia, Daniel Wibbergb, Jörn Kalinowskib, Felix Stehlea, Oliver Kaysera
Journal of Biotechnology Volumes 272–273, 20 April 2018, Pages 40-47
doi: 10.1016/j.jbiotec.2018.03.008
Δ9-Tetrahydrocannabinolic acid (THCA) is a secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain or reducing spasticity associated with multiple sclerosis. Here, we investigated the influence of the co-expression of 12 helper protein genes from Komagataella phaffii(formerly Pichia pastoris) on the functional expression of the Δ9-tetrahydrocannabinolic acid synthase (THCAS) heterologously expressed in K. phaffiiby screening 21 clones of each transformation. Our findings substantiate the necessity of a suitable screening system when interfering with the secretory network of K. phaffii. We found that co-production of the chaperones CNE1p and Kar2p, the foldase PDI1p, the UPR-activator Hac1p as well as the FAD synthetase FAD1p enhanced THCAS activity levels within the K. phaffii cells. The strongest influence showed co-expression of Hac1s - increasing the volumetric THCAS activities 4.1-fold on average. We also combined co-production of Hac1p with the other beneficial helper proteins to further enhance THCAS activity levels. An optimized strain overexpressing Hac1s, FAD1 and CNE1 was isolated that showed 20-fold increased volumetric, intracellular THCAS activity compared to the starting strain. We used this strain for a whole cell bioconversion of cannabigerolic acid (CBGA) to THCA. After 8 h of incubation at 37 °C, the cells produced 3.05 g L−1 THCA corresponding to 12.5% gTHCA gCDW−1.

Plant Regeneration of Cannabis sativa L. through anther culture (in vitro) of the cultivar "USOS"
Ben Wedeking, Ina Pinker, Giampaolo Grassi
Cannabis sativa [L.] – hemp - is an important plant in medicine and pharmacy (Walsh et al., 2003).
https://cin.entecra.it/test/img/1391...rassi_2013.pdf
(if the s is removed from the https it will work, but this link works - after you take out the ‘s’ that the software inserts)
As a plant it is valued for its hallucinogenic and medicinal properties and has been used to treat a variety of ailments including pain, glaucoma, nausea, asthma, depression, insomnia and neuralgia (Mechoulam et al., 1976; Duke and Wain, 1981). In addition to that, fibers are used as raw material for paper and textile production (Alden et al., 1998). There are not many studies on tissue culture of hemp. Hemphill et al., (1978), Fisse et al., (1981) and Feeney and Punja (2003) reported, that callus readily produced roots, but was unreceptive to shoot formation, either directly or indirectly from callus or suspension cultures

Production of ?1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.
Taura, F., Dono, E., Sirikantaramas, S., Yoshimura, K., Shoyama, Y., & Morimoto, S.
Biochemical and Biophysical Research Communications, 361(3), 675–680 (2007)
doi:10.1016/j.bbrc.2007.07.079
D1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of D1 -tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted 1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Production of D9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing D9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Bastian Zirpel . Felix Stehle . Oliver Kayser
Biotechnol Lett (2015) 37:1869–1875
DOI: 10.1007/s10529-015-1853-x
Objective The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of D9- tetrahydrocannabinolic acid (THCA) using whole cells.
Results Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Ko- magataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after 192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1) THCA could be produced by 10.5 gCDW l-1 before enzyme activity was lost.
Conclusion Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production

Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Bastian Zirpel, Felix Stehle, Oliver Kayser
Tu technische universitat Dortmund (poster) Pdf can be obtained by contacting Bastian Zirpel at Reseachgate.net
Increasing pharmaceutical demand for Δ9-tetrahydrocannabinol (THC)
Legislation prevents growing of cannabis
Chemical synthesis and extraction from plant proves problematic
Final goal is production of Δ9-tetrahydro-cannabinolic acid (THCA), precursor of THC, by a microbial host at industrial relevant scale
Aim of this study: THCA synthase, catalyzing final step of THCA synthesis, is functionally expressed in yeasts to investigate the possible application of a whole cell production system for THCA

Production of ?1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.
Taura, F., Dono, E., Sirikantaramas, S., Yoshimura, K., Shoyama, Y., & Morimoto, S.
Biochemical and Biophysical Research Communications, 361(3), 675–680 (2007)
doi:10.1016/j.bbrc.2007.07.079
D1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of D1 -tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted 1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Production of D9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing ?9-tetrahydrocannabinolic acid synthase from Cannabis sativa l.
Zirpel, B., Stehle, F., & Kayser, O.
Biotechnology Letters, 37(9), 1869–1875. (2015).
doi:10.1007/s10529-015-1853-x
Objective The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of D9- tetrahydrocannabinolic acid (THCA) using whole cells.
Results Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after 192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1 ) THCA could be produced by 10.5 gCDW l -1 before enzyme activity was lost.
Conclusion Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production

Progress towards transformation of fibre hemp
L. MacKinnon, G. McDougall, N. Aziz & S. Millam (2000)
https://scri.webarchive.hutton.ac.uk.. .001/11HEMP.PDF
Introduction The last decade has seen the reintroduction to the UK of a crop with a history of triumphs and tragedies; today its very name provokes visions of a drug culture, fuelled by its prohibition in Britain for over 70 years. Historically, cultivation of Cannabis sativa L. (hemp) originated in China around 2700 BC, where its properties as a medicinal plant were first discovered. Cultivation then spread across Asia and through Europe, arriving in the UK some 2000-2200 years ago, by which time it had become widely cultivated as it had so many uses. Hemp fibres were found to be durable and were used in clothing, sailmaking and papermaking. Notably, the first copies of the Bible were composed of hemp paper. Oils from hemp seeds were used for a wide range of
purposes, from cooking to cosmetics, and extracts of hemp were used to treat a wide range of ailments. Queen Victoria is rumoured to have used hemp on a regular basis. This widespread industrial use of hemp continued to the early 20th century, until cheap and plentiful imported jute and cotton made hemp uncompetitive. Around this time, as the legitimate uses of hemp declined, the misuse of hemp as a hallucinogenic agent became more apparent and many countries began to outlaw the cultivation of cannabis. In 1928, an act was passed that finally prohibited hemp cultivation in the UK. Hemp cultivation continued in other European countries, such as The Netherlands and France, and in many Russian states. European hemp breeding programmes produced hemp cultivars for fibre production that were low in the psycho-active constituents, cannabinoids. Eventually, cultivars with ultra-low (<1% wild type) cannabinoid levels were available, which reawakened UK interest in hemp. In 1994, a new hemp-licensing scheme allowed the crop to be cultivated again in the UK and its reintroduction began. Over recent years, hemp cultivation has been increasing steadily (Fig. 1). This renaissance is also attributable to the need for renewable non-food crops in the UK, as outlined by the UK Biodiversity action plan.

Quorum quenching is an antivirulence strategy employed by endophytic bacteria
Parijat Kusari & Souvik Kusari & Marc Lamshöft & Selahaddin Sezgin & Michael Spiteller & Oliver Kayser
APPLIED MICROBIAL AND CELL PHYSIOLOGY 2014
DOI 10.1007/s00253-014-5807-3
Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation,
and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMSn) and matrix-assisted laser desorption ionization imaging highresolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of
bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.

Recent advances in cannabis biotechnology.
Hesami, M., Pepe, M., Alizadeh, M., Rakei, A., Baiton, A., & Phineas Jones, A. M.
Industrial Crops and Products, 158, 113026. (2020)
doi:10.1016/j.indcrop.2020.113026
Cannabis sativa L. is a high-value crop with a multi-billion dollar international market, yet due to the long history of prohibition, there is a significant lack of research on the plant and biotechnological techniques are in their infancy. Developing and applying modern techniques to Cannabis will help overcome some species-specific challenges to increase productivity and improve our knowledge about this plant. With regulatory environments relaxing in many parts of the world, there has been a significant increase in biotechnological research with this species. The current manuscript reviews the advances in Cannabis biotechnology, including molecular markers, microRNA, omics-based methods, and functional genes related to the terpene and cannabinoid biosynthesis as well as fiber quality. The foremost aim of this study is to a comprehensive review of the available literature to guide future cannabis studies in the field of genetic engineering and biotechnology.

Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology
Supaart Sirikantaramas; Futoshi Taura; Satoshi Morimoto; Yukihiro Shoyama
Current Pharmaceutical Biotechnology, Volume 8, Number 4, 2007, pp. 237-243(7)
doi: 10.2174/138920107781387456
Cannabinoids, consiting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate: olivetolate geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

Not Cannabis
Regeneration and Agrobacterium-mediated transformation of hop (Humulus lupulus L.)
C. Horlemann · A. Schwekendiek · M. H_hnle · G. Weber
Plant Cell Rep (2003) 22:210–217
DOI 10.1007/s00299-003-0676-8
An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. “Tettnanger”) was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing
medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (b-glucuronidase). The presence and function of transgenes in plants growing
in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.

Regio- and Substrate-Specific Oxidative Metabolism of Terpinolene by Cytochrome P450 Monooxygenases in Cupressus lusitanica Cultured Cells
Takako Harada, Eriko Harada, Ryoko Sakamoto, Tatsuya Ashitani, Koki Fujita1, Ken-ichi Kuroda
American Journal of Plant Sciences, 2012, 3, 268-275
doi: 10.4236/ajps.2012.32032
Many of monoterpenes produced in plants contribute to defenses against herbivores, insects and microorganisms. Among those compounds, ?-thujaplicin formed in Cupressaceae plants has a unique conjugated seven-membered ring and some useful biological activities, e.g. fungicide, repellent, insecticide and so on. The biosynthesis pathway of ?- thujaplicin has not yet been revealed; we have been trying to uncover it using Cupressus lusitanica cultured cells as a model. In our previous study, terpinolene was identified as a potential ?-thujaplicin intermediate at the branching point to terpenoids. In this article, terpinolene metabolism in C. lusitanica cultured cells was investigated, and it was shown that the microsomal fraction from cells oxidized terpinolene into the hydroxylated compound, 5-isopropylidene-2-met- hylcyclohex-2-enol (IME). Then, IME was further oxidized by microsomal fraction to the epoxidized compound, 1,6- epoxy-4(8)-p-menthen-2-ol (EMO). These were the only two products detected from the microsomal reactions, respect- tively. Moreover, microsomal reactions with monoterpenes other than terpinolene produced nothing detectable. These results show that the enzymes of these reactions had strict substrate specificity and regio-selectivity. Experiments on kinetics and with specific inhibitors confirmed that these reactions were caused by cytochrome P450 monooxygenases, respectively. These results support our hypothesis that terpinolene is a putative intermediate of ?-thujaplicin biosynthe- sis and show that IME and EMO are also putative intermediates.

Scientists brew cannabis using hacked beer yeast
Elie Dolgin
https://www.nature.com/articles/d41586-019-00714-9
doi: 10.1038/d41586-019-00714-9
The yeast that people have used for millennia to brew alcoholic drinks has now been engineered to produce cannabinoids — chemicals with medicinal and sometimes mind-altering properties found in cannabis.
The feat, described on 27 February in Nature, turns a sugar in brewer’s yeast (Saccharomyces cerevisiae) called galactose into tetrahydrocannabinol (THC), the main psychoactive compound in cannabis (Cannabis sativa). The altered yeast can also produce cannabidiol (CBD), another major cannabinoid that’s attracted attention lately for its potential therapeutic benefits, including its anti-anxiety and pain-relief effects.

Structure and Function of ?1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis sativa
Yoshinari Shoyama, Taro Tamada, Kazuo Kurihara, Ayako Takeuchi, Futoshi Taura, Shigeki Arai, Michael Blaber, Yukihiro Shoyama, Satoshi Morimoto and Ryota Kuroki
J. Mol. Biol. (2012) 423, 96–105
doi:10.1016/j.jmb.2012.06.030
?1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ?1 tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure–function relationship of THCA synthase, and the tertiary structure was determined to 2.75 Å resolution by X-ray crystallography (Rcryst=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6? of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active?site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

Subcellular localization defines modification and production of D9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana
Marcus Geissler . Jascha Volk . Felix Stehle . Oliver Kayser . Heribert Warzecha
Biotechnol Lett 30(6), 981-987
doi: 10.1007/s10529-018-2545-0
Objective Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis.
Results Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat gFW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat gFW-1 activity towards CBCA production).
Conclusion Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

Synthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana benthamiana and Saccharomyces cerevisiae
Thies Gülck, J. K. Booth, Â. Carvalho, B. Khakimov, C. Crocoll.
M. S. Motawia, B. L. Møller, J. Bohlmann, and N. J. Gallage
J. Nat. Prod. 2020, XXXX, XXX, XXX-XXX October 1, 2020
https://pubs.acs.org/doi/pdf/10.1021...atprod.0c00241
https://doi.org/10.1021/acs.jnatprod.0c00241
Phytocannabinoids are a group of plant-derived metabolites that display a wide range of psychoactive as well as health-promoting effects. The production of pharmaceutically relevant cannabinoids relies on extraction and purification from cannabis (Cannabis sativa) plants yielding the major constituents, ?9-tetrahydrocannabinol and cannabidiol. Heterologous biosynthesis of cannabinoids in Nicotiana benthamiana or Saccharomyces cerevisiae may provide cost-efficient and rapid future production platforms to acquire pure and high quantities of both the major and the rare cannabinoids as well as novel derivatives. Here, we used a meta-transcriptomic analysis of cannabis to identify genes for aromatic prenyltransferases of the UbiA superfamily and chalcone isomerase-like (CHIL) proteins. Among the aromatic prenyltransferases, CsaPT4 showed CBGAS activity in both N. benthamiana and S. cerevisiae. Coexpression of selected CsaPT pairs and of CHIL proteins encoding genes with CsaPT4 did not affect CBGAS catalytic efficiency. In a screen of different plant UDP-glycosyltransferases, Stevia rebaudiana SrUGT71E1 and Oryza sativa OsUGT5 were found to glucosylate olivetolic acid, cannabigerolic acid, and ?9-tetrahydrocannabinolic acid. Metabolic engineering of N. benthamiana for production of cannabinoids revealed intrinsic glucosylation of olivetolic acid and cannabigerolic acid. S. cerevisiae was engineered to produce olivetolic acid glucoside and cannabigerolic acid glucoside.

The application of plant in vitro cultures in cannabinoid production.
Wróbel, T., Dreger, M., Wielgus, K., & S?omski, R.
Biotechnology Letters, 40(3), 445–454. (2017).
doi:10.1007/s10529-017-2492-1
Cannabinoids have considerable interest in the pharmaceutical industry. However, the production of medicines from hemp (Cannabis sativa L.) in most countries is restricted by law. Large-scale, field cultivation of hemp is difficult to control. Cannabinoid content in plants is variable and depends on multiple factors. Therefore, alternative methods of production have been investigated. The development of micropropagation techniques is a necessary step for genetic modification. Promising results have been obtained for certain narcotic genotypes. However, micropropagation of fibre types requires further research. Hemp can be genetically modified which may contribute to the breeding of new varieties in the future. Cell suspension cultures and hairy root cultures of hemp have been used to produce cannabinoids but obtaining cannabinoids from callus and cell suspension cultures has proved impossible. Adventitious roots can, however, deliver small amounts of these metabolites but production ceases over time and is too low for industrial applications.

The Biotechnology of Cannabis Sativa 1st edition
Sam R. Zwenger
https://issuu.com/weedreads/docs/376...ology-of-canna
Published on Apr 20, 2009
The purpose of this book gives insight into the possibilities that biotechnology can provide to the Cannabis community and the world. It begins with a botanical introduction and continues with an explanation of biotechnology and techniques, examples and purpose for genetically modifying Cannabis, THC biosynthesis and cellular interaction, and information on some necessary molecular biology techniques. The tools of biotechnology, such as DNA sequencing and gene cloning, are speeding up the reality that this highly controversial plant will continue to make an impact on human societies for generations to come. This book covers advances and techniques on how to grow plant tissue in vitro, genetically modify this tissue, and re-grow it in order to produce a transgenic Cannabis plant. Anyone who wants to know what the future holds for Cannabis sativa and marijuana should read this book. Sam R. Zwenger April 2009.

*The Biotechnology of Cannabis sativa 2nd edition
Sam R, Zwenger
EXTREME PUBLICATIONS, INC.
https://www.dmt-nexus.me/users/cosmi...nd_edition.pdf

*The Past, Present and Future of Cannabis sativa Tissue Culture
Adrian S. Monthony , Serena R. Page, Mohsen Hesami, Andrew Maxwell P. Jones
doi: 10.20944/preprints202012.0766.v1
The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect/disease/virus free clonal plants, store germplasm, and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history or prohibitions and restrictions. Existing literature has multiple limitation: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity, studies using drug-type cultivars are predominantly op-timized using a single cultivar, most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems the multiplication phase of micropropagation (stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies.

Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L.
Lata, H., Chandra, S., Khan, I., & ElSohly, M. A.
In Vitro Cellular & Developmental Biology - Plant, 45(1), 12–19.(2008).
doi:10.1007/s11627-008-9167-5
Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yrold mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05– 5.0 ?M thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 ?M thidiazuron) than with benzyladenine or kinetin. Adding 7.0 ?M of gibberellic acid into a medium containing 0.5 ?M thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l?1 activated charcoal and 2.5 ?M indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 ?mol m?2 s ?1 ). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 ?mol m?2 s ?1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 ?mol m?2 s ?1 . Stomatal conductance and transpiration increased with light intensity up to highest level (2000 ?mol m?2 s ?1 ) tested. Intercellular CO2 concentration (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.

Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.).
Feeney, M., & Punja, Z. K.
In Vitro Cellular & Developmental Biology - Plant, 39(6), 578–585. (2003).
doi:10.1079/ivp2003454
Hemp (Cannabis sativa L.) is cultivated in many parts of the world for its fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacteriummediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5mM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1mM kinetin, 3% sucrose, and 8 g l21 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 mM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.

Tissue culture of Cannabis sativa Variables Affecting Shoot Growth and Plantlet Regeneration from Tissue Cultures of Cannabis sativa L
Janesse Holmes, Samantha Lung, Danielle Collyer and Zamir K. Punja
Front. Plant Sci. (2021)
doi: 10.3389/fpls.2021.732344
https://www.frontiersin.org/articles...32344/abstract
Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In the present research, we evaluated the variables that can influence the success of shoot growth plantlet production from tissue cultures of Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused death of explants. Seven commercially grown THC-containing cannabis strains showed significant differences in response to shoot growth from nodal explants and from meristems on Murashige and Skoog (MS) medium containing thidiazuron (1µM) and naphthaleneacetic acid (0.5 µM) plus 1% activated charcoal. The effect of DWK or MS salts in media on shoot length and leaf numbers from nodal explants was compared and showed strain dependency with regards to the growth response. To obtain rooted plantlets, shoots from nodal and meristem explants of strain Moby Dick were evaluated for rooting following addition of sodium metasilicate, silver nitrate, IBA, kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage, and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet recovery using hydroponic culture, peat plugs, and rockwool substrate was 57, 76 and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The response of leaf explants to callus formation in 11 strains on MS medium without activated charcoal was 35 to 100%, depending on the strain; organogenesis was not observed. The success in recovery of plantlets from nodal and meristematic explants is influenced by the cannabis strain and the degree of endophytic contamination of the explants, as well as frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets from tissue cultures of C. sativa.

Towards a platform organism for terpenoid production – in silico analysis of metabolic networks of potential hosts and in vivo validation
Evamaria Gruchattka THESIS
DOI: 10.17877/DE290R-17199
Terpenoids are mostly plant derived compounds with industrial and medicinal applications. These compounds can be provided via biotechnological production as environmental friendly alternative to chemical synthesis or plant extraction. However, productivities from microbial hosts require improvement in order to be economically competitive. Therefore, microbial hosts are compared and new metabolic engineering targets aimed at increasing terpenoid production are investigated by stoichiometric metabolic network analysis and selected strategies are validated in vivo. The in silico analysis of the metabolic networks of Escherichia coli and Saccharomyces cerevisiae (yeast) led to several promising metabolic engineering targets with potential to increase the theoretical maximum terpenoid yield. Production of the sesquiterpenoid patchoulol in yeast was chosen for the in vivo validation study. A two-phase cultivation method for terpenoid capture with dodecane overlay was established and the produced spectrum of sesquiterpenoids was determined. Precursor metabolite flux towards the desired product was increased via overexpression of a truncated HMG-CoA reductase and fusion of FPP synthase with patchoulol synthase. Two in silico identified strategies were implemented: (i) disruption of ?-ketoglutarate dehydrogenase gene redirected the metabolic flux as predicted, however, the intermediate acetate was produced in high amounts instead of the desired product; (ii) expression of ATP-citrate lyase from Arabidopsis did not increase terpenoid production due to insufficient in vivo activity. The findings of this thesis contribute to an increased knowledge about enhancing terpenoid production in both E. coli and S. cerevisiae, as well as metabolic behavior of yeast. The in silico stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. This study highlights that kinetics, regulation and cultivation conditions may interfere with predictions, resulting in poor in vivo performance. These findings promote developments of metabolic modelling to increase the predictive power and accelerate microbial strain improvement.

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids
Simon Dusséauxa, William Thomas Wajna, Yixuan Liua, Codruta Igneaa, and Sotirios C. Kampranis
PNAS (2020)
DOI: 10.1073/pnas.2013968117
Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor transisopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids

Virus-induced gene silencing (VIGS) in Cannabis sativa L.
Schachtsiek, J., Hussain, T., Azzouhri, K., Kayser, O., & Stehle, F.
Plant Methods, 15(1). (2019).
doi:10.1186/s13007-019-0542-5
Background: The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable. Results: For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI), were identifed in C. sativa. Subsequently, the targets of specifc small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach. Here we show for the frst time a gene knockdown in C. sativa using the Cotton leaf crumple virus (CLCrV) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciens strain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The successful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of transcripts from 70 to 73% for ChlI and PDS, respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV-based vector system.
Conclusions: The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between available genomic data and biochemical information of this important medicinal plant

Will Cannabis Producers Ditch Greenhouses for Bioreactors?
Maxx Chatsko
The Fool
https://www.fool.com/investing/2019/...-for-bior.aspx
Plants aren't always the perfect medium for producing high-value compounds. Turns out, cannabinoids can be brewed with microbes instead.
Investors cheered as brewing companies made moves into the nascent and increasingly deregulated cannabis industry in 2018. Canopy Growth(NYSE:CGC) won a $4 billion investment from beer giant Constellation Brands. Cronos Group (NASDAQ:CRON) linked up with Anheuser-Busch InBev to research beverages containing cannabinoids including THC and CBD. Other consumer food brands are sure to follow. While the brewing industry represents an important source of capital for the fledgling cannabis market, brewing science may represent an important source of future production capacity of cannabinoid compounds. Major cannabis producers ranging from Cronos to OrganiGram Holdings(NASDAQ:OGI) have partnered with analytical biotech labs exploring how to ditch inefficient plants grown in greenhouses in favor of genetically engineered microbes brewed in bioreactors. It could prove to be an important relationship for both industries if it works, so a pioneering expert weighed in on whether bioreactors might disrupt the cannabis ecosystem. Kevin Chen is the CEO and cofounder of Canadian startup Hyasynth Biologicals, which has been working on microbial cannabinoids for years.


IC Legal


A body packer with cannabis

Sander Borgsteede, Patricia E B Verwer, Gerrie Prins, Birgit C P Koch
Nederlands tijdschrift voor geneeskunde 163 January 2019
https://www.researchgate.net/publica..._with_cannabis
Background: In recent years, numerous reports have been published on body packers, i.e. people who use their own body to transport drugs. Most body packers swallow small packages of cocaine or heroin. Case description: We describe the case of a patient who, two days after ingestion of packages containing cannabis, was admitted for dizziness and drowsiness. Urinalysis was positive for cannabis and negative for other drugs. The deterioration in clinical status possibly indicated a burst package. The packages were surgically removed and the patient made a full recovery. Conclusion: Some body packers transport other drugs than cocaine or heroin, such as cannabis. In body packers who ingest cannabis, depressed consciousness is a main symptom of intoxication. They should be admitted and monitored for symptoms of intoxication or paralytic ileus. Laboratory tests provide qualitative support when interpreting clinical symptoms. Surgical intervention is indicated in cases of bowel obstruction or when intoxication due to package leakage is suspected.

A comprehensive breath test that confrms recent use of inhaled cannabis within the impairment window
Michael W. De Gregorio, Gregory T. Wurz, Edward Montoya, & Chiao Jung Kao
Scientific Reports | Natire (2021)
DOI: 10.1038/s41598-021-02137-x
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_2137.pdf
Legalization of cannabis for medicinal and/or recreational use is expanding globally. Although cannabis is being regulated country by country, an accurate recent use test with indisputable results correlated with impairment has yet to be discovered. In the present study, a new approach for determining recent cannabis use within the impairment window after smoking was developed by studying 74 subjects with a mean age of 25 years and average use history of 9 years. Horizontal gaze nystagmus was evaluated along with subject self-assessments of impairment, and blood and breath samples were collected before and after smoking cannabis. Breath and blood pharmacokinetic parameters and cannabinoid profles determined recent use within the impairment window. No subjects were positive for recent use pre-smoking, although all subjects had detectable cannabinoids in breath samples. We describe an inhaled cannabis recent use test that correlate

A Guide to the National Academy of Science Report on Cannabis: An Exclusive Discussion with Panel Members
Daniele Piomelli, Ziva Cooper, Donald Abrams, Igor Grant, and Sachin Patel
Cannabis and Cannabinoid Research Volume 2.1, 2017
DOI: 10.1089/can.2017.29009.dpi
The NASEM report1 was developed in response to the significant changes in policy regarding the legalization of medical and recreational cannabis. These changes are happening in the absence of a dialogue regarding the scientific evidence pertaining to both the potential therapeutic effects of cannabis and the health outcomes, both positive and negative, of use. The Health and Medicine Division of the NASEM was asked to convene a committee of experts from a range of fields to conduct a comprehensive review of the literature and develop a consensus report delineating evidence for both the
therapeutic effects and health risks associated with cannabis and cannabinoid use. The committee was also charged with developing a research agenda to address the most critical areas to help guide future highquality research on the effects of cannabis and cannabinoids. Research and recommendations associated with policy was beyond the scope of the report, yet the impact of policy on research was raised in a ‘‘Barriers to Research’’ section that highlighted the difficulty of doing both preclinical and clinical studies with cannabis and cannabinoids due to their Schedule I classification

All Eyes on Europe
As new legal markets rapidly emerge across the globe, attention turns to Europe’s burgeoning market. What lessons can be learnt from those that have gone before?
Luis Merchan
Cannabis Scientist 2022
https://thecannabisscientist.com/business-profession/all-eyes-on-europe?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email
https://thecannabisscientist.com/business-profession/all-eyes-on-europe?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email

Amsterdam mayor plans to press ahead with tourist cannabis cafe ban

April 12, 2022
https://www.dutchnews.nl/news/2022/04/amsterdam-mayor-plans-to-press-ahead-with-tourist-cannabis-cafe-ban/?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email
Amsterdam mayor Femke Halsema wants to press ahead with plans to close the city’s cannabis cafes, or coffee shops, to tourists, saying the move is the only option to get a grip on the local soft drugs market. Halsema told city councillors on Monday afternoon there is a ‘worrying interdependence’ between the soft and hard drug trade and that ‘money from the lucrative cannabis trade easily finds its way into hard drugs’. ‘Many of the major problems in the city are fueled by the cannabis market: from nuisance caused by drug tourism to serious crime and violence,’ she said. ‘Banning sales to tourists is a necessary intervention… and a first step towards regulation.’

Not Cannabis specific
Apoptosis gene EI24, compositions, and methods of use
https://patents.google.com/patent/US5843659A/en
https://patentimages.storage.googleapi s.com/72/63/11/675686448e1963/US5843659.pdf
Disclosed is the isolation and characterization of EI24, a novel gene whose 2.4 kb mRNA is induced following etoposide treatment. Induction of EI24 mRNA by etoposide required expression of wild-type p53. Overexpression of functional p53 was sufficient to induce expression of the EI24 mRNA. The EI24 mRNA was also induced in a p53-dependent manner by ionizing irradiation of primary murine thymocytes. The invention is thus directed to an isolated EI24 protein, nucleotide sequences coding for and regulating expression of the protein, antibodies directed against the protein, and recombinant vectors and host cells containing the genetic sequences coding for and regulating the expression of the protein sequence. The invention is also directed to genomic DNA, cDNA, and RNA encoding the EI24 protein sequence and to corresponding antisense RNA sequences. Antibodies can be used to detect EI24 in biological specimens, including, for example, human tissue samples. The present invention is further directed to methods of treating degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death. The present invention is further directed to methods for diagnosing degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death, as well as methods for monitoring the progress of such degenerative disorders

Are recreational cannabis laws associated with declining medical cannabis program enrollment in the U.S.? An analysis of cardholder enrollment and demographic characteristics from 2013 to 2020
Sarah A.Okey a Saul A.Castro a Jack T.Waddell a Connor B.Jones a Austin J.Blake a Holly P.O'Rourke b Mary C.Davis a Madeline H.Meier
International Journal of Drug Policy Volume 100, February 2022, 103531
DOI: 10.1016/j.drugpo.2021.103531
https://www.sciencedirect.com/scienc...497?via%3Dihub
Background
There is speculation that enrollment in U.S. state medical cannabis programs differs depending on whether adult recreational cannabis use is legal. If true, this could have implications for public health and policy.
Methods
Using medical cannabis registry data from U.S. states with a mandatory registry between 2013 and 2020 (N = 23 states), this study examined time-trends in medical cannabis cardholder enrollment and tested whether enrollment trends differed depending on adult recreational cannabis legalization.
Results
Multilevel models showed that time-trends for registered active medical cardholders differed during years when adult recreational cannabis use was legal versus not legal (time*recreational cannabis law interaction: b = -0.004, p < 0.01, 95% CI = -0.005, -0.003). The population prevalence of registered active medical cardholders increased over time in years when recreational cannabis was not legal (i.e., medical-only years; b = 0.004, p < 0.001, 95% CI = 0.003, 0.004, corresponding to an increase of 380 cardholders per 100,000 people per year), and decreased in years when recreational cannabis was legal (i.e., recreational years; b = -0.001, p < 0.001, 95% CI = -0.002, -0.001, corresponding to a decrease of 100 cardholders per 100,000 people per year). Time-trends were similar for each sex (male, female) and age group (18–30/35, 30/35+), with each cardholder group showing increases in medical-only years and decreases in recreational years. In medical-only years, there were no differences in enrollment time-trends across sex, but older cardholder (30/35+) enrollment increased at a faster rate than younger cardholder enrollment (18–30/35) (F = 16.199, p < 0.001). In recreational years, male cardholder enrollment decreased at a faster rate than female cardholder enrollment (F = 7.347, p < 0.01), but there was no difference in trends across age. Three states, all with medical-only years, provided data on ethnicity/race. Results showed significant increases from 2016 to 2020 in enrollment of White, African-American, and Hispanic individuals.
Conclusions
Findings suggest that recreational cannabis legalization is associated with decreasing enrollment in medical cannabis programs, particularly for males.
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Australian Govt Medicinal Cannabis – TGA Guidance Documents
https://www.tga.gov.au/medicinal-can...#guidance-docs


Cannabinoid Content in Cannabis Flowers and Homemade Cannabis-Based Products Used for Therapeutic Purposes in Argentina
Daniela Sedan, Cristian Vaccarini, Pablo Demetrio, Marcelo Morante, Romina Montiel, Alvaro Saurí, and Dario Andrinolo
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0117
https://www.liebertpub.com/doi/epub/.../can.2020.0117
Introduction: A recent law (DCTO-2020-883-APN-PTE—Law No. 27,350. Regulation) passed in Argentina put an end to the ban imposed for the last 60 years on cannabis cultivation within the country. The law permits restricted access to cannabis derivatives for medicinal, therapeutic, and palliative use by individuals and communities, allowing self- and community based cannabis production. This is cause for concern in view of the lack of quality controls for cannabis derivatives. The several varieties of cannabis grown in Argentina have different chemical pro􀂦les and are processed in a variety of ways— mostly by alcohol extraction or maceration at different temperatures and for different amounts of times—making the cannabinoid content of these preparations highly variable. Determining the characteristics of home- and community-grown cannabis products will facilitate the implementation of public policies conducive to their safety and improvement.
Objective: The aim of this study was to determine the cannabinoid chemotypes used for therapeutic purposes in Argentina and evaluate whether the cannabinoids present in homemade derivatives are comparable to those in commercially available products.
Materials and Methods: High performance liquid chromatography with ultraviolet and diode array detector (HPLC/UV-DAD) analysis of 436 samples (oils, resins, and inflorescences) was carried out to determine the identity and concentration of five cannabinoids: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), and cannabinol (CBN). From three different sources, the samples represent the type of medical cannabis preparations to which patients have access.
Results: The results indicate that the medium-to-low cannabinoid concentration in a significant number of homemade oil samples is similar to that found in commercial products. Most of the samples have a THC/CBD ratio >1 or only contain THC. Acidic cannabinoids were detected in homemade preparations, but were not reported in package inserts of commercial products.
Conclusions: Our results indicate that despite their considerable variability, homemade preparations as a whole show
cannabinoid levels and profiles equivalent to the commercially available products commonly used for medicinal, therapeutic, and palliative purposes in Argentina.
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Cannabis condemned: the proscription of Indian hemp.
Kendell, R.
Addiction, 98(2), 143–151.(2003).
doi:10.1046/j.1360-0443.2003.00273.x
Aims To find out how cannabis came to be subject to international narcotics legislation. Method Examination of the records of the 1925 League of Nations’ Second Opium Conference, of the 1894 Report of the Indian Hemp Drugs Commission and other contemporary documents. Findings Although cannabis (Indian hemp) was not on the agenda of the Second Opium Conference, a claim by the Egyptian delegation that it was as dangerous as opium, and should therefore be subject to the same international controls, was supported by several other countries. No formal evidence was produced and conference delegates had not been briefed about cannabis. The only objections came from Britain and other colonial powers. They did not dispute the claim that cannabis was comparable to opium, but they did want to avoid a commitment to eliminating its use in their Asian and African territories.

Cannabis cultivation in the world: heritages, trends and challenges
Pierre-Arnaud Chouvy
EchoGeo 48 | 2019 : avril / juin 2019
https://journals.openedition.org/echogeo/17591
Despite cannabis being the most common illegal drug crop in the world and its worldwide presence, very little is known about its production, trade, and consumption at the global scale. This is due mostly to over a century of global prohibition and the dangers associated to researching illegal drug crop production. Worse, the limited data available about cannabis cultivation is most often inaccurate, unreliable, and highly controversial. While this has always been problematic, in terms of sheer knowledge and informed policy-making, it has now become even more acute of an issue as global trends towards decriminalisation and legalisation are already provoking negative unintended consequences in poor producing countries. This article is an effort to present the state of the current knowledge and the present and future stakes of the fast-changing cannabis industry and legislation.

Cannabis médical : 1000 patients déjà inclus dans l'expérimentation
https://www.topsante.com/medecine/vo...ntation-634683

‘Cannabis’ ontologies I: Conceptual issues with Cannabis and cannabinoids terminology
Kenzi Riboulet-Zemouli
Drug Science, Policy and Law Volume 6: 1–37
https://journals.sagepub.com/doi/pdf...50324520945797
Objective: Identify a coherent nomenclature for Cannabis sativa L. derived products and their analogues. Design: Research undertaken in parallel to the three-year assessment of Cannabis derivatives by the World Health Organisation. The scope is limited to Cannabis products intended for human incorporation (internal and topical consumption). Primarily embedded in pharmacognosy, the study incorporates a wide range of scholarly and grey literature, folk knowledge, archives, pharmacopœias, international law, field pharmacy, clinical and herbal medicine data, under a philosophical scrutiny. Generic and Cannabis-specific nomenclatural frames are compared to determine the extent to which they coincide or conflict. Results: All lexica reviewed use weak, ambiguous, or inconsistent terms. There is insufficient scientific basis for terms and concepts related to Cannabis at all levels. No sound classification exists: current models conflict by adopting idiosyncratic, partial, outdated, or utilitarian schemes to arrange the extraordinarily numerous and diverse derivatives of the C. sativa plant. In law and policy, no clear or unequivocal boundary between herbal and non-herbal drugs, nor natural and synthetic cannabinoids was found; current nomenclatures need updates. In science, the botanical Cannabis lexicon overlooks parthenocarpy, and wide disagreement remains as to the taxonomy and systematics of the plant; chemical research should address differences in kinds between synthetic cannabinoids; pharmacopœias include little information related to Cannabis, and disagree on broader classes of herbal medicines, virtually failing to embrace many known Cannabis medicines. Since existing products and compounds fail to be categorised in an evidence-based manner, confusions will likely increase as novel cannabinoid compounds, genetic and biotechnological modifications surge. Conclusions: The lack of clarity is comprehensive: for patients, physicians, and regulators. This study proposes an update of terms at several levels. It points at gaps in morphological descriptions in botany and pharmacognosy and a need for a metaphysical address of cannabinoids. Methods of obtention are identified as a common criterion to distinguish products; the way forward suggests a mutually exclusive nomenclatural pattern based on the smallest common denominator of obtention methods. In the context of a swelling number of Cannabis products being consumed (be it via medical prescription, adult-use, ‘hemp’ foodstuff and cosmetics, or other purposes), this study can assist research, contribute to transparent labelling of products, consumer safety and awareness, pharmacovigilance, medical standards of care, and an update of prevention and harm reduction approaches. It can also better inform regulatory policies surrounding C. sativa, its derivatives, and other cannabinoid-containing products.

Cannabis From Bench To Bedside
David Potter
Poster Talk
DOI: 10.1016/j.aimed.2019.03.120
Cannabis History and Legal Issues
GW Pharma – Early Days
Cannabis Botany and Pharmacology
Cannabinoid Biosynthesis
Changes in illicit ‘medicinal’ cannabis
Licensed medicinal cannabis propagation
Processing and formulation
Looking ahead

Cannabis Prohibition in Egypt, 1880–1939: From Local Ban to League of Nations Diplomacy
Liat Kozma
Middle Eastern Studies (2011) Vol. 47, No. 3, 443–460,
DOI: 10.1080/00263206.2011.553890
German criminologist Sebastian Scheer recently challenged the North American focus of drug policy research. Most US and non-US scholarship, he argued, explains drug prohibition, and especially cannabis prohibition, in terms of American domestic policy and the 1937 American Marihuana Tax Act. The history of international cannabis prohibition is often narrated as an extension of the prohibition era, of American relations with its southern neighbours and with its Mexican immigrants. International prohibition, however, dates back to 1925 and to the League of Nations’ Second Opium Convention, in which the US did not play a leading role at all. The role of Italy, South Africa, Egypt and Turkey in international cannabis prohibition, he claimed, is largely overlooked.1 Focusing on Egypt, this article thus fills a gap in drug policy literature. James Mills’ Cannabis Britanica confronted the North American bias of cannabis scholarship by focusing on the Indian case. In one of his chapters, and then in a more recent article, Mills examined League of Nations debates, and thus Egypt’s role in international prohibition. Mills’ argument, in a nutshell, is that the Egyptian delegation’s uncompromising support of prohibition was a direct consequence of British imperial interests in the 1920s. It was also colonial medical doctors’ reports regarding the connection between cannabis and insanity that convinced the Egyptians that hashish was indeed dangerous. The Egyptian stand on cannabis, he claims, had no precedent in Egypt’s international diplomacy in the late nineteenth or early twentieth centuries.2 Relying, as he does, solely on British and League of Nations’ documentation, Mills presents only a partial picture of how cannabis prohibition was conceived of in Egypt itself, sometimes in conflict and sometimes in dialogue with colonial assumptions and policies. Like Ronen Shamir and Daphna Hacker, in their discussion of the 1894 Indian Hemp Commission, I maintain here that elite notions of class distinctions and civilizing of the lower classes were at the heart of indigenous debates on cannabis consumption and prohibition.3 This article, then, queries Mills’ conclusions by going back almost five decades and examining policies, elite discourses and colonial debates within Egypt.

Cannabis reform: home growing and membership associations but no public smoking
Matthew Xuereb
Times Malta
https://timesofmalta.com/articles/vi...urce-to.905887
Cannabis users will be able to grow plants at home or buy the substance from specially set up associations, according to a cannabis reform bill presented to parliament this week, but smoking a joint in public will remain against the law. Giving details about the bill, Equality minister Owen Bonnici said on Wednesday the police will be enforcing the new rules which stipulate that no one can carry more than seven grams of cannabis at any given time. The new rules were drawn up following a public consultation period on the white paper that had been published last March. Bonnici said that more than 300 submissions were sent in during the public consultation period.

2021 Cannabis Regulatory Landscape, Emerging Trends in North America & Europe
New Frontier Data
http://info.newfrontierdata.com/2021...tory-landscape
With the expansion of global legal cannabis markets over the past decade, regulatory frameworks remain fragmented across national and state borders. In our latest report, 2021 Cannabis Regulatory Landscape: Emerging Trends in North America & Europe, we examine how and why regulations are evolving and explore the impact of cannabis consumer sentiment and behavior as a driving force behind regulatory evolution and standardization.
Key Report Findings:
Consumer sentiment will influence regulations with 66% of cannabis consumers agreeing that legal cannabis is safer than illegal cannabis, and 77% of consumers citing potency as a “very” or “extremely” important driver in purchasing decisions.
Potency caps in future legislation will likely include limits on THC to protect younger and inexperienced users, and in the absence of a unified regulatory structure, states are evaluating potency caps individually.
Inconsistent CBD regulations in Europe have created a patchwork landscape for CBD products where average per capita annual spend on CBD was projected to reach €21Billion in 2020.

Cannabis Trademark Strategy in Jeopardy
By Gregory F. Wesner
7/15/2016
https://www.lanepowell.com/portalres...gy-in-Jeopardy
The Trademark Trial and Appeal Board of the U.S. Trademark Office (TTAB) just rejected a popular strategy for protecting cannabis-related marks at the federal level. Many forward-thinking cannabis businesses have applied to register marks for non-cannabis articles that they offer for sale alongside cannabis-containing products: they seek registration for non-cannabis products, and rely on a halo effect to provide protection for the mark when used on cannabis.
That’s out the window now. On July 14, the TTAB ruled in In re Morgan Brown1 that if a mark is used in association with cannabis products, the fact that it is also used in association with non-cannabis products will not protect the mark from refusal. The TTAB’s ruling summary states as follows:
Because the evidence that Applicant’s mark is being used in connection with sales of a specific substance (marijuana) that falls within both the services identification and the prohibitions of the CSA is unrebutted, we find that Applicant’s retail store services include sales of a good that is illegal under federal law, and therefore encompasses a use that is unlawful.

Changes in cannabis policy and prevalence of recreational cannabis use among adolescents and young adults in Europe—An interrupted time-series analysis
Alexander Carl Gabri, Maria Rosaria Galanti, Nicola Orsini, Cecilia Magnusson
Plos One 17(1):e0261885 DOI: 10.1371/journal.pone.0261885 Background Cannabis policy varies greatly across European countries, but evidence of how such policy
impacts on recreational cannabis use among young people is conflicting. This study aimed
to clarify this association by investigating how changes in cannabis legislation influenced
cannabis use.
Methods Available data on self-reports of recreational cannabis use among individuals aged 15–34 years was retrieved from EMCDDA. Information on cannabis policy changes was categorized as more lenient (decriminalisation or depenalisation) or stricter (criminalisation, penalisation). Countries that had implemented changes in cannabis legislation or had information on prevalence of use for at least eight calendar years, were eligible for inclusion. We used interrupted time-series linear models to investigate changes in country specific trajectories of prevalence over calendar time and in relation to policy changes.
Results Data from Belgium, Czech Republic, Germany, Italy, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden and United Kingdom, for 1994–2017 was available for analyses. Cannabis use varied considerably over the study period and between countries. On average, use was stable or weakly increasing in countries where legislation was not changed or changed at the extremes of the study period (+0.08 percent per year [95% CI -0.01, 0.17 percent]). In contrast, the pooled average use decreased after changes in legislation, regardless of whether it had become more lenient (-0.22 [-1.21, 0.77]) or stricter (-0.44 [-0.91, 0.03]).
Conclusions Our findings do not support any considerable impact of cannabis legislation on the prevalence of recreational cannabis use among youth and young adults in Europe.

Debating the legalisation of recreational cannabis
Editorial
The Lancet Regional Health - Europe 10 (2021) 100269
https://doi.org/10.1016/j.lanepe.2021.100269
Cannabis is the most widely used illicit drug globally, particularly in North America and high-income countries in Europe and Oceania. Although the use of medicinal cannabis is legal in many countries, for example to treat chronic pain, poor appetite, or nausea due to chemotherapy, legalisation of non-medicinal or recreational cannabis is a topic of growing public discussion and debate globally. In a 2019 referendum, almost half (484%) of voters in New Zealand supported the proposal to legalise recreational cannabis, and last month the topic became the centre of debate during the formation of a new federal government in Germany. Other European countries, including Luxembourg, Italy, Malta, Switzerland, Spain, France, and the UK, are also discussing decriminalisation and legalisation of recreational use of cannabis. On Oct 22, 2021, Luxembourg announced a proposal to allow adults (aged >18 years) to use cannabis and to grow up to four plants per household, which would make it the first country in Europe to legalise the production and consumption of the drug if the proposal is passed by their Parliament in early 2022. The proposal is aimed to clampdown drug-related crime and the black-market drugs trade. Legalisation of recreational use of cannabis in Canada and some states in the USA has resulted in a booming industry worth CA$26 billion in Canada and US$18 billion in the USA, with tax revenues amounting to US$387 million in 2020 in the state of Colorado alone. Such economic benefits and the potential for tax revenues are also major factors in the political motivation to legalise cannabis. But these financial incentives should be weighed against the public health implications of legalisation.

Declared Rationale for Cannabis Use Before and After Legalization for Nonmedical Use: A Longitudinal Study of Community Adults in Ontario
Mahmood AminiLari, Jason W. Busse, Jasmine Turna, and James MacKillop
https://www.researchgate.net/publication/359486690_Declared_Rationale_f or_Cannabis_Use_Before_and_Aft er_Legalization_for_Nonmedical _Use_A_Longitudinal_Study_of_C ommunity_Adults_in_Ontario
https://www.liebertpub.com/doi/10.10...rid:crossref.o rg&rfr_dat=cr_pub%20%200pubme d
Objectives: To examine the proportion of individuals using cannabis for medical purposes who reported nonmedical use of cannabis after it became legal to do so.
Materials and Methods: We acquired data from the Population Assessment for Tomorrow’s Health, the Cannabis Legalization Surveillance Study on a subpopulation of participants residing in Hamilton, Ontario, Canada, who
reported using cannabis for medical purposes. Specifically, we acquired data 6 months before, and again 6 months after, legalization of cannabis for nonmedical purposes. We constructed a logistic regression model to explore the association between potential explanatory factors and endorsing exclusively nonmedical use after legalization and reported associations as odds ratios and 95% confidence intervals.
Results: Our sample included 254 respondents (mean age 33 – 13; 61% female), of which 208 (82%) reported both medical and nonmedical use of cannabis (dual motives) before legalization for nonmedical purposes, and 46 (18%) reported cannabis use exclusively for medical purposes. Twenty-five percent (n = 63) indicated they had medical authorization to use medical cannabis, of which 37 (59%) also endorsed nonmedical use. After legalization of nonmedical cannabis,*1 in 4 previously exclusive cannabis users for medical purposes declared dual use (medical and nonmedical), and *1 in 4 previously dual users declared exclusively nonmedical use of cannabis. No individual with medical authorization reported a change to exclusively nonmedical use after legalization. Our adjusted regression analysis found that younger age, male sex, and lacking authorization for cannabis use were associated with declaring exclusively nonmedical use of cannabis after legalization. Anxiety, depression, impaired sleep, pain, and headaches were among the most common complaints for which respondents used cannabis therapeutically. Most respondents reported using cannabis as a substitute for prescription medication at least some of the time, and approximately half reported using cannabis as a substitute for alcohol at least some of the time.
Conclusions: In a community sample of Canadian adults reporting use of cannabis for medical purposes, legalization of nonmedical cannabis was associated with a substantial proportion changing to either dual use (using cannabis for both medical and nonmedical purposes) or exclusively nonmedical use. Younger men without medical authorization for cannabis use were more likely to declare exclusively nonmedical use after legalization.
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Diaspora and drug trafficking in West Africa: A case study of Ghana
Emmanuel Akyeampong
African Affairs 104(416):429-447 July 2005
DOI: 10.1093/afraf/adi015
Emmanuel Akyeampong
This article interrogates the emergence of drug trafficking in contemporary Ghana and West Africa within the context of a global political economy, situated within a deeper historical perspective. It examines the earlier trafficking of cannabis along the coast of West Africa in the colonial period, and the later transnational networks that have emerged to promote international drug trafficking (cocaine and heroin). The article probes how the African diaspora and international travel service these emerging drug networks in Ghana, West Africa, Europe and the Americas. It suggests that the concept of an ‘ideological diaspora’ could shed light on a shared global popular culture, which constitutes a counter culture and rationalizes criminal activities.

Drug laws, bioprospecting and the agricultural heritage of Cannabis in Africa
Chris S. Duvall
Space and Polity 2016
DOI: 10.1080/13562576.2016.1138674
For centuries across most of Africa, farmers have valued Cannabis for multiple reasons. Historic crop selection produced genetic diversity that commercial bioprospectors value for marijuana production. African colonial and post-colonial administrations devalued the crop, enacted Cannabis controls earlier than most locations worldwide, and excluded Cannabis from
agricultural development initiatives. Public agricultural institutions exclude Cannabis as an extension of drug-control policies. Only private companies conserve crop genetic diversity for psychoactive Cannabis, without recognizing intellectual property rights embedded in landraces. Cannabis decriminalization initiatives should stimulate evaluation of its roles in African agriculture, and of worldwide control and management of its genetic diversity.

Dutch Cannabis Regulation Statement
Find DOI or Link
Summary and conclusions
The toleration policy for cannabis has been heavily criticised over the past few years. Many municipalities feel that the policy is no longer suitable given the current problems, and makes no contribution to effectively tackling canna¬bis use. At the same time, there are differing opinions on alternatives. Against that background, the Association of Netherlands Municipalities (VNG) established an administrative working group on cannabis policy. The task set the working group was to chart out the problems and possible solutions, and to issue a recommendation to the national political powers. The working group undertook a literature study, spoke to a variety of experts (from practice) and a number of policy officers within municipal authorities.

UK Home Office
Drug Licensing Factsheet- Cannabis, CBD and other cannabinoids
F
This factsheet represents the Home Office’s view on the domestic control measures applicable to cannabis, cannabidiol (CBD) and controlled cannabinoids. It is intended as a resource for existing licensees and prospective licensees who may need to apply for a licence, having fully assessed any proposals they may wish to make in the context of this guidance and that provided by other regulators.
There are two separate licensing regimes relating to cannabis cultivation, according to whether the varieties are high or low THC (as differentiated in the Misuse of Drugs (Fees) Regulations 2010). This factsheet may also be read in conjunction with published guidance relating to low-THC Industrial Hemp cultivation and use of non-controlled hemp products from fibre and seed available at: https://www.gov.uk/guidance/controll...ndustrial-hemp

Editorial: Purple Haze: Issues on Cannabis Legalization
Stéphane Potvin, Yasser Khazaal , Amine Benyamina and Marc N. Potenza
Front. Psychiatry 12:796032.
doi: 10.3389/fpsyt.2021.796032
https://www.frontiersin.org/articles...021.796032/pdf
Considering the progressive legalization of cannabis across jurisdictions, we prepared a special topic that addresses significant issues relevant for future legalization initiatives. This topic seeks to: (i) characterize the personal characteristics of individuals who support recreational and medical cannabis legalization; (ii) characterize the profiles of people who use cannabis and related compounds such as tetrahydrocannabinol (THC) and cannabidiol (CBD); (iii) document the psychiatric and cognitive consequences of cannabis products, used either for recreational or medical purposes; and (iii) define priority areas deserving more research.
Using data from the 2016 National Drug Strategy Household Survey completed by 21,729 participants in Australia, Chiu et al. investigated the relationship between personal characteristics and support for cannabis legalization. Forty percent and 77% of participants supported the legalization of recreational and medical cannabis use, respectively. Cannabis use and high-risk drinking were associated with increased support of recreational and medical cannabis legalization. Nicotine use was only associated with increased support of recreational cannabis legalization. Although younger age was associated with greater support for legalization of recreational cannabis use, there was more support for legalization of medical cannabis use in older individuals. Psychological distress was associated with a higher likelihood of supporting recreational cannabis legalization, whereas support for medical cannabis legalization was stronger amongst individuals with chronic pain. Nevertheless, cannabis-use status was the strongest statistical predictor of support for both recreational and medical cannabis legalization.

Effects of recreational marijuana legalization on clearance rates for violent crimes: Evidence from Oregon
Guangzhen Wu, Yongtao Li, Xiaodong (Eric) Lang
International Journal of Drug Policy 100 (2022)
DOI: 10.1016/j.drugpo.2021.103528
https://authors.elsevier.com/a/1e6KX3PEroVSUj
One important public concern about the impact of recreational marijuana legalization is how legalization may affect police performance in solving serious crimes. Based on Uniform Crime Reports (UCR) data from 2007 to 2017, this study used difference-in-differences (DID) analysis and the synthetic control method (SCM) to examine the effect of recreational marijuana legalization on the clearance rates for multiple types of violent crimes in Oregon (OR), a state that legalized recreational marijuana in late 2014. Results offer some evidence suggesting a beneficial impact of legalization on violent crime clearances, as manifested by significant increases in the clearance rate for overall violent crimes and that for aggravated assault in OR counties relative to those in the non-legalized states following legalization. Results also demonstrate that the positive effect of legalization on violent crime clearance rates appears to reduce over time.

FDA Grants Cannabis-Based Drug (Caflanone) Orphan Status
Terpene and Testing
https://terpenesandtesting.com/fda-g...orphan-status/
Recently, a drug company by the name of Flavocure was granted orphan drug status for flavonoid-based drug caflonone (FBL-03). Pre-clinical studies conducted at Harvard Medical School demonstrated powerful results in difficult-to-treat animal models of pancreatic cancer. [1] This has led to optimism in both the medical research and patient communities.
Caflanone, aka FBL-03G, is derived from a flavonoid-rich Cannabis sativa cultivar that is endemic to Jamaica. The research team created a synthetic version of the bioactive material which is currently available commercially for clinical research.
Flavocure CEO Dr. Ngeh Toyang highlighted that while less than 200,000 are diagnosed with pancreatic cancer in the U.S. each year, pancreatic cancer is the third leading cause of cancer deaths.

France Enters The Medical Cannabis Industry
Dario Sabaghi
Forbes
https://www.forbes.com/sites/dariosabaghi/2022/03/07/france-enters-the-medical-cannabis-industry/?sh=21cedcf01a0d&xnpe_tifc=4DB uhkbXxD_lh.s_hux74jpZhfEWVjQsV uU_O.VjOIolhkUstIYDhIbA4FP_hNl 8bdhNtIHpbDPNxfb7bDHdb9X74uYd4 IBsbDBNxFhL4DhN&utm_source=eNe ws&utm_campaign=TCS%20Newslet t er%20Week%2011%202022&utm_medi um=email https://www.legifrance.gouv.fr/jorf/...XT000045185582
France tiptoes into the medical cannabis industry, but legalization needs to be implemented.
The French government issued a decree last month that authorizes the cultivation of medical cannabis and the development of its industry.
Although the decree entered into force on March 1, 2022, regulators need to implement it.
The legislation only specifies the conditions and procedures for the cultivation and production of cannabis for medical use
The decree has amended some parts of France's Code of Public Health to allow cultivation, production, manufacture, transport, import, export, and possession of cannabis and its derivatives under medical authorization.
The National Agency for the Safety of Medicines and Health Products (ANSM) will supervise the creation of the medical cannabis supply chain.
The agency has also set up a committee made up of 11 members to examine several aspects of the supply chain, including the identification of the cannabis strains available for the medical treatment, the level of THC and CBD, track and trace systems, the methods of ingesting cannabis-based medicines, and the pharmaceutical quality criteria.
The decree follows a medical cannabis pilot project announced in 2020 and launched in March 2021, which is currently targeting up to 3,000 patients suffering from severe conditions, such as chronic pain and epilepsy, and it will continue to operate, supervised by the ANSM, until March 26, 2023.

Germany, should not compromise when it comes to safety, and must aim for a Pharma quality adult use market.
https://businesscann.com/there-shoul...97&mc_eid=e1bd 2996ba
Trends in illicit drugs based on the analysis of seizures from the Tanzania mainland drugs market
John J.Makangara a Elias Z.Mulima
Forensic Science International: Synergy Volume 3, 2021, 100209
DOI: 10.1016/j.fsisyn.2021.100209
This study aimed at investigating the pattern of confiscated illegal drugs in the Tanzania mainland drug market from 2011 to 2016. The samples used in this study were seized by the police force and other law enforcement agents and were analysed at the Government Chemist Laboratory Authority, GCLA. A total of 90,366 samples weighing 17961.5 Kg were seized and analysed during the six years, an average of 15,061 samples corresponding to 2993.6 Kg per year. The overall results indicated cannabis to be the leading drug in terms of number of cases, number of samples and weights with 51.02%, 60.50% and 56.90%, respectively, but from 2011 to 2015 heroin had the highest percentage of both number of cases and samples by 58.46% and 55.91% of all seizures, respectively followed by cannabis. In terms of weight, heroin accounted for 67.55% and 26.32% in 2011 and 2012, respectively, whereas, the rest of the years’ percentage weights were between 0.50 and 6.00% of the total seizures. The trend indicated a steady decrease in heroin seizures over the six years and an increased cannabis seizure. In 2016 cannabis was 78.28% and 80.89% of the total number of cases and samples, respectively. The results also indicated the decrease of cocaine cases and a significant increase in the amount khat. The year 2016 recorded the highest number of cases and number of samples with a total of 1212 and 48,440, respectively, which resulted from the increase in cannabis seizures by 72.28% and 80.89% of the total number of cases and samples, respectively, as compared to 2015. The second and third highest years were 2013 and 2014, respectively. The least was 2011 with 3493 samples. The trend also indicated heroin seizures were highest in 2011 (62.04%), 2014 (75.31%) and 2015 (79.26%) whereas cocaine seizures kept on decreasing gradually from 31.12% in 2011 to 0.05% in 2016. Khat (Catha edulis) had the highest weight in 2014 and 2016 and ranked second after cannabis with 43.63% of the total weight during 2011–2016 period. Benzodiazepinesand amphetamine type stimulants (ATS) accounted the least in terms of number of cases and number of samples.
Find Pdf

Good news for CBD Industry After Frances Highest court suspends ban on flowers

https://businesscann.com/good-news-f...c2&mc_eid=UNIQ ID

Health Canada
Information for Health Care Practitioners - Medical Use of Cannabis
https://www.canada.ca/en/health-canada/services/drugs-medication/cannabis/information-medical-practitioners.html
  • Information - Cannabis (Marihuana, Marijuana) - a summary of essential information about the use of cannabis for medical purposes
  • Information for Health Care Professionals - a comprehensive monograph on the pharmacology, potential therapeutic uses and risks of using cannabis for medical purposes
  • Daily Amount Fact Sheet (Dosage) - information regarding dosing of cannabis for medical purposes
  • And more via the link above….
[Hemp & the Treaties] Scope and definition of the exemption covering “hemp” in the international drug control Conventions. A total exemption – by purpose
Kenzi Riboulet-Zemouli
2019 Independent research
DOI: 10.13140/RG.2.2.15697.28008
The exemption of drugs used in industrial contexts is a core principle of the International Drug Control Conventions (IDCC). 1 Both the 1961 Single Convention on Narcotic drugs as amended by the 1972 Protocol (C61) and its complementary treaty, the 1971 Convention on Psychotropic Substances (C71) only address medicines and the pharmaceutical sector – thus providing repeated statements that their provisions do not apply to “drugs” when used for any other purposes that medical and scientific ones. In addition to numerous general exemptions for all drugs, the writers of the Conventions, respectful of the important uses of “industrial hemp” but also anticipating advancement in technology and industry, secured the non-inclusion of “hemp” by adding specific dispositions.
1. Exemption for products used in industrial settings
1.1. Exemption of non-drug Cannabis products from the Conventions
1.2. Exemption of Cannabis drugs from the Convention, when used in industry
1.3. Trace-amounts do not justify control
2. Exemption for the cultivation of Cannabis to be used for industrial purposes 3. Conclusion
4. References

High compliance, a lex lata legalization for the non-medical cannabis industry: How to regulate recreational cannabis in accordance with the Single Convention on narcotic drugs, 1961 March 2022
Kenzi Riboulet-Zemouli
https://www.researchgate.net/publication/359163413_High_compliance_a_le x_lata_legalization_for_the_no nmedical_cannabis_industry_How _to_regulate_recreational_cann abis_in_accordance_with_the_Si ngle_Convention_on_narcotic_dr ugs_1961
In the current legal landscape, it is possible to craft policy that combats drug abuse and drug harms, protects human rights, and complies with international drug control law in good faith, by regulating the recreational uses of cannabis products rather than outlawing them. This essay proposes exactly that solution. The international drug control Conventions establish the international legal regime for cannabis, but they are silent on “recreational” or “adult use.” However, they do include broad exemptions in the case of “other than medical and scientific uses in the context of industry.” They are not prohibition treaties, but Framework Conventions on the Control of Some Medicines within the Medical and Pharmaceutical Sectors. Shortcomings in the history of the drug control Conventions, and the current hegemony of one particular interpretation (articulated around prohibition), may have impacted our interpretive frames and discouraged legal scholarship from the study of these exemptions for non-medical uses, purposefully added in the treaty. Via an applicatory contestation of the Conventions reliant on classical methods of treaty interpretation, this essay underlines the relevance of these exemptions in the context of domestic “cannabis legalization” efforts. The legal scheme which applies to the Cannabis plant and its derivatives is two-fold: (1) activities related to medical and scientific purposes are under control, (2) activities for “other than medical and scientific purposes” are exempt from control, provided that two requirements are met: implement effective measures to avoid harms & provide reasonable statistical reporting to the INCB. This existing, good faith, legitimate international legal regime for adult-use cannabis opens an alternative pathway for decision-makers, appeasing rule tension and rerouting international relations on Cannabis matters onto less conflictual tracks

Human Olfactory Detection of Packaged Cannabis.
Gilbert, A. N., & DiVerdi, J. A.
Science & Justice.(2019).
doi:10.1016/j.scijus.2019.10.007
Olfactory detection of cannabis aroma by police officers can be the basis for warrantless searches of motor vehicles in many jurisdictions in the United States. The odor source is these cases is often dried cannabis flower contained in various casual wrappings as well as in more elaborate packaging. Here we investigate whether packaging format alters the detectability of the cannabis. Two cannabis strains and five packaging formats were evaluated. Untrained observers were presented with two containers and asked to identify, based only on smell, the container that held a sample of packaged cannabis (the other container held identical, but empty, packaging material). The results showed that open and casually packaged cannabis was identified with high accuracy, while material packaged in doubly vacuum-sealed plastic was correctly identified at rates no different from chance. The results may help address issues involving the detectability of cannabis aroma in law enforcement and other scenarios.

Identification of Δ9-tetrahydrocannabinol (THC) impairment using functional brain imaging
Jodi M. Gilman, William A. Schmitt, Kevin Potter, Brian Kendzior, Gladys N. Pachas, Sarah Hickey, Meena Makary, Marilyn A. Huestis and A. Eden Evins
Neuropsychopharmacology. Nature
https://www.nature.com/articles/s41386-021-01259-0.pdf
The primary cannabinoid in cannabis, Δ9-tetrahydrocannabinol (THC), causes intoxication and impaired function, with implications for traffic, workplace, and other situational safety risks. There are currently no evidence-based methods to detect cannabis-impaired driving, and current field sobriety tests with gold-standard, drug recognition evaluations are resource-intensive and may be prone to bias. This study evaluated the capability of a simple, portable imaging method to accurately detect individuals with THC impairment. In this double-blind, randomized, cross-over study, 169 cannabis users, aged 18–55 years, underwent functional nearinfrared spectroscopy (fNIRS) before and after receiving oral THC and placebo, at study visits one week apart. Impairment was defined by convergent classification by consensus clinical ratings and an algorithm based on post-dose tachycardia and self-rated “high.” Our primary outcome, PFC oxygenated hemoglobin concentration (HbO), was increased after THC only in participants operationalized as impaired, independent of THC dose. ML models using fNIRS time course features and connectivity matrices identified impairment with 76.4% accuracy, 69.8% positive predictive value (PPV), and 10% false-positive rate using convergent classification as ground truth, which exceeded Drug Recognition Evaluator-conducted expanded field sobriety examination (67.8% accuracy, 35.4% PPV, and 35.4% false-positive rate). These findings demonstrate that PFC response activation patterns and connectivity produce a neural signature of impairment, and that PFC signal, measured with fNIRS, can be used as a sole input to ML models to objectively determine impairment from THC intoxication at the individual level. Future work is warranted to determine the specificity of this classifier to acute THC impairment.

If They’re Doling Out Cannabis “Appellations,” L.A.’s Growers Want In
Julia Barajas
https://cannabiswire.com/2019/08/15/...sQhdvHoUcQW5ic
Since 2018, California has been working toward the rollout of its CalCannabis Appellations Project, which, in short, would give the state’s cannabis growers place-of-origin recognition, much like the term “champagne” is reserved for wine produced in a particular province of northeastern France. (Unsurprisingly, one of the first jurisdictions to embrace this idea was Humboldt, in the heart of California’s cannabis cultivation epicenter, in the north, known as the Emerald Triangle.)
But it’s not so clear that the program, which would be a significant differentiating factor and a branding boon in a competitive market, will be open to all cannabis growers, and at least one group—the indoor cannabis growers of Los Angeles— is pushing back.
Earlier this month, the Southern California Coalition, a trade association that represents all sectors of the cannabis industry, issued a formal request to the state’s Department of Food and Agriculture, calling upon the agency to award an appellation of origin to the cultivators of Los Angeles.

International Drug Control and Human rights: Mounting tensions or innovative convergence?
Karen Mamo
Academia Letters, Article 2667. (2021)
DOI: 10.20935/AL2667
www.academia.edu/50968955/International_Drug_Control_and _Human_righys_Mounting_trnsions_or_innobative_convergence
The World Drug Report 2020 highlights that global figures for illicit drug use continued to increase and in 2018 reached 269 million users. This is in stark difference to the number of drug users in the early 2000’s, with the World Drug Report of 2002 estimating 185 million drug users. Interestingly, the World Drug Report 2020 highlights that although prevalence of use is most common in developed and rich countries, it is poor and developing states that carry the heaviest burden of problematic substance use and problems. (World Drug Report, 2002) (World Drug Report, 2020) Some countries, such as Uruguay, fully regulating the consumption, cultivation and sale of the non-medical use of cannabis in 2013, argue that a similar legislative and paradigm shift is based on the recognition that the criminal approach has been causing a number of ‘unintended consequences’ and that principles of human rights and public health prevail on the criminalised aspect required by the UN Drug Control Conventions. (Ramsey, 2016) In the past ten years, various countries in Europe, the Americas, and the Caribbean, have been moving towards a less punitive approach and more towards a human rights-based drug policy. These new legislative changes have been directly challenging the status quo that has relatively dominated the international approach to address and curtail the use and trafficking of illicit substances. This situation is creating an environment of potential tensions or opportunity for innovative convergence between obligations falling under the UN Drug Control Conventions of 1961, 1977 and 1988 and those originating from International Human Rights

Islam and cannabis: Legalisation and religious debate in Iran
Maziyar Ghiabia, Masoomeh Maarefvandb, Hamed Baharib, Zohreh Alavi

International Journal of Drug Policy 56 (2018) 121–127
DOI: 10.1016/j.drugpo.2018.03.009
https://www.researchgate.net/publica...and_cannabis_L egalisation_and_religious_deba te_in_Iran

Iran is currently discussing cannabis and opium regulations, which could bring a legalisation of drug consumption through a state supervised system. The article engages with the question of cannabis by looking at the legal interpretation of religious authorities in the Islamic Republic of Iran. The choice of Iran is justified for several reasons: firstly, Iran has a long history of drug use and cannabis has been part of the country’s intoxicant traditions since times immemorial; secondly, the Iranian state is unique in that it combines religious exegesis with political machination through official channels; finally, among all Middle East and Islamic countries, Iran is at the avant-garde in experimenting in the field of drugs policy which makes an excellent case for the study of cannabis regulation. The article is the result of a direct engagement with Iran’s leading Shi’a authorities, the maraje’-e taqlid, ‘source of emulation’. The authors redacted a list of eight questions (estefta’at) about the status of cannabis in Iranian society. It questioned cannabis’ legality in Islam, its aspects of its social-religious life. Based onthe responses, the authors analysed the difference in opinions among the religious scholars and speculate on the possibility of policy reform. Given the dearth of scholarly work about illicit drugs in the Islamic world, about which many readers might not be familiar, the article opens with an overview of the place of cannabis in the history of Islamic societies. It discusses terminological ambiguities, references in religious texts and traditions, and the general interpretations within Muslim religious schools of thought. Then, it discusses the status of cannabis in contemporary Iran before tackling the responses provided by the religious scholars. Eventually, the paper puts forward reflections about the potential implications for future policy developments on cannabis.


Legalizing Medical Cannabis in Lebanon: the Complex Interface Between Medicine, Law, Ethics, and Economics
Joseph El-Khoury, Rami Bou Khalil, Anthony Nemer, and Sami Richa
Cannabis and Cannabinoid Research
DOI: 10.1089/can.2020.0108
https://www.researchgate.net/publica...zing_Medical_C annabis_in_Lebanon_the_Complex _Interface_Between_Medicine_La w_Ethics_and_Economics
In April 2020, after decades of discussions and controversy, the Lebanese parliament voted a law legalizing the cultivation, production, and sale of cannabis for medicinal purposes. Although the law leaves several unanswered questions and awaits implementation, the symbolic nature of this step in recognizing a positive role of cannabis in the local economy is significant on a regional level. The Arab world has traditionally been conservative when it comes to all drugs-related policies. Cannabis is largely demonized with heavy sentences served to anyone suspected of using selling, let alone planting cannabis. Despite a few countries considered producers and consumers of substances, governing authorities have remained immune to the liberalization trend encountered in western countries. The social experiment taking place in Lebanon is fraught with risks, given the unstable political situation and chronic economic challenges. The reactions to the law have been mixed with several scientific bodies such as the Lebanese Psychiatric Society criticizing the absence of proper consultation of stakeholders. The absence of consistency in enforcing established drugs policies or seriously debating the decriminalization of cannabis use raises concerns over the establishment of a two-tier approach toward drugs, driven solely by economic imperatives

Letter to the Editor: Medical and Industrial Cannabis Legalization in Morocco and the Addictions Care Outlook of Youth: First Prospective Study from African Arabic Experience
Ismail Rammouz and Saïd Boujraf
Cannabis and Cannabinoid Research (2021)
DOI: 10.1089/can.2021.0051
To the Editor,
On March 11, 2021, the Government of Morocco approved a new proclamation allowing the legal use of cannabis. This would allow commercializing and use of cannabis for medical purposes. Besides, the consented law predetermined the submission of all cannabis-related
activities to government including farming, production, manufacturing, transport, marketing, export, and import. The Moroccan government law informed to set up a national agency responsible for the coordination, development, and control of any agricultural and industrial action involving cannabis. The law statement also recommended to farmers to join cannabis agricultural cooperatives with very specific conditions. Finally, the law announced the implementation of sanctions against the offenders of the law. However, still recreational and festive use is legally prohibited. Morocco was ranked the world’s leading producer of cannabis resin by the United Nations Office of Drugs and Crime (UNODC) in the annual report. Indeed, > 37,000 hectares are cultivated in 2016 mainly in the Rif region holding very mountainous provinces in northern Morocco on the Mediterranean Sea. The country is currently the second worldwide producer after Afghanistan. The approved law should certainly bring some economic benefits to the country and improve the social condition of farmers. However, cannabis addiction care and prevention structures dedicated to youth are inquired about their future role.

Limitations to the Dutch cannabis toleration policy: Assumptions underlying the reclassification of cannabis above 15% THC.
DOI: 10.1016/j.drugpo.2016.02.011
The Netherlands has seen an increase in ?9-tetrahydrocannabinol (THC) concentrations from approximately 8% in the 1990s up to 20% in 2004. Increased cannabis potency may lead to higher THC-exposure and cannabis related harm. The Dutch government officially condones the sale of cannabis from so called 'coffee shops', and the Opium Act distinguishes cannabis as a Schedule II drug with 'acceptable risk' from other drugs with 'unacceptable risk' (Schedule I). Even in 1976, however, cannabis potency was taken into account by distinguishing hemp oil as a Schedule I drug. In 2011, an advisory committee recommended tightening up legislation, leading to a 2013 bill proposing the reclassification of high potency cannabis products with a THC content of 15% or more as a Schedule I drug. The purpose of this measure was twofold: to reduce public health risks and to reduce illegal cultivation and export of cannabis by increasing punishment. This paper focuses on the public health aspects and describes the (explicit and implicit) assumptions underlying this '15% THC measure', as well as to what extent these are supported by scientific research. Based on scientific literature and other sources of information, we conclude that the 15% measure can provide in theory a slight health benefit for specific groups of cannabis users (i.e., frequent users preferring strong cannabis, purchasing from coffee shops, using 'steady quantities' and not changing their smoking behaviour), but certainly not for all cannabis users. These gains should be weighed against the investment in enforcement and the risk of unintended (adverse) effects. Given the many assumptions and uncertainty about the nature and extent of the expected buying and smoking behaviour changes, the measure is a political choice and based on thin evidence.

Malta becomes first European nation to approve cannabis for personal use
Reuters
Christopher Scicluna; Editing by Robert Birsel and Crispian Balmer
https://www.reuters.com/markets/comm...se-2021-12-14/
VALLETTA, Dec 14 (Reuters) - Malta became the first European country to allow limited cultivation and possession of cannabis for personal use, following a vote in parliament on Tuesday.
Luxembourg announced similar plans in October but its parliament has yet to give its green light to the measure.
The law, which was approved by 36 votes to 27, allows adults in Malta to possess up to seven grams (0.25 oz) of cannabis and grow up to four plants.
The bill was promoted by Equality Minister Owen Bonnici, who says Malta has adopted a "harm-reduction approach" with the establishment of an authority to regulate the sale of cannabis for personal use by adults from non-profit associations.
Smoking cannabis in public will remain against the law and anyone caught consuming cannabis in front of a child will be liable for fines of between 300 and 500 euros ($340-$564).
The legislation was rushed through parliament in the face of fierce criticism from the centre-right opposition, medical associations and the church who complained that their requests to water down the proposals were ignored.
Bonnici has rejected suggestions that the law will increase drug abuse on the Mediterranean island.
"The government is in no way urging adults to resort to cannabis use or promoting a cannabis culture. The government always urges people to make healthier choices," the minister wrote in an op-ed in the Sunday Times of Malta newspaper.
Malta is also seeking to position itself as a European leader in the production of medical cannabis having approved legislation in 2018 to permit the production of such cannabis for medicinal and research purposes.
The Netherlands also has a liberal attitude towards cannabis, allowing the sale of small quantities in a policy aimed at managing crime and health risks, though officially it is illegal.

Malta Is To Legalize Cannabis For Personal Use, Social Clubs, But Not Sales
https://www.forbes.com/sites/dariosa...e4&mc_eid=UNIQ ID&sh=2e9f03eb6c86

Marijuana and driving impairment
Barry K. Logan .
Chapter 12 in book:
(Marijuana and the Cannabinoids ed. by Mahmoud A ElSohly Pg 277-293)
http://www.datia.org/datia/resources/mjdriving.pdf
After alcohol, Marijuana is the most popular recreational drug in North America. Its effects are largely predictable in type, but not in degree, although they do appear in a roughly does-dependent manner. The effects discussed here make a very convincing case for the potential for marijauan to impare driving, although as noted, the extent to which that potential is realized in a given case will be related to many factors.



Marijuana Moment April 20 2022
https://www.marijuanamoment.net/new-jersey-marijuana-sales-will-start-day-after-4-20-and-ag-says-police-can-use-while-off-duty/?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email
The governor of New Jersey on Thursday announced that adults 21 and older will be able to buy marijuana from select dispensaries starting on April 21—the day after the unofficial cannabis holiday 4/20. And a new memo from the state attorney general’s office says that police can partake, too, as long as they’re off duty.
Gov. Phil Murphy (D) touted the long-awaited first round of adult-use retailer approvals from the New Jersey Cannabis Regulatory Commission (CRC), calling the action a “historic step in our work to create a new cannabis industry.”

Medical Cannabis in Lebanon: History & Therapeutic, Ethical, and Social Challenges. A Narrative Review
Battoul Fakhry, Mostafa Abdulrahim, Mirna N Chahine
DOI: 10.26502/acbr.50170156
https://www.researchgate.net/publication/350043789_Medical_Cannabis_in_ Lebanon_History_Therapeutic_Et hical_and_Social_Challenges_A_ Narrative_Review
March 2021 Legalizing cannabis for both medical and recreational purposes is currently a worldwide trend. Lebanon, a Middle Eastern country, recently became the first Arabic state to legalize the cultivation of medical and industrial cannabis amid a huge economic crisis and the COVID 19 pandemic. However, the state of the art regarding the Lebanese cannabis is still poorly defined. Hence, our aim is to highlight medical and social concerns, and to clearly define the ethical framework for medical cannabis prescription and usage. Studies showed cannabis therapeutic potentials in treating numerous diseases such as epilepsy (high level of evidence), multiple sclerosis(moderate level of evidence), PTSD (low level of evidence), as well as in alleviating symptoms related to other conditions, for instance weight loss in HIV patients and chemotherapy induced nausea and vomiting (high level of evidence in both cases). Nonetheless, legalizing a drug known worldwide for its abuse potential could hinder several ethical principles. Whenever prescribing medical cannabis, the physician should respect patient’s autonomy, assess the risk benefit ratio of his actions, and act by justice. Moreover, authorizing cannabis usage for medical purposes could give rise to stigmatization, and further increase the burden on a society already struggling with the issues of self-medication, unemployment, and drugs diversion phenomena. By legalizing medical cannabis, Lebanon has entered a new chapter that warrants assessments, regulations, and readiness in order to insure a safe and successful experience.

Medico-ethical Considerations for Legalisation of Medicinal Cannabis in Malaysia (thesis 2022)
AIMI BINTI MOHD YUNUS
https://zenodo.org/record/6351516#.YjF_7lzMKqA
There is an abundance of international studies that have lauded the benefits of medicinal cannabis for various illnesses. Likewise, local studies on cannabis in Malaysia that initially revolve around the harmful effects of cannabis misuse are increasingly shifting towards the benefits of medicinal use. Pursuant to scientific discovery of the medicinal benefits of cannabis, a myriad of literature begins to focus on the legality of utilising cannabis for medicinal purposes. Whilst many countries around the world, some of those including South East Asia countries have legalised medicinal cannabis, Malaysia is still lagging behind. As of now, there is no local study focusing on the legalisation of medicinal cannabis, leaving a gap in providing a reference to Malaysia's way forward on this issue. Under the current legislative regime, the use of cannabis is prohibited under the Dangerous Drugs Act (DDA) 1952 [Act 234]. However, the recent case of Muhammad Lukman Mohamad has sparked national interest that triggered calls for decriminalisation and legalisation of cannabis for medical reasons. Following these calls, it is pertinent to examine and critically analyse the medical, ethical, and legal considerations on the legalisation of medicinal cannabis in Malaysia. Such consideration is deemed necessary to protect the health and safety of patients. This research adopts an exploratory research design and employs a qualitative research approach in examining medical, ethical, legal, and theological considerations towards the legalisation of medicinal cannabis. Comparative legal analysis covers substantive and procedural components of jurisdictions in selected countries that have legalised medicinal cannabis such as the United Kingdom (UK), Canada, and Thailand. Examination of medico-ethical considerations is done using Biomedical Ethics Theory and Philosophical Theory. These considerations provide a comprehensive analysis of the legalisation of medicinal cannabis in Malaysia that will contribute to the current body of knowledge. This research proceeds to make several recommendations deemed relevant towards the legalisation of medicinal cannabis in Malaysia.

Modelling opinion on legalization of recreational use of marijuana with a survey of Rio de Janeiro residents.
https://www.academia.edu/46574069/Modelling_opinion_on_legalizat ion_of_recreational_use_of_mar ijuana_with_a_survey_of_Rio_de _Janeiro_residents

More than half of Europeans support cannabis legalization -report
Rithika Krishna

April 7, 2022
https://www.reuters.com/world/europe...rt-2022-04-07/
More than half of the European population support legalization of adult use of cannabis and around 30% of them are interested in purchasing it, according to polling data by industry consultants on Thursday.
Europe's liberal approach could reap multiple financial and economic benefits as seen in the United States, which has witnessed a surge in cannabis use during pandemic-induced lockdowns.
While majority of Europeans support regulated cannabis shops, most do not favor growing the plant at home, according to the report by London-based consultancy Hanway and pot producer Curaleaf International.
The report comes a week after the U.S. House of Representatives on Friday passed a bill to end the federal ban on marijuana, which has created legal headaches for users and businesses in the states that have legalized it.
"We see the European market as three to four years behind (the U.S.), but it actually looks like Europe may initiate sweeping reform before the United States," said Boris Jordan, executive at U.S.-based Curaleaf.
Many European countries, including Germany, have legalized cannabis for limited medicinal purposes, while others have decriminalized its general use. Malta became the first European country to allow limited cultivation and personal use of cannabis. read more
The European cannabis market is expected to cross 3 billion euros ($3.27 billion) in annual revenue by 2025, up from about 400 million euros last year, according to a report from research firm Prohibition Partners. Germany has been the biggest market in the continent so far.
"There is clear political desire and willingness in Germany to legalize recreational use," Joe Bayern, chief executive officer of Curaleaf, told Reuters.
"Given it (Germany) is the largest economy in Europe, we think it will lead the way and create a domino effect for rest of the continent," Bayern said.

Morocco to regulate Legal Cannabis
https://www.africanews.com/2022/06/...=TCS Newsletter Week 23 2022&utm_medium=email

New UAE drug law: Treatment for first-time offenders; deportation, tougher penalties clarified
https://www.khaleejtimes.com/governm...ation-outlined

(Pot)Heads in Beds: The Effect of Marijuana Legalization on Hotel Occupancy in Colorado and Washington
Brian Meehan, Corey J. Rusko, E. Frank Stephenson
Journal of Regional Analysis & Policy 50(1): 46–53 (2020)
https://jrap.scholasticahq.com/artic...and-washington
This paper uses monthly hotel occupancy data to examine the effect of marijuana legalization on tourism in Colorado and Washington. The results indicate a large increase in hotel rooms rented in Colorado, with a considerably smaller effect for Washington. The effects are larger in both states once commercial sale is permitted above and beyond legalizing possession and individual cultivation.

Preliminary Eye-Tracking Data as a Nonintrusive Marker for Blood D-9-Tetrahydrocannabinol
Concentration and Drugged Driving
Ali Shahidi Zandi, Felix J.E. Comeau, Robert E. Mann, Patricia Di Ciano, Eliyas P. Arslan,Thomas Murphy, Bernard Le Foll, and Christine M. Wickens
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0141
https://www.liebertpub.com/doi/10.1089/can.2020.0141
Background: Cannabis is one of the drugs most often found in drivers involved in serious motor vehicle collisions. Validity and reliability of roadside cannabis detection strategies are questioned. This pilot study aimed to investigate the relationship between eye characteristics and cannabis effects in simulated driving to inform potential development of an alternative detection strategy.
Materials and Methods: Multimodal data, including blood samples, eye-tracking recordings, and driving performance
data, were acquired from 10 participants during a prolonged single-session driving simulator experiment. The study session included a baseline driving trial before cannabis exposure and seven trials at various times over *5 h after exposure. The multidimensional eye-tracking recording from each driving trial for each participant was segmented into nonoverlapping epochs (time windows); 34 features were extracted from each epoch. Blood D-9-tetrahydrocannabinol (THC) concentration, standard deviation of lateral position (SDLP), and mean vehicle speed were target variables. The cross-correlation between the temporal profile of each eye-tracking feature and target variable was assessed and a nonlinear regression analysis evaluated temporal trend of features following cannabis exposure.
Results: Mean pupil diameter (r = 0.81–0.86) and gaze pitch angle standard deviation (r = 0.79–0.87) were significantly correlated with blood THC concentration ( p < 0.01) for all epoch lengths. For driving performance variables, saccade-related features were among those showing themost significant correlation (r = 0.61–0.83, p < 0.05). Epoch length significantly affected correlations between eye-tracking features and speed ( p < 0.05), but not SDLP or blood THC concentration ( p > 0.1). Temporal trend analysis of eye-tracking features after cannabis also showed a significant increasing trend ( p < 0.01) in saccade-related features, including velocity, scanpath, and duration, as the influence of cannabis decreased by time. A decreasing trend was observed for fixation percentage and mean pupil diameter. Due to the lack of placebo control in this study, these results are considered preliminary.
Conclusion: Specific eye characteristics could potentially be used as nonintrusive markers of THC presence and driving-related effects of cannabis

Prices and Purchase Sources for Dried Cannabis Flower in the United States, 2019–2020
Elle Wadsworth, Pete Driezen, Rosalie Liccardo Pacula, Beau Kilmer, and David Hammond
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0232
Introduction: The price of cannabis has major implications for public health, public safety, social equity, and government revenues. This article examines prices and sources of purchased dried cannabis flower among consumers
facing different state laws in the United States.
Methods: Repeat cross-sectional survey data were collected from the International Cannabis Policy Study in 2019 and 2020. U.S. respondents were recruited through online commercial panels, ages 16–65, and purchased
dried flower in the past year (n = 9766). Weighted binary logistic regression models examined legal purchasing in states that had legalized recreational cannabis.
Results: Compared with respondents in states with recreational stores, respondents living in ‘‘illegal,’’ ‘‘medical,’’ and ‘‘recreational’’ states without stores were associated with paying a higher unit price of dried flower ( + 20.5%, + 23.6%, + 27.4%, respectively; all p < 0.05). The majority of respondents in states with recreational stores last purchased from stores/dispensaries (2019: 66.6%; 2020: 62.0%) and the odds of purchasing legally was greater with each additional year after stores opened (adjusted odds ratio = 1.48, 95% confidence interval: 1.37, 1.60).
Conclusions: Cannabis prices and purchase behaviors are strongly influenced by its legal status and presence of stores. After states legalize for recreational purposes, it takes multiple years for the legal market to become established
as the number of retail stores increase and prices decrease. The findings demonstrate that consumers use sources that they are legally allowed to access, suggesting an increased number of physical retail stores and online delivery services could expand uptake of legal sources in states with recreational cannabis laws.
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Recreational cannabis use impairs driving performance in the absence of acute intoxication
M. Kathryn Dahlgrena,b,c , Kelly A. Sagara,b,c , Rosemary T. Smitha,b , Ashley M. Lambrosa,b , Madeline K. Kuppea,b , Staci A. Gruber
Drug and Alcohol Dependence (2020)
Background
Across the nation, growing numbers of individuals are exploring the use of cannabis for medical or recreational purposes, and the proportion of cannabis-positive drivers involved in fatal crashes increased from 8 percent in 2013 to 17 percent in 2014, raising concerns about the impact of cannabis use on driving. Previous studies have demonstrated that cannabis use is associated with impaired driving performance, but thus far, research has primarily focused on the effects of acuteintoxication.
Methods
The current study assessed the potential impact of cannabis use on driving performance using a customized driving simulator in non-intoxicated, heavy, recreational cannabis users and healthy controls (HCs) without a history of cannabis use.
Results
Overall, cannabis users demonstrated impaired driving relative to HC participants with increased accidents, speed, and lateral movement, and reduced rule-following. Interestingly, however, when cannabis users were divided into groups based on age of onset of regular cannabis use, significant driving impairment was detected and completely localized to those with early onset (onset before age 16) relative to the late onset group (onset ?16 years old). Further, covariate analyses suggest that impulsivity had a significant impact on performance differences.
Conclusions
Chronic, heavy, recreational cannabis use was associated with worse driving performance in non-intoxicated drivers, and earlier onset of use was associated with greater impairment. These results may be related to other factors associated with early exposure such as increased impulsivity.

Regulating Marijuana in California
Patrick Murphy and John Carnevale
Public Policy Institute of California 2016
https://www.ppic.org/publication/reg...in-california/
California led the nation in 1996 when it declared that marijuana use for medical purposes would no longer be a crime. Twenty years later, four states and the District of Columbia have legalized recreational marijuana use. In November 2016, Californians will be asked whether the state should follow suit. This report does not consider the wisdom of marijuana legalization. Instead, it takes the view that, if recreational marijuana use becomes legally sanctioned, then the debate must turn to how to design regulations that reconcile important, but differing policy goals. These include, among others, limiting the impact of the illegal marijuana market, reducing harm to public health and safety, and raising revenue for the state. The report explores the approach Washington and Colorado have taken to regulating recreational marijuana markets. These two states have histories of legal recreational marijuana that, though brief, are the longest in the nation. What lessons can be gleaned from these experiments? Both states have designed mechanisms to track legal cultivation and production, thereby reducing the diversion of marijuana to the illegal market. They also tax marijuana transactions, collecting tens of millions of dollars in revenue. And it appears that neither overall use nor use by young people has risen dramatically. However, as in California, levels of use were already higher in those states than in the rest of the country.

Regulatory approaches to cannabidiol in the European Union: are market developments sowing the seeds of confusion?
Addiction 2021 editorial
doi:10.1111/add.15587
https://onlinelibrary.wiley.com/doi/...1111/add.15587
The rapid growth in marketing cannabidiol poses a challenge to current European regulatory frameworks that can have important implications for consumer safety, public health, and established industries

Regulatory status of pesticide residues in cannabis: Implications to medical use in neurological diseases
Dorina V. Pinkhasova, Laura E. Jameson, Kendra D. Conrow, Michael P. Simeone, Allan Peter Davis, Thomas C. Wiegers, Carolyn J. Mattingly, Maxwell C.K. Leung
Current Research in Toxicology 2 (2021) 140–148
DOI: 10.1016/j.crtox.2021.02.007
Medical cannabis represents a potential route of pesticide exposure to susceptible populations. We compared the qualifying conditions for medical use and pesticide testing requirements of cannabis in 33 states and
Washington, D.C. Movement disorders were the most common neurological category of qualifying conditions, including epilepsy, certain symptoms of multiple sclerosis, Parkinson’s Disease, and any cause of symptoms leading to seizures or spasticity. Different approaches of pesticide regulation were implemented in cannabis and cannabisderived products. Six states imposed the strictest U.S. EPA tolerances (i.e. maximum residue levels) for food commodities on up to 400 pesticidal active ingredients in cannabis, while pesticide testing was optional in three states. Dimethomorph showed the largest variation in action levels, ranging from 0.1 to 60 ppm in 5 states. We evaluated the potential connections between insecticides, cannabinoids, and seizure using the Comparative Toxicogenomics Database. Twentytwo insecticides, two cannabinoids, and 63 genes were associated with 674 computationally generated chemicalgenephenotypedisease (CGPD) tetramer constructs. Notable functional clusters included oxidationreduction process (183 CGPDtetramers), synaptic signaling pathways (151), and neuropeptide hormone activity (46). Cholinergic, dopaminergic, and retrograde endocannabinoid signaling pathways were linked to 10 genetic variants of epilepsy patients. Further research is needed to assess human health risk of cannabinoids and pesticides in support of a national standard for cannabis pesticide regulations

Should the supply of cannabis be legalised now?
Meacher, M., Nutt, D., Liebling, J., Murray, R. M., & Gridley, A.
BMJ, l4473. (2019).
doi:10.1136/bmj.l4468
Yes—Molly Meacher, David Nutt, Jonathan Liebling
Opponents of legalisation and regulation of the supply of cannabis raise legitimate fears that it could lead to more use and therefore increased mental health harms. However, recent research in the US, where some states have legalised cannabis supply for adult social use, suggests that cannabis consumption has increased irrespective of its legal status in each state.1 Legalising and regulating cannabis confers many benefits over the current illegal and unregulated supply. Here we focus on benefits that directly affect mental and physical health.
No—Robin M Murray, Adam Gridley
A huge pharmacological experiment is currently under way with the brains of young Americans. It would be wise to watch how the results unfold before deciding whether to follow their example

South Dakota: Governor Signs Legislation Preserving Limited Home Grow Rights for Patients
BY NORML MARCH 24, 2022
https://norml.org/news/2022/03/24/so...-for-patients/
Pierre, SD: Republican Gov. Kristi Noem has signed legislation (SB 24) into law preserving the ability of qualified patients to home cultivate limited quantities of cannabis.
Voters in November 2020 overwhelmingly decided in favor of a ballot initiative (Measure 26) permitting qualified patients to possess, grow, and purchase medical cannabis — a measure which Gov. Noem opposed. Since that time, on two occasions, House lawmakers have voted in favor of legislation to eliminate patients’ home cultivation rights.
“Permitting limited home cultivation provides patients with the ability to have reliable, affordable, and consistent access to their medicine,” NORML’s Deputy Director Paul Armentano said. “Seventy percent of voters approved this right at the ballot box and it is reassuring to see that a majority of lawmakers, and the Governor, ultimately decided to respect the voters’ decision.”
Senate Bill 24, as amended in conference committee, permits registered patients to cultivate a total of four marijuana plants (two mature, two immature) and to legally possess the harvest from those plants

Status and Impacts of Recreational and Medicinal Cannabis Policies in Africa: A Systematic Review and Thematic Analysis of Published and ‘‘Gray’’ Literature
Chenai Kitchen, John Alimamy Kabba, and Yu Fang
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0110
Background: Despite cannabis’s societal ubiquity, several African states remain traditional prohibitionists. However, cannabis is becoming a more explored frontier from a health, human rights, and monetary perspective. A number of African countries have taken to tailoring their policies to better engage in emerging global dialogs. Nevertheless, the focus is majorly on the crop’s financial appeal with less consideration on impacts of policies. This review aimed to specifically focus on the identification of existing or pending policies, indicating national positioning in terms of recreational and medicinal cannabis use and summarizing publications addressing related impacts in Africa.
Methods: We systematically searched six academic research databases (including Google Scholar), Google, country specific websites, and websites of relevant organizations. Included publications were in English and published between January 1, 2000, and November 31, 2020 (with exception granted to official legislation not in
English and/or published earlier than 2000, but still in effect). Reference lists of included publications were screened for potentially relevant publications. Results were synthesized thematically and descriptively.
Results: Cannabis is Africa’s most consumed illegal substance, its use entrenched in social, political, historical, economic, and medicinal ties. African users constitute a third of the worldly total and cultivation is a major activity. Policies have led to prison overcrowding, accelerated environmental damage, and sourced regional instability.
South Africa, Seychelles, and Ghana have decriminalized personal use with Egypt and Mozambique exploring similar legislation. Eleven countries have existing or pending medicinal cannabis-specific provisions. South Africa and Seychelles stand out as having regulations for patients to access medicinal cannabis. Other countries have made provisions geared toward creating export markets and economic diversification.
Conclusion: Cannabis policy is a composite and complex issue. Official stances taken are based on long withstanding narratives and characterized by a range of contributing factors. Policy changes based on modern trends should include larger studies of previous policy impacts and future-oriented analysis of country-level goals incorporated with a greater understanding of public opinion.
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Step toward Roadside Sensing: Noninvasive Detection of a THC Metabolite from the Sweat Content of Fingerprints
• Erica Brunelle, Brenna Thibodeau, Alyssa Shoemaker, Jan Halámek
ACS Sens. 2019, 4, 12, 3318-3324
10.1021/acssensors.9b02020
The sudden increase in states legalizing marijuana has forced law enforcement into a situation where the use and consumption are legal, but there are no limitations for what is acceptable for driving or operating machinery. Using ultraviolet–visible (UV–vis) spectroscopy, fingerprints from volunteers who had used marijuana were analyzed via a competitive immunoassay for the detection of ?9-tetrahydrocannabinol (?9-THC), the main psychoactive component of marijuana, and 11-nor-9-carboxy-THC (THC-COOH), one of the main metabolites produced in the body following the use/consumption of THC-related products. In this research, the THC-COOH metabolite and the enzyme-labeled conjugate compete against each other as the antigens for the system. The antibody used in this assay has a greater affinity for the metabolite; so, as its concentration increases, the absorbance of the system decreases due to less binding of the enzyme-labeled conjugate.

Switzerland: Pharmacies sell cannabis
https://www.apotheke-adhoc.de/nachri...ba&mc_eid=UNIQ ID

Tetrahydrocannabinol (THC) Detection using Semiconductor-enriched Single-Walled Carbon Nanotube Chemiresistors
Hwang, S. I., Franconi, N. G., Rothfuss, M. A., Bocan, K., Bian, L., White, D. L., … Star, A.
ACS Sensors. (2019). 4, 2084?2093
doi:10.1021/acssensors.9b00762
Semiconductor-enriched single-walled carbon nanotubes (s-SWCNTs) have potential for application as a chemiresistor for the detection of breath compounds, including tetrahydrocannabinol (THC), the main psychoactive compound found in the marijuana plant. Herein we show that chemiresistor devices fabricated from s-SWCNT ink using dielectrophoresis can be incorporated into a hand-held breathalyzer with sensitivity toward THC generated from a bubbler containing analytical standard in ethanol and a heated sample evaporator that releases compounds from steel wool. The steel wool was used to capture THC from exhaled marijuana smoke. The generation of the THC from the bubbler and heated breath sample chamber was confirmed using ultraviolet?visible absorption spectroscopy and mass spectrometry, respectively. Enhanced selectivity toward THC over more volatile breath components such as CO2, water, ethanol, methanol, and acetone was achieved by delaying the sensor reading to allow for the desorption of these compounds from the chemiresistor surface. Additionally, machine learning algorithms were utilized to improve the selective detection of THC with better accuracy at increasing quantities of THC delivered to the chemiresistor.

The current state of delta-8 THC
American Journal of Emergency Medicine xxx (xxxx) xxx (2021) DOI: 10.1016/j.ajem.2021.06.066 Delta-9 tetrahydrocannabinol (THC) is the main pharmacologically active ingredient of the Cannabis plant and is responsible for the clinical effects associated with the use of this substance.While possession and/or use of cannabis has been legalized in many states, it is still currently illegal to use, sell, or possess cannabis, according to the federal government of the United States. The United States Agriculture Improvement Act of 2018 (“2018 Farm Bill”) was passed with the purpose of supporting agricultural programs in the United States. Legislation included in the 2018 FarmBill approved the legalization of hemp products. Hemp is a variety of the Cannabis sativa plant that is grown specifically for industrial use, including the manufacture of textiles, biodegradable plastics, and paint [1]. Prior to incorporation of the 2018 Farm Bill, the growth and cultivation of hemp was allowed only under state-sanctioned programs [2]. The 2018 Farm Bill removed hemp from the federal list of controlled substances, thereby allowing for the commercial sale and use of hemp-derived products, with the strict definition that hemp cannot contain more than 0.3% delta-9 THC [2]. The 2018 Farm Bill does not specifically address the possession, use, or sale of products that can be derived from hemp, including other biologically active cannabinoids such delta-8 THC

The Rise and Decline of Cannabis Prohibition the History of cannabis in the UN drug control system and options for reform
Dave Bewley-Taylor, Tom Blickman, Martin Jelsma
TRANSNATIONAL I N S T I T U T E March 2014
https://www.tni.org/files/download/r...ecline_web.pdf
Cannabis has long been a substance drawing much attention within the international drug control regime, a system currently based upon the 1961 Single Convention on Narcotic Drugs. Today the regime landscape is changing. Faced with particular challenges and democratic decisions, a number of jurisdictions are moving beyond merely tolerant approaches to the possession of cannabis for personal use to legally regulating markets for the drug. In November 2012 voters within the U.S. states of Colorado and Washington passed ballot initiatives to tax and regulate cannabis cultivation, distribution and consumption for non-medical purposes. Just over a year later, Uruguay legislated state regulation of the entire chain of the domestic cannabis market for medical, industrial and recreational use. These policy shifts go well beyond the permitted prohibitive boundaries of the UN drug control conventions. They represent a break with an historical trajectory founded on dubious science and political imperatives. And they have thrown the global regime into a state of crisis, as this report will argue. This publication is a joint effort of the Transnational Institute in Amsterdam and the Global Drug Policy Observatory at Swansea University. Research has been going on in various stages for about two years, and interim results were presented at the Seventh Annual Conference of the International Society for the Study of Drug Policy at the Universidad de los Andes, in Bogotá, in May 2013 and further discussed in an expert seminar on cannabis regulation in October 2013 in Amsterdam. Many academics, government officials and experts from NGOs and international agencies have provided useful comments on earlier drafts, but needless to say the end result is the sole responsibility of the authors. This final report will be first presented at the 57th session of the UN Commission on Narcotic Drugs (CND) in Vienna, 13-21 March 2014.

The supply of hashish to Europe
Pierre-Arnaud Chouvy
Report prepared for the EMCDDA
https://www.geopium.org/wp-content/u...ish_Europe.pdf
Hashish production in the world is a phenomenon that is far from being satisfactorily studied and understood and it must be stressed from the outset that further research, and especially field-based research in production countries, is needed in order to properly investigate the supply of hashish to Europe. Most of the existing material is actually made of statistical data of unequal value and understanding how the various available estimates are produced, and what their limits are, is a prerequisite to any tentative assessment of production and trafficking trends. As stated by Ted Legget in his review of the world cannabis situation, “Cannabis is the world’s most widely cultivated and consumed illicit drug, but there remain major gaps in our understanding of global cannabis markets” (Legget, 2006: 1). Pretty much everything about the cannabis plant leads to controversial debates, including its taxonomic classification (see below and note 4). Estimates of what is mostly an illegal agricultural production (except in a few states where the fiber-producing plant, but not the drug-producing plant, is permitted) are of course also difficult and controversial. Satellite detection of outdoor cannabis crops prove more difficult, technically2 , than that of opium poppies, and such imagery is rarely resorted to, notably because of its cost. Also, indoor cannabis crops have fast developed during the past decades, whether in the United States, Canada, or Western Europe, and the scale of such crops proves very difficult to assess (estimates and eradication reports actually mention numbers of plants and not cultivated areas). Ground surveys can also be too expensive (or time-consuming, dangerous, etc.) to carry out as cannabis crops can be grown virtually everywhere in the world and are often much more dispersed than coca or opium poppy crops in a given country. In fact, “only a few countries have scientifically valid estimates based on remote sensing technology or based on ground surveys. Most countries provide estimates based on some extrapolations from their cannabis eradication activities” (Legget, Pietschmann, 2008: 191). Of course, yields also need to be estimated, whether herbal cannabis or hashish (the psychoactive drug made by compressing the resin glands, or trichomes, of the female cannabis plant) is concerned. Yet, “given the variability of the plant and the range of cultivation techniques employed, coming up with precise figures such as yield to plot area ratios can be difficult” (Legget, 2006: 22). For example, the United Nations Office on Drugs and Crime (UNODC) makes it clear, in its 2012 Afghan cannabis survey, that “the yield survey is statistically not representative and based on observations rather than measurements” and that they are “exploring methods for more systematically assessing cannabis yields” (UNODC, 2013b: 15). Also, as explained by the UNODC, the comparability of cannabis yield estimates from different countries can be problematic for it can be affected by methodological differences, such as when Afghan yield estimates are based on field observations but Moroccan yield estimates are produced on the basis of off-farm measurement (UNODC, 2010: 23).
Combining sources and disciplinary approaches, including history, geography, geopolitics, economy, botany, agronomy, etc., is imperative for whoever wants to better understand the production of any illegal drug crop. Field-based research, however limited in time, scope, and means, must of course be resorted to in order to draw a better picture of the complex realities and dynamics that drive the cultivation of illegal drug crops. This paper is based upon such an approach but tends to raise more questions than it provides answers

Thailand first in Asia to move to decriminalize marijuana
https://apnews.com/article/health-as...f2bf967d272ab0 98

Thailand plans to make cannabis plant fully usable next year
https://www.nst.com.my/world/region/...able-next-year

The Use of Cannabis for Medical Purposes in the Arab World
Bader Shirah, Mohammed M. Ahmed
Medical Cannabis and Cannabinoids 4(1):1-3 December 2020
DOI: 10.1159/000510824
https://www.researchgate.net/publica...the_Arab_World
The legal landscape of cannabis (marijuana) has dramatically changed over the past few years in several countries worldwide. Many patients now have legal access to products derived from cannabis. In the Middle East, Lebanon became the first Arab country to legalize cannabis for medical and industrial use recently in 2020. Other Middle Eastern and Arab countries continue to completely ban the use of cannabis and products derived from cannabis. This article is a call to conduct medical research in the use of cannabis for medical purposes to determine the suitability and need for this substance in the Arab world. Based on these studies, evidence-based recommendations can be made to the highest authorities in the Arab countries for legalization or continued prohibition. As the international use of cannabis is increasing, the Arab countries may consider legalization of the substance to cover the unmet medical need and offer an additional treatment option for certain conditions.

Trends in intellectual property rights protection for medical cannabis and related products
Joseph Wyse and Gilad Luria
Journal of Cannabis Research (2021) 3:1
DOI: 10.1186/s42238-020-00057-7
https://jcannabisresearch.biomedcent.. .20-00057-7.pdf
The purpose of this review is to advance the field of applied cannabis research by providing insights into the patenting of medical cannabis and current intellectual property rights (IPR) data. Medical cannabis (MC) patent and plant breeders’ rights (PBR) registrations are filed on industrially applicable aspects of research. Studying the filing data and trends informs researchers of both gaps in current applied knowledge in MC (where patents have not been filed) and prior knowledge (where patents have already been filed). Our focus is on those intellectual property rights (IPR) that are registered and germane to technical innovations in MC and related products. These are patents and PBR and thus exclude trade secrets, copyrights, franchises, or trademarks. Methods used for surveying the defined IPR landscape include searches of publicly available patent and PBR data and classifying the data according to the upstream–midstream–downstream innovation paradigm of the MC industry. The findings suggest that the technical knowledge as expressed by patent filings is growing commensurate to the economic and legislative activity. Specific cannabis patents in agricultural technologies directed at improving yield, efficiency, and quality (known as “agritech”) are being filed and granted. These agritech-focused patents represent original novel and applied MC research achievements that address specific problems in cannabis cultivation, such as protection of the cannabis crop, maximizing cannabis yield, harvesting and post-harvesting of cannabis, and new advantageous varieties. Patents on ex planta and in planta cannabis genes expression have been published in recent years while patents on extraction methods for cannabinoids have increased since 2015. Much patent activity is in the downstream category of MC patient-oriented products and delivery systems for a very wide range of medical indications and disease conditions. The emerging importance of access and benefit-sharing treaties and regulations is noted with implications on the industry briefly discussed. Patent data on leading and emerging patentee companies and institutions are shown. We also provide evidence of prior art and freedom to operate.

UK – Cannabis firm launches UK clinic following prescription license allocation

Cantourage has appointed cannabis prescribers and is now officially operational via a new centre

UK - Cannabis firm launches UK clinic following prescription license allocation - RIS.WORLD

European medical cannabis company Cantourage has received two British government licenses that authorise the company to import medical
www.ris.world
European medical cannabis company Cantourage has received two British government licenses that authorise the company to import medical cannabis to the UK.
Additionally, the company has received the required regulatory registration to prescribe medical cannabis products to clients via a new facility.
The centre has duly been named Cantourage Clinic and the company has received regulatory registration for a private tele-healthcare clinic specialised in medical cannabis. Cantourage hopes that offering an alternative to the existing UK medical cannabis clinics will broaden choice and potentially help to push down prices…

Not Cannabis specific
WO2020239984A1 Gene for Parthenogensis Patent
https://patentimages.storage.googleapi s.com/d1/99/59/c922ddef2fd1b8/WO2020239984A1.pdf
https://patents.google.com/patent/WO2020239984A1/en
The invention provides the nucleotide sequence and amino acid sequences of the parthenogenesis gene of Taraxacum as well as (functional) homologues, fragments and variants thereof, which provides parthenogenesis as a part of apomixis. Also parthenogenetic plants and methods for making these are provided, as are molecular markers and methods of using these.
Title: Gene for Parthenogenesis
Field of the invention
The present invention relates to the field of biotechnology and in particular to plant biotechnology including plant breeding. The invention relates in particular to the identification and uses of genes relating to and useful e.g. in apomixis and haploid induction. The invention in particular relates to the gene that is associated with parthenogenesis, as well as the encoded protein, and fragments of both. The invention further relates to methods for suppressing and/or inducing parthenogenesis in plants and crops, to the use of the gene and/or the protein or their fragments for apomixis in particular in combination with apomeiotic gene(s), or for the production of haploid plants of which the chromosomes can be doubled to produce doubled haploids.
 
Last edited:

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Medical Cannabis/Endocannabinoids. Part 1 A-D. RE-ALPHABETIZED


*926 PubMed Cannabidiol Studies
https://medicalmarijuana.procon.org/...%20Studies.pdf

*The List (Granny Storm Crow Bib List) Hundreds of Cannabis articles with links
https://grannystormcrowslist.wordpress.com/the-list/

Cannabis (NATURE SPECIAL)
Vol. 525 No. 7570_supp ppS1-S18
https://www.nature.com/collections/ynbwsfsmjz

Cannabis
Michelle Grayson
Nature 525, S1 (23 September 2015)
https://www.nature.com/articles/525S1a.pdf

The cannabis crop
Julie Gould
Nature 525, S2–S3 (23 September 2015)
https://www.nature.com/articles/525S2a.pdf

Botany: The cultivation of weed
Lucas Laursen
Nature 525, S4–S5 (23 September 2015)
https://www.nature.com/articles/525S4a.pdf

Drug development: The treasure chest
Brian Owens
Nature 525, S6–S8 (23 September 2015)
https://www.nature.com/articles/525S6a.pdf

Perspective: Close the knowledge gap
Jonathan Page
Mark M. Ware
Nature 525, S9 (23 September 2015)
https://www.nature.com/articles/525S9a.pdf

A potted history
Stephanie Pain
Nature 525, S10–S11 (23 September 2015)
https://www.nature.com/articles/525S10a.pdf

Israel: Research without prejudice
Emily Sohn
Nature 525, S12–S13 (23 September 2015)
https://www.nature.com/articles/525S12a.pdf

Perspective: Be clear about the real risks
Matthew Hill
Nature 525, S14 (23 September 2015)
https://www.nature.com/articles/525S14a.pdf

Medical marijuana: Showdown at the cannabis corral
Michael Eisenstein
Nature 525, S15–S17 (23 September 2015)
https://www.nature.com/articles/525S15a.pdf

Cannabis: 4 big questions
Julie Gould
Nature 525, S18 (23 September 2015)
https://www.nature.com/articles/525S18a.pdf

Special Issue:Cannabinoids in Biology and Medicine, Part I. British Journal of Pharmacology
Guest Editors: Itai Bab and Steve Alexander
Volume 163, Issue 7 . British Journal of Pharmacology
https://bpspubs.onlinelibrary.wiley... .14765381/163/7
Free downloads of 19 papers
Themed issue on cannabinoids in biology and medicine
Itai Bab
DOI: 10.1111/j.1476-5381.2011.01536.x
Current cannabinoid research is based on a number of major discoveries made by Professor Raphael Mechoulam. In the mid?1960s, he, together with Professor Yechiel Gaoni, identified in the plant Cannabis sativa, the psychoactive component of marijuana and hashish, ?9?tetrahydrocannabinol (THC), determined its chemical structure (Gaoni and Mechoulam, 1964; Mechoulam and Gaoni, 1967) and synthesized it (Mechoulam et?al., 1967). The pertussis toxin?sensitive inhibition of adenylate cyclase activity by THC and related synthetic cannabimimetic compounds, such as desacetyllevonantradol (Howlett, 1985), led a couple of decades later to the cloning of the first cannabinoid receptor (CB1) in neural cell lines and several regions of the brain (Matsuda et?al., 1990). Reasoning that this receptor is targeted by an endogenous component(s), Professor Mechoulam made his second major breakthrough, the discovery of ligands for this receptor, arachidonyl ethanolamide (anandamide) (Devane et?al., 1992) and 2?arachidonyglycerol (Mechoulam et?al., 1995), thus establishing the presence of the endocannabinoid system. These discoveries were followed by cloning of another, predominantly peripheral cannabinoid receptor (CB2) (Munro et?al., 1993) and the endocannabinoid metabolizing enzymes diacylglycerol lipases, NAPE?selective phospholipase D, monoacylglycerol lipase and fatty acid amide hydrolase (Ligresti et?al., 2005; McKinney and Cravatt, 2005).

Endocannabinoid tone versus constitutive activity of cannabinoid receptors
Allyn C. Howlett, Patricia H. Reggio , Steven R. Childers , Robert E. Hampson , Nadine M. Ulloa, Dale G. Deutsch
British Journal of Pharmacology (2011) 163 1329–1343
doi: 10.1111/j.1476-5381.2011.01364.x
This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2?arachidonoylglycerol (2?AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis?à?vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2?AG is well characterized, and 2?AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid?binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands

Taming THC: potential cannabis synergy and phytocannabinoid terpenoid entourage effects
Ethan B Russo
British Journal of Pharmacology (2011) 163 1344–1364
doi: 10.1111/j.1476-5381.2011.01238.x
Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis chemotypes expressing high titres of each component for future study. This review will explore another echelon of phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, ??pinene, linalool, ??caryophyllene, caryophyllene oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour when inhaled from ambient air at serum levels in the single digits ng·mL?1. They display unique therapeutic effects that may contribute meaningfully to the entourage effects of cannabis?based medicinal extracts. Particular focus will be placed on phytocannabinoid?terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation, depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin?resistant Staphylococcus aureus). Scientific evidence is presented for non?cannabinoid plant components as putative antidotes to intoxicating effects of THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be proposed. Phytocannabinoid?terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new therapeutic products is possible from this venerable plant.

Prospects for cannabinoid therapies in basal ganglia disorders
Javier Fernández?Ruiz , Miguel Moreno?Martet, Carmen Rodríguez?Cueto , Cristina Palomo?Garo , María Gómez?Cañas, Sara Valdeolivas, Manuel Guzmán, Carmen Guaza,, Julián Romero
British Journal of Pharmacology, 163(7), 1365–1378.
doi: 10.1111/j.1476-5381.2011.01365.x
Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinson's disease (PD) and Huntington's disease (HD), two of the most important disorders affecting the basal ganglia. Two pharmacological profiles have been proposed for cannabinoids being effective in these disorders. On the one hand, cannabinoids like ?9?tetrahydrocannabinol or cannabidiol protect nigral or striatal neurons in experimental models of both disorders, in which oxidative injury is a prominent cytotoxic mechanism. This effect could be exerted, at least in part, through mechanisms independent of CB1 and CB2 receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB2receptors leads to a slower progression of neurodegeneration in both disorders. This effect would be exerted by limiting the toxicity of microglial cells for neurons and, in particular, by reducing the generation of proinflammatory factors. It is important to mention that CB2 receptors have been identified in the healthy brain, mainly in glial elements and, to a lesser extent, in certain subpopulations of neurons, and that they are dramatically up?regulated in response to damaging stimuli, which supports the idea that the cannabinoid system behaves as an endogenous neuroprotective system. This CB2receptor up?regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB2 receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB2 receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation

Cannabinoid receptor signalling in neurodegenerative diseases: a potential role for membrane fluidity disturbance
M Maccarrone, G Bernardi, A Finazzi Agrò, D Centonze
British Journal of Pharmacology, 163(7), 1379–1390.
doi: 10.1111/j.1476-5381.2011.01277.x
Type?1 cannabinoid receptor (CB1) is the most abundant G?protein?coupled receptor (GPCR) in the brain. CB1 and its endogenous agonists, the so?called ‘endocannabinoids (eCBs)’, belong to an ancient neurosignalling system that plays important functions in neurodegenerative and neuroinflammatory disorders like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. For this reason, research on the therapeutic potential of drugs modulating the endogenous tone of eCBs is very intense. Several GPCRs reside within subdomains of the plasma membranes that contain high concentrations of cholesterol: the lipid rafts. Here, the hypothesis that changes in membrane fluidity alter function of the endocannabinoid system, as well as progression of particular neurodegenerative diseases, is described. To this end, the impact of membrane cholesterol on membrane properties and hence on neurodegenerative diseases, as well as on CB1 signalling in vitro and on CB1?dependent neurotransmission within the striatum, is discussed. Overall, present evidence points to the membrane environment as a critical regulator of signal transduction triggered by CB1, and calls for further studies aimed at better clarifying the contribution of membrane lipids to eCBs signalling. The results of these investigations might be exploited also for the development of novel therapeutics able to combat disorders associated with abnormal activity of CB1.

The dual neuroprotective–neurotoxic profile of cannabinoid drugs
Yosef Sarne, Fadi Asaf, Miriam Fishbein, Mikhal Gafni, Ora Keren
British Journal of Pharmacology (2011)163 1391–1401
doi: 10.1111/j.1476-5381.2011.01280.x
Extensive in vitro and in vivo studies have shown that cannabinoid drugs have neuroprotective properties and suggested that the endocannabinoid system may be involved in endogenous neuroprotective mechanisms. On the other hand, neurotoxic effects of cannabinoids in vitro and in vivo were also described. Several possible explanations for these dual, opposite effects of cannabinoids on cellular fate were suggested, and it is conceivable that various factors may determine the final outcome of the cannabinoid effect in vivo. In the current review, we focus on one of the possible reasons for the dual neuroprotective/neurotoxic effects of cannabinoids in vivo, namely, the opposite effects of low versus high doses of cannabinoids. While many studies reported neuroprotective effects of the conventional doses of cannabinoids in various experimental models for acute brain injuries, we have shown that a single administration of an extremely low dose of ?9?tetrahydrocannabinol (THC) (3–4 orders of magnitude lower than the conventional doses) to mice induced long?lasting mild cognitive deficits that affected various aspects of memory and learning. These findings led to the idea that this low dose of THC, which induces minor damage to the brain, may activate preconditioning and/or postconditioning mechanisms and thus will protect the brain from more severe insults. Indeed, our recent findings support this assumption and show that a pre? or a postconditioning treatment with extremely low doses of THC, several days before or after brain injury, provides effective long?term cognitive neuroprotection. The future therapeutical potential of these findings is discussed.

Endocannabinoids and traumatic brain injury
Esther Shohami, Ayelet Cohen?Yeshurun, Lital Magid, Merav Algali, Raphael Mechoulam
British Journal of Pharmacology (2011) 163 1402–1410
doi: 10.1111/j.1476-5381.2011.01343.x
Traumatic brain injury (TBI) represents the leading cause of death in young individuals. It triggers the accumulation of harmful mediators, leading to secondary damage, yet protective mechanisms are also set in motion. The endocannabinoid (eCB) system consists of ligands, such as anandamide and 2?arachidonoyl?glycerol (2?AG), receptors (e.g. CB1, CB2), transporters and enzymes, which are responsible for the ‘on?demand’ synthesis and degradation of these lipid mediators. There is a large body of evidence showing that eCB are markedly increased in response to pathogenic events. This fact, as well as numerous studies on experimental models of brain toxicity, neuroinflammation and trauma supports the notion that the eCB are part of the brain's compensatory or repair mechanisms. These are mediated via CB receptors signalling pathways that are linked to neuronal survival and repair. The levels of 2?AG, the most highly abundant eCB, are significantly elevated after TBI and when administered to TBI mice, 2?AG decreases brain oedema, inflammation and infarct volume and improves clinical recovery. The role of CB1 in mediating these effects was demonstrated using selective antagonists or CB1 knockout mice. CB2 were shown in other models of brain insults to reduce white blood cell rolling and adhesion, to reduce infarct size and to improve motor function. This review is focused on the role the eCB system plays as a self?neuroprotective mechanism and its potential as a basis for the development of novel therapeutic modality for the treatment of CNS pathologies with special emphasis on TBI.

Regulation of nausea and vomiting by cannabinoids
Linda A Parker, Erin M Rock
British Journal of Pharmacology, 163(7), 1411–1422.
doi: 10.1111/j.1476-5381.2010.01176.x
Considerable evidence demonstrates that manipulation of the endocannabinoid system regulates nausea and vomiting in humans and other animals. The anti?emetic effect of cannabinoids has been shown across a wide variety of animals that are capable of vomiting in response to a toxic challenge. CB1 agonism suppresses vomiting, which is reversed by CB1 antagonism, and CB1 inverse agonism promotes vomiting. Recently, evidence from animal experiments suggests that cannabinoids may be especially useful in treating the more difficult to control symptoms of nausea and anticipatory nausea in chemotherapy patients, which are less well controlled by the currently available conventional pharmaceutical agents. Although rats and mice are incapable of vomiting, they display a distinctive conditioned gaping response when re?exposed to cues (flavours or contexts) paired with a nauseating treatment. Cannabinoid agonists (?9?THC, HU?210) and the fatty acid amide hydrolase (FAAH) inhibitor, URB?597, suppress conditioned gaping reactions (nausea) in rats as they suppress vomiting in emetic species. Inverse agonists, but not neutral antagonists, of the CB1 receptor promote nausea, and at subthreshold doses potentiate nausea produced by other toxins (LiCl). The primary non?psychoactive compound in cannabis, cannabidiol (CBD), also suppresses nausea and vomiting within a limited dose range. The anti?nausea/anti?emetic effects of CBD may be mediated by indirect activation of somatodendritic 5?HT1A receptors in the dorsal raphe nucleus; activation of these autoreceptors reduces the release of 5?HT in terminal forebrain regions. Preclinical research indicates that cannabinioids, including CBD, may be effective clinically for treating both nausea and vomiting produced by chemotherapy or other therapeutic treatments.

The case for peripheral CB1 receptor blockade in the treatment of visceral obesity and its cardiometabolic complications
George Kunos, Joseph Tam
British Journal of Pharmacology (2011) 163 1423–143
doi: 10.1111/j.1476-5381.2011.01352.x
In this review, we consider the role of endocannabinoids and cannabinoid?1 (CB1) cannabinoid receptors in metabolic regulation and as mediators of the thrifty phenotype that underlies the metabolic syndrome. We survey the actions of endocannabinoids on food intake and body weight, as well as on the metabolic complications of visceral obesity, including fatty liver, insulin resistance and dyslipidemias. Special emphasis is placed on weighing the relative importance of CB1 receptors located in peripheral tissues versus the central nervous system in mediating the metabolic effects of endocannabinoids. Finally, we review recent observations that indicate that peripherally restricted CB1 receptor antagonists retain efficacy in reducing weight and improving metabolic abnormalities in mouse models of obesity without causing behavioural effects predictive of neuropsychiatric side effects in humans.

The endocannabinoid system as a key mediator during liver diseases: new insights and therapeutic openings
A Mallat, F Teixeira?Clerc, V Deveaux, S Manin, S Lotersztajn
British Journal of Pharmacology (2011) 163 1432–1440
doi: 10.1111/j.1476-5381.2011.01397.x
Chronic liver diseases represent a major health problem due to cirrhosis and its complications. During the last decade, endocannabinoids and their receptors have emerged as major regulators of several pathophysiological aspects associated with chronic liver disease progression. Hence, hepatic cannabinoid receptor 2 (CB2) receptors display beneficial effects on alcoholic fatty liver, hepatic inflammation, liver injury, regeneration and fibrosis. Cannabinoid receptor 1 (CB1) receptors have been implicated in the pathogenesis of several lesions such as alcoholic and metabolic steatosis, liver fibrogenesis, or circulatory failure associated with cirrhosis. Although the development of CB1 antagonists has recently been suspended due to the high incidence of central side effects, preliminary preclinical data obtained with peripherally restricted CB1 antagonists give real hopes in the development of active CB1 molecules devoid of central adverse effects. CB2?selective molecules may also offer novel perspectives for the treatment of liver diseases, and their clinical development is clearly awaited. Whether combined treatment with a peripherally restricted CB1 antagonist and a CB2 agonist might result in an increased therapeutic potential will warrant further investigation.

Skeletal lipidomics: regulation of bone metabolism by fatty acid amide family
Itai Bab, Reem Smoum, Heather Bradshaw, Raphael Mechoulam
British Journal of Pharmacology (2011) 163 1441–1446
DOI: 10.1111/j.1476-5381.2011.01474.x
There is increasing evidence demonstrating that fatty acid derivatives play a key regulatory role in a variety of tissues. However, the study of skeletal lipidomics is just emerging and global strategies, such as targeted lipidomics, have not been applied to bone tissue. Such strategies hold great promises as in the case of genomics and proteomics. A partial profile of endocannabinoids and endocannabinoid?like compounds has demonstrated the presence of several long?chain fatty acid amides (FAAs), some of which displaying potent effects on osteoblasts, the bone forming cells and osteoclasts, the bone resorbing cells. In the skeleton, the FAAs activate the CB1 cannabinoid receptor present in sympathetic nerve terminals as well as CB2 cannabinoid receptor, the Gi?protein coupled receptor GPR55, and the transient receptor potential vanilloid type ion channel expressed by osteoblasts and/or osteoclasts. This review on the skeletal FAA system focuses on the production of FAAs in the skeleton and their net bone anabolic and anti?catabolic activity resulting from the stimulation of bone formation and inhibition of bone resorption. As the FAA family holds great promise as a basis for the treatment of osteoporosis and other diseases involving bone, further studies should aim towards the complete profiling of these lipids and their receptors in bone tissue, followed by elucidation of their function and mechanism of action.

The endocannabinoid system and cancer: therapeutic implication
Josée Guindon, Andrea G Hohmann
British Journal of Pharmacology (2011) 163 1447–1463
doi: 10.1111/j.1476-5381.2011.01327.x
The endocannabinoid system is implicated in a variety of physiological and pathological conditions (inflammation, immunomodulation, analgesia, cancer and others). The main active ingredient of cannabis, ?9?tetrahydrocannabinol (?9?THC), produces its effects through activation of CB1 and CB2 receptors. CB1 receptors are expressed at high levels in the central nervous system (CNS), whereas CB2 receptors are concentrated predominantly, although not exclusively, in cells of the immune system. Endocannabinoids are endogenous lipid?signalling molecules that are generated in the cell membrane from phospholipid precursors. The two best characterized endocannabinoids identified to date are anandamide (AEA) and 2?arachidonoylglycerol (2?AG). Here we review the relationship between the endocannabinoid system and anti?tumour actions (inhibition of cell proliferation and migration, induction of apoptosis, reduction of tumour growth) of the cannabinoids in different types of cancer. This review will focus on examining how activation of the endocannabinoid system impacts breast, prostate and bone cancers in both In vito and in vivo systems. The therapeutic potential of cannabinoids for cancer, as identified in clinical trials, is also discussed. Identification of safe and effective treatments to manage and improve cancer therapy is critical to improve quality of life and reduce unnecessary suffering in cancer patients. In this regard, cannabis?like compounds offer therapeutic potential for the treatment of breast, prostate and bone cancer in patients. Further basic research on anti?cancer properties of cannabinoids as well as clinical trials of cannabinoid therapeutic efficacy in breast, prostate and bone cancer is therefore warranted
.

Peripheral antinociceptive effects of inhibitors of monoacylglycerol lipase in a rat model of inflammatory pain
Josée Guindon, Ana Guijarro, Daniele Piomelli, Andrea G. Hohmann
British Journal of Pharmacology (2011) 163 1447–1463
doi: 10.1111/j.1476-5381.2010.01192.x
BACKGROUND AND PURPOSE The endocannabinoid 2?arachidonoylglycerol (2?AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL?preferring inhibitor URB602 and exogenous 2?AG in rats.
EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001–300 mg), URB602 (0.001–600 mg), 2-AG (ED50), 2-AG + JZL184 (at their ED50), 2-AG + URB602 (at their ED50), AM251 (80 mg), AM251 + JZL184 (10 mg), AM630 (25 mg) or AM630 + JZL184 (10 mg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed.
KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED50 Phase 1: 0.06 0.028; Phase 2: 0.03 0.011 mg) produced greater antinociception than URB602 (ED50 Phase 1: 120 51.3; Phase 2: 66 23.9 mg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels.
CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB1 and CB2 receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.

Effects of cannabinoids and cannabinoid?enriched Cannabisextracts on TRP channels and endocannabinoid metabolic enzymes
Luciano De Petrocellis, Alessia Ligresti, Aniello Schiano Moriello, Marco Allarà, Tiziana Bisogno, Stefania Petrosino, Colin G Stott, Vincenzo Di Marzo
British Journal of Pharmacology (2011) 163 1479–1494
doi: 10.1111/j.1476-5381.2010.01166.x
BACKGROUND AND PURPOSE Cannabidiol (CBD) and ?9?tetrahydrocannabinol (THC) interact with transient receptor potential (TRP) channels and enzymes of the endocannabinoid system.
EXPERIMENTAL APPROACH The effects of 11 pure cannabinoids and botanical extracts [botanical drug substance (BDS)] from Cannabis varieties selected to contain a more abundant cannabinoid, on TRPV1, TRPV2, TRPM8, TRPA1, human recombinant diacylglycerol lipase ? (DAGL?), rat brain fatty acid amide hydrolase (FAAH), COS cell monoacylglycerol lipase (MAGL), human recombinant N?acylethanolamine acid amide hydrolase (NAAA) and anandamide cellular uptake (ACU) by RBL?2H3 cells, were studied using fluorescence?based calcium assays in transfected cells and radiolabelled substrate?based enzymatic assays. Cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THCVA) were also tested.
KEY RESULTS CBD, CBG, CBGV and THCV stimulated and desensitized human TRPV1. CBC, CBD and CBN were potent rat TRPA1 agonists and desensitizers, but THCV-BDS was the most potent compound at this target. CBG-BDS and THCV-BDS were the most potent rat TRPM8 antagonists. All non-acid cannabinoids, except CBC and CBN, potently activated and desensitized rat TRPV2. CBDV and all the acids inhibited DAGLa. Some BDS, but not the pure compounds, inhibited MAGL. CBD was the only compound to inhibit FAAH, whereas the BDS of CBC > CBG > CBGV inhibited NAAA. CBC = CBG > CBD inhibited ACU, as did the BDS of THCVA, CBGV, CBDA and THCA, but the latter extracts were more potent inhibitors.
CONCLUSIONS AND IMPLICATIONS These results are relevant to the analgesic, anti-inflammatory and anti-cancer effects of cannabinoids and Cannabis extracts


Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis?like disease in C57BL/6 mice
Ewa Kozela, Nirit Lev, Nathali Kaushansky, Raya Eilam. Neta Rimmerman, Rivka Levy, Avraham Ben?Nun, Ana Juknat, Zvi Vogel
British Journal of Pharmacology (2011) 163 1507–1519
doi: 10.1111/j.1476-5381.2011.01379.x
BACKGROUND AND PURPOSE Cannabis extracts and several cannabinoids have been shown to exert broad anti?inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.
EXPERIMENTAL APPROACH We used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation.
KEY RESULTS Treatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors.
CONCLUSIONS AND IMPLICATIONS CBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways
O Gomez, A Sanchez?Rodriguez, MQU Le, C Sanchez?Caro, F Molina?Holgado, E Molina?Holgado
British Journal of Pharmacology (2011) 163 1520–1532
doi: 10.1111/j.1476-5381.2011.01414.x
BACKGROUND AND PURPOSE The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB1, CB2 and CB1/CB2 receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity.
EXPERIMENTAL APPROACH Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB1, CB2 and CB1/CB2 receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB1 receptor) and AM630 (CB2 receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation.
KEY RESULTS ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation.
CONCLUSIONS AND IMPLICATIONS Activation of cannabinoid CB1 or CB2 receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

Inhibition of COX?2 expression by endocannabinoid 2?arachidonoylglycerol is mediated via PPAR??
Huizhi Du, Xiaolei Chen, Jian Zhang, Chu Chen
British Journal of Pharmacology (2011) 163 1533–1549
doi: 10.1111/j.1476-5381.2011.01444.x
BACKGROUND AND PURPOSE Endocannabinoids have both anti?inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2?arachidonoylglycerol (2?AG) protects hippocampal neurons by limiting the inflammatory response via a CB1 receptor?dependent MAPK/NF??B signalling pathway. The purpose of the present study was to determine whether PPAR?, an important nuclear receptor, mediates 2?AG?induced inhibition of NF??B phosphorylation and COX?2 expression, and COX?2?enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).
EXPERIMENTAL APPROACH By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARg, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.
KEY RESULTS Exogenous and endogenous 2-AG-produced suppressions of NF-kB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1b (IL-1b) and LPS were inhibited by GW9662, a selective PPARg antagonist, in hippocampal neurons in culture. PPARg agonists 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) and rosiglitazone mimicked the effects of 2-AG on NF-kB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARg. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1band LPS-induced reduction of PPARg expression. The 2-AG restoration of the reduced PPARg expression was blocked or attenuated by pharmacological or genetic inhibition of the CB1 receptor. CONCLUSIONS AND IMPLICATIONS Our results suggest that CB1 receptor-dependent PPARg expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

Differential effect of opioid and cannabinoid receptor blockade on heroin?seeking reinstatement and cannabinoid substitution in heroin?abstinent rats[/B]
L Fattore, MS Spano , V Melis, P Fadda, W Fratta
British Journal of Pharmacology (2011) 163 1550–1562
doi: 10.1111/j.1476-5381.2011.01459.x
BACKGROUND AND PURPOSE Opioids and cannabinoids interact in drug addiction and relapse. We investigated the effect of the opioid receptor antagonist naloxone and/or the cannabinoid CB1 receptor antagonist rimonabant on cannabinoid?induced reinstatement of heroin seeking and on cannabinoid substitution in heroin?abstinent rats.
EXPERIMENTAL APPROACH Rats were trained to self-administer heroin (30 mg·kg-1 per infusion) under a fixed-ratio 1 reinforcement schedule. After extinction of self-administration (SA) behaviour, we confirmed the effect of naloxone (0.1–1 mg·kg-1 ) and rimonabant (0.3–3 mg·kg-1 ) on the reinstatement of heroin seeking induced by priming with the CB1 receptor agonist WIN55,212-2 (WIN, 0.15–0.3 mg·kg-1 ). Then, in a parallel set of heroin-trained rats, we evaluated whether WIN (12.5 mg·kg-1 per infusion) SA substituted for heroin SA after different periods of extinction. In groups of rats in which substitution occurred, we studied the effect of both antagonists on cannabinoid intake.
KEY RESULTS Cannabinoid-induced reinstatement of heroin seeking was significantly attenuated by naloxone (1 mg·kg-1 ) and rimonabant (3 mg·kg-1 ) and fully blocked by co-administration of sub-threshold doses of the two antagonists. Moreover, contrary to immediate (1 day) or delayed (90 days) drug substitution, rats readily self-administered WIN when access was given after 7, 14 or 21 days of extinction from heroin, and showed a response rate that was positively correlated with the extinction period. In these animals, cannabinoid intake was increased by naloxone (1 mg·kg-1 ) and decreased by rimonabant (3 mg·kg-1 ).
CONCLUSIONS AND IMPLICATIONS Our findings extend previous research on the crosstalk between cannabinoid and opioid receptors in relapse mechanisms, which suggests a differential role in heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats.

Volume 165, Issue 8 British Journal of Pharmacology
Special Issue:Themed Section: Cannabinoids in Biology and Medicine, Part II.
Guest Editors: Itai Bab and Steve Alexander
26 articles download for FREE
https://bpspubs.onlinelibrary.wiley... .14765381/165/8

Themed Section: Cannabinoids in Biology and Medicine, Part II
VIEWPOINT
Cannabinoid research in the 2010s
Mauro Maccarrone and Steve PH Alexander
bph_1930 2409..2410
DOI:10.1111/j.1476-5381.2012.01930.x
Cannabis sativa is possibly the plant with the longest history of cultivation by man (Russo, 2007). It has long been exploited for its fibre; as a biomass converter, it has exceptional utility. For most people, however, there is the association of cannabis with ‘recreational drugs’, which has lead to the profusion of names associated with the plant and extracts thereof (marijuana, hashish, bhang, weed, grass, etc.). The ‘modern’ scientific era of cannabis research was prompted by the discovery of the major psychoactive ingredient in cannabis extracts (Gaoni and Mechoulam, 1964). This was, of course, D9-tetrahydrocannabinol or THC. Raphael Mechoulam has numerous publications, filled with seminal observations, including the identification of the two ‘best’ candidates for endogenous cannabinoid molecules: anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (Mechoulam et al., 1995). He has become something of an icon in the cannabis field, with this issue of BJP containing a series of original articles prompted by a symposium held in Jerusalem in November 2010 to celebrate his 80th birthday. The first issue, entitled ‘Cannabinoids in Biology and Medicine’, containing primarily reviews, was published in August 2011 (https://onlinelibrary.wiley.com/doi/...sue-7/issuetoc). Current research in cannabinoid-related areas is vibrant, with the added focus of TRPV1 ion channels, PPAR nuclear receptors and the ‘orphan’ G-protein coupled receptors, GPR18, GPR55 and GPR119, as molecular targets of cannabinoids and cannabinoid-like molecules. Furthermore, the identification of endogenous agonists at cannabinoid receptors which lead to the demonstration of multiple routes for synthesis and transformation of these endocannabinoids has added to the molecular targets available for potential exploitation.

So what do we call GPR18 now?
SPH Alexander
DOI:10.1111/j.1476-5381.2011.01731.x
British Journal of Pharmacology (2012) 165 2411–2413 2411
In this issue of the British Journal of Pharmacology, the second associated with a celebration of the 80th birthday of Raphael Mechoulam, Doug McHugh, Heather Bradshaw and colleagues describe their investigations into the orphan GPCR, GPR18 (McHugh et al., 2012). They observed that N-arachidonoylglycine (NAGly, an endogenous fatty acid: amino acid conjugate; see Figure 1A), D9 - tetrahydrocannabinol (THC, the major psychoactive component of the cannabis plant; see Figure 1A) and anandamide (AEA, an endogenous cannabinoid agonist; see Figure 1A) function as ‘full’ agonists at GPR18 expressed heterologously in HEK293 cells, as measured by phosphorylation of ERK1/2, with that rank order of potency. This leads me to pose the question: what do we call GPR18 now? The short answer to that question, of course, is that we wait for a decision from the appropriate Nomenclature Committee of IUPHAR – the Union of Basic and Clinical Pharmacology (https://www.iuphar.org/nciuphar.html). So what options are open to them? Guidance from NC-IUPHAR suggests naming the receptor after the endogenous ligand or group of ligands. Of the ligands examined for activity at recombinant GPR18, NAGly was the most potent in the current study (McHugh et al., 2012). Is GPR18, therefore, a NAGly (or potentially a NAGly1 or NAG1) receptor?

D9 -Tetrahydrocannabinol and N-arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC-1B cells
Douglas McHugh, Jeremy Page, Emily Dunn and Heather B Bradshaw
DOI:10.1111/j.1476-5381.2011.01497.x
BACKGROUND AND PURPOSE Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells.
EXPERIMENTAL APPROACH Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis.
KEY RESULTS AEA, D9 -tetrahydrocannabinol (D9 -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB1 receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB1 and CB2 receptor ligands at GPR18 receptors, including D9 -THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and D9 -THC were antagonized by Pertussis toxin, AM251 and cannabidiol.
CONCLUSIONS AND IMPLICATIONS An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease

Inhibition of monoacylglycerol lipase attenuates vomiting in Suncus murinus and 2-arachidonoyl glycerol attenuates nausea in rats
Martin A Sticht1 , Jonathan Z Long2 , Erin M Rock1 , Cheryl L Limebeer1 , Raphael Mechoulam3 , Benjamin F Cravatt2 and Linda A Parker1
British Journal of Pharmacology (2012) 165 2425–2435 2425
DOI:10.1111/j.1476-5381.2011.01407.x
BACKGROUND AND PURPOSE To evaluate the role of 2-arachidonoyl glycerol (2AG) in the regulation of nausea and vomiting using animal models of vomiting and of nausea-like behaviour (conditioned gaping).
EXPERIMENTAL APPROACH Vomiting was assessed in shrews (Suncus murinus), pretreated with JZL184, a selective monoacylglycerol lipase (MAGL) inhibitor which elevates endogenous 2AG levels, 1 h before administering the emetogenic compound, LiCl. Regulation of nausea-like behaviour in rats by exogenous 2AG or its metabolite arachidonic acid (AA) was assessed, using the conditioned gaping model. The role of cannabinoid CB1 receptors, CB2 receptors and cyclooxygenase (COX) inhibition in suppression of vomiting or nausea-like behaviour was assessed.
KEY RESULTS JZL184 dose-dependently suppressed vomiting in shrews, an effect prevented by pretreatment with the CB1 receptor inverse agonist/antagonist, AM251. In shrew brain tissue, JZL184 inhibited MAGL activity in vivo. In rats, 2AG suppressed LiCl-induced conditioned gaping but this effect was not prevented by AM251 or the CB2 receptor antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented suppression of conditioned gaping by 2AG or AA. However, when rats were pretreated with a high dose of JZL184 (40 mg·kg-1 ), suppression of gaping by 2AG was partially reversed by AM251. Suppression of conditioned gaping was not due to interference with learning because the same dose of 2AG did not modify the strength of conditioned freezing to a shock-paired tone.
CONCLUSIONS AND IMPLICATIONS Our results suggest that manipulations that elevate 2AG may have anti-emetic or anti-nausea potential

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin?1
Neta Rimmerman, Heather B Bradshaw, Ewa Kozela, Rivka Levy, Ana Juknat, Zvi Vogel
British Journal of Pharmacology (2012) 165 2436–2449
DOI:10.1111/j.1476-5381.2011.01380.x
BACKGROUND AND PURPOSE N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line.
EXPERIMENTAL APPROACH BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. KEY RESULTS BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB1 receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change.
CONCLUSIONS AND IMPLICATIONS Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity

?8?Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors
Sándor Bátkai, Partha Mukhopadhyay, B?la Horváth, Mohanraj Rajesh, Rachel Y Gao, Anu Mahadevan, Mukkanti Amere, Natalia Battista, Aron H Lichtman, Lisa A Gauson, Mauro Maccarrone, Roger G Pertwee, Pál Pacher
British Journal of Pharmacology (2012) 165 2450–2461
https://doi.org/10.1111/j.1476-5381.2011.01410.x
BACKGROUND AND PURPOSE Activation of cannabinoid CB2 receptors protects against various forms of ischaemia-reperfusion (I/R) injury. D8 -Tetrahydrocannabivarin (D8 -THCV) is a synthetic analogue of the plant cannabinoid D9 -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB2 receptors. Here, we assessed effects of D8 -THCV and its metabolite 11-OH-D8 -THCV on CB2 receptors and against hepatic I/R injury.
EXPERIMENTAL APPROACH Effects in vitro were measured with human CB2 receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.
KEY RESULTS Displacement of [3 H]CP55940 by D8 -THCV or 11-OH-D8 -THCV from specific binding sites in CHO cell membranes transfected with human CB2 receptors (hCB2) yielded Ki values of 68.4 and 59.95 nM respectively. D8 -THCV or 11-OH-D8 -THCV inhibited forskolin-stimulated cAMP production by hCB2 CHO cells (EC50 = 12.95 and 14.3 nM respectively). D8 -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-a, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of D8 -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB2 receptor antagonist attenuated the protective effects of D8 -THCV, while a CB1 antagonist tended to enhance it.
CONCLUSIONS AND IMPLICATIONS D8 -THCV activated CB2 receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB2 receptor activation.

A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury
Bela Horváth, Lital Magid, Partha Mukhopadhyay, Sándor Bátkail, Mohanraj Rajesh, Ogyi Park, Galin Tanchian, Rachel Y Gao, Catherine E Goodfellow, Michelle Glass, Raphael Mechoulam and Pál Pacher
British Journal of Pharmacology (2012) 165 2462–2478
DOI:10.1111/j.1476-5381.2011.01381.x
BACKGROUND AND PURPOSE Cannabinoid CB2 receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. EXPERIMENTAL APPROACH We have investigated the effects of a novel CB2 receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7- dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. KEY RESULTS Displacement of [3 H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB2 or CB1 receptors (hCB1/2) yielded Ki values of 6 nM and 1.4 mM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB2 CHO cells (EC50 = 162 nM) and yielded EC50 of 26.4 nM in [35S]GTPgS binding assays using hCB2 expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic proinflammatory chemokines (CCL3 and CXCL2), TNF-a, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-a production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-a. Pretreatment with a CB2 receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB1 antagonist tended to enhance them.
CONCLUSION AND IMPLICATIONS HU-910 is a potent CB2 receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury.

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABAA receptors
R Baur, J Gertsch and E Sigel
British Journal of Pharmacology (2012) 165 2479–2484
DOI:10.1111/j.1476-5381.2011.01405.x
BACKGROUND AND PURPOSE Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB1 antagonists / inverse agonists. Concentrations of 0.5–10 mM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABAA receptors but found a significant positive allosteric modulation instead.
EXPERIMENTAL APPROACH Recombinant GABAA receptors were expressed in Xenopus oocytes. Receptors were exposed to AM251 or rimonabant in the absence and presence of GABA. Standard electrophysiological techniques were used to monitor the elicited ionic currents.
KEY RESULTS AM251 dose-dependently potentiated responses to 0.5 mM GABA at the recombinant a1b2g2 GABAA receptor with an EC50 below 1 mM and a maximal potentiation of about eightfold. The Hill coefficient indicated that more than one binding site for AM251 was located in this receptor. Rimonabant had a lower affinity, but a fourfold higher efficacy. AM251 potentiated also currents mediated by a1b2, axb2g2 (x = 2,3,5,6), a1b3g2 and a4b2d GABAA receptors, but not those mediated by a1b1g2. Interestingly, the CB1 receptor antagonists LY320135 and O-2050 did not significantly affect a1b2g2 GABAA receptor-mediated currents at concentrations of 1 mM.
CONCLUSIONS AND IMPLICATIONS This study identified rimonabant and AM251 as positive allosteric modulators of GABAA receptors. Thus, potential GABAergic effects of commonly used concentrations of these compounds should be considered in in vitro experiments, especially at extrasynaptic sites where GABA concentrations are low.

The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice
Lamont Booker, Steven G Kinsey, Rehab A Abdullah, Jacqueline L Blankman, Jonathan Z Long, Cyrine Ezzili, Dale L Boger, Benjamin F Cravatt and Aron H Lichtman
British Journal of Pharmacology (2012) 165 2485–2496
DOI:10.1111/j.1476-5381.2011.01445.x
BACKGROUND AND PURPOSE Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with D9 -tetrahydrocannabinol (THC).
EXPERIMENTAL APPROACH Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses. KEY RESULTS FAAH (-/-) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. m-opioid, TRPV1 and PPARa receptors) had no apparent role.
CONCLUSIONS AND IMPLICATIONS AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/ threonine kinases to activate ERK in neuronal cells
George D Dalton and Allyn C Howlett
British Journal of Pharmacology (2012) 165 2497–2511
DOI:10.1111/j.1476-5381.2011.01455.x
BACKGROUND AND PURPOSE Signalling networks that regulate the progression of cannabinoid CB1 receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB1 receptor-stimulated ERK phosphorylation in a neuronal cell model.
EXPERIMENTAL APPROACH Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB1 receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.
KEY RESULTS The time-course of CB1 receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB1 receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires Gi/o bg subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB1 receptor-stimulated, ligand-independent, transactivation of multiple RTKs.
CONCLUSIONS AND IMPLICATIONS The complex expression of CB1 receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB1-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.

Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and D9 -tetrahydrocannabinol in BV-2 microglial cells
Ana Juknat, Maciej Pietr, Ewa Kozela1, Neta Rimmerman, Rivka Levy, Giovanni Coppola, Daniel Geschwind and Zvi Vogel
British Journal of Pharmacology (2012) 165 2512–2528
DOI:10.1111/j.1476-5381.2011.01461.x
BACKGROUND AND PURPOSE Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid D9 -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways.
EXPERIMENTAL APPROACH To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC.
KEY RESULTS Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 by THC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2a/p8/ATF4/CHOP-TRIB3 pathway. Furthermore, CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. CONCLUSIONS AND IMPLICATIONS These observations indicated that CBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity.

Combined effects of THC and caffeine on working memory in rats
Leigh V Panlilio1 , Sergi Ferré1 , Sevil Yasar2 , Eric B Thorndike1 , Charles W Schindler1 and Steven R Goldberg1
British Journal of Pharmacology (2012) 165 2529–2538
DOI:10.1111/j.1476-5381.2011.01554.x
BACKGROUND AND PURPOSE Cannabis and caffeine are two of the most widely used psychoactive substances. D9 -Tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, induces deficits in short-term memory. Caffeine, a non-selective adenosine receptor antagonist, attenuates some memory deficits, but there have been few studies addressing the effects of caffeine and THC in combination. Here, we evaluate the effects of these drugs using a rodent model of working memory.
EXPERIMENTAL APPROACH Rats were given THC (0, 1 and 3 mg·kg-1 , i.p.) along with caffeine (0, 1, 3 and 10 mg·kg-1 , i.p.), the selective adenosine A1-receptor antagonist CPT (0, 3 and 10 mg·kg-1 ) or the selective adenosine A2A-receptor antagonist SCH58261 (0 and 5 mg·kg-1 ) and were tested with a delayed non-matching-to-position procedure in which behaviour during the delay was automatically recorded as a model of memory rehearsal.
KEY RESULTS THC alone produced memory deficits at 3 mg·kg-1 . The initial exposure to caffeine (10 mg·kg-1 ) disrupted the established pattern of rehearsal-like behaviour, but tolerance developed rapidly to this effect. CPT and SCH58261 alone had no significant effects on rehearsal or memory. When a subthreshold dose of THC (1 mg·kg-1 ) was combined with caffeine (10 mg·kg-1 ) or CPT (10 mg·kg-1 ), memory performance was significantly impaired, even though performance of the rehearsal-like pattern was not significantly altered.
CONCLUSION AND IMPLICATIONS Caffeine did not counteract memory deficits induced by THC but actually exacerbated them. These results are consistent with recent findings that adenosine A1 receptors modulate cannabinoid signalling in the hippocampus.

The anandamide transport inhibitor AM404 reduces the rewarding effects of nicotine and nicotineinduced dopamine elevations in the nucleus accumbens shell in rats
Maria Scherma, Zuzana Justinová, Claudio Zanettini, Leigh V Panlilio, Paola Mascia, Paola Fadda, Walter Fratta, Alexandros Makriyannis, Subramanian K Vadivel, Islam Gamaleddin, Bernard Le Foll and Steven R Goldberg
British Journal of Pharmacology (2012) 165 2539–2548
DOI:10.1111/j.1476-5381.2011.01467.x
BACKGROUND AND PURPOSE The fatty acid amide hydrolase inhibitor URB597 can reverse the abuse-related behavioural and neurochemical effects of nicotine in rats. Fatty acid amide hydrolase inhibitors block the degradation (and thereby magnify and prolong the actions) of the endocannabinoid anandamide (AEA), and also the non-cannabinoid fatty acid ethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). OEA and PEA are endogenous ligands for peroxisome proliferator-activated receptors alpha (PPAR-a). Since recent evidence indicates that PPAR-a can modulate nicotine reward, it is unclear whether AEA plays a role in the effects of URB597 on nicotine reward.
EXPERIMENTAL APPROACH A way to selectively increase endogenous levels of AEA without altering OEA or PEA levels is to inhibit AEA uptake into cells by administering the AEA transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404). To clarify AEA’s role in nicotine reward, we investigated the effect of AM404 on conditioned place preference (CPP), reinstatement of abolished CPP, locomotor suppression and anxiolysis in an open field, and dopamine elevations in the nucleus accumbens shell induced by nicotine in Sprague-Dawley rats.
KEY RESULTS AM404 prevented the development of nicotine-induced CPP and impeded nicotine-induced reinstatement of the abolished CPP. Furthermore, AM404 reduced nicotine-induced increases in dopamine levels in the nucleus accumbens shell, the terminal area of the brain’s mesolimbic reward system. AM404 did not alter the locomotor suppressive or anxiolytic effect of nicotine.
CONCLUSIONS AND IMPLICATIONS These findings suggest that AEA transport inhibition can counteract the addictive effects of nicotine and that AEA transport may serve as a new target for development of medications for treatment of tobacco dependence.

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone
WM Olango, M Roche, GK Ford, B Harhen and DP Finn1
British Journal of Pharmacology (2012) 165 2549–2560
DOI:10.1111/j.1476-5381.2011.01478.x
BACKGROUND AND PURPOSE Endocannabinoids in the midbrain periaqueductal grey (PAG) modulate nociception and unconditioned stress-induced analgesia; however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats.
EXPERIMENTAL APPROACH Rats received intra-dlPAG administration of the CB1 receptor antagonist/inverse agonist rimonabant, or vehicle, before re-exposure to a context paired 24 h previously with foot shock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22 kHz ultrasonic vocalization) were assessed. In a separate cohort, levels of endocannabinoids [2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamide (anandamide; AEA)] and the related N-acylethanolamines (NAEs) [N-palmitoyl ethanolamide (PEA) and N-oleoyl ethanolamide (OEA)] were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone. KEY RESULTS Re-exposure of rats to the context previously associated with foot shock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear without formalin-evoked nociceptive tone was associated with increased levels of 2-AG, AEA, PEA and OEA in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG.
CONCLUSIONS AND IMPLICATIONS Conditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG. These data support a role for endocannabinoids in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts.

AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor
Daniele Bolognini, Maria Grazia Cascio1, Daniela Parolaro and Roger G Pertwee
British Journal of Pharmacology (2012) 165 2561–2574
DOI:10.1111/j.1476-5381.2011.01503.x
BACKGROUND AND PURPOSE We have investigated how pre-incubating hCB2 CHO cells with the CB2 receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB2 receptors in these cells or their membranes.
EXPERIMENTAL APPROACH We tested the ability of AM630, SR144528 and of the CB1/CB2 receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB2 CHO cells or [35S]-GTPgS binding to membranes prepared from these cells, or to displace [3 H]-CP55940 from whole cells and membranes. Assays were also performed with the CB2 receptor partial agonist, D9 -tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively.
KEY RESULTS AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of D9 -tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [3 H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [35S]-GTPgS membrane assay.
CONCLUSIONS AND IMPLICATIONS These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB2 receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528.

D9 -Tetrahydrocannabinol (D9 -THC) attenuates mouse sperm motility and male fecundity
Daniel J Morgan, Charles H Muller, Natalia A Murataeva, Brian J Davis and Ken Mackie
British Journal of Pharmacology (2012) 165 2575–2583
DOI:10.1111/j.1476-5381.2011.01506.x
BACKGROUND AND PURPOSE Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1 cannabinoid receptor agonist, and D9 -tetrahydracannabinol (D9 -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of D9 -THC on male fecundity were determined.
EXPERIMENTAL APPROACH Effects of D9 -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of D9 -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by D9 -THC-treated males.
KEY RESULTS Pretreatment of sperm for 15 min with 1 mM D9 -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 mM WIN 55,212-2 or 10 mM D9 -THC for 30 min reduced sperm ATP levels. In sperm lacking CB1 receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of D9 -THC persisted. Administration of 50 mg·kg-1 D9 -THC to male mice just before mating caused a 20% decrease in embryonic litter size.
CONCLUSIONS AND IMPLICATIONS D9 -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or D9 -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB1 receptordependent whereas that of D9 -THC is not.

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro
LS Whyte, L Ford, SA Ridge, GA Cameron, MJ Rogers and RA Ross
British Journal of Pharmacology (2012) 165 2584–2597
DOI:10.1111/j.1476-5381.2011.01519.x
BACKGROUND AND PURPOSE Both CB1 and CB2 cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro.
EXPERIMENTAL APPROACH Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing avb3 or with F-actin rings, or measurement of resorption area.
KEY RESULTS Human osteoclasts express both CB1 and CB2 receptors. CB2 expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB1 and/or CB2 antagonists.
CONCLUSIONS AND IMPLICATIONS Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB2 receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease.

Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability
A Alhamoruni1 , KL Wright2 , M Larvin1 and SE O’Sullivan
British Journal of Pharmacology (2012) 165 2598–2610
DOI:10.1111/j.1476-5381.2011.01589.x
BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro.
EXPERIMENTAL APPROACH Confluent Caco-2 cell monolayers were treated for 24 h with IFNg and TNFa (10 ng·mL-1 ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB1, CB2, TRPV1, PPARg and PPARa. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids.
KEY RESULTS D9 -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB1 receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB1 antagonism. No role for the CB2 receptor was identified in these studies. Co-application of THC, cannabidiol or a CB1 antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation.
CONCLUSIONS AND IMPLICATIONS These findings suggest that locally produced endocannabinoids, acting via CB1 receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation.

The GPCR-associated sorting protein 1 regulates ligand-induced down-regulation of GPR55
J Kargll, NA Balengal, W Platzer1, L Martini, JL Whistler and M Waldhoer
British Journal of Pharmacology (2012) 165 2611–2619
DOI:10.1111/j.1476-5381.2011.01562.x
BACKGROUND AND PURPOSE Many GPCRs, including the CB1 cannabinoid receptor, are down-regulated following prolonged agonist exposure by interacting with the GPCR-associated sorting protein-1 (GASP-1). The CB1 receptor antagonist rimonabant has also recently been described to be an agonist at GPR55, a cannabinoid-related receptor. Here we investigated the post-endocytic properties of GPR55 after agonist exposure and tested whether GASP-1 is involved in this process.
EXPERIMENTAL APPROACH We evaluated the direct protein-protein interaction of GPR55 with GASP-1 using (i) GST-binding assays and (ii) co-immunoprecipitation assays in GPR55-HEK293 cells with endogenous GASP-1 expression. We further tested the internalization, recycling and degradation of GPR55 using confocal fluorescence microscopy and biotinylation assays in the presence and absence of GASP-1 (lentiviral small hairpin RNA knockdown of GASP-1) under prolonged agonist [rimonabant (RIM), lysophosphatidylinositol (LPI)] stimulation.
KEY RESULTS We showed that the prolonged activation of GPR55 with rimonabant or LPI down-regulates GPR55 via GASP-1. GASP-1 binds to GPR55 in vitro, and this interaction was required for targeting GPR55 for degradation. Disrupting the GPR55-GASP-1 interaction prevented post-endocytic receptor degradation, and thereby allowed receptor recycling.
CONCLUSION AND IMPLICATIONS These data implicate GASP-1 as an important regulator of ligand-mediated down-regulation of GPR55. By identifying GASP-1 as a key regulator of the trafficking and, by extension, functional expression of GPR55, we may be one step closer to gaining a better understanding of this receptor in response to cannabinoid drugs.

Cannabidiol, a nonpsychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT1A somatodendritic autoreceptors in the dorsal raphe nucleus
EM Rock, D Bolognini, CL Limebeer, MG Cascio, S Anavi-Goffer, PJ Fletcher, R Mechoulam, RG Pertwee and LA Parker
British Journal of Pharmacology (2012) 165 2620–2634
DOI:10.1111/j.1476-5381.2011.01621.x
BACKGROUND AND PURPOSE To evaluate the hypothesis that activation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis.
EXPERIMENTAL APPROACH The potential of systemic and intra-DRN administration of 5-HT1A receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT1A receptors and to modify the ability of the 5-HT1A agonist, 8-OH-DPAT, to stimulate [35S]GTPgS binding in rat brainstem membranes.
KEY RESULTS CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg-1 , but not 40 mg·kg-1 )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose–response curve) at enhancing the ability of 8-OH-DPAT to stimulate [ 35S]GTPgS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping.
CONCLUSIONS AND IMPLICATIONS These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT1A autoreceptors in the DRN.

Effects of palmitoylation of Cys415 in helix 8 of the CB1 cannabinoid receptor on membrane localization and signalling
Sergio Oddi, Enrico Dainese, Simone Sandiford, Filomena Fezza, Mirko Lanuti, Valerio Chiurchiù, Antonio Totaro, Giuseppina Catanzaro, Daniela Barcaroli, Vincenzo De Laurenzi, Diego Centonze, Somnath Mukhopadhyay, Jana Selent, Allyn C Howlett and Mauro Maccarrone
British Journal of Pharmacology (2012) 165 2635–2651
DOI:10.1111/j.1476-5381.2011.01658.x
BACKGROUND AND PURPOSE The CB1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB1 receptor by analysing the functional consequences of site-specific mutation of Cys415, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.
EXPERIMENTAL APPROACH The palmitoylation state of CB1 receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys415 was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [35S]GTPgS, cAMP and co-immunoprecipitation assays. KEY RESULTS Endogenous CB1 receptors in rat brain were palmitoylated. Mutation of Cys415 prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB1 receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins.
CONCLUSIONS AND IMPLICATIONS CB1 receptors were post-translationally modified by palmitoylation. Mutation of Cys415 provides a receptor that is functionally impaired in terms of membrane targeting and signalling.

The CB1 cannabinoid receptor C-terminus regulates receptor desensitization in autaptic hippocampal neurones
Alex Straiker, Jim Wager-Miller and Ken Mackie
British Journal of Pharmacology (2012) 165 2652–2659
DOI:10.1111/j.1476-5381.2011.01743.x
BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is the chief mediator of the CNS effects of cannabinoids. In cell culture model systems, CB1 receptors both desensitize and internalize on activation. Previous work suggests that the extreme carboxy-terminus of this receptor regulates internalization via phosphorylation of residues clustered within this region. Mutational analysis of the carboxy-terminus of CB1 receptors has demonstrated that the last six serine/threonine residues are necessary for agonist-induced internalization. However, the structural determinants of CB1 receptor internalization are also dependent on the local cellular environment. The importance of cell context on CB1 receptor function calls for an investigation of the functional roles of these residues in neurones.
EXPERIMENTAL APPROACH To determine the structural requirements of CB1 internalization in neurones, we evaluated the signalling properties of carboxyterminal mutated CB1 receptors expressed in cultured autaptic hippocampal neurones, using electrophysiological methods.
KEY RESULTS CB1 receptors transfected into CB1 knockout neurones signalled and desensitized as did wild-type neurones, allowing us to test specific CB1 receptor mutations. Deletion of the last 13 residues yielded a CB1 receptor that inhibited excitatory postsynaptic currents but did not desensitize. Furthermore, mutation of the final six serine and threonine residues to alanines resulted in a non-desensitizing receptor. In contrast, CB1 receptors lacking residues 419–460, leaving the last 14 residues intact, did desensitize.
CONCLUSIONS AND IMPLICATIONS The distal thirteen residues of CB1 receptors are crucial for their desensitization in cultured neurones. Furthermore, this desensitization is likely to follow phosphorylation of serines and threonines within this region.

Differential signalling in human cannabinoid CB1 receptors and their splice variants in autaptic hippocampal neurones
Alex Straiker, Jim Wager-Miller, Jacqueline Hutchens and Ken Mackie
British Journal of Pharmacology (2012) 165 2660–2671
DOI:10.1111/j.1476-5381.2011.01744.x
BACKGROUND AND PURPOSE Cannabinoids such as D9 - tetrahydrocannabinol, the major psychoactive component of marijuana and hashish, primarily act via cannabinoid CB1 and CB2 receptors to produce characteristic behavioural effects in humans. Due to the tractability of rodent models for electrophysiological and behavioural studies, most of the studies of cannabinoid receptor action have used rodent cannabinoid receptors. While CB1 receptors are relatively well-conserved among mammals, human CB1 (hCB1) differs from rCB1 and mCB1 receptors at 13 residues, which may result in differential signalling. In addition, two hCB1 splice variants (hCB1a and hCB1b) have been reported, diverging in their amino-termini relative to hCB1 receptors. In this study, we have examined hCB1 signalling in neurones.
EXPERIMENTAL APPROACH hCB1, hCB1a hCB1b or rCB1 receptors were expressed in autaptic cultured hippocampal neurones from CB1 -/- mice. Such cells express a complete endogenous cannabinoid signalling system. Electrophysiological techniques were used to assess CB1 receptor-mediated signalling. KEY RESULTS Expressed in autaptic hippocampal neurones cultured from CB1 -/- mice, hCB1, hCB1a and hCB1b signal differentially from one another and from rodent CB1 receptors. Specifically, hCB1 receptors inhibit synaptic transmission less effectively than rCB1 receptors. CONCLUSIONS AND IMPLICATIONS Our results suggest that cannabinoid receptor signalling in humans is quantitatively very different from that in rodents. As the problems of marijuana and hashish abuse occur in humans, our results highlight the importance of studying hCB1 receptors. They also suggest further study of the distribution and function of hCB1 receptor splice variants, given their differential signalling and potential impact on human health.

Slow receptor dissociation is not a key factor in the duration of action of inhaled long-acting b2-adrenoceptor agonists
David A Sykes and Steven J Charlton
British Journal of Pharmacology (2012) 165 2672–2683
DOI:10.1111/j.1476-5381.2011.01639.x
BACKGROUND AND PURPOSE b2-Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on b2-adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of b2-adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic.
EXPERIMENTAL APPROACH [ 3 H]-DHA was used to label b2-adrenoceptors expressed in CHO-cell membranes (Kd of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled b2 agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ¥ Ki) of the unlabelled compound were employed against a fixed concentration of [3 H]-DHA (0.6 nM).
KEY RESULTS The clinically used b2-adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic Kd and the competition Ki values of the eight b2-adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate koff and kon rates. The kinetic on-rate was highly correlated with equilibrium binding affinity.
CONCLUSIONS AND IMPLICATIONS Although the b2-adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing).

Role of neurosteroids in the anticonvulsant activity of midazolam
Ashish Dhir and Michael A Rogawski
British Journal of Pharmacology (2012) 165 2684–2691
DOI:10.1111/j.1476-5381.2011.01733.x
BACKGROUND AND PURPOSE Midazolam is a short-acting benzodiazepine that is widely used as an i.v. sedative and anticonvulsant. Besides interacting with the benzodiazepine site associated with GABAA receptors, some benzodiazepines act as agonists of translocator protein (18 kDa) (TSPO) to enhance the synthesis of steroids, including neurosteroids with positive modulatory actions on GABAA receptors. We sought to determine if neurosteroidogenesis induced by midazolam contributes to its anticonvulsant action.
EXPERIMENTAL APPROACH Mice were pretreated with neurosteroid synthesis inhibitors and potentiators followed by midazolam or clonazepam, a weak TSPO ligand. Anticonvulsant activity was assessed with the i.v. pentylenetetrazol (PTZ) threshold test.
KEY RESULTS Midazolam (500–5000 mg·kg-1 , i.p.) caused a dose-dependent increase in seizure threshold. Pretreatment with the neurosteroid synthesis inhibitors finasteride, a 5a-reductase inhibitor, and a functional TSPO antagonist PK 11195, reduced the anticonvulsant action of midazolam. The anticonvulsant action of midazolam was enhanced by the neurosteroidogenic drug metyrapone, an 11b-hydroxylase inhibitor. In contrast, the anticonvulsant action of clonazepam (100 mg·kg-1 ) was reduced by finasteride but not by PK 11195, indicating a possible contribution of neurosteroids unrelated to TSPO.
CONCLUSION AND IMPLICATIONS Enhanced endogenous neurosteroid synthesis, possibly mediated by an interaction with TSPO, contributed to the anticonvulsant action of midazolam. Enhanced neurosteroidogenesis may also be a factor in the actions of other benzodiazepines, even those that only weakly interact with TSPO

Bilobetin ameliorates insulin resistance by PKA-mediated phosphorylation of PPARa in rats fed a high-fat diet
Xin-Hui Kou, Mei-Feng Zhu, Dai Chen, Yi Lu Hui-Zhu Song Jian-Lin Ye and Lin-Feng Yue
British Journal of Pharmacology (2012) 165 2692–2706
DOI:10.1111/j.1476-5381.2011.01727.x
BACKGROUND AND PURPOSE The amelioration of insulin resistance by bilobetin is closely related to its hypolipidaemic effect. The aim of the present study was to determine the insulin-sensitizing mechanism of bilobetin by elucidating its effect on lipid metabolism.
EXPERIMENTAL APPROACH Rats fed a high-fat diet were treated with bilobetin for either 4 or 14 days before applying a hyperinsulinaemic–euglycaemic clamp. Triglyceride and fatty acids labelled with radioactive isotopes were used to track the transportation and the fate of lipids in tissues. The activity of lipid metabolism-related enzymes and b-oxidation rate were measured. Western blot was used to investigate the phosphorylation, translocation and expression of PPARa in several tissues and cultured cells. The location of amino acid residues subjected to phosphorylation in PPARa was also studied.
KEY RESULTS Bilobetin ameliorated insulin resistance, increased the hepatic uptake and oxidation of lipids, reduced very-low-density lipoprotein triglyceride secretion and blood triglyceride levels, enhanced the expression and activity of enzymes involved in b-oxidation and attenuated the accumulation of triglycerides and their metabolites in tissues. Bilobetin also increased the phosphorylation, nuclear translocation and activity of PPARa accompanied by elevated cAMP level and PKA activity. Threonine-129–alanine and/or serine-163–alanine mutations on the PPARa genes and PKA inhibitors prevented the effects of bilobetin on PPARa. However, cells overexpressing PKA appeared to stimulate the phosphorylation, nuclear translocation and activity of PPARa. CONCLUSIONS AND IMPLICATIONS Bilobetin treatment ameliorates hyperlipidaemia, lipotoxicity and insulin resistance in rats by stimulating PPARa-mediated lipid catabolism. PKA activation is crucial for this process.

Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels
Robiul Islam and Joseph W Lynch
British Journal of Pharmacology (2012) 165 2707–2720
DOI:10.1111/j.1476-5381.2011.01722.x
BACKGROUND AND PURPOSE Docking studies predict that the insecticides, lindane and fipronil, block GABAA receptors by binding to 6? pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABAA receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human a1, a1b, a2 and a3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit a1 glycine receptors.
EXPERIMENTAL APPROACH Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology.
KEY RESULTS Both compounds completely inhibited all tested glycine receptor subtypes with IC50 values ranging from 0.2–2 mM, similar to their potencies at vertebrate GABAA receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6? threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2? level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of b subunits in ab heteromeric glycine receptors.
CONCLUSIONS AND IMPLICATIONS This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6? threonine residues, whereas fipronil may have both pore and non-pore binding sites

Double blockade of angiotensin II (AT1)- receptors and ACE does not improve weight gain and glucose homeostasis better than single-drug treatments in obese rats
Anja Miesel, Helge Müller-Fielitz, Olaf Jöhren, Florian M Vogt and Walter Raasch
British Journal of Pharmacology (2012) 165 2721–2735
DOI:10.1111/j.1476-5381.2011.01726.x
BACKGROUND AND PURPOSE Combination therapies are becoming increasingly important for the treatment of high blood pressure. Little is known about whether double blockade of angiotensin II (AT1) receptors and angiotensin-converting enzyme (ACE) exert synergistic metabolic effects.
EXPERIMENTAL APPROACH Spontaneously hypertensive rats were allowed to choose between palatable chocolate bars and standard chow and were simultaneously treated with the AT1 blocker telmisartan (8 mg·kgbw-1 ·day-1 ), the ACE inhibitor ramipril (4 mg·kgbw-1 ·day-1 ) or a combination of the two (8 + 4 mg·kgbw-1 ·day-1 ) for 12 weeks.
KEY RESULTS Although food-dependent energy intake was increased by telmisartan and telmisartan + ramipril compared with ramipril or controls, body weight gain, abundance of fat and plasma leptin levels were decreased. Increased insulin levels in response to an oral glucose tolerance test were comparably attenuated by telmisartan and telmisartan + ramipril, but not by ramipril. During an insulin tolerance test, glucose utilization was equally as effectively improved by telmisartan and telmisartan + ramipril. In response to a stress test, ACTH, corticosterone and glucose increased in controls. These stress reactions were attenuated by telmisartan and telmisartan + ramipril.
CONCLUSIONS AND IMPLICATIONS The combination of telmisartan + ramipril was no more efficacious in regulating body weight and glucose homeostasis than telmisartan alone. However, telmisartan was more effective than ramipril in improving metabolic parameters and in reducing body weight. The association between the decrease in stress responses and the diminished glucose levels after stress supports our hypothesis that the ability of telmisartan, as an AT1 receptor blocker, to alleviate stress reactions may contribute to its hypoglycaemic actions.

Glucagon and a glucagon-GLP-1 dual-agonist increases cardiac performance with different metabolic effects in insulin-resistant hearts
LN Axelsen, W Keung, HD Pedersen, E Meier, D Riber, AL Kjølbye, JS Petersen, SD Proctor, N-H Holstein-Rathlou and GD Lopaschuk
British Journal of Pharmacology (2012) 165 2736–2748
DOI:10.1111/j.1476-5381.2011.01714.x
BACKGROUND AND PURPOSE The prevalence of heart disease continues to rise, particularly in subjects with insulin resistance (IR), and improved therapies for these patients is an important challenge. In this study we evaluated cardiac function and energy metabolism in IR JCR:LA-cp rat hearts before and after treatment with an inotropic compound (glucagon), a glucagon-like peptide-1 (GLP-1) receptor agonist (ZP131) or a glucagon-GLP-1 dual-agonist (ZP2495).
EXPERIMENTAL APPROACH Hearts from IR and lean JCR:LA rats were isolated and perfused in the working heart mode for measurement of cardiac function and metabolism before and after addition of vehicle, glucagon, ZP131 or ZP2495. Subsequently, cardiac levels of nucleotides and short-chain CoA esters were measured by HPLC.
KEY RESULTS Hearts from IR rats showed decreased rates of glycolysis and glucose oxidation, plus increased palmitate oxidation rates, although cardiac function and energy state (measured by ATP/AMP ratios) was normal compared with control rats. Glucagon increased glucose oxidation and glycolytic rates in control and IR hearts, but the increase was not enough to avoid AMP and ADP accumulation in IR hearts. ZP131 had no significant metabolic or functional effects in either IR or control hearts. In contrast, ZP2495 increased glucose oxidation and glycolytic rates in IR hearts to a similar extent to that of glucagon but with no concomitant accumulation of AMP or ADP.
CONCLUSION AND IMPLICATIONS Whereas glucagon compromised the energetic state of IR hearts, glucagon-GLP-1 dual-agonist ZP2495 appeared to preserve it. Therefore, a glucagon-GLP-1 dual-agonist may be beneficial compared with glucagon alone in the treatment of severe heart failure or cardiogenic shock in subjects with IR.

S(+)amphetamine induces a persistent leak in the human dopamine transporter: molecular stent hypothesis
Aldo A Rodriguez-Menchaca, Ernesto Solis Jr, Krasnodara Cameron and Louis J De Felice
British Journal of Pharmacology (2012) 165 2749–2757
DOI:10.1111/j.1476-5381.2011.01728.x
BACKGROUND AND PURPOSE Wherever they are located, dopamine transporters (DATs) clear dopamine (DA) from the extracellular milieu to help regulate dopaminergic signalling. Exposure to amphetamine (AMPH) increases extracellular DA in the synaptic cleft, which has been ascribed to DAT reverse transport. Increased extracellular DA prolongs postsynaptic activity and reinforces abuse and hedonic behaviour.
EXPERIMENTAL APPROACH Xenopus laevis oocytes expressing human (h) DAT were voltage-clamped and exposed to DA, R(-)AMPH, or S(+)AMPH.
KEY RESULTS At -60mV, near neuronal resting potentials, S(+)AMPH induced a depolarizing current through hDAT, which after removing the drug, persisted for more than 30 min. This persistent leak in the absence of S(+)AMPH was in contrast to the currents induced by R(-)AMPH and DA, which returned to baseline immediately after their removal. Our data suggest that S(+)AMPH and Na+ carry the initial S(+)AMPH-induced current, whereas Na+ and Cl- carry the persistent leak current. We propose that the persistent current results from the internal action of S(+)AMPH on hDAT because the temporal effect was consistent with S(+)AMPH influx, and intracellular S(+)AMPH activated the effect. The persistent current was dependent on Na+ and was blocked by cocaine. Intracellular injection of S(+)AMPH also activated a DA-induced persistent leak current.
CONCLUSIONS AND IMPLICATIONS We report a hitherto unknown action of S(+)AMPH on hDAT that potentially affects AMPH-induced DA release. We propose that internal S(+)AMPH acts as a molecular stent that holds the transporter open even after external S(+)AMPH is removed. Amphetamine-induced persistent leak currents are likely to influence dopaminergic signalling, DA release mechanisms, and amphetamine abuse.

The cytoprotective effects of oleoylethanolamide in insulin-secreting cells do not require activation of GPR119
Virginia M Stone, Shalinee Dhayal, David M Smith, Carol Lenaghan, Katy J Brocklehurst and Noel G Morgan
British Journal of Pharmacology (2012) 165 2758–2770
DOI:10.1111/j.1476-5381.2011.01755.x
BACKGROUND AND PURPOSE b-cells express a range of fatty acid-responsive G protein-coupled receptors, including GPR119, which regulates insulin secretion and is seen as a potential therapeutic target in type 2 diabetes. The long-chain unsaturated fatty acid derivative oleoylethanolamide (OEA) is an endogenous agonist of GPR119 and, under certain conditions, some long-chain unsaturated fatty acids can promote b-cell cytoprotection. It is not known, however, if OEA is cytoprotective in b-cells. The present study has examined this and determined whether GPR119 is involved. METHODS Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1E, were exposed to fatty acids complexed with BSA. cAMP levels, insulin release and cell viability were measured. Protein expression was studied by Western blotting and receptor expression by RT-PCR.
KEY RESULTS GPR119 was expressed in both BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA per se is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate.
CONCLUSIONS AND IMPLICATIONS Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection

Investigation of mechanism of drug-induced cardiac injury and torsades de pointes in cynomolgus monkeys
DL Misner, C Frantz, L Guo, MR Gralinski, PB Senese, J Ly, M Albassam and KL Kolaja
British Journal of Pharmacology (2012) 165 2771–2786
DOI:10.1111/j.1476-5381.2011.01756.x
BACKGROUND AND PURPOSE Drug candidates must be thoroughly investigated for their potential cardiac side effects. During the course of routine toxicological assessment, the compound RO5657, a CCR5 antagonist, was discovered to have the rare liability of inducing torsades de pointes (polymorphic ventricular arrhythmia) in normal, healthy animals. Studies were conducted to determine the molecular mechanism of this arrhythmia.
EXPERIMENTAL APPROACH Toxicological effects of repeat dosing were assessed in naïve monkeys. Cardiovascular effects were determined in conscious telemetry-implanted monkeys (repeat dosing) and anaesthetized instrumented dogs (single doses). Mechanistic studies were performed in guinea-pig isolated hearts and in cells recombinantly expressing human cardiac channels.
KEY RESULTS In cynomolgus monkeys, RO5657 caused a low incidence of myocardial degeneration and a greater incidence of ECG abnormalities including prolonged QT/QTc intervals, QRS complex widening and supraventricular tachycardia. In telemetry-implanted monkeys, RO5657 induced arrhythmias, including torsades de pointes and in one instance, degeneration to fatal ventricular fibrillation. RO5657 also depressed both heart rate (HR) and blood pressure (BP), with no histological evidence of myocardial degeneration. In the anaesthetized dog and guinea-pig isolated heart studies, RO5657 induced similar cardiovascular effects. RO5657 also inhibited Kv11.1 and sodium channel currents.
CONCLUSIONS AND IMPLICATIONS The molecular mechanism of RO5657 is hypothesized to be due to inhibition of cardiac sodium and Kv11.1 potassium channels. These results indicate that RO5657 is arrhythymogenic due to decreased hemodynamic function (HR/BP), decreased conduction and inhibition of multiple cardiac channels, which precede and are probably the causative factors in the observed myocardial degeneration.

Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates
AM Filppula1 , J Laitila1 , PJ Neuvonen1,2 and JT Backman
British Journal of Pharmacology (2012) 165 2787–2798
DOI:10.1111/j.1476-5381.2011.01732.x
BACKGROUND AND PURPOSE Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro. EXPERIMENTAL APPROACH Amodiaquine N-deethylation and midazolam 1?-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC50-shift, and time-dependent inhibition were assessed with human liver microsomes.
KEY RESULTS Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables KI and kinact were 14.3 mM and 0.072 min-1 respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a Ki of 8.4 and 12.8 mM, respectively, and midazolam 1?-hydroxylation with a Ki of 23.3 and 18.1 mM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.
CONCLUSIONS AND IMPLICATIONS Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib–simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.

Agonists at GPR119 mediate secretion of GLP-1 from mouse enteroendocrine cells through glucose independent pathways
H Lan, HV Lin, CF Wang, MJ Wright, S Xu, L Kang, K Juhl, JA Hedrick and TJ Kowalski
British Journal of Pharmacology (2012) 165 2799–2807
DOI:10.1111/j.1476-5381.2011.01754.x
BACKGROUND AND PURPOSE The G protein-coupled receptor 119 (GPR119) mediates insulin secretion from pancreatic b cells and glucagon-like peptide 1 (GLP-1) release from intestinal L cells. While GPR119-mediated insulin secretion is glucose dependent, it is not clear whether or not GPR119-mediated GLP-1 secretion similarly requires glucose. This study was designed to address the glucosedependence of GPR119-mediated GLP-1 secretion, and to explore the cellular mechanisms of hormone secretion in L cells versus those in b cells.
EXPERIMENTAL APPROACH GLP-1 secretion in response to GPR119 agonists and ion channel modulators, with and without glucose, was analysed in the intestinal L cell line GLUTag, in primary intestinal cell cultures and in vivo. Insulin secretion from Min6 cells, a pancreatic b cell line, was analysed for comparison.
KEY RESULTS In GLUTag cells, GPR119 agonists stimulated GLP-1 secretion both in the presence and in the absence of glucose. In primary mouse colon cultures, GPR119 agonists stimulated GLP-1 secretion under glucose-free conditions. Moreover, a GPR119 agonist increased plasma GLP-1 in mice without a glucose load. However, in Min6 cells, GPR119-mediated insulin secretion was glucose-dependent. Among the pharmacological agents tested in this study, nitrendipine, an L-type voltage-dependent calcium channel blocker, dose-dependently reduced GLP-1 secretion from GLUTag cells, but had no effect in Min6 cells in the absence of glucose.
CONCLUSIONS AND IMPLICATIONS Unlike that in pancreatic b cells, GPR119-mediated GLP-1 secretion from intestinal L cells was glucose-independent in vitro and in vivo, probably because of a higher basal calcium tone in the L cells.

Corrigendum (error)
In Ueng et al. (2011) there were errors in the y-axes of Figure 1D–F. The corrected panels appear below
British Journal of Pharmacology (2012) 165 2808
DOI:10.1111/j.1476-5381.2012.01915.x
As a result of these errors the values of the inactivation rates (Kapp) at different chalepensin concentrations, kinact (maximal inactivation rate constant of chalepensin) and KI (chalepensin concentration required for half-maximal inactivation) were incorrect. Further errors appeared on pages 1250, 1254 and 1256 and are corrected by the following: In the Key Results section of the abstract on page 1250 the sentence beginning ‘This time-dependent inactivation . . .’ should read ‘This time-dependent inactivation (kinact 0.298 min-1 ; KI 4.8 mM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation.’ In the Data and kinetic analyses section of the Methods on page 1254 ‘Lineweaver-Burke plots’ should be ‘Lineweaver-Burk plots’. In the same section the sentence beginning ‘The kinact . . .’ should read ‘The kinact (maximal inactivation rate constant of chalepensin) and KI (chalepensin concentration required for half-maximal inactivation) values were estimated from nonlinear regression from the equation Kapp = (kinactI)/(I+KI) with the initial values calculated from the double reciprocal plot of inactivation rate versus chalepensin concentration (Silverman, 1995).’ In the The metabolism-dependence and kinetic analysis of the CYP2A6 inhibition by chalepensin section of the Results on page 1254 the paragraph beginning ‘Pre-incubation with NADPH and chalepensin . . .’ should read ‘Pre-incubation with NADPH and chalepensin induced a time-dependent inactivation of coumarin 7-hydroxylation activity with kinact of 0.298 0.044 min-1 and apparent KI of 4.8 1.8 mM (Figures 1D,E). Linear regression analysis of the double reciprocal plots for inactivation rate and chalepensin concentration generated the kinact and apparent KI values for CYP2A6 of 0.233 min-1 and 2.6 mM respectively (Figure 1F).’ In the Discussion and conclusions on page 1256 the sentence beginning ‘In the presence of NADPH . . .’ should read ‘In the presence of NADPH, the efficiency of enzyme inactivation by chalepensin, calculated as the ratio of kinact to KI was 62.1 min-1 mM-1 .’ The errors in the figure and text do not change the conclusion of the study.

7-HYDROXY CANNABIDIOL (7-OH-CBD) FOR USE IN THE TREATMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD)

Applicant: GW PHARMA LIMITED
PCT/GB20 15/05 1893
Inventors: STOTT, Colin; Sover DUNCAN, Marnie; DI MARZO, Vincenzo;
MARTELLA, Andrea;
https://patents.google.com/patent/EP3160456A1
The liver plays a key role in regulating total body energy homeostasis and its ability to do so is greatly affected by the o occurrence of pathological conditions such as hepatosteatosis or non-alcoholic fatty liver disease (NAFLD), which contributes to hepatic insulin resistance and potentially end-stage liver disease-related mortality. Triglyceride accumulation in hepatocytes of steatotic livers results from the incorporation of plasma free fatty acids as well as de novo fat synthesis. The present invention relates to the use of 7-hydroxy-cannabidol (7-OH-CBD) in the treatment of non-alcoholic fatty liver disease (NAFLD). Treatment of NAFLD involves lowering the triglyceride levels in a patient's blood stream.

A124 SYNERGISTIC INTERACTION OF CANNABINOIDS AND OPIOIDS REDUCES PAIN SIGNALING IN COLONIC NOCICEPTIVE NERVES.
Tsang, Q., Yu, Y., Lomax, A. E., Vanner, S., & Reed, D. E.
Journal of the Canadian Association of Gastroenterology, 3(Supplement_1), 144–145.(2020).
doi:10.1093/jcag/gwz047.123
Background: With the recent legalization of recreational marijuana in Canada, increasing numbers of patients with gastrointestinal (GI) disorders are using cannabis to treat their pain, either alone or together with opioids. However, little is known about potential benefits of cannabinoids for treating visceral pain originating within the GI tract and whether the combined use of cannabinoids and opioids could enable the reduction or even discontinuation of opioids.
Aims: To investigate the effects of cannabinoids alone or in combination with opioids on colonic nociceptive nerves.
Methods: Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of male C57BL/6 mouse distal colons. Single unit analysis discriminated individual afferent neuron responses to mechanical probing of the colon with a 1g von Frey hair before and after superfusion of HU-210, a selective CB1 receptor agonist, HU-308, a selective CB2 receptor agonist, DAMGO, a selective muopioid receptor (MOR) agonist, or a combination. In parallel studies, perforated patch clamp techniques were employed to assess the rheobase as a measure of neuronal excitability in acutely dissociated dorsal root ganglia (DRG) neurons in the presence of one or more of these agonists. Data were analyzed using a one-way ANOVA with Bonferroni multiple comparisons test.
Results: Superfusion of HU-210 (1 ?M), caused significant inhibition in afferent nerve mechanosensitivity compared to control (6.2±1.1 vs. 13.7±2.5 Hz, p=0.005, n=10); lower concentrations (10 nM and 100 nM) had no effect (p>0.99, n=11; p=0.600, n=10 respectively). Conversely, the CB2 agonist HU-308 (1 ?M and 10 ?M), did not alter mechanosensitivity (p>0.9, n=8 for both concentrations). Superfusion of HU-210 alone (100 nM) or DAMGO (1 nM) alone in the same recording had no effect, but when both agonists were superfused together, there was a significant reduction in mechanosensitivity (8.1±1.7 vs. 14.8±2.3 Hz, p0.99, n=9; HU-210: p>0.99, n=10), whereas the combination of both agonists significantly decreased excitability (123.0±13.4 vs. 62.7±6.4 pA, p<0.01, n=10).
Conclusions: Activation of CB1 receptors inhibits mechanosensitivity of colonic afferent nerves while a CB2 agonist had no effect. Interestingly, combination of subthreshold concentrations of CB1 and MOR agonists inhibited colonic afferent nerves and thus, may suggest that cannabinoids could enable opioid dose reduction or discontinuation.

A Call for Person-Centered Language and Consistent Terminology Regarding Cannabis Use
Anna Kabakov, Radhika S. Polisetty, and Milena Murray
DOI: 10.1089/can.2021.0073
https://www.liebertpub.com/doi/epub/.../can.2021.0073
https://www.researchgate.net/publica...e_and_Consiste nt_Terminology_Regarding_Canna bis_Use
The increased availability of medical and recreational cannabis has facilitated a need for a change in health care practices. The language surrounding substance use disorders (SUDs) needs to be destigmatized. The necessity for utilizing “person-first” or “person-centered language” is fundamental to ensure that there is consistency among health care personnel for treating patients with an SUD and providing quality patient care. There also lies an enhanced need to more clearly define recreational cannabis use on a state versus federal level, in the workplace, and within higher education.
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A cannabigerol-rich Cannabis sativa extract, devoid of [INCREMENT]9-tetrahydrocannabinol, elicits hyperphagia in rats.
Brierley, D. I., Samuels, J., Duncan, M., Whalley, B. J., & Williams, C. M. Behavioural Pharmacology, 28(4), 280–284. (2017).
doi:10.1097/fbp.0000000000000285
Nonpsychoactive phytocannabinoids (pCBs) from Cannabis sativa may represent novel therapeutic options for cachexia because of their pleiotropic pharmacological activities, including appetite stimulation. We have recently shown that purified cannabigerol (CBG) is a novel appetite stimulant in rats. As standardized extracts from Cannabis chemotypes dominant in one pCB [botanical drug substances (BDSs)] often show greater efficacy and/or potency than purified pCBs, we investigated the effects of a CBG-rich BDS, devoid of psychoactive ?9 -tetrahydrocannabinol, on feeding behaviour. Following a 2 h prefeed satiation procedure, 16 male Lister-hooded rats were administered CBG-BDS (at 30–240 mg/kg) or vehicle. Food intake, meal pattern microstructure and locomotor activity were recorded over 2 h. The total food intake was increased by 120 and 240 mg/kg CBG-BDS (1.53 and 1.36 g, respectively, vs. 0.56 g in vehicletreated animals). Latency to feeding onset was dose dependently decreased at all doses, and 120 and 240 mg/kg doses increased both the number of meals consumed and the cumulative size of the first two meals. No significant effect was observed on ambulatory activity or rearing behaviour. CBG-BDS is a novel appetite stimulant, which may have greater potency than purified CBG, despite the absence of ?9 - tetrahydrocannabinol in the extract.

A Case of Vaping THC Oil Leading to Vaping Associated Pulmonary Injury: Our Approach to Its Diagnosis, Management, and Recommendations.
Singh, A., Tan, Q., Saccone, N. M., & Lindner, D. H.
Case Reports in Pulmonology, 2020, 1–5. (2020).
doi:10.1155/2020/6138083
Vaping’s popularity has grown exponentially since its introduction to the US market in 2003. Its use has sky-rocketed since the unveiling of the vaping pods in 2017 which may account for the advent of the vaping related illnesses we are now seeing. Substances such as nicotine solution, tetrahydrocannabinol (THC) oil, cannabidiol (CBD) oil, and butane hash oil (BHC) packaged in cartridges available in various ‘avors and concentrations are aerosolized by the heating of metal coils in the e-cigarette/ vaping devices. Cases from all over the country have recently been coming to light in which vaping has led to severe acute pulmonary disease or vaping-associated-pulmonary-injury (VAPI). A vast majority of the presenting patients in the reported cases have required hospitalization and intensive care, needing supplemental oxygen and even endotracheal intubation and mechanical ventilation. 98% of patients present with respiratory symptoms (dyspnea, hypoxia, chest pain, cough, hemoptysis), 81% of patients have gastrointestinal symptoms (nausea, vomiting, diarrhea, and abdominal pain), and 100% of patients have constitutional symptoms such as fever, chills, and fatigue/malaise on presentation. Although based on history and clinical presentation it is reasonable to have a high suspicion for VAPI, diagnostic workup to rule out alternative underlying causes such as infection, malignancy, or autoimmune process should be performed before establishing the diagnosis. Computed Tomography (CT) scans of the chest have predominantly shown ground-glass opacity in the lungs, ošen with areas of lobular or subpleural sparing. Although lung biopsies have been performed on a relatively low number of cases, lung injury patterns so far have shown acute ›brinous pneumonitis, diœuse alveolar hemorrhage, or organizing pneumonia, usually bronchiolocentric, and accompanied by bronchiolitis. Treatment plans that have led to clinical improvement in the reported cases center around high-dose systemic steroids, although there are a lack of data regarding the best regimen and the absolute need for corticosteroids. e role of antibiotics appears to be limited once infection has de›nitively been ruled out. We present the case of a young male who vaped THC oil and developed severe acute pulmonary injury requiring mechanical ventilation and showed a remarkable response to high dose steroid therapy with improvement in clinical symptoms and resolution of diœuse ground glass opacity on repeat HRCT scan.

A case study for the use of medical cannabis in generalized anxiety disorder
Chad Walkaden
DISCOVERIES 2019, 7(2): e92
DOI: 10.15190/d.2019.5
Despite the increasing prevalence and acceptance of the medical cannabis use among the general public, the evidence required by physicians to use cannabis as a treatment is generally lacking. Research on the health effects of cannabis and cannabinoids has been limited worldwide, leaving patients, health care professionals, and policymakers without the evidence they need to make sound decisions regarding the use of cannabis and cannabinoids. This case study outlines an intervention that involved a patient integrating medical cannabis into her treatment to better manage a generalized anxiety disorder and the debilitating symptoms of vertigo. This case demonstrates how the patient drastically improved her quality of life and reinforces the need for more rigorous testing on the use of medical cannabis to support patients and better manage the symptoms associated with their medical conditions.

A double-blind, randomized, crossover trial protocol of whole hemp seed protein and hemp seed protein hydrolysate consumption for hypertension.
Samsamikor, M., Mackay, D., Mollard, R. C., & Aluko, R. E.
Trials, 21(1). (2020).
doi:10.1186/s13063-020-4164-z
Background: Primary hypertension accounts for almost 95% of all cases of high blood pressure and is a major modifiable risk factor for cardiovascular diseases. Lifestyle interventions have been shown to prevent hypertension. One of the prominent potential therapeutic lifestyle strategies to prevent or manage hypertension is increasing dietary protein as a macronutrient or as bioactive peptides. An emerging plant-based protein source that may have anti-hypertensive properties is hemp seed. Methods/design: A randomized, double-blind, crossover clinical trial will be conducted on 35 hypertensive participants aged 18–75 years, with a BMI between 18.5 and 40 kg/m2 , systolic blood pressure (SBP) between 130 and 160 mmHg and diastolic blood pressure (DBP) ? 110 mmHg. The trial will be conducted for a period of 22 weeks and will consist of three treatment periods of 6 weeks, separated by 2-week washout periods. The treatments will be consumed twice a day and consist of 25 g casein, hemp seed protein (HSP), or HSP plus HSP hydrolysate (HSP+). The primary outcome of this trial is 24-h SBP, measured on the first day of first phase and the last day of each phase. Office-measured blood pressure, pulse-wave velocity and augmentation index and anthropometrics will be determined at the first and last days of each period. Also, body composition will be assessed by dual x-ray absorptiometry (DXA) scan on the first day of the first phase and within the last 2 days of each treatment period. Blood samples will be collected on the first and last 2 days of each treatment phase whereas urine samples will be collected on the first day of the first phase plus the last day of each phase to be analyzed for specific biomarkers. Discussion: This trial protocol is designed to evaluate the hypotensive potential of consuming whole HSP, and HSP+, in comparison to casein protein. This study will be the first trial investigating the potential anti-hypertensive benefit of dietary hemp protein plus bioactive peptide consumption in humans

A large-scale genome-wide association study meta-analysis of cannabis use disorder
Emma C Johnson, Ditte Demontis, Thorgeir E Thorgeirsson, et al
The Lancet psychiatry (2020)
https://www.thelancet.com/action/sho...2820%2930339-4
DOI: 10.1016/S2215-0366(20)30339-4
Background Variation in liability to cannabis use disorder has a strong genetic component (estimated twin and family heritability about 50–70%) and is associated with negative outcomes, including increased risk of psychopathology. The aim of the study was to conduct a large genome-wide association study (GWAS) to identify novel genetic variants associated with cannabis use disorder.
Methods To conduct this GWAS meta-analysis of cannabis use disorder and identify associations with genetic loci, we used samples from the Psychiatric Genomics Consortium Substance Use Disorders working group, iPSYCH, and deCODE (20916 case samples, 363 116 control samples in total), contrasting cannabis use disorder cases with controls. To examine the genetic overlap between cannabis use disorder and 22 traits of interest (chosen because of previously published phenotypic correlations [eg, psychiatric disorders] or hypothesised associations [eg, chronotype] with cannabis use disorder), we used linkage disequilibrium score regression to calculate genetic correlations. Findings We identified two genome-wide significant loci: a novel chromosome 7 locus (FOXP2, lead single-nucleotide polymorphism [SNP] rs7783012; odds ratio [OR] 1·11, 95% CI 1·07–1·15, p=1·84×10? ?) and the previously identified chromosome 8 locus (near CHRNA2 and EPHX2, lead SNP rs4732724; OR 0·89, 95% CI 0?86–0?93, p=6·46×10? ?). Cannabis use disorder and cannabis use were genetically correlated (rg 0·50, p=1·50×10? ²¹), but they showed significantly different genetic correlations with 12 of the 22 traits we tested, suggesting at least partially different genetic underpinnings of cannabis use and cannabis use disorder. Cannabis use disorder was positively genetically correlated with other psychopathology, including ADHD, major depression, and schizophrenia.
Interpretation These findings support the theory that cannabis use disorder has shared genetic liability with other psychopathology, and there is a distinction between genetic liability to cannabis use and cannabis use disorder.

A Literature Analysis on Medicinal Use and Research of Cannabis in the Meiji Era of Japan
Byung-Soo Ahn, Seokhyun Kang, Kyung Hoon Lee, Seoyoon Kim, Jin Sung Park, Hyung-Sik Seo
Journal of Pharmacopuncture 2020;23(3):142-157
DOI: 10.3831/KPI.2020.23.3.142
Cannabis is a historical plant which has been used as a medicine in East Asia. These days, there are active debates about using cannabis in clinical field. Collecting and comparing cannabis research articles which had been published in the Opening of Japan to spot the interactions between the traditional medicine of Japan, Rangaku which was established in Edo Period and the European medicine which is transferred after Perry Expedition is academically meaningful. This study searched publications, which were listed on Open-Access databases by Dec. 11th, 2019. We collected research articles which had been published from January 3rd, 1867 to July 30th, 1912 also known as Meiji era and uploaded on OpenAccess databases. Our searching databases were J-stage, CiNii (Scholarly and Academic Information Navigator), Tokyo Metropolitan Library, The National Diet Library, IRDB (Institutional Repositories DataBase) and KAKEN (Grant-in-Aid for Scientific Research Database). Searching keywords were cannabis, hemp and all their Japanese synonyms and available combinations. We selected final 15 studies which met every selection criteria in the 346,393 collected studies. Cannabis was prescribed in Meiji era of Japan to alleviate pain and cure the digestive, respiratory, urinary, and nervous system diseases such as indigestion, asthma, tuberculosis, gonorrhea and its complications, insomnia, and nervous prostration. Cannabis was medically used in Meiji era of Japan and the reporting and sharing of its clinical effect was published on the medical journals like present days. There were already Cannabis regulations in that era, but its medicinal use was more liberated than nowadays. It may be a chance to reconsider the current legal system, which strictly controls the use of Cannabis

A Molecular Link Between the Active Component of Marijuana and Alzheimer's Disease Pathology
Lisa M. Eubanks, Claude J. Rogers, Albert E. Beuscher IV, George F. Koob, Arthur J.
Olson, Tobin J. Dickerson, and Kim D. Janda
December 2006 Molecular Pharmaceutics 3(6):773-7
DOI: 10.1021/mp060066m
Alzheimer's disease is the leading cause of dementia among the elderly, and with the ever-increasing size of this population, cases of Alzheimer's disease are expected to triple over the next 50 years. Consequently, the development of treatments that slow or halt the disease progression have become imperative to both improve the quality of life for patients and reduce the health care costs attributable to Alzheimer's disease. Here, we demonstrate that the active component of marijuana, Delta9-tetrahydrocannabinol (THC), competitively inhibits the enzyme acetylcholinesterase (AChE) as well as prevents AChE-induced amyloid beta-peptide (Abeta) aggregation, the key pathological marker of Alzheimer's disease. Computational modeling of the THC-AChE interaction revealed that THC binds in the peripheral anionic site of AChE, the critical region involved in amyloidgenesis. Compared to currently approved drugs prescribed for the treatment of Alzheimer's disease, THC is a considerably superior inhibitor of Abeta aggregation, and this study provides a previously unrecognized molecular mechanism through which cannabinoid molecules may directly impact the progression of this debilitating disease.

A Multicenter Dose-escalation Study of the Analgesic and Adverse Effects of an Oral Cannabis Extract (Cannador) for Postoperative Pain Management Anita Holdcroft, M.D.,* Mervyn Maze, F.R.C.P., F.R.C.A., F.Med.Sci.,† Caroline Dore´ , B.Sc.,‡ Susan Tebbs, M.Sc.,§ Simon Thompson, D.Sc.
Anesthesiology 2006; 104:1040–6
https://watermark.silverchair.com/00...oRmNkBV8S7YilW
Background: Cannabinoids have dose-related antinociceptive effects in animals. This clinical study aimed to investigate whether a single oral dose of cannabis plant extract (Cannador; Institute for Clinical Research, IKF, Berlin, Germany) could provide pain relief with minimal side effects for postoperative pain. Methods: Patients (aged 18–75 yr) were recruited and consented before surgery if patient-controlled analgesia was planned for provision of postoperative pain relief. Each patient received a single dose of 5, 10, or 15 mg Cannador if he or she had at least moderate pain after stopping patient-controlled analgesia. Starting with 5 mg, dose escalation was based on the number of patients requesting rescue analgesia and adverse effects. Pain relief, pain intensity, and side effects were recorded over 6 h and analyzed using tests for trend with dose. Results: Rescue analgesia was requested by all 11 patients (100%) receiving 5 mg, 15 of 30 patient (50%) receiving 10 mg, and 6 of 24 patients (25%) receiving 15 mg Cannador (log rank test for trend in time to rescue analgesia with dose P < 0.001). There were also significant trends across the escalating dose groups for decreasing pain intensity at rest (P 0.01), increasing sedation (P 0.03), and more adverse events (P 0.002). The number needed to treat to prevent one rescue analgesia request for the 10-mg and 15-mg doses, relative to 5 mg, were 2.0 (95% confidence interval, 1.5–3.1) and 1.3 (95% confidence interval, 1.1–1.7), respectively. The study was terminated because of a serious vasovagal adverse event in a patient receiving 15 mg. Conclusion: These significant dose-related improvements in rescue analgesia requirements in the 10 mg and 15 mg groups provide a number needed to treat that is equivalent to many routinely used analgesics without frequent adverse effects.

A National Survey of Marijuana Use Among US Adults With Medical Conditions, 2016-2017.
Dai, H., & Richter, K. P.
JAMA Network Open, 2(9), e1911936. (2019).
doi:10.1001/jamanetworkopen.2019.11936
IMPORTANCE The number of states legalizing marijuana for medical and recreational use is increasing. Little is known regarding how or why adults with medical conditions use it.
OBJECTIVES To report the prevalence and patterns of marijuana use among adults with and without medical conditions, overall and by sociodemographic group, and to further examine the associations between current marijuana use and the types and number of medical conditions.
DESIGN, SETTING, AND PARTICIPANTS This survey study used a probability sample of US adults aged 18 years and older from the 2016 and 2017 Behavioral Risk Factor Surveillance System, a telephone-administered survey that collects data from a representative sample of US adult residents across the states regarding health-related risk behaviors, chronic health conditions, and use of preventive services.
MAIN OUTCOMES AND MEASURES Current (past month) and daily (20 days in the last 30 days) marijuana use.
RESULTS The study sample included 169 036 participants (95 780 female [weighted percentage, 52.0%]). Adults with medical conditions had higher odds of reporting current marijuana use than those without medical conditions (age 18-34 years: adjusted odds ratio, 1.8 [95% CI, 1.5-2.1]; age 35-54 years: adjusted odds ratio, 1.4 [95% CI, 1.2-1.7]; age 55 years: adjusted odds ratio, 1.6 [95% CI, 1.3-2.0]), especially among those with asthma, chronic obstructive pulmonary disease, arthritis, cancer, and depression. Among those with medical conditions, the prevalence of marijuana use decreased with increasing age, ranging from 25.2% (95% CI, 22.0%-28.3%) for those aged 18 to 24 years to 2.4% (95% CI, 2.0%-2.8%) for those aged 65 years or older for current marijuana use and from 11.2% (95% CI, 8.7%-13.6%) to 0.9% (95% CI, 0.7%-1.2%), respectively, for daily marijuana use. Most adults who used marijuana (77.5%; 95% CI, 74.7%-80.3%), either with or without medical conditions, reported smoking as their primary method of administration. Adults with medical conditions were more likely than those without medical conditions to report using marijuana for medical reasons (45.5% [95% CI, 41.1%-49.8%] vs 21.8% [95% CI, 17.8%-25.7%]; difference, 23.7% [95% CI, 17.8%-29.6%]) and less likely to report using marijuana for recreational purposes (36.2% [95% CI, 32.1%-40.3%] vs 57.7% [95% CI, 52.6%-62.9%]; difference, ?21.5% [95% CI, ?28.1% to 14.9%]). CONCLUSIONS AND RELEVANCE This study found that marijuana use was more common among adults with medical conditions than those without such conditions. Notably, 11.2% of young adults with medical conditions reported using marijuana on a daily basis. Clinicians should screen for marijuana use among patients, understand why and how patients are using marijuana, and work with patients to optimize outcomes and reduce marijuana-associated risks.

A New Method of Cannabis Ingestion: The Dangers of Dabs?
Mallory Loflin, Mitch Earleywine
Addictive Behaviors (2014), Oct;39(10):1430-3.
doi: 10.1016/j.addbeh.2014.05.013
A new method for administering cannabinoids, called butane hash oil (“dabs”), is gaining popularity among marijuana users. Despite press reports that suggest that “dabbing” is riskier than smoking flower cannabis, no data address whether dabs users experience more problems from use than those who prefer flower cannabis.
Objective: The present study aimed to gather preliminary information on dabs users and test whether dabs use is associated with more problems than using flower cannabis.
Method: Participants (n = 357) reported on their history of cannabis use, their experience with hash oil and the process of “dabbing,” reasons for choosing “dabs” over other methods, and any problems related to both flower cannabis and butane hash oil.
Results: Analyses revealed that using “dabs” created no more problems or accidents than using flower cannabis. Participants did report that “dabs” led to higher tolerance and withdrawal (as defined by the participants), suggesting that the practice might be more likely to lead to symptoms of addiction or dependence.
Conclusions: The use of butane hash oil has spread outside of the medical marijuana community and users view it as significantly more dangerous than other forms of cannabis use.

A Novel Observational Method for Assessing Acute Responses to Cannabis: Preliminary Validation Using Legal Market Strains.
Bidwell, L. C., Mueller, R., YorkWilliams, S. L., Hagerty, S., Bryan, A. D., & Hutchison, K. E.
Cannabis and Cannabinoid Research, 3(1), 35–44.
doi:10.1089/can.2017.0038
Background: The development of novel cannabis research methods that are compatible with current federal regulations is imperative to conduct studies of the effects of legal market cannabis. There is very little research on higher strength, higher D9-tetrahydrocannabinol (THC), which has become increasingly available since legalization. Research on strains containing cannabidiol (CBD), a second primary, but nonpsychotomimetic, cannabinoid, is very limited.
Materials and Methods: Using a novel observational methodology, regular cannabis users were asked to use one of two legal market cannabis strains that they purchased from a local dispensary (one strain containing 8% THC and 16% CBD (THC+CBD) and one containing a 17% THC concentration, but no CBD (THC). After using their suggested cannabis strain as they typically would for a 3-day period, participants returned to the laboratory immediately after their final use. Measures included a blood draw to measure cannabinoid blood levels and circulating cytokines, self-reported subjective drug effects, and verbal recall memory.
Results: Analysis of CBD/THC concentration levels in the blood following the 3-day strain manipulation suggests that all, but one participant (n = 23/24) followed instructions and used their assigned strain. Individuals in the THC group (n = 11) smoked no more than their usual amount, and participants who used the THC+CBD (n = 12) strain smoked more than their reported usual amount, but did not have significantly different THC+metabolite blood levels from the THC group. The THC+CBD strain was also associated with less desire to smoke, lower levels of subjective drug effects, and lower levels of circulating cytokines (TNF-a, IL-6, and IL-1b) immediately after use. Conclusions: Initial results support the feasibility of this novel observational methodology involving brief manipulation of strain use. Preliminary findings indicate that participants may self-titrate cannabis use based on cannabinoid concentration and the THC+CBD strain was associated with lower levels of cannabis craving, subjective intoxication, and circulating cytokines.

A narrative review of the ethnomedicinal usage of Cannabis sativa Linnaeus as traditional phytomedicine by folk medicine practitioners of Bangladesh
Shahriar S. M. Shakil, Matt Gowan, Kerry Hughes, Md. Nur Kabidul Azam, Md. Nasir Ahmed
Journal of Cannabis Research 3(1) December 2021
DOI: 10.1186/s42238-021-00063-3
Background There is a worldwide interest in the use of Cannabis sativa for biomedicine purposes. Cannabis has ethnomedicinal usage as a natural medicine in Bangladesh and cultivated during the British Empire period for revenues. Objective Folk medicine practitioners (FMPs) from different districts of Bangladesh have been using Cannabis sativa , but until now there have not been any compiled studies particularly regarding this practice. Hence, this review is an effort to retrieve the traditional usage of Cannabis sativa as a phytomedicine from published ethnomedicinal studies. Methods and materials Information was searched by using the search terms “ethnomedicinal Cannabis sativa and Bangladesh”; “Bangladesh cannabaceae and ethnomedicinal survey”; “ganja, bhang and folk medicine Bangladesh”; “tetrahydrocannabinol (THC), cannabinoid and therapeutic, clinical trial”; and “cannabis and pharmacological/biological” and retrieved from ethnobotanical articles available on PubMed, Scopus, Science Direct, and Google Scholar databases. A search of the relevant scientific literature also was conducted to assess the efficacy of the ethnomedicinal usage of Cannabis sativa. Results While reviewing over 200 ethnomedicinal plants’ survey articles, we found that FMPs of Bangladesh from 12 different districts used Cannabis sativa to treat cited ailments like sleep-associated problems ( n =5), neuropsychiatric and CNS problems ( n =5), and infections and respiratory problems ( n =5) followed by rheumatism, gastrointestinal, gynecological ( n =4 each), cancer, sexual, and other ailments including hypertension, headache, itch, increases bile secretion, abortifacient, dandruff, fever, and urinary problems ( n =1 each). There are a total of 15 formulations identified from the 11 out of 18 ethnomedicinal plant survey reports. The leaf was the main plant part used (53.8%), followed by root (23%), seed (7.7%) and flower, inflorescence, resin, and all parts 3.8% respectively. Conclusions Sales and cultivation of Cannabis are illegal at present in Bangladesh, but the use of Cannabis sativa as a natural phytomedicine has been practiced traditionally by folk medicine practitioners of Bangladesh for many years and validated through relevant pharmacological justification. Although Cannabis sativa possesses ethnomedicinal properties in the folk medicine of Bangladesh, it is, furthermore, needed to conduct biological research to consolidate pharmacological justification about the prospects and challenges of Cannabis and cannabinoids’ use in Bangladesh as safer biomedicine in the future.

A Network Pharmacology Approach to Identify Potential Molecular Targets for Cannabidiol’s Anti-Inflammatory Activity
Hang Ma,1,* Feng Xu,1,2 Chang Liu,1 and Navindra P. Seeram
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0025
Introduction: Published preclinical and clinical studies support the anti-inflammatory activity of CBD, but the molecular targets (e.g., genes and proteins) that are involved in its mechanisms of action remain unclear. Herein, a network-based pharmacology analysis was performed to aid in the identification of potential molecular targets for CBD’s anti-inflammatory activity. Materials and Methods: Target genes and proteins were obtained from several online databases, including Swiss target prediction, Online Mendelian Inheritance in Man, and the DrugBank database. A compound-targetdisease network was constructed with Cytoscape tool, and a network of protein–protein interactions was established with the Search Tool for the Retrieval of Interacting Genes/Proteins database. Lead proteins identified from the compound-target-disease network were further studied for their interactions with CBD by computational docking. In addition, biological pathways involved in CBD’s anti-inflammatory activity were identified with the Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes analysis. Results: A panel of proteins, including cellular tumor antigen p53, NF-kappa-B essential modulator, tumor necrosis factor (TNF) receptor, transcription factor p65, NF-kappa-B p105, NF-kappa-B inhibitor alpha, inhibitor of nuclear factor kappa-B kinase subunit alpha, and epidermal growth factor receptor, were identified as lead targets involved in CBD’s anti-inflammatory activity. This finding was further supported by molecular docking, which showed interactions between the lead proteins and CBD. In addition, several signaling pathways, including TNF, toll-like receptor, mitogen-activated protein kinases, nuclear factor kappa-light-chain-enhancer of activated B cells, and nucleotide-binding oligomerization domain-like receptors, were identified as key regulators in the mediation of CBD’s anti-inflammatory activity. Conclusion: A network-based pharmacology analysis identified potential molecular targets and signaling pathways for CBD’s anti-inflammatory activity. Findings from this study add to the growing body of data supporting the utilization of CBD as a promising anti-inflammatory natural product.


A Phase I, Randomized, Double?Blind, Placebo?Controlled, Single Ascending
Application of Hempseed (Cannabis sativa L.) Oil in the Treatment of Ear, Nose and Throat (ENT) Disorders

Oleg V. Grigoriev
June 2002 Journal of Industrial Hemp 7(2):5-15
DOI: 10.1300/J237v07n02_02

A Phase I Trial of the Safety, Tolerability, and Pharmacokinetics of Cannabidiol Administered as Single-dose Oil Solution and Single and Multiple doses of a Sublingual Wafer in Healthy Volunteers
Adele Hosseini, Andrew J McLachlan, Jason D Lickliter
????FIND JOURNAL
DOI: 10.1111/bcp.14617
Aims: This study investigated the safety, tolerability, and PK after administration of a specific Cannabis sativa cultivar extract, standardised to CBD content as sublingual wafer or oil formulation compared to nabiximols oromucosal spray.
Methods: For the single-dose study, the design was an open-label, four-way crossover in 12 healthy volunteers randomised to receive a sequence of four different single doses of CBD as a sublingual wafer (25 or 50 mg CBD), oil solution (50 mg CBD), or nabiximols oromucosal spray (20 mg CBD, 21.6 mg THC). For the multiple-dose study, sublingual wafer (50 mg CBD) was administered twice a day for five days.
Results: The extract was generally well tolerated by participants when administered in either wafer or oil form, with some adverse events, including mild or moderate somnolence, sedation and altered mood. The relative bioavailability of CBD after administration as a sublingual wafer was comparable with that of oil solution with 90% confidence interval of 83–131%. The median maximum concentrations of CBD after administration of oil solution and wafer was 9.4 and 11.9 ng mL-1 , respectively. Maximum concentrations of CBD occurred 4 hours after administration, with an estimated terminal elimination half-life of 6 hours. There was no statistically significant difference between the AUC0-t of CBD after administration of oil solution or wafer compared with nabiximols oromucosal spray.
Conclusion: Oil solution and sublingual wafer formulations of the extract standardised with CBD were well tolerated and achieved equivalent concentrations of CBD when compared to an available commercial nabiximols formulation.
Conclusion CBD was generally well tolerated. Most AEs were mild in severity; none were severe or serious. The safety and PK profle support twice-daily administration of CBD.

A pilot study of the effects of cannabis on appetite hormones in HIV-infected adult men
Patricia K. Riggsa, Florin Vaidaa, Steven S. Rossib, Linda S. Sorkinc, Ben Gouauxa, Igor Granta, Ronald J. Ellisa
B R A I N R E S E A R C H 1 4 3 1 ( 2 0 1 2 ) 4 6 – 5 2
doi:10.1016/j.brainres.2011.11.001
Rationale: The endocannabinoid system is under active investigation as a pharmacological target for obesity management due to its role in appetite regulation and metabolism. Exogenous cannabinoids such as tetrahydrocannabinol (THC) stimulate appetite and food intake. However, there are no controlled observations directly linking THC to changes of most of the appetite hormones.
Objectives: We took the opportunity afforded by a placebo-controlled trial of smoked medicinal cannabis for HIV-associated neuropathic pain to evaluate the effects of THC on the appetite
hormones ghrelin, leptin and PYY, as well as on insulin.
Methods: In this double-blind cross-over study, each subject was exposed to both active cannabis (THC) and placebo.
Results: Compared to placebo, cannabis administration was associated with significant increases in plasma levels of ghrelin and leptin, and decreases in PYY, but did not significantly influence insulin levels.
Conclusion: These findings are consistent with modulation of appetite hormones mediated through endogenous cannabinoid receptors, independent of glucose metabolism

A Placebo-Controlled Trial of Atomoxetine in Marijuana-Dependent Individuals with Attention Deficit Hyperactivity Disorder.
McRae-Clark, A. L., Carter, R. E., Killeen, T. K., Carpenter, M. J., White, K. G., & Brady, K. T.
The American Journal on Addictions, 19(6), 481–489. (2010).
doi:10.1111/j.1521-0391.2010.00076.x
This study evaluated the effects of atomoxetine on the symptoms of attention deficit hyperactivity disorder (ADHD) and marijuana use in marijuana-dependent adults. In conjunction with motivational interviewing, participants received either atomoxetine (n = 19) or matching placebo (n = 19) for 12 weeks. Participants randomized to atomoxetine had greater improvement in ADHD on the Clinical Global Impression-Improvement scale than participants treated with placebo. No treatment group differences in selfrated ADHD symptoms, overall Wender-Reimherr Adult Attention Deficit Disorder Scale scores, or marijuana use outcomes were noted. These results suggest that atomoxetine may improve some ADHD symptoms but does not reduce marijuana use in this population

A potential role for cannabichromene in modulating TRP channels during acute respiratory distress syndrome
Hesam Khodadadi, Évila Lopes Salles, Eunice Shin, Abbas Jarrahi, Vincenzo Costigliola, Pritesh Kumar, Jack C. Yu, John C. Morgan, David C. Hess, Kumar Vaibhav, Krishnan M. Dhandapani and Babak Baban
J Cannabis Res (2021) 3:45
DOI: 10.1186/s42238-021-00101-0
https://jcannabisresearch.biomedcent.. .21-00101-0.pdf
Background: Acute respiratory distress syndrome (ARDS) is a life-threatening clinical syndrome whose potential to become one of the most grievous challenges of the healthcare system evidenced by the COVID-19 pandemic. Considering the lack of target-specifc treatment for ARDS, it is absolutely exigent to have an efective therapeutic modality to reduce hospitalization and mortality rate as well as to improve quality of life and outcomes for ARDS patients. ARDS is a systemic infammatory disease starting with the pulmonary system and involves all other organs in a morbid
bidirectional fashion. Mounting evidence including our fndings supporting the notion that cannabinoids have potential to be targeted as regulatory therapeutic modalities in the treatment of infammatory diseases. Therefore, it is plausible to test their capabilities as alternative therapies in the treatment of ARDS. In this study, we investigated the potential protective efects of cannabichromene (CBC) in an experimental model of ARDS.
Methods: We used, for the frst time, an inhalant CBC treatment as a potential therapeutic target in a murine model of ARDS-like symptoms. ARDS was induced by intranasal administration of Poly(I:C), a synthetic mismatched doublestranded RNA, into the C57BL/6 mice (6–10 male mice/group, including sham, placebo, and CBC treated), three once-daily doses followed by a daily dose of inhalant CBC or placebo for the period of 8 days starting the frst dose 2 h after the second Poly(I:C) treatment. We employed histologic, immunohistochemistry, and fow cytometry methods to assess the fndings. Statistical analysis was performed by using one way analysis of variance (ANOVA) followed by Newman–Keuls post hoc test to determine the diferences among the means of all experimental groups and to establish signifcance (p < 0.05) among all groups.
Results: Our data showed that CBC was able to reverse the hypoxia (increasing blood O2 saturation by 8%), ameliorate the symptoms of ARDS (reducing the pro-infammatory cytokines by 50% in lung and blood), and protect the lung tissues from further destruction. Further analysis showed that CBC may wield its protective efects through transient receptor potential (TRP) cation channels, TRPA1 and TRPV1, increasing their expression by 5-folds in lung tissues compared to sham and untreated mice, re-establishing the homeostasis and immune balance.
Conclusion: Our fndings suggest that inhalant CBC may be an efective alternative therapeutic target in the treatment of ARDS. In addition, Increased expression of TRPs cation channels after CBC treatment proposes a novel role for TRPs (TRPA1 and TRPV2) as new potential mechanism to interpret the benefcial efects of CBC as well as other cannabinoids in the treatment of ARDS as well as other infammatory diseases. Importantly, delivering CBC through an inhaler device is a translational model supporting the feasibility of trial with human subjects, authorizing further research.

A preliminary controlled study to determine whether whole-plant cannabis extracts can improve intractable neurogenic symptoms.
Wade, D. T., Robson, P., House, H., Makela, P., & Aram, J.
Clinical Rehabilitation, 17(1), 21–29.(2003).
doi:10.1191/0269215503cr581oa
Objectives: To determine whether plant-derived cannabis medicinal extracts (CME) can alleviate neurogenic symptoms unresponsive to standard treatment, and to quantify adverse effects.
Design: A consecutive series of double-blind, randomized, placebo-controlled single-patient cross-over trials with two-week treatment periods. Setting: Patients attended as outpatients, but took the CME at home. Subjects: Twenty-four patients with multiple sclerosis (18), spinal cord injury (4), brachial plexus damage (1), and limb amputation due to neuro?bromatosis (1). Intervention: Whole-plant extracts of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), 1:1 CBD:THC, or matched placebo were self-administered by sublingual spray at doses determined by titration against symptom relief or unwanted effects within the range of 2.5–120 mg/24 hours. Measures used: Patients recorded symptom, well-being and intoxication scores on a daily basis using visual analogue scales. At the end of each two-week period an observer rated severity and frequency of symptoms on numerical rating scales, administered standard measures of disability (Barthel Index), mood and cognition, and recorded adverse events. Results: Pain relief associated with both THC and CBD was significantly superior to placebo. Impaired bladder control, muscle spasms and spasticity were improved by CME in some patients with these symptoms. Three patients had transient hypotension and intoxication with rapid initial dosing of THC-containing CME.
Conclusions: Cannabis medicinal extracts can improve neurogenic symptoms unresponsive to standard treatments. Unwanted effects are predictable and generally well tolerated. Larger scale studies are warranted to confirm these fndings

A Phase 2 Randomized Controlled Trial of the Efficacy and Safety of Cannabidivarin as Add-on Therapy in Participants with Inadequately Controlled Focal Seizures
Martin J. Brodie, Piotr Czapinski, Ladislav Pazdera, Josemir W. Sander, Manuel Toledo, Mariana Napoles, Farhad Sahebkar, and Ashley Schreiber
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0075
https://www.liebertpub.com/doi/pdf/1.../can.2020.0075
Objective: We assessed the efficacy, safety, and tolerability of cannabidivarin (CBDV) as add-on therapy in adults with inadequately controlled focal seizures.
Materials and
Methods: One hundred and sixty-two participants (CBDV n = 81; placebo n = 81) were enrolled. After a 4-week baseline, participants titrated from 400 to 800 mg CBDV twice daily (b.i.d.) (or placebo) over 2 weeks, followed by 6 weeks stable dosing (at 800 mg b.i.d.) and a 12-day taper period. The primary endpoint was the change from baseline in focal seizure frequency during the 8-week treatment period. Secondary endpoints
included additional efficacy measures relating to seizures, physician- and participant-reported outcomes, change in the use of rescue medication, cognitive assessments, and safety.
Results: Median baseline focal seizure frequencies were 17–18 per 28 days in both groups, and similar reductions in frequency were observed in the CBDV (40.5%) and placebo (37.7%) groups during the treatment period (treatment ratio [% reduction] CBDV/placebo: 0.95 [4.6]; confidence interval: 0.78–1.17 [_16.7 to 21.9]; p = 0.648). There were no differences between the CBDV and placebo groups for any seizure subtype. There were no significant treatment differences between CBDV and placebo groups for any of the secondary efficacy outcome measures. Overall, 59 (72.8%) of participants in the CBDV group and 39 (48.1%) in the placebo group had ‡ 1 treatment-emergent adverse event (AE); the 3most common were diarrhea, nausea, and somnolence. The incidence of serious AEs was low (3.7% in the CBDV group vs. 1.2% in the placebo group). There was little or no effect of CBDV on vital signs, physical examination, or electrocardiogram findings. Elevations in serum transaminases (alanine aminotransferase or aspartate aminotransferase) to levels > 3 · upper limit of normal occurred in three participants taking CBDV (two discontinued as a result) and one taking placebo; however, none met the criteria for potential Hy’s Law cases.
Conclusion: It is likely the 40.5% seizure reduction with CBDV represents an appropriate pharmacological response in this population with focal

A primer on medicinal cannabis safety and potential adverse effects
Jonathon C Arnold
AJGP Vol. 50, No. 6, June 2021
https://www1.racgp.org.au/getattachm...-potentia.aspx
Background
Medicinal cannabis prescriptions are on the rise in Australia, and general practitioners will increasingly encounter patients using cannabis-based products.
Objective
The aim of this review is to provide a primer on the safety issues that need to be considered with medicinal cannabis.
Discussion
Medicinal cannabis is generally well tolerated when dosed appropriately. It is important for doctors to consider carefully the ∆9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD) content of the products. CBD is not intoxicating and has fewer safety concerns than THC. When commencing a new medicinal cannabis product, the recommendation is to prescribe relatively low doses and slowly up-titrate the dose. This aims to minimise dose-related toxicities and the potential for drug–drug interactions with concomitant medications. THC found in medicinal cannabis may acutely impair cognitive function and is best not prescribed to children or adolescents unless the benefits outweigh the risks. THC-containing cannabis products should not be prescribed to individuals with angina or a history of myocardial infarction, or to those who have a personal or family history of psychosis.

A Putative ‘Pre-Nervous’ Endocannabinoid System in Early Echinoderm Development G.A. Buznikov, L.A. Nikitina, V.V. Bezuglov, M.E.Y. Francisco, G. Boysen, Obispo-Peak, R.E. Peterson, E.R. Weiss, H. Schuel, B.R.S. Temple, A.L. Morrow, J.M. Lauder
Dev Neurosci 2010;32:1–18 DOI: 10.1159/000235758
https://www.karger.com/Article/PDF/235758
Embryos and larvae of sea urchins ( Lytechinus variegatus, Strongylocentrotus droebachiensis , Strongylocentrotus purpuratus, Dendraster excentricus), and starfish ( Pisaster ochraceus ) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8–16 cell and mid-blastula 2 stage. ‘Perturb-andrescue’ experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [ 3 H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [ 3 H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate or thologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D 1 , D 2 or 1 -adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences


A randomized trial of medical Cannabis in patients with stage IV cancers to assess feasibility, dose requirements, impact on pain and opioid use, safety, and overall patient satisfaction
Zylla, Dylan M., Eklund, Justin, Gilmore, Grace, Gavenda, Alissa, Guggisberg, Jordan, VazquezBenitez, Gabriela, Pawloski, Pamala A., Arneson, Tom, Richter, Sara, Birnbaum, Angela K., Dahmer, Stephen, Tracy, Matthew, Dudek, Arkadiusz
Supportive Care in Cancer ( IF 3.603 ) Pub Date : 2021-06-04 ,
DOI: 10.1007/s00520-021-06301-x
Purpose
The prevalence of medical cannabis (MC) use in patients with cancer is growing, but questions about safety, efficacy, and dosing remain. Conducting randomized, controlled trials (RCTs) using state-sponsored MC programs is novel and could provide data needed to guide patients and providers.
Methods
A pilot RCT of patients with stage IV cancer requiring opioids was conducted. Thirty patients were randomized 1:1 to early cannabis (EC, n = 15) versus delayed start cannabis (DC, n = 15). The EC group obtained 3 months (3 M) of MC through a state program at no charge, while the DC group received standard oncology care without MC for the first 3 M. Patients met with licensed pharmacists at one of two MC dispensaries to determine a suggested MC dosing, formulation, and route. Patients completed surveys on pain levels, opioid/MC use, side effects, and overall satisfaction with the study.
Results
Interest in the study was high as 36% of patients who met eligibility criteria ultimately enrolled. The estimated mean daily THC and CBD allotments at 3 M were 34 mg and 17 mg, respectively. A higher proportion of EC patients achieved a reduction in opioid use and improved pain control. No serious safety issues were reported, and patients reported high satisfaction.
Conclusion
Conducting RCTs using a state cannabis program is feasible. The addition of MC to standard oncology care was well-tolerated and may lead to improved pain control and lower opioid requirements. Conducting larger RCTs with MC in state-sponsored programs may guide oncology providers on how to safely and effectively incorporate MC for interested patients.

A Report of Adverse Effects Associated With the Administration of Cannabidiol in Healthy Dogs
Stephanie McGrath, Lisa R. Bartner Sangeeta Rao, Lori R. Kogan, Peter W. Hellyer
Journal of the American Holistic Veterinary Medical Association (JAHVMA). Volume 52, Fall Issue, 2018.
https://www.ahvma.org/wp-content/upl...rseEffects.pdf
Cannabis-based therapies have been used for centuries for various medicinal purposes. They have recently gained recognition as an effective treatment for medical conditions in humans; and, as such, awareness is increasing among veterinarians and pet owners. However, side effects, pharmacokinetics, and effi¬cacy in dogs are not known. The purpose of this study was to determine the tolerability of cannabidiol (CBD) by healthy dogs. We hypothesized that
CBD would be tolerated in a healthy population of dogs. A group of 30 healthy Beagle dogs were randomly assigned to receive CBD in the form of microencapsulated oil beads (capsule), CBD-infused oil, or CBD-infused
transdermal cream at a dose of 10 mg/kg/day or 20 mg/ kg/day for 6 weeks. Complete blood counts, chemistry panels, urinalysis, and bile acids were performed at 0, 2, 4, and 6 weeks. Elevations in serum ALP occurred in some
dogs. All of the dogs in the study experienced diarrhea that was not associated with the formulation or dose of CBD that they received. CBD appeared to be well tolerated in dogs. However, a more extensive safety study is necessary to
determine if there are long-term effects of CBD on the liver and an association with diarrhea.

A Review of Scientific Evidence for THC:CBD Oromucosal Spray (Nabiximols) in the Management of Chronic Pain
Michael A Überall
Journal of Pain Research 2020:13 399–410
doi: 10.2147/JPR.S240011
The 20% prevalence of chronic pain in the general population is a major health concern given the often profound associated impairment of daily activities, employment status, and health-related quality of life in sufferers. Resource utilization associated with chronic pain represents an enormous burden for healthcare systems. Although analgesia based on the World Health Organization’s pain ladder continues to be the mainstay of chronic pain management, aside from chronic cancer pain or end-of-life care, prolonged use of non-steroidal anti-inflammatory drugs or opioids to manage chronic pain is rarely sustainable. As the endocannabinoid system is known to control pain at peripheral, spinal, and supraspinal levels, interest in medical use of cannabis is growing. A proprietary blend of cannabis plant extracts containing delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) as the principal cannabinoids is formulated as an oromucosal spray (USAN name: nabiximols) and standardized to ensure quality, consistency and stability. This review examines evidence for THC:CBD oromucosal spray (nabiximols) in the management of chronic pain conditions. Cumulative evidence from clinical trials and an exploratory analysis of the German Pain e-Registry suggests that add-on THC:CBD oromucosal spray (nabiximols) may have a role in managing chronic neuropathic pain, although further precise clinical trials are required to draw definitive conclusions.

A review of the cultivation and processing of cannabis (Cannabis sativa L.) for production of prescription medicines in the UK
David J. Potter
Drug Testing and Analysis, 2013
DOI 10.1002/dta.1531
The quality demands of the pharmaceutical industry require prescription medicines to be consistent in their active ingredient content. Achieving this, using raw cannabis as a feedstock, is especially challenging. The plant material is extremely inhomogeneous, and the ratios of active ingredients are affected by a range of factors. These include the genetics of the plant, the growing and storage conditions, the state of maturity at harvest, and the methods used to process and formulate the material. The reasons for this variability are described, with particular emphasis on the botanical considerations. To produce the complex botanical medicine Sativex®, which contains the cannabinoids ?(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) and a range of other ingredients, GW Pharmaceuticals had to manage these variables. This medicine, for the treatment of spasticity due to multiple sclerosis, is the first cannabis-based medicine to be approved in the UK. The company's methodology for producing this and other chemotypes is described.

A Review on Studies of Marijuana for Alzheimer’s Disease – Focusing on CBD, THC
Seok Hee Kim, Jin Won Yang, Kyung Han Kim, Jong Uk Kim, Tae Han Yook
Journal of Pharmacopuncture 2019;22(4):225-230
Objectives: This study was to discuss the research trend of dementia treatment using cannabis for the purpose of providing the basis of cannabis use for medical purposes in the future.
Methods: This study searched publications, which were registered to databases or published by Aug 22, 2019, and targeted the full-text or abstracts of these publications. We selected the final nine studies met all selection criteria.
Results: These results implied that the CBD components of cannabis might be useful to treat and prevent AD because CBD components could suppress the main causal factors of AD. Moreover, it was suggested that using CBD and THC together could be more useful than using CBD or THC alone. Conclusion: We hope that there will be a solid foundation to use cannabis for medical use by continuously evaluating the possibility of using cannabis for clinical purposes as a dementia treatment substance and cannabis can be used as a positive tool

A systematic review and meta-analysis of cannabis-based medicines, cannabinoids and endocannabinoid system modulators tested for antinociceptive effects in animal models of injury-related or pathological persistent pain
Soliman N, Haroutounian S, Hohmann AG, Krane E, Liao J, Macleod M, Segelcke D, Sena C, Thomas J, Vollert J, Wever K, Alaverdyan H, Barakat A, Barthlow T, Harris Bozer AL, Davidson A, Diaz-delCastillo M, Dolgorukova A, Ferdousi MI, Healy C, Hong S, Hopkins M, James A, Leake HB, Malewicz NM, Mansfield M, Mardon AK, Mattimoe D, McLoone DP, Noes-Holt G, Pogatzki-Zahn EM, Power E, Pradier B, Romanos-Sirakis E, Segelcke A, Vinagre R, Yanes JA, Zhang J, Zhang XY, Finn DP and Rice ASC
Pain (2021)
DOI: 10.1097/j.pain.0000000000002269
https://www.researchgate.net/publica..._or_pathologic
We report a systematic review and meta-analysis of studies which assessed the antinociceptive efficacy of cannabinoids, cannabis-based medicines, and endocannabinoid system modulators on pain-associated behavioural outcomes in animal models of pathological or injury-related persistent pain. In April 2019, we systematically searched 3 online databases and used crowd science and machine learning to identify studies for inclusion. We calculated a standardised mean difference (SMD) effect size for each comparison and performed a random effects meta-analysis. We assessed the impact of study design characteristics and reporting of mitigations to reduce the risk of bias. We meta-analysed 374 studies in which 171 interventions were assessed for antinociceptive efficacy in rodent models of pathological or injury-related pain. Most experiments were conducted in male animals (86 %). Antinociceptive efficacy was most frequently measured by attenuation of hypersensitivity to evoked limb withdrawal. Selective CB1, CB2, non-selective cannabinoid receptor agonists (including delta-9-tetrahydrocannabinol; THC), and PPAR-alpha agonists (predominantly palmitoylethanolamide; PEA) significantly attenuated pain-associated behaviours in a broad range of inflammatory and neuropathic pain models. Fatty acid amide hydrolase (FAAH) inhibitors, monoacylglycerol lipase (MGL) inhibitors and cannabidiol (CBD) significantly attenuated pain-associated behaviours in neuropathic pain models but yielded mixed results in inflammatory pain models. The reporting of criteria to reduce the risk of bias was low, therefore the studies have an unclear risk of bias. The value of future studies could be enhanced by improving the reporting of methodological criteria, the clinical relevance of the models and behavioural assessments. Notwithstanding, the evidence supports the hypothesis of cannabinoid-induced analgesia.

A Systematic Review on Cannabis Hyperemesis Syndrome and Its Management Options
Helen Senderovich, Preet Patel, Briam Jimenez Lopez, Sarah Waicus
Med Princ Pract (2021)
DOI: 10.1159/000520417
https://www.karger.com/Article/Pdf/520417
Introduction: Several forms of cannabinoids are currently being used to manage nausea and vomiting (N/V). Emerging cases of refractory N/V associated with chronic cannabis use among adults and older patients have been reported named cannabis hyperemesis syndrome (CHS). CHS is a condition that leads to repeated and severe N/V in long-term users of cannabinoids.
Objective: The aim of this study was to outline current treatments for the management of CHS. Methods: A systematic review was conducted using PubMed, Ovid MEDLINE, Cochrane Central, EMBASE, and Google Scholar. Databases were used to search for articles on CHS published from January 2009 to June 2021, yielding 225 results of which 17 were deemed relevant and underwent review by 2 separate reviewers.
Results: The duration of cannabis administration ranged between 6 months to 11 years may precipitate symptoms of CHS. The Rome IV diagnostic criteria of CHS require cannabinoid use and persistence of N/V symptoms for at least the past 6 months. Cannabis cessation is noted to be the most successful management, but other treatments also demonstrated symptom relief; these include hot water hydrotherapy, topical capsaicin cream, haloperidol, droperidol, benzodiazepines, propranolol, and aprepitant administration. Conclusion: More research on CHS is needed to enhance knowledge translation, education, and create awareness in the medical community on the side effects of cannabinoids and to propose the best treatment options.

A Systematic Review on the Pharmacokinetics of Cannabidiol in Humans
Sophie A. Millar1, Nicole L. Stone, Andrew S. Yates, Saoirse E. O'Sullivan
Frontiers in Pharmacology 9:1365 November 2018
DOI: 10.3389/fphar.2018.01365
Background: Cannabidiol is being pursued as a therapeutic treatment for multiple conditions, usually by oral delivery. Animal studies suggest oral bioavailability is low, but literature in humans is not sufficientlacking. The aim of this review was to collate published data in this area.
Methods: A systematic search of PubMed and EMBASE (including MEDLINE) was conducted to retrieve all articles reporting pharmacokinetic data of CBD in humans.
Results: Of 792 articles retireved, 24 included pharmacokinetic parameters in humans. The half-life of cannabidiol was reported between 1.4-10.9 hours after oromucosal spray, 2-5 days after chronic oral administration, 24 hours after i.v., and 31 hours after smoking. Bioavailability following smoking was 31% however no other studies attempted to report the absolute bioavailability of CBD following other routes in humans, despite i.v formulations being available. The area-under-the-curve and Cmax increase in dose-dependent manners and arereached quicker following smoking/inhalation compared to oral/oromucosal routes. Cmax is increased during fed states and in lipid formulations. Tmax is reached between 0-4 hours.
Conclusions: This review highlights the paucity in data and some discrepancy in the pharmacokinetics of cannabidiol, is area despite its widespread use of cannabidiol in humans. Analysis and understanding of these properties such as bioavailability and half-life is critical to future therapeutic success, and robust data from a variety of formulations is required.

Abrupt withdrawal of cannabidiol (CBD): A randomized trial.
Taylor, L., Crockett, J., Tayo, B., Checketts, D., & Sommerville, K.
Epilepsy & Behavior, 104, 106938. (2020).
doi:10.1016/j.yebeh.2020.106938
Rationale: The rationale of this study was to assess occurrence of withdrawal symptoms induced by abrupt cessation of cannabidiol (CBD) after prolonged administration in healthy volunteers. Methods: Thirty volunteers were randomized to receive 750 mg of a plant-derived pharmaceutical formulation of highly purified CBD in oral solution (100 mg/mL; Epidiolex® in the United States and Epidyolex® in Europe) twice daily (b.i.d.) for 4 weeks (Part 1) followed by 2 weeks of 750 mg b.i.d. CBD (Part 2, Arm 1) or matched placebo (Part 2, Arm 2). All volunteers completed the Cannabis Withdrawal Scale (CWS) and the 20-item Penn Physician Withdrawal Checklist (PWC-20) on days ?1, 21, 28, 31, 35, 42, and at follow-up. Results: Median CWS and PWC-20 scores slightly decreased from Part 1 to Part 2. Median CWS scores ranged from 0.0 to 4.0 (out of a possible 190) in Arm 1 and 0.0 to 0.5 in Arm 2. Median PWC-20 scores were 0.0 (out of a possible 60) in both arms. Twenty-nine (97%) volunteers in Part 1 reported all-causality treatment-emergent adverse events (AEs); the most commonly reported was diarrhea (63%). In Part 2, Arm 1, 6 (67%) volunteers reported all-causality AEs; the most commonly reported was diarrhea (44%). In Part 2, Arm 2, 9 (75%) volunteers reported all-causality AEs; the most commonly reported was headache (58%). Nine volunteers withdrew because of AEs in Part 1; 1 withdrew in Part 2, Arm 2, because of an AE that began in Part 1. Four severe AEs were reported in Part 1; the remainder were mild or moderate. No serious AEs were reported. Conclusion: In healthy volunteers, no evidence of withdrawal syndrome was found with abrupt discontinuation of short-term treatment with CBD.

Activation of cannabinoid receptors in breast cancer cells improves osteoblast viability in cancer‑bone interaction model while reducing breast cancer cell survival and migration
Tueanjai Khunluck, Kornkamon Lertsuwan, Chartinun Chutoe, Supagarn Sooksawanwit, Ingon Inson, Jarinthorn Teerapornpuntakit, Rutaiwan Tohtong & Narattaphol Charoenphandhu
NATURE Scientifc Reports (2022) 12:7398
DOI: 10.1038/s41598-022-11116-9
https://www.nature.com/articles/s41598-022-11116-9.pdf
The endocannabinoid system has been postulated to help restrict cancer progression and maintain osteoblastic function during bone metastasis. Herein, the efects of cannabinoid receptor (CB) type 1 and 2 activation on breast cancer cell and osteoblast interaction were investigated by using ACEA and GW405833 as CB1 and CB2 agonists, respectively. Our results showed that breast cancer cell (MDA-MB-231)-derived conditioned media markedly decreased osteoblast-like UMR-106 cell viability. In contrast, media from MDA-MB-231 cells pre-treated with GW405833 improved UMR-106 cell viability. MDA-MB-231 cells were apparently more susceptible to both CB agonists than UMR-106 cells. Thereafter, we sought to answer the question as to how CB agonists reduced MDA-MB-231 cell virulence. Present data showed that co-activation of CB1 and CB2 exerted cytotoxic efects on MDA-MB-231 cells by increasing apoptotic cell death through suppression of the NF-κB signaling pathway in an ROS-independent mechanism. ACEA or GW405833 alone or in combination also inhibited MDA-MB-231 cell migration. Thus, it can be concluded that the endocannabinoid system is able to provide protection during breast cancer bone metastasis by interfering cancer and bone cell interaction as well as by the direct suppression of cancer cell growth and migration.

Action of Cannabidiol on the Anxiety and Other Effects Produced by A 9-THC in Normal Subjects
A. W. Zuardi, I. Shirakawa, E. Finkelfarb, and I. G. Karniol
Psychopharmacology (1982) 76:245-250
https://www.researchgate.net/publica...ormal_subjects
The object of the experiment was to verify whether cannabidiol (CBD) reduces the anxiety provoked by A 9-THC in normal volunteers, and whether this effect occurs by a general block of the action of A g-THC or by a specific
anxiolytic effect. Appropriate measurements and scales were utilized and the eight volunteers received, the following treatments in a double-blind procedure: 0.5 mg/kg A9-THC, 1 mg/kg CBD, a mixture containing 0.5 mg/kg A9-THC and I mg/kg CBD and placebo and diazepam (10 rag) as controls. Each volunteer received the treatments in a different sequence. It was verified that CBD blocks the anxiety provoked by A9-THC, however this effect also extended to marihuana like effects and to other subjective alterations induced by A 9-THC. This antagonism does not appear to be caused by a general block of A9-THC effects, since no change was detected in the pulse-rate measurements. Several further effects were observed typical of CBD and of an opposite nature to those of A 9-THC. These results suggest that the effects of CBD, as opposed to those of A9-THC, might be involved in the antagonism of effects between the two cannabinoids.

Actions of Cannabis sativa L. fixed oil and nano-emulsion on venom-induced inflammation of Bothrops moojeni snake in rats
Helison Carvalho, Danna Emanuelle Santos Gonçalves, Karyny Roberta Tavares Picanço, Abrahão Victor Tavares de Lima Teixeir, Maria Lucia, Xuebo Hu, Caio Pinho Fernandes, Irlon Ferreira, Jose Carlos Tavares Carvalho
Inflammopharmacology 29(2) February 2021
DOI: 10.1007/s10787-020-00754-y
https://www.researchgate.net/publica..._snake_in_rats
Background: Bothrops moojeni snake venom (VBm) has toxins that cause pronounced tissue damage and exacerbated inflammatory reaction. Cannabis sativa L. is a plant species that produces an oil (CSO) rich in unsaturated fatty acids. Nano-emulsions have several advantages, such as better stability and higher penetrating power in membranes. Therefore, this study evaluated the effect of a nano-emulsion based on this herbal derivative (NCS) against VBm-induced inflammation in Wistar rats. Methods: The CSO and NCS were submitted to physicochemical characterization. The inflammatory process was induced by the VBm (0.10 mg/kg) as follows: rat paw edema, peritonitis, analysis of leukocyte infiltrate in gastrocnemius muscle of rats and formation of granulomatous tissue. Results: No significant changes were observed when the NCS was submitted to the centrifugation and thermal stress tests. There was no phase separation, changes in density (0.978 ± 0.01 g/cm3) and viscosity (0.889 ± 0.15). The droplet diameter ranged from 119.7 ± 065 to 129.3 ± 0.15 nm and the polydispersity index ranged from 0.22 ± 0.008 to 0.23 ± 0.011. The results showed that treatments with CSO (200 and 400 mg/kg) and NCS (100 mg/kg) were able to decrease significantly (p < 0.001) the formation of edema and granulomatous tissue. The CSO and NCS groups significantly attenuated (p < 0.001) the recruitment of inflammatory cells in the tests for peritonitis and leukocyte infiltrate. The histopathological analysis of the gastrocnemius muscle showed a reduction in tissue damage caused by VBm. Conclusion: The results obtained in this study showed anti-inflammatory activity of the CSO which may be due to a high UFA content. The nanosizing, as evidenced by the incorporation of the CSO in the NCS improved the effect and opens the perspective for the obtainment of a nanomedicine in which a kinetic stable phytotherapic can be used at low doses

Activation of Cannabinoid Receptor 2 Prevents Colitis-Associated Colon Cancer through Myeloid Cell De-activation Upstream of IL-22 Production.
Becker, W., Alrafas, H. R., Wilson, K., Miranda, K., Culpepper, C., Chatzistamou, I., … Nagarkatti, P. S.
iScience, 23(9), 101504.(2020).
doi:10.1016/j.isci.2020.101504
Intestinal disequilibrium leads to inflammatory bowel disease (IBD), and chronic inflammation predisposes to oncogenesis. Antigen-presenting dendritic cells (DCs) and macrophages can tip the equilibrium toward tolerance or pathology. Here we show that delta-9-tetrahydrocannabinol (THC) attenuates colitis-associated colon cancer and colitis induced by anti-CD40. Working through cannabinoid receptor 2 (CB2), THC increases CD103 expression on DCs and macrophages and upregulates TGF-b1 to increase T regulatory cells (Tregs). THC-induced Tregs are necessary to remedy systemic IFNg and TNFa caused by anti-CD40, but CB2- mediated suppression of APCs by THC quenches pathogenic release of IL-22 and IL-17A in the colon. By examining tissues from multiple sites, we confirmed that THC affects DCs, especially in mucosal barrier sites in the colon and lungs, to reduce DC CD86. Using models of colitis and systemic inflammation we show that THC, through CB2, is a potent suppressor of aberrant immune responses by provoking coordination between APCs and Tregs.

Acute and chronic effects of cannabinoids on effort-related decision-making and reward learning: an evaluation of the cannabis ‘amotivational’ hypotheses
Will Lawn & Tom P Freeman & Rebecca A Pope & Alyssa Joye & Lisa Harvey & Chandni Hindocha & Claire Mokrysz & Abigail Moss & Matthew B Wall & Michael AP Bloomfield & Ravi K Das & Celia JA Morgan & David J Nutt & H Valerie Curran
Psychopharmacology September 2016
DOI 10.1007/s00213-016-4383-x
Rationale Anecdotally, both acute and chronic cannabis use have been associated with apathy, amotivation, and other reward processing deficits. To date, empirical support for these effects is limited, and no previous studies have assessed both acute effects of Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), as well as associations with cannabis dependence. Objectives The objectives of this study were (1) to examine acute effects of cannabis with CBD (Cann + CBD) and without CBD (Cann-CBD) on effort-related decision-making and (2) to examine associations between cannabis dependence, effort-related decision-making and reward learning. Methods In study 1, 17 participants each received three acute vaporized treatments, namely Cann-CBD (8 mg THC), Cann + CBD (8 mg THC + 10 mg CBD) and matched placebo, followed by a 50 % dose top-up 1.5 h later, and completed the Effort Expenditure for Rewards Task (EEfRT). In study 2, 20 cannabis-dependent participants were compared with 20 non-dependent, drug-using control participants on the EEfRT and the Probabilistic Reward Task (PRT) in a nonintoxicated state. Results Cann-CBD reduced the likelihood of high-effort choices relative to placebo (p = 0.042) and increased sensitivity to expected value compared to both placebo (p = 0.014) and Cann + CBD (p = 0.006). The cannabis-dependent and control groups did not differ on the EEfRT. However, the cannabis-dependent group exhibited a weaker response bias than the control group on the PRT (p = 0.007). Conclusions Cannabis acutely induced a transient amotivational state and CBD influenced the effects of THC on expected value. In contrast, cannabis dependence was associated with preserved motivation alongside impaired reward learning, although confounding factors, including depression, cannot be disregarded. This is the first well powered, fully controlled study to objectively demonstrate the acute amotivational effects of THC.

Acute and Severe Acute Pancreatitis and the Effect of Cannabis in States Before and After Legalization Compared With States Without Legalized Cannabis
Luis F. Lara, Laura Nemer, Alice Hinton, Gokulakrishnan Balasubramanian, Darwin L. Conwell, Krishna, Somashekar
Pancreas: May 19, 2021
doi: 10.1097/MPA.0000000000001830
Objectives
Cannabis legalization has increased its use. The incidence of acute pancreatitis (AP) and severe acute pancreatitis (SAP) has also increased. In this study, data on pancreatitis were obtained from 2 states before and after cannabis legalization and compared with 2 states without legalized cannabis.
Methods
Data were extracted from State Inpatient Databases from the states of Colorado and Washington before recreational cannabis legalization (2011) and after legalization (2015). Arizona and Florida were used as the nonlegalized cannabis states. Multivariable logistic regression models were fit for AP and SAP to determine a trend difference between legalized and nonlegalized cannabis states.
Results
Cannabis use, AP, and SAP increased in all states. The increase in AP and SAP was not significantly different between the states that legalized cannabis use and those that did not. Legalized cannabis states had lower charges for AP and SAP and shorter length of hospitalizations.
Conclusions
The trend of AP and SAP increased during the study period, but this was not correlated to cannabis use. Cannabis users had lower hospitalization costs and hospital stay. The effects of other confounders such as cannabis dose and delivery methods, alcohol, tobacco, and others need to be studied further as use increases.
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Acute cannabis toxicity
Clinical Toxicology 57(8):1-8 January 2019
DOI: 10.1080/15563650.2018.1548708
Matthew J. Noble, Katrina Hedberg, Robert G Hendrickson
Objective: We describe the clinical effects of, and products associated with, acute exposures to cannabis during the early legalization period of recreational cannabis in Oregon and Alaska. Methods: This was an observational study of Oregon/Alaska Poison Center data between 4 December 2015 and 15 April 2017. A standardized data collection instrument was created for this study that captured information about cannabis product description, route of exposure, intentional vs unintentional exposure, product dose, product manufacture source, product ownership source, initial vital signs, clinical signs and symptoms, and subject disposition. Subjects were included if the Poison Center received a call about an acute exposure to cannabis from the subject, subject’s family member or friend, or healthcare worker participating in the subject’s care. Subjects were excluded if there was no evident exposure, the exposure was chronic, there were co-ingestants, or the subject was non-human (e.g. pet). Results: Two hundred fifty three individuals were acutely exposed to cannabis (median age 20 years; range 8 months – 96 years; 54.2% males): 71 (28.1%) children (<12 years), 42 (16.6%) adolescents (12–17 years), and 140 (55.3%) adults (?18 years). Children were most likely to unintentionally (98.6%) ingest (97.2%) homemade (35.2%) edibles (64.8%) belonging to a family member (73.2%) and experience sedation (52.1%). Adults were most likely to intentionally (88.6%) ingest (66.4%) retail (40.0%) edibles (48.6%) and experience neuroexcitation (47.1%). Adolescents’ exposures had similarities to both adult and children; they were most likely to intentionally (81.0%) ingest (50.0%) homemade (23.8%) edibles (45.2%) belonging to a friend (47.3%) and to experience either neuroexcitation (42.9%) or sedation (40.5%). Among all ages, tachycardia and neuroexcitation were more likely following inhalation exposures compared to ingestions. Eight subjects were admitted to an intensive care unit, including three patients who were intubated; one subject died. Edibles were the most common products to cause symptoms in all age groups, while concentrated products were more likely to lead to intubation, especially when ingested. Children in particular had a higher likelihood of intensive care unit admission and intubation following exposure to concentrated products. Conclusions: Neurotoxicity is common after acute cannabis exposures. Children experienced unintentional exposures, particularly within the home and occasionally with major adverse outcomes. Concentrated products such as resins and liquid concentrates were associated with greater toxicity than other cannabis products. These findings may help guide other states during the early retail cannabis legalization period.

Acute Pharmacokinetic Profile of Smoked and Vaporized Cannabis in Human Blood and Oral Fluid
Tory R. Spindle, Edward J. Cone, Nicolas J. Schlienz,
John M. Mitchell, George E. Bigelow, Ronald Flegel, Eugene Hayes, and Ryan Vandrey
Journal of Analytical Toxicology, 2019;1–26
doi: 10.1093/jat/bky104
Currently, an unprecedented number of individuals can legally access cannabis. Vaporization is increasingly popular as a method to self-administer cannabis, partly due to perception of reduced harm compared with smoking. Few controlled laboratory studies of cannabis have used vaporization as a delivery method or evaluated the acute effects of cannabis among infrequent cannabis users. This study compared the concentrations of cannabinoids in whole blood and oral fluid after administration of smoked and vaporized cannabis in healthy adults who were infrequent users of cannabis. Seventeen healthy adults, with no past-month cannabis use, self-administered smoked or vaporized cannabis containing ?9-tetrahydrocannabinol (THC) doses of 0, 10 and 25 mg in six double-blind outpatient sessions. Whole blood and oral fluid specimens were obtained at baseline and for 8 h after cannabis administration. Cannabinoid concentrations were assessed with enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC–MS-MS) methods. Sensitivity, specificity and agreement between ELISA and LC–MS-MS results were assessed. Subjective, cognitive performance and cardiovascular effects were assessed. The highest concentrations of cannabinoids in both whole blood and oral fluid were typically observed at the first time point (+10 min) after drug administration. In blood, THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide concentrations were dose-dependent for both methods of administration, but higher following vaporization compared with smoking. THC was detected longer in oral fluid compared to blood and THCCOOH detection in oral fluid was rare and highly erratic. For whole blood, greater detection sensitivity for ELISA testing was observed in vaporized conditions. Conversely, for oral fluid, greater sensitivity was observed in smoked sessions. Blood and/or oral fluid cannabinoid concentrations were weakly to moderately correlated with pharmacodynamic outcomes. Cannabis pharmacokinetics vary by method of inhalation and biological matrix being tested. Vaporization appears to be a more efficient method of delivery compared with smoking.

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts ?9-Tetrahydrocannabinol-Induced Cognitive Impairment.
Aso E, Fernández-Dueñas V, López-Cano M, Taura J, Watanabe M, Ferrer I, Luján R, Ciruela F
Mol Neurobiol. 2019 Jan 4.
doi: 10.1007/s12035-018-1456-3.
At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid ?9-tetrahydrocannabinol (?9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with ?9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the ?9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.

Adherence, Safety, and Effectiveness of Medical Cannabis and Epidemiological Characteristics of the Patient Population: A Prospective Study
Lihi Bar-Lev Schleider, Raphael Mechoulam, Inbal Sikorin, Timma Naftali and Victor Novack
Front. Med., 09 February 2022 DOI: 10.3389/fmed.2022.827849
https://www.frontiersin.org/articles...22.827849/full
Background: Despite the absence of rigorous prospective studies, there has been an increase in the use of cannabis-based medicinal products. During the study period, the use of medical cannabis in Israel was tightly regulated by national policy. Through a prospective study of approximately 10,000 patients, we aimed to characterize the medical cannabis patient population as well as to identify treatment adherence, safety, and effectiveness.
Methods and Findings: In this study of prescribed medical cannabis patients, adherence, safety, and effectiveness were assessed at 6 months. Treatment adherence was assessed by the proportion of patients purchasing the medication out of the total number of patients (excluding deceased cases and patients transferred to another cannabis clinic). Safety was assessed by the frequency of the side-effects, while effectiveness was defined as at least moderate improvement in the patient condition without treatment cessation or serious side-effects. The most frequent primary indications requiring therapy were cancer (49.1%), followed by non-specific pain (29.3%). The average age was 54.6 ± 20.9 years, 51.1% males; 30.2% of the patients reported prior experience with cannabis. During the study follow-up, 1,938 patients died (19.4%) and 1,735 stopped treatment (17.3%). Common side-effects, reported by 1,675 patients (34.2%), were: dizziness (8.2%), dry mouth (6.7%), increased appetite (4.7%), sleepiness (4.4%), and psychoactive effect (4.3%). Overall, 70.6% patients had treatment success at 6 months. Multivariable logistic regression analysis revealed that the following factors were associated with treatment success: cigarette smoking, prior experience with cannabis, active driving, working, and a young age. The main limitation of this study was the lack of data on safety and effectiveness of the treatment for patients who refused to undergo medical assessment even at baseline or died within the first 6 months.
Conclusions: We observed that supervised medical-cannabis treatment is associated with high adherence, improvement in quality of life, and a decrease in pain level with a low incidence of serious adverse events.

Adjunctive Management of Opioid Withdrawal with the Nonopioid Medication Cannabidiol
Christopher Kudrich , Yasmin L Hurd , Edwin Salsitz , An-Li Wang
Cannabis and Cannabinoid Research Oct (2021)
DOI: 10.1089/can.2021.0089
Introduction: Opioid use disorder (OUD) is a major public health crisis worldwide. Patients with OUD inevitably experience withdrawal symptoms when they attempt to taper down on their current opioid use, abstain completely from opioids, or attempt to transition to certain medications for opioid use disorder. Acute opioid withdrawal can be debilitating and include a range of symptoms such as anxiety, pain, insomnia, and gastrointestinal symptoms. Whereas acute opioid withdrawal only lasts for 1-2 weeks, protracted withdrawal symptoms can persist for months after the cessation of opioids. Insufficient management of opioid withdrawal often leads to devastating results including treatment failure, relapse, and overdose. Thus, there is a critical need for cost-effective, nonopioid medications, with minimal side effects to help in the medical management of opioid withdrawal syndrome. We discuss the potential consideration of cannabidiol (CBD), a nonintoxicating component of the cannabis plant, as an adjunctive treatment in managing the opioid withdrawal syndrome.
Materials and Methods:A review of the literature was performed using keywords related to CBD and opioid withdrawal syndrome in PubMed and Google Scholar. A total of 144 abstracts were identified, and 41 articles were selected where CBD had been evaluated in clinical studies relevant to opioid withdrawal.
Results: CBD has been reported to have several therapeutic properties including anxiolytic, antidepressant, anti-inflammatory, anti-emetic, analgesic, as well as reduction of cue-induced craving for opioids, all of which are highly relevant to opioid withdrawal syndrome. In addition, CBD has been shown in several clinical trials to be a well-tolerated with no significant adverse effects, even when co-administered with a potent opioid agonist.
Conclusions: Growing evidence suggests that CBD could potentially be added to the standard opioid detoxification regimen to mitigate acute or protracted opioid withdrawal-related symptoms. However, most existing findings are either based on preclinical studies and/or small clinical trials. Well-designed, prospective, randomized-controlled studies evaluating the effect of CBD on managing opioid withdrawal symptoms are warranted.
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Adolescent cannabis use and adult psychoticism: A longitudinal co-twin control analysis using data from two cohorts

Schaefer JD, Jang SK, Vrieze S, Iacono WG, McGue M, Wilson S.
American Psychological Association Sept 2021
DOI: 10.1037/abn0000701
https://www.researchgate.net/publica...cent_cannabis_ use_and_adult_psychoticism_A_l ongitudinal_co-twin_control_analysis_using_da ta_from_two_cohorts
Observational studies have repeatedly linked cannabis use and increased risk of psychosis. We sought to clarify whether this association reflects a causal effect of cannabis exposure or residual confounding. We analyzed data from two cohorts of twins who completed repeated, prospective measures of cannabis use (N = 1544) and cannabis use disorder symptoms (N = 1458) in adolescence and a dimensional measure of psychosis-proneness (the Personality Inventory for DSM-5 Psychoticism scale) in adulthood. Twins also provided molecular genetic data, which were used to estimate polygenic risk of schizophrenia. Both cumulative adolescent cannabis use and use disorder were associated with higher Psychoticism scores in adulthood. However, we found no evidence of an effect of cannabis on Psychoticism or any of its facets in co-twin control models that compared the greater-cannabis-using twin to the lesser-using co-twin. We also observed no evidence of a differential effect of cannabis on Psychoticism by polygenic risk of schizophrenia. Although cannabis use and disorder are consistently associated with increased risk of psychosis, the present results suggest this association is likely attributable to familial confounds rather than a causal effect of cannabis exposure. Efforts to reduce the prevalence and burden of psychotic illnesses thus may benefit from greater focus on other therapeutic targets.
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Alternative Targets Within the Endocannabinoid System for Future Treatment of Gastrointestinal Diseases Rudolf Schicho, Martin Storr can J gastroenterol 2011;25(7):377-383. doi: 10.1155/2011/953975
https://www.researchgate.net/publica...tinal_Diseases
Many beneficial effects of herbal and synthetic cannabinoids on gut motility and inflammation have been demonstrated, suggesting a vast potential for these compounds in the treatment of gastrointestinal disorders. These effects are based on the so-called ‘endocannabinoid system’ (ECS), a cooperating network of molecules that regulate the metabolism of the body’s own and of exogenously administered cannabinoids. The ECS in the gastrointestinal tract quickly responds to homeostatic disturbances by de novo synthesis of its components to maintain homeostasis, thereby offering many potential targets for pharmacological intervention. Of major therapeutic interest are nonpsychoactive cannabinoids or compounds that do not directly target cannabinoid receptors but still possess cannabinoid-like properties. Drugs that inhibit endocannabinoid degradation and raise the level of endocannabinoids are becoming increasingly promising alternative therapeutic tools to manipulate the ECS.

An integrative overview of the cannabinergic system and mental health Oscar E. Prospéro García, Pavel E. Rueda Orozco, Octavio Amancio Belmont, Alejandra E. Ruiz Contreras, Mónica Méndez Díaz Salud Mental Vol. 40, No. 3, mayo-junio 2017 doi:10.17711/SM.0185-3325.2017.015 https://www.academia.edu/34243948/An...download-paper The endocannabinoid system (eCBs) is one of the modulatory systems widely expressed in the brain. It consists of receptors expressed in the cytoplasmic (CB1 and CB2), the mitochondrial membrane (CB1), and the endogenous ligands known as endocannabinoids, such as anandamide, 2AG and oleamide. CB1 has been found in excitatory and inhibitory neurons in the pre- and post-synaptic membranes. It is expressed in several brain areas such as the hippocampus, dorsal, and ventral striatum, amygdala and prefrontal cortex. The eCBs has been involved in the regulation of learning and memory, mood, energy balance, sleep, and drug addiction. Objective. Integrate existing information about the eCBs and its role in brain function and mental health. Method. Review of the information of basic and clinical relevance obtained from indexed scientific journals (PubMed/Medline, Scopus). Results. Basic and clinical research on eCBs related to central nervous system function is described. Discussion and conclusion. At present, the study of eCBs is of importance. The development of drugs that affect this system may be clinically useful to control different debilitating diseases. This is an area of interest to the scientific community and health care providers.

An investigation of cannabis use for insomnia in depression and anxiety in a naturalistic sample

Nirushi Kuhathasan, Luciano Minuzzi, James MacKillop and Benicio N. Frey

BMC Psychiatry (2022) 22:303

DOI: 10.1186/s12888-022-03948-6

https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/s12888-022-03948-6.pdf

Background: Little is known about cannabis use for insomnia in individuals with depression, anxiety, and comorbid depression and anxiety. To develop a better understanding of distinct profles of cannabis use for insomnia management, a retrospective cohort study was conducted on a large naturalistic sample.
Methods: Data were collected using the medicinal cannabis tracking app, Strainprint®, which allows users to monitor and track cannabis use for therapeutic purposes. The current study examined users managing insomnia symptoms in depression (n=100), anxiety (n=463), and comorbid depression and anxiety (n=114), for a total of 8476 recorded sessions. Inferential analyses used linear mixed efects modeling to examine self-perceived improvement across demographic variables and cannabis product variables.
Results: Overall, cannabis was perceived to be efcacious across all groups, regardless of age and gender. Dried fower and oral oil were reported as the most used and most efcacious product forms. In the depression group, all strains were perceived to be efcacious and comparisons between strains revealed indica-dominant (Mdif=1.81, 95% CI 1.26–2.36, Padj<.001), indica hybrid (Mdif=1.34, 95% CI 0.46–2.22, Padj=.045), and sativa-dominant (Mdif=1.83, 95% CI 0.68–2.99, Padj=.028) strains were signifcantly more efcacious than CBD-dominant strains. In anxiety and comorbid conditions, all strain categories were perceived to be efcacious with no signifcant diferences between strains.
Conclusions: In terms of perceptions, individuals with depression, anxiety, and both conditions who use cannabis for insomnia report signifcant improvements in symptom severity after cannabis use. The current study highlights the need for placebo-controlled trials investigating symptom improvement and the safety of cannabinoids for sleep in individuals with mood and anxiety disorders .

An Updated Analysis of Clinical Outcome Measures Across Patients From the UK Medical Cannabis Registry
Mehmet Ergisi, Simon Erridge, Michael Harris, Michal Kawka, Devaki Nimalan, Oliver Salazar, Katerina Loupasaki, Rayyan Ali, Carl Holvey, Ross Coomber, Azfer Usmani, Mohammed Sajad, Sushil Beri, Jonathan Hoare, Shaheen A. Khan, Mark W. Weatherall, Michael Platt, James J. Rucker, and Mikael H. Sodergren
Cannabis and Cannabinoid Research
Volume X, Number X, 2022
DOI: 10.1089/can.2021.0145
https://www.liebertpub.com/doi/pdf/1.../can.2021.0145
Introduction: There is a growing body of literature supporting the efficacy of cannabis-based medicinal products (CBMPs). Despite an increase in prescribing globally, there is a paucity of high-quality clinical data on the efficacy of CBMPs for many conditions. This study aims to detail the changes in health-related quality of life (HRQoL) and associated clinical safety in patients prescribed CBMPs for any clinical indication from the UK Medical Cannabis Registry (UKMCR).
Methods: An uncontrolled prospective case series of the UKMCR was analyzed. Primary outcomes included change from baseline in patient-reported outcome measures collected across all patients (the Generalized Anxiety Disorder Scale [GAD-7], EQ-5D-5L, and Sleep Quality Scale [SQS]) at 1, 3, and 6 months. Secondary outcomes included the self-reported incidence and severity of adverse events. Statistical significance was defined as p < 0.050.
Results: Three hundred twelve patients were included in the final analysis, with a mean age of 44.8. The most common primary diagnoses were chronic pain of undefined etiology (n = 102, 32.7%), neuropathic pain (n = 43, 13.8%), and fibromyalgia (n = 31, 9.9%). Before enrolment, 112 (35.9%) patients consumed cannabis daily. The median cannabidiol and (_)-trans-D9-tetrahydrocannabinol doses prescribed at baseline were 20.0 mg (0.0 510.0mg) and 3.0mg (0.0–660.0 mg), respectively. Statistically significant improvements were observed in GAD-7, EQ-5D-5L Index, EQ-5D Visual Analog Scale and SQS scores at 1, 3, and 6 months ( p < 0.050). There were 94 (30.1%) reported adverse events, of which nausea (n = 12, 3.8%), dry mouth (n = 10, 3.2%), dizziness (n = 7, 2.2%), and somnolence (n = 7, 2.2%) were the most common.
Conclusion: This study demonstrated CBMP treatment to be associated with a relatively low incidence of severe adverse events in the medium-term. Positive changes following treatment were observed in general, as well as
anxiety and sleep-specific, HRQoL outcomes. Randomized controlled trials are still awaited to assess causation; however, real-world evidence can help inform current clinical practice, future trials, and is an important component
of pharmacovigilance.

Anandamide Activity and Degradation Are Regulated by Early Postnatal Aging and Follicle-Stimulating Hormone in Mouse Sertoli Cells
MAURO MACCARRONE, SANDRA CECCONI, GIANNA ROSSI, NATALIA BATTISTA, RICCARDO PAUSELLI, AND ALESSANDRO FINAZZI-AGRO Endocrinology 144(1):20–28
doi: 10.1210/en.2002-220544
Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to adversely affect female fertility. However, a potential role of AEA in male reproductive functions is unknown. Here we report evidence that immature mouse Sertoli cells have the biochemical tools to bind and inactivate AEA, i.e. a functional type-2 cannabinoid receptor (CB2R), a selective AEA membrane transporter, and an AEA-degrading enzyme fatty acid amide hydrolase. We show that, unlike CB2R, the activity of AEA membrane transporter and the activity and expression of FAAH decrease, whereas the apoptosis-inducing activity of AEA increases with age during the neonatal period. We also show that FSH reduces the apoptotic potential of AEA, but not that of its nonhydrolyzable analog methanandamide. Concomitantly, FSH enhances FAAH activity in a manner dependent on mRNA transcription and protein synthesis and apparently involving cAMP. These data demonstrate that Sertoli cells partake in the peripheral endocannabinoid system, and that FSH reduces the apoptotic potential of AEA by activating FAAH. Taken together, it can be suggested that the endocannabinoid network plays a role in the hormonal regulation of male fertility. .

Antidepressant and Anxiolytic Effects of Medicinal Cannabis Use in an Observational Trial
Erin L. Martin, Justin C. Strickland, Nicolas J. Schlienz, Joel Munson, Heather Jackson, Marcel O. Bonn-Miller and Ryan Vandrey
Front. Psychiatry 12:729800. (2021)
doi: 10.3389/fpsyt.2021.729800
https://www.frontiersin.org/articles...21.729800/full
Background: Anxiety and depressive disorders are highly prevalent. Patients are increasingly usingmedicinal cannabis products to treat these disorders, but little is known about the effects of medicinal cannabis use on symptoms of anxiety and depression. The Aim of the present observational study was to assess general health inmedicinal cannabis users and non-using controls with anxiety and/or depression.
Methods: Participants (368 Cannabis Users; 170 Controls) completed an online survey assessing anxiety and depressive symptoms, cannabis product use, sleep, quality of life, and comorbid chronic pain. Participants that completed this baseline survey were then invited to complete additional follow-up surveys at 3-month intervals. Baseline differences between Cannabis Users and Controls were assessed using independent samples t-tests and generalized linear mixed effects models were used to assess the impact of initiating cannabis product use, sustained use, or discontinuation of use on anxiety and depressive symptoms at follow-up.
Results: Medicinal cannabis use was associated with lower self-reported depression, but not anxiety, at baseline.Medicinal cannabis users also reported superior sleep, quality of life, and less pain on average. Initiation of medicinal cannabis during the follow-up period was associated with significantly decreased anxiety and depressive symptoms, an effect that was not observed in Controls that never initiated cannabis use.
Conclusions: Medicinal cannabis use may reduce anxiety and depressive symptoms in clinically anxious and depressed populations. Future placebo-controlled studies are necessary to replicate these findings and to determine the route of administration, dose, and product formulation characteristics to optimize clinical outcomes.

Antinociceptive and chondroprotective effects of prolonged β-caryophyllene treatment in the animal model of osteoarthritis: Focus on tolerance development
Jakub Mlost, Przemysław Kac, Marta Kędziora, Katarzyna Starowicz
Neuropharmacology 204 (2022) 108908
DOI: 10.1016/j.neuropharm.2021.108908
https://reader.elsevier.com/reader/s...2?token=E5A5B9 D844495EC5179E81ABE13F6899C218 143DEAEF1B65DC45C148EF1B6A903A 1B50484FD006980DE391BC7002D30B &originRegion=us-east-1&originCreation=20220103044 60 4
Osteoarthritis (OA) is a chronic joint disease in which cartilage degeneration leads to chronic pain. The endocannabinoid system has attracted attention as an emerging drug target for OA. However, the therapeutic potential of cannabinoids is limited by psychoactive side-effects related to CB1 activation and tolerance development for analgesic effects. β-Caryophyllene (BCP) is a low-efficacy natural agonist of CB2 and a common constituent of human diet with well-established anti-inflammatory properties. The results presented herein show the anti-nociceptive and chondroprotective potential of BCP in an animal model of OA induced by intra-articular injection of monoiodoacetate (MIA). Behavioural assessment included pressure application measurement and kinetic weight bearing tests. Histological assessment of cartilage degeneration was quantified using OARSI scoring. Experiments established the dose-response effects of BCP and pharmacological mechanisms of the antinociceptive action dependent on CB2 and opioid receptors. Chronic BCP treatment was able to hamper cartilage degeneration without producing tolerance for the analgesic effects. The data presented herein show that BCP is able to produce both acute and prolonged antinociceptive and chondroprotective effects. Together with the safety profile and legal status of BCP, these results indicate a novel and promising disease-modifying strategy for treating OA.

Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma
Alessia Ligresti, Aniello Schiano Moriello, Katarzyna Starowicz, Isabel Matias, Simona Pisanti, Luciano De Petrocellis, Chiara Laezza, Giuseppe Portella, Maurizio Bifulco, and Vincenzo Di Marzo
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 3
Doi: 10.1124/jpet.106.105247
9 -Tetrahydrocannabinol (THC) exhibits antitumor effects on various cancer cell types, but its use in chemotherapy is limited by its psychotropic activity. We investigated the antitumor activities of other plant cannabinoids, i.e., cannabidiol, cannabigerol, cannabichromene, cannabidiol acid and THC acid, and assessed whether there is any advantage in using Cannabis extracts (enriched in either cannabidiol or THC) over pure cannabinoids. Results obtained in a panel of tumor cell lines clearly indicate that, of the five natural compounds tested, cannabidiol is the most potent inhibitor of cancer cell growth (IC50 between 6.0 and 10.6 M), with significantly lower potency in noncancer cells. The cannabidiol-rich extract was equipotent to cannabidiol, whereas cannabigerol and cannabichromene followed in the rank of potency. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of xenograft tumors obtained by s.c. injection into athymic mice of human MDA-MB-231 breast carcinoma or rat v-K-ras-transformed thyroid epithelial cells and reduced lung metastases deriving from intrapaw injection of MDA-MB-231 cells. Judging from several experiments on its possible cellular and molecular mechanisms of action, we propose that cannabidiol lacks a unique mode of action in the cell lines investigated. At least for MDA-MB-231 cells, however, our experiments indicate that cannabidiol effect is due to its capability of inducing apoptosis via: direct or indirect activation of cannabinoid CB2 and vanilloid transient receptor potential vanilloid type-1 receptors and cannabinoid/vanilloid receptor-independent elevation of intracellular Ca2 and reactive oxygen species. Our data support the further testing of cannabidiol and cannabidiol-rich extracts for the potential treatment of cancer.

Are Sweet Dreams Made of These? Understanding the Relationship Between Sleep and Cannabis Use
Dalton Edwards, Francesca M Filbey
Cannabis Cannabinoid Res. 2021 Jun 18.
doi: 10.1089/can.2020.0174
Introduction: There is widespread literature on the interaction between cannabis use and sleep, yet the mechanisms that underlie this relationship are not well understood. Several factors lead to inconsistencies in this relationship suggesting a nuanced interaction between cannabis and sleep. An important question that remains to be addressed is the temporal relationship between disrupted sleep and cannabis use. This literature review summarizes the existing literature on the association between disrupted sleep and cannabis toward the goal of addressing the question of the chronology of these reported effects.
Materials and Methods: We conducted a review of the literature using PubMed to summarize current knowledge on the association between cannabis use and sleep in humans.
Results: We identified 31 studies on the association of cannabis use and sleep. The findings from these studies were mixed. Cannabis was associated with a variety of impacts on sleep ranging from beneficial effects, such as reduced sleep-onset latency, to negative outcomes, such as reduced sleep duration and suppressed rapid eye movement oscillations. The chronology of the interaction of cannabis and sleep was unclear, although much of the current literature focus on factors that modulate how cannabis impairs sleep after initial use.
Conclusion: There was sufficient evidence to suggest that cannabis use alters circadian rhythms, and hence, negatively impacts sleep. The current literature is largely from studies utilizing self-report measures of sleep; thus, objective measures of sleep are needed. In addition, although there were no empirical studies on the temporal relationship between cannabis use and sleep, the majority of the literature focused on characterizing sleep impairment after cannabis use.

Administration of ?9?Tetrahydrocannabinol (THC) Post?Staphylococcal Enterotoxin B Exposure Protects Mice From Acute Respiratory Distress Syndrome and Toxicity.
Mohammed, A., Alghetaa, H., Sultan, M., Singh, N. P., Nagarkatti, P., & Nagarkatti, M.
Frontiers in Pharmacology, 11.(2020).
doi:10.3389/fphar.2020.00893
Acute Respiratory Distress Syndrome (ARDS) is a life-threatening complication that can ensue following Staphylococcus aureus infection. The enterotoxin produced by these bacteria (SEB) acts as a superantigen thereby activating a large proportion of T cells leading to cytokine storm and severe lung injury. D9Tetrahydrocannabinol (THC), a psychoactive ingredient found in Cannabis sativa, has been shown to act as a potent anti-inflammatory agent. In the current study, we investigated the effect of THC treatment on SEB-induced ARDS in mice. While exposure to SEB resulted in acute mortality, treatment with THC led to 100% survival of mice. THC treatment significantly suppressed the inflammatory cytokines, IFN-g and TNF-a. Additionally, THC elevated the induction of regulatory T cells (Tregs) and their associated cytokines, IL-10 and TGF-b. Moreover, THC caused induction of Myeloid-Derived Suppressor Cells (MDSCs). THC acted through CB2 receptor as pharmacological inhibitor of CB2 receptors blocked the anti-inflammatory effects. THC-treated mice showed significant alterations in the expression of miRNA (miRs) in the lung-infiltrated mononuclear cells (MNCs). Specifically, THC caused downregulation of let7a-5p which targeted SOCS1 and downregulation of miR-34-5p which caused increased expression of FoxP3, NOS1, and CSF1R. Together, these data suggested that THC-mediated alterations in miR expression in the lungs may play a critical role in the induction of immunosuppressive Tregs and MDSCs as well as suppression of cytokine storm leading to attenuation of SEB-mediated lung injury.

Adolescent cannabis use and adult psychoticism: A longitudinal co-twin control analysis using data from two cohorts
Schaefer JD, Jang SK, Vrieze S, Iacono WG, McGue M, Wilson S.
American Psychological Association Sept 2021
DOI: 10.1037/abn0000701
https://www.researchgate.net/publica...om_two_cohorts
Observational studies have repeatedly linked cannabis use and increased risk of psychosis. We sought to clarify whether this association reflects a causal effect of cannabis exposure or residual confounding. We analyzed data from two cohorts of twins who completed repeated, prospective measures of cannabis use (N = 1544) and cannabis use disorder symptoms (N = 1458) in adolescence and a dimensional measure of psychosis-proneness (the Personality Inventory for DSM-5 Psychoticism scale) in adulthood. Twins also provided molecular genetic data, which were used to estimate polygenic risk of schizophrenia. Both cumulative adolescent cannabis use and use disorder were associated with higher Psychoticism scores in adulthood. However, we found no evidence of an effect of cannabis on Psychoticism or any of its facets in co-twin control models that compared the greater-cannabis-using twin to the lesser-using co-twin. We also observed no evidence of a differential effect of cannabis on Psychoticism by polygenic risk of schizophrenia. Although cannabis use and disorder are consistently associated with increased risk of psychosis, the present results suggest this association is likely attributable to familial confounds rather than a causal effect of cannabis exposure. Efforts to reduce the prevalence and burden of psychotic illnesses thus may benefit from greater focus on other therapeutic targets.
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Adolescent Treatment Admissions for Marijuana Following Recreational Legalization in Colorado and Washington.
Mennis, J., & Stahler, G. J.
Drug and Alcohol Dependence, (2020). doi:10.1016/j.drugalcdep.2020.107960
Introduction: There is concern that recreational marijuana legalization (RML) may lead to increased cannabis use disorder (CUD) among youth due to increased marijuana use. This study investigates whether adolescent substance use disorder treatment admissions for marijuana use increased in Colorado and Washington following RML.
Methods: Annual data on 2008–2017 treatment admissions for marijuana use from the SAMHSA TEDS-A dataset for adolescents age 12–17 were used to model state treatment admissions trends. Difference-in-differences models were used to investigate whether treatment admissions increased following RML in Colorado/ Washington compared to non-RML states, after adjusting for socioeconomic characteristics and treatment availability.
Results: Over all states in the analysis, the rate of adolescent treatment admissions for marijuana use declined significantly over the study period (?=?3.375, 95 % CI=?4.842, ?1.907), with the mean rate falling nearly in half. The decline in admissions rate was greater in Colorado and Washington compared to non-RML states following RML, though this difference was not significant (?=-7.671, 95 % CI=-38.798, 23.456).
Conclusion: Adolescent treatment admissions for marijuana use did not increase in Colorado and Washington following RML. This may be because youth marijuana use did not increase, CUD did not increase (even if use did increase), or treatment seeking behaviors changed due to shifts in attitudes and perceptions of risk towards marijuana use.

Adverse Outcome Pathway of Developmental Neurotoxicity Resulting from Prenatal Exposures to Cannabis Contaminated with Organophosphate Pesticide Residues
Maxwell C. K. Leung, Marilyn H. Silva, Amanda J. Palumbo, Peter N. Lohstroh, Svetlana E. Koshlukova, Shelley B. DuTeaux
Reproductive Toxicology
Doi: 10.1016/j.reprotox.2019.01.004
There is growing concern that increased use of medical and recreational cannabis may result in increased exposure to contaminants on the cannabis, such as pesticides. Several states are moving towards implementing robust regulation of the sales, cultivation, and manufacture of cannabis products. However, there are challenges with creating health-protective regulations in an industry that, to date, has been largely unregulated. The focus of this publication is a theoretical examination of what may happen when women are exposed pre-conceptually or during pregnancy to cannabis contaminated with pesticides. We propose an adverse outcome pathway of concomitant prenatal exposure to cannabinoids and theorganophosphate pesticide chlorpyrifos by curating what we consider to be the key events at the molecular, cellular, and tissue levels that result in developmental neurotoxicity. The implications of this adverse outcome pathway underscore the need to elucidate the potential developmental neurotoxicity that may result from prenatal exposure to pesticide-contaminated cannabis.

Age-dependent Alteration in Mitochondrial Dynamics and Autophagy in Hippocampal Neuron of Cannabinoid CB1 receptor-deficient Mice.
Kataoka, K., Bilkei-Gorzo, A., Nozaki, C., Togo, A., Nakamura, K., Ohta, K., … Asahi, T.
Brain Research Bulletin. (2020).
doi:10.1016/j.brainresbull.2020.03.014
Endocannabinoid system activity contributes to the homeostatic defense against aging and thus may counteract the progression of brain aging. The cannabinoid type 1 (CB1) receptor activity declines with aging in the brain, which impairs neuronal network integrity and cognitive functions. However, the underlying mechanisms that link CB1 activity and memory decline remain unknown. Mitochondrial activity profoundly influences neuronal function, therefore age-dependent mitochondrial activity change is one of the known hallmarks of brain aging. As CB1 receptor is expressed on mitochondria and
may regulate neuronal energy metabolism in hippocampus, we hypothesized that CB1 receptors might influence mitochondria in hippocampal neurons. We found that CB1 receptor significantly affected mitochondrial autophagy (mitophagy) and morphology in an age-dependent manner. We also found that Serine 65-phosphorylated ubiquitin, a key marker for mitophagy, was reduced in adult CB1-deficient mice (CB1-KO) compared to those in wild type controls, particularly in CA1 pyramidal cell layer. Transmission electron microscopy (TEM) analysis showed reduced mitophagy-like events in hippocampus of adult CB1-KO. TEM analysis also showed an increase in thin and elongated mitochondria in hippocampal neurons of adult CB1-KO. 3D reconstruction revealed that mitochondrial morphology in adult CB1-KO was altered as represented by an enhanced density of elongated and interconnected mitochondria. Altogether, these findings suggest that reduced CB1 signaling in CB1-KO mice leads to reduced mitophagy and abnormal mitochondrial morphology in hippocampal neurons during aging. These mitochondrial changes might be due to the impairments in mitochondrial quality control system, which links age-related decline in CB1 activity and impaired memory.

AKT1 genotype moderates the acute psychotomimetic effects of naturalistically smoked cannabis in young cannabis smokers
CJA Morgan, TP Freeman, J Powell and HV Curran
Transl Psychiatry (2016) 6, e738;
doi:10.1038/tp.2015.219
Smoking cannabis daily doubles an individual’s risk of developing a psychotic disorder, yet indicators of specific vulnerability have proved largely elusive. Genetic variation is one potential risk modifier. Single-nucleotide polymorphisms in the AKT1 and catechol-O-methyltransferase (COMT) genes have been implicated in the interaction between cannabis, psychosis and cognition, but no studies have examined their impact on an individual’s acute response to smoked cannabis. A total 442 healthy young cannabis users were tested while intoxicated with their own cannabis—which was analysed for delta-9-tetrahydrocannbinol (THC) and cannabidiol content—and also ± 7 days apart when drug-free. Psychotomimetic symptoms and working memory were assessed on both the sessions. Variation at the rs2494732 locus of the AKT1 gene predicted acute psychotic response to cannabis along with
dependence on the drug and baseline schizotypal symptoms. Working memory following cannabis acutely was worse in females, with some suggestion of an impact of COMT polymorphism on working memory when drug-free. These findings are the first to demonstrate that AKT1 mediates the acute response to cannabis in otherwise healthy individuals and implicate the AKT1 pathway as a possible target for prevention and treatment of cannabis psychosis.

Allergic and Respiratory Symptoms in Employees of Indoor Cannabis Grow Facilities.
Sack, C., Ghodsian, N., Jansen, K., Silvey, B., & Simpson, C. D.
Annals of Work Exposures and Health.(2020).
doi:10.1093/annweh/wxaa050
Background: While little is known about the occupational hazards associated with Cannabis cultivation, both historical research in the hemp industry and preliminary data from modern grow houses, suggest that Cannabis workers may be at increased risk of respiratory and allergic diseases.
Objectives: We sought to investigate the association between workplace exposures and health symptoms in an indoor Cannabis grow facility in Washington State, USA.
Methods: We performed a cross-sectional study with all consenting employees in an indoor Cannabis grow facility in Seattle, WA using a questionnaire. The questionnaire gathered data on respiratory, ocular, nasal, and dermal symptoms. A subset of employees with work-related symptoms underwent repeated cross-shift and cross-week measurement of spirometry, fractional exhaled nitrogen oxide (FeNO), and skin prick testing for Cannabis sensitization. Exposure to Cannabis dust was classified based on self-described tasks, expert opinion, and exposure monitoring of particulate matter. Multivariable logistic regression was undertaken to examine associations between exposure to Cannabis dust (classified as low, medium, and high) and health symptoms. Linear mixed effects models examined the relationship between cross-shift and cross-week changes in spirometry and FeNO.
Results: Ninety-seven percent (97%) of the employees (n = 31) surveyed were recreational cannabis users, with 81% (n = 25) smoking cannabis multiple times per day. Twenty-two (71%) employees reported one or more work-related symptoms: 65% respiratory, 39% ocular, 32% nasal, and 26% dermal symptoms. There was a trend toward increased likelihood of work-related symptoms with increasing exposure to Cannabis dust, although none of these results were statistically significant. Of the 10 employees with work-aggravated symptoms, 5 had borderline-high or high FeNO, 7 had abnormal spirometry, and 5 had evidence of Cannabis sensitization on skin prick testing. FeNO increased by 3.78 ppb (95% confidence interval 0.68–6.88 ppb) across the work-week and there was a trend toward cross-week and cross-shift reduced airflow.
Conclusions: We found a high prevalence of work-related allergic- and particularly respiratory symptoms in the employees of one indoor Cannabis grow facility in Washington State. A high proportion of employees with work-aggravated symptoms had findings consistent with probable work-related asthma based on high FeNO, airflow obstruction on spirometry, and Cannabis sensitization on skin prick testing. However, due to the high incidence of recreational cannabis use among these workers, the relative influence of occupational versus recreational exposure to Cannabis dust on the respiratory health and sensitization status of these workers could not be resolved in this study.

Amyloid proteotoxicity initiates an inflammatory response blocked by cannabinoids

Antonio Currais, Oswald Quehenberger, Aaron M Armando, Daniel Daugherty, Pam Maher & David Schubert
Aging and Mechanisms of Disease 2, Article number: 16012 (2016)
DOI: 10.1038/npjamd.2016.12
he beta amyloid (A?) and other aggregating proteins in the brain increase with age and are frequently found within neurons. The mechanistic relationship between intracellular amyloid, aging and neurodegeneration is not, however, well understood. We use a proteotoxicity model based upon the inducible expression of A? in a human central nervous system nerve cell line to characterize a distinct form of nerve cell death caused by intracellular A?. It is shown that intracellular A? initiates a toxic inflammatory response leading to the cell's demise. A? induces the expression of multiple proinflammatory genes and an increase in both arachidonic acid and eicosanoids, including prostaglandins that are neuroprotective and leukotrienes that potentiate death. Cannabinoids such as tetrahydrocannabinol stimulate the removal of intraneuronal A?, block the inflammatory response, and are protective. Altogether these data show that there is a complex and likely autocatalytic inflammatory response within nerve cells caused by the accumulation of intracellular A?, and that this early form of proteotoxicity can be blocked by the activation of cannabinoid receptors.

An efficient new cannabinoid antiemetic in pediatric oncology.
Abrahamov, A., Abrahamov, A., & Mechoulam, R.
Life Sciences, 56(23-24), 2097–2102. (1995).
doi:10.1016/0024-3205(95)00194-b
Delta-%tetrahydrocannabinol (delta-%THC), a cannabinoid with lower psychotropic potency than the main Cannabis constituent, delta-9-tetrahydrocannabinol (delta-9-THC), was administerd (18 mg/m2 in edible oil,p.o.) to eight children, aged 3-33 years with various hematologic cancers, treated
with different antineoplastic drugs for up to 8 months. The total number of treatments with delta-8-THC so far is 480. The THC treatment started two hours before each antineoplastic treatment and was continued every 6 hrs for 24 hours,Vomiting was completely prevented. The side effects observed were negligible.

An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity.
Ben-Shabat, S., Fride, E., Sheskin, T., Tamiri, T., Rhee, M.-H., Vogel, Z., … Mechoulam, R.
European Journal of Pharmacology, 353(1), 23–31.(1998).
doi:10.1016/s0014-2999(98)00392-6
2-Arachidonoyl-glycerol 2-Ara-Gl has been isolated from various tissues and identified as an endogenous ligand for both Ž . cannabinoid receptors, CB and CB . Here we report that in spleen, as in brain and gut, 2-Ara-Gl is accompanied by several 1 2 2-acyl-glycerol esters, two major ones being 2-linoleoyl-glycerol 2-Lino-Gl and 2-palmitoyl-glycerol 2-Palm-Gl . These two esters do Ž. Ž . not bind to the cannabinoid receptors, nor do they inhibit adenylyl cyclase via either CB or CB ; however, they significantly potentiate 1 2 the apparent binding of 2-Ara-Gl and its apparent capacity to inhibit adenylyl cyclase. Together these esters also significantly potentiate 2-Ara-Gl inhibition of motor behavior, immobility on a ring, analgesia on a hot plate and hypothermia caused by 2-Ara-Gl in mice. 2-Lino-Gl, but not 2-Palm-Gl, significantly inhibits the inactivation of 2-Ara-Gl by neuronal and basophilic cells. These data indicate that the biological activity of 2-Ara-Gl can be increased by related, endogenous 2-acyl-glycerols, which alone show no significant activity in any of the tests employed. This effect ‘entourage effect’ may represent a novel route for molecular regulation of endogenous Ž . cannabinoid activity

An evaluation of the anti?hyperalgesic effects of cannabidiolic acid?methyl ester (CBDA?ME) in a preclinical model of peripheral neuropathic pain.
Zhu, Y. F., Linher?Melville, K., Niazmand, M. J., Sharma, M., Shahid, A., Zhu, K. L., … Singh, G.
British Journal of Pharmacology. (2020).
doi:10.1111/bph.14997
Background and purpose Chronic neuropathic pain (NEP) is associated with growing therapeutic cannabis use. To promote quality of life without psychotropic effects, cannabinoids other than ?9- tetrahydrocannabidiol, including cannabidiol and its precursor cannabidiolic acid (CBDA), are being evaluated. Due to its instability, CBDA has been understudied, particularly as an anti-nociceptive agent. Adding a methyl ester group (CBDA-ME) significantly enhances its stability, facilitating analyses of its analgesic effects in vivo. This study examines early treatment efficacy of CBDA-ME in a rat model of peripherally induced NEP and evaluates sex as a biological variable. Experimental approach After 14 consecutive days of intraperitoneal CBDA-ME administration at 0.01, 0.1, and 1 µg/kg, commencing one day after surgically implanting a sciatic nerve-constricting cuff to induce NEP, the anti-nociceptive efficacy of this cannabinoid was assessed in male and female Sprague-Dawley rats relative to vehicle-treated counterparts. In females, 2 and 4 µg/kg daily doses of CBDA-ME were also evaluated. Behavioural tests were performed for hind paw mechanical and thermal withdrawal thresholds once a week for eight weeks. At endpoint, in vivo electrophysiological recordings were obtained to characterize soma threshold changes in primary sensory neurons. Key results In males, CBDA-ME elicited a significant concentration-dependent chronic anti-hyperalgesic effect, also influencing both nociceptive and non-nociceptive mechanoreceptors, which were not observed in females at any of the concentrations tested. Conclusion and implications Initiating treatment of a peripheral nerve injury with CBDA-ME at an early stage postsurgery provides anti-nociception in males, warranting further investigation into potential sexual dimorphisms underlying this response.

An Exploratory Human Laboratory Experiment Evaluating Vaporized Cannabis in the Treatment of Neuropathic Pain from Spinal Cord Injury and Disease
Barth Wilsey, MD, Thomas D. Marcotte, Reena Deutsch, Holly Zhao, Hannah Prasad, Amy Phan,
Journal of Pain (2016), 17(9), 982–1000.
10.1016/j.jpain.2016.05.010
Using eight hour human laboratory experiments, we evaluated the analgesic efficacy of vaporized cannabis in patients with neuropathic pain related to injury or disease of the spinal cord, the majority of whom were experiencing pain despite traditional treatment. After obtaining baseline data, 42 participants underwent a standardized procedure for inhaling 4 puffs of vaporized cannabis containing either placebo, 2.9%, or 6.7% delta-9-tetrahydrocannabinol on three separate occasions. A second dosing occurred 3 hours later; participants chose to inhale 4 to 8 puffs. This flexible dosing was utilized to attempt to reduce the placebo effect. Using an 11- point numerical pain intensity rating scale as the primary outcome, a mixed effects linear regression model demonstrated a significant analgesic response for vaporized cannabis. When subjective and psychoactive side effects (e.g., good drug effect, feeling high, etc.) were added as covariates to the model, the reduction in pain intensity remained significant above and beyond any effect of these measures (all p<0.0004). Psychoactive and subjective effects were dose dependent. Measurement of neuropsychological performance proved challenging because of various disabilities in the population studied. As the two active doses did not significantly differ from each other in terms of analgesic potency, the lower dose appears to offer the best riskbenefit ratio in patients with neuropathic pain associated with injury or disease of the spinal cord

An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol
Paula Morales, Patricia H. Reggio and Nadine Jagerovic
Front. Pharmacol. 8:422.
doi: 10.3389/fphar.2017.00422
Cannabidiol (CBD) has been traditionally used in Cannabis -based preparation, however historically, it has received far less interest as a single drug than the other components of Cannabis . Currently, CBD generates considerable interest due to its beneficial neuroprotective, antiepileptic, anxiolytic, antipsychotic, and anti-inflammatory properties. Therefore, the CBD scaffold becomes of increasing interest for medicinal chemists. This review provides an overview of the chemical structure of natural and synthetic CBD derivatives including the molecular targets associated with these compounds. A clear identification of their biological targets has been shown to be still very challenging.

An Unusual Case of Ventricular Tachycardia in a Young Patient Associated with Cannabis Use
Parth J Sampat , Sana Riaz , Maneesh Bisen , Robert Carhart
Case Rep Cardiol. 2020 Dec 1;2020:8813930.
DOI: 10.1155/2020/8813930
Marijuana has become the most widely used illicit drug in the United States. Approximately 43.5 million Americans aged 12 or above have reported the use of marijuana in the last year. The use of cannabinoids and its relationship with cardiac effects are not well known. Many types of arrhythmias have been noted with the use of cannabis products with atrial fibrillation being the most common arrhythmia associated with the use of cannabis. We present a case of a 36-year-old male who presented with pounding chest pain, dyspnea, and diaphoresis following marijuana use. He was found to be in ventricular tachycardia which responded to amiodarone. Workup done was negative for any structural disease, and cardiac catheterization was negative for coronary artery disease. He was ultimately discharged on metoprolol. In this report, we focus on how marijuana can be associated with many arrhythmias including ventricular tachycardia with focus on mechanisms by which it can occur. We believe a detailed social history with screening for cannabis use can identify more cases of arrhythmias that can be potentially associated with marijuana use.

Analysis of 2-Arachidonoylglycerol Levels in Polycystic Ovary Syndrome in the Context of Hormonal and Metabolic Alterations and Across the Classical Phenotypes
Plamena Kabakchieva, Antoaneta Gateva, Julieta Hristova, Tsvetoslav Georgiev, and Zdravko Kamenov
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0183
Introduction: Ovarian folliculogenesis requires a fine balance between extra- and intra-ovarian factors. Endocannabinoids are found in the female reproductive system and are essential for a normal follicular growing process and ovulation. First, our study aimed to analyze levels of the endocannabinoid—2-arachidonoylglycerol (2-AG)— in patients with polycystic ovary syndrome (PCOS) and to compare with healthy controls. In addition, the study aimed to explore the association of 2-AG with hormonal and metabolic alterations, ovulatory dysfunction, and the presence of polycystic ovarian morphology (PCOM) across the classical PCOS phenotypes.
Methods: Fifty-four women with PCOS were compared with 26 healthy controls. PCOS patients were diagnosed and phenotyped according to the Rotterdam criteria. Further analyses were performed with the classical PCOS phenotypes A and B comprising hyperandrogenism with oligo-anovulation with or without PCOM, respectively. Full medical history, clinical investigations, anthropometric measurements, laboratory tests, and ultrasound investigations were carried out in the follicular phase. Serum levels of 2-AG were measured by enzyme-linked immunosorbent assay.
Results: PCOS patients (n = 54) and healthy controls (n = 26) showed similar metabolic parameters and anthropometric characteristics. PCOS patients were more hirsute than healthy women ( p = 0.001). Luteinizing hormone/ follicle-stimulating hormone ratio and serum levels of androgens were significantly higher in the patient than in the control group ( p = 0.035, p < 0.001, respectively). Free androgen index was also higher in the patient group ( p = 0.002). Serum levels of 2-AG did not significantly differ when comparing all PCOS patients versus healthy controls; however, further analysis of individual phenotype groups revealed that 2-AG levels in PCOS patients with phenotyope A (n = 30) were significantly lower when compared with PCOS patients with phenotype B (n = 20) and healthy controls (n = 26).
Conclusion: Serum levels of 2-AG were similar between PCOS patients and healthy controls. Nevertheless, phenotype A PCOS patients had significantly lower levels of the endocannabinoid compared with phenotype B patients
and healthy controls. Collectively, these results suggest that overall serum levels of 2-AG are not a diagnostic marker for PCOS; however, their altered secretion or activity may influence normal follicular processes.
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Anandamide, an endogenous cannabinoid receptor ligand, also interacts with 5-hydroxytryptamine (5-HT) receptor.

Kimura T, Ohta T, Watanabe K, Yoshimura H, Yamamoto I.
Biol Pharm Bull. 1998 Mar;21(3):224-6.
DOI:10.1248/bpb.21.224
https://www.jstage.jst.go.jp/article.../21_3_224/_pdf

Anandamide hydrolase: A guardian angel of human reproduction?
Mauro Maccarrone, Alessandro Finazzi Agrò
Trends in Pharmacological Sciences 25(7):353-7 August 2004
DOI: 10.1016/j.tips.2004.05.002
Anandamide (AEA) is the best-studied member of the endocannabinoids, a group of bioactive fatty acid amides and esters that activate cannabinoid receptors and have several roles in both the CNS and the periphery. The tone, signaling and activity of AEA in vivo are terminated by a specific fatty acid amide hydrolase (FAAH), whose inhibition has potential therapeutic applications in pain, neurodegenerative disorders, cancer and anxiety. In this article, we discuss the participation of AEA in hormone-cytokine networks that are essential for reproduction, and support the view that FAAH plays a key role in regulating this activity of AEA. We underline the fact that FAAH in maternal lymphocytes is a molecular integrator of signals that are crucial for human gestation, and that its downregulation to date is the only early marker of spontaneous abortion, both in vivo and in vitro. We propose that drugs that are able to enhance FAAH activity might become useful therapeutic tools for the management of human infertility.


Annotated bibliography of research on marijuana and health
Assembled by the Colorado School of Public Health, intended as background for the Marijuana and Public Health Symposium on March 7, 2014.
https://csph.ucdenver.edu/cphp/mj_bib.pdf
General reviews of marijuana and health
Marijuana and cancer
Marijuana and heart diseases
Marijuana and stroke
Marijuana and lung diseases
Marijuana and mental health
Marijuana and drug dependency
Marijuana and pregnancy, breast feeding
Marijuana and traffic safety
Avoiding unintentional marijuana exposures

Antibacterial Cannabinoids from Cannabis sativa: A Structure?Activity Study.
Appendino, G., Gibbons, S., Giana, A., Pagani, A., Grassi, G., Stavri, M., Rahman M. M.
Journal of Natural Product, 71(8), 1427–1430. (2008)
doi:10.1021/np8002673
Marijuana (Cannabis satiVa) has long been known to contain antibacterial cannabinoids, whose potential to address antibiotic resistance has not yet been investigated. All five major cannabinoids (cannabidiol (1b), cannabichromene (2), cannabigerol (3b), ?9-tetrahydrocannabinol (4b), and cannabinol (5)) showed potent activity against a variety of methicillin-resistant Staphylococcus aureus (MRSA) strains of current clinical relevance. Activity was remarkably tolerant to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids), and to carboxylation of the resorcinyl moiety (pre-cannabinoids). Conversely, methylation and acetylation of the phenolic hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were all detrimental for antibacterial activity. Taken together, these observations suggest that the prenyl moiety of cannabinoids serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore, while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.

Anticancer and Antioxidant Properties of Terpinolene in Rat Brain Cells.
Aydin, E., Türkez, H., & Taşdemir, Ş.
Archives of Industrial Hygiene and Toxicology, 64(3), 415–424.(2013).
doi:10.2478/10004-1254-64-2013-2365
Terpinolene (TPO) is a natural monoterpene present in essential oils of many aromatic plant species. Although various biological activities of TPO have been demonstrated, its neurotoxicity has never been explored. In this in vitro study we investigated TPO’s antiproliferative and/or cytotoxic properties using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test, genotoxic damage potential using the single-cell gel electrophoresis (SCGE), and oxidative effects through total antioxidant capacity (TAC) and total oxidative stress (TOS) in cultured primary rat neurons and N2a neuroblastoma cells. Dose-dependent effects of TPO (at 10 mg L-1, 25 mg L-1, 50 mg L-1, 100 mg L-1, 200 mg L-1, and 400 mg L-1) were tested in both cell types. Significant (P<0.05) decrease in cell proliferation were observed in cultured primary rat neurons starting with the dose of 100 mg L-1 and in N2a neuroblastoma cells starting with 50 mg L-1. TPO was not genotoxic in either cell type. In addition, TPO treatment at 10 mg L-1, 25 mg L-1, and 50 mg L-1 increased TAC in primary rat neurons, but not in N2a cells. However, at concentrations above 50 mg L-1 it increased TOS in both cell types. Our fi ndings clearly demonstrate that TPO is a potent antiproliferative agent for brain tumour cells and may have potential as an anticancer agent, which needs to be further studied.

Antidepressant-like effect of cannabidiol injection into the ventral medial prefrontal cortex - possible involvement of 5-HT1A and CB1 receptors.
Sartim AG1, Guimarães FS2, Joca SR3.
Behavioural Brain Research, 303, 218–227. *
DOI: 10.1016/j.bbr.2016.01.033
RATIONALE:
systemic administration of Cannabidiol (CBD), the main non-psychotomimetic constituent of Cannabis sativa, induces antidepressant-like effects. The mechanism of action of CBD is thought to involve the activation of 5-HT1A receptors and the modulation of endocannabinoid levels with subsequent CB1 activation. The brain regions involved in CBD-induced antidepressant-like effects remain unknown. The ventral medial prefrontal cortex (vmPFC), which includes the infralimbic (IL) and prelimbic (PL) subregions, receives dense serotonergic innervation and plays a significant role in stress responses.

Antifungal activity of the volatiles of high potency Cannabis sativa L. against Cryptococcus neoformans.
Wanas, A. S., Radwan, M. M., Mehmedic, Z., Jacob, M., Khan, I. A., and Elsohly, M. A.
Rec. Nat. Prod. 10, 214–220.
https://www.researchgate.net/publica...cus_neoformans
The n-hexane extracted volatile fraction of high potency Cannabis sativa L (Cannabaceae). was assessed in vitro for antifungal, antibacterial and antileishmanial activities. The oil exhibited selective albeit modest, antifungal activity against Cryptococcus neoformans with an IC50 value of 33.1 ?g/mL. Biologically- guided fractionation of the volatile fraction resulted in the isolation of three major compounds (1-3) using various chromatographic techniques. The chemical structures of the isolated compounds were identified as ?- humulene (1), ?-caryophyllene (2) and caryophyllene oxide (3) using GC/FID, GC/MS, 1D- and 2D-NMR analyses, respectively. Compound 1 showed potent and selective antifungal activity against Cryptococcus neoformans with IC50 and MIC values of 1.18 ?g/mL and 5.0 ?g/mL respectively. Whereas compound 2 showed weak activity (IC50 19.4 ?g/mL), while compound 3 was inactive against C. neoformans.

Anti-inflammatory activity and accelerated stability studies of crude extract syrup of Cannabis sativa
Wilfred O Obonga, Charles O Nnadi, Sunday N Okafor, Edwin O Omeje,
Patrick Igbinnaduwa, Charles O Okoli, Patience O Osadebe
Tropical Journal of Pharmaceutical Research February 2019; 18 (2): 295-301
Doi: 10.4314/tjpr.v18i2.11
Purpose: To formulate Cannabis sativa-based syrup and investigate its anti-inflammatory potential and the stability of the formulation under stress conditions.
Methods: The syrup was prepared using different combinations of crude C. sativa resin, propylene glycol, aspartame, sucrose, sodium metabisulphite (SMBs) and ethylenediaminetetraacetic acid (EDTA). The stability of the formulations was determined under accelerated temperature conditions.
The anti-inflammatory activity of the resin and different formulations were evaluated by the egg albumin induced paw edema model in rats. Biochemical assay was determined by Reitman and Frankel colorimetric assay method while hematological assay was evaluated by standard protocols.
Results: EDTA-containing syrup (CE) was the most stable with estimated shelf-life of 2204 days (K25ºC, 4.78 x 10-5/day). Higher propylene glycol levels significantly improved anti-inflammatory activity compared to those containing a lower amount. All the formulations showed anti-inflammatory activity higher than the crude resin with a dose-dependent inhibition of paw edema compared with the control. There was no significant difference (p < 0.05) between the serum glutamate-oxaloacetate transaminase (SGOT, 13.821 ± 0.190 - 16.008 ± 1.012), serum glutamate-pyruvate transaminase (SGPT, 19.241 ± 1.027 - 22.901 ± 1.093) and urea (9.812 ± 0.252 - 10.054 ± 0.252) levels of the treated and 16.856 ± 1.053, 24.960 ± 0.101 and 10.654 ± 0.925 units/L of the control animals respectively. With the exception of eosinophil that disappeared from the blood in the third week, all the hematological parameters showed a gradual increase in lymphocytes, neutrophils, monocytes, packed cell volume (PCV), white and red blood cell counts in the third week compared to control.
Conclusion: Formulation of C. sativa as syrup using efficient carriers improves the pharmacological activity of the crude extract. SMBs and EDTA significantly enhance the stability of the syrup with no observable biochemical and hematological changes in treated animals

Anti-Inflammatory Activity in Colon Models Is Derived from D9-Tetrahydrocannabinolic Acid That Interacts with Additional Compounds in Cannabis Extracts
Rameshprabu Nallathambi, Moran Mazuz, Aurel Ion, Gopinath Selvaraj, Smadar Weininger, Marcelo Fridlender, Ahmad Nasser, Oded Sagee, Puja Kumari, Diana Nemichenizer, Maayan Mendelovitz, Nave Firstein, Orly Hanin, Fred Konikoff, Yoram Kapulnik, Timna Naftali, and Hinanit Koltai
Cannabis and Cannabinoid Research, 2(1), 167–182.
DOI: 10.1089/can.2017.0027
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner. Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell cultureand colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

Anti-Inflammatory Activity in Colon Models Is Derived from D9-Tetrahydrocannabinolic Acid That Interacts with Additional Placebo Effects of Edible Cannabis: Reported Intoxication Effects at a 30-Minute Delay
Mallory J. E. Loflin, Ph.D., Mitch Earleywine, Ph.D., Stacey Farmer, M.A., Melissa Slavin M.A., Rachel Luba, B.S. and Marcel Bonn-Miller, Ph.D.
DOI: 10.1080/02791072.2017.1354409
Previous research has demonstrated the ability of non-active smoked cannabis cigarettes to induce subjective effects of intoxication (i.e., placebo effect). No studies have been conduced to test whether edible forms of cannabis, which are associated with a significant delay in onset of effect, are able to induce a placebo effect. In the present study, 20 participants were told that they would receive an edible cannabis lollipop containing a high dose of tetrahydrocannabinol (THC), but were instead given a placebo control. Measures of intoxication and mood were taken at baseline, 30 minutes, and 60 minutes post-ingestion of the placebo lollipop. Results of four repeated-measures ANOVAs found significant and quadratic changes across time in cannabis (ARCI m-scale) intoxication (F(2,18) = 4.90, p = .01, ?2 = .22) and negative mood (F(2,18) = 3.99, p = .05, ?2 = .19). Changes in positive mood and the overall measure of general intoxication (ARCI) failed to reach significance. The present study provides preliminary evidence that a placebo effect can be induced with inert edible agents when participants are told that they are receiving active THC. This is the first known study to demonstrate an edible cannabis intoxication placebo effect.
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner.
Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of
concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

Anti-inflammatory Potential of Terpenes Present in Cannabis sativa L.
Eric J. Downer
ACS Chem. Neurosci. XXXX, XXX, XXX?XXX
Doi: 10.1021/acschemneuro.0c00075
Cannabis sativa L. (C. sativa) contains an array of plant-derived (phyto) cannabinoids and terpenes that are predominantly located in the trichome cavity of the plant. Terpenes, aromatic organic hydrocarbons characterized for their role in plant protection/pollination, are gaining attention for their potential as novel therapeutics in many areas of biomedicine. This Viewpoint will explore the exciting recent evidence that terpenes have anti-inflammatory/antioxidant propensity by targeting inflammatory signaling mechanisms relevant to human disease. Given their anti-inflammatory properties, terpenes may contribute to the effects of current cannabinoid-based therapies.

ANTINEOPLASTIC AND APOPTOTIC EFFECTS OF CANNABINOIDS. N-ACYLETHANOLAMINES: PROTECTORS OR KILLERS? V.M. Pushkarev, O.I. Kovzun, M.D. Тronkо
Exp Oncol 2008 30, 1, –000
http://vpushkarev.narod.ru/doc/cannabin.pdf
The proapoptotic and antineoplastic properties of cannabinoids with emphasis on effects of N-acylethanolamines were analyzed. Cannabinoids enhanced apoptotic and necrotic processes in many types of tumour cells and tissues. Involvement of different types of receptors and signaling pathways in mediating the proapoptotic effects of cannabinoids are discussed. The evidences in favour of both proapoptotic, pronecrotic and protective, antiapoptotic effects of cannabinoids and, especially N-acylethanolamines, are evaluated. The hypothesis is suggested that N-acylethanolamines, formed in some tissues under strong stress conditions, can be not a consequence of tissue damage but cause such damage. The conclusion is made on promising of cannabinoids as potential anticancer agents. Key Words: Cannabinoids, N-acylethanolamines, apoptosis, necrosis, tumours, ischemia.

Antioxidant and antimicrobial activity of two standardized extracts from a new Chinese accession of non?psychotropic Cannabis sativa L.
Muscarà, C., Smeriglio, A., Trombetta, D., Mandalari, G., La Camera, E., Occhiuto, C., Circosta, C.
Phytotherapy Research. (2020).
doi:10.1002/ptr.6891
The purpose of this study was to evaluate the antioxidant and antimicrobial properties of two extracts from a new Chinese accession (G-309) of Cannabis sativa L. (?9 - tetrahydrocannabinol <0.2% with high content of propyl side chain phytocannabinoids. Dried flowering tops, as such and after hydrodistillation of the essential oil, were extracted with acidic hexane to produce the Cannabis Chinese hexane extract 1 (CChHE1) and 2 (CChHE2), respectively. The phytochemical profile of CChHE1 and CChHE2 was investigated by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–diode array detector–electrospray ionization–tandem mass spectrometry (LC-DAD-ESI-MS/MS) analyses. The antioxidant properties were assessed by several in vitro cell-free assays. The antimicrobial activity was evaluated against Gram-positive and Gram-negative bacteria and the yeast Candida albicans. Phytochemical analyses highlighted a high content of cannabidivarinic acid (CBDVA) and tetraydrocannabivarinic acid (THCVA) in CChHE1, and cannabidivarin (CBDV) and tetraydrocannabivarin (THCV) in CChHE2. Both extracts showed remarkable antioxidant activity and strong antimicrobial properties (MIC 39.06 and MBC 39.06–78.13 ?g/ml) against both ATCC and methicillin-resistant clinical strains of Staphylococcus aureus. In conclusion, standardized extracts of C. sativa Chinese accession could be promising for their possible use as novel antibacterial agents for the treatment of widespread S. aureus infections.

Antioxidative and Anti-Inflammatory Properties of Cannabidiol
Atalay, S.; Jarocka-Karpowicz, I.; Skrzydlewska, E.
Antioxidants 2020, 9, 21
DOI: 10.3390/antiox9010021
https://mdpi-res.com/d_attachment/an...9-00021-v3.pdf
Cannabidiol (CBD) is one of the main pharmacologically active phytocannabinoids of Cannabis sativa L. CBD is non-psychoactive but exerts a number of beneficial pharmacological effects, including anti-inflammatory and antioxidant properties. The chemistry and pharmacology of CBD, as well as various molecular targets, including cannabinoid receptors and other components of the endocannabinoid system with which it interacts, have been extensively studied. In addition, preclinical and clinical studies have contributed to our understanding of the therapeutic potential of CBD for many diseases, including diseases associated with oxidative stress. Here, we review the main biological effects of CBD, and its synthetic derivatives, focusing on the cellular, antioxidant, and anti-inflammatory properties of CBD


Antineoplastic Activity of Cannabinoids
Munson, A. E., Harris, L. S., Friedman, M. A., Dewey, W. L., & Carchman, R. A.
JNCI: Journal of the National Cancer Institute, 55(3), 597–602.(1975).
doi:10.1093/jnci/55.3.597
Lewis lung adenocarcinoma growth was retarded by the oral administration of ~9-tetrahydrocannabinol (~9-THC), ~S-tetrahydrocannabinol (As.THC), and cannabinol (CBN), but not cannabidiol (CBD). Animals treated for 10 consecutive days with ~9-THC, beginning the day after tumor implantation, demonstrated a dose-dependent action of retarded tumor growth. Mice treated for 20 consecutive days with ~s-THC and CBN had reduced primary tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days. ~9-THC, ~s·THC, and CBN increased the mean survival time (36% at 100 mg/kg, 25% at 200 mg/kg, and 27% at SO mg/kg, respectively), whereas CBD did not. ~9-THC administered orally daily until death in doses of SO, 100, or 200 mg/kg did not increase the life-spans of (C57BL/6 x DBA/2)Fl (BDFl) mice hosting the L1210 murine leukemia. However, ~9-THC administered daily for 10 days Significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and isolated Lewis lung cells incubated in vitro with ~9-THC and ~s-THC showed a dose-dependent (10-4-10-7) inhibition (80-20%, respectively) of tritiated thymidine and 14C-uridine uptake into these cells. CBD was active only in high concentrations (10-4

Antitumor Cannabinoid Chemotypes: Structural Insights.
Morales, P., & Jagerovic, N.
Frontiers in Pharmacology, 10. (2019).
doi:10.3389/fphar.2019.00621
Cannabis has long been known to limit or prevent nausea and vomiting, lack of appetite, and pain. For this reason, cannabinoids have been successfully used in the treatment of some of the unwanted side effects caused by cancer chemotherapy. Besides their palliative effects, research from the past two decades has demonstrated their promising potential as antitumor agents in a wide variety of tumors. Cannabinoids of endogenous, phytogenic, and synthetic nature have been shown to impact the proliferation of cancer through the modulation of different proteins involved in the endocannabinoid system such as the G protein–coupled receptors CB1, CB2, and GRP55, the ionotropic receptor TRPV1, or the fatty acid amide hydrolase (FAAH). In this article, we aim to structurally classify the antitumor cannabinoid chemotypes described so far according to their targets and types of cancer. In a drug discovery approach, their in silico pharmacokinetic profile has been evaluated in order to identify appropriate drug-like profiles, which should be taken into account for further progress toward the clinic. This analysis may provide structural insights into the selection of specific cannabinoid scaffolds for the development of antitumor drugs for the treatment of particular types of cancer.

Anxiety and cognitive-related effects of Δ 9-tetrahydrocannabinol (THC) are differentially mediated through distinct GSK-3 vs. Akt-mTOR pathways in the nucleus accumbens of male rats
Roger Hudson, Christopher Norris, Hanna J. Szkudlarek, Dinat Khan, Susanne Schmid, Walter J. Rushlow & Steven R. Laviolette
Psychopharmacology (Dec. 2021)
DOI: 10.1007/s00213-021-06029-w
https://link.springer.com/article/10...13-021-06029-w
Rationale
Δ9-tetrahydrocannabinol (THC) is the primary psychoactive compound in cannabis and is responsible for cannabis-related neuropsychiatric side effects, including abnormal affective processing, cognitive and sensory filtering deficits and memory impairments. A critical neural region linked to the psychotropic effects of THC is the nucleus accumbens shell (NASh), an integrative mesocorticolimbic structure that sends and receives inputs from multiple brain areas known to be dysregulated in various disorders, including schizophrenia and anxiety-related disorders. Considerable evidence demonstrates functional differences between posterior vs. anterior NASh sub-regions in the processing of affective and cognitive behaviours influenced by THC. Nevertheless, the neuroanatomical regions and local molecular pathways responsible for these psychotropic effects are not currently understood.
Objectives
The objectives of this study were to characterize the effects of intra-accumbens THC in the anterior vs. posterior regions of the NASh during emotional memory formation, sensorimotor gating and anxiety-related behaviours.
Methods
We performed an integrative series of translational behavioural pharmacological studies examining anxiety, sensorimotor gating and fear-related associative memory formation combined with regionally specific molecular signalling analyses in male Sprague Dawley rats.
Results
We report that THC in the posterior NASh causes distortions in emotional salience attribution, impaired sensory filtering and memory retention and heightened anxiety, through a glycogen-synthase-kinase-3 (GSK-3)-β-catenin dependent signalling pathway. In contrast, THC in the anterior NASh produces anxiolytic effects via modulation of protein kinase B (Akt) phosphorylation states.
Conclusions
These findings reveal critical new insights into the neuroanatomical and molecular mechanisms associated with the differential neuropsychiatric side effects of THC in dissociable nucleus accumbens sub-regions.
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Are cannabidiol and ?9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review
John M McPartland, Marnie Duncan, Vincenzo Di Marzo and Roger G Pertwee
British Journal of Pharmacology
Doi: 10.1111/bph.12944

2-Arachidonoylglycerol Attenuates Myocardial Fibrosis in Diabetic Mice Via the TGF-β1/Smad Pathway
Zhengjie Chen & Liangyu Zheng & Gang Chen
Cardiovascular Drugs and TherapyAccepted: 21 December 2021
https://link.springer.com/content/pd...21-07307-7.pdf
Purpose Diabetic cardiomyopathy (DM) is the cause of late cardiac dysfunction in diabetic patients. Myocardial fibrosis is the main pathological mechanism, and it is associated with transforming growth factor-β1(TGF-β1) expression up-regulation. 2- Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that can effectively improve myocardial cell energy metabolism and cardiac function. Here, we evaluated the protective effect of 2-AG on diabetic cardiomyopathy. Methods Male C57BL/6 mice were injected with 2-AG intraperitoneally for 4 weeks (10 micro g/kg/day) after 12 weeks of diabetic modeling. After 4 weeks, heart function was evaluated by echocardiography. Heart structure was assessed by hematoxylin and eosin staining. Cardiac fibrosis was analyzed using immunohistochemistry, Sirius red stain, and western blot. Results After modeling in diabetic mice, cardiac ultrasonography showed decreased cardiac function and pathological findings showed myocardial fibrosis. 2-AG could effectively inhibit the up-regulation of TGF-β1 and Smad2/3, reduce myocardial fibrosis, and ultimately improve cardiac function in diabetic mice. Conclusion 2-AG reduces cardiac fibrosis via the TGF-β1/Smad2/3 pathway and is a potential pathway for the treatment of cardiac dysfunction in diabetic mice. .

Are recreational cannabis laws associated with declining medical cannabis program enrollment in the U.S.? An analysis of cardholder enrollment and demographic characteristics from 2013 to 2020
Sarah A.Okey, Saul A. Castro, Jack T. Waddell, Connor B. Jones, Austin J. Blake, Holly P. O' Rourke, Mary C. Davis, Madeline H. Meier
Int J Drug Policy 2021 Nov 21;100:103531.
10.1016/j.drugpo.2021.103531
https://www.sciencedirect.com/scienc...497?via%3Dihub
Background: There is speculation that enrollment in U.S. state medical cannabis programs differs depending on whether adult recreational cannabis use is legal. If true, this could have implications for public health and policy.
Methods: Using medical cannabis registry data from U.S. states with a mandatory registry between 2013 and 2020 (N = 23 states), this study examined time-trends in medical cannabis cardholder enrollment and tested whether enrollment trends differed depending on adult recreational cannabis legalization.
Results: Multilevel models showed that time-trends for registered active medical cardholders differed during years when adult recreational cannabis use was legal versus not legal (time*recreational cannabis law interaction: b = -0.004, p < 0.01, 95% CI = -0.005, -0.003). The population prevalence of registered active medical cardholders increased over time in years when recreational cannabis was not legal (i.e., medical-only years; b = 0.004, p < 0.001, 95% CI = 0.003, 0.004, corresponding to an increase of 380 cardholders per 100,000 people per year), and decreased in years when recreational cannabis was legal (i.e., recreational years; b = -0.001, p < 0.001, 95% CI = -0.002, -0.001, corresponding to a decrease of 100 cardholders per 100,000 people per year). Time-trends were similar for each sex (male, female) and age group (18-30/35, 30/35+), with each cardholder group showing increases in medical-only years and decreases in recreational years. In medical-only years, there were no differences in enrollment time-trends across sex, but older cardholder (30/35+) enrollment increased at a faster rate than younger cardholder enrollment (18-30/35) (F = 16.199, p < 0.001). In recreational years, male cardholder enrollment decreased at a faster rate than female cardholder enrollment (F = 7.347, p < 0.01), but there was no difference in trends across age. Three states, all with medical-only years, provided data on ethnicity/race. Results showed significant increases from 2016 to 2020 in enrollment of White, African-American, and Hispanic individuals.
Conclusions: Findings suggest that recreational cannabis legalization is associated with decreasing enrollment in medical cannabis programs, particularly for males.
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Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?
Lachenmeier DW, Habel S, Fischer B*et al.*
F1000Research*2020,*8:1394
DOI: 10.12688/f1000research.19931.2
Cannabidiol (CBD)-containing products are widely marketed as over the counter products, mostly as food supplements, to avoid the strict rules of medicinal products. Side-effects reported in anecdotal consumer reports or during clinical studies were first assumed to be due to hydrolytic conversion of CBD to psychotropic ?9-tetrahydrocannabinol (THC) in the stomach after oral consumption. However, research of pure CBD solutions stored in simulated gastric juice or subjected to various storage conditions such as heat and light with specific liquid chromatographic/tandem mass spectrometric (LC/MS/MS) and ultra-high pressure liquid chromatographic/quadrupole time-of-flight mass spectrometric (UPLC-QTOF) analyses was unable to confirm THC formation. Another
hypothesis for the side-effects of CBD products may be residual THC concentrations in the products as contamination, because most of them are based on crude hemp extracts containing the full spectrum of cannabinoids besides CBD. Analyses of 67 food products of the German market (mostly CBD oils) confirmed this hypothesis: 17 products (25%) contained THC above the lowest observed adverse effects level (2.5 mg/day). Inversely, CBD was present in the products below the no observed adverse effect level. Hence, it may be assumed that the adverse effects of some commercial CBD products are based on a low-dose effect of THC and not due to effects of CBD itself. The safety, efficacy and purity of commercial CBD products is highly questionable, and all of the products in our sample collection showed various non-conformities to European food law such as unsafe THC levels, full-spectrum hemp extracts as non-approved novel food ingredients, non-approved health claims, and deficits in mandatory food labelling requirements. In view of the growing market for such lifestyle products, the effectiveness of the instrument of food business operators' own responsibility for product safety must obviously be challenged.

Are vaporizers a lower-risk alternative to smoking cannabis?
Michael Chaiton, Anasua Kundu, Sergio Rueda , Patricia Di Ciano
Can J Public Health. 2021 Aug 26.
doi: 10.17269/s41997-021-00565-w.
Cannabis use is associated with various adverse physical and mental health outcomes as well as increased risk of motor vehicle collision. Many organizations and the "Lower-Risk Cannabis Use Guidelines" have recommended to use cannabis vaporizers instead of smoking to reduce the associated health risk. This commentary draws attention to the present evidence regarding harm reduction potential of cannabis vaping. Cannabis vaporizer use can reduce the emission of carbon monoxide, chronic respiratory symptoms, and exposure to several toxins while producing similar subjective effects and blood THC concentration compared with smoking cannabis, holding potential for harm reduction among habitual cannabis smokers. However, new cannabis users, regardless of method of administration of cannabis, may experience intense subjective effects and cognitive impairment with increased susceptibility to dependence. Hence, policy makers should consider limiting access to cannabis among young people and adopting strategies to reduce impaired driving under influence of cannabis. Future research should focus on impact of switching from cannabis smoking to dried herb vaping using cannabis vaporizers among chronic cannabis smokers, and long-term outcomes of medical cannabis vaping, and further explore association of vaping-associated lung injury with THC-containing e-liquids.
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Arrhythmic Effects of Cannabis in Ischemic Heart Disease

Leah Gillett, Cecelia Johnson-Sasso, Brian Miller, Courtney Shakowski, Lori A. Walker, and Christine Tompkins,
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0188
Rationale: Cannabis use is increasing worldwide, especially among older individuals at risk for chronic ischemic heart disease (IHD). However, little is known about the arrhythmic effects of cannabis use in IHD. Accordingly,
we prospectively assessed the relationship between cannabis use, heart rate (HR), and arrhythmias in healthy age-matched controls and subjects with IHD.
Methods: Healthy controls (n = 37, 57% men) and subjects with IHD (myocardial infarction ‡ 3 months ago; n = 24, 58% men) who used cannabis wore a Zio_ (iRhythm Technologies) monitor for 14 days. Noncannabis
using ischemic subjects (n = 35, 51% males) wore Zio monitors for standard clinical indications. Baseline HR was compared with average HR measured for 4 h following consumption and changes in HR and frequency of arrhythmias were correlated with cannabis use.
Results: In controls, HR increased 20 min (4.99 – 6.7 bpm, p = 0.08) after use, then declined 4 h following use (_7.4 – 7.7, p < 0.001). Conversely, subjects with IHD showed minimal HR increase (1.6 – 3.9 bpm) and blunted
HR decline (_3.4 – 5.6 bpm, p < 0.001). Supraventricular tachycardia (SVT) (29.7% vs. 58.3%; p = 0.04) and nonsustained ventricular tachycardia (NSVT) (5.6% vs. 47.8%, p = 0.01) were the most frequently occurring arrhythmias in
controls and IHD subjects, respectively. Incidence of SVT decreased as cannabis use increased in both groups. Conversely, NSVT tended to increase with increased use in controls, and was significantly more prevalent in
IHD. However, overall arrhythmia burden did not differ between cannabis users and nonusers with IHD.
Conclusion: Our findings demonstrate that chronic cannabis use is associated with only mild HR changes, which are blunted in IHD. In addition, our data suggest that among cannabis users, arrhythmias are more frequent in IHD subjects that in healthy subjects .

Ask Your Provider About Cannabis: Increasing Nurse Practitioner Knowledge and Confidence
Tracy A. Klein1, and Ross Bindler
Cannabis and Cannabinoid Research
Volume X, Number X, 2021
DOI: 10.1089/can.2021.0061
Introduction: Nurse practitioners (NPs) are authorizing providers for medical cannabis in many states, and may serve as a primary care clinician. We report findings from a nationally distributed 2-h continuing education (CE) module aimed to improve knowledge, confidence, and willingness to communicate with patients about cannabis.
Methods: Data were electronically obtained from the CE platform pre- and post-test (n = 289) and a follow-up survey sent within 3 months postcompletion (n = 184, 63%). Pre- and post-testing assessed cannabis pharmacodynamics, law, evidence-based use, metabolism, pharmacokinetics, laboratory testing, adverse reactions, and drug–drug interactions. The subsequent survey asked about changes in practice behavior, including willingness and self-identified recommendations for use. Quantitative and qualitative descriptive analysis and repeatedmeasures analysis of variance were used to analyze CE impact.
Results: Significant improvement in scores was noted from pretest to post-test for all content with a mean improvement of 39.3% (95% CI: 30.6–47.9%). The greatest increases were for metabolism, pharmacokinetics, and drug–drug
interaction content. At follow-up, 52.2% reported that the CE changed their attitudes about cannabis and although 86% had rarely or never applied it yet in practice, 92% reported they were now likely to inquire about cannabis use in
their patients and 84% were likely to counsel patients about it. Although self-identified recommendations overlapped by conditions, some were unique to CBD (complex regional pain syndrome, migraine, mood disorder, smoking cessation) and THC products (appetite, cachexia, depression, fibromyalgia, HIV, seizure disorder, stress, and weight loss). Pain was the most common condition for recommendation of both CBD and THC, followed by anxiety and arthritis.
Conclusions: NPs gained key knowledge about cannabis, which may impact patient care and prescribing practices. The educational module resulted in more willingness to discuss and counsel patients about cannabis, even if practitioner attitudes did not change.

Assessing Health Care Providers’ Knowledge of Medical Cannabis
Daniel J. Kruger, Majd A. Mokbel, Daniel J. Clauw, and Kevin F. Boehnke
Cannabis and Cannabinoid Research
Volume X, Number X, 2021
DOI: 10.1089/can.2021.0032
https://www.liebertpub.com/doi/pdf/1.../can.2021.0032
Introduction: Many health care providers would benefit from greater knowledge and awareness of medical cannabis, even if they choose not to integrate it into their medical practice. Unfortunately, health care providers
generally report low knowledge of medical cannabis and cite this lack of knowledge as a barrier to making patient recommendations. It is important to understand health care providers’ medical cannabis knowledge and its correlates. However, few studies have rigorously assessed clinically relevant cannabis-related knowledge, instead typically focusing on attitudes toward cannabis and perceived knowledge.
Methods: Physicians in a university-affiliated health system completed an anonymous online survey. The survey assessed participants’ basic demographics and medical experience, experiences with cannabis education, beliefs about their knowledge of and competency regarding medical cannabis, and knowledge of medical cannabis in relation to the current scientific evidence.
Results: The average level of medical cannabis knowledge was 58% correct, with scores ranging from 39% to 78% correct. Perceived cannabis knowledge predicted actual knowledge, and those who pursued self-initiated study or attended a lecture on medical cannabis had higher knowledge levels.
Conclusion: Levels of factual knowledge about medical cannabis among physicians were moderate. Our results highlight the mismatch between physician knowledge and cannabis policy. We offer our brief, 10-min assessment as a baseline for characterizing cannabis knowledge, acknowledging that the content and interpretation may change as knowledge advances.

Assessment of Worker Exposure to Occupational Organic Dust in a Hemp Processing Facility.
Gardner, M., Reed, S., & Davidson, M.
Annals of Work Exposures and Health.(2020).
doi:10.1093/annweh/wxaa065
The cultivation and processing of industrial hemp, Cannabis sativa L., is a developing industry in Australia. Exposure to hemp dust is demonstrated as producing reactive and respiratory health effects, potentially causing permanent lung disease or damage. The aim of this study was to assess the airborne organic dust concentrations generated in an Australian hemp processing facility. Personal sampling, in the breathing zone of exposed workers was undertaken for exposure to respirable dust, along with parallel static sampling for airborne concentrations of inhalable and respirable dust fractions. Both static and personal sampling showed that respirable dust concentrations (mg m?3) exceeded the Australian Institute of Occupational Hygienists (AIOH) recommended maximum exposure limit of 1 mg m?3 (respirable fraction) for dusts not otherwise specified, with mean exposures (mg m?3) of M = 1.33, standard deviation (SD) = 1.09 (range 0.07–3.67 mg m?3) and M = 4.49, SD = 4.49 (range 0.77–11.08 mg m?3). The results of the investigation indicate that workers in the hemp processing industry are at risk of developing permanent and disabling respiratory disease due to high dust exposure. There is no Australian occupational exposure limit specifically for hemp dust. It is recommended further research is needed and industry-specific guidance material or model code of practice developed to effectively control exposures.

Association Between Marijuana Exposure and Pulmonary Function Over 20 Years.
Pletcher, M. J., Vittinghoff, E., Kalhan, R., Richman, J., Safford, M., Sidney, S., … Kertesz, S.
JAMA, 307(2), 173. (2012).
doi:10.1001/jama.2011.1961
Context Marijuana smoke contains many of the same constituents as tobacco smoke, but whether it has similar adverse effects on pulmonary function is unclear. Objective To analyze associations between marijuana (both current and lifetime exposure) and pulmonary function. Design, Setting, and Participants The Coronary Artery Risk Development in Young Adults (CARDIA) study, a longitudinal study collecting repeated measurements of pulmonary function and smoking over 20 years (March 26, 1985-August 19, 2006) in a cohort of 5115 men and women in 4 US cities. Mixed linear modeling was used to account for individual age-based trajectories of pulmonary function and other covariates including tobacco use, which was analyzed in parallel as a positive control. Lifetime exposure to marijuana joints was expressed in joint-years, with 1 joint-year of exposure equivalent to smoking 365 joints or filled pipe bowls. Main Outcome Measures Forced expiratory volume in the first second of expiration (FEV1) and forced vital capacity (FVC). Results Marijuana exposure was nearly as common as tobacco exposure but was mostly light (median, 2-3 episodes per month). Tobacco exposure, both current and lifetime, was linearly associated with lower FEV1 and FVC. In contrast, the association between marijuana exposure and pulmonary function was nonlinear (P.001): at low levels of exposure, FEV1 increased by 13 mL/joint-year (95% CI, 6.4 to 20; P.001) and FVC by 20 mL/joint-year (95% CI, 12 to 27; P.001), but at higher levels of exposure, these associations leveled or even reversed. The slope for FEV1 was ?2.2 mL/joint-year (95% CI, ?4.6 to 0.3; P=.08) at more than 10 joint-years and ?3.2 mL per marijuana smoking episode/mo (95% CI, ?5.8 to ?0.6; P=.02) at more than 20 episodes/mo. With very heavy marijuana use, the net association with FEV1 was not significantly different from baseline, and the net association with FVC remained significantly greater than baseline (eg, at 20 joint-years, 76 mL [95% CI, 34 to 117]; P.001). Conclusion Occasional and low cumulative marijuana use was not associated with adverse effects on pulmonary function. JA

Association between marijuana use and electrocardiographic abnormalities by middle age The Coronary Artery Risk Development in Young Adults (CARDIA) Study.
Jakob, J., Stalder, O., Syrogiannouli, L., Pletcher, M. J., Vittinghoff, E., Ning, H., … Auer, R.
Addiction.(2020).
doi:10.1111/add.15188
Aims: To evaluate the prevalence of electrocardiogram (ECG) abnormalities in marijuana users as an indirect measure of subclinical cardiovascular disease (CVD).
Design: Longitudinal and cross-sectional secondary data analysis from the CARDIA (Coronary Artery Risk Development in Young Adults) study.
Setting: 4 communities in the United States.
Participants: A total of 2,585 participants from the 5,115 black and white men and women recruited at age 18 to 30 years in 1985 to 1986 in CARDIA.
Measurements: ECG abnormalities coded as minor and major abnormalities with the Minnesota code of electrocardiographic findings at Year 20. Self-reported current (past 30 days) and computed cumulative lifetime marijuana use (one “marijuana-year” corresponds to 365 days of use) through assessments every 2-5 years. We fitted logistic regression models adjusting for sex, race, center, education, age, tobacco smoking, physical activity, alcohol use, and body mass index.
Findings: Among the 2,585 participants with an ECG at Year 20, mean age was 46, 57% were women, 45% were black. 83% had past exposure to marijuana and 11% were using marijuana currently. One hundred and seventy-three participants (7%) had major abnormalities and 944 (37%) had minor abnormalities. Comparing current with never use in
multivariable-adjusted models, the OR for major ECG abnormalities was 0.60 (95% CI: 0.32 to 1.15) and for minor ECG abnormalities 1.21 (95% CI: 0.87 to 1.68). Results did not change after stratifying by sex and race. Cumulative marijuana use was not associated with ECG abnormalities.
Conclusion: In a middle-aged US population, lifetime cumulative and occasional current marijuana use were not associated with increases in electrocardiogram abnormalities. This adds to the growing body of evidence that occasional marijuana use and cardiovascular disease events and markers of subclinical atherosclerosis are not associated.

Association of a Variant of CNR1 Gene Encoding Cannabinoid Receptor 1 With Gilles de la Tourette Syndrome.
Szejko, N., Fichna, J. P., Safranow, K., Dziuba, T., ?ekanowski, C., & Janik, P.
Frontiers in Genetics, 11.(2020).
doi:10.3389/fgene.2020.00125
Background: Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder of unknown etiology, although a major role of genetic factors has been established. Cannabis-based medicines may alleviate GTS-associated tics and variants of CNR1 gene encoding central cannabinoid receptor (CB1) are believed to be a risk factor for the development of some neurodevelopmental diseases. Our aim was to test the association of selected CNR1 gene variants with GTS. Material and Methods: The cohort of GTS cases comprised 262 unrelated patients aged 3–53 years (mean age: 18.3 ± 9.1 years; 204 males (77.9%), 126 (48.1%) adults defined as ?18 years). As a control group we enrolled 279 unrelated, ethnically and gender matched individuals with no diagnosed mental, neurological or general disorder, aged 13– 54 years (mean age: 22.5 ± 3.0 years; 200 males, (74.1%). Both study and control groups were selected from Polish population, which is ethnically homogenous subgroup of Caucasian population. Four single nucleotide polymorphisms (SNPs) in CNR1 were selected: rs2023239, rs2180619, rs806379, and rs1049353 based on minor allele frequency in general population >15%. These variants were genotyped using a realtime quantitative polymerase chain reaction system (TaqMan SNP genotyping assay). Results: We found significant association of GTS clinical phenotype with rs2023239 variant. Minor allele C and CT+CC genotypes were found significantly more often in GTS patients compared to controls (17.4 vs 11.1%, p=0.003 and 32.8 vs 20.4%, p=0.001, respectively), and the difference remained significant after correction for multiple testing. C allele of rs2023239 polymorphism of the CNR1 gene was associated with the occurrence of tics. There were no statistically significant associations for rs806379, rs1049353 or rs2180619 variants. Conclusion: Our findings suggest that C allele of rs2023239 polymorphism of the CNR1 gene is a risk factor of GTS in Polish population. The variant can be potentially associated with abnormal endocannabinoid transmission, which is suspected to be one of the causes of GTS.

Association of cannabinoid receptor modulation with normal and abnormal skeletal remodelling: A systematic review and meta-analysis of in vitro,in vivo and human studies
Antonia Sophocleous a Michael Yiallourides b Feier Zeng b Pantelis Pantelas b Eleni Stylianou a Boya Li b Giovana Carrasco b Aymen I.Idris
Pharmacological Research Volume 175, January 2022, 105928
DOI: 10.1016/j.phrs.2021.105928
https://www.sciencedirect.com/scienc...120?via%3Dihub
To address the inconsistent findings from studies that used different models to explore the role of classical cannabinoid type 1 (CB1) and 2 (CB2) receptors in skeletal remodelling, we searched Medline, Web of Science and Embase for relevant studies from inception to June 23, 2020. We identified 38 in vitro, 34 in vivo and 9 human studies. A meta-analysis of in vitro studies showed that exposure to the inverse-agonists AM251 (mean difference [MD]:−26.75, 95% confidence interval [CI]:−45.36,−8.14, p = 0.005), AM630 (standardised[std.] MD:−3.11, CI:−5.26,−0.97, p = 0.004;SR144528, std.MD:−4.88, CI −7.58,−2.18, p = 0.0004) and CBD (std.MD:−1.39, CI −2.64,−0.14, p = 0.03) is associated with reduced osteoclastogenesis, whereas theendocannabinoid 2-AG (std.MD:2.00, CI:0.11–3.89, p = 0.04) and CB2-selective agonist HU308 (MD:19.38, CI:11.75–27.01, p < 0.00001) were stimulatory. HU308 also enhanced osteoblast differentiation (std.MD:2.22, CI:0.95–3.50, p = 0.0006) and activity (std.MD:2.97, CI:1.22–4.71, p = 0.0008). In models of bone loss, CB1/2 deficiency enhanced peak bone volume (std.MD:3.70, CI:1.77–5.63, p = 0.0002) but reduced bone formation (std.MD:−0.54, CI:−0.90,−0.17, p = 0.004) in female mice. In male rats, CB1/2 deficiency (std.MD:2.31, CI:0.30–4.33, p = 0.02) and AM251 or CBD treatments (std.MD:2.19, CI:0.46–3.93, p = 0.01) enhanced bone volume. CB1/2 deficiency (std.MD:9.78, CI:4.96–14.61, p < 0.0001) and AM251 or AM630 treatments (std.MD:28.19, CI:19.13–37.25, p < 0.0001) were associated with osteoprotection. The CB2-selective agonists JWH133 and 4Q3C enhanced bone volume in arthritic rodents (std.MD:14.45, CI:2.08–26.81, p = 0.02). In human, CB2 SNPs (AA:rs2501431, MD:−0.28, CI:−0.55,−0.01, p = 0.04; CC:rs2501432, MD:−0.29, CI:−0.56,−0.02, p = 0.03) were associated with reduced bone mineral density, however the association of Marijuana use remains unclear. Thus, CB1/2 modulation is associated with altered bone metabolism, however findings are confounded by low study number and heterogenicity of models.
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Association of cannabis with glutamatergic levels in patients with early psychosis: Evidence for altered volume striatal glutamate relationships in patients with a history of cannabis use in early psychosis.
Sami, M., Worker, A., Colizzi, M., Annibale, L., Das, D., … Bhattacharyya, S.
Translational Psychiatry, 10(1). (2020).
doi:10.1038/s41398-020-0790-1
The associative striatum, an established substrate in psychosis, receives widespread glutamatergic projections. We sought to see if glutamatergic indices are altered between early psychosis patients with and without a history of cannabis use and characterise the relationship to grey matter. 92 participants were scanned: Early Psychosis with a history of cannabis use (EPC = 29); Early Psychosis with minimal cannabis use (EPMC = 25); Controls with a history of cannabis use (HCC = 16) and Controls with minimal use (HCMC = 22). Whole brain T1 weighted MR images and localised proton MR spectra were acquired from head of caudate, anterior cingulate and hippocampus. We examined relationships in regions with known high cannabinoid 1 receptor (CB1R) expression (grey matter, cortex, hippocampus, amygdala) and low expression (white matter, ventricles, brainstem) to caudate Glutamine+Glutamate (Glx). Patients were well matched in symptoms, function and medication. There was no significant group difference in Glx in any region. In EPC grey matter volume explained 31.9% of the variance of caudate Glx (p = 0.003) and amygdala volume explained 36.9% (p = 0.001) of caudate Glx. There was no significant relationship in EPMC. The EPC vs EPMC interaction was significant (p = 0.042). There was no such relationship in control regions. These results are the first to demonstrate association of grey matter volume and striatal glutamate in the EPC group. This may suggest a history of cannabis use leads to a conformational change in distal CB1 rich grey matter regions to influence striatal glutamatergic levels or that such connectivity predisposes to heavy cannabis use

Association of Naturalistic Administration of Cannabis Flower and Concentrates With Intoxication and Impairment.
Bidwell, L. C., Ellingson, J. M., Karoly, H. C., YorkWilliams, S. L., Hitchcock, L. N., Tracy, B. L., … Hutchison, K. E. ).
JAMA Psychiatry. (2020) doi:10.1001/jamapsychiatry.2020.0927
IMPORTANCE The rapidly growing legal cannabis market includes new and highly potent products, the effects of which, to our knowledge, have not previously been examined in biobehavioral research studies because of federal restrictions on cannabis research.
OBJECTIVE To use federally compatible, observational methods to study high-?9- tetrahydrocannabinol (THC) legal market forms of cannabis. DESIGN, SETTING, AND PARTICIPANTS In this cohort study with a between-groups design that was conducted in a community and university setting, cannabis flower users and concentrate users were randomly assigned to higher- vs lower-THC products within user groups. Participants completed a baseline and an experimental mobile laboratory assessment that included 3 points: before, immediately after, and 1 hour after ad libitum legal market flower and concentrate use. Of the 133 individuals enrolled and assessed, 55 regular flower cannabis users (41.4%) and 66 regular concentrate cannabis users (49.6%) complied with the study’s cannabis use instructions and had complete data across primary outcomes.
EXPOSURES Flower users were randomly assigned to use either 16% or 24% THC flower and concentrate users were randomly assigned to use either 70% or 90% THC concentrate that they purchased from a dispensary. MAIN OUTCOMES AND MEASURES Primary outcome measures included plasma cannabinoids, subjective drug intoxication, and neurobehavioral tasks testing attention, memory, inhibitory control, and balance.
RESULTS A total of 121 participants completed the study for analysis: 55 flower users (mean [SD] age, 28.8 [8.1] years; 25 women [46%]) and 66 concentrate users (mean [SD] age, 28.3 [10.4] years; 30 women [45%]). Concentrate users compared with flower users exhibited higher plasma THC levels and 11-hydroxy?9 -THC (THC’s active metabolite) across all points. After ad libitum cannabis administration, mean plasma THC levels were 1016 (SE = 1380) ?g/mL in concentrate users (to convert to millimoles per liter, multiply by 3.18) and 455 (SE = 503) ?g/mL in flower users. Most neurobehavioral measures were not altered by short-term cannabis consumption. However, delayed verbal memory (F1,203 = 32.31; P < .001) and balance function (F1,203 = 18.88; P < .001) were impaired after use. Differing outcomes for the type of product (flower vs concentrate) or potency within products were not observed.
CONCLUSIONS AND RELEVANCE This study provides information about the association of pharmacological and neurobehavioral outcomes with legal market cannabis. Short-term use of concentrates was associated with higher levels of THC exposure. Across forms of cannabis and potencies, users’ domains of verbal memory and proprioception-focused postural stability were primarily associated with THC administration.

Attenuation of Oxidative Stress by Cannabinoids and Cannabis Extracts in Differentiated Neuronal Cells
Aruna Raja, Soha Ahmadi, Fernanda de Costa, Nan Li *and Kagan Kerman
Pharmaceuticals*2020, 13, 328;
doi:10.3390/ph13110328
In this proof-of-concept study, the antioxidant activity of phytocannabinoids, namely cannabidiol (CBD) and ?9- tetrahydrocannabinol (THC), were investigated using an in vitro system of differentiated human neuronal SY-SH5Y cells. The oxidative stress was induced by hydrogen peroxide, as reactive oxygen species (ROS). Alzheimer’s disease (AD)-like pathological conditions were mimicked in vitro by treating the differentiated neuronal cells with amyloid-?1–42 (A?1–42) in the presence of Cu(II). We showed that THC had a high potency to combat oxidative stress in both in vitro models, while CBD did not show a remarkable antioxidant activity. The cannabis extracts also exhibited a significant antioxidant activity, which depended on the ratio of the THC and CBD. However, our results did not suggest any antagonist effect of the CBD on the antioxidant activity of THC. The effect of cannabis extracts on the cell viability of differentiated human neuronal SY-SH5Y cells was also investigated, which emphasized the differences between the bioactivity of cannabis extracts due to their composition. Our preliminary results demonstrated that cannabis extracts and phytocannabinoids have a promising potential as antioxidants, which can be further investigated to develop novel pharmaceuticals targeting oxidative stress therapy.

Behavioural and pharmacological effects of cannabidiol (CBD) and the cannabidiol analogue KLS-13019 in mouse models of pain and reinforcement
Jeffery D. Foss, Daniel J. Farkas, Lana M. Huynh, William A. Kinney, Douglas E. Brenneman, Sara Jane Ward
British Journal of Pharmacology 06-Apr-2021
DOI: 10.1111/bph.15486
Background and Purpose
Cannabidiol (CBD) is a non-euphorigenic component of Cannabis sativa that prevents the development of paclitaxel-induced mechanical sensitivity in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). We recently reported that the CBD structural analogue KLS-13019 shows efficacy in an in vitro model of CIPN. The present study was to characterize the behavioural effects of KLS-13019 compared to CBD and morphine in mouse models of CIPN, nociceptive pain and reinforcement.
Experimental Approach
Prevention or reversal of paclitaxel-induced mechanical sensitivity were assessed following intraperitoneal or oral administration of CBD, KLS-13019 or morphine. Antinociceptive activity using acetic acid-induced stretching and hot plate assay, anti-reinforcing effects on palatable food or morphine self-administration and binding to human opioid receptors were also determined.
Key Results
Like CBD, KLS-13019 prevented the development of mechanical sensitivity associated with paclitaxel administration. In contrast to CBD, KLS-13019 was also effective at reversing established mechanical sensitivity. KLS-13019 significantly attenuated acetic acid-induced stretching and produced modest effects in the hot plate assay. KLS-13019 was devoid of activity at μ-, δ- or κ-opioid receptors. Lastly, KLS-13019, but not CBD, attenuated the reinforcing effects of palatable food or morphine.
Conclusions and Implications
KLS-13019 like CBD, prevented the development of CIPN, while KLS-13019 uniquely attenuated established CIPN. Because KLS-13019 binds to fewer biological targets, this will help to identifying molecular mechanisms shared by these two compounds and those unique to KLS-13019. Lastly, KLS-13019 may possess the ability to attenuate reinforced behaviour, an effect not observed in the present study with CBD.

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease.
Borrelli, F., Fasolino, I., Romano, B., Capasso, R., Maiello, F., Coppola, D., … Izzo, A. A.
Biochemical Pharmacology, 85(9), 1306–1316.(2013).
doi:10.1016/j.bcp.2013.01.017
Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people in industrialized countries. Anecdotal and scientific evidence suggests that Cannabis use may have a positive impact in IBD patients. Here, we investigated the effect of cannabigerol (CBG), a nonpsychotropic Cannabis-derived cannabinoid, in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulphonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and immunohistochemistry; interleukin-1b, interleukin-10 and interferon-g levels by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the effect of CBG on nitric oxide production and oxidative stress, respectively. CBG reduced colon weight/colon length ratio, myeloperoxidase activity, and iNOS expression, increased SOD activity and normalized interleukin- 1b, interleukin-10 and interferon-g changes associated to DNBS administration. In macrophages, CBG reduced nitric oxide production and iNOS protein (but not mRNA) expression. Rimonabant (a CB1 receptor antagonist) did not change the effect of CBG on nitric oxide production, while SR144528 (a CB2 receptor antagonist) further increased the inhibitory effect of CBG on nitric oxide production. In conclusion, CBG attenuated murine colitis, reduced nitric oxide production in macrophages (effect being modulated by the CB2 receptor) and reduced ROS formation in intestinal epithelial cells. CBG could be considered for clinical experimentation in IBD patients.

Beneficial Effects of Cannabis on Blood Brain Barrier Function in HIV.
Ellis, R. J., Peterson, S., Cherner, M., Morgan, E., Schrier, R., Tang, B., … Iudicello, J.
Clinical Infectious Diseases. (2020).
doi:10.1093/cid/ciaa437
Background. HIV infection leads to blood-brain barrier (BBB) dysfunction that does not resolve despite viral suppression on antiretroviral therapy and is associated with adverse clinical outcomes. In preclinical models, cannabis restores BBB integrity.
Methods. We studied people with HIV (PWH) and HIV- individuals who had used cannabis recently. We assessed two biomarkers of BBB permeability: the cerebrospinal fluid [CSF]-to-serum albumin ratio [CSAR], and CSF levels of soluble urokinase plasminogen activator receptor [suPAR], a receptor for uPA, a matrix-degrading proteolytic enzyme that disrupts the BBB. A composite index of the BBB markers was created using principal components analysis. Neural injury was assessed using neurofilament light (NFL) in CSF by immunoassay.
Results. Participants were 45 PWH and 30 HIV- individuals of similar age and ethnicity. Among PWH, higher CSF suPAR levels correlated with higher CSAR values (r=0.47; p,0.001). PWH had higher (more abnormal) BBB index values than HIV- individuals (mean +/- SD 0.361 +/-1.20 versus -0.501 +/- 1.11; p=0.0214). HIV serostatus interacted with cannabis use frequency such that more frequent use of cannabis was associated with lower BBB index values in PWH but not in HIV-. Worse BBB Index values was associated with higher NFL in CSF (r=0.380,p=0.0169).
Conclusions. Cannabis may have a beneficial impact on HIV-associated BBB injury. Since BBB disruption may permit increased entry of toxins such as microbial antigens and inflammatory mediators, with consequent CNS injury, these results support a potential therapeutic role of cannabis among PWH and may have important treatment implications for ART effectiveness and toxicity.

Beta-Caryophyllene, a CB2-Receptor-Selective Phytocannabinoid, Suppresses Mechanical Allodynia in a Mouse Model of Antiretroviral-Induced Neuropathic Pain.
Aly, E., Khajah, M. A., & Masocha, W.
Molecules, 25(1), 106. (2019).
doi:10.3390/molecules25010106
Neuropathic pain associated with nucleoside reverse transcriptase inhibitors (NRTIs), therapeutic agents for human immunodeficiency virus (HIV), responds poorly to available drugs. Smoked cannabis was reported to relieve HIV-associated neuropathic pain in clinical trials. Some constituents of cannabis (Cannabis sativa) activate cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors. However, activation of the CB1 receptor is associated with side effects such as psychosis and physical dependence. Therefore, we investigated the effect of ?-caryophyllene (BCP), a CB2-selective phytocannabinoid, in a model of NRTI-induced neuropathic pain. Female BALB/c mice treated with 20 -30 -dideoxycytidine (ddC, zalcitabine), a NRTI, for 5 days developed mechanical allodynia, which was prevented by cotreatment with BCP, minocycline or pentoxifylline. A CB2 receptor antagonist (AM 630), but not a CB1 receptor antagonist (AM 251), antagonized BCP attenuation of established ddC-induced mechanical allodynia. ?-Caryophyllene prevented the ddC-induced increase in cytokine (interleukin 1 beta, tumor necrosis factor alpha and interferon gamma) transcripts in the paw skin and brain, as well as the phosphorylation level of Erk1/2 in the brain. In conclusion, BCP prevents NRTI-induced mechanical allodynia, possibly via reducing the inflammatory response, and attenuates mechanical allodynia through CB2 receptor activation. Therefore, BCP could be useful for prevention and treatment of antiretrovi

Beta-caryophyllene attenuates short-term recurrent seizure activity and blood-brain-barrier breakdown after pilocarpine-induced status epilepticus in rats
Michele Pereira Mallmann, Fernanda Kulinski Mello, Bruna Neuberger, Karine Gabriela da Costa Sobral, Michele Rechia Fighera, Luiz Fernando Freire Royes, Ana Flávia Furian, Mauro Schneider Oliveira
Brain Res. 2022 Mar 14;1784:147883.
doi: 10.1016/j.brainres.2022.147883
Background: Status epilepticus (SE) is a neurological life-threatening condition, resulting from the failure of the mechanisms responsible for seizure termination. SE is often pharmacoresistant and associated with significant morbidity and mortality. Hence, ceasing or attenuating SE and its consequences is of fundamental importance. Beta-caryophyllene is a functional CB2 receptor agonist and exhibit a good safety profile. Besides, it displays beneficial effects in several experimental conditions, including neuroprotective activity. In the present study we aimed to investigate the effects of beta-caryophyllene on pilocarpine-induced SE.
Methods: Wistar rats were submitted to pilocarpine-induced SE and monitored for 24 h by video and EEG for short-term recurrence of seizure activity (i.e. seizures occurring within 24 h after termination of SE). Rats received beta-caryophyllene (100 mg/kg, ip) at 1, 8- and 16-hours after SE. Twenty-four hours after SE we evaluated sensorimotor response, neuronal damage (fluoro jade C staining) and serum albumin infiltration into brain parenchyma.
Results: Beta-caryophyllene-treated animals presented fewer short-term recurrent seizures than vehicle-treated counterparts, suggesting an anticonvulsant effect after SE. Behavioral recovery from SE and the number of fluoro jade C positive cells in the hippocampus and thalamus were not modified by beta-caryophyllene. Treatment with beta-caryophyllene attenuated the SE-induced increase of albumin immunoreactivity in the hippocampus, indicating a protective effect against blood-brain-barrier breakdown.
Conclusions: Given the inherent difficulties in the treatment of SE and its consequences, present results suggest that beta-caryophyllene deserve further investigation as an adjuvant therapeutic strategy for SE.
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Beta-caryophyllene is a dietary cannabinoid.
Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, Altmann KH, Karsak M, Zimmer A.
PNAS July 1, 2008 105 (26) 9099-9104
doi: 10.1073/pnas.0803601105
The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Beyond Cannabis: Plants and the Endocannabinoid System
Ethan B. Russo
Trends in Pharmacological Sciences
DOI: 10.1016/j.tips.2016.04.005
Plants have been the predominant source of medicines throughout the vast majority of human history, and remain so today outside of industrialized societies. One of the most versatile in terms of its phytochemistry is cannabis, whose investigation has led directly to the discovery of a unique and widespread homeostatic physiological regulator, the endocannabinoid system. While it had been the conventional wisdom until recently that only cannabis harbored active agents affecting the endocannabinoid system, in recent decades the search has widened and identified numerous additional plants whose components stimulate, antagonize, or modulate different aspects of this system. These include common foodstuffs, herbs, spices, and more exotic ingredients: kava, chocolate, black pepper, and many others that are examined in this review

Bibliography: Cannabis Canadensis
Jeremy Burman
AHP 2007
https://ahp.apps01.yorku.ca/2007/07/...is-canadensis/

Bidirectional Longitudinal Associations Between Cannabis Use and Body Mass Index Among Adolescents.
Ross, J. M., Pacheco-Colón, I., Hawes, S. W., & Gonzalez, R.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2019.0091
Introduction: Literature on the association between cannabis use and body mass index (BMI) among adults suggests that greater cannabis use is associated with a lower BMI. However, results are mixed among adolescents, with both cross-sectional and longitudinal studies finding positive, negative, and nonsignificant associations between cannabis use and BMI. This longitudinal study aims to shed light on these associations by prospectively examining the associations between cannabis use and BMI across a 2-year window in a large sample of adolescent cannabis users. Methods: Participants were 401 adolescents ages 14–17 at baseline who were at risk for escalation in their use of cannabis. We conducted a parallel process latent growth curve model to examine associations between the cannabis use intercept, BMI intercept, cannabis use slope, and BMI slope. Results: Results showed that baseline BMI predicted a positive and significant association with cannabis use slope. In addition, there was a significant and negative correlation between the cannabis use slope and the BMI slope. These significant associations remained after controlling for relevant covariates. Conclusions: Results are consistent with the adult literature that reports a negative association between cannabis use and BMI. Future research should focus on uncovering the mechanisms that may drive the association between cannabis use and BMI.

Biological Activity of Cannabis sativa L. Extracts Critically Depends on Solvent Polarity and Decarboxylation
Guillermo Moreno-Sanz, Carlos Ferreiro Vera, Carolina Sá nchez-Carnerero,
Xavier Nadal Roura and Veró nica Sá nchez de Medina Baena
Separations 2020, 7(4), 56;
https://www.mdpi.com/2297-8739/7/4/56
DOI: 10.3390/separations7040056
Minor cannabinoid and non-cannabinoid molecules have been proposed to significantly contribute to the pharmacological profile of cannabis extracts. Phytoplant Research has developed highly productive cannabis cultivars with defined chemotypes, as well as proprietary methods for the extraction and purification of cannabinoids. Here, we investigate the effect of solvent
selection and decarboxylation on the composition and pharmacological activity of cannabis extracts. A library of forty cannabis extracts was generated from ten different cannabis cultivars registered by Phytoplant Research at the EU Community Plant Variety Office. Plant material was extracted using two different solvents, ethanol and hexane, and crude extracts were subsequently decarboxylated or not. Cannabinoid content in the resulting extracts was quantified, and biological activity was screened in vitro at three molecular targets involved in hypoxia and inflammation (NF-_ B, HIF-1_
and STAT3). Changes in transcriptional activation were strongly associated to solvent selection and decarboxylation. Two decarboxylated extracts prepared with hexane were the most potent at inhibiting NF-_ B transcription, while HIF-1_ activation was preferentially inhibited by ethanolic extracts, and decarboxylated extracts were generally more potent at inhibiting STAT3 induction. Our results indicate that solvent selection and proper decarboxylation represent key aspects of the standardized production of cannabis extracts with reproducible pharmacological activity.

Biological bases for a possible effect of cannabidiol in Parkinson’s disease.
Ferreira-Junior, N. C., Campos, A. C., Guimarães, F. S., Del-Bel, E., Zimmermann, P. M. da R., Brum Junior, L.,Zuardi, A. W.
Brazilian Journal of Psychiatry. (2019).
doi:10.1590/1516-4446-2019-0460
Objective: Current pharmacotherapy of Parkinson’s disease (PD) is palliative and unable to modify the progression of neurodegeneration. Treatments that can improve patients’ quality of life with fewer side effects are needed, but not yet available. Cannabidiol (CBD), the major non-psychotomimetic constituent of cannabis, has received considerable research attention in the last decade. In this context, we aimed to critically review the literature on potential therapeutic effects of CBD in PD and discuss clinical and preclinical evidence supporting the putative neuroprotective mechanisms of CBD.
Methods: We searched MEDLINE (via PubMed) for indexed articles published in English from inception to 2019. The following keywords were used: cannabis; cannabidiol and neuroprotection;
endocannabinoids and basal ganglia; Parkinson’s animal models; Parkinson’s history; Parkinson’s and cannabidiol.
Results: Few studies addressed the biological bases for the purported effects of CBD on PD. Six preclinical studies showed neuroprotective effects, while three targeted the antidyskinetic effects of
CBD. Three human studies have tested CBD in patients with PD: an open-label study, a case series, and a randomized controlled trial. These studies reported therapeutic effects of CBD on non-motor
symptoms.
Conclusions: Additional research is needed to elucidate the potential effectiveness of CBD in PD and the underlying mechanisms involved.

Biosynthesis of Endocannabinoids and Their Modes of Action in Neurodegenerative Diseases
MARIO van der STELT, HENRIK H. HANSEN, WOUTER B. VELDHUIS, PETER R. BAR, KLAAS NICOLAY, GERRIT A.VELDINK, JOHANNES F.G. VLIEGENTHART and HARALD S. HANSEN
Neurotoxicity Research, 2003, Vol. 5 (3). pp. 183-200
doi: 10.1007/BF03033139
https://dspace.library.uu.nl/bitstre...pdf?sequence=1
Endocannabinoids are thought to function as retrograde messengers, which modulate neurotransmitter release by activating presynaptic cannabinoid receptors. Anandamide and 2-arachidonoylglycerol (2-AG) are the two best studied endogenous lipids which can act as endocannabinoids. Together with the proteins responsible for their biosynthesis, inactivation and the cannabinoid receptors, these lipids constitute the endocannabinoid system. This system is proposed to be involved in various neurodegenerative diseases such as Parkinson’s and Huntington’s diseases as well as Multiple Sclerosis. It has been demonstrated that the endocannabinoid system can protect neurons against glutamate excitotoxicity and acute neuronal damage in both in vitro and in vivo models. In this paper we review the data concerning the involvement of the endocannabinoid system in neurodegenerative diseases in which neuronal cell death may be elicited by excitotoxicity. We focus on the biosynthesis of endocannabinoids and on their modes of action in animal models of these neurodegenerative diseases.

Biotransformation of Tetrahydrocannabinol
Muhammad T. Akhtar . Khozirah Shaari . Robert Verpoorte
Phytochem Rev
DOI: 10.1007/s11101-015-9438-9
Cannabinoids are terpenophenolic com- pounds consisting of an aromatic polyketide and derived from the geranyl diphosphate C10 terpenoid unit. They are the active constituents in Cannabis sativa and have been utilized in a number of cannabis- based medicines. Biotransformation of cannabinoids is an important field of xenobiochemistry and toxicology and the study of the metabolism of these compounds can lead to the discovery of new com- pounds, unknown metabolites with unique structures and new therapeutic entities. Different fungi, bacteria, plants and animal cells have been used for the regio- and stereoselective transformation of cannabinoids. All of the above mentioned organisms have distinct enzymes which catalyze the conversion of a specific cannabinoid at different positions and thus provide a variety of derivatives. All organisms are able to transform the alkyl side chain where as mammalians are unique in the formation of the carboxy derivatives. This review article assesses the current knowledge on the biotransformation of tetrahydrocannabinol and with particular focus on D9-THC.

Blockage of Ventrolateral Periaqueductal Gray Matter Cannabinoid 1 Receptor Increases Dental Pulp Pain and Pain-Related Subsequent Learning and Memory Deficits in Rats
Behav Pharmacol 2020 May 29.
doi: 10.1097/FBP.0000000000000566.
Cannabinoid 1 receptor (CB1R) signaling has a pivotal role in the modulation of both pain and cognitive responses. This study aims at investigating the role of CB1R in the ventrolateral periaqueductal gray matter (vlPAG) on both pulpal pain and pain-related subsequent changes in learning and memory performances in rats. The adult male Wistar rats were cannulated in the vlPAG. The rats were pretreated by intra-vlPAG administration of selective CB1R antagonist AM-251 (2, 4 and 8 µg/rat) and vehicle dimethylsulfoxide. The drugs were microinjected 20 min before the induction of capsaicin-induced pulpalgia. The nociceptive behaviors were recorded for 40 min. Then, passive avoidance and spatial learning and memory were assessed using the shuttle box and Morris water maze tests, respectively. Following the administration of intradental capsaicin, there was a significant nociceptive response that increased after an induced blockage of CB1R by AM-251 at 4 and 8 µg. In addition, capsaicin impaired passive avoidance and spatial memory performance of rats. Microinjection of AM-251, prior to capsaicin, could dose-dependently exaggerate capsaicin-related learning and memory deficits in both tests. The present data indicated that the vlPAG endocannabinoid system is involved in the modulation of pain signals from dental pulp. It was also accompanied by learning and memory impairments.

Blood endocannabinoid levels in patients with panic disorder
Katja Petrowski, Clemens Kirschbaum, Wei Gao d, Jochen Hardt, Rupert Conrad
Psychoneuroendocrinology 122 (2020) 104905
DOI: 10.1016/j.psyneuen.2020.104905
Background: The development and maintenance of anxiety disorders is not fully understood. There is consensus in the literature that in addition to genetic factors, social, psychological and neurobiological factors are of crucial importance. The present exploratory study investigates the influence of the endocannabinoids (EC) and related N-acylethanolamines (NA) on the maintenance of panic disorder (PD).
Methods: A total of n = 36 PD and n = 26 healthy controls (HC) were included in the study. Baseline characteristics showed no differences between the two groups. The participants were exposed to the Trier Social Stress Test (TSST) for reliable laboratory stress induction. Blood samples were taken during the TSST by an intravenous catheter to examine the endocannabinoid (EC) stress response. Repeated measures ANOVA was conducted to test for main effects of time and group as well as the respective interaction.
Results: Participants with PD consistently had significantly higher EC and NA blood levels than HC. The consistently high EC and NA levels barely showed any reactivity as indicated by a lack of statistical variance. In line with these findings no reaction to the psychosocialstressor TSST could be detected.
Conclusion: Our main results show significant differences in EC concentrations between participants with PD and HC. These findings suggest that an imbalance in the ECS contributes to the maintenance of PD. Increased endocannabinoid levels may have important implications for organic diseases such as cardiovascular disorders. The limitations of the study as well as implications for further investigations are discussed.


Blunted Psychotomimetic and Amnestic Effects of ?-9-Tetrahydrocannabinol in Frequent Users of Cannabis.
D’Souza, D. C., Ranganathan, M., Braley, G., Gueorguieva, R., Zimolo, Z., Cooper, T., … Krystal, J
Neuropsychopharmacology, 33(10), 2505–2516. (2008).
doi:10.1038/sj.npp.1301643
Cannabis is one of the most widely used illicit substances and there is growing interest in the association between cannabis use and psychosis. Delta-9-Tetrahydrocannabinol (D-9-THC) the principal active ingredient of cannabis has been shown to induce psychotomimetic and amnestic effects in healthy individuals. Whether people who frequently use cannabis are either protected from or are tolerant to these effects of D-9-THC has not been established. In a 3-day, double-blind, randomized, placebo-controlled study, the dose-related effects of 0, 2.5, and 5 mg intravenous D-9-THC were studied in 30 frequent users of cannabis and compared to 22 healthy controls. D-9-THC (1) produced transient psychotomimetic effects and perceptual alterations; (2) impaired memory and attention; (3) increased subjective effects of ‘high’; (4) produced tachycardia; and (5) increased serum cortisol in both groups. However, relative to controls, frequent users showed blunted responses to the psychotomimetic, perceptual altering, cognitive impairing, anxiogenic, and cortisol increasing effects of D-9-THC but not to its euphoric effects. Frequent users also had lower prolactin levels. These data suggest that frequent users of cannabis are either inherently blunted in their response to, and/or develop tolerance to the psychotomimetic, perceptual altering, amnestic, endocrine, and other effects of cannabinoids.

Brain Anatomical Alterations in Young Cannabis Users: Is it All Hype? A Meta-Analysis of Structural Neuroimaging Studies
Valentina Lorenzetti, Magdalena Kowalczyk, Leonie Duehlmeyer, Lisa-Marie Greenwood, Yann Chye, Murat Yucel, Sarah Whittle, and Carl A Roberts
Cannabis and Cannabinoid Research
Volume X, Number X, 2022
DOI: 10.1089/can.2021.0099 www.liebertpub.com/doi/10.1089/can.2021.0099 Introduction: Cannabis use has a high prevalence in young youth and is associated with poor psychosocial outcomes. Such outcomes have been ascribed to the impact of cannabis exposure on the developing brain. However, findings from individual studies of volumetry in youth cannabis users are equivocal.
Objectives: Our primary objective was to systematically review the evidence on brain volume differences between young cannabis users and nonusers aged 12–26 where profound neuromaturation occurs, accounting for the role of global brain volumes (GBVs). Our secondary objective was to systematically integrate the findings on the association between youth age and volumetry in youth cannabis users. Finally, we aimed to evaluate the quality of the evidence.
Materials and Methods: A systematic search was run in three databases (PubMed, Scopus, and PsycINFO) and was reported using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We run meta-analyses (with and without controlling for GBV) of brain volume differences between young cannabis users and nonusers. We conducted metaregressions to explore the role of age on volumetric differences.
Results: Sixteen studies were included. The reviewed samples included 830 people with mean age 22.5 years (range 14–26 years). Of these, 386 were cannabis users (with cannabis use onset at 15–19 years) and 444 were controls. We found no detectable group differences in any of the GBVs (intracranium, total brain, total white matter, and total gray matter) and regional brain volumes (i.e., hippocampus, amygdala, orbitofrontal cortex, and total cerebellum). Age and cannabis use level did not predict (standardized mean) volume group differences in metaregression. We found little evidence of publication bias (Egger’s test p > 0.1).
Conclusions: Contrary to evidence in adult samples (or in samples mixing adults and youth), previous single studies in young cannabis users, and meta-analyses of brain function in young cannabis users, this early evidence suggests nonsignificant volume differences between young cannabis users and nonusers. While prolonged and long-term exposure to heavy cannabis use may be required to detect gross volume alterations, more studies in young cannabis users are needed to map in detail cannabis-related neuroanatomical changes.
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Bridging the Accessibility Gap of Cannabinoid Medicine and Arabic Culture
Dror Robinson, Sivan Ritter, Lilach Zadik-Weiss, Hadile Ounallah-Saad, Nour AbuAhmad, Rashid Kashkoosh, Mustafa Yassin, and Reuven Or
Rambam Maimonides Med J January 2020 ? Volume 11 ? Issue 1
DOI:*10.5041/RMMJ.10392
Arabs are a large minority group in the Israeli society. With the increasing use of medical cannabis throughout Israel due to changing governmental policies, the interactions of the Arab society with medical cannabis becomes of scientific and medical relevance. Recreational cannabis use is considered haram (forbidden) in Islam. However, most religious scholars agree that medical cannabis usage might be justified as zarurat(emergency and life-saving, therefore allowed) use. Obstacles to medical cannabis use within the Arabic population may relate to language barrier and/or cultural barriers. There are few Arabic-speaking web-based medical-cannabis support groups, and little official information about it is available in the Arabic language. In order for the full benefits of medical cannabis to reach the entire Israeli population, a government-sponsored web-based educational program is necessary in Hebrew and Arabic, both of which are among the nation’s official languages, thereby contributing to the equalization of health resource accessibility

Broadly Neutralizing Synthetic Cannabinoid Vaccines
Mingliang Lin, Jinny Claire Lee, Steven Blake, Beverly Ellis, Lisa M. Eubanks, and Kim D. Janda
JACS Au 2021, 1, 31−40
https://pubs.acs.org/doi/pdf/10.1021/jacsau.0c00057
Synthetic cannabinoids (SCs) constitute a signifi- cant portion of psychoactive substances forming a major public health risk. Due to the wide variety of SCs, broadly neutralizing antibodies generated by active immunization present an intriguing pathway to combat cannabinoid use disorder. Here, we probed hapten design for antibody affinity and cross reactivity against two classes of SCs. Of the 10 haptens screened, 3 vaccine groups revealed submicromolar IC50, each targeting 5−6 compounds in our panel of 22 drugs. Moreover, SCs were successfully sequestered when administered by vaping or intraperitoneal injection, which was confirmed within animal models by observing locomotion, body temperature, and pharmacokinetics. We also discovered synergistic effects to simultaneously blunt two drug classes through an admixture vaccine approach. Collectively, our study provides a comprehensive foundation for the development of vaccines against SCs

Byssinosis in Hemp Mill Workers
John McPartland
January 2003 Journal of Industrial Hemp 8(1):33-44
DOI: 10.1300/J237v08n01_04
This review article concerns byssinosis, a respiratory disease that affects workers in textile mills. According to experts in the field of occupational medicine, hemp mill workers suffer worse than workers in flax, cotton, jute, and sisal mills. The causative factor in hemp dust has not been determined with certainty. However, this review assembles evidence that implicates hemp dust contaminated by bacterial endotoxins, rather than fungal toxins or constituents in hemp itself. Endotoxins are expressed by Gram negative bacteria, and Enterobacter cloacae is a prime suspect. It is proposed that endotoxin contamination occurs during the biological retting process, and not before (in living hemp plants) or after (within the textile mill). Methods of preventing and treating byssinosis are assessed, including some new proposals for management.

Blockage of Ventrolateral Periaqueductal Gray Matter Cannabinoid 1 Receptor Increases Dental Pulp Pain and Pain-Related Subsequent Learning and Memory Deficits in Rats
Behav Pharmacol 2020 May 29.
doi: 10.1097/FBP.0000000000000566.
Cannabinoid 1 receptor (CB1R) signaling has a pivotal role in the modulation of both pain and cognitive responses. This study aims at investigating the role of CB1R in the ventrolateral periaqueductal gray matter (vlPAG) on both pulpal pain and pain-related subsequent changes in learning and memory performances in rats. The adult male Wistar rats were cannulated in the vlPAG. The rats were pretreated by intra-vlPAG administration of selective CB1R antagonist AM-251 (2, 4 and 8 µg/rat) and vehicle dimethylsulfoxide. The drugs were microinjected 20 min before the induction of capsaicin-induced pulpalgia. The nociceptive behaviors were recorded for 40 min. Then, passive avoidance and spatial learning and memory were assessed using the shuttle box and Morris water maze tests, respectively. Following the administration of intradental capsaicin, there was a significant nociceptive response that increased after an induced blockage of CB1R by AM-251 at 4 and 8 µg. In addition, capsaicin impaired passive avoidance and spatial memory performance of rats. Microinjection of AM-251, prior to capsaicin, could dose-dependently exaggerate capsaicin-related learning and memory deficits in both tests. The present data indicated that the vlPAG endocannabinoid system is involved in the modulation of pain signals from dental pulp. It was also accompanied by learning and memory impairments.

Can Cannabis Cure Cancer?
Abrams, D. I., & Guzmán, M.
JAMA Oncology. (2020).
doi:10.1001/jamaoncol.2019.5983
Not long ago, discussion was focused on whether cannabis caused cancer.A thorough review by theUSNational Academies of Sciences, Engineering, and Medicine found moderate evidence of no statistical association between cannabis use and the development of lung and head and neck cancers.1 Limited evidence of a statistical association was found between cannabis use and the development of nonseminomatous testicular carcinomas without good support for a causative effect. Throughout the past fewyears, the pendulum has swung to the point where many patients with cancer diagnoses are convinced, mainly by internet testimonials, that cannabis, particularly highly concentrated oils or tinctures of ?9 -tetrahydrocannabinol (THC) and/or cannabidiol (CBD),may actually cure their cancers.What is the basis of this belief?

Can CBD Cure What Ails You?
Carolyn Barber
Scientific American Medicine / Opinion Oct 2020
https://www.scientificamerican.com/a...071a-365750754
When a shop selling cannabidiol (CBD), a compound found in marijuana, opened near the place where I get coffee in my Southern California neighborhood, I was of two minds. Actually, for several weeks I was of one mind, which was to wish for the speedy demise of the business. As a physician, I just know too much about some of the deleterious consequences associated with marijuana use: Memory issues, altered judgment, addiction, longer-term cognitive issues, chronic bronchitis symptoms, and so on.

Can we make cannabis safer?
Amir Englund, Tom P Freeman, Robin M Murray, Philip McGuire
Lancet Psychiatry 2017
Doi: 10.1016/S2215-0366(17)30075-5
Cannabis use and related problems are on the rise globally alongside an increase in the potency of cannabis sold on both black and legal markets. Additionally, there has been a shift towards abandoning prohibition for a less punitive and more permissive legal stance on cannabis, such as decriminalisation and legalisation. It is therefore crucial that we explore new and innovative ways to reduce harm. Research has found cannabis with high concentrations of its main active ingredient, ?-9-tetrahydrocannabinol (THC), to be more harmful (in terms of causing the main risks associated with cannabis use, such as addiction, psychosis, and cognitive impairment) than cannabis with lower concentrations of THC. By contrast, cannabidiol, which is a non-intoxicating and potentially therapeutic component of cannabis, has been found to reduce the negative effects of cannabis use. Here, we briefly review findings from studies investigating various types of cannabis and discuss how future research can help to better understand and reduce the risks of cannabis use

Canadian clinical practice guidelines for the use of plant-based cannabis and cannabinoid-based products in the management of chronic non-cancer pain and co-occurring conditions: protocol for a systematic literature review.
Wright, P., Walsh, Z., Margolese, S., Sanchez, T., Arlt, S., Belle-Isle, L., … Costiniuk, C.
BMJ Open, 10(5), e036114. (2020).
doi:10.1136/bmjopen-2019-036114
Introduction Chronic pain and co-occurring disorders, such as sleep disorders, anxiety, depression, posttraumatic stress disorder and substance use disorders, are among the most common conditions for which cannabis and cannabinoid-based products derived from the cannabis plant (CBP) are used for therapeutic purposes. However, healthcare providers report that they lack sufficient information on the risks, benefits and appropriate use of cannabis and CBP derived from the cannabis plant for therapeutic purposes.
Methods and analysis We will conduct a systematic review of studies investigating the use of cannabis and CBP derived from the cannabis plant for the treatment of chronic pain and co-occurring conditions. Randomised controlled trials, meta-analyses and observational studies will be prioritised. We will exclude reviews of cannabinoid mechanisms of actions, commentary articles and narrative reviews. The primary outcome of interest will be efficacy in relieving chronic pain. Secondary outcomes will be efficacy in ameliorating conditions such as sleep disorders, anxiety, depression, post-traumatic stress disorder and substance use disorders. We will search electronic bibliographic databases including Academic Search Complete, Cochrane Database of Systematic Reviews, Evidence based Medicine Reviewes, OVID Medline, PsychINFO, PubMed, CINAHL and Web of Science. Two reviewers will conduct screening and data collection independently. Study level of bias will be assessed using the Cochrane Risk of Bias Assessment Tool for randomised controlled trials and non-randomised studies. Narrative analysis will be utilised to interpret the data.

Cannabichromene, Related Phytocannabinoids, and 5-Fluoro-cannabichromene Have Anticonvulsant Properties On A Mouse Model Of Dravet Syndrome
Lyndsey L. Anderson, Adam Ametovski, Jia Lin Luo, Declan Everett-Morgan, Iain S. McGregor, Samuel D. Banister, and Jonathon C. Arnold
ACS Chem. Neurosci. XXXX, X (2020)
DOI: 10.1021/acschemneuro.0c00677
Cannabis-based products are increasingly being used to treat refractory childhood epilepsies such as Dravet syndrome. Cannabis contains at least 140 terpenophenolic compounds known as phytocannabinoids. These include the
known anticonvulsant compound cannabidiol (CBD) and several molecules showing emergent anticonvulsant properties in animal models. Cannabichromene (CBC) is a phytocannabinoid frequently detected in artisanal cannabis oils used in the community by childhood epilepsy patients. Here we examined the brain and plasma pharmacokinetic profi les of CBC, cannabichromenic acid (CBCA), cannabichromevarin (CBCV), and cannabichromevarinic acid (CBCVA) following intraperitoneal administration in mice. The anticonvulsant potential of each was then tested against hyperthermia-induced seizures in the Scn1a+/? mouse model of Dravet syndrome. All phytocannabinoids within the CBC series were readily absorbed and showed substantial brain penetration (brain? plasma ratios ranging from 0.2 to 5.8). Anticonvulsant effi cacy was evident with CBC, CBCA, and CBCVA, each signifi cantly increasing the temperature threshold at which Scn1a+/? mice had a generalized tonic? clonic seizure. We synthesized a fl uorinated derivative of CBC (5-fl uoro-CBC), which showed improved brain penetration relative to the parent CBC molecule but not any greater anticonvulsant eff ect. Since CBC and derivatives are anticonvulsant in a model of intractable pediatric epilepsy, they may constitute part of the mechanism through which artisanal cannabis oils are anticonvulsant in patients.

Cannabimimetic Effects of Osteopathic Manipulative Treatment
John M. McPartland, Andrea Giuffrida, Jeremy King, Evelyn Skinner,
John Scotter, and Richard E. Musty
Journal of Osteopathic Medicine
DOI: 10.7556/jaoa.2005.105.6.283
Endogenous cannabinoids activate cannabinoid receptors in the brain and elicit mood-altering effects. Parallel effects (eg, anxiolysis, analgesia, sedation) may be elicited by osteopathic manipulative treatment (OMT), and previous research has shown that the endorphin system is not responsible for OMT’s mood-altering effects. The authors investigate whether OMT generated cannabimimetic effects for 31 healthy subjects in a dual-blind, randomized controlled trial that measured changes in subjects’ scores on the 67-item Drug Reaction Scale (DRS). Chemical ionization gas chromatography and mass spectrometry were also used to determine changes in serum levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), and oleylethanolamide (OEA). In subjects receiving OMT, posttreatment DRS scores increased significantly for the cannabimimetic descriptors good, high, hungry, lightheaded, and stoned, with significant score decreases for the descriptors inhibited, sober, and uncomfortable. Mean posttreatment AEA levels (8.01 pmol/mL) increased 168% over pretreatment levels (2.99 pmol/mL), mean OEA levels decreased 27%, and no changes occurred in 2-AG levels in the group receiving OMT. Subjects in the sham manipulative treatment group recorded mixed DRS responses, with both increases and decreases in scores for cannabimimetic and noncannabimimetic descriptors and no changes in sera levels. When changes in serum AEA were correlated with changes in subjects’ DRS scores, increased AEA correlated best with an increase for the descriptors cold and rational, and decreased sensations for the descriptors bad, paranoid, and warm. The authors propose that healing modalities popularly associated with changes in the endorphin system, such as OMT, may actually
be mediated by the endocannabinoid system

Cannabinol and Sleep: Separating Fact from Fiction
Jamie Corroon
Cannabis and Cannabinoid Research
Volume X, Number X, 2021
DOI: 10.1089/can.2021.0006
https://pubmed.ncbi.nlm.nih.gov/34468204/
https://www.liebertpub.com/doi/pdf/1.../can.2021.0006
In recent years, marketers of cannabis (i.e., marijuana) products have claimed that cannabinol (CBN) has unique sleep-promoting effects. Despite a plausible mechanism, it is possible that such claims are merely rooted in cannabis lore. The aim of this narrative review was to answer the question: ‘‘Is there sufficient clinical evidence to support claims that CBN has sleep-promoting effects?’’ A systematic search of PubMed/-MEDLINE was performed to evaluate the published evidence. The abstracts of 99 human studies were screened for relevance by the author and reviewed for compliance with the inclusion criteria. The characteristics and principal findings were extracted from eight full-text articles that met inclusion criteria for detailed review. Pre-clinical and clinical research investigating the effects of CBN is dated and limited, with the preponderance of human studies occurring in the 1970–1980s with small sample sizes lacking diversity in sociodemographic characteristics. Studies specifically assessing subjective effects associated with sleep, such as sedation or fatigue, are rare. Most importantly, published clinical trials investigating associations between CBN and validated sleep questionnaires and/or formal polysomnography were not identified in this
review. In addition, evidence demonstrating that CBN itself elicits cannabis-like effects in humans is mixed, with the majority of available evidence demonstrating a lack of such an effect. Consequently, there is insufficient published evidence to support sleep-related claims. Randomized controlled trials are needed to substantiate claims made by manufacturers of cannabis products containing CBN. These studies should specifically evaluate its effects on sleep through polysomnography, or at minimum, through validated sleep questionnaires, and use dosages significantly higher than those found in currently available cannabis products marketed for sleep (typically £ 5 mg). Individuals seeking cannabis-derived sleep aids should be skeptical of manufacturers’ claims of sleep-promoting effects

Cannabidiol, a Regulator of Intracellular Calcium and Calpain
Kyung-Hwa Jeon, Sang-Hyuck Park, Woong Jin Bae, Sae Woong Kim, Hyo Jung Park, Soomin Kim, Tae Hyung Kim, Seung Hwan Jeon, Ilbum Park, Hyun-Je Park, and Youngjoo Kwon
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0197
Cannabidiol (CBD) is one of the most abundant components of Cannabis and has long been used in Cannabisbased preparations. Recently, CBD has become a promising pharmacological agent because of its beneficial properties in the pathophysiology of several diseases. Although CBD is a kind of cannabinoid and acts on cannabinoid receptors (CB1 and CB2), molecular targets involved in diverse therapeutic properties of CBD have not been identified because CBD also interacts with other molecular targets. Considering that CBD alters the intracellular calcium level by which calpain activity is controlled, and both CBD and calpain are associated with various diseases related to calcium signaling, including neurological disorders, this review provides an overview of calpain and calcium signaling as possible molecular targets of CBD. As calpain is known to play an important role in the pathophysiology of neurological disease, a deeper understanding of its relationship with CBD will be meaningful. To understand the role of CBD as a calpain regulator, in silico structural analysis on the binding mode of CBD with calpain was performed.
Find Pdf .

Cannabidiol as potential anticancer drug
Paola Massi, Marta Solinas, Valentina Cinquina & Daniela Parolaro
Br J Clin Pharmacol / 75:2 / 303–312 / (2012)
DOI:10.1111/j.1365-2125.2012.04298.x
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3579246/pdf/bcp0075-0303.pdf
Over the past years, several lines of evidence support an antitumourigenic effect of cannabinoids including D9 -tetrahydrocannabinol (D9 -THC), synthetic agonists, endocannabinoids and endocannabinoid transport or degradation inhibitors. Indeed, cannabinoids possess anti-proliferative and pro-apoptotic effects and they are known to interfere with tumour neovascularization, cancer cell migration, adhesion, invasion and metastasization. However, the clinical use of D9 -THC and additional cannabinoid agonists is often limited by their unwanted psychoactive side effects, and for this reason interest in non-psychoactive cannabinoid compounds with structural affinity for D9 -THC, such as cannabidiol (CBD), has substantially increased in recent years. The present review will focus on the efficacy of CBD in the modulation of different steps of tumourigenesis in several types of cancer and highlights the importance of exploring CBD/CBD analogues as alternative therapeutic agents.

Cannabidiol but not cannabidiolic acid reduces behavioural sensitisation to methamphetamine in rats, at pharmacologically effective doses
Laísa De Siqueira Umpierrez, Priscila Almeida Costa, Eden A. Michelutti, Sarah J Baracz, Melanie Sauer, Anita Jillian Turner, Nicholas A Everett, Jonathon C. Arnold, Iain S. McGregor, Jennifer L Cornish Psychopharmacology April 2022
DOI: 10.1007/s00213-022-06119-3
https://www.researchgate.net/public..._in_rats_at_pharmacologically_effective_doses
Rationale Cannabidiol (CBD) and cannabidiolic acid (CBDA) are non-psychoactive components of the cannabis plant. CBD has been well characterised to have anxiolytic and anticonvulsant activity, whereas the behavioural effects of CBDA are less clear. Preclinical and clinical data suggests that CBD has antipsychotic properties and reduces methamphetamine self-administration in rats. An animal model that is commonly used to mimic the neurochemical changes underlying psychosis and drug dependence is methamphetamine (METH) sensitisation, where repeated administration of the psychostimulant progressively increases the locomotor effects of METH. Objective The aim of this study was to determine whether CBD or CBDA attenuate METH-induced sensitisation of locomotor hyperactivity in rats. Methods Eighty-six male Sprague Dawley rats underwent METH sensitisation protocol where they were subjected to daily METH (1 mg/kg on days 2 and 8, 5 mg/kg on days 3–7; i.p.) injections for 7 days. After 21 days of withdrawal, rats were given a prior injection of CBD (0, 40 and 80 mg/kg; i.p.) or CBDA (0, 0.1, 10 and 1000 µg/kg; i.p.) and challenged with acute METH (1 mg/kg; i.p.). Locomotor activity was then measured for 60 min. Results Rats displayed robust METH sensitisation as evidenced by increased locomotor activity to METH challenge in METH-pretreated versus SAL-pretreated rats. CBD (40 and 80 mg/kg) reduced METH-induced sensitisation. There was no effect of any CBDA doses on METH sensitisation or acute METH-induced hyperactivity. Conclusion These results demonstrate that CBD, but not CBDA, reduces METH sensitisation of locomotor activity in rats at pharmacologically effective doses, thus reinforcing evidence that CBD has anti-addiction and antipsychotic properties.

Cannabidiol converts NF-κB into a tumor suppressor in glioblastoma with defined antioxidative properties
Marie N. M. Volmar, Jiying Cheng, Haitham Alenezi, Sven Richter, Alisha Haug, Zonera Hassan, Maria Goldberg, Yuping Li, Mengzhuo Hou, Christel Herold-Mende, Cecile L. Maire, Katrin Lamszus, Charlotte Flüh, Janka Held-Feindt, Gaetano Gargiulo, Geoffrey J. Topping, Franz Schilling, Dieter Saur, Günter Schneider, Michael Synowitz, Joel A. Schick, Roland E. Kälin, and Rainer Glass
Neuro-Oncology 23(11), 1898–1910, 2021
doi:10.1093/neuonc/noab095
https://pdfs.semanticscholar.org/bc7...7ab532242f94b1 cf26d1.pdf?_ga=2.156822075.180 5733955.1645801231-873149879.1582090724
Background. The transcription factor NF-κB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma [GBM]). Precise therapeutic modulation of NF-κB activity can suppress central oncogenic signaling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive.
Methods. In a pharmacogenomics study with a panel of transgenic glioma cells, we observed that NF-κB can be converted into a tumor suppressor by the non-psychotropic cannabinoid cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study, we performed pharmacological assays, gene expression profiling, biochemical, and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM datasets. Results. We found that CBD promotes DNA binding of the NF-κB subunit RELA and simultaneously prevents RELA phosphorylation on serine-311, a key residue that permits genetic transactivation. Strikingly, sustained DNA binding by RELA-lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen species (ROS), while high ROS content in other tumors blocked CBD-induced hGSC death. Consequently, ROS levels served as a predictive biomarker for CBD-sensitive tumors.
Conclusions. This evidence demonstrates how a clinically approved drug can convert NF-κB into a tumor suppressor and suggests a promising repurposing option for GBM therapy

Cannabidiol decreases motivation for cocaine in a behavioral economics paradigm but does not prevent incubation of craving in mice
Laia Alegre-Zurano, Paula Berbegal-S´aez, Miguel ´A. Lujan, Lídia Cantacorps, Ana Martín-S´anchez, Alba García-Baos, Olga Valverde Biomedicine & Pharmacotherapy 148 (2022)
DOI: 10.1016/j.biopha.2022.112708
Cocaine is a highly consumed drug worldwide which directly targets brain areas involved in reinforcement processing and motivation. Cannabidiol is a phytocannabinoid that exerts protecting effects upon cocaine-induced addictive behavior, although many questions about the mechanisms of action and the specific affected processes remain unknown. Moreover, its effects on cue-induced cocaine-craving incubation have never been addressed. The present study aimed to assess the effects of cannabidiol (20 mg/kg, i.p.) administered during the acquisition of cocaine self-administration (0.75 mg/kg/infusion) and demand task or during cocaine abstinence and craving. Moreover, we measured the alterations in expression of AMPAR subunits and ERK1/2 phosphorylation due to cannabidiol treatment or cocaine withdrawal. Our results showed that cannabidiol reduced cocaine intake when administered during the acquisition phase of the self-administration paradigm, increased behavioral elasticity and reduced motivation for cocaine in a demand task. Cannabidiol also reduced GluA1/2 ratio and increased ERK1/2 phosphorylation in amygdala. No effects over cocaine-craving incubation were found when cannabidiol was administered during abstinence. Furthermore, cocaine withdrawal induced changes in GluA1 and GluA2 protein levels in the prelimbic cortex, ventral striatum and amygdala, as well as a decrease in ERK1/2 phosphorylation in ventral striatum. Taken together, our results show that cannabidiol exerts beneficial effects attenuating the acquisition of cocaine self-administration, in which an operant learning process is required. However, cannabidiol does not affect cocaine abstinence and craving FIND PDF .

Cannabidiol enhances the inhibitory effects of Δ9- tetrahydrocannabinol on human glioblastoma cell proliferation and survival
Jahan P. Marcu, Rigel T. Christian, Darryl Lau, Anne J. Zielinski, Maxx P. Horowitz, Jasmine Lee, Arash Pakdel, Juanita Allison, Chandani Limbad, Dan H. Moore, Garret L. Yount, Pierre-Yves Desprez, and Sean D. McAllister
Mol Cancer Ther. 2010 January ; 9(1): 180–189.
doi:10.1158/1535-7163.MCT-09-0407
https://www.ncbi.nlm.nih.gov/labs/pm....pdf/?tool=EBI
The cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor agonist, Δ 9 -tetrahydrocannabinol (THC), has been shown to be a broad range inhibitor of cancer in culture and in vivo, and is currently being used in a clinical trial for the treatment of glioblastoma. It has been suggested that other plant-derived cannabinoids, which do not interact efficiently with CB1 and CB2 receptors, can modulate the actions of Δ 9 -THC. However, there are conflicting reports as to what extent other cannabinoids can modulate Δ 9 -THC activity, and most importantly, it is not clear whether other cannabinoid compounds can either potentiate or inhibit the actions of Δ 9 -THC. We therefore tested cannabidiol (CBD), the second most abundant plant derived cannabiniod, in combination with Δ 9 -THC. In U251 and SF126 glioblastoma cell lines, Δ 9 -THC and CBD acted synergistically to inhibit cell proliferation. The treatment of glioblastoma cells with both compounds led to significant modulations of the cell cycle and induction of reactive oxygen species (ROS) and apoptosis as well as specific modulations of extracellular signal-regulated kinase (ERK) and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. Our results suggest that the addition of CBD to Δ 9 -THC may improve the overall effectiveness of Δ 9 -THC in the treatment of glioblastoma in cancer patients.


Cannabidiol for COVID-19 Patients with Mild to Moderate Symptoms (CANDIDATE Study): A Randomized, Double-Blind, Placebo-Controlled Clinical Trial
Jose´ Alexandre S. Crippa, Julia Cozar Pacheco, Antonio W. Zuardi, Francisco S. Guimara˜es, Alline Cristina Campos, Fla´via de Lima Oso´rio, Sonia Regina Loureiro, Rafael G. dos Santos, Jose´ Diogo S. Souza, Juliana Mayumi Ushirohira, Rafael Rinaldi Ferreira, Karla Cristinne Mancini Costa, Davi Silveira Scomparin, Franciele Franco Scarante, Isabela Pires-Dos-Santos, Raphael Mechoulam, Fla´vio Kapczinski, Benedito A.L. Fonseca, Danillo L.A. Esposito, Afonso Dinis Costa Passos, Amaury Lelis Dal Fabbro, Fernando Bellissimo-Rodrigues, Eurico Arruda, Sandro Scarpelini, Maristela Haddad Andraus, Julio Cesar Nather Junior, Danilo Tadao Wada, Marcel Koenigkam-Santos, Antonio Carlos Santos, Geraldo Busatto Filho, and Jaime E.C. Hallak
Cannabis and Cannabinoid Research
Volume X, Number X, 2021
DOI: 10.1089/can.2021.0093
https://www.liebertpub.com/doi/pdfpl.../can.2021.0093
Importance: Owing to its anti-inflammatory properties and antiviral ‘‘in vitro’’ effect against severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), cannabidiol (CBD) has been proposed as a potential treatment for
coronavirus disease 2019 (COVID-19).
Objective: To investigate the safety and efficacy of CBD for treating patients with mild to moderate COVID-19.
Design: Randomized, parallel-group, double-blind, placebo-controlled clinical trial conducted between July 7 and October 16, 2020, in two sites in Brazil.
Setting: Patients were recruited in an emergency room.
Participants: Block randomized patients (1:1 allocation ratio—by a researcher not directly involved in data collection) with mild and moderate COVID-19 living in Ribeira˜o Preto, Brazil, seeking medical consultation, and those who voluntarily agreed to participate in the study.
Interventions: Patients received 300 mg of CBD or placebo added to standard symptomatic care during 14 days.
Main Outcome and Measure: The primary outcome was reduction or prevention of the deterioration in clinical status from mild/moderate to severe/critical measured with the COVID-19 Scale or the natural course of the resolution of typical clinical symptoms. Primary study outcome was assessed on days 14, 21, and 28 after enrollment.
Results: A total of 321 patients were recruited and assessed for eligibility, and 105 were randomly allocated either in CBD (n = 49) or in placebo (n = 42) group. Ninety-one participants were included in the analysis of efficacy.
There were no baseline between-group differences regarding disease severity (v2 = 0.025, p = 0.988) and median time to symptom resolution (12 days [95% confidence interval, CI, 6.5–17.5] in the CBD group, 9 days [95% CI, 4.8–13.2] in the placebo group [v2 = 1.6, p = 0.205 by log-rank test]). By day 28, 83.3% in the CBD group and 90.2% in the placebo group had resolved symptoms. There were no between-group differences on secondary measures.
CBD was well tolerated, producing mostly mild and transient side effects (e.g., somnolence, fatigue, changes in appetite, lethargy, nausea, diarrhea, and fever), with no significant differences between CBD and placebo treatment groups.
Conclusions and Relevance: Daily administration of 300 mg CBD for 14 days failed to alter the clinical evolution of COVID-19. Further trials should explore the therapeutic effect of CBD in patients with severe COVID-19, possibly trying higher doses than the used in our study.

(German)
Cannabidiol bei Tumorerkrankungen.
Likar, R., Köstenberger, M., & Nahler, G.
Der Schmerz.(2020).
doi:10.1007/s00482-019-00438-9
Cannabis erlebt derzeit eine Renaissance. Die beiden von der Pflanze Cannabis sativa L. produzierten Hauptcannabinoide sind Delta-9-Tetrahydrocannabinol (THC, synthetische Form: Dronabinol) und Cannabidiol (CBD). Von beiden sind seit mehr als 40 Jahren tumorinhibierende Effekte bekannt. Erst in den letzten Jahren wurde diesem Aspekt, vor allem im Zusammenhang mit der Erforschung des – im Gegensatz zu THC – nicht psychotomimetischen CBD, wieder vermehrt Aufmerksamkeit geschenkt. Untersuchungen in vitro und an verschiedenen Tiermodellen sowie vereinzelte Beobachtungen am Menschen deuten auf eine mögliche therapeutische Rolle von CBD bei Tumorerkrankungen hin.

Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts.
Lowin, T., Tingting, R., Zurmahr, J., Classen, T., Schneider, M., & Pongratz, G.
Cell Death & Disease, 11(8). (2020).
doi:10.1038/s41419-020-02892-1
Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated antiinflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltagedependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses

Cannabidiol and Cannabidiol Metabolites: Pharmacokinetics, Interaction with Food, and Influence on Liver Function
Kieran Shay Struebin Abbotts, Taylor Russell Ewell, Hannah Michelle Butterklee, Matthew Charles Bomar, Natalie Akagi, Gregory P. Dooley and Christopher Bell.
Nutrients 2022, 14, 2152.
DOI: 10.3390/nu14102152
Cannabidiol (CBD) is widely available and marketed as having therapeutic properties. Over-the-counter CBD is unregulated, many of the therapeutic claims lack scientific support, and controversy exists as to the safety of CBD-liver interaction. The study aims were to compare the pharmacokinetics of commercial CBD and CBD metabolites following the ingestion of five different CBD formulations, determine the influence of CBD on food induced thermogenesis, determine the influence of food on CBD pharmacokinetics, and determine the influence of CBD on markers of liver function. Fourteen males (body mass index _ 25 kg/m2) were studied in a placebo-controlled, randomized, crossover design. On five occasions, different CBD formulations were ingested (one per visit). On two additional occasions, CBD or placebo was ingested following a meal. CBD servings were standardized to 30 mg. Considerable pharmacokinetic variability existed between formulations; this pharmacokinetic variability transferred to several of the metabolites. CBD did not influence food induced thermogenesis but did favorably modify early insulin and triglyceride responses. Food appreciably altered the pharmacokinetics of CBD. Finally, CBD did not evoke physiologically relevant changes in markers of liver function. Collectively, these data suggest that consumers should be aware of the appreciable pharmacokinetic differences between commercial CBD formulations, CBD is unlikely to influence the caloric cost of eating but may prove to be of some benefit to initial metabolic responses, consuming CBD with food alters the dynamics of CBD metabolism and increases systemic availability, and low-dose CBD probably does not represent a risk to normal liver function. .

Cannabidiol and Sports Performance: a Narrative Review of Relevant Evidence and Recommendations for Future Research
Danielle McCartney, Melissa J. Benson, Ben Desbrow, Christopher Irwin, Anastasia Suraev and Iain S. McGregor
Sports Medicine - Open (2020) 6:27
Doi: 10.1186/s40798-020-00251-0
https://sportsmedicine-open.springer...20-00251-0.pdf
Cannabidiol (CBD) is a non-intoxicating cannabinoid derived from Cannabis sativa. CBD initially drew scientific interest due to its anticonvulsant properties but increasing evidence of other therapeutic effects has attracted the attention of additional clinical and non-clinical populations, including athletes. Unlike the intoxicating cannabinoid, Δ9 -tetrahydrocannabinol (Δ9 -THC), CBD is no longer prohibited by the World Anti-Doping Agency and appears to be safe and well-tolerated in humans. It has also become readily available in many countries with the introduction of over-the-counter “nutraceutical” products. The aim of this narrative review was to explore various physiological and psychological effects of CBD that may be relevant to the sport and/ or exercise context and to identify key areas for future research. As direct studies of CBD and sports performance are is currently lacking, evidence for this narrative review was sourced from preclinical studies and a limited number of clinical trials in non-athlete populations. Preclinical studies have observed robust anti-inflammatory, neuroprotective and analgesic effects of CBD in animal models. Preliminary preclinical evidence also suggests that CBD may protect against gastrointestinal damage associated with inflammation and promote healing of traumatic skeletal injuries. However, further research is required to confirm these observations. Early stage clinical studies suggest that CBD may be anxiolytic in “stress-inducing” situations and in individuals with anxiety disorders. While some case reports indicate that CBD improves sleep, robust evidence is currently lacking. Cognitive function and thermoregulation appear to be unaffected by CBD while effects on food intake, metabolic function, cardiovascular function, and infection require further study. CBD may exert a number of physiological, biochemical, and psychological effects with the potential to benefit athletes. However, well controlled, studies in athlete populations are required before definitive conclusions can be reached regarding the utility of CBD in supporting athletic performance.

Cannabidiol as a potential treatment for psychosis
C.D. Schubartc , I.E.C. Sommera , P. Fusar-Polib , L. de Wittea , R.S. Kahnc , M.P.M. Boks
European Neuropsychopharmacology (2014) 24, 51–64
DOI: 10.1016/j.euroneuro.2013.11.002
Although cannabis use is associated with an increased risk of developing psychosis, the cannabis constituent cannabidiol (CBD) may have antipsychotic properties. This review concisely describes the role of the endocannabinoid system in the development of psychosis and provides an overview of currently available animal, human experimental, imaging, epidemiological and clinical studies that investigated the antipsychotic properties of CBD. In this targeted literature review we performed a search for English articles using Medline and EMBASE. Studies were selected if they described experiments with psychosis models, psychotic symptoms or psychotic disorders as outcome measure and involved the use of CBD as intervention. Evidence from several research domains suggests that CBD shows potential for antipsychotic treatment

Cannabidiol (CBD) enhanced the hippocampal immune response and autophagy of APP/PS1 Alzheimer’s mice uncovered by RNA-seq.
Hao, F., & Feng, Y.
Life Sciences, 118624.(2020).
doi:10.1016/j.lfs.2020.118624
Alzheimer’s disease (AD) is a central nervous system disease characterized by dementia, which has now become a major threat to global health. Cannabidiol (CBD) is a natural component extracted from the hemp plant and exhibits multiple mechanisms to improve the pathological process of AD in vitro and in vivo. However, its underlying molecular mechanism is still unclear. This study attempts to reveal its common mechanism through transcriptome sequence. We performed transcriptome sequence in the hippocampus of 6 month old APP/PS1 mice chronically treated with CBD for one months or 30 days. In addition, A? plaques were investigated by Immunohistochemistry. Autophagosomes were observed by transmission electron microscopy and the expression of autophagy related proteins was examined by Western blot. GO_BP and KEGG enriched analysis showed that the immune system response was up-regulation significantly. Both KEGG pathway analysis and GSEA analysis showed that autophagy was significantly up-regulated. Finally, the autophagy of hippocampal neurons in APP/PS1 mice treated with CBD was significantly enhanced by transmission electron microscopy. This study illustrated that CBD may improve the pathological process of AD by enhancing immune system response and autophagy pathway

Cannabidiol Cigarettes as Adjunctive Treatment for Psychotic Disorders –A Randomized, Open-Label Pilot-Study
Patrick Köck, Elisabeth Lang, Valerie-Noelle Trulley, Frieder Dechent, Katja Mercer-Chalmers-Bender, Priska Frei, Christian Huber and Stefan Borgwardt
Front. Psychiatry November 2021 | Volume 12 | Article 736822
doi: 10.3389/fpsyt.2021.736822
https://www.frontiersin.org/articles...21.736822/full
Background: Psychotic disorders are associated with high rates of comorbid substanceuse disorders. Use of cannabis rich in tetrahydrocannabinol (THC) is linked to an increased risk of psychosis, worsening of psychotic symptoms, and an adverse course of psychotic disorders. Previous studies suggest oral cannabidiol (CBD) as possible novel antipsychotic agent; however, no studies evaluated the effects of smoked CBD.
Objective: The main aim of the study was to clarify the antipsychotic potential of CBD used as adjunctive therapy simulating a naturalistic setting. Our trial is the first study evaluating the effects of smoked CBD-cigarettes as adjunctive therapy for psychotic symptoms.
Methods: A randomized, placebo-controlled open-label trial of cigarettes containing CBD-rich cannabis (THC < 1%) as adjunctive therapy to standard psychiatric treatment was conducted (ClinicalTrials.gov identifier NCT04700930). Primary outcomes were mean scores of Positive and Negative Syndrome Scale (PANSS), Brøset Violence Checklist, the Beck’s Depression Inventory (BDI), the Subjective Well-Being Under Neuroleptics Scale short form (SWN-K), and antipsychotic medication equivalent doses. Outcomes were assessed after 4 weeks of acute treatment and long-term follow-up after discontinuation of CBD-cigarettes after 25 weeks. Participants were 31 acutely psychotic patients with tobacco use disorder and a mean age of 35.1 ± 10.58 years (71% male). Comorbid cannabis use was diagnosed in 51.6%.
Results: A discontinuous multilevel model revealed no significant group differences for primary outcomes. After 4 weeks of acute treatment, mean PANSS and BDI decreased in both groups, while an increase of antipsychotic medication equivalent was observed in the placebo group.
Conclusions: The presented findings might suggest an antipsychotic medication sparing effect of CBD-cigarettes as adjunctive treatment of acute psychosis. However, the low number of participants did not allow for further statistical analysis. Hence, a larger study sample and a more rigorous study design (blinding of the interventional product, fixed dosing regimen) may reveal different results.

Cannabidiol Does Not Impact Acute Anabolic or Inflammatory Signaling in Skeletal Muscle In Vitro
Henning T. Langer, Alec Avey, and Keith Baar
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0132
https://www.liebertpub.com/doi/epdf/.../can.2021.0132
Background: Cannabidiol (CBD) is becoming increasingly popular for the treatment of clinical conditions including as an aid for muscle recovery. Previous work demonstrated that CBD exhibited mild effects on skeletal muscle, with a tendency to increase anabolic signaling and decrease inflammatory signaling.
Methods: To gain mechanistic insight and extend these findings, we conducted a set of experiments using C2C12 myotubes.
Results: Increasing the dose of CBD (1–5 lM) provided with insulin-like growth factor 1 (IGF-1) showed no effect on anabolic signaling through mTORC1 (S6K1 [Thr389], p = 0.27; rpS6 [Ser240/244], p = 0.81; or 4E-BP1 [Thr37/46], p = 0.87). Similarly, inflammatory signaling through nuclear factor kappa B (NF-jB) (p105, p = 0.88; p50, p = 0.93; or phosphorylated
p65 [Ser536], p = 0.84) in response to tumor necrosis factor a (TNFa) was unaffected by CBD (2.5 lM), whereas dioscin, a natural product that blocks NF-jB signaling, reduced p105 and phosphorylated p65 (Ser536) compared with the TNFa and the TNFa + CBD condition ( p < 0.01 and p < 0.05, respectively). Finally, cannabinoid receptor type 1 (CB1) receptor levels were measured in C2C12 cells, murine skeletal muscle, cortex, and hippocampus. Although CB1 was not detectable in muscle cells or muscle tissue, high levels were observed in brain tissue.
Conclusion: In conclusion, CBD does not directly modulate anabolic or inflammatory signaling in myotubes in vitro, which can likely be explained by the lack of functional receptors.

Cannabidiol effects on cognition in individuals with cocaine use disorder: Exploratory results from a randomized controlled trial
Elie Rizkallah, Violaine Mongeau-Perusse, Leonardo Lamanuzzi, Sol’Abraham Castenada-Ouellet, Emmanuel Stip, Louis-Christophe Juteau, Suzanne Brissette, Julie Bruneau, Simon Dubreucq, Didier Jutras-Aswa
Pharmacology, Biochemistry and Behavior 216 (2022)
https://reader.elsevier.com/reader/s...20220411012302
Background: Cocaine use disorder (CUD) is associated with various cognitive deficits that impede patients' functionality, prognosis and therapeutic outcomes. New pharmacological treatments for CUD that could improve cognition are needed.
Objective: To explore whether cannabidiol (CBD) is superior to placebo to improve cognitive functioning in individuals with CUD.
Methods: We conducted an exploratory analysis of a single site, randomized, double-blind, placebo-controlled trial evaluating CBD's efficacy in reducing craving, cocaine use and relapse in individuals with CUD. Seventy- eight individuals diagnosed with CUD were randomized to receive either CBD (800 mg) or placebo for 92 days. We used the Cambridge Neuropsychological Test Automated Battery (CANTAB) to assess inhibition (Stop Signal Task; SST), risky decision making (Cambridge Gambling Task; CGT) and visual memory (Pattern Recognition Memory; PRM). This assessment was made on day 1, day 7 and at week 6. We controlled for sex, severity of dependence and baseline cognitive scores in our generalized estimating equation models.
Results: Both groups performed similarly on the PRM (correct answers: p = 0.080), SST (stop signal reaction time: p = 0.644) and CGT (quality of decision making: p = 0.994; deliberation time: p = 0.507; delay aversion: p = 0.968; risk taking: p = 0.914) tests.
Conclusions: We found no evidence for 800 mg of CBD to be more efficacious than placebo for improving cognitive outcomes. Clinical trials evaluating pharmacological treatments for CUD should continue to be a research priority

Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation
Shaobin Yang, Yaqin Du, Xiaoqian Zhao, Qi Tang, Wei Su, Yuemeng Hu and Peng Yu
Int. J. Mol.Sci. 2022, 23, 5367.
DOI: 10.3390/ijms23105367 Alzheimer’s disease (AD) is associated with the accumulation and aggregation of amyloid in the brain. The cation channel TRPV2 may mediate the pathological changes in mild cognitive impairment. A high-affinity agonist of TRPV2 named cannabidiol is one of the candidate drugs for AD.
However, the molecular mechanism of cannabidiol via TRPV2 in AD remains unknown. The present study investigated whether cannabidiol enhances the phagocytosis and clearance of microglial A_ via the TRPV2 channel. We used a human dataset, mouse primary neuron and microglia cultures, and AD model mice to evaluate TRPV2 expression and the ability of microglial amyloid-_ phagocytosis in vivo and in vitro. The results revealed that TRPV2 expression was reduced in the cortex and hippocampus of AD model mice and AD patients. Cannabidiol enhanced microglial amyloid-_phagocytosis through TRPV2 activation, which increased the mRNA expression of the phagocytosisrelated receptors, but knockdown of TRPV2 or Trem2 rescued the expression. TRPV2-mediated effects were also dependent on PDK1/Akt signaling, a pathway in which autophagy was indispensable. Furthermore, cannabidiol treatment successfully attenuated neuroinflammation while simultaneously improving mitochondrial function and ATP production via TRPV2 activation. Therefore, TRPV2 is proposed as a potential therapeutic target in AD, while CBD is a promising drug candidate for AD.

Cannabidiol Improves Antioxidant Capacity and Reduces Inflammation in the Lungs of Rats with Monocrotaline-Induced Pulmonary Hypertension
Anna Krzyz ̇ewska, Marta Baranowska-Kuczko, Anna Jastrza, Irena Kasacka and Hanna Kozłowska
Cannabidiol (CBD) is a plant-derived compound with antioxidant and anti-inflammatory properties. Pulmonary hypertension (PH) is still an incurable disease. CBD has been suggested to ameliorate monocrotaline (MCT)-induced PH, including reduction in right ventricular systolic pressure (RVSP), a vasorelaxant effect on pulmonary arteries and a decrease in the white blood cell count. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on the parameters of oxidative stress and inflammation in the lungs of rats with MCT-induced PH. In MCT-induced PH, we found a decrease in total antioxidant capacity (TAC) and glutathione level (GSH), an increase in inflammatory parameters, e.g., tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), nuclear factor kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1), and cluster of differentiation 68 (CD68), and the overexpression of cannabinoid receptors type 1 and 2 (CB-Rs, CB-Rs). Administration of CBD increased TAC and GSH concentrations, glutathione reductase (GSR) activity, and decreased CB-Rs expression and levels of inflammatory mediators such as TNF-α, IL -1β, NF-κB, MCP-1 and CD68. In conclusion, CBD has antioxidant and anti-inflammatory effects in MCT-induced PH. CBD may act as an adjuvant therapy for PH, but further detailed preclinical and clinical studies are recommended to confirm our promising results. .
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Cannabidiol in breast cancer.
Mohamad Elbaza, Mohd W. Nassera, Janani Ravia, Nissar A. Wania, Dinesh K. Ahirwara, Helong Zhaoa, Steve Oghumua, Abhay R. Satoskara, Konstantin Shiloa, William E. Carson IIIb, Ramesh K. Ganju
MOLECULAR ONCOLOGY 9 (2015) 906 e919
https://www.ncbi.nlm.nih.gov/labs/pm...es/PMC4387115/
doi: 10.1016/j.molonc.2014.12.010

The anti‐tumor role and mechanisms of Cannabidiol (CBD), a non‐psychotropic cannabinoid compound, are not well studied especially in triple‐negative breast cancer (TNBC). In the present study, we analyzed CBD's anti‐tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here ‐for the first time‐that CBD significantly inhibits epidermal growth factor (EGF)‐induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF‐induced activation of EGFR, ERK, AKT and NF‐kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor‐associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD‐treated cancer cells. The conditioned medium of CBD‐treated cancer cells also showed lower levels of GM‐CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows ‐for the first time‐that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

Cannabidiol‐induced activation of the metallothionein pathway impedes anticancer effects of disulfiram and its metabolite CuET
Tereza Buchtova, Zdenek Skrott, Katarina Chroma, Jiri Rehulka, Petr Dzubak, Marian Hajduch, David Lukac, Stefanos Arampatzis, Jiri Bartek,and Martin Mistrik
Mol Oncol. 2022 Apr; 16(7): 1541–1554.
doi: 10.1002/1878-0261.13114
https://www.ncbi.nlm.nih.gov/pmc/art...L2-16-1541.pdf
Disulfiram (DSF), an established alcohol‐aversion drug, is a candidate for repurposing in cancer treatment. DSF’s antitumor activity is supported by preclinical studies, case reports, and small clinical trials; however, ongoing clinical trials of advanced‐stage cancer patients encounter variable results. Here, we show that one reason for the inconsistent clinical effects of DSF may reflect interference by other drugs. Using a high‐throughput screening and automated microscopy, we identify cannabidiol, an abundant component of the marijuana plant used by cancer patients to mitigate side effects of chemotherapy, as a likely cause of resistance to DSF. Mechanistically, in cancer cells, cannabidiol triggers the expression of metallothioneins providing protective effects by binding heavy metal‐based substances including the bis‐diethyldithiocarbamate‐cop per complex (CuET). CuET is the documented anticancer metabolite of DSF, and we show here that the CuET’s anticancer toxicity is effectively neutralized by metallothioneins. Overall, this work highlights an example of undesirable interference between cancer therapy and the concomitant usage of marijuana products. In contrast, we report that insufficiency of metallothioneins sensitizes cancer cells toward CuET, suggesting a potential predictive biomarker for DSF repurposing in oncology.

Cannabidiol Induces Cell Death in Human Lung Cancer Cells and Cancer Stem Cells
Hussein Hamad and Birgitte Brinkmann Olsen
Pharmaceuticals 2021, 14(11), 1169;
doi: 10.3390/ph14111169
https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC8624 994&blobtype=pdf
Currently, there is no effective therapy against lung cancer due to the development of resistance. Resistance contributes to disease progression, recurrence, and mortality. The presence of so-called cancer stem cells could explain the ineffectiveness of conventional treatment, and the development of successful cancer treatment depends on the targeting also of cancer stem cells. Cannabidiol (CBD) is a cannabinoid with anti-tumor properties. However, the effects on cancer stem cells are not well understood. The effects of CBD were evaluated in spheres enriched in lung cancer stem cells and adherent lung cancer cells. We found that CBD decreased viability and induced cell death in both cell populations. Furthermore, we found that CBD activated the effector caspases 3/7, increased the expression of pro-apoptotic proteins, increased the levels of reactive oxygen species, as well as a leading to a loss of mitochondrial membrane potential in both populations. We also found that CBD decreased self-renewal, a hallmark of cancer stem cells. Overall, our results suggest that CBD is effective against the otherwise treatment-resistant cancer stem cells and joins a growing list of compounds effective against cancer stem cells. The effects and mechanisms of CBD in cancer stem cells should be further explored to find their Achilles heel.

Cannabidiol Inhibits Multiple Ion Channels in Rabbit Ventricular Cardiomyocytes
Dmytro Isaev, Waheed Shabbir, Ege Y. Dinc, Dietrich E Lorke, Georg Petroianu and Murat Oz
Front. Pharmacol., 03 February 2022 https://doi.org/10.3389/fphar.2022.821758
https://www.frontiersin.org/articles...kP_huxJxubZOfo ZxFVDOypZhfEWVjQsVuU_O.VjOIolh kUstIYDhIbA4FP_hNl8bdhNtIHpbDP Nxfb7bDHdb9XZ4uodx.U_4uo74DsLx .hu&utm_source=eNews&utm_campa ign=TCS%20Newsletter%20week%20 08%202022&utm_medium=email
Cannabidiol (CBD), a major non-psychotropic cannabinoid found in the Cannabis plant, has been shown to exert anti-nociceptive, anti-psychotic, and anti-convulsant effects and to also influence the cardiovascular system. In this study, the effects of CBD on major ion currents were investigated using the patch-clamp technique in rabbit ventricular myocytes. CBD inhibited voltage-gated Na+ and Ca2+ channels with IC50 values of 5.4 and 4.8 µM, respectively. In addition, CBD, at lower concentrations, suppressed ion currents mediated by rapidly and slowly activated delayed rectifier K+ channels with IC50 of 2.4 and 2.1 µM, respectively. CBD, up to 10 μM, did not have any significant effect on inward rectifier IK1 and transient outward Ito currents. The effects of CBD on these currents developed gradually, reaching steady-state levels within 5–8 min, and recoveries were usually slow and partial. Hill coefficients higher than unity in concentration-inhibition curves suggested multiple CBD binding sites on these channels. These findings indicate that CBD affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when CBD is administered to carriers of cardiac channelopathies or to individuals using drugs known to affect the rhythm or the contractility of the heart.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response
Long Chi Nguyen, Dongbo Yang, Vlad Nicolaescu, Thomas J. Best, Takashi Ohtsuki, Shao-Nong Chen, J. Brent Friesen, Nir Drayman, Adil Mohamed, Christopher Dann, Diane Silva, Haley Gula, Krysten A. Jones, J. Michael Millis, Bryan C. Dickinson, Savaş Tay, Scott A. Oakes, Guido F. Pauli, David O. Meltzer, Glenn Randall and Marsha Rich Rosner
Version 1. bioRxiv. Preprint. 2021 Mar 10.
doi: 10.1101/2021.03.10.432967
https://www.ncbi.nlm.nih.gov/pmc/art....10.432967.pdf
The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV- 2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARSCoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

Cannabidiol (CBD) for Treatment of Neurofibromatosis-related Pain and Concomitant Mood Disorder: A Case Report .
Hegazy O, Platnick H
Cureus 11(12): e6312. (2019)
DOI 10.7759/cureus.6312
Neurofibromatosis type 1 (NF1) is a common genetic disorder. Pain is a major symptom of this disease which can be secondary to the development of plexiform and subcutaneous neurofibromas, musculoskeletal symptoms (such as scoliosis and pseudoarthrosis), and headaches. Visible neurofibromas add significant psychosocial distress for NF1 patients. Along with the chronic pain, psychosocial distress contributes to associated mood disorders, such as depression and anxiety. Cannabis has been the focus of many studies for treating multiple conditions, including epilepsy, multiple sclerosis, Parkinsonism disease, and many chronic pain conditions. Cannabidiol (CBD) is the major non-psychotropic component of cannabis. CBD has shown anti-inflammatory and analgesic properties, as well as having mood stabilizer and anxiolytic effects. In this report, we present the use of cannabidiol (CBD) for the management of chronic pain and concomitant mood disorder in an NF1 patient.

Cannabidiol (CBD) modulation of apelin in acute respiratory distress syndrome
Évila Lopes Salles | Hesam Khodadadi | Abbas Jarrahi | Meenakshi Ahluwalia | Valdemar Antonio Paffaro Jr | Vincenzo Costigliola | Jack C. Yu | David C. Hess | Krishnan M. Dhandapani | Babak Baban
J Cell Mol Med. 2020;00:1–4
DOI: 10.1111/jcmm.15883
Considering lack of target-specific antiviral treatment and vaccination for COVID-19, it is absolutely exigent to have an effective therapeutic modality to reduce hospitalization and mortality rate as well as to improve COVID-19-infected patient outcomes. In a follow-up study to our recent findings indicating the potential of Cannabidiol (CBD) in the treatment of acute respiratory distress syndrome (ARDS), here we show for the first time that CBD may ameliorate the symptoms of ARDS through up-regulation of apelin, a peptide with significant role in the central and peripheral regulation of immunity, CNS, metabolic and cardiovascular system. By administering intranasal Poly (I:C), a synthetic viral dsRNA, while we were able to mimic the symptoms of ARDS in a murine model, interestingly, there was a significant decrease in the expression of apelin in both blood and lung tissues. CBD treatment was able to reverse the symptoms of ARDS towards a normal level. Importantly, CBD treatment increased the apelin expression significantly, suggesting a potential crosstalk between apelinergic system and CBD may be the therapeutic target in the treatment of inflammatory diseases such as COVID-19 and many other pathologic conditions.

Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines.
Feinshtein et al
PeerJ 1:e153; (2013)
DOI: 10.7717/peerj.153
Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison).
Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h) exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3) and rhodamine123(rh123)). Cyclosporine A (CsA) served as a positive control.
Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3) and rh123) was inhibited upon short-term exposure to CBD.
Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

Cannabidiol Counteracts the Psychotropic Side-Effects of ?-9-Tetrahydrocannabinol in the Ventral Hippocampus Through Bi-Directional Control of ERK1-2 Phosphorylation
Roger Hudson, Justine Renard, Christopher Norris, Walter J. Rushlow, and Steven R. Laviolette
JNEUROSCI (2019)
10.1523/JNEUROSCI.0708-19.2019
Evidence suggests that the phytocannabinoids ?-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) differentially regulate salience attribution and psychiatric risk. The ventral hippocampus (vHipp) relays emotional salience via control of dopamine (DA) neuronal activity states, which are dysregulated in psychosis and schizophrenia. Using in-vivo electrophysiology in male Sprague Dawley rats, we demonstrate that intra-vHipp THC strongly increases ventral tegmental area (VTA) DA neuronal frequency and bursting rates, decreases GABA frequency, and amplifies VTA beta gamma and epsilon oscillatory magnitudes via modulation of local extracellular signal-regulated kinase phosphorylation (pERK1-2). Remarkably, whereas intra-vHipp THC also potentiates salience attribution in morphine place-preference and fear conditioning assays, CBD co-administration reverses these changes by down-regulating pERK1-2 signaling, as pharmacological re-activation of pERK1-2 blocked the inhibitory properties of CBD. These results identify vHipp pERK1-2 signaling as a critical neural nexus point mediating THC-induced affective disturbances and suggest a potential mechanism by which CBD may counteract the

Cannabidiol Does Not Cause Significant Changes to Working Memory Performance in the N-Back Task
Éamon Jones and Styliani Vlachou
Pharmaceuticals 2021, 14, 1165.
DOI: 10.3390/ph14111165
Cannabis use can be traced back to several centuries before the Common Era, when it was used for industrial, medicinal and recreational purposes. More recently, over 100 different cannabinoid compounds have been identified, one of which is cannabidiol (CBD), a compound widely used for anti-inflammatory and anxiolytic treatment. The literature surrounding the cognitive effects of CBD is limited, with most studies focusing on the effects of other cannabinoids on cognition. To expand this literature, this study investigated whether CBD causes significant differences to working memory (WM) functioning, as measured by the N-back task. It was hypothesised that CBD does not cause statistically significant differences to WM. In all, 54 participants, 33 females and 21 males, were recruited, with a mean age of 32.63 years. Of these 54 participants, 26 reported using CBD and no other cannabinoids, while 28 reported not using any cannabinoid. The participants were instructed to answer a short online survey to gather basic demographic data and to complete an online N-back task to measure WM. For the computerised N-back task, the participants completed a practice and three test blocks, where they were instructed to respond to whether a series of letter stimuli were presented one trial back (1-back), two trials back (2-back) or three trials back (3-back). Multivariate analysis of covariance yielded no statistically significant difference on either response time or response accuracy data between groups after controlling for how long the participants use CBD and for what reason they use CBD. These results support our hypothesis that CBD does not cause significant changes to WM functioning. Further research is greatly needed to investigate the long-term effects of CBD use on WM and on general cognitive functioning

Cannabidiol Does Not Impact Acute Anabolic or Inflammatory Signaling in Skeletal Muscle In Vitro
Henning T. Langer, Alec Avey, and Keith Baar
Cannabis and Cannabinoid Research Nov 2021
DOI: 10.1089/can.2021.0132
https://www.liebertpub.com/doi/10.1089/can.2021.0132
Background: Cannabidiol (CBD) is becoming increasingly popular for the treatment of clinical conditions including as an aid for muscle recovery. Previous work demonstrated that CBD exhibited mild effects on skeletal muscle, with a tendency to increase anabolic signaling and decrease inflammatory signaling.
Methods: To gain mechanistic insight and extend these findings, we conducted a set of experiments using C2C12 myotubes.
Results: Increasing the dose of CBD (1–5 μM) provided with insulin-like growth factor 1 (IGF-1) showed no effect on anabolic signaling through mTORC1 (S6K1 [Thr389], p=0.27; rpS6 [Ser240/244], p=0.81; or 4E-BP1 [Thr37/46], p=0.87). Similarly, inflammatory signaling through nuclear factor kappa B (NF-κB) (p105, p=0.88; p50, p=0.93; or phosphorylated p65 [Ser536], p=0.84) in response to tumor necrosis factor α (TNFα) was unaffected by CBD (2.5 μM), whereas dioscin, a natural product that blocks NF-κB signaling, reduced p105 and phosphorylated p65 (Ser536) compared with the TNFα and the TNFα + CBD condition (p<0.01 and p<0.05, respectively). Finally, cannabinoid receptor type 1 (CB1) receptor levels were measured in C2C12 cells, murine skeletal muscle, cortex, and hippocampus. Although CB1 was not detectable in muscle cells or muscle tissue, high levels were observed in brain tissue.
Conclusion: In conclusion, CBD does not directly modulate anabolic or inflammatory signaling in myotubes in vitro, which can likely be explained by the lack of functional receptors.
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Cannabidiol exerts protective effects in an in vitro model of Parkinson’s disease activating AKT/mTOR pathway.
Gugliandolo, A., Pollastro, F., Bramanti, P., & Mazzon, E.
Fitoterapia, 104553. (2020).
doi:10.1016/j.fitote.2020.104553
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of the nigrostriatal dopaminergic pathway with loss of substantia nigra pars compacta neurons and dopamine depletion. Various natural compounds showed protective actions against PD. In this work, the protective effects of cannabidiol (CBD), obtained from Cannabis sativa, were evaluated in retinoic acid differentiated SH-SY5Y cells exposed to 1-methyl-4 phenylpyridinium (MPP+), an in vitro PD model. In order to evaluate which receptor is involved in CBD actions CB1, CB2 and TRPV1 receptor antagonists were used. CBD counteracted the loss of cell viability caused by MPP+, reducing apoptosis as demonstrated by the reduction of Bax and caspase 3. Moreover, CBD reduced the nuclear levels of PARP-1. The protective effects of CBD seem to be mediated by the activation of ERK and AKT/mTOR pathways. The treatment with AKT1/2 inhibitor and the mTOR inhibitor rapamycin abolished CBD protective effects. The CBD-induced ERK activation may be mediated by CBD interaction with CB2 and TRPV1. We also investigated the protein levels of the autophagic proteins LC3 and beclin 1. CBD reduced the MPP+-induced increase of LC3 by CB2 and TRPV1 receptors. These data suggested the involvement of ERK in the modulation of autophagy. However, beclin 1 levels were not modified neither by MPP+ nor by CBD. These results indicated that CBD may exert preventive and protective actions in PD

Cannabidiol for the Management of Endometriosis and Chronic Pelvic Pain
Megha Mistry, Paul Simpson, Edward Morris, Ann-Katrin-Fritz, Babu Karavadra, Carole Lennox , Ed Prosser-Snelling
JMIG 2021
DOI: 10.1016/j.jmig.2021.11.017
https://www.jmig.org/action/showPdf?...2821%2901286-3
Objective
To review the available literature on the effect of cannabis-based products on the female reproductive system and establish if there is any evidence that they benefit or harm patients with endometriosis and therefore if there is sufficient evidence to recommend them.
Data Sources
An electronic-based search was performed in PubMed, Embase and the Cochrane Database. Reference lists of articles retrieved were reviewed and a grey literature search was also performed.
Methods of Study Selection
The original database search yielded 264 articles from PubMed, Embase and the Cochrane Database, of which forty-one were included. One hundred and sixty-one studies relating to gynaecological malignancy, conditions unrelated to endometriosis or therapies unrelated to cannabis-based products were excluded. Twelve articles were included from a grey literature search and review of references.
Results
The majority of available evidence is from laboratory studies aiming to simulate the effects of cannabis-based products on preclinical endometriosis models. Some show evidence of benefit with cannabis-based products. However, results are conflicting and the impact in humans cannot necessarily be extrapolated from this data. Few studies exist looking at the effect of cannabis or its derived products in women with endometriosis – the majority are in the form of surveys and are affected by bias. National guidance was also reviewed: at present this dictates that cannabis-based products can only be prescribed for conditions where there is clear published evidence of benefit and only when all other treatment options have been exhausted.
Conclusion
Current treatment options for endometriosis often affect fertility and/or have undesirable side effects that impede long-term management. Cannabis-based products have been suggested as a novel therapeutic option that may circumvent these issues. However, there is a paucity of welldesigned, robust studies and randomised controlled trials looking at their use in the treatment of endometriosis. In addition, cannabis use has a potential for harm in the long term; with a possible association with ‘cannabis use disorder’, psychosis and mood disturbances. At present, national guidance cannot recommend cannabis-based products to patients in the UK due to lack of clear evidence of benefit. More comprehensive research into the impact of endocannabinoids in the context of endometriosis is required before their use can be recommended or prescribed.

Cannabidiol for the treatment of cannabis use disorder: a phase 2a, double-blind, placebo-controlled, randomised, adaptive Bayesian trial.
Freeman, T. P., Hindocha, C., Baio, G., Shaban, N. D. C., Thomas, E. M., Astbury, D., … Curran, H. V.
The Lancet Psychiatry. (2020).
doi:10.1016/s2215-0366(20)30290-x
Background A substantial and unmet clinical need exists for pharmacological treatment of cannabis use disorders. Cannabidiol could offer a novel treatment, but it is unclear which doses might be efficacious or safe. Therefore, we aimed to identify efficacious doses and eliminate inefficacious doses in a phase 2a trial using an adaptive Bayesian design. Methods We did a phase 2a, double-blind, placebo-controlled, randomised, adaptive Bayesian trial at the Clinical Psychopharmacology Unit (University College London, London, UK). We used an adaptive Bayesian dose-finding design to identify efficacious or inefficacious doses at a-priori interim and final analysis stages. Participants meeting cannabis use disorder criteria from DSM-5 were randomly assigned (1:1:1:1) in the first stage of the trial to 4-week treatment with three different doses of oral cannabidiol (200 mg, 400 mg, or 800 mg) or with matched placebo during a cessation attempt by use of a double-blinded block randomisation sequence. All participants received a brief psychological intervention of motivational interviewing. For the second stage of the trial, new participants were randomly assigned to placebo or doses deemed efficacious in the interim analysis. The primary objective was to identify the most efficacious dose of cannabidiol for reducing cannabis use. The primary endpoints were lower urinary 11-nor-9-carboxy-?-9-tetrahydrocannabinol (THC-COOH):creatinine ratio, increased days per week with abstinence from cannabis during treatment, or both, evidenced by posterior probabilities that cannabidiol is better than placebo exceeding 0·9. All analyses were done on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov (NCT02044809) and the EU Clinical Trials Register (2013-000361-36). Findings Between May 28, 2014, and Aug 12, 2015 (first stage), 48 participants were randomly assigned to placebo (n=12) and to cannabidiol 200 mg (n=12), 400 mg (n=12), and 800 mg (n=12). At interim analysis, cannabidiol 200 mg was eliminated from the trial as an inefficacious dose. Between May 24, 2016, and Jan 12, 2017 (second stage), randomisation continued and an additional 34 participants were allocated (1:1:1) to cannabidiol 400 mg (n=12), cannabidiol 800 mg (n=11), and placebo (n=11). At final analysis, cannabidiol 400 mg and 800 mg exceeded primary endpoint criteria (0·9) for both primary outcomes. For urinary THC-COOH:creatinine ratio, the probability of being the most efficacious dose compared with placebo given the observed data was 0·9995 for cannabidiol 400 mg and 0·9965 for cannabidiol 800 mg. For days with abstinence from cannabis, the probability of being the most efficacious dose compared with placebo given the observed data was 0·9966 for cannabidiol 400 mg and 0·9247 for cannabidiol 800 mg. Compared with placebo, cannabidiol 400 mg decreased THC-COOH:creatinine ratio by –94·21 ng/mL (95% interval estimate –161·83 to –35·56) and increased abstinence from cannabis by 0·48 days per week (0·15 to 0·82). Compared with placebo, cannabidiol 800 mg decreased THC-COOH:creatinine ratio by –72·02 ng/mL (–135·47 to –19·52) and increased abstinence from cannabis by 0·27 days per week (–0·09 to 0·64). Cannabidiol was well tolerated, with no severe adverse events recorded, and 77 (94%) of 82 participants completed treatment. Interpretation In the first randomised clinical trial of cannabidiol for cannabis use disorder, cannabidiol 400 mg and 800 mg were safe and more efficacious than placebo at reducing cannabis use

Cannabidiol for the treatment of crack-cocaine craving: an exploratory double-blind study
Carolina de Meneses-Gaya, Jose´ A. Crippa, Jaime E. Hallak, Andre´ Q. Miguel, Ronaldo Laranjeira, Rodrigo A. Bressan, Antonio W. Zuardi, Acioly L. Lacerda
Braz J Psychiatry. 2020 xxx-xxx;00(00):000-000
doi:10.1590/1516-4446-2020-1416
Objective: To assess the efficacy of cannabidiol (CBD) in the management of crack-cocaine craving and the treatment of frequent withdrawal symptoms. Methods: Thirty-one men with a diagnosis of crack-cocaine dependence were enrolled in a randomized, double-blind, placebo-controlled trial. We applied neuropsychological tests and assessed craving intensity, anxiety and depression symptoms, and substance use patterns at baseline and at the end of the trial. The participants were treated with CBD 300 mg/day or placebo for 10 days. During this period, we used a technique to induce craving and assessed the intensity of symptoms before and after the induction procedure. Results: Craving levels reduced significantly over the 10 days of the trial, although no differences were found between the CBD and placebo groups. Craving induction was successful in both groups, with no significant differences between them. Indicators of anxiety, depression, and sleep alterations before and after treatment also did not differ across groups. Conclusion: Under the conditions of this trial, CBD was unable to interfere with symptoms of crackcocaine withdrawal. Further studies with larger outpatient samples involving different doses and treatment periods would be desirable and timely to elucidate the potential of CBD to induce reductions in crack-cocaine self-administration.

Cannabidiol: From Drug Interaction Potential to Modulation of the Gut Microbiome.
Kortubash, I., Skinner, C., Ewing, L., McGill, M., Kennon-McGill, S., Nookaew, I., … Gurley, B. ).
Current Developments in Nutrition, 4(Supplement_2), 418–418.(2020) doi:10.1093/cdn/nzaa045_051 NOT FULL PAPER
Objectives: Cannabidiol (CBD) is the major non-psychotropic phytocannabinoid present in Cannabis sativa. In 2018, Congress designated select C. sativa cultivars as “hemp” removing them from the DEA’s list of controlled substances. As a result, CBD-containing hemp extracts and other CBD products are now widely available and heavily marketed, yet their FDA regulatory status is still hotly debated. Further complicating the debate is CBD’s vastly under-researched safety profile. Safety concerns yet to be adequately addressed include CBD’s drug interaction potential and its effect on the gut microbiome.
Methods: Using acetaminophen (APAP), the most commonly ingested over-the-counter pain medication, we demonstrated that CBD-rich cannabis extract (CRCE) poses a significant drug interaction risk.
Results: Mice exposed to both CRCE and APAP developed severe liver injury. This hepatotoxicity, however, was not observed when either CRCE or APAP were administered separately. Importantly, this injury was observed in two different strains of mice with susceptibilities seemingly linked to sex (female) and age (older animals). Furthermore, both beneficial and adverse effects of CRCE on the gut microbiome were observed. Specifically, CRCE exposure increased the relative abundance of the beneficial gut microbe, Akkermansia muciniphila, however, an overall decrease in the relative abundance of all gut bacterial species was noted. This decrease was paralleled by numerous pro-inflammatory responses in the proximal jejunum and colon. Conclusions: Taken together, these findings raise significant concerns about the safety of widespread CBD usage and underlines the need for additional well-designed studies into its safety and efficacy.

Cannabidiol in Anxiety and Sleep: A Large Case Series
Scott Shannon, Nicole Lewis, Heather Lee, Shannon Hughes
The Permanente journal 2019;23:18-041.
doi:10.7812/TPP/18-041
Context: Cannabidiol (CBD) is one of many cannabinoid compounds found in cannabis. It does not appear to alter consciousness or trigger a “high.” A recent surge in scientific publications has found preclinical and clinical evidence documenting value for CBD in some neuropsychiatric disorders, including epilepsy, anxiety, and schizophrenia. Evidence points toward a calming effect for CBD in the central nervous system. Interest in CBD as a treatment of a wide range of disorders has exploded, yet few clinical studies of CBD exist in the psychiatric literature.
Objective: To determine whether CBD helps improve sleep and/ or anxiety in a clinical population.
Design: A large retrospective case series at a psychiatric clinic involving clinical application of CBD for anxiety and sleep complaints as an adjunct to usual treatment. The retrospective chart review included monthly documentation of anxiety and sleep quality in 103 adult patients.
Main Outcome Measures: Sleep and anxiety scores, using validated instruments, at baseline and after CBD treatment.
Results: The final sample consisted of 72 adults presenting with primary concerns of anxiety (n = 47) or poor sleep (n = 25). Anxiety scores decreased within the first month in 57 patients (79.2%) and remained decreased during the study duration. Sleep scores improved within the first month in 48 patients (66.7%) but fluctuated over time. In this chart review, CBD was well tolerated in all but 3 patients.
Conclusion: Cannabidiol may hold benefit for anxiety-related disorders. Controlled clinical studies are needed.

Cannabidiol interferes with the effects of ?9tetrahydrocannabinol in man
IsacG.Karniol,ItiroShirakawa,N elsonKasinski,AbrahamPfeferman ,Elisaldo A.Carlini
European Journal of Pharmacology Vol 28, 1 , Sept 1974,172-177
doi.org/10.1016/0014-2999(74)90129-0
Based on previous observations that cannabidiol (CBD) blocks some effects of ?9-tetrahydrocannabinol (?9-THC) in laboratory animals, the present work was carried out to study possible interaction between CBD and ?9-THC in human beings. In a double blind procedure, 40 healthy male volunteers were assigned to 1 of 8 experimental groups, receiving per oral route, placebe, 30 mg ?9-THC, 15 30 or 60 mg of CBD, and mixtures of 30 mg of ?9-THC plus either 15, 30 or 60 mg of CBD respectively. Pulse rate, time production tasks and psychological logical reactions were measured at several time intervals after drug ingestion. 30 mg ?9-THC alone increased pulse rate, disturbed time tasks and induced strong psychological reactions in the subjects. 15–60 mg of CBD alone provoked no effects. On the other hand, CBD was efficient in blocking most of the effects of ?9-THC when both drugs were given together. CBD also decreased the anxiety component of ?9-THC effects, in such a way that the subjects reported more pleasurable effects.

Cannabidiol in treatment of refractory epileptic spasms: An open-label study.
Herlopian, A., Hess, E. J., Barnett, J., Geffrey, A. L., Pollack, S. F., Skirvin, L., … Thiele, E. A.
Epilepsy & Behavior, 106, (2020).
doi:10.1016/j.yebeh.2020.106988
Objective: This study aimed to evaluate clinical efficacy and safety of purified pharmaceutical cannabidiol (CBD) as an adjunctive therapy in refractory childhood-onset epileptic spasms (ES). Methods: Nine patients with ES were enrolled in an Institutional Review Board (IRB)- and Food and Drug Administration (FDA)-approved expanded access investigational new drug trial. Patients received plant-derived highly purified CBD in oral solution in addition to their baseline medications at an initial dosage of 5 mg/kg/day, which was increased by 5 mg/kg/day every week to an initial target dosage of 25 mg/kg/day. Seizure frequency, adverse event, and parents' subjective reports of cognitive and behavioral changes were recorded after 2 weeks and 1, 2, 3, 6, 9, and 12 months of CBD treatment. Responder rates (percent of patients with N50% reduction in ES frequency from baseline) were calculated. Electrographic changes were studied in relation to CBD initiation and clinical response. Results: Overall, the responder rates in 9 patients were 67%, 78%, 67%, 56%, 78%, 78%, and 78% after 2 weeks and 1, 2, 3, 6, 9, and 12 months of CBD treatment, respectively. Three out of nine patients (33%) were ES free after two months of treatment. Parents reported subjective improvements in cognitive and behavioral domains. Side effects, primarily drowsiness, were seen in 89% of patients (n = 8). Eight of the nine (89%) patients had electroencephalographic (EEG) studies prior to and after initiation of CBD. Three out of five patients (60%) had resolution in their hypsarrhythmia pattern. Significance: Purified pharmaceutical CBD may be an effective and safe adjunctive therapy in refractory ES and may also be associated with improvements in electrographic findings.

Cannabidiol in the Treatment of Epilepsy: Current Evidence and Perspectives for Further Research
Valentina Franco , Meir Bialer, Emilio Perucca
Neuropharmacology. 2020 Dec 18;108442.
doi: 10.1016/j.neuropharm.2020.108442
The therapeutic potential of cannabidiol (CBD) in seizure disorders has been known for many years, but it is only in the last decade that major progress has been made in characterizing its preclinical and clinical properties as an antiseizure medication. The mechanisms responsible for protection against seizures are not fully understood, but they are likely to be multifactorial and to include, among others, antagonism of G protein-coupled receptor, desensitization of transient receptor potential vanilloid type 1 channels, potentiation of adenosine-mediated signaling, and enhancement of GABAergic transmission. CBD has a low and highly variable oral bioavailability, and can be a victim and perpetrator of many drug-drug interactions. A pharmaceutical-grade formulation of purified CBD derived from Cannabis sativa has been evaluated in several randomized placebo-controlled adjunctive-therapy trials, which resulted in its regulatory approval for the treatment of seizures associated with Dravet syndrome, Lennox-Gastaut syndrome and tuberous sclerosis complex. Interpretation of results of these trials, however, has been complicated by the occurrence of an interaction with clobazam, which leads to a prominent increase in the plasma concentration of the active metabolite N-desmethylclobazam in CBD-treated patients. Despite impressive advances, significant gaps in knowledge still remain. Areas that require further investigation include the mechanisms underlying the antiseizure activity of CBD in different syndromes, its pharmacokinetic profile in infants and children, potential relationships between plasma drug concentration and clinical response, interactions with other co-administered medications, potential efficacy in other epilepsy syndromes, and magnitude of antiseizure effects independent from interactions with clobazam.

Cannabidiol metabolism revisited: tentative identifcation of novel decarbonylated metabolites of cannabidiol formed by human liver microsomes and recombinant cytochrome P450 3A4
Kazuhito Watanabe, Noriyuki Usami, Shigehiro Osada1, Shizuo Narimatsu, Ikuo Yamamoto, Hidetoshi Yoshimura
Forensic Toxicology (2019) 37:449–455
doi: 10.1007/s11419-019-00467-0
Purpose The purpose of the present study was to identify the structures of cannabidiol (CBD) metabolites during CO formation by human liver microsomes and human recombinant cytochrome P450 (CYP) enzymes.
Methods CBD was NADPH-dependently metabolized by human liver microsomes and human recombinant CYP enzymes. Less-polar metabolites were analyzed by gas chromatography–mass spectrometry monitoring, and their estimated molecular ions were m/z 286, 358 and 481 after non-derivatization, trimethylsilylation and pentafuorobenzyl oxime formation, respectively.
Results We tentatively identifed novel decarbonylated metabolites of CBD as keto-enol tautomers. Among eight major recombinant human CYP enzymes, only CYP3A4 catalyzed the formation of decarbonylated metabolites.
Conclusions was biotransformed to two decarbonylated metabolites, an enol-form (cyclopentadienol structure), and a keto-form (cyclopentenone structure) by human liver microsomes and CYP3A4.

Cannabinoid Metabolites as Inhibitors of Major Hepatic CYP450 Enzymes, with Implications for Cannabis-Drug Interactions
Shamema Nasrin, Christy J.W. Watson, Yadira X Perez-Paramo, and Philip Lazarus
Drug Metab Dispos 49:1070–1080, December 2021
Doi: 10.1124/dmd.121.000442
https://dmd.aspetjournals.org/conten...00442.full.pdf
The legalization of cannabis in many parts of the United States and other countries has led to a need for a more comprehensive understanding
of cannabis constituents and their potential for drug-drug interactions. Although (2)-trans-D9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most abundant cannabinoids present in cannabis, THC metabolites are found in plasma at higher concentrations and for a longer duration than that of the parent cannabinoids. To understand the potential for drugdrug interactions, the inhibition potential of major cannabinoids and their metabolites on major hepatic cytochrome P450 (P450) enzymes was examined. In vitro assays with P450-overexpressing
cell microsomes demonstrated that the major THC metabolites 11-hydroxy-D9-tetra-hydrocannabinol and 11-nor-9-carboxy-D9 THCglucuronide competitively inhibited several major P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6 (apparent Ki,u values 5 0.086 ± 0.066 mM and 0.90 ± 0.54 mM, 0.057 ± 0.044 mM and 2.1 ± 0.81 mM, 0.15 ± 0.067 mM and 2.3 ± 0.54 mM, respectively). 11-Nor-9-carboxy- D9- tetrahydrocannabinol exhibited no inhibitory activity against any CYP450 tested. THC competitively inhibited CYP1A2,
CYP2B6, CYP2C9, and CYP2D6; CBD competitively inhibited CYP3A4, CYP2B6, CYP2C9, CYP2D6, and CYP2E1; and CBN competitively inhibited CYP2B6, CYP2C9, and CYP2E1. THC and CBD showed mixed-type inhibition for CYP2C19 and CYP1A2, respectively. These data suggest that cannabinoids and major THC metabolites are able to inhibit the activities of multiple P450 enzymes, and basic static modeling of these data suggest the possibility of pharmacokinetic interactions between these cannabinoids and xenobiotics extensively metabolized by CYP2B6, CYP2C9, and CYP2D6.

Cannabidiol Modulates Cytokine Storm in Acute Respiratory Distress Syndrome Induced by Simulated Viral Infection Using Synthetic RNA.
Khodadadi, H., Lopes Salles, É., Jarrahi, A., Chibane, F., Costigliola, V., Yu, J. C., … Baban, B.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0043
Introduction: In the absence of effective antivirals and vaccination, the pandemic of COVID-19 remains the most significant challenge to our health care system in decades. There is an urgent need for definitive therapeutic intervention. Clinical reports indicate that the cytokine storm associated with acute respiratory distress syndrome (ARDS) is the leading cause of mortality in severe cases of some respiratory viral infections, including COVID-19. In recent years, cannabinoids have been investigated extensively due to their potential effects on the human body. Among all cannabinoids, cannabidiol (CBD) has demonstrated potent anti-inflammatory effects in a variety of pathological conditions. Therefore, it is logical to explore whether CBD can reduce the cytokine storm and treat ARDS. Materials and Methods: In this study, we show that intranasal application of Poly(I:C), a synthetic analogue of viral double-stranded RNA, simulated symptoms of severe viral infections inducing signs of ARDS and cytokine storm. Discussion: The administration of CBD downregulated the level of proinflammatory cytokines and ameliorated the clinical symptoms of Poly I:C-induced ARDS. Conclusion: Our results suggest a potential protective role for CBD during ARDS that may extend CBD as part of the treatment of COVID-19 by reducing the cytokine storm, protecting pulmonary tissues, and re-establishing inflammatory homeostasis

Cannabinol modulates neuroprotection and intraocular pressure: A potential multi-target therapeutic intervention for glaucoma
Rishi K Somvanshi , Shenglong Zou , Salam Kadhim , Sapna Padania , Eric Hsu , Ujendra Kumar
Biochim Biophys Acta Mol Basis Dis. 2021 Dec 16;1868(3):166325.
doi: 10.1016/j.bbadis.2021
https://pubmed.ncbi.nlm.nih.gov/34921975/
Objectives: Glaucoma is characterized by progressive damage of the retinal ganglion cells (RGCs), resulting in irreversible vision loss. Cannabinoids (CBs) ameliorate several factors that contribute to the progression of glaucoma, including increased intraocular pressure (IOP), degeneration of RGC and optical nerve (ON) damage. However, a direct correlation of specific CBs with the molecular events pertaining to glaucoma pathology is not well established. Therefore, this study aims to evaluate the role of cannabinol (CBN) on RGC protection, modulation of IOP, and its effects on the level of extracellular matrix (ECM) proteins using both in vitro and in vivo models of glaucoma.
Methods and results: When exposed to elevated hydrostatic pressure, CBN, in a dose-dependent manner, protected differentiated mouse 661W retinal ganglion precursor-like cells from pressure-induced toxicity. In human trabecular meshwork cells (hTM), CBN attenuated changes in the ECM proteins, including fibronectin and α-smooth muscle actin (α-SMA), as well as mitogen-activated protein kinases (phospho-ERK1/2) in the presence or absence of transforming growth factor-beta 2 (TGF-β2) induced stress. Ocular pharmacokinetic parameters were evaluated post-intravitreal (IVT) CBN delivery in vivo. Furthermore, we demonstrated that IVT-administered CBN improved pattern electroretinogram (pERG) amplitudes and reduced IOP in a rat episcleral vein laser photocoagulation model of glaucoma.
Conclusion: CBN promotes neuroprotection, abrogates changes in ECM protein, and normalizes the IOP levels in the eye. Therefore, our observations in the present study indicate a therapeutic potential for CBN in the treatment of glaucoma.
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Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain
Krishna Ghosh, Guang-Fen Zhang, Hong Chen, Shao-Rui Chen, Hui-Lin Pan
Journal of Biological Chemistry (2022),
doi: 10.1016/j.jbc.2022.101999
https://www.jbc.org/action/showPdf?pii=S0021-9258(22)00439-2
Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord. .

Cannabinoid modulation of mother-infant interaction: is it just about milk?
Antonia Manduca, Patrizia Campolongo and Viviana Trezza
Rev. Neurosci. 2012; 23(5-6): 707–722
DOI 10.1515/revneuro-2012-0074
Mother-infant interactions are essential for proper neurobehavioral development of the offspring, and disruptions in those relationships may result in neuroendocrine, neurochemical and behavioral alterations at adulthood. The neural circuitries involved in mother-infant interactions have not been completely elucidated yet. The brain endocannabinoid system plays an essential role in prenatal and postnatal neurobehavioral development. Here, we will summarize and discuss the available findings about the role of endocannabinoids in three key aspects of mother-infant interactions in rodents: suckling, maternal behavior and separationinduced ultrasonic vocalizations (USVs). The studies reviewed here show that endocannabinoids are not only involved in suckling initiation and, therefore, in the feeding and growth of the offspring, but also regulate the emotional reactivity of rodent pups, as measured by the rate of isolation-induced USVs. Conversely, less information is available about endocannabinoid modulation of maternal behavior, and therefore more research in this direction is warranted. Indeed, since Cannabis sativa preparations are widely used by young people, including pregnant and lactating women, it is important to understand whether developmental exposure to cannabinoids interferes with mother-infant bond formation, potentially leading to neurodevelopmental alterations and increased vulnerability to psychopathology later in life.

Cannabidiol or CBD Oil: Help, Hope, and Hype for Psychiatric and Neurologic Conditions.
Newton, M., & Newton, D. W. (2020).
Journal of the American Psychiatric Nurses Association, (2020).
doi:10.1177/1078390320929410
OBJECTIVE: This article presents proven, promising, and potential therapeutic uses for cannabidiol (CBD) in the treatment of psychiatric and neurologic conditions and diseases. It presents popular, but scientifically unproven health and therapeutic claims of CBD supporting the beneficial homeostatic effects of the intrinsic or endogenous cannabinoid system. It includes a review of cannabinoid pharmacology; it compares properties and the legal status of CBD and THC (delta 9-tetrahydrocannabinol) as well as the hemp and marijuana varieties of Cannabis, and it reviews the historic 2018 U.S. Food and Drug Administration approval of Epidiolex, an oral solution of cannabidiol for two rare treatment-resistant childhood epilepsies, as the first Cannabis-derived drug.
METHOD: We reviewed literature on cannabidiol, CBD, the endocannabinoid neuropharmacology system, and hemp and marijuana varieties of Cannabis sativa. RESULTS: The proven and promising medical uses and deficiencies of unproven health claims for CBD, legal implications for Cannabis-derived drugs, and comparisons of CBD and THC and hemp and marijuana are summarized objectively with pertinent references. CONCLUSION: CBD and CBD and THC combinations have potential to provide safe, effective therapy for several psychiatric and neurologic conditions and diseases. However, such achievement will require a uniform standard of CBD purity and strength, and corroboration from adequately large and rigorously controlled clinical research studies.

Cannabidiol: Pharmacology and potential therapeutic role in epilepsy and other neuropsychiatric disorders
Orrin Devinsky, Maria Roberta Cilio, Helen Cross, Javier Fernandez-Ruiz, Jacqueline French, Charlotte Hill, Russell Katz, Vincenzo Di Marzo, Didier Jutras-Aswad, William George Notcutt, Jose Martinez-Orgado, Philip J. Robson, Brian G. Rohrback, Elizabeth Thiele, Benjamin Whalley, and Daniel Friedman
Epilepsia. 2014 June ; 55(6): 791–802.
doi:10.1111/epi.12631
https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC4707 667&blobtype=pdf
Objective—To present a summary of current scientific evidence about the cannabinoid, cannabidiol (CBD) with regards to their relevance to epilepsy and other selected neuropsychiatric disorders. Methods—We summarize the presentations from a conference in which invited participants reviewed relevant aspects of the physiology, mechanisms of action, pharmacology and data from studies with animal models and human subjects. Results—Cannabis has been used to treat disease since ancient times. Δ9 -THC is the major psychoactive ingredient and cannabidiol (CBD) is the major non-psychoactive ingredient in cannabis. Cannabis and Δ9 -THC are anticonvulsant in most animal models but can be proconvulsant in some healthy animals. Psychotropic effects of Δ9 -THC limit tolerability. CBD is anticonvulsant in many acute animal models but there is limited data in chronic models. The antiepileptic mechanisms of CBD are not known, but may include effects on the equilibrative nucleoside transporter; the orphan G-protein-coupled receptor GPR55; the transient receptor potential of melastatin type 8 channel; the 5-HT1a receptor; the α3 and α1 glycine receptors; and the transient receptor potential of ankyrin type 1 channel. CBD has neuroprotective and antiinflammatory effects. CBD appears to be well tolerated in humans but small and methodologically limited studies of CBD in human epilepsy have been inconclusive. More recent anecdotal reports of high-ratio CBD:Δ9 -THC medical marijuana have claimed efficacy, but studies were not controlled. Significance—CBD bears investigation in epilepsy and other neuropsychiatric disorders, including anxiety, schizophrenia, addiction and neonatal hypoxic-ischemic encephalopathy. However, we lack data from well-powered double-blind randomized, controlled studies on the efficacy of pure CBD for any disorder. Initial dose-tolerability and double-blind randomized, controlled studies focusing on target intractable epilepsy populations such as patients with Dravet and Lennox-Gastaut syndromes are being planned. Trials in other treatment-resistant epilepsies may also be warranted. .

Cannabidiol prevents disruptions in sensorimotor gating induced by psychotomimetic drugs that last for 24-h with probable involvement of epigenetic changes in the ventral striatum
Prog Neuropsychopharmacol Biol Psychiatry. 2021 Dec 20;111:110352.
doi: 10.1016/j.pnpbp.2021.110352
Cannabidiol (CBD), a major non-psychotomimetic component of the Cannabis sativa plant, shows therapeutic potential in several psychiatric disorders, including schizophrenia. The molecular mechanisms underlying the antipsychotic-like effects of CBD are not fully understood. Schizophrenia and antipsychotic treatment can modulate DNA methylation in the blood and brain, resulting in altered expression of diverse genes associated with this complex disorder. However, to date, the possible involvement of DNA methylation in the antipsychotic-like effects of CBD has not been investigated. Therefore, this study aimed at evaluating in mice submitted to the prepulse inhibition (PPI) model: i) the effects of a single injection of CBD or clozapine followed by AMPH or MK-801 on PPI and global DNA methylation changes in the ventral striatum and prefrontal cortex (PFC); and ii). if the acute antipsychotic-like effects of CBD would last for 24-h. AMPH (5 mg/kg) and MK-801 (0.5 mg/kg) impaired PPI. CBD (30 and 60 mg/kg), similar to clozapine (5 mg/kg), attenuated AMPH- and MK801-induced PPI disruption. AMPH, but not MK-801, increased global DNA methylation in the ventral striatum, an effect prevented by CBD. CBD and clozapine increased, by themselves, DNA methylation in the prefrontal cortex. The acute effects of CBD (30 or 60 mg/kg) on the PPI impairment induced by AMPH or MK-801 was also detectable 24 h later. Altogether, the results show that CBD induces acute antipsychotic-like effects that last for 24-h. It also modulates DNA methylation in the ventral striatum, suggesting a new potential mechanism for its antipsychotic-like effects.
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Cannabidiol protects keratinocyte cell membranes following exposure to UVB and hydrogen peroxide.
Atalay, S., Dobrzy?ska, I., G?gotek, A., & Skrzydlewska, E.
Redox Biology, 36, 101613.(2020).
doi:10.1016/j.redox.2020.101613
Keratinocytes, the major cell type of the epidermis, are particularly sensitive to environmental factors including exposure to sunlight and chemical agents. Since oxidative stress may arise as a result of these factors, compounds are actively sought that can act as protective agents. Recently, cannabidiol (CBD), a phytocannabinoid found in Cannabis Sativa L., has gained increased interest due to its anti-inflammatory and antioxidant properties, and absence of psychoactive effects. This prompted us to analyze the protective effects of CBD on keratinocytes exposed to UVB irradiation and hydrogen peroxide. Here we show, using liquid chromatography mass spectrometry, that CBD was able to penetrate keratinocytes, and accumulated within the cellular membrane. CBD reduced redox balance shift, towards oxidative stress, caused by exposure UVB/hydrogen peroxide, estimated by superoxide anion radical generation and total antioxidant status and consequently lipid peroxidation level. CBD was found to protect keratinocytes by preventing changes in the composition of the cellular membrane, associated with UVB/hydrogen peroxide damages which included reduced polyunsaturated fatty acid levels,
increased sialic acid and lipid peroxidation products (malondialdehyde and 8-isoprostanes) levels. This maintains cell membranes integrity and prevents the release of lactate dehydrogenase. In addition, CBD prevented UVB/hydrogen peroxide-induced reduction of keratinocyte size and zeta potential, and also decreased activity of ATP-binding cassette membrane transporters. Together, these findings suggest that CBD could be a potential protective agent for keratinocytes against the harmful effects of irradiation and chemical environmental factors that cause oxidative stress

Cannabidiol improves survival and behavioural co-morbidities of Dravet syndrome in mice.
Patra, P. H., Serafeimidou-Pouliou, E., Bazelot, M., Whalley, B. J., Williams, C. M., & McNeish, A. J.
British Journal of Pharmacology. (2020).
doi:10.1111/bph.15003
Background and Purpose: Dravet syndrome is a severe, genetic form of paediatric epilepsy associated with premature mortality and co-morbidities such as anxiety, depression, autism, motor dysfunction and memory deficits. Cannabidiol is an approved anticonvulsive drug in the United States and Europe for seizures associated with Dravet syndrome in patients 2 years of age and older. We investigated its potential to prevent premature mortality and improve associated co-morbidities. Experimental Approach: The efficacy of sub-chronic cannabidiol administration in two mouse models of Dravet syndrome was investigated. The effect of cannabidiol on neonatal welfare and survival was studied using Scn1a?/? mice. We then used a hybrid, heterozygote Scn1a+/? mouse model to study the effect of cannabidiol on survival and behavioural co-morbidities: motor deficits (rotarod and static-beam test), gait abnormality (gait test), social anxiety (social interaction test), anxiety-like (elevated plus maze) and depressive-like behaviours (sucrose preference test) and cognitive impairment (radial arm maze test). Key Results: In Scn1a?/? mice, cannabidiol increased survival and delayed worsening of neonatal welfare. In Scn1a+/? mice, chronic cannabidiol administration did not show any adverse effect on motor function and gait, reduced premature mortality, improved social behaviour and memory function, and reduced anxiety-like and depressive-like behaviours. Conclusion and Implications: We are the first to demonstrate a potential diseasemodifying effect of cannabidiol in animal models of Dravet syndrome. Cannabidiol treatment reduced premature mortality and improved several behavioural comorbidities in Dravet syndrome mice. These crucial findings may be translated into human therapy to address behavioural co-morbidities associated with Dravet syndrome.

Cannabidiol is an effective helper compound in combination with bacitracin to kill Gram-positive bacteria.
Wassmann, C. S., Højrup, P., & Klitgaard, J. K.
Scientific Reports, 10(1).(2020).
doi:10.1038/s41598-020-60952-0
The cannabinoid cannabidiol (CBD) is characterised in this study as a helper compound against resistant bacteria. CBD potentiates the efect of bacitracin (BAC) against Gram-positive bacteria (Staphylococcus species, Listeria monocytogenes, and Enterococcus faecalis) but appears inefective against Gramnegative bacteria. CBD reduced the MIC value of BAC by at least 64-fold and the combination yielded an FIC index of 0.5 or below in most Gram-positive bacteria tested. Morphological changes in S. aureus as a result of the combination of CBD and BAC included several septa formations during cell division along with membrane irregularities. Analysis of the muropeptide composition of treated S. aureus indicated no changes in the cell wall composition. However, CBD and BAC treated bacteria did show a decreased rate of autolysis. The bacteria further showed a decreased membrane potential upon treatment with CBD; yet, they did not show any further decrease upon combination treatment. Noticeably, expression of a major cell division regulator gene, ezrA, was reduced two-fold upon combination treatment emphasising the impact of the combination on cell division. Based on these observations, the combination of CBD and BAC is suggested to be a putative novel treatment in clinical settings for treatment of infections with antibiotic resistant Gram-positive bacteria.

Cannabidiol selectively modulates interleukin (IL)-1β and IL-6 production in toll-like receptor activated human peripheral blood monocytes
Sera Sermet , Jinpeng Li , Anthony Bach , Robert B Crawford , Norbert E Kaminski
Toxicology . 2021 Dec;464:153016.
doi: 10.1016/j.tox.2021.153016
Cannabidiol (CBD) is a major non-euphoric cannabis-derived compound that has become popular in its over-the-counter use. CBD possesses low affinity for cannabinoid receptors, while the primary molecular target(s) by which it mediates biological activity remain poorly defined. Individuals commonly self-medicate using CBD products with little knowledge of its specific immunopharmacological effects on the human immune system; however, research has established primarily in rodent models that CBD possesses immune modulating properties. The objective of this study was to evaluate whether CBD modulates the innate immune response by human primary monocytes activated through toll-like receptors (TLR) 1-9. Monocytes were activated through each TLR and treated with CBD (0.5-10 μM) for 22 h. Monocyte secretion profiles for 13 immune mediators were quantified including: IL-4, IL-2, IP-10, IL-1β, TNFα, MCP-1, IL-17a, IL-6, IL-10, IFNγ, IL-12p70, IL-8, and TGF-β1. CBD treatment significantly suppressed secretion of proinflammatory cytokine IL-1β by monocytes activated through most TLRs, apart from TLRs 3 and 8. Additionally, CBD treatment induced significant modulation of IL-6 production by monocytes activated through most TLRs, except for TLRs 1 and 3. Most other monocyte-derived factors assayed were refractory to CBD modulation. Overall, CBD selectively altered monocyte-derived IL-1β and IL-6 when activated through most TLRs. This study is of particular importance as it provides a direct and comprehensive assessment of the effects of CBD on TLR-activated primary human monocytes at a time when CBD containing products are being widely used by the public.
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Cannabidiol Suppresses Angiogenesis and Stemness of Breast Cancer Cells by Downregulation of Hypoxia-Inducible Factors-1α

Min Jee Jo, Bu Gyeom Kim, Woo Young Kim, Dae-Hee Lee, Hye Kyeong Yun, Soyeon Jeong, Seong Hye Park, Bo Ram Kim, Jung Lim Kim, Dae Yeong Kim, Sun Il Lee and Sang Cheul Oh
Cancers 2021, 13, 5667.
Doi: 10.3390/cancers13225667
https://www.ncbi.nlm.nih.gov/labs/pm...s-13-05667.pdf
Cannabidiol (CBD), one of the compounds present in the marijuana plant, has antitumor properties. However, the effect of CBD on breast cancer remains unclear. The aim of this study was to assess the effects of CBD for the angiogenesis and stemness of breast cancer cells by decreasing the expression of hypoxia-induced factor-1_ (HIF-1_) through the Src/von Hippel– Lindau tumor suppressor protein (VHL) interaction. CBD can suppress angiogenesis and stem cell-like properties of breast cancer through Src/VHL/HIF-1_ signaling.
To assess the effect of Cannabidiol (CBD) on the angiogenesis and stemness of breast cancer cells as well as proliferation. Methods: mRNA level and the amount of protein of vascular endothelial growth factor (VEGF) were determined by qRT-PCR and ELISA. The angiogenic potential of breast cancer cells under hypoxic conditions was identified by the HUVEC tube formation assay. The degradation of HIF-1_ by CBD and the Src/von Hippel–Lindau tumor suppressor protein (VHL)
interaction were assessed by a co-immunoprecipitation assay andWestern blotting. To identify the stemness of mamospheres, they were evaluated by the sphere-forming assay and flow cytometry.
Results: CBD can suppress angiogenesis and stem cell-like properties of breast cancer through Src/VHL/HIF-1_ signaling. CBD may potentially be utilized in the treatment of refractory or recurrent breast cancer.

Cannabidiol Therapy for Refractory Epilepsy and Seizure Disorders
Victoria Golub , D Samba Reddy
Adv Exp Med Biol. 2021;1264:93-110.
doi: 10.1007/978-3-030-57369-0_7.
Cannabis-derived cannabinoids have neuroactive properties. Recently, there has been emerging interest in the use of cannabidiol (CBD)-enriched products for treatment of drug-resistant epilepsy. In 2018, the FDA approved the use of CBD-rich Epidiolex for two severe forms of epilepsy in children (Lennox-Gastaut and Dravet syndromes). Experimental research supports the use of CBD in many CNS disorders, though the mechanisms underlying its anticonvulsant and neuroprotective effects remain unclear. CBD has been shown to reduce inflammation, protect against neuronal loss, normalize neurogenesis, and act as an antioxidant. These actions appear to be due to the multimodal mechanism of action of CBD in the brain. This chapter briefly describes the current information on cannabis pharmacology with an emphasis on the clinical utility of CBD in the treatment of refractory epilepsies and other related seizure conditions. Clinical trials are ongoing for other forms of epilepsy and refractory seizures associated with infantile spasms, tuberous sclerosis, and Rett syndrome. Overall, adjunct CBD has been found to be generally safe and effective for treatment-resistant seizures in children with severe early-onset epilepsy. Whether an add-on CBD is efficacious for the long-term treatment of various epilepsy and seizure types in adults being tested in various clinical trials.

Cannabidiol treatment in hand osteoarthritis and psoriatic arthritis a randomized, double-blind placebo-controlled trial
Vela, Jonathan Dreyer, Lene Petersen, Kristian Kjæ Lars, Arendt Nielsen Duch, Kirsten Skjærbæk Kristensen, Salome
J Pain. 2021 Aug 27
DOI: 10.1097/j.pain.0000000000002466
Cannabidiol (CBD) is increasingly used as analgesic medication even though the recent International Association for the Study of Pain presidential task force on cannabis and cannabinoid analgesia found a lack of trials examining CBD for pain management. The present trial examines CBD as add on analgesic therapy in patients with hand osteoarthritis or psoriatic arthritis experiencing moderate pain intensity despite therapy. Using a randomized double-blind, placebo-controlled design, patients received synthetic CBD 20-30mg or placebo daily for 12 weeks. Primary outcome was pain intensity during the last 24 hours (0-100mm); safety outcomes were percentage of patients experiencing adverse events and a characterization of serious adverse events. Explorative outcomes included change in Pittsburgh Sleep Quality Index (PSQI), Hospital Anxiety and Depression Scale (HADS), Pain Catastrophizing Scale (PCS) and Health Assessment Questionnaire (HAQ-DI). One hundred and thirty-six patients were randomized 129 were included in the primary analysis. Between group difference in pain intensity at 12 weeks was 0.23mm (95%CI -9.41 to 9.90; p = 0.96). 22% patients receiving CBD and 21% receiving placebo experienced a reduction in pain intensity of more than 30mm. We found neither clinically nor statistically significant effect of CBD for pain intensity in patients with hand osteoarthritis and psoriatic arthritis when compared to placebo. Additionally, no statistically significant effects were found on sleep quality, depression, anxiety, or pain catastrophizing scores.
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Cannabidiol use and effectiveness: realworld evidence from a Canadian medical cannabis clinic
Lucile Rapin, Rihab Gamaoun, Cynthia El Hage, Maria Fernanda Arboleda and Erin Prosk
Journal of Cannabis Research (2021) 3:19
DOI: 10.1186/s42238-021-00078-w
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223341/
Background: Cannabidiol (CBD) is a primary component in the cannabis plant; however, in recent years, interest in CBD treatments has outpaced scientific research and regulatory advancement resulting in a confusing landscape of misinformation and unsubstantiated health claims. Within the limited results from randomized controlled trials, and lack of trust in product quality and known clinical guidelines and dosages, real-world evidence (RWE) from countries with robust regulatory frameworks may fill a critical need for patients and healthcare professionals. Despite growing evidence and interest, no real-world data (RWD) studies have yet investigated patients’ reports of CBD impact on symptom control in the common expression of pain, anxiety, depression, and poor wellbeing. The objective of this study is to assess the impact of CBD-rich treatment on symptom burden, as measured with a specific symptom assessment scale (ESAS-r).
Methods: This retrospective observational study examined pain, anxiety, depression symptoms, and wellbeing in 279 participants over 18 years old, prescribed with CBD-rich treatment at a network of clinics dedicated to medical cannabis in Quebec, Canada. Data were collected at baseline, 3 (FUP1), and 6 (FUP2) month after treatment initiation. Groups were formed based on symptom severity (mild vs
moderate/severe) and based on changes to treatment plan at FUP1 (CBD vs THC:CBD). Two-way mixed ANOVAs were used to assess ESAS-r scores differences between groups and between visits.
Results: All average ESAS-r scores decreased between baseline and FUP1 (all ps < 0.003). The addition of delta-9- tetrahydrocannabinol (THC) during the first follow-up had no effect on symptom changes. Patients with moderate/ severe symptoms experienced important improvement at FUP1 (all ps < 0.001), whereas scores on pain, anxiety, and wellbeing of those with mild symptoms actually increased. Differences in ESAS-r scores between FUP1 and FUP2 were not statistically different.
Conclusion: This retrospective observational study suggests CBD-rich treatment has a beneficial impact on pain, anxiety, and depression symptoms as well as overall wellbeing only for patients with moderate to severe symptoms; however, no observed effect on mild symptoms. The results of this study contribute to address the myths and misinformation about CBD treatment and demand further investigation

Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism
T D M Hill, M-G Cascio, B Romano, M Duncan, R G Pertwee, C M Williams, B J Whalley and A J Hill
British Journal of Pharmacology (2013) 170 679–692
https://www.ncbi.nlm.nih.gov/pmc/art...h0170-0679.pdf
BACKGROUND AND PURPOSE Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis-derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB1 receptors.
EXPERIMENTAL APPROACH The anticonvulsant profiles of two CBDV BDSs (50–422 mg·kg−1 ) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB1 receptors was evaluated using displacement binding assays.
KEY RESULTS CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg−1 ) and audiogenic seizure models (≥87 mg·kg−1 ), and suppressed pilocarpine-induced convulsions (≥100 mg·kg−1 ). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ9 -tetrahydrocannabinol and Δ9 -tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB1 cannabinoid receptors than purified CBDV. CONCLUSIONS AND IMPLICATIONS CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB1 cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy

Cannabidivarin (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in epilepsy-related gene expression.
Amada et al. (2013),
PeerJ 1:e214;
DOI 10.7717/peerj.214
To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV), have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o.) on acute, pentylenetetrazole (PTZ: 95 mg/kg, i.p.)-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1) by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00) and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25) and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ?3.25) and non-responders (criterion: seizure severity >3.25), PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV responders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy
Lyndsey L Anderson , Marika Heblinski , Nathan L Absalom , Nicole A Hawkins , Michael T Bowen, Melissa J Benson , Fan Zhang, Dilara Bahceci, Peter T Doohan, Mary Chebib, Iain S McGregor , Jennifer A Kearney, Jonathon C Arnold
Br J Pharmacol . 2021 Dec;178(24):4826-4841.
doi: 10.1111/bph.15661
Background and purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties.
Experimental approach: We used the Scn1a+/- mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/- mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets.
Key results: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/- mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors.
Conclusion and implications: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class.
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Cannabigerol Is a Potential Therapeutic Agent in a Novel Combined Therapy for Glioblastoma
Tamara T. Lah, Metka Novak, Milagros A. Pena Almidon, Oliviero Marinelli, Barbara Žvar Baškovi?, Bernarda Majc, Mateja Mlinar, Roman Bošnjak, Barbara Breznik, Roby Zomer and Massimo Nabissi
Cells (2021 Feb)
DOI: 10.3390/cells10020340
Among primary brain tumours, glioblastoma is the most aggressive. As early relapses are unavoidable despite standard-of-care treatment, the cannabinoids delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) alone or in combination have been suggested as a combined treatment strategy for glioblastomas. However, the known psychoactive effects of THC hamper its medical applications in these patients with potential cognitive impairment due to the progression of the disease. Therefore, nontoxic cannabigerol (CBG), being recently shown to exhibit anti-tumour properties in some carcinomas, is assayed here for the first time in glioblastoma with the aim to replace THC. We indeed found CBG to effectively impair the relevant hallmarks of glioblastoma progression, with comparable killing effects to THC and in addition inhibiting the invasion of glioblastoma cells. Moreover, CBG can destroy therapy-resistant glioblastoma stem cells, which are the root of cancer development and extremely resistant to various other treatments of this lethal cancer. CBG should present a new yet unexplored adjuvant treatment strategy of glioblastoma.
Glioblastoma is the most aggressive cancer among primary brain tumours. As with other cancers, the incidence of glioblastoma is increasing; despite modern therapies, the overall mean survival of patients post-diagnosis averages around 16 months, a
figure that has not changed in many years. Cannabigerol (CBG) has only recently been reported to prevent the progression of certain carcinomas and has not yet been studied in glioblastoma. Here, we have compared the cytotoxic, apoptotic, and anti-invasive effects of the purified natural cannabinoid CBG together with CBD and THC on established differentiated glioblastoma tumour cells and glioblastoma stem cells. CBG and THC reduced the viability of both types of cells to a similar extent, whereas combining CBD with CBG was more efficient than with THC. CBD and CBG, both alone and in combination, induced caspase-dependent cell apoptosis, and there was no additive THC effect. Of note, CBG inhibited glioblastoma invasion in a similar manner to CBD and the chemotherapeutic temozolomide. We have demonstrated that THC has little added value in combined-cannabinoid glioblastoma treatment, suggesting that this psychotropic cannabinoid should be replaced with CBG in future clinical studies of glioblastoma therapy.

Cannabinoid and Terpenoid Doses are Associated with Adult ADHD Status of Medical Cannabis Patients
Jeffrey Y. Hergenrather, Joshua Aviram, Yelena Vysotski, Salvatore Campisi-Pinto, Gil M. Lewitus, and David Meiri
Rambam Maimonides Med J January 2020 Volume 11 Issue 1
doi: 10.5041/RMMJ.10384.
https://www.rmmj.org.il/userimages/1...038Article.pdf
Objective: The aim of this cross-sectional questionnaire-based study was to identify associations between the doses of cannabinoids and terpenes administered, and symptoms of attention deficit hyperactivity
disorder (ADHD).

Cannabinoid 1 (CB-1) receptor antagonists: a molecular approach to treating acute cannabinoid overdose
Journal of Neural Transmission. (2019).
doi:10.1007/s00702-019-02132-7
The legalization of cannabis for both recreational and medical use in the USA has resulted in a dramatic increase in the number of emergency department visits and hospital admissions for acute cannabinoid overdose (also referred to as cannabis intoxication and cannabis poisoning). Both “edibles” (often sold as brownies, cookies, and candies) containing large amounts of ?9 -tetrahydrocannabinol and synthetic cannabinoids (many possessing higher potencies and efcacies than ?9 -tetrahydrocannabinol) are responsible for a disproportionate number of emergency department visits relative to smoked cannabis. Symptoms of acute cannabinoid overdose range from extreme lethargy, ataxia, and generalized psychomotor impairment to feelings of panic and anxiety, agitation, hallucinations, and psychosis. Treatment of acute cannabinoid overdose is currently supportive and symptom driven. Converging lines of evidence indicating many of the symptoms which can precipitate an emergency department visit are mediated through activation of cannabinoid1 receptors. Here, we review the evidence that cannabinoid1 receptor antagonists, originally developed for indications ranging from obesity to smoking cessation and schizophren

Cannabinoid CB2 receptor: a new target for controlling neural cell survival?
Javier Ferna´ndez-Ruiz, Julia´n Romero, Guillermo Velasco, Rosa M. Tolo´n , Jose´ A. Ramos and Manuel Guzma´n
TRENDS in Pharmacological Sciences Vol.28 No.1
DOI:10.1016/j.tips.2006.11.001
Two types of cannabinoid receptor have been cloned and characterized. Whereas CB1 receptors are ubiquitously expressed in neurons of the CNS, CB2 receptors have been thought to be absent from the CNS. Recent data now question this notion and support the expression of CB2 receptors in microglial cells, astrocytes and even some neuron subpopulations. This discrete distribution makes CB2 receptors interesting targets for treating neurological disorders because CB2-selective agonists lack psychoactivity. Here, we review evidence supporting the idea that CB2 receptors are implicated in the control of fundamental neural cell processes, such as proliferation and survival, and that their pharmacological manipulation might be useful for both delaying the progression of neurodegenerative disorders and inhibiting the growth of glial tumors.

Cannabinoid Combination Induces Cytoplasmic Vacuolation in MCF-7 Breast Cancer Cells.
Schoeman, R., Beukes, N., & Frost, C.
Molecules, 25(20), 4682.(2020).
doi:10.3390/molecules25204682
This study evaluated the synergistic anti-cancer potential of cannabinoid combinations across the MDA-MB-231 and MCF-7 human breast cancer cell lines. Cannabinoids were combined and their synergistic interactions were evaluated using median effect analysis. The most promising cannabinoid combination (C6) consisted of tetrahydrocannabinol, cannabigerol (CBG), cannabinol (CBN), and cannabidiol (CBD), and displayed favorable dose reduction indices and limited cytotoxicity against the non-cancerous breast cell line, MCF-10A. C6 exerted its effects in the MCF-7 cell line by inducing cell cycle arrest in the G2 phase, followed by the induction of apoptosis. Morphological observations indicated the induction of cytoplasmic vacuolation, with further investigation suggesting that the vacuole membrane was derived from the endoplasmic reticulum. In addition, lipid accumulation, increased lysosome size, and significant increases in the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) expression were also observed. The selectivity and ability of cannabinoids to halt cancer cell proliferation via pathways resembling apoptosis, autophagy, and paraptosis shows promise for cannabinoid use in standardized breast cancer treatment.

Cannabinoid Control of Neurogenic Inflammation.
McKenna, M., & McDougall, J. J.
British Journal of Pharmacology. (2020).
doi:10.1111/bph.15208
A significant number of cannabinoids are known to have analgesic and anti-inflammatory properties in various diseases. Due to their prejunctional location, cannabinoid receptors can inhibit synaptic transmission and have the potential to regulate neurogenic inflammation. Neurogenic inflammation occurs when a noxious signal is detected in the periphery initiating an antidromic axon reflex in the same sensory neurone leading to depolarisation of the afferent terminal. Neuropeptides are subsequently released and contribute to vasodilation, plasma extravasation and modulation of immune cells. Endo-, synthetic and phytocannabinoids can reduce neuroinflammation by inhibiting afferent firing and inflammatory neuropeptide release. Thus, in addition to a direct effect on vascular smooth muscle and inflammatory cells, cannabinoids can reduce inflammation by silencing small diameter neurones. This review interrogates the neuropharmacological processes involved in regulating antidromic depolarisation of afferent nerve terminals by cannabinoids and the control of neurogenic inflammation in different diseases.

Cannabinoid-deficient Benin republic hemp (Cannabis sativa L.) improves semen parameters by reducing prolactin and enhancing anti-oxidant status.
Alagbonsi, A. I., Olayaki, L. A., Abdulrahim, H. A., Adetona, T. S., & Akinyemi, G. T. (2019).
BMC Complementary and Alternative Medicine, 19(1).
doi:10.1186/s12906-019-2541-5
Background: Nigerian Cannabis sativa (hemp) causes male gonadotoxicity by inducing hyperprolactinemia, down-regulation of hypothalamic-pituitary-testicular axis, and oxidative stress. Benin republic hemp has been preferred by illicit users in Nigeria but its effect on male fertility is not understood. This study determined and compared the compositions of Benin republic hemp ethanol extract (BHE) and Nigerian hemp. The effects of BHE on semen parameters, reproductive hormones, and anti-oxidant status, and the possibility of bromocriptine (prolactin inhibitor) to abolish hemp-induced toxicities in rats were also investigated. Methods: Thirty-six male Wistar rats were blindly randomized into 6 oral treatment groups (n = 6 each). Groups I (control) and II received normal saline and bromocriptine (3 mg/kg) respectively. Groups III and IV received 2 mg/kg of BHE alone and in combination with bromocriptine respectively, while groups V and VI received 10 mg/kg BHE alone and in combination with bromocriptine respectively. Comparisons among the groups were done by one-way analysis of variance, followed by post-hoc Tukey multiple comparison test. Statistical significance was considered at p < 0.05. Results: The BHE has no cannabichromene and tetrahydrocannabinol but a very small quantity of cannabinol and higher quantity of fatty acids when compared to Nigerian hemp. Both doses of BHE increased sperm count, morphology and viability but not motility. Co-administration of BHE with bromocriptine lowered sperm count but increased sperm morphology and viability. Bromocriptine and/or BHE caused reduction in the plasma prolactin level, increase in the plasma superoxide dismutase activity, but no significant change in the plasma gonadotropin releasing hormone, follicle stimulating hormone (except for the increase in rats that received bromocriptine+ 10 mg/kg BHE), luteinizing hormone, estradiol, malondialdehyde and glutathione peroxidase. The 10 mg/kg BHE or bromocriptine+BHE (both doses) increased total anti-oxidant capacity and catalase. Conclusions: The BHE improves semen parameters by reducing plasma prolactin and enhancing plasma anti-oxidant status.

Cannabinoid Delivery Systems for Pain and Inflammation Treatment
Natascia Bruni, Carlo Della Pepa, Simonetta Oliaro-Bosso, Enrica Pessione,
Daniela Gastaldi and Franco Dosio
Molecules 2018, 23, 2478;
doi:10.3390/molecules23102478
There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

Cannabinoid derivatives acting as dual PPAR?/CB2 agonists as therapeutic agents for Systemic Sclerosis.
García-Martín, A., Garrido-Rodríguez, M., Navarrete, C., Caprioglio, D., Palomares, B., DeMesa, J., … Muñoz, E.
Biochemical Pharmacology.(2019).*
doi:10.1016/j.bcp.2019.02.029*
The endocannabinoid system (ECS) may play a role in the pathophysiology of systemic sclerosis (SSc). Cannabinoids acting as dual PPAR?/CB2 agonists, such as VCE-004.8 and Ajulemic acid (AjA), have been shown to alleviate skin fibrosis and inflammation in SSc models. Since both compounds are being tested in humans, we compared their activities in the bleomycin (BLM) SSc model. Specifically, the pharmacotranscriptomic signature of the compounds was determined by RNA-Seq changes in the skin of BLM mice treated orally with AjA or EHP- 101, a lipidic formulation of VCE-004.8. While both compounds down-regulated the expression of genes involved in the inflammatory and fibrotic components of the disease and the pharmacotranscriptomic signatures were similar for both compounds in some pathways, we found key differences between the compounds in vasculogenesis. Additionally, we found 28 specific genes with translation potential by comparing with a list of human scleroderma genes. Immunohistochemical analysis revealed that both compounds prevented fibrosis, collagen accumulation and Tenascin C (TNC) expression. The endothelial CD31+/CD34+ cells and telocytes were reduced in BLM mice and restored only by EHP-101 treatment. Finally, differences were found in plasmatic biomarker analysis; EHP-101, but not AjA, enhanced the expression of some factors related to angiogenesis and vasculogenesis. Altogether the results indicate that dual PPAR?/CB2 agonists qualify as a novel therapeutic approach for the treatment of SSc and other fibrotic diseases. EHP-101 demonstrated unique mechanisms of action related to the pathophysiology of SSc that could be beneficial in the treatment of this complex disease without current therapeutic options.

Cannabinoid–hormone interactions in the regulation of motivational processes
Hassan H. LópezHormones and Behavior 58 (2010) 100–110
doi:10.1016/j.yhbeh.2009.10.005 https://d1wqtxts1xzle7.cloudfront.ne.. .ne%3B+filename %3DCannabinoid_hormone_interac tions_in_the.pdf&Expires=16488 46795&Signature=M6uyVrfsBxCNVQ X7Rn83jlahHuNeeggboBMUGOc-QmKt034vtUmssSJBYdOCUkK-YIZZhE~Z44rLDEkk8G7YrmnQJMXVnP hXBVQksS8m80EjtSzcIK8In3pRUkRu x85IB7wscY5bnRF5QHquW6Dct~8wQo m6jw6uRxmWkZKzeC89MAfZ~dBRslif hA51Pzrgdw6215fQx23tPvWGddB7fd FZsLZXtELqWC9uLkK4dfu0U81FMBqg wtU2FQBtbrVjO93D3e9vFX3RjWfSw2 yHf8FlSSBifv2k-NT2NqUIS5RR4W2je1VBQWDSr4g-sr~a0GNfWT-5sd0qb1PBdY65gg__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA
There is a bi-directionality in hormone–cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic–pituitary–gonadal ), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a “case study.” I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction

Cannabinoid Hyperemesis
Joseph V. Pergolizzi Jr. Jo Ann LeQuang John F. Bisney
Med Cannabis Cannabinoids November 15, 2018
DOI: 10.1159/000494992
Cannabinoid hyperemesis syndrome (CHS) is a paradoxical condition in which a long-term cannabis user suffers an episode of intractable vomiting that may last days separated by longer asymptomatic periods of weeks or months. Cannabinoids are often utilized for their antiemetic properties, so CHS can be a puzzling condition, and the diagnosis of CHS may be disputed by patients. Unlike other cyclic vomiting syndromes, CHS can be relieved by hot showers or topical capsaicin. Abstinence from cannabinoids causes CHS to resolve, sometimes in a matter of days or hours. Marijuana users as well as many clinicians are not aware of CHS, and patients may undergo unnecessary tests, scans, and other procedures to get an accurate diagnosis. Symptoms may be severe enough to require hospitalization. With liberalization of marijuana laws and favorable public opinion about the healing properties of cannabis, CHS may be more frequently observed in clinical practice

Cannabinoid Hyperemesis Syndrome: A Case Report and Review of Pathophysiology.
Iacopetti, C. L., & Packer, C. D.
Clinical Medicine & Research, 12(1-2), 65–67. (2014)
doi:10.3121/cmr.2013.1179
Cannabis is the most widely used illicit drug in the U nited States, w ith lifetime prevalence of use estim ated at 42% to 46%. The antiemetic properties o f cannabis are w ell-known by the medical community and the general public; however, less well-recognized is the paradoxical potential for certain chronic users to develop hyperemesis. We describe in this case a patient with prior extensive work-up for nausea and vomiting and previous diagnosis of cyclic vomiting syndrome who presented with characteristic features of cannabinoid hyperemesis syndrome. We review the current literature for this condition and highlight potential mechanisms for its pathogenesis.

Cannabinoid hyperemesis syndrome: Clinical diagnosis of an underrecognised manifestation of chronic cannabis abuse
Siva P Sontineni, Sanjay Chaudhary, Vijaya Sontineni, Stephen J Lanspa
World J Gastroenterol March 14, 2009 Volume 15 Number 10
doi:10.3748/wjg.15.1264
World J Gastroenterol March 14, 2009 Volume 15 Number 10
Cannabis is a common drug of abuse that is associated with various long-term and short-term adverse effects. The nature of its association with vomiting after chronic abuse is obscure and is underrecognised by clinicians. In some patients this vomiting can take on a pattern similar to cyclic vomiting syndrome with a peculiar compulsive hot bathing pattern, which relieves intense feelings of nausea and accompanying symptoms. In this case report, we describe a twentytwo year-old-male with a history of chronic cannabis abuse presenting with recurrent vomiting, intense nausea and abdominal pain. In addition, the patient reported that the hot baths improved his symptoms during these episodes. Abstinence from cannabis led to resolution of the vomiting symptoms and abdominal pain. We conclude that in the setting of chronic cannabis abuse, patients presenting with chronic severe nausea and vomiting that can sometimes be accompanied by abdominal pain and compulsive hot bathing behaviour, in the absence of other obvious causes, a diagnosis of cannabinoid hyperemesis syndrome should be considered.

Cannabinoid Hyperemesis Syndrome: Public Health Implications and a Novel Model Treatment Guideline
Jeff Lapoint, Seth Meyer, Charles K. Yu, Kristi L. Koenig, Roneet Lev, Sayone Thihalolipavan, Katherine Staats, Christopher A. Kahn,
Western Journal of Emergency Medicine 380 Volume 19, no. 2: March 2018
DOI: 10.5811/westjem.2017.11.36368
Introduction: Cannabinoid hyperemesis syndrome (CHS) is an entity associated with cannabinoid overuse. CHS typically presents with cyclical vomiting, diffuse abdominal pain, and relief with hot
showers. Patients often present to the emergency department (ED) repeatedly and undergo extensive evaluations including laboratory examination, advanced imaging, and in some cases unnecessary procedures. They are exposed to an array of pharmacologic interventions including opioids that not only lack evidence, but may also be harmful. This paper presents a novel treatment guideline that highlights the identification and diagnosis of CHS and summarizes treatment strategies aimed at resolution of symptoms, avoidance of unnecessary opioids, and ensuring patient safety.
Methods: The San Diego Emergency Medicine Oversight Commission in collaboration with the County of San Diego Health and Human Services Agency and San Diego Kaiser Permanente Division of Medical Toxicology created an expert consensus panel to establish a guideline to unite the ED community in the treatment of CHS.
Results: Per the consensus guideline, treatment should focus on symptom relief and education on the need for cannabis cessation. Capsaicin is a readily available topical preparation that is reasonable to use as first-line treatment. Antipsychotics including haloperidol and olanzapine have been reported to provide complete symptom relief in limited case studies. Conventional antiemetics including antihistamines, serotonin antagonists, dopamine antagonists and benzodiazepines may have limited effectiveness. Emergency physicians should avoid opioids if the diagnosis of CHS is certain and educate patients thatcannabis cessation is the only intervention that will provide complete symptom relief.
Conclusion: An expert consensus treatment guideline is provided to assist with diagnosis and appropriate treatment of CHS. Clinicians and public health officials should identity and treat CHS patients with strategies that decrease exposure to opioids, minimize use of healthcare resources, and maximize patient safety.

Cannabinoid Hyperemesis Syndrome Survey and Genomic Investigation
Ethan B. Russo, Chris Spooner, Len May, Ryan Leslie, and Venetia L. Whiteley
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0046
https://www.liebertpub.com/doi/pdf/1.../can.2021.0046
Background: Cannabinoid hyperemesis syndrome (CHS) is a diagnosis of exclusion with intractable nausea, cyclic vomiting, abdominal pain, and hot bathing behavior associated with ongoing tetrahydrocannabinol (THC) exposure. Increasing cannabis use may elevate CHS prevalence, exacerbating a public health issue with attendant costs and morbidity.
Objective, Design, and Data Source: This study, the largest contemporaneous database, investigated genetic mutations underlying CHS. Patients with CHS diagnosis and ongoing symptoms were compared with current cannabis users lacking symptoms.
Target Population: A screening questionnaire was posted online. Of 585 respondents, 205 qualified as the CHS pool and 54 as controls; a reduced pool of 28 patients and 12 controls ultimately completed genomic testing. Results: Patients and controls were high-frequency users of cannabis flower or concentrates (93%), using multiple grams/day of THC-predominant material. Among patients, 15.6% carried diagnoses of cannabis dependency or addiction, and 56.6% experienced withdrawal symptoms. About 87.7% of patients improved after cannabis cessation, most suffering recurrence rapidly after resumption. Findings in patients included mutations in genes COMT {odds ratio, 12 (95% confidence limit [CL], 1.3–88.1) p = 0.012}, transient receptor potential vanilloid receptor 1 (TRPV1) (odds ratio, 5.8 [95% CL, 1.2–28.4] p = 0.015), CYP2C9 (odds ratio, 7.8 [95% CL, 1.1–70.1] p = 0.043), gene coding dopamine-2 receptor (DRD2) (odds ratio, 6.2 [95% CL, 1.1–34.7] p = 0.031), and ATPbinding cassette transporter gene (ABCA1) (odds ratio, 8.4 [95% CL, 1.5–48.1] p = 0.012).
Limitations: Some participants were reluctant to undergo genetic testing; only 28 of 99 CHS patients who agreed to testing ultimately returned a kit.
Conclusion: This is the largest patient cohort of CHS examined to date, and first to note associated mutations in genes affecting neurotransmitters, the endocannabinoid system, and the cytochrome P450 complex associated with cannabinoid metabolism. Although the sample size was smaller than desired, these preliminary findings may contribute to the growing body of knowledge, stimulate additional investigation, help elucidate the pathophysiology of CHS, and, ultimately, direct future treatment

Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after ?9-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716.
Higgs, S., Williams, C. M., & Kirkham, T. C.
Psychopharmacology, 165(4), 370–377. (2003).
doi:10.1007/s00213-002-1263-3
Rationale: Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability. Objectives: The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution. Methods: Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats. Results: D9 -tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/ kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a nonsignificant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent. Conclusions: These data are consistent with endocannabinoid involvement in the mediation of food palatability

Cannabinoid Interactions with Cytochrome P450 Drug Metabolism: a Full-Spectrum Characterization
Peter T Doohan, Lachlan D Oldfield, Jonathon C Arnold, Lyndsey L Anderson
AAPS J. 2021 Jun 28;23(4):91.
DOI: 10.1208/s12248-021-00616-7
Medicinal cannabis use has increased exponentially with widespread legalization around the world. Cannabis-based products are being used for numerous health conditions, often in conjunction with prescribed medications. The risk of clinically significant drug-drug interactions (DDIs) increases in this setting of polypharmacy, prompting concern among health care providers. Serious adverse events can result from DDIs, specifically those affecting CYP-mediated drug metabolism. Both cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC), major constituents of cannabis, potently inhibit CYPs. Cannabis-based products contain an array of cannabinoids, many of which have limited data available regarding potential DDIs. This study assessed the inhibitory potential of 12 cannabinoids against CYP-mediated drug metabolism to predict the likelihood of clinically significant DDIs between cannabis-based therapies and conventional medications. Supersomes™ were used to screen the inhibitory potential of cannabinoids in vitro. Twelve cannabinoids were evaluated at the predominant drug-metabolizing isoforms: CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2B6, and CYP2C19. The cannabinoids exhibited varied effects and potencies across the CYP isoforms. CYP2C9-mediated metabolism was inhibited by nearly all the cannabinoids with estimated Ki values of 0.2-3.2 μM. Most of the cannabinoids inhibited CYP2C19, whereas CYP2D6, CYP3A4, and CYP2B6 were either not affected or only partially inhibited by the cannabinoids. Effects of the cannabinoids on CYP2D6, CYP1A2, CYP2B6, and CYP3A4 metabolism were limited so in vivo DDIs mediated by these isoforms would not be predicted. CYP2C9-mediated metabolism was inhibited by cannabinoids at clinically relevant concentrations. In vivo DDI studies may be justified for CYP2C9 substrates with a narrow therapeutic index.
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Cannabinoid modulation of corticolimbic activation to threat in trauma-exposed adults: a preliminary study.
Rabinak, C. A., Blanchette, A., Zabik, N. L., Peters, C., Marusak, H. A., Iadipaolo, A., & Elrahal, F.
Psychopharmacology.(2020).
doi:10.1007/s00213-020-05499-8
Rationale Excessive fear and anxiety, coupled with corticolimbic dysfunction, are core features of stress- and trauma-related psychopathology, such as posttraumatic stress disorder (PTSD). Interestingly, low doses of ?9 -tetrahydrocannabinol (THC) can produce anxiolytic effects, reduce threat-related amygdala activation, and enhance functional coupling between the amygdala and medial prefrontal cortex and adjacent rostral cingulate cortex (mPFC/rACC) during threat processing in healthy adults. Together, these findings suggest the cannabinoid system as a potential pharmacological target in the treatment of excess fear and anxiety. However, the effects of THC on corticolimbic functioning in response to threat have not be investigated in adults with traumarelated psychopathology. Objective To address this gap, the present study tests the effects of an acute low dose of THC on corticolimbic responses to threat in three groups of adults: (1) non-trauma-exposed healthy controls (HC; n = 25), (2) trauma-exposed adults without PTSD (TEC; n = 27), and (3) trauma-exposed adults with PTSD (n = 19). Methods Using a randomized, double-blind, placebo-controlled, between-subjects design, 71 participants were randomly assigned to receive either THC or placebo (PBO) and subsequently completed a well-established threat processing paradigm during functional magnetic resonance imaging. Results In adults with PTSD, THC lowered threat-related amygdala reactivity, increased mPFC activation during threat, and increased mPFC-amygdala functional coupling. Conclusions These preliminary data suggest that THC modulates threat-related processing in trauma-exposed individuals with PTSD, which may prove advantageous as a pharmacological approach to treating stress- and trauma-related psychopathology.

Cannabinoid Modulation of Neuroinflammatory Disorders
Viviane M. Saito, Rafael M. Rezende and Antonio L. Teixeira
Current Neuropharmacology, 2012, 10, 159-166
https://www.ncbi.nlm.nih.gov/pmc/art.../CN-10-159.pdf
In recent years, a growing interest has been dedicated to the study of the endocannabinoid system. The isolation of Cannabis sativa main psychotropic compound, Δ9-tetrahydrocannabinol (THC), has led to the discovery of an atypical neurotransmission system that modulates the release of other neurotransmitters and participates in many biological processes, including the cascade of inflammatory responses. In this context, cannabinoids have been studied for their possible therapeutic properties in neuroinflammatory diseases. In this review, historic and biochemical aspects of cannabinoids are discussed, as well as their function as modulators of inflammatory processes and therapeutic perspectives for neurodegenerative disorders, particularly, multiple sclerosis.

Cannabinoid receptor expression in non-small cell lung cancer. Effectiveness of tetrahydrocannabinol and cannabidiol inhibiting cell proliferation and epithelial-mesenchymal transition in vitro.
Milian, L., Mata, M., Alcacer, J., Oliver, M., Sancho-Tello, M., Martín de Llano, J. J., … Carda, C.
PLOS ONE, 15(2), e0228909. (2020).
doi:10.1371/journal.pone.0228909*
Background/Objective Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines. Methods Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10–100 ?M), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF). Results The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increasedsurvival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used. Conclusions The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.

Cannabinoid receptor 2 activation alleviates septic lung injury by promoting autophagy via inhibition of inflammatory mediator release.
Liu, A. P., Yuan, Q. H., Zhang, B., Yang, L., He, Q. W., Chen, K., … Zhan, J.
Cellular Signalling, 109556. (2020).
doi:10.1016/j.cellsig.2020.109556
Septic lung injury is one of main causes of high mortality in severe patients. Inhibition of excessive inflammatory response is considered as an effective strategy for septic lung injury. Previous studies have shown that cannabinoid receptor 2 (CB2), a G protein-coupled receptor, play an important role in immunosuppression. Whether CB2 can be used as a therapeutic target for septic lung injury is unclear. The aim of this study is to explore the role of CB2 in sepsis and its potential mechanism. In this study, treatment with HU308, a specific agonist of CB2, could reduce lung pathological injury, decrease the level of inflammatory cytokines and strengthen the expression of autophagy-related gene after cecal ligation puncture (CLP)-induced sepsis in mice. Similar results were obtained in RAW264.7 macrophages after LPS treatment. Furthermore, the effect of HU308 could be blocked by autophagy blocker 3-MA in vivo and in vitro. These results suggest that CB2 serves as a protective target for septic lung injury by decreasing inflammatory factors, which is associated with the enhancement of autophagy.

Cannabinoid signalling and effects of cannabis on the male reproductive system.
Maccarrone, M., Rapino, C., Francavilla, F., & Barbonetti, A.
Nature Reviews Urology. (2020).
doi:10.1038/s41585-020-00391-8
Marijuana is the most widely consumed recreational drug worldwide, which raises concerns for its potential effects on fertility. Many aspects of human male reproduction can be modulated by cannabis-derived extracts (cannabinoids) and their endogenous counterparts, known as endocannabinoids (eCBs). These latter molecules act as critical signals in a variety of physiological processes through receptors, enzymes and transporters collectively termed the endocannabinoid system (ECS). Increasing evidence suggests a role for eCBs, as well as cannabinoids, in various aspects of male sexual and reproductive health. Although preclinical studies have clearly shown that ECS is involved in negative modulation of testosterone secretion by acting both at central and testicular levels in animal models, the effect of in vivo exposure to cannabinoids on spermatogenesis remains a matter of debate. Furthermore, inconclusive clinical evidence does not seem to support the notion that plant-derived cannabinoids have harmful effects on human sexual and reproductive health. An improved understanding of the complex crosstalk between cannabinoids and eCBs is required before targeting of ECS for modulation of human fertility becomes a reality.

Cannabinoid Signaling and Neuroinflammatory Diseases: A Melting pot for the Regulation of Brain Immune Responses
Valerio Chiurchiù, Alessandro Leuti, Mauro Maccarrone
Journal of Neuroimmune Pharmacology 10(2) January 2015
DOI: 10.1007/s11481-015-9584-2
https://www.researchgate.net/publica...mune_Responses
The concept of the central nervous system (CNS) as an immune-privileged site, essentially due to the presence of the blood brain barrier, appears to be overly simplistic. Indeed, within healthy CNS immune activities are permitted and are required for neuronal function and host defense, not only due to the presence of the resident innate immune cells of the brain, but also by virtue of a complex cross-talk of the CNS with peripheral immune cells. Nonetheless, long-standing and persisting neuroinflammatory responses are most often detrimental and characterize several neuroinflammatory diseases, including multiple sclerosis, Alzheimer's disease and amyotrophic lateral sclerosis. A growing body of evidence suggests that Cannabis sativa-derived phytocannabinoids, as well as synthetic cannabinoids, are endowed with significant immunoregulatory and anti-inflammatory properties, both in peripheral tissues and in the CNS, through the activation of cannabinoid receptors. In this review, the immunomodulatory effects of cannabinoid signaling on the most relevant brain immune cells will be discussed. In addition, the impact of cannabinoid regulation on the overall integration of the manifold brain immune responses will also be highlighted, along with the implication of these compounds as potential agents for the management of neuroinflammatory disorders.

Cannabinoid system in the skin - a possible target for future therapies in dermatology
Piotr Kupczyk , Adam Reich, Jacek C Szepietowski
Exp Dermatol. 2009 Aug.
DOI: 10.1111/j.1600-0625.2009.00923.x
https://www.researchgate.net/publica..._P_Reich_A_Sze pietowski_JCCannabinoid_system _in_the_skin_-_a_possible_target_for_future_ therapies_in_dermatology_Exp_D ermatol_18669-679
Cannabinoids and their derivatives are group of more than 60 biologically active chemical agents, which have been used in natural medicine for centuries. The major agent of exogenous cannabinoids is Delta(9)-tetrahydrocannabinol (Delta(9)-THC), natural psychoactive ingredient of marijuana. However, psychoactive properties of these substances limited their use as approved medicines. Recent discoveries of endogenous cannabinoids (e.g. arachidonoylethanolamide, 2-arachidonoylglycerol or palmithyloethanolamide) and their receptors initiated discussion on the role of cannabinoid system in physiological conditions as well as in various diseases. Based on the current knowledge, it could be stated that cannabinoids are important mediators in the skin, however their role have not been well elucidated yet. In our review, we summarized the current knowledge about the significant role of the cannabinoid system in the cutaneous physiology and pathology, pointing out possible future therapeutic targets.

Cannabinoid use and effects in patients with epidermolysis bullosa: an international cross-sectional survey study
Nicholas H B Schräder , Emily S Gorell , Roy E Stewart , José C Duipmans , Nicole Harris , Victoria A Perez , Jean Y Tang , André P Wolff , Marieke C Bolling
Orphanet J Rare Dis. 2021 Sep 6;16(1):377.
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_2010.pdf
doi: 10.1186/s13023-021-02010-0
Background: Epidermolysis bullosa (EB) patient anecdotes and case reports indicate that cannabinoid-based medicines (CBMs) may alleviate pain and pruritus and improve wound healing. CBM use has not been characterized in the EB patient population.
Objectives: To evaluate CBM use among EB patients, including CBM types, efects on symptoms (e.g., pain and pruritus), disease process (e.g., blistering, wounds, and infammation), well-being (e.g., sleep, appetite) and concomitant medications.
Methods: English-speaking EB patients or caregivers completed an online international, anonymous, cross-sectional survey regarding CBM use. Respondents reported the types of CBMs, subsequent effects including perceived EB symptom alteration, changes in medication use, and side effects.
Results: Seventy-one EB patients from five continents reported using or having used CBMs to treat their EB. Missing question responses ranged between 0 (0%) and 33 (46%). Most used more than one CBM preparation (mean: 2.4±1.5) and route of administration (mean: 2.1±1.1). Topical and ingested were the most common routes. Pain and pruritus were reported retrospectively to decrease by 3 points (scale: 0–10; p<0.001 for both) after CBM use. Most reported that CBM use improved their overall EB symptoms (95%), pain (94%), pruritus (91%) and wound healing (81%). Most participants (79%) reported decreased use of pain medications. The most common side-effect was dry mouth (44%).
Conclusions: CBMs improve the perception of pain, pruritus, wound healing, and well-being in EB patients and reduced concomitant medication use. Nevertheless, a direct relation between the use of CBMs and reduction of the above-mentioned symptoms cannot be proven by these data. Therefore, future controlled studies using pharmaceutically standardised CBM preparations in EB are warranted to delineate the risks and benefts of CBMs

Cannabinoids: A new hope for breast cancer therapy?
María M. Caffarel, Clara Andradas, Eduardo Pérez-Gómez, Manuel Guzmán, Cristina Sánchez
Cancer Treatment reviews Vol 38, Issue 7, Nov 1, 2012 Pg 911-918
doi: 10.1016/j.ctrv.2012.06.005
Breast cancer is a very common disease that affects approximately 1 in 10 women at some point in their lives. Importantly, breast cancer cannot be considered a single disease as it is characterized by distinct pathological and molecular subtypes that are treated with different therapies and have diverse clinical outcomes. Although some highly successful treatments have been developed, certain breast tumors are resistant to conventional therapies and a considerable number of them relapse. Therefore, new strategies are urgently needed, and the challenge for the future will most likely be the development of individualized therapies that specifically target each patient’s tumor. Experimental evidence accumulated during the last decade supports that cannabinoids, the active components of Cannabis sativa and their derivatives, possess anticancer activity. Thus, these compounds exert anti-proliferative, pro-apoptotic, anti-migratory and anti-invasive actions in a wide spectrum of cancer cells in culture. Moreover, tumor growth, angiogenesis and metastasis are hampered by cannabinoids in xenograft-based and genetically-engineered mouse models of cancer. This review summarizes our current knowledge on the anti-tumor potential of cannabinoids in breast cancer, which suggests that cannabinoid-based medicines may be useful for the treatment of most breast tumor subtypes.


Cannabinoids and appetite: Food craving and food pleasure
TIM C. KIRKHAM
International Review of Psychiatry, April 2009; 21(2): 163–171
DOI: 10.1080/09540260902782810.
The ability of Cannabis sativa to promote eating has been documented for many centuries, with the drug reported by its users to promote strong cravings for, and an intensification of the sensory and hedonic properties of food. These effects are now known to result from the actions of cannabinoid molecules at specific cannabinoid receptor sites within the brain, and to reflect the physiological role of their natural ligands, the endocannabinoids, in the control of appetite. Recent developments in the biochemistry and pharmacology of endocannabinoid systems have generated convincing evidence from animal models for a normal role of endocannabinoids in the control of eating motivation. The availability of specific cannabinoid receptor agonists and antagonists raises the possibility of improved therapies for disorders of eating and body weight: not only in the suppression of appetite to counter our susceptibility to the over-consumption of highly pleasurable and energy- dense foods; but also in the treatment of conditions that involve reduced appetite and weight loss. Here, we outline some of the findings of the past decade that link endocannabinoid function appetite control, and the possible clinical applications of that knowledge.
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Cannabinoids and cancer: causation, remediation, and palliation
Wayne Hall, MacDonald Christie, David Currow
Lancet Vol 6 January, 2005
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.841.5709&rep=rep1&type=pdf
This review discusses three different associations between cannabinoids and cancer. First, it assesses evidence that smoking of cannabis preparations may cause cancers of the aerodigestive and respiratory system. There have been case reports of upper-respiratory-tract cancers in young adults who smoke cannabis, but evidence from a few epidemiological cohort studies and case-control studies is inconsistent. Second, there is mixed evidence on the effects of THC and other cannabinoids on cancers: in some in vitro and in vivo studies THC and some synthetic cannabinoids have had antineoplastic effects, but in other studies THC seems to impair the immune response to cancer. As yet there is no evidence that THC or other cannabinoids have anticancer effects in humans. Third, 9 -tetrahydrocannabinol (THC) may treat the symptoms and side-effects of cancer, and there is evidence that it and other cannabinoids may be useful adjuvant treatments that improve appetite, reduce nausea and vomiting, and alleviate moderate neuropathic pain in patients with cancer. The main challenge for the medical use of cannabinoids is the development of safe and effective methods of use that lead to therapeutic effects but that avoid adverse psychoactive effects. Furthermore, medical, legal, and regulatory obstacles hinder the smoking of cannabis for medical purposes. These very different uses of cannabinoids are in danger of being confused in public debate, especially in the USA where some advocates for the medical use of cannabinoids have argued for smoked cannabis rather than pharmaceutical cannabinoids. We review the available evidence on these three issues and consider their implications for policy. .

Cannabinoids and Cancer Chemotherapy-Associated Adverse Effects
Sara Jane Ward, Aron H Lichtman, Daniele Piomelli, Linda A Parker,
J Natl Cancer Inst Monogr (2021) 2021(58): lgab007
doi: 10.1093/jncimonographs/lgab007
https://academic.oup.com/jncimono/ar.../58/78/6446185
The use of cannabis is not unfamiliar to many cancer patients, as there is a long history of its use for cancer pain and/or pain, nausea, and cachexia induced by cancer treatment. To date, the US Food and Drug Administration has approved 2 cannabisbased pharmacotherapies for the treatment of cancer chemotherapy-associated adverse effects: dronabinol and nabilone. Over the proceeding decades, both research investigating and societal attitudes toward the potential utility of cannabinoids for a range of indications have progressed dramatically. The following monograph highlights recent preclinical research focusing on promising cannabinoid-based approaches for the treatment of the 2 most common adverse effects of cancer chemotherapy: chemotherapy-induced peripheral neuropathy and chemotherapy-induced nausea and vomiting. Both plant-derived and synthetic approaches are discussed, as is the potential relative safety and effectiveness of these approaches in relation to current treatment options, including opioid analgesics.

Cannabinoids and cancer: pros and cons of an antitumour strategy
Maurizio Bifulco, Chiara Laezza, Simona Pisanti, and Patrizia Gazzerro
Br J Pharmacol 148(2); 2006 May
doi: 10.1038/sj.bjp.0706632
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1617062/
In the last two decades, research has dramatically increased the knowledge of cannabinoids biology and pharmacology. In mammals, compounds with properties similar to active components of Cannabis sativa, the so called ‘endocannabinoids', have been shown to modulate key cell-signalling pathways involved in cancer cell growth, invasion and metastasis. To date, cannabinoids have been licensed for clinical use as palliative treatment of chemotherapy, but increased evidences showed direct antiproliferative actions of cannabinoid agonists on several tumour cells in vitro and in animal models. In this article, we will review the principal molecular pathways modulated by cannabinoids on cancer and summarize pros and consevidence on the possible future use of endocannabinoid-based drugs in cancer therapy.

Cannabinoids and cell fate
Manuel Guzma´n, Cristina Sa´nchez, Ismael Galve-Roperh
Pharmacology & Therapeutics 95 (2002) 175 – 184
DOI: 10.1016/s0163-7258(02)00256-5
Cannabinoids recently have been shown to control the cell survival/death decision. Thus, cannabinoids induce growth arrest or apoptosis in a number of transformed neural and non-neural cells in culture. In addition, cannabinoid administration induces regression of malignant gliomas in rodents by a mechanism that may involve sustained ceramide generation and extracellular signal-regulated kinase activation. In contrast, most of the experimental evidence indicates that cannabinoids may protect normal neurons from toxic insults, such as glutamatergic overstimulation, ischaemia, and oxidative damage. Regarding immune cells, low doses of cannabinoids may enhance proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis. The potential therapeutic applications of these findings are discussed.

Cannabinoids and Inflammations of the Gut-Lung-Skin Barrier
Cristian Scheau, Constantin Caruntu, Ioana Anca Badarau, Andreea-Elena Scheau, Anca Oana Docea, Daniela Calina, and Ana Caruntu
J. Pers. Med. 2021, 11, 494.
DOI: 10.3390/jpm11060494
Recent studies have identified great similarities and interferences between the epithelial layers of the digestive tract, the airways and the cutaneous layer. The relationship between these structures seems to implicate signaling pathways, cellular components and metabolic features, and has led to the definition of a gut-lung-skin barrier. Inflammation seems to involve common features in these tissues; therefore, analyzing the similarities and differences in the modulation of its biomarkers can yield significant data promoting a better understanding of the particularities of specific signaling pathways and cellular effects. Cannabinoids are well known for a wide array of beneficial effects, including anti-inflammatory properties. This paper aims to explore the effects of natural and synthetic cannabinoids, including the components of the endocannabinoid system, in relation to the inflammation of the gut-lung-skin barrier epithelia. Recent advancements in the use of cannabinoids as anti-inflammatory substances in various disorders of the gut, lungs and skin are detailed. Some studies have reported mixed or controversial results, and these have also been addressed in our paper.

Cannabinoids and Neurodegenerative Diseases
CNS & Neurological Disorders - Drug Targets (Formerly Current Drug Targets - CNS & Neurological Disorders) 8(6):440-50 October 2009
DOI: 10.2174/187152709789824589
Although significant advances have taken place in recent years on our understanding of the molecular mechanisms of different neurodegenerative diseases, its translation into effective therapeutic treatments has not been as successful as could be expected. There is still a dramatic lack of curative treatments for the most frequent disorders and only symptomatic relief for many others. Under this perspective, the search for novel therapeutic approaches is demanding and significant attention and efforts have been directed to studying additional neurotransmission systems including the endocannabinoid system (ECS). The neuroprotective properties of exogenous as well as endogenous cannabinoids have been known for years and the underlying molecular mechanisms have been recently unveiled. As discussed later, antioxidative, antiglutamatergic and antiinflammatory effects are now recognized as derived from cannabinoid action and are known to be of common interest for many neurodegenerative processes. Thus, these characteristics make cannabinoids attractive candidates for the development of novel therapeutic strategies [1]. The present review will focus on the existing data regarding the possible usefulness of cannabinoid agents for the treatment of relevant neurological pathologies for our society such as Alzheimer's disease, multiple sclerosis, Huntington's disease and amyotrophic lateral sclerosis.

Cannabinoids and Neuroprotection in Basal Ganglia Disorders
Onintza Sagredo & Moisés García-Arencibia & Eva de Lago & Simone Finetti & Alessandra Decio & Javier Fernández-Ruiz
Mol Neurobiol (2007) 36:82–91
DOI 10.1007/s12035-007-0004-3
Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson’s disease and Huntington’s chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.

Cannabinoids and Neuropsychiatric Disorders.
Murillo-Rodriguez, E., Pandi-Perumal, S. R., & Monti, J. M. (Eds.).
Advances in Experimental Medicine and Biology. (2021).
doi:10.1007/978-3-030-57369-0
For most researchers, and certainly for the general population, “cannabis” relates to the plant and its constituents alone. However, since the mid-1980s and early 1990s, research has expanded our knowledge.*Today, the cannabinoid field of science covers the cannabinoid receptors, the endocannabinoids (particularly anandamide and 2-AG), their synthetic and degradation pathways, and endogenous anandamide-like compounds, which are fatty acid amides with amino acids or ethanolamines. All these entities are parts of a major new physiological system—the endocannabinoid one. Most probably, the field will expand further.

Cannabinoids and psychotic symptoms: A potential role for a genetic variant in the P2X purinoceptor 7 (P2RX7) gene.
Boks, M. P., He, Y., Schubart, C. D., van Gastel, W., Elkrief, L., Huguet, G., … de Witte, L. D.
Brain, Behavior, and Immunity. (2020).
doi:10.1016/j.bbi.2020.04.051
To investigate the biological mechanisms underlying the higher risk for psychosis in 3 those that use cannabis, we conducted a genome-wide environment-interaction study 4 (GWEIS). In a sample of individuals without a psychiatric disorder (N=1262), we analyzed 5 the interactions between regular cannabis use and genotype with psychotic-like 6 experiences (PLE) as outcome. PLE were measured using the Community Assessment of 7 Psychic Experiences (CAPE). The sample was enriched for those at the extremes of both 8 cannabis use and PLE to increase power. A single nucleotide polymorphism in the P2RX7 9 gene (rs7958311) was associated with risk for a high level of psychotic experiences in 10 regular cannabis users (p=1.10 x10-7) and in those with high levels of lifetime cannabis 11 use (p= 4.5 x 10-6). This interaction was replicated in individuals with high levels of 12 lifetime cannabis use in the IMAGEN cohort (N=1217, p=0.020). Functional relevance of 13 P2RX7 in cannabis users was suggested by in vitro experiment on activated monocytes. 14 Exposure of these cells to tetrahydrocannabinol (THC) or cannabidiol (CBD) reduced the 15 immunological response of the P2X7 receptor, which was dependent on the identified 16 genetic variant. P2RX7 variants have been implicated in psychiatric disorders before and 17 the P2X7 receptor is involved in pathways relevant to psychosis, such as 18 neurotransmission, synaptic plasticity and immune regulation. We conclude that P2RX7 19 plays a role in vulnerability to develop psychotic symptoms when using cannabis and 20 point to a new pathway that can potentially be targeted by newly developed P2X7 21 antagonists.

Cannabinoids and Reproduction: A Lasting and Intriguing History
Giovanna Cacciola, Rosanna Chianese, Teresa Chioccarelli, Vincenza Ciaramella,Silvia Fasano, Riccardo Pierantoni, Rosaria Meccariello, and Gilda Cobellis
Pharmaceuticals 2010, 3, 3275-3323;
doi:10.3390/ph3103275
https://pdfs.semanticscholar.org/f78...879.1582090724
Starting from an historical overview of lasting Cannabis use over the centuries, we will focus on a description of the cannabinergic system, with a comprehensive analysis of chemical and pharmacological properties of endogenous and synthetic cannabimimetic analogues. The metabolic pathways and the signal transduction mechanisms, activated by cannabinoid receptors stimulation, will also be discussed. In particular, we will point out the action of cannabinoids and endocannabinoids on the different neuronal networks involved in reproductive axis, and locally, on male and female reproductive tracts, by emphasizing the pivotal role played by this system in the control of fertility.

Cannabinoids and the Brain Attila Kofalvi Editor (2008) DOI: 10.1007/978-0-387-74349-3_17 https://www.researchgate.net/publication/227141784_Targeting_Cannabinoi d_Receptors_in_Brain_Tumors Did you know that if you take aspirin or some other type of painkillers, you simply upregulate your endocannabinoid system against your endovanilloid system? If it happens to be a completely new piece of information to you, then this book is for you! Seriously speaking, the first part of the book you are holding in your hands is an exhaustive source of scientific reviews on the molecular biology, pharmacology, anatomy, and physiology of the endocannabinoid and related lipid mediator systems.
The second part of the book, however, covers the involvement of these signaling systems in metabolic, neurological, and psychiatric disorders, and gives an overview on clinical trials and on recent advances in cannabinoid-based medicine. Therefore, the target audience for this book are (a) physicians, especially endocrinologists, neurologists, psychiatrists, and neuroscientists who want to update their knowledge about metabolism, basic brain physiology, molecular biology, and pathology and about novel therapeutic opportunities; (b) graduate and undergraduate students who also wish to broaden their knowledge about endocrinology, neuroscience, neurology, and psychiatry, or may need orientation to determine their future scientific goals; (c) politicians and health care employers who hesitate whether marijuana or cannabinoid-based medications should be legalized; and last but not least, (d) journalists who can help the scientists to convey their message to a larger audience. All the authors of the present volume are world’s leading neuroscientists and physicians, who are also regarded to be pioneers in the cannabinoid research area. Here I would like to gratefully thank them for all their altruistic contributions, and for sparing their precious time on this work.

Cannabinoids and the Coronavirus.
Hill, K. P.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0035
These are uncertain times as we attempt to manage our lives during the Coronavirus Covid-19 pandemic. It is not surprising, as possible treatments for Covid-19 are discussed, that people wonder about a role for cannabinoids, another topic associated with a lack of clarity about their therapeutic efficacy. Patients commonly ask clinicians about the benefits and risks of cannabinoids and now patients have begun to ask about cannabinoids as they relate to Covid-19. This interest creates an opportunity to strengthen the therapeutic alliance between patients and clinicians and to increase the likelihood that patients receive evidence-based treatments that may help them

Cannabinoids and the immune system: potential for the treatment of inflammatory diseases?
J Ludovic Croxford , Takashi Yamamura
J Neuroimmunol . 2005 Sep;166(1-2):3-18.
DOI:10.1016/j.jneuroim.2005.04.023
Since the discovery of the cannabinoid receptors and their endogenous ligands, significant advances have been made in studying the physiological function of the endocannabinoid system. The presence of cannabinoid receptors on cells of the immune system and anecdotal and historical evidence suggesting that cannabis use has potent immuno-modulatory effects, has led to research directed at understanding the function and role of these receptors within the context of immunological cellular function. Studies from chronic cannabis smokers have provided much of the evidence for immunomodulatory effects of cannabis in humans, and animal and in vitro studies of immune cells such as T cells and macrophages have also provided important evidence. Cannabinoids can modulate both the function and secretion of cytokines from immune cells. Therefore, cannabinoids may be considered for treatment of inflammatory disease. This review article will highlight recent research on cannabinoids and how they interact with the immune system and also their potential use as therapeutic agents for a number of inflammatory disorders.

Cannabinoids and the Microbiota-Gut-Brain-Axis: Emerging Effects of Cannabidiol and Potential Applications to Alcohol Use Disorders
Hollis C. Karoly1 , Raeghan L. Mueller, L. Cinnamon Bidwell, & Kent E. Hutchison
Alcohol Clin Exp Res. 2020 Feb;44(2):340-353.
DOI: 10.1111/acer.14256
The endocannabinoid system (ECS) has emerged in recent years as a potential treatment target for alcohol use disorders (AUD). In particular, the non-psychoactive cannabinoid cannabidiol (CBD) has shown preclinical promise in ameliorating numerous clinical symptoms of AUD. There are several proposed mechanism(s) through which cannabinoids (and CBD in particular) may confer beneficial effects in the context of AUD. First, CBD may directly impact specific brain mechanisms underlying AUD to influence alcohol consumption and the clinical features of AUD. Second, CBD may influence AUD symptoms through its actions across the digestive, immune, and central nervous systems, collectively known as the microbiota-gut-brain-axis (MGBA). Notably, emerging work suggests that alcohol and cannabinoids exert opposing effects on the MGBA. Alcohol is linked to immune dysfunction (e.g., chronic systemic inflammation in the brain and periphery) as well as disturbances in gut microbial species (microbiota) and increased intestinal permeability. These MGBA disruptions have been associated with AUD symptoms such as craving and impaired cognitive control. Conversely, existing preclinical data suggest that cannabinoids may confer beneficial effects on the gastrointestinal and immune system, such as reducing intestinal permeability, regulating gut bacteria and reducing inflammation. Thus, cannabinoids may exert AUD harm-reduction effects, at least in part, through their beneficial actions across the MGBA. This review will provide a brief introduction to the ECS and the MGBA, discuss the effects of cannabinoids (particularly CBD) and alcohol in the brain, gut, and immune system (i.e., across the MGBA), and put forth a theoretical framework to inform future research questions.

Cannabinoids and Tremor Induced by Motor-related Disorders: Friend or Foe?
Shokouh Arjmand, Zohreh Vaziri, Mina Behzadi, Hassan Abbassian, Gary J. Stephens, and Mohammad Shabani Neurotherapeutics. 2015 Oct; 12(4): 778–787. 2015 Jul 8.
doi: 10.1007/s13311-015-0367-5
https://www.ncbi.nlm.nih.gov/labs/pm...rticle_367.pdf
Tremor arises from an involuntary, rhythmic muscle contraction/relaxation cycle and is a common disabling symptom of many motor-related diseases such as Parkinson disease, multiple sclerosis, Huntington disease, and forms of ataxia. In the wake of anecdotal, largely uncontrolled, observations claiming the amelioration of some symptoms among cannabis smokers, and the high density of cannabinoid receptors in the areas responsible for motor function, including basal ganglia and cerebellum, many researchers have pursued the question of whether cannabinoid-based compounds could be used therapeutically to alleviate tremor associated with central nervous system diseases. In this review, we focus on possible effects of cannabinoid-based medicines, in particular on Parkinsonian and multiple sclerosis-related tremors and the common probable molecular mechanisms. While, at present, inconclusive results have been obtained, future investigations should extend preclinical studies with different cannabinoids to controlled clinical trials to determine potential benefits in tremor.

Cannabinoids and Vanilloids in Schizophrenia: Neurophysiological Evidence and Directions for Basic Research
Rafael N. Ruggiero, Matheus T. Rossignoli, Jana B. De Ross, Jaime E. C. Hallak, Joao P. Leite and Lezio S. Bueno-Junior
Front. Pharmacol., 21 June 2017 | DOI: 10.3389/fphar.2017.00399
https://www.frontiersin.org/articles...017.00399/full
Much of our knowledge of the endocannabinoid system in schizophrenia comes from behavioral measures in rodents, like prepulse inhibition of the acoustic startle and open-field locomotion, which are commonly used along with neurochemical approaches or drug challenge designs. Such methods continue to map fundamental mechanisms of sensorimotor gating, hyperlocomotion, social interaction, and underlying monoaminergic, glutamatergic, and GABAergic disturbances. These strategies will require, however, a greater use of neurophysiological tools to better inform clinical research. In this sense, electrophysiology and viral vector-based circuit dissection, like optogenetics, can further elucidate how exogenous cannabinoids worsen (e.g., tetrahydrocannabinol, THC) or ameliorate (e.g., cannabidiol, CBD) schizophrenia symptoms, like hallucinations, delusions, and cognitive deficits. Also, recent studies point to a complex endocannabinoid-endovanilloid interplay, including the influence of anandamide (endogenous CB1 and TRPV1 agonist) on cognitive variables, such as aversive memory extinction. In fact, growing interest has been devoted to TRPV1 receptors as promising therapeutic targets. Here, these issues are reviewed with an emphasis on the neurophysiological evidence. First, we contextualize imaging and electrographic findings in humans. Then, we present a comprehensive review on rodent electrophysiology. Finally, we discuss how basic research will benefit from further combining psychopharmacological and neurophysiological tools.

Cannabinoids as novel anti-inflammatory drugs
Prakash Nagarkatti, Rupal Pandey, Sadiye Amcaoglu Rieder, Venkatesh L Hegde, and Mitzi Nagarkatti
Future Med Chem. 2009 October ; 1(7): 1333–1349.
doi:10.4155/fmc.09.93
https://europepmc.org/backend/ptpmcr...?accid=PMC2828 614&blobtype=pdf
Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of Δ9 -tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to a significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found on the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids, led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivo can constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components

Cannabinoids as pharmacotherapies for neuropathic pain: From the bench to the bedside.
Rahn, E. J., & Hohmann, A. G.
Neurotherapeutics, 6(4), 713–737. (2009).
doi:10.1016/j.nurt.2009.08.002
Neuropathic pain is a debilitating form of chronic pain resulting from nerve injury, disease states, or toxic insults. Neuropathic pain is often refractory to conventional pharmacotherapies, necessitating validation of novel analgesics. Cannabinoids, drugs that share the same target as 9 -tetrahydrocannabinol (9 -THC), the psychoactive ingredient in cannabis, have the potential to address this unmet need. Here, we review studies evaluating cannabinoids for neuropathic pain management in the clinical and preclinical literature. Neuropathic pain associated with nerve injury, diabetes, chemotherapeutic treatment, human immunodeficiency virus, multiple sclerosis, and herpes zoster infection is considered. In animals, cannabinoids attenuate neuropathic nociception produced by traumatic nerve injury, disease, and toxic insults. Effects of mixed cannabinoid CB1/CB2 agonists, CB2 selective agonists, and modulators of the endocannabinoid system (i.e., inhibitors of transport or degradation) are compared. Effects of genetic disruption of cannabinoid receptors or enzymes controlling endocannabinoid degradation on neuropathic nociception are described. Specific forms of allodynia and hyperalgesia modulated by cannabinoids are also considered. In humans, effects of smoked marijuana, synthetic 9 -THC analogs (e.g., Marinol, Cesamet) and medicinal cannabis preparations containing both 9 -THC and cannabidiol (e.g., Sativex, Cannador) in neuropathic pain states are reviewed. Clinical studies largely affirm that neuropathic pain patients derive benefits from cannabinoid treatment. Subjective (i.e., rating scales) and objective (i.e., stimulusevoked) measures of pain and quality of life are considered. Finally, limitations of cannabinoid pharmacotherapies are discussed together with directions for future research. Key Words: Endocannabinoid, marijuana, neuropathy, multiple sclerosis, chemotherapy, diabetes.

Cannabinoids: Between Neuroprotection and Neurotoxicity
Sarne, Yosef; Mechoulam, Raphael
Current Drug Targets-CNS & Neurological Disorders, Volume 4, Number 6, 2005, pp. 677-684
DOI: 10.2174/156800705774933005
http://www.eurekaselect.com/article/35396
Cannabinoids, such as the Δ9- tetrahydrocannabinol (THC), present in the cannabis plant, as well as anandamide and 2-arachidonoyl glycerol, produced by the mammalian body, have been shown to protect the brain from various insults and to improve several neurodegenerative diseases. The current review summarizes the evidence for cannabinoid neuroprotection in vivo, and refers to recent in vitro studies, which help elucidate possible molecular mechanisms underlying this protective effect. Some of these mechanisms involve the activation of CB1 and CB2 cannabinoid receptors, while others are not dependent on them. In some cases, protection is due to a direct effect of the cannabinoids on neuronal cells, while in others, it results from their effects on non-neuronal elements within the brain. In many experimental set-ups, cannabinoid neurotoxicity, particularly by THC, resides side by side with neuroprotection. The current review attempts to shed light on this dual activity, and to dissociate between the two contradictory effects.
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Cannabinoids Biology: The Search for New Therapeutic Targets

Christian C Felder, Amy K Chesterfield, Steve Moore
Molecular interventions 6(3):149-61
DOI: 10.1124/mi.6.3.6
https://www.researchgate.net/publica...peutic_Targets
Cannabinoids, in the form of marijuana plant extracts, have been used for thousands of years for a wide variety of medical conditions, ranging from general malaise and mood disorders to more specific ailments, such as pain, nausea, and muscle spasms. The discovery of tetrahydrocannabinol, the active principal in marijuana, and the identification and cloning of two cannabinoid receptors (i.e., CB 1 and CB 2) has subsequently led to biomedical appreciation for a family of endocannabinoid lipid transmitters. The biosynthesis and catabolism of the endocannabinoids and growing knowledge of their broad physiological roles are providing insight into potentially novel therapeutic targets. Compounds directed at one or more of these targets may allow for cannabinoid-based therapeutics with limited side effects and abuse liability.

Cannabinoids Block Cellular Entry of SARS-CoV‑2 and the Emerging Variants
Richard B. van Breemen, Ruth N. Muchiri, Timothy A. Bates, Jules B. Weinstein, Hans C. Leier, Scotland Farley, and Fikadu G. Tafesse
J. Nat. Prod. 2022, 85, 176−184
https://pubs.acs.org/doi/pdf/10.1021...atprod.1c00946
As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection−mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2. .

Cannabinoids Decrease the Th17 Inflammatory Autoimmune Phenotype
Ewa Kozela & Ana Juknat & Nathali Kaushansky & Neta Rimmerman & Avraham Ben-Nun & Zvi Vogel
J Neuroimmune Pharmacol
DOI 10.1007/s11481-013-9493-1
Cannabinoids, the Cannabis constituents, are known to possess anti-inflammatory properties but the mechanisms involved are not understood. Here we show that the main psychoactive cannabinoid, ?-9-tetrahydrocannabinol (THC), and the main nonpsychoactive cannabinoid, cannabidiol (CBD), markedly reduce the Th17 phenotype which is known to be increased in inflammatory autoimmune pathologies such as Multiple Sclerosis.We found that reactivation byMOG35-55 of MOG35-55-specific encephalitogenic T cells (cells that induce Experimental Autoimmune Encephalitis when injected to mice) in the presence of spleen derived antigen presenting cells led to a large increase in IL-17 production and secretion. In addition, we found that the cannabinoids CBD and THC dose-dependently (at 0.1–5 ?M) suppressed the production and secretion of this cytokine. Moreover, the mRNA and protein of IL-6, a key factor in Th17 induction, were also decreased. Pretreatment with CBD also resulted in increased levels of the anti-inflammatory cytokine IL-10. Interestingly, CBD and THC did not affect the levels of TNF? and IFN?. The downregulation of IL-17 secretion by these cannabinoids does not seem to involve the CB1, CB2, PPAR?, 5-HT1A or TRPV1 receptors. In conclusion, the results show a unique cannabinoid modulation of the autoimmune cytokine milieu combining suppression of the pathogenic IL-17 and IL-6 cytokines along with boosting the expression of the anti-inflammatory cytokine IL-10.

Cannabinoids, Endocannabinoids, and Related Analogs in Inflammation
Sumner H. Burstein, and Robert B. Zurier
The AAPS Journal, Vol. 11, No. 1, March 2009 (# 2009)
DOI: 10.1208/s12248-009-9084-5
This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation. A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process

Cannabinoids/Endocannabinoids as Possible Antineoplastic Therapy in Comparisson to Cancer Pharmacological Treatments Used Today: Narrative Review
Fabio Mayorga Niño, Nelson Camilo, Gutierrez Alvarado
Cancer is a complex pathophysiological condition that produces an important number of death around the world. At present, there are different ways to treat cancer: chemotherapy, radiotherapy and surgery. Cancer chemotherapy used today in many cases is effective, but it is very toxic too. The endocannabinoid system is implicated in a variety of physiological and pathological processes, including cancer. Many studies have shown, since 1975, that both phy- tocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) offer an antioneoplastic activity. Latter, oth- er researchers have displayed that endocannabinoids as anandamide (ANA) and 2-arachidonoylglycerol (2-AG) also present the same potential activity. Phytocannabinoids and endocannabinoids act through CB1 and CB2 receptors to produce that effect. However, THC -the main phytocannabinoid presenting anticancer action- as well as anandamide employed in pharmacological doses, produce important phycotropic effects, but these cannabinoid compounds do not produce major adverse reactions like conventional antineoplastic drugs. On this basis, scientists have to develop analogs or derivatives of cannabinoids/endocannabinoids that cannot induce psychotropic effects. It is important to study more deeply chronopharmacological aspects of cannabinoids/endocannabinoids in cancer therapy, although some is known today.
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Cannabinoid system in the skin – a possible target for future therapies in dermatology
Piotr Kupczyk, Adam Reich and Jacek C. Szepietowski
Experimental Dermatology, 18, 669–679
DOI:10.1111/j.1600-0625.2009.00923.x
https://sci-hub.se/10.1111/j.1600-0625.2009.00923.x
Cannabinoids and their derivatives are group of more than 60 biologically active chemical agents, which have been used in natural medicine for centuries. The major agent of exogenous cannabinoids is D9 -tetrahydrocannabinol (D9 -THC), natural psychoactive ingredient of marijuana. However, psychoactive properties of these substances limited their use as approved medicines. Recent discoveries of endogenous cannabinoids (e.g. arachidonoylethanolamide, 2-arachidonoylglycerol or palmithyloethanolamide) and their receptors initiated discussion on the role of cannabinoid system in physiological conditions as well as in various diseases. Based on the current knowledge, it could be stated that cannabinoids are important mediators in the skin, however their role have not been well elucidated yet. In our review, we summarized the current knowledge about the significant role of the cannabinoid system in the cutaneous physiology and pathology, pointing out possible future therapeutic targets .

Cannabinoids for the treatment mental disorders and symptoms of mental disorders: a systematic review and meta-analysis
Nicola Black, Emily Stockings, Gabrielle Campbell, Lucy T Tran, Dino Zagic, Wayne D Hall, Michael Farnell, Louisa Degenhardt
The Lancet Psychiatry Oct 2019
DOI: 10.1016/S2215-0366(19)30401-8
Background Medicinal cannabinoids, including medicinal cannabis and pharmaceutical cannabinoids and their synthetic derivatives, such as tetrahydrocannabinol (THC) and cannabidiol (CBD), have been suggested to have a therapeutic role in certain mental disorders. We analysed the available evidence to ascertain the effectiveness and safety of all types of medicinal cannabinoids in treating symptoms of various mental disorders.
Methods For this systematic review and meta-analysis we searched MEDLINE, Embase, PsycINFO, the Cochrane Central Register of Controlled Clinical Trials, and the Cochrane Database of Systematic Reviews for studies published between Jan 1, 1980, and April 30, 2018. We also searched for unpublished or ongoing studies on ClinicalTrials.gov, the EU Clinical Trials Register, and the Australian and New Zealand Clinical Trials Registry. We considered all studies examining any type and formulation of a medicinal cannabinoid in adults (?18 years) for treating depression, anxiety, attention-deficit hyperactivity disorder (ADHD), Tourette syndrome, post-traumatic stress disorder, or psychosis, either as the primary condition or secondary to other medical conditions. We placed no restrictions on language, publication status, or study type (ie, both experimental and observational study designs were included). Primary outcomes were remission from and changes in symptoms of these mental disorders. The safety of medicinal cannabinoids for these mental disorders was also examined. Evidence from randomised controlled trials was synthesised as odds ratios (ORs) for disorder remission, adverse events, and withdrawals and as standardised mean differences (SMDs) for change in symptoms, via random-effects meta-analyses. The quality of the evidence was assessed with the Cochrane risk of bias tool and Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. This study is registered with PROSPERO (CRD42017059372, CRD42017059373, CRD42017059376, CRD42017064996, and CRD42018102977).
Findings 83 eligible studies (40 randomised controlled trials, n=3067) were included: 42 for depression (23 randomised controlled trials; n=2551), 31 for anxiety (17 randomised controlled trials; n=605), eight for Tourette syndrome (two randomised controlled trials; n=36), three for ADHD (one randomised controlled trial; n=30), 12 for post-traumatic stress disorder (one randomised controlled trial; n=10), and 11 for psychosis (six randomised controlled trials; n=281). Pharmaceutical THC (with or without CBD) improved anxiety symptoms among individuals with other medical conditions (primarily chronic non-cancer pain and multiple sclerosis; SMD ?0·25 [95% CI ?0·49 to ?0·01]; seven studies; n=252), although the evidence GRADE was very low. Pharmaceutical THC (with or without CBD) worsened negative symptoms of psychosis in a single study (SMD 0·36 [95% CI 0·10 to 0·62]; n=24). Pharmaceutical THC (with or without CBD) did not significantly affect any other primary outcomes for the mental disorders examined but did increase the number of people who had adverse events (OR 1·99 [95% CI 1·20 to 3·29]; ten studies; n=1495) and withdrawals due to adverse events (2·78 [1·59 to 4·86]; 11 studies; n=1621) compared with placebo across all mental disorders examined. Few randomised controlled trials examined the role of pharmaceutical CBD or medicinal cannabis.
Interpretation There is scarce evidence to suggest that cannabinoids improve depressive disorders and symptoms, anxiety disorders, attention-deficit hyperactivity disorder, Tourette syndrome, post-traumatic stress disorder, or psychosis. There is very low quality evidence that pharmaceutical THC (with or without CBD) leads to a small improvement in symptoms of anxiety among individuals with other medical conditions. There remains insufficient evidence to provide guidance on the use of cannabinoids for treating mental disorders within a regulatory framework. Further high-quality studies directly examining the effect of cannabinoids on treating mental disorders are needed.

Cannabinoids for the treatment of cannabis use disorder: New avenues for reaching and helping youth?
Kristine Rømer Thomsen, BirgitteThylstrup, Emily A.Kenyon, Rachael Lee, Lone Baandrup, Sarah W. Feldstein Ewing, Tom P. Freeman
Neuroscience & Biobehavioral Reviews Volume 132, January 2022, Pages 169-180
https://www.sciencedirect.com/scienc...273?via%3Dihub
Cannabis use peaks during adolescence and emerging adulthood, and cannabis use disorder (CUD) is associated with a wide range of adverse outcomes. This is particularly pertinent in youth, because the developing brain may be more vulnerable to adverse effects of frequent cannabis use. Combining evidence-based psychosocial interventions with safe and effective pharmacotherapy is a potential avenue to improve youth outcomes, but we lack approved CUD pharmacotherapies. Here, we review new potential avenues for helping youth with CUD, with a particular focus on cannabinoid-based treatments. Evidence from placebo-controlled RCTs suggests synthetic delta-9-tetrahydrocannabinol (THC) decreases withdrawal symptoms, but not cannabis use, in adults with daily cannabis use/CUD, while findings regarding formulations containing THC combined with cannabidiol (CBD) are mixed. Preliminary evidence from two placebo-controlled RCTs in adults with CUD suggests that both Fatty Acid Amide Hydrolase inhibitors and CBD can reduce cannabis use. However, larger trials are needed to strengthen the evidence. Findings from adults point to cannabinoid-based treatments as a potential strategy that should be examined in youth with CUD.

Cannabinoids in cancer treatment: Therapeutic potential and legislation
Barbara Dariš, Mojca Tancer Verboten, Željko Knez, and Polonca Ferk
Bosn J Basic Med Sci. 2019 Feb; 19(1): 14–23.
doi: 10.17305/bjbms.2018.3532
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387667/
The plant Cannabis sativa L. has been used as an herbal remedy for centuries and is the most important source of phytocannabinoids. The endocannabinoid system (ECS) consists of receptors, endogenous ligands (endocannabinoids) and metabolizing enzymes, and plays an important role in different physiological and pathological processes. Phytocannabinoids and synthetic cannabinoids can interact with the components of ECS or other cellular pathways and thus affect the development/progression of diseases, including cancer. In cancer patients, cannabinoids have primarily been used as a part of palliative care to alleviate pain, relieve nausea and stimulate appetite. In addition, numerous cell culture and animal studies showed antitumor effects of cannabinoids in various cancer types. Here we reviewed the literature on anticancer effects of plant-derived and synthetic cannabinoids, to better understand their mechanisms of action and role in cancer treatment. We also reviewed the current legislative updates on the use of cannabinoids for medical and therapeutic purposes, primarily in the EU countries. In vitro and in vivocancer models show that cannabinoids can effectively modulate tumor growth, however, the antitumor effects appear to be largely dependent on cancer type and drug dose/concentration. Understanding how cannabinoids are able to regulate essential cellular processes involved in tumorigenesis, such as progression through the cell cycle, cell proliferation and cell death, as well as the interactions between cannabinoids and the immune system, are crucial for improving existing and developing new therapeutic approaches for cancer patients. The national legislation of the EU Member States defines the legal boundaries of permissible use of cannabinoids for medical and therapeutic purposes, however, these legislative guidelines may not be aligned with the current scientific knowledge.

Cannabinoids in epilepsy: clinical efficacy and pharmacological considerations
C.Espinosa-Jovel
Neurología (English Edition) 22 November 2021
https://www.sciencedirect.com/scienc...826?via%3Dihub
https://doi.org/10.1016/j.nrleng.2020.02.012
Introduction
Advances in the development of drugs with novel mechanisms of action have not been sufficient to significantly reduce the percentage of patients presenting drug-resistant epilepsy. This lack of satisfactory clinical results has led to the search for more effective treatment alternatives with new mechanisms of action.
Development
The aim of this study is to examine epidemiological aspects of the use of cannabis-based products for the treatment of epilepsy, with particular emphasis on the main mechanisms of action, indications for use, clinical efficacy, and safety. We conducted a narrative review of articles gathered from the PubMed, EMBASE, and Google Scholar databases and from the reference sections of relevant publications.
Conclusions
In recent years there has been growing interest in the use of cannabis-based products for the treatment of a wide range of diseases, including epilepsy. The cannabis plant is currently known to contain more than 100 terpenophenolic compounds, known as cannabinoids. The 2 most abundant are delta-9-tetrahydrocannabinol and cannabidiol. Studies of preclinical models of epilepsy have shown that these cannabinoids have anticonvulsant properties, and 100% purified cannabidiol and cannabidiol-enriched cannabis extracts are now being used to treat epilepsy in humans. Several open-label studies and randomisedcontrolled clinical trials have demonstrated the efficacy and safety of these products

Cannabinoids in multiple sclerosis: A neurophysiological analysis.
Vecchio, D., Varrasi, C., Virgilio, E., Spagarino, A., Naldi, P., & Cantello, R.**
Acta Neurologica Scandinavica. (2020).
doi:10.1111/ane.13313
Objectives. To investigate the action of cannabinoids on spasticity and pain in secondary progressive multiple sclerosis, by means of neurophysiological indexes.
Material and Methods. We assessed 15 patients with progressive MS (11 females) using clinical scales for spasticity and pain, as well as neurophysiological variables (H/M ratio, cutaneous silent period or CSP). Testing occurred before (T0) and during (T1) a standard treatment with an oral spray containing delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD). Neurophysiological measures at T0 were compared with those of 14 healthy controls of similar age and sex (HC). We then compared the patient results at the 2 time points (T1 vs T0).
Results. At T0, neurophysiological variables did not differ significantly between patients and controls. At T1, spasticity and pain scores improved, as detected by the Modified Ashworth Scale or MAS (p=0.001), 9- Hole Peg Test or 9HPT (p=0.018), numeric rating scale for spasticity or NRS (p=0.001), and visual analogue scale for pain or VAS (p=0.005). At the same time, the CSP was significantly prolonged (p=0.001).
Conclusions. The THC-CBD spray improved spasticity and pain in secondary progressive MS patients. The spray prolonged CSP duration, which appears a promising tool for assessing and monitoring the analgesic effects of THC-CBD in MS.

Cannabinoids in neurodegeneration and neuroprotection
Javier Fernández-Ruiz1, Sara González1, Julián Romero and José Antonio
Ramos
In book Cannabinoids as Therapeutics Edited by R. Mechoulam
DOI: 10.1007/3-7643-7358-X_5
https://www.researchgate.net/publica...inoids_in_neur odegeneration_and_neuroprotect ion
Among a variety of cellular and tissue functions, it has been suggested that the endocannabinoid system might also exert an important function in the cellular decision about death or survival (for review see [1–3]). This finding has derived from several experimental observations indicating that cannabinoids combine at the same time neuroprotective [4–6] and anti-proliferative [1, 3] properties. Thus, over the last decade, a considerable volume of work has accumulated evidence to assume that the endocannabinoid system plays a role in the protection against acute or chronic brain damage [4–6]. This fact is particularly relevant considering the postmitotic characteristics of neuronal cells, which makes repair processes after several types of brain injury extremely difficult. For instance, plant-derived, synthetic and/or endogenous cannabinoids provide neuroprotection in in vitro and in vivo models that replicate cytotoxic events, mainly energy failure and excitotoxicity, occurring during several types of accidental brain injury (i.e. ischemia and head trauma), that acutely trigger degeneration (see [4–6] for recent reviews). In addition, cannabinoids are also neuroprotective in several chronic neurodegenerative pathologies that also involve the occurrence of excitotoxicity, mitochondrial dysfunction, inflammation and/or oxidative stress, such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and multiple sclerosis (MS); see [4, 7] for review.

Cannabinoids in Neurodegenerative Disorders and Stroke/Brain Trauma: From Preclinical Models to Clinical Applications
Javier Fernández-Ruiz, & María A. Moro, & José Martínez-Orgado
Neurotherapeutics (2015) 12:793–806
DOI 10.1007/s13311-015-0381-7
https://www.ncbi.nlm.nih.gov/pmc/art...rticle_381.pdf
Cannabinoids form a singular family of plantderived compounds (phytocannabinoids), endogenous signaling lipids (endocannabinoids), and synthetic derivatives with multiple biological effects and therapeutic applications in the central and peripheral nervous systems. One of these properties is the regulation of neuronal homeostasis and survival, which is the result of the combination of a myriad of effects addressed to preserve, rescue, repair, and/or replace neurons, and also glial cells against multiple insults that may potentially damage these cells. These effects are facilitated by the location of specific targets for the action of these compounds (e.g., cannabinoid type 1 and 2 receptors, endocannabinoid inactivating enzymes, and nonendocannabinoid targets) in key cellular substrates (e.g., neurons, glial cells, and neural progenitor cells). This potential is promising for acute and chronic neurodegenerative pathological conditions. In this review, we will collect all experimental evidence, mainly obtained at the preclinical level, supporting that different cannabinoid compounds may be neuroprotective in adult and neonatal ischemia, brain trauma, Alzheimer’s disease, Parkinson’s disease, Huntington’s chorea, and amyotrophic lateral sclerosis. This increasing experimental evidence demands a prompt clinical validation of cannabinoid-based medicines for the treatment of all these disorders, which, at present, lack efficacious treatments for delaying/arresting disease progression, despite the fact that the few clinical trials conducted so far with these medicines have failed to demonstrate beneficial effects.

Cannabinoids in the management of difficult to treat pain
Ethan B Russo
Therapeutics and Clinical Risk Management 2008:4(1) 245–259
doi: 10.2147/tcrm.s1928
This article reviews recent research on cannabinoid analgesia via the endocannabinoid system and non-receptor mechanisms, as well as randomized clinical trials employing cannabinoids in pain treatment. Tetrahydrocannabinol (THC, Marinol®) and nabilone (Cesamet®) are currently approved in the United States and other countries, but not for pain indications. Other synthetic cannabinoids, such as ajulemic acid, are in development. Crude herbal cannabis remains illegal in most jurisdictions but is also under investigation. Sativex®, a cannabis derived oromucosal spray containing equal proportions of THC (partial CB1 receptor agonist ) and cannabidiol (CBD, a non-euphoriant, anti-infl ammatory analgesic with CB1 receptor antagonist and endocannabinoid modulating effects) was approved in Canada in 2005 for treatment of central neuropathic pain in multiple sclerosis, and in 2007 for intractable cancer pain. Numerous randomized clinical trials have demonstrated safety and effi cacy for Sativex in central and peripheral neuropathic pain, rheumatoid arthritis and cancer pain. An Investigational New Drug application to conduct advanced clinical trials for cancer pain was approved by the US FDA in January 2006. Cannabinoid analgesics have generally been well tolerated in clinical trials with acceptable adverse event profi les. Their adjunctive addition to the pharmacological armamentarium for treatment of pain shows great promise

Cannabinoids, Inner Ear, Hearing, and Tinnitus: A Neuroimmunological Perspective.
Perin, P., Mabou Tagne, A., Enrico, P., Marino, F., Cosentino, M., Pizzala, R., & Boselli, C. (2020).**
Frontiers in Neurology, 11.
doi:10.3389/fneur.2020.505995
Cannabis has been used for centuries for recreational and therapeutic purposes. Whereas, the recreative uses are based on the psychotropic effect of some of its compounds, its therapeutic effects range over a wide spectrum of actions, most of which target the brain or the immune system. Several studies have found cannabinoid receptors in the auditory system, both at peripheral and central levels, thus raising the interest in cannabinoid signaling in hearing, and especially in tinnitus, which is affected also by anxiety, memory, and attention circuits where cannabinoid effects are well described. Available studies on animal models of tinnitus suggest that cannabinoids are not likely to be helpful in tinnitus treatment and could even be harmful. However, the pharmacology of cannabinoids is very complex, and most studies focused on neural CB1R-based responses. Cannabinoid effects on the immune system (where CB2Rs predominate) are increasingly recognized as essential in understanding nervous system pathological responses, and data on immune cannabinoid targets have emerged in the auditory system as well. In addition, nonclassical cannabinoid targets (such as TRP channels) appear to play an important role in the auditory system as well. This review will focus on neuroimmunological mechanisms for cannabinoid effects and their possible use as protective and therapeutic agents in the ear and auditory system, especially in tinnitus.

Cannabinoids as anticancer therapeutic agents.
Kovalchuk, O., & Kovalchuk, I.
Cell Cycle, 1–29. (2020).
doi:10.1080/15384101.2020.1742952
The recent announcement of marijuana legalization in Canada spiked many discussions about potential health benefits of Cannabis sativa. Cannabinoids are active chemical compounds produced by cannabis, and their numerous effects on the human body are primarily exerted through interactions with cannabinoid receptor types 1 (CB1) and 2 (CB2). Cannabinoids are broadly classified as endo-, phyto-, and synthetic cannabinoids. In this review, we will describe the activity of cannabinoids on the cellular level, comprehensively summarize the activity of all groups of cannabinoids on various cancers and propose several potential mechanisms of action of cannabinoids on cancer cells.

Cannabinoids as pharmacotherapies for neuropathic pain: From the bench to the bedside.
Rahn, E. J., & Hohmann, A. G.
Neurotherapeutics, 6(4), 713–737. (2009).
doi:10.1016/j.nurt.2009.08.002
Neuropathic pain is a debilitating form of chronic pain resulting from nerve injury, disease states, or toxic insults. Neuropathic pain is often refractory to conventional pharmacotherapies, necessitating validation of novel analgesics. Cannabinoids, drugs that share the same target as
9 -tetrahydrocannabinol (9 -THC), the psychoactive ingredient in cannabis, have the potential to address this unmet need. Here, we review studies evaluating cannabinoids for neuropathic pain management in the clinical and preclinical literature. Neuropathic pain associated with nerve injury, diabetes, chemotherapeutic treatment, human immunodeficiency virus, multiple sclerosis, and herpes zoster infection is considered. In animals, cannabinoids attenuate neuropathic nociception produced by traumatic nerve injury, disease, and toxic insults. Effects of mixed cannabinoid CB1/CB2 agonists, CB2 selective agonists, and modulators of the endocannabinoid system (i.e., inhibitors of transport or degradation) are compared. Effects of genetic disruption of cannabinoid receptors or enzymes controlling endocannabinoid degradation on neuropathic nociception are described. Specific forms of allodynia and hyperalgesia modulated by cannabinoids are also considered. In humans, effects of smoked marijuana, synthetic
9 -THC analogs (e.g., Marinol, Cesamet) and medicinal cannabis preparations containing both
9 -THC and cannabidiol (e.g., Sativex, Cannador) in neuropathic pain states are reviewed. Clinical studies largely affirm that neuropathic pain patients derive benefits from cannabinoid treatment. Subjective (i.e., rating scales) and objective (i.e., stimulusevoked) measures of pain and quality of life are considered. Finally, limitations of cannabinoid pharmacotherapies are discussed together with directions for future research. Key Words: Endocannabinoid, marijuana, neuropathy, multiple sclerosis, chemotherapy, diabetes.

Cannabinoids, Endocannabinoids, and Related Analogs in Inflammation
Sumner H. Burstein1,2,3 and Robert B. Zurier
The AAPS Journal, Vol. 11, No. 1, March 2009 (# 2009)
DOI: 10.1208/s12248-009-9084-5.
This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation. A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process.

Cannabinoids for Treatment of MS Symptoms: State of the Evidence
Rice J, Cameron M.
Curr Neurol Neurosci Rep. 2018 Jun 19;18(8):50.
doi: 10.1007/s11910-018-0859-x.
PURPOSE OF REVIEW:
Cannabis and cannabinoids have been used medically and recreationally for thousands of years and recently there has been a growing body of research in this area. With increased access now that medical marijuana is available in many jurisdictions, patients and providers want to know more about the evidence for benefits and risks of cannabinoid use. This paper provides an overview of the available cannabinoid-based formulations, a summary of the highest quality evidence for the use of cannabinoids for treating spasticity and pain associated with multiple sclerosis (MS), and a discussion of possible dosing regimens based on information from these studies.
RECENT FINDINGS:
Two recent high-quality systematic reviews concluded that the only strong evidence for medical marijuana in neurological disorders was for reducing the symptoms of patient-reported spasticity and central pain in MS and that the only complementary and alternative medicine (CAM) intervention in MS with strong supportive evidence was cannabinoids. Based on this review, they concluded that nabiximols (Sativex oral spray), oral cannabis extract (OCE), and synthetic tetrahydrocannabinol (THC) are probably effective at reducing patient-reported symptoms of spasticity in people with MS, but OCE and synthetic THC were not found to be effective for reducing physician-administered measures of spasticity. In addition, nabiximols, OCE, and synthetic THC are probably effective at reducing MS-related pain. Cannabinoids were generally well-tolerated. However, cannabis use has been associated with an increased risk of psychosis and schizophrenia in at-risk individuals, there is growing evidence that cannabis can increase the risk for cardiovascular diseases, including myocardial infarction (MI), hypertension, heart failure, and stroke, and a recently recognized adverse effect of cannabis is cannabinoid hyperemesis syndrome. The medical use of cannabinoids remains controversial. While cannabinoids have been studied for a variety of neurologic disorders, there is strongest evidence to indicate benefits in treatment of spasticity and neuropathic pain in multiple sclerosis. Although the best dose for an individual remains uncertain, most participants in the studies discussed in this paper used between 20 and 40 mg of THC a day in divided doses. Adverse events in studies were generally more common in the groups using cannabinoid products but serious adverse events were rare and cannabis products were generally well-tolerated. Cannabis use does appear to be associated with increased risk of certain adverse events, including psychosis, cardiovascular diseases, and cannabinoid hyperemesis syndrome.

Cannabinoids Improve Gastrointestinal Symptoms in a Parenteral Nutrition–Dependent Patient With Chronic Intestinal Pseudo?Obstruction.
Zemrani, B., Lambe, C., & Goulet, O.
Journal of Parenteral and Enteral Nutrition. (2020).
doi:10.1002/jpen.1821
Chronic intestinal pseudo-obstruction (CIPO) is a rare and challenging cause of pediatric intestinal failure, requiring long-term parenteral nutrition in most cases. Despite optimal management, some patients experience chronic abdominal pain and recurrent obstructive episodes with a major impact on their quality of life. Cannabinoids have been successfully used in some conditions. However, their use in CIPO has never been reported in the literature. We report a case of successful use of medicinal cannabinoids in a patient with CIPO, resulting in a significant reduction of abdominal pain, vomiting, and subocclusive episodes and increased appetite and weight, without major adverse events. Although further observations are required to consolidate these findings, this case may be helpful for other patients suffering from the same condition.

Cannabinoids in Clinical Practice.
Williamson, E. M., & Evans, F. J.
Drugs, 60(6), 1303–1314. (2000).
doi:10.2165/00003495-200060060-00005
Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (?9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC REVIEW ARTICLE Drugs 2000 Dec; 60 (6): 1303-1314 0012-6667/00/0012-1303/$25.00/0 © Adis International Limited. All rights reserved. rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties of THC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapyinduced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.

Cannabinoids in neurodegeneration and neuroprotection
In book Cannabinoids as Therapeutics Edited by R. Mechoulam 2005 Pg 79
Javier Fernández-Ruiz, Sara González, Julián Romero and José Antonio Ramos
Among a variety of cellular and tissue functions, it has been suggested that the endocannabinoid system might also exert an important function in the cellular decision about death or survival (for review see [1–3]). This finding has derived from several experimental observations indicating that cannabinoids combine at the same time neuroprotective [4–6] and anti-proliferative [1, 3] properties. Thus, over the last decade, a considerable volume of work has accumulated evidence to assume that the endocannabinoid system plays a role in the protection against acute or chronic brain damage [4–6]. This fact is particularly relevant considering the postmitotic characteristics of neuronal cells, which makes repair processes after several types of brain injury extremely difficult. For instance, plant-derived, synthetic and/or endogenous cannabinoids provide neuroprotection in in vitro and in vivo models that replicate cytotoxic events, mainly energy failure and excitotoxicity, occurring during several types of accidental brain injury (i.e. ischemia and head trauma), that acutely trigger degeneration (see [4–6] for recent reviews). In addition, cannabinoids are also neuroprotective in several chronic neurodegenerative pathologies that also involve the occurrence of excitotoxicity, mitochondrial dysfunction, inflammation and/or oxidative stress, such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and multiple sclerosis (MS); see [4, 7] for review. On the other hand, the activation of different elements of the endocannabinoid
system, as part of an endogenous protectant response, has been documented in different experimental paradigms of neurodegeneration, although with variable results, depending on age, species, type and severity of injury, and mechanism(s) activated for cell death (reviewed in [5, 8, 9]). Thus, several studies have demonstrated that neuronal damage is accompanied by an increase in the production of endocannabinoids (see [5, 8] for recent reviews), although other authors did not find this response [10]. For instance, Hansen and coworkers described an increase in the levels of anandamide (N-arachidonoylethanolamine, AEA) and its phospholipid precursor, but not of 2-arachidonoyl glycerol (2-AG), during acute degeneration in the neonatal rat brain [11, 12]. Similar results, increases in AEA with no changes in 2-AG, were found by Marsicano et al. [13] in a mouse model of kainate-induced excitotoxicity, and by Gubellini et al. [14] in a rat model of PD. However, Panikashvili et al. [15] showed that 2-AG is massively produced in the mouse brain after closed head injury. In addition, they found that this endocannabinoid has neuroprotective effects, as indicated by a reduction in edema and infarct volume and by improved clinical recovery after being administered to animals. This endogenous response has been also found in humans since elevated levels of AEA and other fatty acid amides have been also measured around the site of damage in a microdialysis study perfomed on a single stroke patient [16].
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Cannabinoids in Parkinson’s Disease
Mario Stampanoni Bassi, Andrea Sancesario, Roberta Morace, Diego Centonze, and Ennio Iezzi
Cannabis and Cannabinoid Research Volume 2.1, 2017
DOI: 10.1089/can.2017.0002
The endocannabinoid system plays a regulatory role in a number of physiological processes and has been found altered in different pathological conditions, including movement disorders. The interactions between cannabinoids and dopamine in the basal ganglia are remarkably complex and involve both the modulation of other neurotransmitters (c-aminobutyric acid, glutamate, opioids, peptides) and the activation of different receptors subtypes (cannabinoid receptor type 1 and 2). In the last years, experimental studies contributed to enrich this scenario reporting interactions between cannabinoids and other receptor systems (transient receptor potential vanilloid type 1 cation channel, adenosine receptors, 5-hydroxytryptamine receptors). The improved knowledge, adding new interpretation on the biochemical interaction between cannabinoids and other signaling pathways, may contribute to develop new pharmacological strategies. A number of preclinical studies in different experimental Parkinson’s disease (PD) models demonstrated that modulating the cannabinoid system may be useful to treat some motor symptoms. Despite new cannabinoid-based medicines have been proposed for motor and nonmotor symptoms of PD, so far, results from clinical studies are controversial and inconclusive. Further clinical studies involving larger samples of patients, appropriate molecular targets, and specific clinical outcome measures are needed to clarify the effectiveness of cannabinoid based therapies

Cannabinoids in multiple sclerosis: A neurophysiological analysis.
Vecchio, D., Varrasi, C., Virgilio, E., Spagarino, A., Naldi, P., & Cantello, R.**
Acta Neurologica Scandinavica. (2020).
doi:10.1111/ane.13313
Objectives. To investigate the action of cannabinoids on spasticity and pain in secondary progressive multiple sclerosis, by means of neurophysiological indexes.
Material and Methods. We assessed 15 patients with progressive MS (11 females) using clinical scales for spasticity and pain, as well as neurophysiological variables (H/M ratio, cutaneous silent period or CSP). Testing occurred before (T0) and during (T1) a standard treatment with an oral spray containing delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD). Neurophysiological measures at T0 were compared with those of 14 healthy controls of similar age and sex (HC). We then compared the patient results at the 2 time points (T1 vs T0).
Results. At T0, neurophysiological variables did not differ significantly between patients and controls. At T1, spasticity and pain scores improved, as detected by the Modified Ashworth Scale or MAS (p=0.001), 9- Hole Peg Test or 9HPT (p=0.018), numeric rating scale for spasticity or NRS (p=0.001), and visual analogue scale for pain or VAS (p=0.005). At the same time, the CSP was significantly prolonged (p=0.001).
Conclusions. The THC-CBD spray improved spasticity and pain in secondary progressive MS patients. The spray prolonged CSP duration, which appears a promising tool for assessing and monitoring the analgesic effects of THC-CBD in MS.

Cannabinoids in the Management of Acute Pain: A Systematic Review and Meta-analysis.
Gazendam, A., Nucci, N., Gouveia, K., Abdel Khalik, H., Rubinger, L., & Johal, H.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2019.0079
Objective: To synthesize the best evidence surrounding the efficacy of cannabinoids for acute pain in the clinical setting based on subjective pain scores and observed adverse effects. Design: Systematic review with meta-analysis. Data Sources: PubMed, Embase, Cochrane Databases, and Google Scholar. Eligibility Criteria: English-language randomized-controlled clinical trials comparing cannabinoids with placebo in patients with acute pain. Data Extraction and Synthesis: Study quality was assessed using the Cochrane risk of bias tool. All stages were conducted independently by a team of three reviewers. Data were pooled through meta-analysis and stratified by route of administration. Primary Outcomes and Measures: Patient-reported pain and adverse events (AEs). Results: Six trials (678 participants) were included examining oral (5 trials) and intramuscular (1 trial) cannabinoids. Overall, there was a small but statistically significant treatment effect favoring the use of cannabinoids over placebo (0.90, 95% confidence interval [CI]1.69 to0.1, i 2 = 65%, p = 0.03). When stratified by route of administration, intramuscular cannabinoids were found to have a significant reduction in pain relative to placebo (2.98, 95% CI4.09 to1.87, i 2 = 0%, p < 0.0001). No difference in effect was observed between oral cannabinoids and placebo (0.21, 95% CI0.64 to 0.22, i 2 = 3%, p = 0.34). Serious AEs were rare, and similar across the cannabinoid (14/374, 3.7%) and placebo groups (8/304, 2.6%). Conclusions: There is low-quality evidence indicating that cannabinoids may be a safe alternative for a small but significant reduction in subjective pain score when treating acute pain, with intramuscular administration resulting in a greater reduction relative to oral. Higher quality, long-term randomized-controlled trials examining whether there may be a role for cannabinoids in treating acute pain are required.

Cannabinoids in the Treatment of Epilepsy: Current Status and Future Prospects
Morano, A., Fanella, M., Albini, M., Cifelli, P., Palma, E., Giallonardo, A. T., & Di Bonaventura, C.
Neuropsychiatric Disease and Treatment, Volume 16, 381–396 (2020).
doi:10.2147/ndt.s203782*
Cannabidiol (CBD) is one of the prominent phytocannabinoids found in Cannabis sativa, differentiating from ?9 -tetrahydrocannabinol (THC) for its non-intoxicating profile and its antianxiety/antipsychotic effects. CBD is a multi-target drug whose anti-convulsant properties are supposed to be independent of endocannabinoid receptor CB1 and might be related to several underlying mechanisms, such as antagonism on the orphan GPR55 receptor, regulation of adenosine tone, activation of 5HT1A receptors and modulation of calcium intracellular levels. CBD is a lipophilic compound with low oral bioavailability (6%) due to poor intestinal absorption and high first-pass metabolism. Its exposure parameters are greatly influenced by feeding status (ie, high fatcontaining meals). It is mainly metabolized by cytochrome P 450 (CYP) 3A4 and 2C19, which it strongly inhibits. A proprietary formulation of highly purified, plant-derived CBD has been recently licensed as an adjunctive treatment for Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS), while it is being currently investigated in tuberous sclerosis complex. The regulatory agencies’ approval was granted based on four pivotal double-blind, placebocontrolled, randomized clinical trials (RCTs) on overall 154 DS patients and 396 LGS ones, receiving CBD 10 or 20 mg/kg/day BID as active treatment. The primary endpoint (reduction in monthly seizure frequency) was met by both CBD doses. Most patients reported adverse events (AEs), generally from mild to moderate and transient, which mainly consisted of somnolence, sedation, decreased appetite, diarrhea and elevation in aminotransferase levels, the last being documented only in subjects on concomitant valproate therapy. The interaction between CBD and clobazam, likely due to CYP2C19 inhibition, might contribute to some AEs, especially somnolence, but also to CBD clinical effectiveness. Cannabidivarin (CBDV), the propyl analogue of CBD, showed anti-convulsant properties in pre-clinical studies, but a plant-derived, purified proprietary formulation of CBDV recently failed the Phase II RCT in patients with uncontrolled focal seizures

Cannabinoids in the treatment of pain and spasticity in multiple sclerosis.
Smith PF.
Curr Opin Investig Drugs. 2002 Jun;3(6):859-64.
FIND DOI or LINK
There is a large amount of evidence to support the view that the psychoactive ingredient in cannabis, delta9-tetrahydrocannabinol (delta9-THC), and cannabinoids in general, can reduce muscle spasticity and pain under some circumstances. Cannabinoid (CB1) receptors in the CNS appear to mediate both of these effects and endogenous cannabinoids may fulfil these functions to some extent under normal circumstances. However, in the context of multiple sclerosis (MS), it is still questionable whether cannabinoids are superior to existing, conventional medicationsfor the treatment of spasticity and pain. In the case of spasticity, there are too few controlled clinical trials to draw any reliable conclusion at this stage. In the case of pain, most of the available trials suggest that cannabinoids are not superior to existing treatments; however, few trials have examined chronic pain syndromes that are relevant to MS. Whether or not cannabinoids do have therapeutic potential in the treatment of MS, a further issue will be whether synthetic cannabinoids should be used in preference to cannabis itself. Smoking cannabis is associated with significant risks of lung cancer and other respiratory dysfunction. Furthermore, delta9-THC, as a broad-spectrum cannabinoid receptor agonist, will activate both CB1 and CB2 receptors. Synthetic cannabinoids, which target specific cannabinoid receptor subtypes in specific parts of the CNS, are likely to be of more therapeutic use than delta9-THC itself. If rapid absorption is necessary, such synthetic drugs could be delivered via aerosol formulations.

Cannabinoids, Inner Ear, Hearing, and Tinnitus: A Neuroimmunological Perspective.
Perin, P., Mabou Tagne, A., Enrico, P., Marino, F., Cosentino, M., Pizzala, R., & Boselli, C. (2020).**
Frontiers in Neurology, 11.
doi:10.3389/fneur.2020.505995
Cannabis has been used for centuries for recreational and therapeutic purposes. Whereas, the recreative uses are based on the psychotropic effect of some of its compounds, its therapeutic effects range over a wide spectrum of actions, most of which target the brain or the immune system. Several studies have found cannabinoid receptors in the auditory system, both at peripheral and central levels, thus raising the interest in cannabinoid signaling in hearing, and especially in tinnitus, which is affected also by anxiety, memory, and attention circuits where cannabinoid effects are well described. Available studies on animal models of tinnitus suggest that cannabinoids are not likely to be helpful in tinnitus treatment and could even be harmful. However, the pharmacology of cannabinoids is very complex, and most studies focused on neural CB1R-based responses. Cannabinoid effects on the immune system (where CB2Rs predominate) are increasingly recognized as essential in understanding nervous system pathological responses, and data on immune cannabinoid targets have emerged in the auditory system as well. In addition, nonclassical cannabinoid targets (such as TRP channels) appear to play an important role in the auditory system as well. This review will focus on neuroimmunological mechanisms for cannabinoid effects and their possible use as protective and therapeutic agents in the ear and auditory system, especially in tinnitus.
Cannabinoids modulate associative cerebellar learning via alterations in behavioral state
Catarina Albergaria, N. Tatiana Silva, Dana Darmohray, Megan R. Carey
bioRxiv (2020)
https://www.biorxiv.org/content/10.1...113v2.full.pdf
DOI: 10.1101/511113
Cannabinoids are notorious and profound modulators of behavioral state. In the brain, endocannabinoids act via Type 1-cannabinoid receptors (CB1) to modulate synaptic transmission and mediate multiple forms of synaptic plasticity. CB1 knockout (CB1KO) mice display a range of behavioral phenotypes, in particular hypoactivity and various deficits in learning and memory, including cerebellumdependent delay eyeblink conditioning. Here we find that the apparent effects of CB1 deletion on cerebellar learning are not due to direct effects on CB1-dependent plasticity, but rather, arise as a secondary consequence of altered behavioral state. Hypoactivity of CB1KO mice accounts for their impaired eyeblink conditioning across both animals and trials. Moreover, learning in these mutants is rescued by walking on a motorized treadmill during training. Finally, cerebellar granule cell-specific CB1KOs exhibit normal eyeblink conditioning, and both global and granule cell-specific CB1KOs display normal cerebellum-dependent locomotor coordination and learning. These findings highlight the modulation of behavioral state as a powerful independent means through which individual genes contribute to complex behaviors.

CANNABINOIDS: POTENTIAL ANTICANCER AGENTS
Manuel Guzmán
Nature reviews VOLUME 3 | OCTOBER 2003 | 745
https://www.cannabis-med.org/data/pdf/en_2006_02_1.pdf
The active components of Cannabis sativa and their derivatives — exert palliative effects in cancer patients by preventing nausea, vomiting and pain and by stimulating appetite. In addition, these compounds have been shown to inhibit the growth of tumour cells in culture and animal models by modulating key cell-signalling pathways. Cannabinoids are usually well tolerated, and do not produce the generalized toxic effects of conventional chemotherapies. So, could cannabinoids be used to develop new anticancer therapies?

Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores
Shou-Yuan Zhuang, Daniel Bridges, Elena Grigorenko, Stephen McCloud,
Andrew Boon, Robert E. Hampson, Sam A. Deadwyler
Neuropharmacology 48 (2005) 1086e1096
doi:10.1016/j.neuropharm.2005.01.005
Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 mM). WIN 55,212-2 was also shown to inhibit the NMDAinduced increase in intracellular calcium concentration ([Ca2C]i) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2C]i occurred with similar concentrations (25e100 nM) and in the same time-dependent manner (preexposure 1e15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2C]i was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositoltrisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2C]i via a cAMP/PKA-dependent process during the neurotoxic event.

Cannabis: A Neurological Remedy or a Drug of Abuse in India
Pronit Biswas, Pooja Mishra, Devasish Bose and Abhilasha Durgbanshi,
CNS & Neurological Disorders - Drug Targets, 2017, 16,
Since ancient times, the use of cannabis as a medicine is well documented due to its potential therapeutic activity while subsequently its use as drug of abuse spread increasingly. The present review sought to give an insight in the history of medical and recreational use of cannabis in India. Indian use of cannabis dates back to Vedic time, mostly for the ritualistic and religious purposes, as documented in the ancient literature. It was India that introduced the medical use of cannabis to neighboring countries. Nevertheless, in the same India, medical use did not propagate due to religious and social stigma related to the plant itself. The pharmacoactive constituents of cannabis and their therapeutic values in Ayurvetic medicine have been here described together with the adverse effects they can cause with special reference to neurological ones, including withdrawal symptoms. Finally, how cannabis made its route to the Indian society has also been discussed
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Cannabis adulterated with the synthetic cannabinoid receptor agonist MDMB-4en-PINACA and the role of European drug checking services
Pieter E. Oomen, Dominique Schori, Karsten Tögel-Lins, Dean Acreman, Sevag Chenorhokian, Anton Luf, Alexandra Karden, Carlos Paulos, Elisa Fornero, Enrico Gerace, Raoul P.J. Koning, Liliana Galindo, Laura A. Smit-Rigter, Fiona Measham, Mireia Ventura
International Journal of Drug Policy ◽ 2022 ◽ Vol 100 ◽ pp. 103493
DOI: 10.1016/j.drugpo.2021.103493
Background: European drug checking services exchange information on drug trends within the Trans European Drug Information (TEDI) network, allowing monitoring and coordination of responses. Starting in Spring 2020, several services detected the synthetic cannabinoid receptor agonist MDMB-4en-PINACA in adulterated low-THC cannabis products.
Methods: Cannabis products suspected of adulteration were analyzed for the presence of MDMB-4en-PINACA by 9 services in 8 countries within the TEDI network. If available, phytocannabinoid analysis was also performed.
Results: 1142 samples sold as cannabis in herbal, resin and e-liquid form were analyzed, of which 270 were found to contain MDMB-4en-PINACA. All cannabis samples contained low THC (<1%), except the e-liquids which contained no phytocannabinoids. Three serious health incidents requiring hospitalization after use of an adulterated cannabis sample were reported.
Conclusion: Adulteration of cannabis with synthetic cannabinoid receptor agonists is a new phenomenon that carries risk for people who use it. Given that cannabis consumers are not a usual target group for drug checking services, services and associated harm reduction interventions could be reconfigured to include them.
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Cannabis and Its Potential Protective Role Against Inflammatory Bowel Disease: A Scoping Review

Nso Nso , Akwe Nyabera , Mahmoud Nassar , Mohsen S. Alshamam , Vikram Sumbly , Mallorie Vest , Nehal Patel , Gilbert Ojong , Vincent Rizzo
Cureus 13(10): e18841.
DOI: 10.7759/cureus.18841
https://www.ncbi.nlm.nih.gov/pmc/art...0000018841.pdf
Globally, around 15%-40% of patients suffering from inflammatory bowel disease (IBD) use Cannabis for pain reduction, increased appetite, and reduced need for other medications. Although many patients report having benefited by using Cannabis in IBD, there is still a lack of consensus regarding the use of Cannabis in IBD. The aim is to identify, explore and map literature on the potential protective role of Cannabis against IBD through this scoping review. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed during the search to answer the focal question: (1) Does Cannabis play a protective role against IBD as assessed by clinical remission; (2) If yes, what is the mechanism of action for this protective role. There were only three randomized controlled trials (RCTs) and three observational studies that satisfied the selection criteria of this scoping review. Although promising results including the improvement in general well-being/ Harvey-Bradshaw Index, health perception enhancement [4.1±1.43 to 7±1.42 (p = 0.0002)], weight gain, Crohn's Disease Activity Index (CDAI) score

Cannabis allergy: A diagnostic challenge. Allergy.
Decuyper, I. I., Faber, M. A., Lapeere, H., Mertens, C., Rihs, H. P., Van Gasse, A. L., … Ebo, D. G.
Allergy (2018)
doi:10.1111/all.13491
In the absence of a validated confirmatory test to document cannabis allergy, physicians use skin prick-prick testing with cannabis buds or leaves to confirm their clinical suspicion of a cannabis allergy (1-4). Obviously, this approach is difficult to standardize as results are dependent on the source material. Our earlier study (5) revealed that a basophil activation test (BAT) with an nsLTP
rich cannabis extract could reliably diagnose cannabis allergy but leaves room for improvement. Here, we take advantage of the newly expressed Can s 3 protein (6) to develop three flow cytometric diagnostics for cannabis allergy; a BAT with rCan s 3, a BAT with a crude CS extract and a bead assay (CBA) quantifying sIgE to rCan s 3.

Cannabis Allergy: A Melting “Pot” of Clinical Manifestations.
Wong, D. D., Abbas, K. F., Grunberg, R., & Sussman, G. L.
Journal of Allergy and Clinical Immunology, 141(2), AB255. (2018).
doi:10.1016/j.jaci.2017.12.810
RATIONALE: Cannabis allergy is currently on the rise, and the symptoms of cannabis allergy can vary from mild rhinitis to life threatening anaphylactic reactions. We investigated the relationship
between the routes of exposure and clinical manifestations in 11 patients with cannabis allergy.
METHODS: 11 patients who presented to an ambulatory allergy clinic with cannabis (marijuana or hempseed) allergy were evaluated. Skin prick test with a cannabis extract was performed to confirm diagnosis. A questionnaire was administered to document the routes of exposure, timeline of symptoms development, reaction types and cross-sensitizations.
RESULTS: The mean age of the patients were 27 years (range 18 – 37), and the mean duration of cannabis use is 7 years (range 1 – 13). 3 patients had reaction to cannabis with first exposure. 3 patients reported GI symptoms (abdominal pain and vomiting) with ingestion of marijuana, and 2 of these 3 patients also reported the same reactions with hempseed ingestion. 8 patients reported rhinitis and ocular symptoms with smoking marijuana, and 7 patients reported urticaria and pruritus with touching cannabis. Two patients developed GI symptoms and anaphylactic reaction to ingestion of hempseed after 1 – 2 years of smoking marijuana. One patient also have allergic reaction to tomato which is known to cross-react with cannabis. One patient reported exercise-induced anaphylaxis dependent on cannabis ingestion and smoking.
CONCLUSIONS: Cannabis allergy symptoms vary depending on the routes of exposure. Anaphylactic reactions to ingestion of hempseeds can develop after marijuana exposure. Cannabis can be a co-factor in fooddependent exercise-induced anaphylaxis.

CANNABIS ALLERGY IN A YOUNG CHILD WITH SEVERE ASTHMA EXPOSED TO SECONDHAND MARIJUANA SMOKE.
Hoffman, B., Kuhl, M., Knight, V., Phillips, M., & Rabinovitch, N.
Annals of Allergy, Asthma & Immunology, 121(5), S82. (2018).
doi:10.1016/j.anai.2018.09.268 (Paper)
https://www.epostersonline.com/acaai...1599?view=true (Poster)
Introduction: Secondhand cannabis exposure is expected to increase significantly as personal cannabis use becomes legalized in more states and countries. Cannabis allergy from firsthand use has been reported in adults but allergy in young children exposed to secondhand smoke has not been previously reported. We present a case of cannabis allergy associated with secondhand cannabis exposure and highlight its potential impact on childhood asthma.
Case Description: A 6-year-old boy with severe asthma was poorly controlled on a regimen of high-dose inhaled mometasone/formoterol, inhaled tiotropium, and montelukast. Additional history taking revealed that family members were frequently smoking cannabis in the household, and his maternal grandmother reported a history of urticaria after personal use of cannabis. Skin prick testing to a cannabis puddle (in saline) resulted in wheals of 9 £ 8 and 15 £ 8 mm for patient and grandmother respectively, while a control individual was negative. We then performed a basophil activation test in which the patient demonstrated significant activation to cannabis (figure 1). After removal of cannabis from the home environment, the patient's asthma improved by subjective (Asthma Control Test questionnaire) and objective (impulse oscillometry) measures.
Discussion: There have been several reports associating cannabis use with allergic symptoms including rhinitis, conjunctivitis, asthma, and anaphylaxis. This is the first report of cannabis allergy in a young child and suggests that exposure to secondhand marijuana smoke may contribute to difficult-to-control asthma in such children.

Cannabis Allergy: More Than a Bad Trip.
Van Gasse, A. L., Sabato, V., Faber, M. M., Bridts, C. H., & Ebo, D. G.
Handbook of Cannabis and Related Pathologies, 517–526. (2017).
doi:10.1016/b978-0-12-800756-3.00063-6
An allergy is defined as an immune-pathological, mostly IgE-mediated, reaction that ultimately results into a hypersensitivity reaction toward one or more foreign compounds, that is, an allergen.
The exact prevalence of cannabis allergy is not known, it seems to be an increasing problem in the past few years.
Active use, as well as passive exposure to cannabis could lead to sensitization to cannabis allergens, and eventually to cannabis allergy if this sensitization is of clinical relevance.
The clinical manifestations of cannabis allergy can vary from mild to life-threatening reactions, often depending on the route of exposure.
Sensitization to cannabis could also lead to secondary allergies, such as allergies to fruit and vegetables, designated as the “cannabis-fruit/ vegetable syndrome,” but also to Hevea latex, cereals, tobacco, and alcoholic beverages, such as beer and wine.
At present, diagnosis generally rests upon history, skin testing, and an IgE test with an industrial hemp variety.
Recently, the basophil activation test (BAT) has been proven to be a reliable additional diagnostic.
For the time being, there is no cure for cannabis related allergies. Strict avoidance of cannabis and other allergens implicated in the individual crossreactivity syndrome is imperative.

Cannabis Allergy: What do We Know Anno 2015.
Decuyper, I., Ryckebosch, H., Van Gasse, A. L., Sabato, V., Faber, M., Bridts, C. H., & Ebo, D. G. (2015).
Archivum Immunologiae et Therapiae Experimentalis, 63(5), 327–332.
doi:10.1007/s00005-015-0352-z
For about a decade, IgE-mediated cannabis (marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may lead to a cannabis sensitization and/or allergy. The clinical manifestations of a cannabis allergy can vary from mild to life threatening reactions, often depending on the route of exposure. In addition, sensitization to cannabis allergens can trigger various secondary cross-allergies, mostly for plant-derived food. This clinical entity, which we have designated as the ‘‘cannabis-fruit/vegetable syndrome’’ might also imply cross-reactivity with tobacco, latex and plant-food derived alcoholic beverages. These secondary cross-allergies are mainly described in Europe and appear to result from cross-reactivity between non-specific lipid transfer proteins or thaumatin-like proteins present in Cannabis sativa and their homologues that are ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies rests upon a thorough history completed with skin testing using native extracts from buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures including a stop of any further cannabis (ab)use.

Cannabis allergy: what the clinician needs to know in 2019.
Decuyper, I. I., Rihs, H.-P., Van Gasse, A. L., Elst, J., De Puysseleyr, L., Faber, M. A., … Ebo, D. G. (2019).
Expert Review of Clinical Immunology, 1–8. (2019).
doi:10.1080/1744666x.2019.1600403
Introduction: Although the use of cannabis dates back millennia, the first description of cannabis allergy is relatively recent (1971). Recent large-scale data show that cannabis allergy can manifest severe and generalized symptoms with extensive cross-reactions. Thus, it is essential to become familiarized with its clinical presentation, diagnostic aids, and adequate therapeutic guidance.
Areas covered: Here we provide a hands-on overview on cannabis allergy focusing on symptomatology and the reliability of diagnostic options. Recent advances in proteomics are discussed in detail,
elucidating the link with nsLTP-related allergies. The proteomics advancements have paved the way for more reliable diagnostics, especially component-based tools. Finally, the current experience in treatment options is highlighted.
Expert opinion: Cannabis allergy is an allergy entity which can significantly impact the quality of life. For optimal diagnosis, we advise to start with a validated and standardized crude-extract based test such as sIgE hemp complemented by component-based diagnostics such as sIgE Can s 3 quantifications where available. Future research should lift the veil on the true prevalence of cannabis allergy and the importance of other cannabis allergens to further guide our practice.

Cannabis and the Cancer Patient
Ilana M. Braun, Donald I. Abrams, Stacey E. Blansky, Steven A. Pergam
J Natl Cancer Inst Monogr (2021) 2021(58): lgab012
doi: 10.1093/jncimonographs/lgab012
https://academic.oup.com/jncimono/ar.../58/68/6446214
Session 2 of the National Cancer Institute’s Cannabis, Cannabinoids, and Cancer Research Workshop opened with testimony from a lymphoma survivor who detailed medicinal cannabis-related improvements in nausea, low appetite, insomnia, and mental health and the limited clinical counsel she received regarding cannabis use. Discussion next turned to the evolution of the legal landscape of cannabis in the United States, one in which state and federal laws frequently conflict and the Controlled Substance Act renders cannabis Schedule I. This legal climate creates conundrums for US medicinal cannabis researchers who contend with limited funding opportunities, avenues to source trial drug, and procedural red tape and for oncology clinicians who recommend medicinal cannabis to patients with some frequency while perceiving themselves as ill equipped to make such clinical recommendations. Ultimately, it creates challenges for cancer patients who find themselves turning to nonmedical and anecdotal information sources. The risks of cannabis use by the cancer patient were discussed next. These include infection, pharmacodynamic and pharmacokinetic drug-botanical interactions, cyclic nausea and vomiting, e-cigarette or vaping product use–associated illness, legal issues, and high cost. The session concluded with a broad survey of the research supporting oncologic cannabinoid use, conclusive evidence for chemotherapy-induced nausea and vomiting, and suggestive evidence for cancer-related pain.

Cannabis and the developing brain: Insights from behavior
Viviana Trezza, Vincenzo Cuomo, Louk J.M.J. Vanderschuren
European Journal of Pharmacology 585 (2008) 441-452
DOI: 10.1016/j.ejphar.2008.01.058
The isolation and identification, in 1964, of delta-9-tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis, opened the door to a whole new field of medical research. The exploration of the therapeutic potential of THC and other natural and synthetic cannabinoid compounds was paralleled by the discovery of the endocannabinoid system, comprising cannabinoid receptors and their endogenous ligands, which offered exciting new insights into brain function. Besides its well-known involvement in specific brain functions, such as control of movement, memory and emotions, the endocannabinoid system plays an important role in fundamental developmental processes such as cell proliferation, migration and differentiation. For this reason, changes in its activity during stages of high neuronal plasticity, such as the perinatal and the adolescent period, can have long-lasting neurobehavioral consequences. Here, we summarize human and animal studies examining the behavioral and neurobiological effects of in utero and adolescent exposure to cannabis. Since cannabis preparations are widely used and abused by young people, including pregnant women, understanding how cannabinoid compounds affect the developing brain, leading to neurobehavioral alterations or neuropsychiatric disorders later in life, is a serious health issue. In addition, since the endocannabinoid system is emerging as a novel therapeutic target for the treatment of several neuropsychiatric diseases, a detailed investigation of possible adverse effects of cannabinoid compounds on the central nervous system (CNS) of immature individuals is warranted .

Cannabis as an Anticancer Agent: A Review of Clinical Data and Assessment of Case Reports
Jordan Guggisberg, Megan Schumacher, Grace Gilmore, and Dylan M. Zylla
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0045
https://www.researchgate.net/publica...is_as_an_Antic ancer_Agent_A_Review_of_Clinic al_Data_and_Assessment_of_Case _Reports
https://www.liebertpub.com/doi/epub/.../can.2021.0045
Background: Pre-clinical studies have demonstrated the potential anticancer activity of cannabinoids, yet little clinical data exist to support this. Nearly 40% of patients with cancer using cannabis believe it will treat their cancer with numerous anecdotal reports shared online through social media platforms. Case reports have been published in peer-reviewed journals, but often lack key clinical information to validate anticancer claims.
Methods: We reviewed literature in PubMed and EBSCO databases that evaluated the relationship between cannabis or the endocannabinoid system and potential anticancer activity. We also reviewed online sources, books, and ClinicalTrials.gov for reports or studies on using cannabis as cancer treatment. All case reports published in peer-reviewed journals were compiled and appraised as weak, moderate, or strong based on the quality of evidence provided supporting an anticancer effect. Strong reports met three criteria; (a) active cancer at time of cannabis administration, (b) validated laboratory or radiographic responses were reported, and (c) cannabis used without concurrent anticancer treatments.
Results: Of the 207 pre-clinical articles reviewed, 107 (52%) were pre-clinical studies with original data. A total of 77 unique case reports described patients with various cancers (breast, central nervous system, gynecological, leukemia, lung, prostate, and pancreatic) using cannabis. Our appraisal showed 14% of the case reports were considered strong, 5% moderate, and the remaining 81% were weak. Ten percent of cases were in pediatric patients. Cannabidiol use was most often reported as the anticancer cannabinoid with daily doses ranging from 10 to 800 mg. Tetrahydrocannabinol use was reported in six studies, with doses ranging from 4.8 to 7.5 mg. Two small trials published data on survival in patients with recurrent glioblastoma multiforme.
Conclusion: This review of clinical data suggests most published, peer-reviewed case reports provide insufficient data to support the claim for cannabis as an anticancer agent, and should not be used in place of evidence-based, traditional treatments outside of a clinical trial. No strong clinical trial data exist to confirm the pre-clinical studies that suggest cannabinoids may have an anticancer benefit. Future studies exploring anticancer potential of cannabis in patients with metastatic cancers who have not responded to traditional therapy are needed.
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Cannabis: An Emerging Occupational Allergen?
Sussman, G. L., Beezhold, D. H., Cohn, J. R., Silvers, W. S., Zeiger, J. S., & Nayak, A. P.
Annals of Work Exposures and Health.(2020).
doi:10.1093/annweh/wxaa043
Cannabis is the most commonly used psychoactive drug. In recent years, Cannabis access has expanded for both medicinal and non-medicinal has grown. This is also marked with an increasing number of individuals gaining employment in this emerging industry. In this article, we briefly discuss the health hazards associated with Cannabis exposure with an emphasis on the potential for allergic reactions in workers who handle and process Cannabis plant

Cannabis And Athletic Performance
Jamie F Burr, Christian Cheung, Andreas Kasper, Graeme Close
Sports Medicine April 2021
Cannabis is widely used for both recreational and medicinal purposes on a global scale. There is accumulating interest in the use of cannabis and its constituents for athletic recovery, and in some instances, performance. Amidst speculation of potential beneficial applications, the effects of cannabis and its two most abundant constituents, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), remain largely un-investigated. The purpose of this review was to critically evaluate the literature describing the effects of whole cannabis, THC, and CBD, on athletic performance and recovery. While investigations of whole cannabis and THC have generally shown either null or detrimental effects on exercise performance, studies of sufficient rigor and validity to conclusively declare ergogenic or ergolytic potential in athletes are lacking. The ability of cannabis and THC to perturb cardiovascular homeostasis warrants further investigation regarding mechanisms by which performance may be affected across different exercise modalities and energetic demands. In contrast to cannabis and THC, CBD has largely been scrutinized for its potential to aid in recovery. The beneficial effects of CBD on sleep quality, pain, and mild traumatic brain injury may be of particular interest to certain athletes. However, research in each of these respective areas has yet to be thoroughly investigated in athletic populations. Elucidating the effects of whole cannabis, THC, and CBD is pertinent for both researchers and practitioners given the widespread use of these products, and their potential to interact with athletes’ performance and recovery.
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Cannabis and Cannabidiol (CBD) for the Treatment of Fibromyalgia.
Berger, A. A., Keefe, J., Winnick, A., Gilbert, E., Eskander, J. P., Yazdi, C., … Urits, I.
Best Practice & Research Clinical Anaesthesiology.(2020).
doi:10.1016/j.bpa.2020.08.010
Fibromyalgia is a complex disease process that is as prevalent as it is poorly understood. Research into the pathophysiology is ongoing, and findings will likely assist in identifying new therapeutic options to augment those in existence*today*that are still insufficient for the care of a large population of patients. Recent evidence describes the use of cannabinoids in the treatment of fibromyalgia. This study provides a systematic, thorough review of the evidence alongside a review of the seminal data regarding the pathophysiology, diagnosis, and current treatment options. Fibromyalgia is characterized by widespread chronic pain, fatigue, and depressive episodes without an organic diagnosis, which may be prevalent in up to 10% of the population and carries a significant cost in healthcare utilization, morbidity, a reduced quality of life, and productivity. It is frequently associated with psychiatric comorbidities. The diagnosis is clinical and usually prolonged, and diagnostic criteria continue to evolve. Some therapies have been previously described, including neuropathic medications, milnacipran, and antidepressants. Despite some level of efficacy, only physical exercise has strong evidence to support it. Cannabis has been used historically to treat different pain conditions since ancient times. Recent advances allowed for the isolation of the active substances in cannabis and the production of cannabinoid products that are nearly devoid of psychoactive influence and provide pain relief and alleviation of other symptoms. Many of these, as well as cannabis itself, are approved for use in chronic pain conditions. Evidence supporting cannabis in chronic pain conditions is plentiful; however, in fibromyalgia, they are mostly limited. Only a handful of randomized trials exists, and their objectivity has been questioned. However, many retrospective trials and patient surveys suggest the significant alleviation of pain, improvement in sleep, and abatement of associated symptoms. Evidence supporting the use of cannabis in chronic pain and specifically in fibromyalgia is being gathered as the use of cannabis increases with current global trends. While the current evidence is still limited, emerging data do suggest a positive effect of cannabis in fibromyalgia. Cannabis use is not without risks, including psychiatric, cognitive, and developmental as well as the risks of addiction. As such, clinical judgment is warranted to weigh these risks and prescribe to patients who are more likely to benefit from this treatment. Further research is required to define appropriate patient selection and treatment regimens.

Cannabis and Cannabinoid Intoxication and Toxicity
Ziva Cooper, Arthur Robin Williams
In book: Cannabis Use Disorders January 2019
DOI: 10.1007/978-3-319-90365-1_12
Adverse effects related to cannabis are an emerging health risk exemplified by reliable increases in calls to poison control centers and emergency department visits due to both unintentional and abuse-related exposures. Toxicity as a consequence of synthetic cannabinoid use (i.e., Spice, K2) has also become an apparent clinical issue with sometimes severe adverse consequences. This chapter will describe the prevalence of over-intoxication, or toxicity by cannabis, cannabis-derived products, and synthetic cannabinoids, with attention to understanding populations most at risk. The constellation of physiological and psychiatric symptoms of intoxication and severe adverse reactions according to cannabis, cannabis-derived products, and synthetic cannabinoids will be described. While there is currently no standard course of action to treat cannabis and cannabinoid exposures, the current treatments reported in the literature will be summarized. As cannabis availability grows and strength of products increases, clinician awareness of the signs and symptoms of cannabis toxicity is critical for undertaking the optimal course of patient treatment. Additionally, knowledge of the risks associated with synthetic cannabinoid use and most frequent treatment approaches will also prepare clinicians for the most effective strategies to addressing their physiological and psychiatric adverse effects.

Cannabis and cannabinoids in cancer pain management
Howard Meng, Tianyang Dai, John G. Hanlon, James Downar, Shabbir M.H. Alibhai, Hance Clarke
Current Opinion in Supportive and Palliative Care April 2020
DOI: 10.1097/SPC.0000000000000493
Purpose of review: An increasing number of patients are turning to cannabis and cannabinoids for management of their palliative and nonpalliative cancer pain and other cancer-related symptoms. Canadians have a legal framework for access to medical cannabis, which provides a unique perspective in a setting lacking robust clinical evidence. This review seeks to delineate the role of cannabis and cannabinoids in cancer pain management and offers insight into the Canadian practice.
Recent findings: A cohort study using nabiximols on advanced cancer pain in patients already optimized on opioids, over 3 weeks, demonstrated improved average pain score. A large observational study of cancer patients using cannabis over 6 months demonstrated a decreased number of patients with severe pain and decreased opioid use, whereas the number of patients reporting good quality of life increased.
Summary: Good preclinical animal data and a large body of observational evidence point to the potential efficacy of cannabinoids for cancer pain management. However, there are relatively weak data pointing to clinical efficacy from clinical trial data to date. In Canada, the burgeoning cannabis industry has driven the population to embrace a medicine before clinical evidence. There remains a need for high-quality randomized controlled trials to properly assess the effectiveness and safety of medical cannabis, compared with placebo and standard treatments for cancer-related symptoms.

Cannabis and Canabidinoids on the Inflammatory Bowel Diseases: Going Beyond Misuse.
Carvalho, A. C. A. de, Souza, G. A. de, Marqui, S. V. de, Guiguer, É. L., Araújo, A. C., Rubira, C. J., … Barbalho, S. M.
International Journal of Molecular Sciences, 21(8), (2020).
doi:10.3390/ijms21082940
Inflammatory bowel diseases (IBD) are characterized by a chronic and recurrent gastrointestinal condition, including mainly ulcerative colitis (UC) and Crohn’s disease (CD). Cannabis sativa (CS) is widely used for medicinal, recreational, and religious purposes. The most studied compound of CS is tetrahydrocannabinol (THC) and cannabidiol (CBD). Besides many relevant therapeutic roles such as anti-inflammatory and antioxidant properties, there is still much controversy about the consumption of this plant since the misuse can lead to serious health problems. Because of these reasons, the aim of this review is to investigate the effects of CS on the treatment of UC and CD. The literature search was performed in PubMed/Medline, PMC, EMBASE, and Cochrane databases. The use of CS leads to the improvement of UC and CD scores and quality of life. The medical use of CS is on the rise. Although the literature shows relevant antioxidant and anti-inflammatory effects that could improve UC and CD scores, it is still not possible to establish a treatment criterion since the studies have no standardization regarding the variety and part of the plant that is used, route of administration and doses. Therefore, we suggest caution in the use of CS in the therapeutic approach of IBD until clinical trials with standardization and a relevant number of patients are performed.

Cannabis and cannabinoids on treatment of inflammation: a patent review.
Modesto Nascimento Menezes P, Chiara Valença Pereira E, Eduarda Gomes da Cruz Silva M, Augusto Oliveira da Silva B, Antonio Miranda de Souza Duarte Filho L, Cícero de Lima Araújo T, Simoni Bezerra Lima K, Souza Silva F, Araújo Rolim L.
Recent Pat Biotechnol. 2019 Jun 18.
doi: 10.2174/1872208313666190618124
BACKGROUND: The inflammatory process is a physiological response to a vast number harmful stimulus that takes place in order to restore homeostasis. Many drugs used in pharmacotherapy are effective to control inflammatory responses, however there is a range of adverse effects attributed to steroidal and non-steroidal anti-inflammatory drugs (NSAIDs). In this sense, herbal medicine and derivatives gain more adepts because of their effectiveness and safety, showing the importance of medicinal plants, especially the Cannabis genus and the cannabinoid derivatives.
OBJECTIVE: The aim of this prospection was to identify data related to patents involving Cannabis and cannabinoids for the treatment of inflammation.
METHOD: To do so, a search for patents was conducted to evaluate the anti-inflammatory activity of Cannabis and cannabinoids. Four specialized databases for patent research were consulted using the terms "cannabis", "cannabidiol", "cannabinoids" and "THC" associated with "inflammation".
RESULTS: A total of 370 patents were found, of which 17 patents met the inclusion criteria. Although reports show synergistic effects of the plant components, patents involving Cannabis and cannabinoids focus on isolated substances (CBD e THC).
CONCLUSION: However, patents related to Cannabis and cannabinoids are promising for future use of the plant or its derivatives on the treatment of inflammation.

Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?
McPartland, J. M., & Russo, E. B.
Journal of Cannabis Therapeutics, 1(3-4), 103–132. (2001).
doi:10.1300/j175v01n03_08
SUMMARY. A central tenet underlying the use of botanical remedies is that herbs contain many active ingredients. Primary active ingredients may be enhanced by secondary compounds, which act in beneficial synergy. Other herbal constituents may mitigate the side effects of dominant active ingredients. We reviewed the literature concerning medical cannabis and its primary active ingredient, ?9-tetrahydrocannabinol (THC). Good evidence shows that secondary compounds in cannabis may enhance the beneficial effects of THC. Other cannabinoid and non-cannabinoid compounds in herbal cannabis or its extracts may reduce THC-induced anxiety, cholinergic deficits, and immunosuppression. Cannabis terpenoids and flavonoids may also increase cerebral blood flow, enhance cortical activity, kill respiratory pathogens, and provide anti-inflammatory activity.

Cannabis and Male Fertility: A Systematic Review
Kelly S. Payne, Daniel J. Mazur, James M. Hotaling, Alexander W Pastuszak
The Journal of Urology March 2019
DOI: 10.1097/JU.0000000000000248
Introduction: With cannabis consumption on the rise, and use prominent among males of reproductive age, it is essential to understand the potential impact of cannabis on male fertility. Here we review the literature regarding cannabis' effects on male fertility.
Materials and methods: A literature search was conducted using PubMed/MEDLINE to identify relevant studies examining the effects of cannabis on male fertility. Relevant studies were identified and reviewed. Results: The strongest evidence for cannabis-induced alterations in male fertility is in the category of semen parameters. Research supports a role for cannabis in reducing sperm count and concentration, inducing abnormalities in sperm morphology, reducing sperm motility and viability, and inhibiting capacitation and fertilizing capacity. Animal models demonstrate a role for cannabis in testicular atrophy and reduced libido and sexual function, but these results have not yet been replicated in human studies. Studies examining hormonal changes suggest inconclusive effects on testosterone levels, lowered luteinizing hormone (LH) levels, and unchanged follicle stimulating hormone (FSH) levels.
Conclusions: Current research suggests that cannabis may negatively impact male fertility. Further studies are needed to validate that robust findings in animal models will carry over into human experience. Clinicians should be aware of these potential effects in prescribing medical marijuana therapies to men of reproductive age, and should consider degree of cannabis use as a possible component of a complete male infertility workup.

Cannabis and multiple sclerosis
Gillian Ingram, Owen R Pearson
Practical Neurology June 2019
DOI: 10.1136/practneurol-2018-002137
Patients with multiple sclerosis have long turned to complementary therapies to manage symptoms that licensed products can only partially control. Around half of patients with multiple sclerosis admit to previous or current cannabis use for medicinal purposes and would endorse legalisation. Despite many governments worldwide relaxing regulations around medicinal cannabis, there remain many unanswered questions as to how clinicians should prescribe or recommend products, and access to pharmaceutical-grade products remains highly restricted. Here we address what adult neurologists need to know about cannabis and its use in multiple sclerosis.

Cannabis and Natural Cannabis Medicines
Robert C. Clarke and David P. Watson
In: MARIJUANA AND THE CANNABINOIDS
Edited by Mahmoud A. ElSohly
CHAPTER 1 · NOVEMBER 2007
DOI: 10.1007/978-1-59259-947-9_1
Cannabis plants produce many compounds of possible medical importance. This chapter briefly explains the life cycle, origin, early evolution, and domestication of Cannabis, plus provides a brief history of drug Cannabis breeding and looks into the future of Cannabis as a source of medicines. Cannabis is among the very oldest of economic plants providing humans with fiber for spinning, weaving cloth, and making paper; seed for human foods and animal feeds; and aromatic resin containing compounds of recreational and medicinal value. Human selection for varying uses and natural selection pressures imposed by diverse introduced climates have resulted in a wide variety of growth forms and chemical compositions. Innovative classical breeding techniques have been used to improve recreational drug forms of Cannabis, resulting in many cannabinoid-rich cultivars suitable for medical use. The biosynthesis of cannabinoid compounds is unique to Cannabis, and cultivars with specific chemical profiles are being developed for diverse industrial and pharmaceutical uses.

Cannabis and psychosis/schizophrenia: human studies.
D’Souza, D. C., Sewell, R. A., & Ranganathan, M
European Archives of Psychiatry and Clinical Neuroscience, 259(7), 413–431. (2009).
doi:10.1007/s00406-009-0024-2
The association between cannabis use and psychosis has long been recognized. Recent advances in knowledge about cannabinoid receptor function have renewed interest in this association. Converging lines of evidence suggest that cannabinoids can produce a full range of transient schizophrenia-like positive, negative, and cognitive symptoms in some healthy individuals. Also clear is that in individuals with an established psychotic disorder, cannabinoids can exacerbate symptoms, trigger relapse, and have negative consequences on the course of the illness. The mechanisms by which cannabinoids produce transient psychotic symptoms, while unclear may involve dopamine, GABA, and glutamate neurotransmission. However, only a very small proportion of the general population exposed to cannabinoids develop a psychotic illness. It is likely that cannabis exposure is a ‘‘component cause’’ that interacts with other factors to ‘‘cause’’ schizophrenia or a psychotic disorder, but is neither necessary nor sufficient to do so alone. Nevertheless, in the absence of known causes of schizophrenia, the role of component causes remains important and warrants further study. Dose, duration of exposure, and the age of first exposure to cannabinoids may be important factors, and genetic factors that interact with cannabinoid exposure to moderate or amplify the risk of a psychotic disorder are beginning to be elucidated. The mechanisms by which exposure to cannabinoids increase the risk for developing a psychotic disorder are unknown. However, novel hypotheses including the role of cannabinoids on neurodevelopmental processes relevant to psychotic disorders are being studied

Cannabis and public health: a global experiment without control
Jürgen Rehm, Jakob Manthey
World Psychiatry 11 May 2020
https://onlinelibrary.wiley.com/doi/...R_NXroe8S07BM8
10.1002/wps.20740
Every few weeks, new findings on the effects of legalizing recreational cannabis use are published. Thus, the review of Hall and Lynskey – or any review for that matter – can only provide a preliminary summary of the collected evidence to date.
Looking into public health effects of le¬galization, two seemingly easy indicators may be prevalence and patterns of canna¬bis use, as both are potentially linked to health and social problems in the presence or absence of legalization. However, the main source for both indicators are surveys, with their severe limitations, as today's surveys are neither based on representative sampling frames nor on high response rates. In addition, in the case of cannabis, we are dealing with a (formerly) illicit and stigmatized substance, making comparisons over time even more challenging. More reliable measures such as wastewater analyses are needed here, but these measures cannot assess patterns of use or individual behaviors.

Cannabis and the Anxiety of Fragmentation—A Systems Approach for Finding an Anxiolytic Cannabis Chemotype
Brishna S. Kamal, Fatima Kamal, Daniel Lantela
Frontiers in Neuroscience 12:730 October 2018
DOI: 10.3389/fnins.2018.00730
Cannabis sativa is a medicinal herb with a diverse range of chemotypes that can exert both anxiolytic and anxiogenic effects on humans. Medical cannabis patients receiving organically grown cannabis from a single source were surveyed about the effectiveness of cannabis for treating anxiety. Patients rated cannabis as highly effective overall for treating anxiety with an average score of 8.03 on a Likert scale of 0 to 10 (0 = not effective, 10 = extremely effective). Patients also identified which strains they found the most or least effective for relieving their symptoms of anxiety. To find correlations between anxiolytic activity and chemotype, the top four strains voted most and least effective were analyzed by HPLC-MS/MS to quantify cannabinoids and GC-MS to quantify terpenes. Tetrahydrocannabinol (THC) and trans-nerolidol have statistically significant correlations with increased anxiolytic activity. Guiaol, eucalyptol, ?-terpinene, ?-phellandrene, 3-carene, and sabinene hydrate all have significant correlations with decreased anxiolytic activity. Further studies are needed to better elucidate the entourage effects that contribute to the anxiolytic properties of cannabis varieties.

Cannabis and the brain.
Iversen, L.
Brain, 126(6), 1252–1270. (2003).
doi:10.1093/brain/awg143
The active compound in herbal cannabis, D9-tetrahydrocannabinol, exerts all of its known central effects through the CB1 cannabinoid receptor. Research on cannabinoid mechanisms has been facilitated by the availability of selective antagonists acting at CB1 receptors and the generation of CB1 receptor knockout mice. Particularly important classes of neurons that express high levels of CB1 receptors are GABAergic interneurons in hippocampus, amygdala and cerebral cortex, which also contain the neuropeptides cholecystokinin. Activation of CB1 receptors leads to inhibition of the release of amino acid and monoamine neurotransmitters. The lipid derivatives anandamide and 2-arachidonylglycerol act as endogenous ligands for CB1 receptors (endocannabinoids). They may act as retrograde synaptic mediators of the phenomena of depolarizationinduced suppression of inhibition or excitation in hippocampus and cerebellum. Central effects of cannabinoids include disruption of psychomotor behaviour, shortterm memory impairment, intoxication, stimulation of appetite, antinociceptive actions (particularly against pain of neuropathic origin) and anti-emetic effects. Although there are signs of mild cognitive impairment in chronic cannabis users there is little evidence that such impairments are irreversible, or that they are accompanied by drug-induced neuropathology. A proportion of regular users of cannabis develop tolerance and dependence on the drug. Some studies have linked chronic use of cannabis with an increased risk of psychiatric illness, but there is little evidence for any causal link. The potential medical applications of cannabis in the treatment of painful muscle spasms and other symptoms of multiple sclerosis are currently being tested in clinical trials. Medicines based on drugs that enhance the function of endocannabinoids may offer novel therapeutic approaches in the future

Cannabis and the Gut–Brain Axis Communication in HIV Infection.
Wilson, N. L., Peterson, S. N., & Ellis, R. J.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0037
People living with HIV infection (PWH) disclose that cannabis is an effective strategy for alleviating symptoms associated with HIV disease. However, some medical providers feel ill-informed to engage in evidence-based conversations. HIV leads to alterations in the gut microbiome, gut–brain axis signaling, and chronic inflammation. The endocannabinoid system regulates homeostasis of multiple organ systems. When deficient, dysregulation of the gut–brain axis can result in chronic inflammation and neuroinflammation. Cannabis along with the naturally occurring endocannabinoids has antioxidant and anti-inflammatory properties that can support healing and restoration as an adjunctive therapy. The purpose of this literature review is to report the physiologic mechanisms that occur in the pathology of HIV and discuss potential benefits of cannabinoids in supporting health and reducing the negative effects of comorbidities in PWH.

Cannabis and the Health and Performance of the Elite Athlete
Mark A. Ware, Dennis Jensen, Amy Barrette, Alan Vernec, and Wayne Derman,
Clinical Journal of Sport Medicine, 28(5), 480–484. (2018)
DOI: 10.1097/JSM.0000000000000650
Objective: Cannabis (marijuana) is undergoing extensive regulatory review in many global jurisdictions for medical and nonmedical access. Cannabis has potential impact on the health of athletes as well as on performance in both training and in competition. The aim of this general review is to identify and highlight the challenges in interpreting information with respect to elite athletic performance, and to point to important research areas that need to be addressed.
Data Sources: A nonsystematic literature review was conducted using Medline and PubMed for articles related to cannabis/marijuana use and sports/athletic performance; abstracts were reviewed by lead author and key themes identified and explored.
Main Results: Cannabis may be primarily inhaled or ingested orally for a range of medical and nonmedical reasons; evidence for efficacy is limited but promising for chronic pain management. Although evidence for serious harms from cannabis use on health of athletes is limited, one should be cognizant of the potential for abuse and mental health issues. Although the prevalence of cannabis use among elite athletes is not well-known, use is associated with certain high-risk sports. There is no evidence for cannabis use as a performance-enhancing drug.
Conclusions: Medical and nonmedical cannabis use among athletes reflects changing societal and cultural norms and experiences. Although cannabis use is more prevalent in some athletes engaged in high-risk sports, there is no direct evidence of performance-enhancing effects in athletes. The potential beneficial effects of cannabis as part of a pain management protocol, including reducing concussion-related symptoms, deserve further attention.

Cannabis as a Cause of Death: A Review
Olaf H. Drummer, Dimitri Gerostamoulos, Noel W. Woodford
Forensic Science International 298 (2019)
Doi:10.1016/j.forsciint.2019.03.007
Synthetic cannabinoids have caused a large number of emergency presentations to hospitals for adverse cardiovascular events including numerous deaths, particularly for the more potent analogs acting on the CB1 receptor. While smoked cannabis use is often associated with significant changes in heart rate and cardiac output, amongst other physiological changes, it has been rarely considered in the forensic literature as a significant contributory or causal factor in sudden unexpected death. A review of case reports of admissions to hospitals for cardiovascular events was undertaken
together with a review of epidemiological studies, and case reports of sudden death attributed, at least in part, to use of this drug. These publications show that use of cannabis is not without its risks of occasional serious medical emergencies and sudden death, with reports of at least 35 persons presenting with significant cardiovascular emergencies who had recently smoked a cannabis preparation. At least 13 deaths from a cardiovascular mechanism have been reported from use of this drug which is very likely to be an under-estimate of the true incidence of its contribution to sudden death. In addition, many cases of stroke and vascular arteritis have also been reported with the latter often involving a limb amputation. While it is a drug with widespread usage among the community with relatively few deaths when faced with a circumstance of very recent use (within a few hours), a positive blood concentration of THC and a possible cardiac-related or cerebrovascular cause of death this drug should be considered, at least, a contributory cause of death in cases of sudden or unexpected death.

Cannabis-Based Medicines and Medical Cannabis in the Treatment of Nociplastic Pain
Mary-Ann Fitzcharles, Frank Petzke, Thomas R. Tölle & Winfried Häuser
Drugs volume 81, pages2103–2116 (2021)
doi: 10.1007/s40265-021-01602-1
https://www.researchgate.net/publica...and_Medical_Ca nnabis_in_the_Treatment_of_Noc iplastic_Pain
Nociplastic pain is defined as pain due to sensitization of the nervous system, without a sufficient underlying anatomical abnormality to explain the severity of pain. Nociplastic pain may be manifest in various organ systems, is often perceived as being more widespread rather than localized and is commonly associated with central nervous system symptoms of fatigue, difficulties with cognition and sleep, and other somatic symptoms; all features that contribute to considerable suffering. Exemplified by fibromyalgia, nociplastic conditions also include chronic visceral pain, chronic headaches and facial pain, and chronic musculoskeletal pain. It has been theorized that dysfunction of the endocannabinoid system may contribute to persistent pain in these conditions. As traditional treatments for chronic pain in general and nociplastic pain in particular are imperfect, there is a need to identify other treatment options. Cannabis-based medicines and medical cannabis (MC) may hold promise and have been actively promoted by the media and advocacy. The medical community must be knowledgeable of the current evidence in this regard to be able to competently advise patients. This review will briefly explain the understanding of nociplastic pain, examine the evidence for the effect of cannabinoids in these conditions, and provide simplified guidance for healthcare providers who may consider prescribing cannabinoids for these conditions.

Cannabis-based medicines prolong survival time in patients treated under the Specialized Palliative Outpatient Care
Gastmeier K, Gastmeier A, Böhm R, Bimberg R, Herdegen T
POSTER second listed
http://www.cannabis-med.org/english/...kel.php?id=630
BACKGROUND
Although the 2017 Act Amending Narcotic Drugs Provisions and Other Related Provisions (Gesetz zur Änderung betäubungsmittelrechtlicher und anderer Vorschriften) explicitly aimed at improving provision of care for palliative patients, the use of cannabis-based medicines (CBM) in palliative care remains controversial due to insufficient evidence. However, many Specialized Palliative Outpatient Care (SAPV: Spezielle Ambulante Palliativversorgung) team members see in their daily practice a positive impact of the use of CBM on the four factors defi ned by the Institute
for Quality and Efficiency in Health Care (IQWIG: Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen) as patient-relevant outcomes: morbidity, mortality, adverse events and quality of life. This led us to analyze our practice data for a potential impact of CBM therapy on the survival time of our patients. Due to the pressing relevance of our observations for palliative care patients, we are making the preliminary data from our ongoing retrospective study available in this pre-publication. Based on the current trend in the evaluation, we consider that patients could promptly benefit from a low and slowly increasing dose.
METHODS
Data of all patients under the SAPV, treated and deceased in the period from 01.04.2017 to 30.09.2021, were extracted from the Pallidoc database and analyzed statistically using R. Collected data included survival time (initiation of SAPV treatment until date of death), age, gender, and treatment with CBM. Patients with a treatment duration of less than 10 days or more than three years were excluded from the analysis.
RESULTS
A total of 800 SAPV patients were included in the analysis, of which 137 received THC (Δ-9-Tetrahydrocannabinol)- containing CBM (17.1 %, female: 45.3 %; > 75 years of age: 44.5 %), and 663 patients (82.9 %, female: 51.3 %; > 75 years of age: 62.7 %) were provided SAPV without the use of CBM (Table 1). The therapy with CBM was associated with prolonged median survival after initiation of SAPV treatment from 44
to 65 days (log rank p = 0.0046) (Figure 1). The assessment of the infl uence of gender and age on prolonged median survival time with CBM therapy showed that women (64 days with CBM vs 43 days without CBM; log rank p = 0.036; Figure 2) and elderly patients (> 75 years
of age: 62 days with CBM vs 37.5 days without CBM; log rank p = 0.022; women > 75 years of age: 62.5 days with CBM vs 33 days without CBM; log rank p = 0.041) particularly benefi ted from CBM therapy (Table 2, Figure 3). For men, prolonged median survival time was observed with CBM therapy but this did not reach statistical significance (Table 2). A multivariate analysis confirmed the observed positive association between CBM therapy and median survival time of SAPV patients (REF/CBM HR: 1.29; p = 0.008) when age and gender were included as influencing factors (Table 2). CBM therapy was initiated at the lowest dose which was then gradually increased until the first effect was observed. After a short observation period, a decision was made whether a further dose increase was required. By using this approach, patients generally received low-dose CBM therapy. A subdivision of CBM therapy into low-dose (< 7.5 mg THC / day; median daily dose 3.2 ± 2.1 mg THC) and patients with higher-dose (> 7.5 mg THC / day; median daily dose 12.0 ± 8.4 mg THC) suggested a superiority of the higher-dose therapy in terms of median survival time (104 days > 7.5 mg THC vs 57 days < 7.5 mg THC) (Table 3). It is worth noting that even in the patient group with > 7.5 mg THC / day, the median daily dose was relatively low. Men under 75 years of age took the highest median daily doses (6.4 ± 9.08 mg THC / day), whereas for the other groups the median daily dose was 4.8 mg THC / day
CONCLUSION
The current trend in the evaluation of patient data from our SAPV team indicates an overall positive association between CBM therapy and prolonged median survival time of SAPV patients. Women and elderly patients appear to be particularly likely to benefit from such therapy. From the available data, we can conclude that current prescribing practices deprive patients of days of life and that CBM therapy should be included as first line therapy for the patient groups considered due to the significant prolongation of survival time. Since these observations are highly relevant for palliative care clinical practice, we are currently working in cooperation with other SAPV teams to expand and validate the present dataset

Cannabis-Based Oral Formulations for Medical Purposes: Preparation, Quality and Stability
Francesca Baratta, Marco Simiele, Irene Pignata, Lorenzo Ravetto Enri, Antonio D’Avolio, Riccardo Torta, Anna De Luca, Massimo Collino and Paola Brusa
Pharmaceuticals 2021, 14(2), 171;
DOI:10.3390/ph14020171
Current legislation in Italy provides that medical Cannabis may be administered orally or by inhalation. One of the fundamental criteria for the administration of oral formulations is that they deliver a known consistent quantity of the active ingredients to ensure uniform therapies leading to the optimisation of the risks/benefits. In 2018, our group developed an improved Cannabis oil extraction technique. The objective of the present work was to carry out a stability study for the oil extracts obtained by this method. Furthermore, in order to facilitate the consumption of the prescribed medical Cannabis therapy by patients, a standard procedure was defined for the preparation of a single-dose preparation for oral use (hard capsules) containing the oil extract; thereafter, the quality and stability were evaluated. The hard capsules loaded with the oil extract were analysed and found to be uniform in content. The encapsulation process did not alter the quantity of the active molecule present in the oil. The stability tests yielded excellent results. Since the capsule dosage form is easily transported and administered, has pleasant organoleptic properties and is stable at room temperature for extended periods of time, this would facilitate the adherence to therapy by patients in treatment.

Cannabis: Chemistry, extraction and therapeutic applications
Falguni Pattnaik, Sonil Nanda, Shobhangam Mohanty, Ajay K. Dalai, Vivek Kumar, Senthil Kumar Ponnusamy, Satyanarayan Naik
Chemosphere Volume 289, February 2022, 133012
DOI: 10.1016/j.chemosphere.2021.133012
Cannabis, a genus of perennial indigenous plants is well known for its recreational and medicinal activities. Cannabis and its derivatives have potential therapeutic activities to treat epilepsy, anxiety, depression, tumors, cancer, Alzheimer's disease, Parkinson's disease, to name a few. This article reviews some recent literature on the bioactive constituents of Cannabis, commonly known as phytocannabinoids, their interactions with the different cannabinoids and non-cannabinoid receptors as well as the significances of these interactions in treating various diseases and syndromes. The biochemistry of some notable cannabinoids such as tetrahydrocannabinol, cannabidiol, cannabinol, cannabigerol, cannabichromene and their carboxylic acidderivatives is explained in the context of therapeutic activities. The medicinal features of Cannabis-derived terpenes are elucidated for treating several neuro and non-neuro disorders. Different extraction techniques to recover cannabinoids are systematically discussed. Besides the medicinal activities, the traditional and recreational utilities of Cannabis and its derivatives are presented. A brief note on the legalization of Cannabis-derived products is provided. This review provides comprehensive knowledge about the medicinal properties, recreational usage, extraction techniques, legalization and some prospects of cannabinoids and terpenes extracted from Cannabis.
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Cannabis compounds exhibit anti infammatory activity in vitro in COVID 19 related infammation in lung epithelial cells and pro infammatory activity in macrophages
Seegehalli M.Anil, Nurit Shalev, Ajjampura C.Vinayaka, Stalin Nadarajan, Dvora Namdar, Eduard Belausov, Irit Shoval, KarthikAnanth Mani, Guy Mechrez & Hinanit Koltai
Nature Scientifc Reports | (2021) 11:1462
DOI: 10.1038/s41598-021-81049-2
https://www.nature.com/articles/s41598-021-81049-2.pdf
Cannabis sativa is widely used for medical purposes and has anti-infammatory activity. This study intended to examine the anti-infammatory activity of cannabis on immune response markers associated with coronavirus disease 2019 (COVID-19) infammation. An extract fraction from C. sativa Arbel strain (FCBD) substantially reduced (dose dependently) interleukin (IL)-6 and -8 levels in an alveolar epithelial (A549) cell line. FCBD contained cannabidiol (CBD), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with FCBD and a FCBD formulation using phytocannabinoid standards (FCBD:std) reduced IL-6, IL-8, C–C Motif Chemokine Ligands (CCLs) 2 and 7, and angiotensin I converting enzyme 2 (ACE2) expression in the A549 cell line. Treatment with FCBD induced macrophage (diferentiated KG1 cell line) polarization and phagocytosis in vitro, and increased CD36 and type II receptor for the Fc region of IgG (FcγRII) expression. FCBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. FCBD:std, while maintaining antiinfammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-infammatory IL secretion in macrophages in comparison to FCBD. The phytocannabinoid formulation may show superior activity versus the cannabis-derived fraction for reduction of lung infammation, yet there is a need of caution proposing cannabis as treatment for COVID-19.

Cannabis constituents interact at the drug efux pump BCRP to markedly increase plasma cannabidiolic acid concentrations
Lyndsey L.Anderson, MaiaG. Etchart, Dilara Bahceci, TaliesinA. Golembiewski & Jonathon C.Arnold
Nature Scientifc Reports | (2021) 11:14948
https://www.nature.com/articles/s41598-021-94212-6.pdf
DOI: 10.1038/s41598-021-94212-6
Cannabis is a complex mixture of hundreds of bioactive molecules. This provides the potential for pharmacological interactions between cannabis constituents, a phenomenon referred to as “the entourage efect” by the medicinal cannabis community. We hypothesize that pharmacokinetic interactions between cannabis constituents could substantially alter systemic cannabinoid concentrations. To address this hypothesis we compared pharmacokinetic parameters of cannabinoids administered orally in a cannabis extract to those administered as individual cannabinoids at equivalent doses in mice. Astonishingly, plasma cannabidiolic acid (CBDA) concentrations were 14-times higher following administration in the cannabis extract than when administered as a single molecule. In vitro transwell assays identifed CBDA as a substrate of the drug efux transporter breast cancer resistance protein (BCRP), and that cannabigerol and Δ9 -tetrahydrocannabinol inhibited the BCRP-mediated transport of CBDA. Such a cannabinoid-cannabinoid interaction at BCRP transporters located in the intestine would inhibit efux of CBDA, thus resulting in increased plasma concentrations. Our results suggest that cannabis extracts provide a natural vehicle to substantially enhance plasma CBDA concentrations. Moreover, CBDA might have a more signifcant contribution to the pharmacological efects of orally administered cannabis extracts than previously thought.

Cannabis Contaminants Limit Pharmacological Use of Cannabidiol
Zackary Montoya, Matthieu Conroy, Brian D. Vanden Heuvel, Christopher S. Pauli and Sang-Hyuck Park
Front. Pharmacol. 11:571832.
doi: 10.3389/fphar.2020.571832
For nearly a century, Cannabis has been stigmatized and criminalized across the globe, but in recent years, there has been a growing interest in Cannabis due to the therapeutic potential of phytocannabinoids. With this emerging interest in Cannabis, concerns have arisen about the possible contaminations of hemp with pesticides, heavy metals, microbial pathogens, and carcinogenic compounds during the cultivation, manufacturing, and packaging processes. This is of particular concern for those turning to Cannabis for medicinal purposes, especially those with compromised immune systems. This review aims to provide types of contaminants and examples of Cannabis contamination using case studies that elucidate the medical consequences consumers risk when using adulterated Cannabis products. Thus, it is imperative to develop universal standards for cultivation and testing of products to protect those who consume Cannabis.

Cannabis-derived cannabidiol and nanoselenium improve gut barrier function and affect bacterial enzyme activity in chickens subjected to C. perfringens challenge.
Konieczka, P., Szkopek, D., Kinsner, M., Fotschki, B., Ju?kiewicz, J., & Banach, J.
Veterinary Research,*51(1). (2020).
doi:10.1186/s13567-020-00863-0
Revealing the multifocal mechanisms afecting cross-talk between Clostridium perfringens pathogenesis and the host response is an urgent need in the poultry industry. Herein, the activity of Cannabis sativa-derived cannabidiol (CBD) and selenium nanoparticles (Nano-Se) in modulating the host response to Clostridium perfringens challenge was investigated in broiler chickens subjected to a mild infection model. The infected chickens exhibited no clinical manifestations, confrming the potential hazard of pathogen transmission to the food chain in the commercial sector. However, both CBD and Nano-Se afected the responses of chickens to C. perfringens challenge. The benefcial actions of both agents were manifested in the upregulated expression of genes determining gut barrier function. Both CBD and Nano-Se promoted shifts in gut bacterial enzyme activity to increased energy uptake in challenged chickens and upregulated potential collagenase activity. There was no opposite efect of CBD and Nano-Se in mediating the host response to challenge, whereas an additive efect was evidenced on the upregulation of gene determining gut integrity. Collectively, these fndings indicate that understanding the action mechanisms of CBD and Nano-Se is of great interest for developing a preventive strategy for C. perfringens infection in broilers.

Cannabis-Derived Compounds Cannabichromene and ∆9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization
Omer Anis, Ajjampura C. Vinayaka, Nurit Shalev, Dvora Namdar, Stalin Nadarajan, Seegehalli M. Anil, Ofer Cohen, Eduard Belausov, Jacob Ramon, Einav Mayzlish Gati and Hinanit Koltai
Molecules 2021, 26, 465.
DOI: 10.3390/molecules26020465
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-00465.pdf
Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and ∆9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.

Cannabis edibles already harming kids,new data show
Lauren Vogel
CMAJ 2019 July 15;191:E801.
doi: 10.1503/cmaj.109-5789
More than a dozen children suffered serious or life-threatening events linked to recreational cannabis in the months around legalization, according to preliminary data from a Canadian Paediatric Surveillance Program (CPSP) study. “These numbers are really the tip of the iceberg,” said Dr. Christina Grant, co-principal investigator. The study captures only the most serious adverse events involving cannabis that resulted in hospitalizations, permanent disability and death, she explained. It doesn’t include visits to family doctors or emergency departments, which are likely many more.
Between September and December 2018, 16 cases of serious adverse events involving cannabis were reported to CPSP. Researchers verified 11 of these cases, six of which involved young children
accidentally consuming cannabis edibles that belonged to a parent or grandparent. All involved hospitalizations. At least one involved an infant.

Cannabis Ethnomedicine
Stephen Dahmer, Michael Balick
In book: Understanding Medical Cannabis November 2020
DOI: 10.4324/9780429343803-3
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Cannabis Exposure Decreases Need for Blood Pressure Support During General Anesthesia in Orthopedic Trauma Surgery
Brent G. Yeung, Michael W. Ma, John A. Scolaro, and Ariana M. Nelson
Cannabis and Cannabinoid Research
Volume X, Number X, 2021
DOI: 10.1089/can.2021.0009
https://www.liebertpub.com/doi/pdf/1.../can.2021.0009
Introduction: As cannabis use continues to increase in popularity, it is important to investigate how it impacts public health in all sectors of the population, including patients undergoing anesthetic management. This retrospective study focuses on the orthopedic trauma population presenting through an emergency department (ED) and receiving a urine drug screen (UDS) with subsequent urgent surgical intervention. We aimed to evaluate
differences in response to general anesthesia in patients with exposure to THC, a major cannabinoid, compared to controls that screened negative for THC.
Materials and Methods: All ED visits at UC Irvine, a level 1 trauma center between November 4, 2017 and January 7, 2020, were evaluated in this study. Only adult patients who received a UDS and underwent urgent orthopedic
trauma surgery within 48 h of ED visit were included in this study. Additional inclusion criteria required an anesthesia time greater than 1 h as well as anesthesia induction and intubation while in the operating room. Overall, we analyzed a total of 221 adult patients.
Discussion: When adjusting for demographic variability, there were statistically significant differences in response to general anesthesia between these two groups. The THC-positive (THC( + )) group was less likely to receive intraoperative vasopressors, had higher mean arterial blood pressure and mean diastolic blood pressure, needed less total fluid input and had a lower overall fluid balance. Chronic exposure to THC has been shown
to downregulate cannabinoid 1 receptors and cause alterations in endocannabinoid tone. These are two potential mechanisms by which the THC( + ) group in our study may have become more resistant to the typically observed hypotensive effects of general anesthesia.
Conclusion: The present study suggests that prior use of cannabis, objectively assessed by urinalysis, results in a decreased need for blood pressure support during general anesthesia. The physiological basis for this phenomenon is unclear, but possible causes might include the downregulation of vascular cannabinoid receptor 1 and/or altered endocannabinoid levels after exposure to cannabis.

Cannabis exposure is associated with a lower likelihood of neurocognitive impairment in people living with HIV.
Caitlin Wei-Ming Watson, Emily W. Paolillo, Erin E. Morgan, Anya Umlauf, Erin E. Sundermann, Ronald J. Ellis, Scott Letendre, Thomas D. Marcotte, Robert K. Heaton, Igor Grant
J Acquir Immune Defic Syndr. 2020 January 01; 83(1): 56–64.
doi:10.1097/QAI.0000000000002211.
Background: Aging and HIV have adverse effects on the CNS, including increased inflammation and neural injury, and confer risk for neurocognitive impairment (NCI). Prior research suggests the non-acute neurocognitive effects of cannabis in the general population are adverse or null. However, in the context of aging and HIV, cannabis use may exert beneficial effects due to its anti-inflammatory properties. In the current study, we examined the independent and interactive effects of HIV and cannabis on NCI, and the potential moderation of these effects by age. :
Methods: Participants included 679 people living with HIV (PLHIV) and 273 people living without HIV (HIV−) (18-79 years old) who completed neurocognitive, neuromedical, and substance use assessments. NCI was defined as demographically-corrected global deficit score ≥0.5. Logistic regression models examined the effects of age, HIV, cannabis (history of cannabis substance use disorder and cannabis use in past year), and their two-way and three-way interactions on NCI. Results: In logistic regression models, only a significant interaction of HIV X cannabis was detected (p=0.02). Among PLHIV, cannabis was associated with a lower proportion of NCI (OR=0.53, 95%CI=0.33–0.85), but not among HIV− individuals (p=0.40). These effects did not vary by age.
Conclusion: Findings suggest cannabis exposure is linked to a lower odds of NCI in the context of HIV. A possible mechanism of this result is the anti-inflammatory effect of cannabis, which may be particularly important for PLHIV. Further investigations are needed to refine the effects of dose, timing, and cannabis compound on this relationship, which could inform guidelines for cannabis use among populations vulnerable to cognitive decline.


Cannabis Extract Has a Positive-Immunostimulating Effect through Proteolytic System and Metabolic Compounds of Honey Bee ( Apis mellifera) Workers
Patrycja Skowronek , Łukasz Wójcik , Aneta Strachecka
Animals (Basel). 2021 Jul 23;11(8):2190.
doi: 10.3390/ani11082190
https://www.ncbi.nlm.nih.gov/pmc/art...s-11-02190.pdf
In the study, we assessed the effect of hemp extract on activities of resistance parameters and the metabolic compound concentration in adult workers' hemolymph. Bees were divided into the following groups: (1) control group fed with mixture of sugar and water-glycerine solution, (2) experimental group with pure sugar syrup and inside with cotton strips soaked with hemp extract, (3) experimental group with a mixture of sugar syrup with hemp extract. Hemp extracts caused an increase in the protein concentrations and reduced the protease activities regardless of the administration method. The protease inhibitor activities were decreased only in the group that received hemp extract on the strips. The biomarker activities (ALP, ALT, AST) increased from the control group and workers feeding extract in syrup and decreased in workers supplemented with the extract on strips. In young, 2-day-old workers, the glucose concentration was higher in the groups feeding with the extract than in the control. Hemp extract influenced an increase in urea concentrations in workers' hemolymph in comparison with the control. The hemp supplementation positively influences the immune system of workers, and the appropriate method of administration may be adapted to the health problems of bees.

Cannabis; extracting the medicine
Arno Hazekamp
5 september 2007 Thesis
https://www.researchgate.net/publica...g_the_medicine
It is hard to think of a medical topic that so strongly divides the research community as the medicinal use of cannabis. It can probably be said that cannabis is the most controversial plant in the history of mankind. But surely, if the plant Cannabis sativa would be discovered today, growing in some remote spot of the world, it would be hailed as a wonder of nature; a new
miracle plant with the potential to treat anything ranging from headaches to neurological disorders to cancer. It is therefore interesting to notice that, even after decades of research, cannabis is probably most well known for causing anxiety, agitation and paranoia among politicians, while its medicinal potential continues to be disputed. Interestingly, delta-9-tetrahydrocannabinol (THC), the main component of the cannabis plant, and one of the most renowned plant compounds of the world, is in fact already acknowledged as a medicine. It has been available to patients since 1986 under the name Marinol., which is prescribed to treat nausea, pain and loss of appetite. So even if cannabis was nothing more than an herbal receptacle of THC, it should at least be accepted as some generic form of this registered medicine. However, on multiple levels (in vivo, in vitro, in clinical trials) it is becoming increasingly clear that THC alone does not equal cannabis [Williamson 2000; Russo 2003], pointing out that other components are necessary to explain
the claimed medicinal effects.

Cannabis for Chronic Pain: A Rapid Systematic Review of Randomized Control Trials
Riana Longo , Abe Oudshoorn , Deanna Befus
Pain Manag Nurs. 2020*Dec 19;S1524-9042(20)30225-3.
doi: 10.1016/j.pmn.2020.11.006
Background: The high prevalence of inadequately managed chronic pain indicates the need for alternative and multimodal treatment options. Use of cannabinoids in medicine is becoming a growing area of interest, specifically in the context of chronic pain. The efficacy of cannabinoids for the treatment of chronic pain is not well established.
Aims: The objectives of this rapid systematic literature review are to summarize the efficacy and secondary effects of cannabinoids for chronic pain management.
Design: Rapid systematic review of randomized control trials.
Participants: Individuals with chronic pain (n = 1352).
Methods: Embase, Cochrane, PubMed, and CINAHL databases were searched. Inclusion criteria included cannabis of any formulation used to treat chronic pain of any origin.
Results: Thirteen randomized controlled trials met the inclusion criteria. Five demonstrated moderate analgesic effects of cannabis for chronic pain, and eight concluded there were no significant impacts on pain in the cannabis-treated group versus the control group.
Conclusions: Evidence on the efficacy of cannabinoids for chronic pain shows patient-perceived benefit but inconsistent other treatment effects. These findings indicate cannabinoids may have a modest analgesic effect for chronic neuropathic pain conditions, and that the use of cannabinoids is relatively safe, with few severe adverse events. This review concludes that cannabinoids may have a potential role in chronic pain management. Inconsistent evidence on the efficacy of cannabis to treat chronic pain indicates the need for more studies on a larger scale. Clinicians should draw on available evidence and consider cannabinoids as a potential approach to chronic pain management.

Cannabis for COVID-19: can cannabinoids quell the cytokine storm?
Emmanuel Shan Onaivi & Venkatanarayanan Sharma
Future Sci. OA (2020) 6(8), FSO625
DOI: 10.2144/fsoa-2020-0124
https://www.ncbi.nlm.nih.gov/pmc/art...soa-06-625.pdf
The recent emergence of SARS coronavirus 2 (SARS-CoV-2) has resulted in the alarming spread of the highly infectious and contagious COVID-19 pandemic that is causing catastrophic damage and affecting health, life and death around the world [1,2]. SARS-CoV-2 has also created a COVID-19 ‘infodemic’ crisis with an overabundance of information and misinformation about the origin of the virus, potential therapies, and whether it was engineered in the laboratory. However, the global pace of SARS-CoV-2 and COVID-19 research is providing rapid and critical advances in comparison to that of the previous SARS-CoV, Middle East respiratory syndrome (MERS) and HIV [1,3,4]. COVID-19 has a protean manifestation, and the cryptic transmission of SARS-CoV-2 is characterized by multiple chains of transmission, unlike the SARS-CoV and MERS-CoV viruses that have been reported to occur mainly through nosocomial transmission [3,4]. There are still many unknowns regarding COVID-19, but there are also important lessons to be gleaned from AIDS that are applicable to the COVID-19 pandemic. They are both zoonotic diseases with different mode of transmission, with no vaccine or cure yet; however, there is an effective antiretroviral therapy for AIDS [5,6]. Furthermore, cannabis and cannabinoids have been proposed and used as adjunctive treatment for AIDS-associated cachexia, and in reduction of disease symptoms [7,8]. The processes of inflammation are important in both the pathogenesis of AIDS and COVID-19 [6,8]. Cannabinoids are effective at suppressing immune and inflammatory functions [7–9], and their potential as an anti-inflammatory treatment in COVID-19 has been suggested

Cannabis for Pain and Headaches: Primer
Philip S. Kim, Michael A. Fishman
Current Pain and Headache Reports 21(4) April 2017
DOI: 10.1007/s11916-017-0619-7
Purpose of review: Marijuana has been used both medicinally and recreationally since ancient times and interest in its compounds for pain relief has increased in recent years. The identification of our own intrinsic, endocannabinoid system has laid the foundation for further research. Recent findings: Synthetic cannabinoids are being developed and synthesized from the marijuana plant such as dronabinol and nabilone. The US Food and Drug Administration approved the use of dronabinol and nabilone for chemotherapy-associated nausea and vomiting and HIV (Human Immunodeficiency Virus) wasting. Nabiximols is a cannabis extract that is approved for the treatment of spasticity and intractable pain in Canada and the UK. Further clinical trials are studying the effect of marijuana extracts for seizure disorders. Phytocannabinoids have been identified as key compounds involved in analgesia and anti-inflammatory effects. Other compounds found in cannabis such as flavonoids and terpenes are also being investigated as to their individual or synergistic effects. This article will review relevant literature regarding medical use of marijuana and cannabinoid pharmaceuticals with an emphasis on pain and headaches.

Cannabis for rheumatic pain: hope or hype?
Hazlewood, G. S., Niaki, O. Z., & Fitzcharles, M.-A.
Clinical Rheumatology. (2019).
doi:10.1007/s10067-019-04658-3
Even when inflammatory rheumatic diseases are “well controlled” by current standards, most patients continue to suffer ongoing pain, as well as other concomitant symptoms such as sleep disturbance, difficulty with mood, and impaired overallhealth. Moreover, in the absence of any disease-modifying treatment for osteoarthritis, a condition likely to afflict most persons in the later years of life, chronic pain remains a prevailing symptom. The prevalence estimates for chronic pain in populations is in the order of 31%. Chronic pain is often due to musculoskeletal conditions, with limited effectiveness and/or high rates of adverse events related to current pharmacological treatments. There is therefore an urgent need to identify new treatment strategies to alleviate pain. Could medical cannabis fill this void?

Cannabis for the Treatment of Inflammatory Bowel Disease: A True Medicine or a False Promise?
Timna Naftali, and*Michael Dor
Rambam Maimonides Med J. 2020 Jan; 11(1):
doi:*10.5041/RMMJ.10390
https://www.ncbi.nlm.nih.gov/pmc/art...11-1-e0008.pdf
Cannabis is the most widely used recreational drug worldwide and is used by some patients with inflammatory bowel disease (IBD) to ameliorate their disease. Whereas epidemiological studies indicate that as many as 15% of IBD patients use cannabis, studies inspecting cannabis use in IBD are few and small. We have conducted several studies looking at the use of cannabis in IBD. In Crohn’s disease, we demonstrated that cannabis reduces the Crohn’s disease activity index (CDAI) by >100 points (on a scale of 0–450).Two small studies in ulcerative colitis showed a marginal benefit. However, no improvement was observed in inflammatory markers or in endoscopic score in either disease. Many questions regarding cannabis use in IBD remain unanswered. For example, cannabis is a complex plant containing many ingredients, and the synergism or antagonism between them likely plays a role in the relative efficacy of various cannabis strains. The optimal doses and mode of consumption are not determined, and the most common form of consumption, i.e. smoking, is unacceptable for delivering medical treatment. Cannabis is a psychotropic drug, and the consequences of long-term use are unknown. Despite all these limitations, public opinion regards cannabis as a harmless drug with substantial medical efficacy. In Israel, the number of licenses issued for the medical use of cannabis is rising rapidly, as are the acknowledged indications for such use, but good-quality evidence for the effectiveness of cannabis is still lacking. Further studies investigating the medical use of cannabis are urgently needed.

Cannabis (hemp) positive skin tests and respiratory symptoms
Jeffrey R Stokes, Rita Hartel, Linda B Ford, and Thomas B Casale,
Ann Allergy Asthma Immunol 2000;85:238–240.
DOI:10.1016/S1081-1206(10)62473-8
Background: We have noted several patients who had rhinitis and/or asthma
symptoms when exposed to Cannabis plants in the summer months. Cannabis plants are common in the Midwest.
Objectives: To examine whether Cannabis might be a clinically important
allergen, we determined Cannabis pollination patterns in the Omaha area for 5 years, the prevalence of skin test positivity, and the association with respiratory symptoms.
Methods: Airborne Cannabis (and other weed) pollens were collected using a
Rotorod air impactor, and pollen counts were done using a standardized protocol.
Results: Measurable Cannabis pollen count was not recorded until the last 2
weeks of July. Peak pollination typically occurred during mid- to late-August, and comprised up to 36% of the total pollen counts. Cannabis pollen was not observed after mid-September. To determine the prevalence of skin test positivity, we added Cannabis to the multi-test routine skin test battery. Seventy-eight of 127 patients tested (61%) were skin test positive. Thirty of the 78 patients were randomly selected to determine if they had allergic rhinitis and/or asthma symptoms during the Cannabis pollination period. By history, 22 (73%) claimed respiratory symptoms in the July through September period. All 22 of these subjects were also skin test positive to weeds pollinating during the same period as Cannabis (ragweed, pigweed,
cocklebur, Russian thistle, marsh elder, or kochia).
Conclusions: The strong association between skin test reactivity, respiratory
symptoms, and pollination period suggests that Cannabis could be a clinically
important aeroallergen for certain patients and should be further studied.

Cannabis hyperemesis syndrome: still under recognised after all these years
Joanne Lua, Lauren Olney, Chris Isles
J R Coll Physicians Edinb 2019; 49: 132–4
doi: 10.4997/JRCPE.2019.210.
Cannabis hyperemesis syndrome (CHS) is characterised by cyclical vomiting, relieved by hot water bathing, in the context of chroniccannabis consumption. Many cannabis users find this hard to accept given that cannabis is often promoted as a treatment for vomiting. Stopping cannabis is the best and only effective way to manage the condition. We report a case showing that CHS remains under recognised 15 years after it was first described.

Cannabis improves night vision: a case study of dark adaptometry and scotopic sensitivity in kif smokers of the Rif mountains of northern Morocco.
Russo, E. B., Merzouki, A., Mesa, J. M., Frey, K. A., & Bach, P. J.
Journal of Ethnopharmacology, 93(1), 99–104. (2004).
doi:10.1016/j.jep.2004.03.029
Previous reports have documented an improvement in night vision among Jamaican fishermen after ingestion of a crude tincture of herbal cannabis, while two members of this group noted that Moroccan fishermen and mountain dwellers observe an analogous improvement after smoking kif, sifted Cannabis sativa mixed with tobacco (Nicotiana rustica). Field-testing of night vision has become possible with a portable device, the LKC Technologies Scotopic Sensitivity Tester-1 (SST-1). This study examines the results of double-blinded graduated THC administration 0–20 mg (as Marinol®) versus placebo in one subject on measures of dark adaptometry and scotopic sensitivity. Analogous field studies were performed in Morocco with the SST-1 in three subjects before and after smoking kif. In both test situations, improvements in night vision measures were noted after THC or cannabis. It is believed that this effect is dose-dependent and cannabinoid-mediated at the retinal level. Further testing may assess possible clinical application of these results in retinitis pigmentosa or other conditions.

Cannabis Improves Stuttering: Case Report and Interview with the Patient
Natalia Szejko, Carolin Fremer, Franziska Baacke, Martin Ptok, and Kirsten R. Müller-Vahl
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0060
https://www.liebertpub.com/doi/epub/.../can.2021.0060
https://www.researchgate.net/publica...is_Improves_St uttering_Case_Report_and_Inter view_with_the_Patient
Introduction: Speech dysfluency, often referred to as stuttering, is a frequent speech disorder encountered in about 5% of children. Although in the majority of people affected, symptoms improve in adulthood, in some patients, stuttering persists and significantly impairs everyday functioning and quality of life. Treatment for stuttering includes speech therapy, cognitive behavioral therapy, and relaxation techniques. However, a substantial number of patients do not benefit sufficiently from these treatment strategies or are even treatment resistant.
Methods: We present the case of a 20-year-old male with treatment-resistant stuttering, who markedly improved after treatment with medicinal cannabis.
Results: Besides improved speech fluency as assessed by several phoniatric tests, we observed remission of (social) anxiety, improved mood, and reduced stress, resulting in an overall improvement of quality of life after cannabis therapy. The patient, in addition, reported improved attention, concentration, and sleep, increased self-confidence, and better social life. No side effects occurred. Over a time period of more than a year, treatment was equally effective. In an interview, the patient describes his personal view and the influence of cannabis-based treatment on his life.
Conclusions: Medicinal cannabis could be effective in treatment of refractory stuttering, but these preliminary data have to be confirmed in controlled studies.
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Cannabis in Medicine An Evidence-Based Approach
Finn, K. (Ed.). (2020).
doi:10.1007/978-3-030-45968-0
Part I Basic Science
1 The Properties and Use of Cannabis sativa Herb and Extracts . . . . . 3 Richard L. Hilderbrand
2 Cannabinoid and Marijuana Neurobiology . . . . . . . . . . . . . . . . . . . . . 25 Bertha K. Madras
3 The Pharmacodynamics, Pharmacokinetics, and Potential Drug Interactions of Cannabinoids . . . . . . . . . . . . . . . . 49 Grace S. Chin, Robert L. Page II, and Jacquelyn Bainbridge
Part II Clinical Evidence
4 Cannabis and Neuropsychiatric Effects . . . . . . . . . . . . . . . . . . . . . . . . 65 David C. Rettew, Doris C. Gundersen, Erica Kirsten Rapp, Paula Riggs, Christine L. Miller, Monica C. Jackson, Kevin Sabet, Ben Cort, and LaTisha L. Bader
5 Cannabis and the Impact on the Pediatric and Adolescent Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 George Sam Wang, Donald E. Greydanus, and Maria Demma Cabral
6 Acute Emergency Department Presentations Related to Cannabis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Karen Randall, Brad Roberts, and John Cienki
7 Evidence of Cannabinoids in Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Peter R. Wilson and Sanjog Pangarkar
8 Cannabis in Pulmonary Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Christopher M. Merrick and Jesse J. LeBlanc III
9 Clinical Cardiovascular Effects of Cannabis Use . . . . . . . . . . . . . . . . 209 Cynthia Philip, Rebecca Seifried, Marcio Sommer Bittencourt, and Edward Hulten xxvi
10 Cannabinoids in Neurologic Conditions . . . . . . . . . . . . . . . . . . . . . . . . 231 Tyler E. Gaston, Jerzy P. Szaflarski, Allen C. Bowling, Ying Liu, Tristan Seawalt, Maureen A. Leehey, E. Lee Nelson, Sharad Rajpal, Alan T. Villavicencio, Andrew Bauer, and Sigita Burneikiene
11 Ocular Conditions and the Endocannabinoid System . . . . . . . . . . . . . 313 Finny T. John and Jean R. Hausheer
12 Cannabis in Oncology and Symptom Management . . . . . . . . . . . . . . 325 Matthew Chung and Salahadin Abdi
13 Cannabis in Palliative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Fabienne Saint-Preux, Arpit Arora, Derek Moriyama, Esther Kim, and Arum Kim
14 Charting the Pathways Taken by Older Adults Who Use Cannabis: Where Are the Baby Boomers Going Now?. . . . 373 Brian P. Kaskie and Amanjot Mona Sidhu
15 Cannabis in Dermatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Catherine Murer Antley, Reagan Anderson, and Judith Margulies
16 Fetal and Neonatal Marijuana Exposure . . . . . . . . . . . . . . . . . . . . . . . 401 Leeann M. Blaskowsky
17 Cannabinoids in Gastrointestinal Disorders . . . . . . . . . . . . . . . . . . . . 415 Michelle Kem Su Hor, Monica Dzwonkowski, Tesia Kolodziejczyk, Lorne Muir, Nazar Dubchak, Sabina Hochroth, Bhaktasharan Patel, Aaron Wu, Sean Knight, Garrett Smith, Uday Patel, Quentin Remley, and Cicily Hummer
18 Looking at Marijuana Through the Lens of Public Health . . . . . . . . 453 Elizabeth Brooks and Stig Erik Sørheim
19 Cannabis-Impaired Driving: Evidence and the Role of Toxicology Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 Edward C. Wood and Robert L. Dupont
20 The Legal Aspects of Marijuana as Medicine . . . . . . . . . . . . . . . . . . . 515 David G. Evans Epilogue: The Medical Cannabis Landscape . . . . . . . . . . 535 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

Cannabis in neurology—a potted review
Richard Hosking & John Zajicek
Nature Reviews Neurology 10(8) July 2014
DOI: 10.1038/nrneurol.2014.122
Discovery of the endogenous cannabinoid signalling system unleashed substantial new research into several neurological conditions. A recent systematic review suggests that medical marijuana can improve a number of symptoms—particularly spasticity—in multiple sclerosis, but cannabinoids can have adverse psychological effects and their comparative effectiveness is unknown.

Cannabis in Pain
January 2021
DOI: 10.1016/B978-0-323-72216-2.00011-9
In book: Pain Care Essentials and Innovations
Kenneth Finn
Cannabis (marijuana) has been used for its purported benefits on pain for millennia. It has been listed in the Chinese pharmacopeia to treat a variety of medical conditions for at least 1800 years. The potency of the cannabinoids has been relatively stable during this time. Marijuana products available now, however, are markedly different than before and with the expansion of medical marijuana programs across the United States, the focus of whether this drug is effective has been called into question. Despite public belief that marijuana cannot be researched, there have been thousands of studies on this drug over the last several decades, even since the Marihuana Tax Act of 1937.¹,² With the dawn of the opioid epidemic, there has been a coincidental expansion of marijuana programs for medical use, followed by recreational use. In response to the opioid epidemic, the view that cannabis could replace opioids for pain control has become appealing. This chapter will review the pharmacology of cannabis along with the clinical evidence for pain.
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Cannabis Indica in 19th-Century Psychiatry.
CARLSON, E. T.
American Journal of Psychiatry, 131(9), 1004–1007. (1974).
doi:10.1176/ajp.131.9.1004
The author presents a stuck of the history and usage of cannabis indicus (the 19th-century pharmacological term referring to the plant we today call cannabis sativa indica). His review of the drug’s physiological and psychological effects reveals that most of the effects reported in the 1960s were known to writers of the 19th century, when the drug was alternate/v considered a cure for and a cause of insanity.

Cannabis Indica speeds up Recovery from Coronavirus
Gershom Zajicek
YouTube March 2020
DOI: 10.26303/p9dq-ns94
https://www.youtube.com/watch?v=da_5...PrKGvs&index=2
Cannabis Indica Speeds up Recovery from Coronavirus Severe acute respiratory syndrome (SARS) is a viral respiratory disease caused by the SARS coronavirus (SARS-CoV). As of 2017, there is no cure or protective vaccine for SARS that has been shown to be both safe and effective in humans. Curing coronavirus infection prevents clinical SARS . Cannabis indica speeds up recovery. Recovered individuals do not infect others Phytocannabinoids (Cannabinoids) are the naturally occurring cannabinoids found in the cannabis plant. Endocannabinoids are substances produced from within the body that activate cannabinoid receptors Two plants Cannabis sativa and Cannabis indica Their resins contain hundreds of cannabinoids C. Sativa has also THC C. Indica has also CBD Avoid C. Sativa (THC) !! Unfortunately extracts of both plants are called medical marijuana. Since only cannabis indica extracts are not psychoactive I shall now define what we are after. Cannabis indica resin is antiviral and inhibits cell proliferation. It contains about 60 cannabinoids. It has a higher efficacy than any single compound like THC or CBD Cannabis indica resin is antiviral and inhibits cell proliferation

Cannabis-induced altered states of consciousness are associated with specific dynamic brain connectivity states
Yuliya Zaytseva1–3 , Jiří Horáček1,2, Jaroslav Hlinka1,4, Iveta Fajnerová1,2, Renata Androvičová1,2, Jaroslav Tintěra1, Virginio Salvi5, Marie Balíková6, Tomáš Hložek6, Filip Španiel1,2 and Tomáš Páleníček1,2
Journal of Psychopharmacology 2019, Vol. 33(7) 811–821
DOI: 10.1177/0269881119849814
Background: Cannabis, and specifically one of its active compounds delta-9-tetrahydrocannabinol in recreational doses, has a variety of effects on cognitive processes. Most studies employ resting state functional magnetic resonance imaging techniques to assess the stationary effects of cannabis and to-date one report addressed the impact of delta-9-tetrahydrocannabinol on the dynamics of whole-brain functional connectivity.
Methods: Using a repeated-measures, within-subjects design, 19 healthy occasional cannabis users (smoking cannabis ⩽2 per week) underwent resting state functional magnetic resonance imaging scans. Each subject underwent two scans: in the intoxicated condition, shortly after smoking a cannabis cigarette, and in the non-intoxicated condition, with the subject being free from cannabinoids for at least one week before. All sessions were randomized and performed in a four-week interval. Data were analysed employing a standard independent component analysis approach with subsequent tracking of the functional connectivity dynamics, which allowed six connectivity clusters (states) to be individuated. Results: Using standard independent component analysis in resting state functional connectivity, a group effect was found in the precuneus connectivity. With a dynamic independent component analysis approach, we identified one transient connectivity state, characterized by high connectivity within and between auditory and somato-motor cortices and anti-correlation with subcortical structures and the cerebellum that was only found during the intoxicated condition. Behavioural measures of the subjective experiences of changed perceptions and tetrahydrocannabinol plasma levels during intoxication were associated with this state.
Conclusions: With the help of the dynamic connectivity approach we could elucidate neural correlates of the transitory perceptual changes induced by delta-9-tetrahydrocannabinol in cannabis users, and possibly identify a biomarker of cannabis intoxication.

Cannabis-induced dysphoria/paranoia mediates the link between childhood trauma and psychotic-like experiences in young cannabis users
Molly Carlyle, Toby Constable, Zoe C Walter, Joanna Wilson, Grace Newland, Leanne Hides
Schizophr Res . 2021 Dec;238:178-184.
doi: 10.1016/j.schres.2021.10.011
Childhood trauma (abuse and neglect) is a major risk factor for cannabis use disorder and psychotic-spectrum disorders. Psychotic-like experiences (PLEs) in young people who use cannabis may be an early indicator of psychosis risk following cannabis use. We examined whether (i) childhood trauma moderates the association between cannabis use and PLEs, (ii) the association between childhood trauma and cannabis use is mediated by subjective effects of cannabis (euphoria and dysphoria/paranoia), and (iii) the association between childhood trauma and PLEs is also mediated by these subjective effects. Participants were 2630 cannabis users (aged 16-25) recruited online. They were asked to complete a cross-sectional survey measuring cannabis and other substance use, childhood trauma, PLEs, and the subjective effects of cannabis (euphoria and dysphoria/paranoia). A significant interaction indicated that the effect of cannabis on PLE frequency was stronger for individuals with more severe childhood trauma. Childhood trauma was also associated with greater cannabis use and PLE frequency, both of which were mediated by subjective dysphoria/paranoia when using the drug. This suggests childhood trauma is associated with greater PLEs in young people who use cannabis, which may be linked with an increased susceptibility to the dysphoric/paranoid subjective effects when using the drug. Childhood trauma should be addressed early in young people who use cannabis to mitigate the psychosis-associated harms of the drug.
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Cannabis-Induced Neuroactivity: Research Trends and Commercial Prospects

Rajiv Kumar Chaturvedi, Dinesh Chandra Agrawal
Medicinal Herbs and Fungi pp 159-185
DOI: 10.1007/978-981-33-4141-8_6
https://link.springer.com/chapter/10...81-33-4141-8_6
https://www.researchgate.net/publica...ivity_Research _Trends_and_Commercial_Prospec ts
The worldwide interest and push for the legalization of cannabis/marijuana, especially in the United States, are increasing with each passing day. The present article deals with the concise yet broad review of chemical, medicinal (neuroprotection), and adverse psychotic aspects of cannabis (marijuana or marihuana). The emphasis is made to understand the influence of tetrahydrocannabinol (THC) on a broad spectrum of properties ranging from psychosis, neuroprotection, neurotoxicity to medicinal. The reason why THC shows psychoactivity, but cannabidiol (CBD) does not, has been elucidated based on the minor difference in their chemical structures inhibiting CBD to bind with cannabinoid receptors due to steric hindrance. The distribution of cannabinoid receptors (namely, CB1 and CB2) in the human body and the role of endocannabinoids (namely, anandamide and 2-arachidonoyl glycerol) throughout the human system are described. The effect of the method of consumption (inhalation vs. ingestion) on the psychotropicity of cannabis/THC has also been discussed. Additionally, the effect of the use of synthetic endocannabinoid receptor blocker (antagonist) as a drug molecule for a specific purpose, such as for reducing the appetite, to treat obesity, or for the treatment of tobacco, alcohol, and other hard drugs induced addiction, and their potential adverse effects are also the focus of the article. Both the benefits and the risks of consuming cannabinoids are mainly dose-dependent, just like any other legal or prescription pharma products or regulated/unregulated psychotropic substances. Moderation is the right old prescription for a healthy and long productive life, and it applies to the use of medicinal, cultural, and/or recreational products like cannabis/cannabinoids. The traditional use of cannabis leaves (bhang) in India for medical as well as cultural purposes has been discussed from the modern scientific perspective. Lastly, the rapidly growing trend of the number of the publication of both the scientific research papers and the patent applications on cannabis, along with the market trend of cannabis-derived products, has been provided, showing quite high and promising growth.
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Cannabis-induced oceanic boundlessness

Mitch Earleywine, Luna F Ueno, Maha Mian, Brianna R Altman
Journal of Psychopharmacology March 2021
DOI: 10.1177/0269881121997099
Background Despite tetrahydrocannabinol (THC)’s reputation for creating dramatic effects at high doses, empirical work rarely addresses cannabis’s impact on subjective responses common to the tryptamine psychedelics. We focused on these effects because they have preceded and covaried with the therapeutic impact of psilocybin in previous work.
Aims The current study examined if self-reported responses to cannabis products might parallel those found in clinical trials of psilocybin administration. We also investigated if measures of demographics and cannabis use might correlate with these responses.
Methods Participants reported the subjective effect of their highest THC experience using 27 items that assess oceanic boundlessness, a correlate of mystical experiences. They also answered infrequency items and questions on demographics and cannabis consumption.
Results In an effort to address concerns about replication, we divided respondents who passed infrequency items into two random samples. Self-reported “breakthrough” experiences were significantly greater than zero but significantly lower than those reported in randomized clinical trials of psilocybin (17–19% vs. 59%). Total scores covaried with perceived dosages of THC, but only in one sample. Heavier users of cannabis reported lower scores.
Conclusions Self-report data suggest that high doses of cannabis can create subjective effects comparable to those identified in trials of psilocybin that precede relief from cancer-related distress, treatment-resistant depression, alcohol problems, and cigarette dependence. Given the disparate mechanisms of action, comparing THC-induced to psilocybin-induced effects might improve our understanding of the mechanisms underlying subjective experiences. This work might also support the development of a cannabis-assisted psychotherapy comparable to psilocybin-assisted psychotherapy.
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Cannabis-Induced Psychosis
Oldani, L., Benedetta Grancini
In book: Clinical Cases in Psychiatry: Integrating Translational Neuroscience Approaches January 2019
DOI: 10.1007/978-3-319-91557-9_7
The association between mental illness and drug abuse is complex and has been investigated by a wide scientific literature. Such comorbidity has a negative impact on both persistence and severity of illness. Nonetheless, a causal relationship still needs to be univocally defined, and the sole chronological criterion is not sufficient to determine a cause-effect relationship. Among different illicit drugs, cannabis is the most commonly used in Europe, with almost 26.3% of adults using it in their lifetime. Although cannabis is generally regarded as a substance with low acute toxicity, its THC content and relative potency have increased over time, leading to a more frequent onset of psychotic pictures and an increased number of hospital admissions and of anxiety symptoms. As many experimental studies on healthy humans report, THC cannot only induce transient, dose-dependent psychotic symptoms but also affective, behavioral, cognitive, neurovegetative, and psychophysiological effects. This chapter will present a case of a young adult with a history of cannabis abuse and who was admitted to the psychiatric ward for an acute psychotic symptomatology. His clinical picture was characterized by psychomotor agitation, aggressiveness, logorrhea, and a florid psychotic dimension. A toxicology screen on his urine resulted positive for cannabinoids. He underwent EKG, blood tests, magnetic resonance imaging, and positron emission tomography, all resulted in normal range. In clinical cases similar to the current one, it seems crucial to distinguish between a primary psychiatric syndrome and a substance-induced disorder in order to establish an adequate acute treatment and follow-up.

Cannabis is associated with clinical but not endoscopic remission in ulcerative colitis: A randomized controlled trial
Timna Naftali, Lihi Bar-Lev Schleider, Fabiana Scklerovsky Benjaminov, Fred Meir Konikoff, Shelly Tartakover Matalon, Yehuda Ringel
PLOS One Feb 2021
Background Cannabis is often used by patients with ulcerative colitis, but controlled studies are few. We aimed to assess the effect of cannabis in improving clinical and inflammatory outcomes in ulcerative colitis patients. Methods In a double-blind, randomized, placebo-controlled trial, patients received either cigarettes containing 0.5 g of dried cannabis flowers with80mgTetrahydrocannabinol (THC)or placebo cigarettes for 8 weeks. Parameters of disease including Lichtiger disease activity index, C reactive protein (CRP), calprotectin, Mayo endoscopic score and quality of life (QOL) were assessed before, during and after treatment.
Results The study included 32 patients. Mean age was 30 years, 14 (43%) females. Lichtiger index improved in the cannabis group from 10.9 (IQR 9–14) to5 (IQR 1–7), (p<0.000), and in the placebo group from 11 (IQR 9–13) to 8 (IQR 7–10)(p = 0.15, p between groups 0.001). QOL improved in the cannabis group from 77±4 to 98±20 (p = 0.000) but not in the placebo group (78±3 at week 0 and 78±17 at week 8;p = 0.459; p between groups 0.007). Mayo endoscopic score changed in the cannabis group from 2.13±1 to 1.25±2 (p = 0.015) and in the placebo group from 2.15±1to 1.69±1 (p = 0.367, p between groups 0.17).
Conclusion Short term treatment with THC rich cannabis induced clinical remission and improved quality of life in patients with mild to moderately active ulcerative colitis. However, these beneficial clinical effects were not associated with significant anti-inflammatory improvement in the Mayo endoscopic score or laboratory markers for inflammation

Cannabis is more than simply D9-tetrahydrocannabinol
Ethan B. Russo · John M. McPartland
Psychopharmacology (2003) 165:431–432
DOI 10.1007/s00213-002-1348-z
In response to your recent publication comparing subjective effects of D9-tetrahydrocannabinol and herbal cannabis (Wachtel et al. 2002), a number of comments are necessary. The first concerns the suitability of the chosen “marijuana” to assay the issues at hand. NIDA cannabis has been previously characterized in a number of studies (Chait and Pierri 1989; Russo et al. 2002), as a crude lowgrade product (2–4% THC) containing leaves, stems and
seeds, often 3 or more years old after processing, with a stale odor lacking in terpenoids. This contrasts with the more customary clinical cannabis employed by patients in Europe and North America, composed solely of unseeded flowering tops with a potency of up to 20% THC. Cannabis-based medicine extracts (CBME) (Whittle et al. 2001), employed in clinical trials in the UK (Notcutt 2002; Robson et al. 2002), are extracted from flowering
tops with abundant glandular trichomes, and retain full terpenoid and flavonoid components.

Cannabis (Marijuana) — Effects on Human Behavior and Performance
M. A. Huestis (in Researchgate)
Forensic Science Review • Volume Fourteen Number One/Two • Jan 2002
Cannabis is one of the oldest and most commonly abused drugs in the world. Recently, tremendous advances have been made in our understanding of the endogenous cannabinoid system with the identification of cannabinoid receptors, cannabinoid receptor antagonists, endogenous neurotransmitters, metabolic enzymes, and reuptake mechanisms. These advances have helped us to elucidate the mechanisms of action of cannabis and the side effects and toxicities associated with its use. In addition, potential therapeutic applications are being investigated for the use of smoked cannabis and synthetic THC (dronabinol). Most workplace, military, and criminal justice positive urine drug tests are due to the use of cannabis. In addition, alternative matrices, including saliva, sweat, and hair, are being utilized for monitoring cannabis use in treatment, employment, and criminal justice settings. Experimental laboratory studies have identified cognitive, physiological, and psychomotor effects following cannabis. Epidemiological studies reveal that cannabis is the most common illicit drug world-wide in impaired drivers, and in motor vehicle injuries and fatalities. Driving simulator studies also indicate performance impairment following cannabis use; however, the results of open- and closed-road driving studies and of culpability studies do not consistently document increased driving risk. Clearly a combination of ethanol and cannabis use significantly increases risks. This article reviews the pharmacokinetics and pharmacodynamics of cannabis and places special emphasis on the effects of cannabis on complex tasks such as driving and flying.
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Cannabis (Marijuana) - Effects on Human Performance and Behavior
Marilyn A Huestis
Forensic Science Review · February 2002
https://www.researchgate.net/publica...e_and_Behavior
Cannabis is one of the oldest and most commonly abused drugs in the world. Recently, tremendous advances have been made in our understanding of the endogenous cannabinoid system with the identification of cannabinoid receptors, cannabinoid receptor antagonists, endogenous neurotransmitters, metabolic enzymes, and reuptake mechanisms. These advances have helped us to elucidate the mechanisms of action of cannabis and the side effects and toxicities associated with its use. In addition, potential therapeutic applications are being investigated for the use of smoked cannabis and synthetic THC (dronabinol). Most workplace, military, and criminal justice positive urine drug tests are due to the use of cannabis. In addition, alternative matrices, including saliva, sweat, and hair, are being utilized for monitoring cannabis use in treatment, employment, and criminal justice settings. Experimental laboratory studies have identified cognitive, physiological, and psychomotor effects following cannabis. Epidemiological studies reveal that cannabis is the most common illicit drug world-wide in impaired drivers, and in motor vehicle injuries and fatalities. Driving simulator studies also indicate performance impairment following cannabis use; however, the results of open- and closed-road driving studies and of culpability studies do not consistently document increased driving risk. Clearly a combination of ethanol and cannabis use significantly increases risks. This article reviews the pharmacokinetics and pharmacodynamics of cannabis and places special emphasis on the effects of cannabis on complex tasks such as driving and flying.

Cannabis (marijuana): Psychoactive properties, Addiction, Therapeudic uses and toxicity
Sumanasekera WK and Spio KJ
Addict Behav Ther Rehabil 2016, 5:2
DOI: 10.4172/2324-9005.1000156
https://www.researchgate.net/publica...erties_Addicti on_Therapeutic_Uses_and_Toxici ty
Background and Objectives: To analyze the existing literature on cannabis (marijuana) under several sub topics; history of use, addiction, mechanism of action, therapeutic uses, and adverse effects.
Methods: A comprehensive literature search was conducted. Data sources include original research articles, systematic reviews and meta- analysis, and web-based references such as Drug strategy monographs and Merck Index. Data bases such as PubMed and Cinhal was used. References were from 1979 -2016.
Hypothesis: Based on the available literature it can be hypothesized that despite the addictive properties and adverse effects, marijuana has a therapeutic potential and should be used with caution.
Results: The available literature on cannabis smoke was discussed under five major categories, which are history of use, addiction, mechanism of action, therapeutic uses, and adverse effects.
Discussion and Conclusions: Marijuana (preparations of the hemp plant, cannabis sativa), is usually consumed as a smoke. It is an addictive compound that has been consumed for medical ailments for generations. The mechanism of action of cannabis was not known until the recent discovery of the endocannabinoid system.
Endocannabinoids are released from the post-synaptic neurons and act on the pre-synaptic neurons. Δ-9 tetrahydrocannabinol (THC), the psychoactive component present in Cannabis behaves similar to endocannabinoids. In addition to psychoactive modulations, cannabis consumption leads to cognitive impairments. Despite its’ medicinal and recreational value, due to its’ addictive properties cannabis consumption has been banned by several countries. In US, recreational and therapeutic use of cannabis is recently allowed in some states.
Scientific Significance: Despite the psych activity associated with cannabis consumption, it can be used therapeutically for many diseases including epilepsy and to alleviate chronic neuropathic pain associated with many sicknesses. Cannabis also possesses anti-cancer properties. However, strict laws should be enforced to avoid its’ abuse while allowing its’ medicinal use..

Cannabis Microbiome Sequencing: Implications for Cannabis Safety Testing
Kyle Boyar
Conference: Cannabis Science Conference East April 2019
DOI: 10.13140/RG.2.2.18308.17288
Project: Medicinal Genomics
The cannabis plant and cannabis products are highly varied and complex matrices with each different product and route of administration having its own considerations for microbial testing. In the absence of rigorous study, this immature industry has decided to adopt methods commonly used in food testing to obtain information about the potential microbial hazards present. However, DNA sequencing of both the cannabis microbiome and the conditions before and after culturing tell a tale of inaccurate methodology. Many of the methods that are currently being employed are leading the cannabis industry astray, while blinding them to the real hazards that could be present. This presentation will walk you through the data that shows this and the discoveries we've made along the way that will hopefully open fresh discussions with new perspective on how to tackle microbiological contaminants in cannabis.

Cannabis, One Health, and Veterinary Medicine: Cannabinoids’ Role in Public Health, Food Safety, and Translational Medicine
Sivan Ritter, Lilach Zadik-Weiss, Osnat Almogi-Hazan, and*Reuven Or,
Rambam Maimonides Med J. 2020 Jan; 11(1):
doi:*10.5041/RMMJ.10388
https://www.ncbi.nlm.nih.gov/pmc/art...11-1-e0006.pdf
Public health is connected to cannabis with regard to food, animal feed (feed), and pharmaceuticals. Therefore, the use of phytocannabinoids should be examined from a One Health perspective. Current knowledge on medical cannabis treatment (MCT) does not address sufficiently diseases which are of epidemiological and of zoonotic concern. The use of cannabinoids in veterinary medicine is illegal in most countries, mostly due to lack of evidence-based medicine. To answer the growing need of scientific evidence-based applicable medicine in both human and veterinary medicine, a new approach for the investigation of the therapeutic potential of cannabinoids must be adopted. A model that offers direct study of a specific disease in human and veterinary patients may facilitate development of novel therapies. Therefore, we urge the regulatory authorities—the ministries of health and agriculture (in Israel and worldwide)—to publish guidelines for veterinary use due to its importance to public health, as well as to promote One Health-related preclinical translational medicine studies for the general public health.

Cannabis, Pain, and Sleep: Lessons from Therapeutic Clinical Trials of Sativex, a Cannabis-Based Medicine
Ethan B. Russo, Geoffrey W. Guy, and Philip J. Robson
CHEMISTRY & BIODIVERSITY (2007) 4(8):1729-43
DOI: 10.1002/cbdv.200790150
Cannabis sativa L. has been utilized for treatment of pain and sleep disorders since ancient times. This review examines modern studies on effects of Delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on sleep. It goes on to report new information on the effects on sleep in the context of medical treatment of neuropathic pain and symptoms of multiple sclerosis, employing standardized oromucosal cannabis-based medicines containing primarily THC, CBD, or a 1 : 1 combination of the two (Sativex). Sleep-laboratory results indicate a mild activating effect of CBD, and slight residual sedation with THC-predominant extracts. Experience to date with Sativex in numerous Phase I-III studies in 2000 subjects with 1000 patient years of exposure demonstrate marked improvement in subjective sleep parameters in patients with a wide variety of pain conditions including multiple sclerosis, peripheral neuropathic pain, intractable cancer pain, and rheumatoid arthritis, with an acceptable adverse event profile. No tolerance to the benefit of Sativex on pain or sleep, nor need for dosage increases have been noted in safety extension studies of up to four years, wherein 40-50% of subjects attained good or very good sleep quality, a key source of disability in chronic pain syndromes that may contribute to patients' quality of life.

105,000 cannabis patients in Israel are entitled to 4 tons of cannabis a month
Monthly status report: The number of licensees for the use of medical cannabis in Israel is back to climbing and currently reaches more than 105,000 who are entitled to more than 4 tons of cannabis per month
Cannabis Magazine Posted on: 29.11.21,
https://www.xn--4dbcyzi5a.com/105000...95%D7%A4%D7%9C %D7%99-%D7%A7%D7%A0%D7%90%D7%91%D7%99 %D7%A1-%D7%A8%D7%A4%D7%95%D7%90%D7%99-%D7%91%D7%99%D7%A9%D7%A8%D7%90 %D7%9C-%D7%96%D7%9B%D7%90%D7%99%D7%9D-%D7%9C-4-%D7%98/
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After the first slowdown in the rate of increase in the number of cannabis patients in Israel was recorded in August and a decrease in the number of patients was first recorded in September , in October the rate of increase returned to normal.
According to official data, published by the Ministry of Health only today, at the end of November and much later than usual and only following a request from Cannabis Magazine, there are more than 104,000 patients in Israel.
More precisely, as of the end of October 2021, there were 104,907 medical cannabis licenses in Israel, and if we take into account that October is now over, it can be estimated that today there are even more than 106,000, although these data will only be revealed next month.
In terms of the monthly amount of cannabis to which patients are entitled, the data in the monthly report ( PDF ) show that it continues to rise and stood at 3,961 kg in total in October, which means that today, the end of November, eligibility must already exceed the 4 tonne threshold per month. As usual, more than 88% of it in inflorescence and less than 12% in extract (oil).
Keep in mind that these are eligibility data, not actual "withdrawal" data - data that the Ministry of Health has so far refused to disclose despite repeated requests from Cannabis Magazine. It is likely that these data will soon be revealed.According to estimates, the actual utilization is about 80% of the total eligibility.
As usual, the majority of those entitled to a license are entitled to it on the basis of the label "pain", which constitutes more than 50% of the total number of patients. At the end of October more than 58,000 of the 105,000 patients hold a license because of this indication.
In second place, also as usual, is an "other" section , with more than 15,000 patients, ie about 15% of all licenses. This section addresses health issues that are not on the list of contraindications. The Ministry of Health also refuses to disclose in this case which diseases are included in this section.
Most patients, about 23% of them, hold a license for 30 grams of cannabis a month and next to them, 21% of the patients hold a license for 20 grams a month and 20% for 40 grams. The rest hold 50 grams (13%), 60 grams (8%), 70 grams a month (4%) and the rest with 80 grams or more.
Almost half of the patients, 51,062 of them to be exact, are associated with Clalit Health Insurance Fund, 27,208 with the Maccabi Health Insurance Fund, 9,563 with the United Health Insurance Fund, 7,725 with the National Health Insurance Fund and 9,249 with the "other"
Police have stopped issuing licenses for cannabis businesses across the country

Cannabis-Responsive Biomarkers: A Pharmacometabolomics-Based Application to Evaluate the Impact of Medical Cannabis Treatment on Children with Autism Spectrum Disorder
Michael Siani-Rose, Stephany Cox, Bonni Goldstein, Donald Abrams, Myiesha Taylor, and Itzhak Kurek
Cannabis and Cannabinoid Research 2021
Doi: 10.1089/can.2021.0129
https://www.liebertpub.com/doi/10.1089/can.2021.0129
Introduction: Autism spectrum disorder (ASD) is a group of neurodevelopmental conditions that impact behavior, communication, social interaction, and learning abilities. Treatment of ASD with medical cannabis (MC) shows promising results in reducing the severity of certain behavioral aspects. The goals of this observational study are to demonstrate the potential of metabolic biomarkers to (1) objectively determine the impact on metabolites of MC treatment and (2) suggest the metabolic pathways of children with ASD, who respond to MC treatment.
Materials and Methods: The impact of effective physician-supervised MC treatment on children with ASD (n=15), compared with an age-matched group of typically developing (TD; n=9) children, was evaluated in an observational study design. Each child followed a unique MC regimen determined by their specific response over at least 1 year of treatment, which included the following: tetrahydrocannabinol-dominant MC (dosing range 0.05–50 mg per dose) in 40% of children and cannabidiol-dominant MC (dosing range 7.5–200 mg per dose) in 60% of children. Samples from the ASD group collected pre-MC treatment and at time of maximal impact, and from the TD group, were subjected to salivary metabolomics analysis. Ten minutes before saliva sampling, parents filled out behavioral rating surveys.
Results: Sixty-five potential cannabis-responsive biomarkers exhibiting a shift toward the TD physiological levels were identified in children with ASD after MC treatment. For each biomarker, the physiological levels were determined based on the values detected in the TD group. A similar qualitative improvement trend in children with ASD treated with MC was also observed in the behavioral surveys. Twenty-three potential Cannabis-Responsive biomarkers exhibiting change toward TD mean were categorized as anti-inflammatory, bioenergy associated, neurotransmitters, amino acids, and endocannabinoids. The changes in the levels of the Cannabis-Responsive biomarkers N-acetylaspartic acid, spermine, and dehydroisoandrosterone 3-sulfate have been previously linked to behavioral symptoms commonly observed in individuals with ASD.
Conclusions: Our results suggest Cannabis-Responsive biomarkers shift toward the TD mean after MC treatment and can potentially quantify benefit at the metabolic level. These changes appear to be similar to the trend described in behavior surveys. Larger trials are needed to confirm these preliminary findings.

Cannabis Roots: A Traditional Therapy with Future Potential for Treating Inflammation and Pain
Natasha Ryz, David J. Remillard, Ethan Budd Russo
August 2017 Cannabis and Cannabinoid Research, 2(1), 210–216.
DOI: 10.1089/can.2017.0028
The roots of the cannabis plant have a long history of medical use stretching back millennia. However, the therapeutic potential of cannabis roots has been largely ignored in modern times. Discussion: In the first century, Pliny the Elder described in Natural Histories that a decoction of the root in water could be used to relieve stiffness in the joints, gout, and related conditions. By the 17th century, various herbalists were recommending cannabis root to treat inflammation, joint pain, gout, and other conditions. There has been a subsequent paucity of research in this area, with only a few studies examining the composition of cannabis root and its medical potential. Active compounds identified and measured in cannabis roots include triterpenoids, friedelin (12.8 mg/kg) and epifriedelanol (21.3 mg/kg); alkaloids, cannabisativine (2.5 mg/kg) and anhydrocannabisativine (0.3 mg/kg); carvone and dihydrocarvone; N-(p-hydroxy-?-phenylethyl)-p-hydroxy-trans-cinnamamide (1.6 mg/kg); various sterols such as sitosterol (1.5%), campesterol (0.78%), and stigmasterol (0.56%); and other minor compounds, including choline. Of note, cannabis roots are not a significant source of ??-tetrahydrocannabinol (THC), cannabidiol, or other known phytocannabinoids. Conclusion: The current available data on the pharmacology of cannabis root components provide significant support to the historical and ethnobotanical claims of clinical efficacy. Certainly, this suggests the need for reexamination of whole root preparations on inflammatory and malignant conditions employing modern scientific techniques.

Cannabis sativa allergy: looking through the fog.
Decuyper, I. I., Van Gasse, A. L., Cop, N., Sabato, V., Faber, M. A., Mertens, C., … Ebo, D. G.
Allergy, 72(2), 201–206. (2016).
doi:10.1111/all.13043
IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross -allergies, mostly for plant foods. This clinical entity, designated as the “cannabis-fruit/vegetable syndrome” might also imply cross-reactivity with tobacco, natural latex and plant -food derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between non-specific lipid transfer proteins (ns
-LTPs) or thaumatin-like proteins (TLPs) present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE (sIgE) antibodies and basophil activation tests (BAT) can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Cannabis sativa L. and Nonpsychoactive Cannabinoids: Their Chemistry and Role against Oxidative Stress, Inflammation, and Cancer
Federica Pellati, Vittoria Borgonetti, Virginia Brighenti, Marco Biagi, Stefania Benvenuti, and Lorenzo Corsi
Hindawi BioMed Research International Volume 2018, doi: 10.1155/2018/1691428
In the last decades, a lot of attention has been paid to the compounds present in medicinal Cannabis sativa L., such as ?9 - tetrahydrocannabinol (?9 -THC) and cannabidiol (CBD), and their efects on infammation and cancer-related pain. Te National Cancer Institute (NCI) currently recognizes medicinal C. sativa as an efective treatment for providing relief in a number of symptoms associated with cancer, including pain, loss of appetite, nausea and vomiting, and anxiety. Several studies have described CBD as a multitarget molecule, acting as an adaptogen, and as a modulator, in diferent ways, depending on the type and location of disequilibrium both in the brain and in the body, mainly interacting with specifc receptor proteins CB1 and CB2. CBD is present in both medicinal and fbre-type C. sativa plants, but, unlike ?9 -THC, it is completely nonpsychoactive. Fibre-type C. sativa (hemp) difers from medicinal C. sativa, since it contains only few levels of ?9 -THC and high levels of CBD and related nonpsychoactive compounds. In recent years, a number of preclinical researches have been focused on the role of CBD as an anticancer molecule, suggesting CBD (and CBD-like molecules present in the hemp extract) as a possible candidate for future clinical trials. CBD has been found to possess antioxidant activity in many studies, thus suggesting a possible role in the prevention of both neurodegenerative and cardiovascular diseases. In animal models, CBD has been shown to inhibit the progression of several cancer types. Moreover, it has been found that coadministration of CBD and ?9 -THC, followed by radiation therapy, causes an increase of autophagy and apoptosis in cancer cells. In addition, CBD is able to inhibit cell proliferation and to increase apoptosis in diferent types of cancer models. Tese activities seem to involve also alternative pathways, such as the interactions with TRPV and GRP55 receptor complexes. Moreover, the fnding that the acidic precursor of CBD (cannabidiolic acid, CBDA) is able to inhibit the migration of breast cancer cells and to downregulate the proto-oncogene c-fos and the cyclooxygenase-2 (COX-2) highlights the possibility that CBDA might act on a common pathway of infammation and cancer mechanisms, which might be responsible for its anticancer activity. In the light of all these fndings, in this review we explore the efects and the molecular mechanisms of CBD on infammation and cancer processes, highlighting also the role of minor cannabinoids and noncannabinoids constituents of ?9 -THC deprived hemp.

Cannabis sativa as a Treatment for Obesity: From Anti-Inflammatory Indirect Support to a Promising Metabolic Re-Establishment Target
Larissa Espindola da Silva, Cristini da Rosa Turatti, Rafael Mariano de Bitencourt, and Gislaine Tezza Rezin
Cannabis and Cannabinoid Res 2021
DOI: 10.1089/can.2021.0016
https://www.liebertpub.com/doi/epub/.../can.2021.0016
https://www.researchgate.net/publica...is_sativa_as_a _Treatment_for_Obesity_From_An ti-Inflammatory_Indirect_Support_ to_a_Promising_Metabolic_Re-Establishment_Target
Introduction: Obesity is de􀂦ned as an excess of accumulation of fat that can be harmful to health. Storage of excess fat in the adipose tissue triggers an inflammatory process, which makes obesity a low-grade chronic inflammatory disease. Obesity is considered a complex and multifactorial disease; hence, no intervention strategy appears to be an ideal treatment for all individuals. Therefore, new therapeutic alternatives are often studied for the treatment of this disease. Currently, herbal medicines are gaining ground in the treatment of obesity and its comorbidities. In this context, much attention is being paid to Cannabis sativa derivatives, and their therapeutic functions are being widely studied, including in treating obesity.
Objective: Highlight the pharmacological properties of Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabidinol (THC), and cannabidiol (CBD), the predominant isolated components of Cannabis sativa, as well as its therapeutic potential in the treatment of obesity.
Methods: This is a narrative review that shows the existing scientific evidence on the clinical application of Cannabis sativa as a possible treatment for obesity. Data collection was performed in the PubMed electronic database. The following word combinations were used: Cannabis and obesity, Cannabis sativa and obesity, THCV and obesity, THC and obesity, CBD and obesity, and Cannabis sativa and inflammation.
Results: Evidence shows that Cannabis sativa derivatives have therapeutic potential due to their anti-inflammatory
properties. In addition, people who use cannabis have a lower body mass index than those who do not, making the plant anoption to reduce and reverse in􀂧ammation and comorbidities in obesity.
Conclusion: It is concluded that phytocannabinoids derived from Cannabis sativa have therapeutic potential due to its antiinflammatory, antioxidant, and neuroprotective properties, making the plant a study option to reduce and reverse inflammation and comorbidities associated with obesity.
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Cannabis sativa L. extract and cannabidiol inhibit in vitro mediators of skin inflammation and wound injury.
Sangiovanni, E., Fumagalli, M., Pacchetti, B., Piazza, S., Magnavacca, A., Khalilpour, S., … Dell’Agli, M.
Phytotherapy Research. (2019)
doi:10.1002/ptr.6400
Skin inflammatory diseases result from complex events that include dysregulation and abnormal expression of inflammatory mediators or their receptors in skin cells. The present study investigates the potential effect of a Cannabis sativa L. ethanolic extract standardized in cannabidiol as antiinflammatory agent in the skin, unraveling the molecular mechanisms in human keratinocytes and fibroblasts. The extract inhibited the release of mediators of inflammation involved in wound healing and inflammatory processes occurring in the skin. The mode of action involved the impairment of the nuclear factor?kappa B (NF??B) pathway since the counteracted the tumor necrosis factor?alpha?induced NF??B?driven transcription in both skin cell lines. Cannabis extract and cannabidiol showed different effects on the release of interleukin?8 and vascular endothelial growth factor, which are both mediators whose genes are dependent on NF??B. The effect of cannabidiol on the NF??B pathway and metalloproteinase?9 (MMP?9) release paralleled the effect of the extract thus making cannabidiol the major contributor to the effect observed. Down?regulation of genes involved in wound healing and skin inflammation was at least in part due to the presence of cannabidiol. Our findings provide new insights into the potential effect of Cannabis extracts against inflammation?based skin diseases

Cannabis sativa extracts protect LDL from Cu2+-mediated oxidation
Bruno Musetti, Helena González-Ramos, Mercedes González, Edward M. Bahnson, Javier Varela, and Leonor Thomson
Journal of Cannabis Research (2020) 2:37
doi 10.1186/s42238-020-00042-0
Background: Multiple therapeutic properties have been attributed to Cannabis sativa. However, further research is required to unveil the medicinal potential of Cannabis and the relationship between biological activity and chemical profile.
Objectives: The primary objective of this study was to characterize the chemical profile and antioxidant properties of three varieties of Cannabis sativa available in Uruguay during progressive stages of maturation.
Methods: Fresh samples of female inflorescences from three stable Cannabis sativa phenotypes, collected at different time points during the end of the flowering period were analyzed. Chemical characterization of chloroform extracts was performed by 1 H-NMR. The antioxidant properties of the Cannabis sativa extracts, and pure cannabinoids, were measured in a Cu2+-induced LDL oxidation assay.
Results: The main cannabinoids in the youngest inflorescences were tetrahydrocannabinolic acid (THC-A, 242 ± 62 mg/g) and tetrahydrocannabinol (THC, 7.3 ± 6.5 mg/g). Cannabinoid levels increased more than twice in two of the mature samples. A third sample showed a lower and constant concentration of THC-A and THC (177 ± 25 and 1 ± 1, respectively). The THC-A/THC rich cannabis extracts increased the latency phase of LDL oxidation by a factor of 1.2– 3.5 per ?g, and slowed down the propagation phase of lipoperoxidation (IC50 1.7–4.6 ?g/mL). Hemp, a cannabidiol (CBD, 198 mg/g) and cannabidiolic acid (CBD-A, 92 mg/g) rich variety, also prevented the formation of conjugated dienes during LDL oxidation. In fact, 1 ?g of extract was able to stretch the latency phase 3.7 times and also to significantly reduce the steepness of the propagation phase (IC50 of 8 ?g/mL). Synthetic THC lengthened the duration of the lag phase by a factor of 21 per ?g, while for the propagation phase showed an IC50 ? 1 ?g/mL. Conversely, THC-A was unable to improve any parameter. Meanwhile, the presence of 1 ?g of pure CBD and CBD-A increased the initial latency phase 4.8 and 9.4 times, respectively, but did not have an effect on the propagation phase.
Conclusion: Cannabis whole extracts acted on both phases of lipid oxidation in copper challenged LDL. Those effects were just partially related with the content of cannabinoids and partially recapitulated by isolated pure cannabinoids. Our results support the potentially beneficial effects of Cannabis sativa whole extracts on the initial phase of atherosclerosis.

Cannabis sativa: much more beyond ?9-tetrahydrocannabinol.
Alves, P., Amaral, C., Teixeira, N., & Correia-da-Silva, G.
Pharmacological Research, (2020)
doi:10.1016/j.phrs.2020.104822
Cannabis is the most used illicit drug worldwide and its medicinal use is under discussion, being regulated in several countries. However, the psychotropic effects of ?9-tetrahydrocannabinol (THC), the main
psychoactive compound of Cannabis sativa, are of concern. Thus, the interest in the isolated constituents without psychotropic activity, such as cannabidiol (CBD) and cannabidivarin (CBDV) is growing. CBD and
CBDV are lipophilic molecules with poor oral bioavailability and are mainly metabolized by cytochrome P450 (CYP450) enzymes. The pharmacodynamics of CBD is the best explored, being able to interact with diverse molecular targets, like cannabinoid receptors, G protein-coupled receptor-55, transient receptor potential vanilloid 1 channel and peroxisome proliferator-activated receptor-?. Considering the therapeutic potential, several clinical trials are underway to study the efficacy of CBD and CBDV in different pathologies, such as neurodegenerative diseases, epilepsy, autism spectrum disorders and pain conditions. The anti-cancer properties of CBD have also been demonstrated by several pre-clinical studies in different types of tumour cells. Although less studied, CBDV, a structural analogue of CBD, is receiving attention in the last years. CBDV exhibits anticonvulsant properties and, currently, clinical trials are underway for the treatment of autism spectrum disorders. Despite the benefits of these phytocannabinoids, it is important to highlight their potential interference with relevant physiologic mechanisms. In fact, CBD interactions with CYP450 enzymes and with drug efflux transporters may have serious consequences when co-administered with other drugs. This review summarizes the therapeutic advances of CBD and CBDV and explores some aspects of their pharmacokinetics, pharmacodynamics and possible interactions. Moreover, it also highlights the therapeutic potential of CBD and CBDV in several medical conditions and clinical applications

Cannabis sativa L. protects against oxidative injury in kidney (vero) cells by mitigating perturbed metabolic activities linked to chronic kidney diseases

Ochuko L.Erukainure, SunelleRademan, Joseph O.Erhabor, Chika I.Chukwuma, Adeline LumNde, Motlalepula G.Matsabisa

Journal of Ethnopharmacology
Volume 293
, 15 July 2022, 115312
https://www.sciencedirect.com/science/article/abs/pii/S0378874122003518?via=ihub#screen-reader-main-content

Ethnopharmacological relevance​

Cannabis sativa L. is among numerous medicinal plants widely used in traditional medicine in treating various ailments including kidney diseases.

Aims​

The protective effect of C. sativa on oxidative stress, cholinergic and purinergic dysfunctions, and dysregulated glucogenic activities were investigated in oxidative injured kidney (Vero) cell lines.

Methods​

Fixed Vero cells were treated with sequential extracts (hexane, dichloromethane [DCM] and ethanol) of C. sativa leaves for 48 h before subjecting to MTT assay. Vero cells were further incubated with FeSO4 for 30 min, following pretreatment with C. sativa extracts for 25 min. Normal control consisted of Vero cells not treated with the extracts and/or FeSO4, while untreated (negative) control consisted of cells treated with only FeSO4.

Results​

MTT assay revealed the extracts were slightly cytotoxic at the highest concentrations (250 μg/mL). There was a significant depletion in glutathione level and catalase activity on induction of oxidative stress, with significant elevation in malondialdehyde level, acetylcholinesterase, ATPase, ENTPDase, fructose-1,6-biphosphatase, glucose 6-phosphatase and glycogen phosphorylase activities. These activities and levels were significantly reversed following pretreatment with C. sativa extracts.

Conclusion​

These results portray the protective potentials of C. sativa against iron-mediated oxidative renal injury as depicted by the ability of its extracts to mitigate redox imbalance and suppress acetylcholinestererase activity, while concomitantly modulating purinergic and glucogenic enzymes activities in Vero cells.
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Cannabis suppresses antitumor immunity by inhibiting JAK/ STAT signaling in T cells through CNR2
Xinxin Xiong, Siyu Che, Jianfei She, Hua You, Han Yan, Chao Yan, Ziqian Fan, Jianeng Zhan, Xiuyu Ca, Xingjun Don, Tiebang Kan, Wende Li and Penghui Zhou
Signal Transduction and Targeted Therapy
DOI: 10.1038/s41392-022-00918-y
https://www.nature.com/articles/s41392-022-00918-y.pdf
The combination of immune checkpoint blockade (ICB) with chemotherapy significantly improves clinical benefit of cancer treatment. Since chemotherapy is often associated with adverse events, concomitant treatment with drugs managing side effects of chemotherapy is frequently used in the combination therapy. However, whether these ancillary drugs could impede immunotherapy remains unknown. Here, we showed that Δ 9-tetrahydrocannabinol (THC), the key ingredient of drugs approved for the treatment of chemotherapy-caused nausea, reduced the therapeutic effect of PD-1 blockade. The endogenous cannabinoid anandamide (AEA) also impeded antitumor immunity, indicating an immunosuppressive role of the endogenous cannabinoid system (ECS). Consistently, high levels of AEA in the sera were associated with poor overall survival in cancer patients. We further found that cannabinoids impaired the function of tumor-specific T cells through CNR2. Using a knock-in mouse model expressing a FLAG-tagged Cnr2 gene, we discovered that CNR2 binds to JAK1 and inhibits the downstream STAT signaling in T cells. Taken together, our results unveiled a novel mechanism of the ECS-mediated suppression on T-cell immunity against cancer, and suggest that cannabis and cannabinoid drugs should be avoided during immunotherapy.

Cannabis – the Israeli perspective
Raphael Mechoulam
J Basic Clin Physiol Pharmacol 1;27(3):181-7. 2015
DOI 10.1515/jbcpp-2015-0091
Short overviews are presented on the historical uses of cannabis in the Middle East and on the more recent scientific and medical research on phytocannabinoids and the endocannabinoid system, with emphasis on research contributions from Israel. These are followed by examples of research projects and clinical trials with cannabinoids and by a short report on the regulation of medical marijuana in Israel, which at present is administered to over 22,000 patients.

Cannabis the polypharmaceutical herb a review of the endocannabinoid system and Cannabis plants
Stephanie R. Hruza
Thesis Dec 11 2015 https://www.academia.edu/28772390/Ca...ypharmaceutica l_herb_A_review_of_the_endocan nabinoid_system_and_Cannabis_p lant_constituents The plant, Cannabis sativa, has shown great promise as a polypharmaceutical herb with diverse medicinal applications. This review first gives an overview of the pharmacokinetics and pharmacodynamics of Cannabis. Next, it explains the components of the endocannabinoid system, including receptors and endocannabinoids, and then closely examines the function of specific Cannabis constituents including the phytocannabinoids, THC, CBD, CBC, CBG, THCV, CBDV, and CBN, the terpenoids, limonene, myrcene, pinene, linalool, caryophyllene, nerolidol, caryophyllene oxide, phytol, pulegone, eucalyptol, terpeneols, cymene, borneol, careen, the flavonoids, naringenin, apigenin, quercein, and cannflavin, and the phytosterol, sitosterol. Emphasis is placed on the role of synergy between Cannabis constituents that results in an entourage effect, whereby the phytocannabinoids, terpenoids, and flavonoids interact to mitigate side effects and enhance therapeutic benefits of the active components.

Cannabis Therapeutics and the Future of Neurology
Ethan B. Russo
Front Integr Neurosci. 2018; 12: 51.
doi: 10.3389/fnint.2018.00051
Neurological therapeutics have been hampered by its inability to advance beyond symptomatic treatment of neurodegenerative disorders into the realm of actual palliation, arrest or reversal of the attendant pathological processes. While cannabisbased medicines have demonstrated safety, efficacy and consistency sufficient for regulatory approval in spasticity in multiple sclerosis (MS), and in Dravet and Lennox-Gastaut Syndromes (LGS), many therapeutic challenges remain. This review will examine the intriguing promise that recent discoveries regarding cannabis-based medicines offer to neurological therapeutics by incorporating the neutral phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), their acidic precursors, tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), and cannabis terpenoids in the putative treatment of five syndromes, currently labeled recalcitrant to therapeutic success, and wherein improved pharmacological intervention is required: intractable epilepsy, brain tumors, Parkinson disease (PD), Alzheimer disease (AD) and traumatic brain injury (TBI)/chronic traumatic encephalopathy (CTE). Current basic science and clinical investigations support the safety and efficacy of such interventions in treatment of these currently intractable conditions, that in some cases share pathological processes, and the plausibility of interventions that harness endocannabinoid mechanisms, whether mediated via direct activity on CB1 and CB2 (tetrahydrocannabinol, THC, caryophyllene), peroxisome proliferator-activated receptorgamma (PPAR?; THCA), 5-HT1A (CBD, CBDA) or even nutritional approaches utilizing prebiotics and probiotics. The inherent polypharmaceutical properties of cannabis botanicals offer distinct advantages over the current single-target pharmaceutical model and portend to revolutionize neurological treatment into a new reality of effective interventional and even preventative treatment

Cannabis treatment in hospitalized patients using the SYQE inhaler: Results of a pilot open-label study.
Vulfsons, S., Ognitz, M., Bar-Sela, G., Raz-Pasteur, A., & Eisenberg, E.
Palliative and Supportive Care, 1–6. (2019).
doi:10.1017/s147895151900021x
Objective. The objectives were to evaluate t
he, usability, feasibility of use, satisfaction, and safety of the Syqe Inhaler Exo (Syqe Inhaler), a metered dose, Pharmacokinetics-validated, cannabis inhaler device in a cohort of hospitalized patients that were using medical cannabis under license as a part of their ongoing medical treatment. Method. Before and after inhaling from the Syqe Inhaler, participants were asked to fill a questionnaire regarding pain reduction on a visual analog scale from 0 to 10 and, if relevant, reduction in chemotherapy-induced nausea and vomiting and/or spasticity. A patient satisfaction questionnaire and a usability questionnaire were filled in following the last use. Prescribed treatment included 4 daily doses of 500 ?g tetrahydrocannabinol each delivered from 16 mg cannabis flos per inhalation plus up to an additional four SOS (distress code for more doses of cannabis) doses. Result. Daily cannabis dose consumed during hospitalization with the Syqe Inhaler was 51 mg (20–96) versus 1,000 mg (660-3,300) consumed prehospitalization. Patients were easily trained and continued to use Syqe Inhaler for the duration of their hospitalization (5 [3–7] days). Pain intensity 30–60 minutes following inhalations was reported to be significantly lower than preinhalation 4 [1–5] versus 7 [2–9]). Participants ranked their satisfaction with Syqe Inhaler as 6 (5–7). Three participants reported mild cough, which resolved spontaneously. Significance of results. Cannabis inhalation by combustion is not feasible for hospitalized patients. The use of Syqe Inhaler during hospitalization yielded high levels of patients and staff satisfaction with no complications.

Cannabis Use among Patients with Alopecia Areata: A Cross-Sectional Survey Study
Jane J Han, Adam Faletsky, Arash Mostaghimi, Kathie P Huang
Int J Trichology. Jan-Feb 2022;14(1):21-24.
DOI: 10.4103/ijt.ijt_96_21
https://www.ncbi.nlm.nih.gov/pmc/art...er#!po=83.3333
Importance: Little is known about cannabis use among patients with alopecia areata (AA). These patients often experience significant psychosocial burden and may seek alternative therapies beyond that of traditional medical treatments, such as cannabis.
Objective: To characterize cannabis use among patients with AA.
Design: This was a cross-sectional study conducted from March 9, 2021, to March 22, 2021, using a web-based survey distributed to adult patients with AA using the National AA Foundation's email listserv and social media platforms.
Results: 1,087 participants completed the survey (completion rate: 88.1%). Most participants were female (n = 870, 83.3%) and Caucasian (n = 771, 73.8%), with a mean age of 47.6 ± 15.5 years. 65.9% (n = 689) of participants with AA had a history of cannabis use and among those, 51.8% (n = 357) were current cannabis users. The most common reason for cannabis use among current users was for AA-related symptoms (n = 199, 55.7%), with the greatest perceived improvement in symptoms of stress (n = 261, 73.1%) and anxiety, sadness, and depression (n = 234, 65.6%). 80.4% (n = 287) indicated that cannabis had no impact on their hair loss.
Conclusion: Cannabis use is common among patients with AA and is often used to alleviate the psychosocial symptoms related to AA, despite the lack of perceived improvement in hair regrowth
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Cannabis use and cyclical vomiting
Enriqueta Ochoa-Mangado, Agustín Madoz-Gúrpide
adicciones vol.xx, nºx · 2020
https://adicciones.es/index.php/adic...view/1581/1182
Cannabis has traditionally been associated with antiemetic effects and used by some patients to control chemotherapy-induced vomiting. In 2004, however, cannabinoid hyperemesis or cyclic vomiting secondary to cannabis use was described (Allen, de Moore, Heddle & Twartz, 2004), and this has since been confirmed in a variety of studies (Contreras Narváez et al., 2016; Ochoa-Mangado, Madoz-Gúrpide, Jiménez Giménez & Salvador Vadillo, 2009; Simonetto, Oxentenko, Herman & Szostek, 2012)

Cannabis use and medication nonadherence in bipolar disorder: A nationwide inpatient sample database analysis
Adeolu Funso Oladunjoye a Syed Zane Kaleem d Aishwarya Suresh d Vikram Sahni d Matthew J.Thoonkuzhy d Gibson Anugwom e Olubunmi Oladunjoyef g David Otuada c Joseph Ikekwere h Eduardo D Espiridion
Journal of Affective Disorders Volume 299, 15 February 2022, Pages 174-179
DOI: 10.1016/j.jad.2021.11.067
Background
Medication nonadherence among bipolar disorder (BD) is often linked with comorbid substance use disorders. This study aims to investigate cannabis use disorder (CUD) association with medication noncompliance in hospitalized BD patients.
Methods
Using data on 266,303 BD hospitalizations between 2010 and 2014 from the US Nationwide Inpatient Sample database, we obtained medication noncompliance rates stratified by demographics and CUD. Logistic regression was used to evaluate factors associated with medication noncompliance.
Results
Overall mean age, the prevalence of CUD, and medication nonadherence were 41.58 (± 0.11) years, 15.0% and 16.1%, respectively. There were 56.6% females in the overall population. There was a significant difference in the characteristics of those in the medication nonadherence vs adherence groups, including age, sex, race, comorbid substance use, income, insurance type, hospital region, and hospital teaching status (p < 0.001). After adjusting for other variables using multivariate analysis, there remained a statistically significant association of medication nonadherence in BD hospitalization and CUD (OR 1.42, 95% CI 1.36–1.48).
Limitation
Confounding multiple substance use could not be accounted for, and the retrospective nature of the database which includes only inpatients is prone to possible selection and reporting bias.
Conclusion
CUD statistically predicts increased rates of medication nonadherence among patients with BD. Given the possible association of CUD with medication nonadherence among BD patients, collaborative work between general adult psychiatry and addiction services is imperative in improving the management outcome of patients with BD and comorbid CUD.
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Cannabis Use and Risk of Acute Kidney Injury in Patients with Advanced Chronic Kidney Disease Transitioning to Dialysis

Praveen K. Potukuchi, Hamid Moradi, Frank Park, Cameron Kaplan, Fridtjof Thomas, Ankur A. Dashputre, Keiichi Sumida, Miklos Z. Molnar, Abduzhappar Gaipov, Justin D. Gatwood, Connie Rhee, Elani Streja, Kamyar Kalantar-Zadeh, and Csaba P. Kovesdy
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0044
https://www.liebertpub.com/doi/pdf/1.../can.2021.0044
Background: The current social and legal landscape is likely to foster the medicinal and recreational use of cannabis. Synthetic cannabinoid use is associated with acute kidney injury (AKI) in case reports; however, the association between natural cannabis use and AKI risk in patients with advanced chronic kidney disease (CKD) is unknown.
Materials and Methods: From a nationally representative cohort of 102,477 U.S. veterans transitioning to dialysis between 2007 and 2015, we identified 2215 patients with advanced CKD who had undergone urine toxicology (UTOX) tests within a year before dialysis initiation and had inpatient serial serum creatinine levels measured within 7 days after their UTOX test. The exposure of interest was cannabis use compared with no use as ascertained by the UTOX test. We examined the association of this exposure with AKI using logistic regression and inverse probability of treatment weighting with extensive adjustment for potential confounders.
Results: The mean age of the overall cohort was 61 years; 97% were males, 51% were African Americans, 97% had hypertension, 76% had hyperlipidemia, and 75% were diabetic. AKI occurred in 56% of the cohort, and in multivariable-adjusted analysis, cannabis use (when compared with no substance use) was not associated with significantly higher odds of AKI (odds ratio 0.85, 95% confidence interval 0.38–1.87; p = 0.7). These results were robust to various sensitivity analyses.
Conclusions: In this observational study examining patients with advanced CKD, cannabis use was not associated with AKI risk. Additional studies are needed to characterize the impact of cannabis use on risk of kidney disease and injury.

Cannabis use and the sperm epigenome: a budding concern?
Schrott, R., & Murphy, S. K.
Environmental Epigenetics, 6(1). (2020).
doi:10.1093/eep/dvaa002
The United States is swiftly moving toward increased legalization of medical and recreational cannabis. Currently considered the most commonly used illicit psychoactive drug, recreational cannabis is legal in 11 states and Washington, DC, and male use is an important and understudied concern. Questions remain, however, about the potential long-term consequences of this exposure and how cannabis might impact the epigenetic integrity of sperm in such a way that could influence the health and development of offspring. This review summarizes cannabis use and potency in the USA, provides a brief overview of DNA methylation as an epigenetic mechanism that is vulnerable in sperm to environmental exposures including cannabis, and summarizes studies that have examined the effects of parental exposure to cannabis or delta-9 tetrahydrocannabinol (THC, the main psychoactive component of cannabis) on the epigenetic profile of the gametes and behavior of offspring. These studies have demonstrated significant changes to the sperm DNA methylome following cannabis use in humans, and THC exposure in rats. Furthermore, the use of rodent models has shown methylation and behavioral changes in rats born to fathers exposed to THC or synthetic cannabinoids, or to parents who were both exposed to THC. These data substantiate an urgent need for additional studies assessing the effects of cannabis exposure on childhood health and development. This is especially true given the current growing state of cannabis use in the USA.

Cannabis use as a moderator of cognitive behavioral therapy for insomnia
Mary Beth Miller, Ryan W. Carpenter, Lindsey K. Freeman, Ashley F. Curtis, Ali M. Yurasek, Christina S. McCrae,
Journal of Clinical Sleep Medicine 2021
https://jcsm.aasm.org/doi/pdf/10.5664/jcsm.9796
DOI: 10.5664/jcsm.9796
STUDY OBJECTIVES:
Cannabis use is common among young adults and has been proposed as a potential treatment for insomnia. However, controlled studies examining the impact of cannabis use on insomnia symptoms are rare. This secondary analysis of published trial data tested cannabis use during cognitive behavioral treatment for insomnia (CBT-I) as a moderator of treatment efficacy.
METHODS:
Young adults (ages 18-30 years) who reported past-month binge drinking (4/5+ drinks for women/men) and met DSM-5 and research diagnostic criteria for Insomnia Disorder were randomized to CBT-I (n=28) or sleep hygiene (SH; n=28). Interaction effects were tested using multilevel models. Outcomes included insomnia severity, actigraphy-assessed sleep efficiency, diary-assessed sleep quality, drinking quantity, and alcohol-related consequences.
RESULTS:
Twenty-six participants (46%; 12 SH and 14 CBT-I) reported using cannabis during the treatment phase of the study, on an average of 23% of treatment days (range=3-100%). Relative to those who did not use cannabis, participants who used cannabis during treatment reported heavier drinking and more frequent cigarette use. Approximately 1 in 4 cannabis users (27%) reported using cannabis to help with sleep; however, cannabis and non-cannabis users did not differ in use of alcohol as a sleep aid. Controlling for sex, race, drinking quantity, cigarette use, symptoms of depression, and symptoms of anxiety, use of cannabis during treatment did not moderate CBT-I effects on insomnia severity (b= -.002, p=.99) or other outcomes (all p >.20).
CONCLUSIONS:
CBT-I is effective in reducing insomnia symptoms among young adult drinkers with insomnia, regardless of cannabis use.
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Cannabis use assessment and its impact on pain in rheumatologic diseases: a systematic review and meta-analysis
M. Guillouard, N. Authier, B. Pereira, M. Soubrier and S. Mathieu
Rheumatology 2020;00:1–8
Doi:10.1093/rheumatology/keaa534
Objectives. Despite classic analgesic or effective treatments in rheumatic diseases, such as synthetic DMARDs in RA, patients remain in pain and often turn to non-prescribed pharmacological alternatives, such as cannabis self-therapeutic use. However, this medical use of cannabis has not been thoroughly studied.
Methods. We performed a systematic literature review up to*June 2020. The incidence of cannabis consumption was calculated by metaproportion. Differences between cannabis users and non-users were expressed as standardized mean differences using the inverse-variance method. We also assessed the effects of cannabis on pain.
Results. A total of 2900 patients reported cannabis consumption in a sample of 10 873 patients [incidence 40.4% (95% confidence interval (CI): 0.28, 0.54)], and 15.3% (95% CI: 0.07, 0.27) specified that they were currently taking cannabis. Cannabis use was higher in the four fibromyalgia studies [68.2% (95% CI: 0.41, 0.90), n¼ 611] compared with seven articles concerning RA or lupus [26.0% (95% CI: 0.14, 0.41), n¼ 8168]. Cannabis consumption was associated with a decrease in pain intensity [VAS pain at baseline 8.2 (2.9) vs 5.6 (3.5)mm over time; pooled effect size 1.75 (95% CI: 2.75, 0.76)]. Cannabis users were younger [58.4 (11.4) vs 63.6(12.1)years; P**
<0.001, *more often smokers [OR 2.91 (95% CI: 1.84, 4.60)] or unemployed [OR 2.40 (95% CI: 1.31, 4.40)], and had higher pain intensity [5.0 (2.4) vs 4.1(2.6)mm; P <0.001) than non-users.
Conclusion. Nearly 20% of patients suffering from rheumatologic diseases actively consume cannabis, with an improvement in pain. The issue of cannabis use in the management of these patients should be addressed during medical consultation, essentially with cannabis-based standardized pharmaceutical products.

Cannabis use disorder and the lungs.
Gracie, K., & Hancox, R. J.
Addiction. (2020).
doi:10.1111/add.15075
Cannabis is one of the world’s most widely used recreational drugs and the second most commonly smoked substance. Research on cannabis and the lungs has been limited by its illegal status, the variability in strength and size of cannabis cigarettes (joints), and the fact that most cannabis users also smoke tobacco making the effects hard to separate. Despite these difficulties, the available evidence indicates that smoking cannabis causes bronchitis and is associated with changes in lung function. The pattern of effects is surprisingly different from that of tobacco. Whereas smoking cannabis appears to increase the risk of severe bronchitis at quite low exposure, there is no convincing evidence that this leads to chronic obstructive pulmonary disease. Instead cannabis use is associated with increased central airway resistance, lung hyperinflation, and higher vital capacity with little evidence of airflow obstruction or impairment of gas transfer. There are numerous reports of severe bullous lung disease and pneumothorax among heavy cannabis users, but convincing epidemiological data of an increased risk of emphysema or alveolar destruction are lacking. An association between cannabis and lung cancer remains unproven with studies providing conflicting findings

Cannabis use during pregnancy and its relationship with fetal developmental outcomes and psychiatric disorders. A systematic review.
Roncero, C., Valriberas-Herrero, I., Mezzatesta-Gava, M., Villegas, J. L., Aguilar, L., & Grau-López, L.
Reproductive Health, 17(1).*(2020).
doi:10.1186/s12978-020-0880-9*
Introduction: This study analyze factors associated to cannabis use in pregnant women, its perceived availability, its risk perception and the relationship between prenatal exposure to cannabis and developmental and mental disorders. Objectives: We present a review of the literature on cannabis use among pregnant women. The objective is to analyze factors associated to cannabis use during pregnancy and assess the potential effects of prenatal exposure to cannabis on the development of the fetus and the mental health of those exposed. Methods: Systematic review of studies on the maternal use of cannabis and the relationship between early exposure and the development of psychiatric disorders in the PubMed database until July 2018 in English and Spanish with the following keywords: Marijuana, Cannabinoids, Mental disorders, Pregnancy, Prenatal Cannabis Exposure, Risk factors. Results: The use of cannabis among pregnant women is frequent but it has not been extensively researched. Prenatal exposure to cannabis may be associated with affective symptoms and ADHD. Conclusions: Mental healthcare professionals who treat women during their fertile life need to be able to explain the relationship between prenatal exposure to cannabis and the presence of developmental and mental disorders.

Cannabis Use in People with Multiple Sclerosis and Spasticity: A Cross-Sectional Analysis.
Rice, J., Hugos, C., Hildebrand, A., & Cameron, M.
Multiple Sclerosis and Related Disorders, (2020).
doi:10.1016/j.msard.2020.102009
Background: Growing evidence supports that cannabinoids relieve MS-related spasticity but little is known about cannabis use among people with MS (PwMS) and spasticity.
Objective: To characterize cannabis use among PwMS and spasticity.
Methods: As part of baseline data collection for a spasticity intervention trial in Oregon, PwMS and self-reported spasticity answered questions about cannabis use.
Results: 54% reported ever using cannabis and 36% currently use. 79% use multiple routes of administration, 58% use at least daily. 79% find cannabis helpful for spasticity and 26% use cannabis and prescribed oral antispasticity medications.
Conclusions: Many PwMS and spasticity use cannabis and report it helps their spasticity.

Cannabis use in youth is associated with limited alterations in brain structure
J. Cobb Scott, Adon F. G. Rosen, Tyler M. Moore, David R. Roalf, Theodore D. Satterthwaite, Monica E. Calkins, Kosha Ruparel, Raquel E. Gur, and Ruben C. Gur
Neuropsychopharmacology (2019) 44:1362–1369; DOI: 10.1038/s41386-019-0347-2 https://www.ncbi.nlm.nih.gov/labs/pm...rticle_347.pdf Frequent cannabis use during adolescence has been associated with alterations in brain structure. However, studies have featured relatively inconsistent results, predominantly from small samples, and few studies have examined less frequent users to shed light on potential brain structure differences across levels of cannabis use. In this study, high-resolution T1-weighted MRIs were obtained from 781 youth aged 14–22 years who were studied as part of the Philadelphia Neurodevelopmental Cohort. This sample included 147 cannabis users (109 occasional [≤1–2 times per week] and 38 frequent [≥3 times per week] users) and 634 cannabis non-users. Several structural neuroimaging measures were examined in whole brain analyses, including gray and white matter volumes, cortical thickness, and gray matter density. Established procedures for stringent quality control were conducted, and two automated neuroimaging software processing packages were used to ensure robustness of results. There were no significant differences by cannabis group in global or regional brain volumes, cortical thickness, or gray matter density, and no significant group by age interactions were found. Follow-up analyses indicated that values of structural neuroimaging measures by cannabis group were similar across regions, and any differences among groups were likely of a small magnitude. In sum, structural brain metrics were largely similar among adolescent and young adult cannabis users and non-users. Our data converge with prior largescale studies suggesting small or limited associations between cannabis use and structural brain measures in youth. Detailed studies of vulnerability to structural brain alterations and longitudinal studies examining long-term risk are clearly indicated

Cannabis use is associated with reduced prevalence of non-alcoholic fatty liver disease: A cross-sectional study.
Adejumo, A. C., Alliu, S., Ajayi, T. O., Adejumo, K. L., Adegbala, O. M., Onyeakusi, N. E., … Bukong, T. N.
PLOS ONE, 12(4), e0176416.(2017).
doi:10.1371/journal.pone.0176416
Cannabis use is associated with reduced prevalence of obesity and diabetes mellitus (DM) in humans and mouse disease models. Obesity and DM are a well-established independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most prevalent liver disease globally. The effects of cannabis use on NAFLD prevalence in humans remains ill-defined. Our objective is to determine the relationship between cannabis use and the prevalence of NAFLD in humans. We conducted a population-based case-control study of 5,950,391 patients using the 2014 Healthcare Cost and Utilization Project (HCUP), Nationwide Inpatient Survey (NIS) discharge records of patients 18 years and older. After identifying patients with NAFLD (1% of all patients), we next identified three exposure groups: non-cannabis users (98.04%), non-dependent cannabis users (1.74%), and dependent cannabis users (0.22%). We adjusted for potential demographics and patient related confounders and used multivariate logistic regression (SAS 9.4) to determine the odds of developing NAFLD with respects to cannabis use. Our findings revealed that cannabis users (dependent and nondependent) showed significantly lower NAFLD prevalence compared to non-users (AOR: 0.82[0.76–0.88]; p

Cannabis Use, Lung Cancer, and Related Issues.
Jett, J., Stone, E., Warren, G., & Cummings, K. M.
Journal of Thoracic Oncology, 13(4), 480–487.(2018).
doi:10.1016/j.jtho.2017.12.013
The cannabis plant and its derivatives have been exploited for centuries for recreational and medicinal purposes with millions of regular users around the world. The recreational use of cannabis is reflective of its neuropsychiatric effects such as anxiolysis and euphoria. However, cannabis appears to have an emerging therapeutic role, especially in chronic disease and as an adjunct to cancer treatment. Increasing evidence supports cannabis in the management of chemotherapy induced nausea and vomiting and for pain management, but studies are limited particularly by difficulties associated with standardized dosing estimates and inability to accurately assess biologic activities of compounds in cannabis and derivative products. Smoking cannabis has not been proven to be a risk factor in the development of lung cancer but the data are limited by small studies, misclassification due to self-reporting of usage, small numbers of heavy cannabis smoking and confounding of risk associated with known causative agents for lung cancer such as parallel chronic tobacco use. Cannabis and its biologically effective derivatives warrant additional research, ideally controlled trials where the CBD and the THC strength and usage are controlled and documented.

Cannabis Use May Reduce Healthcare Utilization and Improve Hospital Outcomes in Patients with Cirrhosis.
Sobotka, L. A., Mumtaz, K., Hinton, A., Kelly, S. G., Conteh, L. F., Michaels, A. J., … Wellner, M. R.
Annals of Hepatology.(2020).
doi:10.1016/j.aohep.2020.10.008
Introduction and Objectives: Previous studies reveal conflicting data on the effect of cannabis use in patients with cirrhosis. This researchevaluates the impact of cannabis on hepatic decompensation, health care utilization, and mortality in patients with cirrhosis.
Material and Methods: A retrospective analysis of the State Inpatient Database (SID) was performed evaluating patients from Colorado and Washington in 2011 to represent pre-cannabis legalization and 2015 to represent post-cannabis legalization. Multivariable analysis was performed to study the impact of cannabis on the rate of admissions with hepatic decompensations, healthcare utilization, and mortality in patients with cirrhosis.
Results: Cannabis use was detected in 370 (2.1%) of 17,520 cirrhotics admitted in 2011 and in 1,162 (5.3%) of 21,917 cirrhotics in 2015 (pvalue
<0.001) On multivariable analysis, cirrhotics utilizing cannabis after its legalization experienced a decreased rate of admissions related to hepatorenal syndrome (Odds Ratio (OR): 0.51; 95% Confidence Interval (CI): 0.34-0.78) and ascites (OR: 0.73; 95% CI: 0.63-0.84). Cirrhotics with an etiology of disease other than alcohol and hepatitis C had a higher risk of
admission for hepatic encephalopathy if they utilized cannabis [OR: 1.57; 95% CI: 1.16-2.13]. Decreased length of stay (-1.15 days; 95% CI: -1.62, -0.68), total charges (-$15,852; 95% CI: -$21,009, -$10,694), and inpatient mortality (OR: 0.68; 95% CI: 0.51-0.91) were also observed in cirrhotics utilizing cannabis after legalization compared to cirrhotics not utilizing cannabis or utilizing cannabis prior to legalization.
Conclusion: Cannabis use in patients with cirrhosis resulted in mixed outcomes regarding hospital admissions with hepatic decompensation. A trend towards decreased hospital utilization and mortality was noted in cannabis users after legalization. These observations need to be confirmed with a longitudinal randomized study.

Cannabis use, pain interference, and prescription opioid receipt among persons with HIV: a target trial emulation study
William C. Becker, Yu Li, Ellen C. Caniglia, Rachel Vickers-Smith, Termeh Feinberg, Brandon D.L. Marshall & E. Jennifer Edelman
AIDS Care Psychological and Socio-medical Aspects of AIDS/HIV
https://www.tandfonline.com/doi/abs/...nalCode=caic20
DOI: 10.1080/09540121.2021.1944597
Concomitant with expanded legalization, cannabis is increasingly used to treat chronic pain among persons with HIV (PWH), despite equivocal benefit in research limited by small sample sizes and short duration of follow-up. To address these limitations, among a sample of PWH with pain interference enrolled in the Veterans Aging Cohort Study, we performed a target trial emulation study to compare the impact of four cannabis use strategies on pain interference. Among those receiving long-term opioid therapy (LTOT), we also explored impact of these strategies on ≥ 25% LTOT dose reduction. Among the analytic sample (N = 1284), the majority were men with a mean age of 50. Approximately 31% used cannabis and 12% received LTOT at baseline. Adjusting for demographic and clinical factors, cannabis use in any of 4 longitudinal patterns was not associated with resolved pain interference over 12- to 24-month follow-up. Among 153 participants receiving LTOT at baseline, cannabis use at both baseline and followup was negatively associated with LTOT dose reduction compared to no use at both baseline and follow-up. These findings support other observational studies finding no association between cannabis use and improved chronic pain or LTOT reduction among PWH.

Cannabis (marijuana): Psychoactive properties, Addiction, Therapeudic uses and toxicity
Sumanasekera WK and Spio KJ
Addict Behav Ther Rehabil 2016, 5:2
DOI: 10.4172/2324-9005.1000156
https://www.researchgate.net/publica...erties_Addicti on_Therapeutic_Uses_and_Toxici ty
Background and Objectives: To analyze the existing literature on cannabis (marijuana) under several sub topics; history of use, addiction, mechanism of action, therapeutic uses, and adverse effects.
Methods: A comprehensive literature search was conducted. Data sources include original research articles, systematic reviews and meta- analysis, and web-based references such as Drug strategy monographs and Merck Index. Data bases such as PubMed and Cinhal was used. References were from 1979 -2016.
Hypothesis: Based on the available literature it can be hypothesized that despite the addictive properties and adverse effects, marijuana has a therapeutic potential and should be used with caution.
Results: The available literature on cannabis smoke was discussed under five major categories, which are history of use, addiction, mechanism of action, therapeutic uses, and adverse effects.
Discussion and Conclusions: Marijuana (preparations of the hemp plant, cannabis sativa), is usually consumed as a smoke. It is an addictive compound that has been consumed for medical ailments for generations. The mechanism of action of cannabis was not known until the recent discovery of the endocannabinoid system.
Endocannabinoids are released from the post-synaptic neurons and act on the pre-synaptic neurons. Δ-9 tetrahydrocannabinol (THC), the psychoactive component present in Cannabis behaves similar to endocannabinoids. In addition to psychoactive modulations, cannabis consumption leads to cognitive impairments. Despite its’ medicinal and recreational value, due to its’ addictive properties cannabis consumption has been banned by several countries. In US, recreational and therapeutic use of cannabis is recently allowed in some states.
Scientific Significance: Despite the psych activity associated with cannabis consumption, it can be used therapeutically for many diseases including epilepsy and to alleviate chronic neuropathic pain associated with many sicknesses. Cannabis also possesses anti-cancer properties. However, strict laws should be enforced to avoid its’ abuse while allowing its’ medicinal use..

Cannabis use, subsequent other illicit drug use and drug use disorders: A 16-year follow-up study among Swedish adults.
Rabiee, R., Lundin, A., Agardh, E., Forsell, Y., Allebeck, P., & Danielsson, A.-K.
Addictive Behaviors, 106, (2020).
doi:10.1016/j.addbeh.2020.106390
Aims: To examine the association between cannabis use and subsequent other illicit drug use and drug use disorders (harmful use and dependence). Design, Setting, Participants: We used survey data from a population-based cohort in Stockholm County (collected 1998–2000), with linkage to the National Patient Register. The study base comprised participants aged 20–64 years (N = 10 345), followed-up until 2014. Cox and logistic regression analyses were conducted to test associations between self-reported cannabis use and risk of subsequent other illicit drug use (three-year followup) and drug use disorders (16-year follow-up). Findings: The odds ratio (OR) for other illicit drug use onset at three-year follow-up for lifetime cannabis users was 7.00 (4.47–10.35, 95% CI) and for recent cannabis users 34.41 (19.14–61.88, 95% CI). Adjusting for age and AUDIT score attenuated the association, for lifetime users: OR = 5.48 (3.69–8.13, 95% CI) and OR = 5.65 (3.80–8.41, 95% CI), and for recent users: OR = 18.32 (9.88–33.99, 95% CI) and OR = 20.88 (11.19–38.95, 95% CI). For cannabis users only, the hazard ratio (HR) for drug use disorders at 16-year follow-up was 0.89 (0.31–2.61, 95%CI). For cannabis and other illicit drug users, the corresponding HR was 7.27 (3.85–13.75, 95% CI). Conclusions: There was no independent association between cannabis use and subsequent drug use disorders. The association with subsequent drug use disorders was rather explained by other illicit drug use, which cannabis users were at higher risk of at the three-year follow-up.

Cannabis Withdrawal
Schlienz, N. J., & Vandrey
In book: Cannabis Use Disorders R. 93–102. (2018)
doi:10.1007/978-3-319-90365-1_11
Drug withdrawal refers to a constellation of symptoms that occur following abrupt cessation of chronic drug use. Though drug withdrawal can occur from stopping use of medication, it is most often encountered within the context of illicit, nonmedicinal, drug use. The withdrawal symptoms that emerge following extended and frequent use of abused drugs are a key feature of what define substance use disorders. Further, there is accumulating neurobiological evidence that withdrawal drives the maintenance of problematic substance misuse through a mechanism of reward dysfunction and negative reinforcement. Historically, there was debate and controversy regarding the existence of a valid and clinically meaningful cannabis withdrawal syndrome. However, extensive translational research has now firmly established that cannabis withdrawal occurs reliably in a subset of cannabis users, that it is pharmacologically specific to the use of cannabis, and that it is clinically meaningful within the contextof treating cannabis use disorder (CUD). As a result, mitigating cannabis withdrawal has been targeted in several studies aiming to develop improved treatments for CUD (discussed in detail in other chapters of this book). There are also individual characteristics, such as sex, genetics, and co-occurring psychiatric disorders that have been associated with differences in the type or severity of cannabis withdrawal. This chapter will provide a detailed overview of the etiology and characterization of cannabis withdrawal.
Cessation from daily cannabis use reliably elicits a valid withdrawal syndrome characterized by a constellation of behavioral, somatic, and mood symptoms. The onset of these symptoms closely follows cessation, mostly persists for 2–3 weeks, and can produce clinically significant distress that contributes to the maintenance of frequent cannabis use. Cannabis withdrawal develops as a result of neurobiological changes in the endogenous cannabinoid system that result from prolonged cannabis use and are pharmacologically specific to activity at the CB1 receptor. Individual differences in the expression of cannabis withdrawal are apparent, and several experiments have demonstrated robust sex differences in the frequency and severity of withdrawal. The presence of psychiatric comorbidity may complicate symptom intensity and time course. Cannabis withdrawal significantly influences treatment outcomes and is a prime target of continued efforts to develop effective pharmacological interventions for cannabis use disorder.

Cannmed conference 2019 presentations videos
https://cannmedevents.com/2019-video...NeHzlS3qJ33HpA

Video #2 Dr David Meiri Laboratory of Cancer biology & Cannabinoid Research, Technion, From the patient to the petri dish and back, a glimpse to the future of medical cannabis
There are many more interesting talks, 27 in all.

Cultivation Panel: Breeders Rights, Cannabis IP, Blockchains: Their role in Appellations and Provena

Cannabis Tissue Culture and Germplasm Storage

Microbial Inoculation of Clonally Propagated Cannabis Plant Starts: Effects on Harvest Quality and Secondary metabolites

Quantitative Chemomics: A novel genomic selection approach for precision breeding of high quality recreational and Medcinal Cannabis varieties

Effect of Container Size on Plant Growth and Development of Cannabis ruderalis in Controlled Greenhouse

Cardiac sudden death in a young cannabis user
Shogo Kasudaa Takeshi Kondob Ikuko Terazawaa Mami Morimotoa Katsuya Yuuia Risa Kudoa
Legal Medicine Volume 53, November 2021
DOI: 10.1016/j.legalmed.2021.101955
https://www.ncbi.nlm.nih.gov/pmc/art...1626-2-208.pdf
We report a case of sudden death due to acute coronary syndrome (ACS) in a young cannabis user. A man in his late thirties died at home, and marijuana was found. The autopsy revealed severe occlusion by an atherosclerotic plaque in the left anterior descending artery. The histopathological examination revealed ischemic changes, likely caused by cannabis-induced sympathetic β-adrenergic stimulation. Both cannabinoid receptors (CB1 and CB2) were expressed in the atherosclerotic lesions. The CB2 expression was higher than CB1 expression in the atherosclerotic plaque, corresponding to macrophage infiltration. Since cannabis is regarded as a casual drug due to its lower levels of dependency, some individuals have supported legalized marijuana use. However, this case report will provide cautions on the casual use of cannabis.
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Care and Feeding of the Endocannabinoid System: A Systematic Review of Potential Clinical Interventions that Upregulate the Endocannabinoid System
John M. McPartland1, Geoffrey W. Guy, Vincenzo Di Marzo
PLOS ONE | March 2014 | Volume 9 | Issue 3
doi: 10.1371/journal.pone.0089566
Background: The ‘‘classic’’ endocannabinoid (eCB) system includes the cannabinoid receptors CB1 and CB2, the eCB ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and their metabolic enzymes. An emerging literature documents the ‘‘eCB deficiency syndrome’’ as an etiology in migraine, fibromyalgia, irritable bowel syndrome, psychological disorders, and other conditions. We performed a systematic review of clinical interventions that enhance the eCB system—ways to upregulate cannabinoid receptors, increase ligand synthesis, or inhibit ligand degradation.
Methodology/Principal Findings: We searched PubMed for clinical trials, observational studies, and preclinical research. Data synthesis was qualitative. Exclusion criteria limited the results to 184 in vitro studies, 102 in vivo animal studies, and 36 human studies. Evidence indicates that several classes of pharmaceuticals upregulate the eCB system, including analgesics (acetaminophen, non-steroidal anti-inflammatory drugs, opioids, glucocorticoids), antidepressants, antipsychotics, anxiolytics, and anticonvulsants. Clinical interventions characterized as ‘‘complementary and alternative medicine’’ also upregulate the eCB system: massage and manipulation, acupuncture, dietary supplements, and herbal medicines. Lifestyle modification (diet, weight control, exercise, and the use of psychoactive substances—alcohol, tobacco, coffee, cannabis) also modulate the eCB system.
Conclusions/Significance: Few clinical trials have assessed interventions that upregulate the eCB system. Many preclinical
studies point to other potential approaches; human trials are needed to explore these promising interventions.

Case in Context: Tourette Syndrome
Kirsten Mu¨ller-Vahl
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0005
https://www.liebertpub.com/doi/pdf/1.../can.2021.0005
Definition and clinical course Tourette syndrome is a chronic neuropsychiatric disorder. Tics are the hallmark of the disease. Motor tics are involuntary movements that are most commonly located at the face and head such as eye blinking, grimacing, and head jerking. However, in more severely affected patients, motor tics may also involve other parts of the body and become more complex. In addition to motor tics, patients with Tourette syndrome suffer from one or more vocal tics, most often coughing, sniffling, or clearing one’s throat. With increasing severity of the disease, vocal tics may become louder and more complex. In *20–30% of patients, obscene vocal tics occur, called coprolalia. Although the age at onset is in early childhood around the age of 6–8 years, the maximum of tics is typically reached at age 10–12 years. Although tics often improve during the later course of the disease, complete recovery is rare. Depending on the number, frequency, intensity, location, and complexity of the tics, quality of life is significantly impaired. However, even mild tics may cause reduced self-esteem and result in social withdrawal, teasing, and bullying. About 80% of patients with Tourette syndrome suffer in addition from psychiatric comorbidities such as attention defi- cit/hyperactivity disorder, obsessive compulsive disorder, depression, anxiety, rage attacks, self-injurious behavior, and sleeping problems, which impacts quality of life in many cases as well.

CBD CANNABINOID AND TERPENE FORMULATIONS ELICIT DISTINCT MOOD EFFECTS
Jonathan R. Martin, Robert F. Roscow, Martin Enmark, Sean M. Conrad and Brian G. Reid
Ebbu poster at ICRS 2018
Find link or DOI
Conclusions
Two cannabinoid, terpene formulations each elicited subjectively distinct mood effects in participants.
Differences in mood effects were measured between differing cannabinoid, terpene formulations.
Results encourage formulating of cannabinoids and terpenes for eliciting specific effects in persons.
Purpose
To identify and measure potentially distinct mood effects that are elicited by vaping different cannabinoid, terpene formulations

CBD-enriched cannabis for autism spectrum disorder: an experience of a single center in Turkey and reviews of the literature
Serap Bilge, and Barış Ekic
Journal of Cannabis Research (2021) 3:53
DOI: 10.1186/s42238-021-00108-7
https://jcannabisresearch.biomedcentra l.com/articles/10.1186/s42238-021-00108-7
Introduction: Autism spectrum disorder is a neurodevelopmental disorder characterized by deficits in communication, social interaction, restricted interest, and repetitive behaviors. Although more cases are being diagnosed, no drugs are approved to treat the core symptoms or cognitive and behavioral problems associated with autism. Therefore, there is an urgent need to develop an effective and safe treatment.
Objective: In this study, we aim to share our 2-year experience with CBD-enriched cannabis treatment in autism and review the latest studies.
Materials and methods: The study included 33 (27 males, six females) children diagnosed with autism spectrum disorder who were followed up between January 2018 and August 2020. The mean age was 7.7 ± 5.5 years. The average daily dosage of cannabidiol (CBD) was 0.7 mg/kg/day (0.3–2 mg/kg/day). The median duration of treatment was 6.5 months (3–28 months). The preparations used in this study contained full-spectrum CBD and trace elements tetrahydrocannabinol (THC) of less than 3%.
Results: The outcomes were evaluated before and after treatment based on clinical interviews. At each follow-up visit, parents were asked to evaluate the effectiveness of the CBD-enriched cannabis treatment. According to the parents’ reports, no change in daily life activity was reported in 6 (19.35%) patients. The main improvements of the treatment were as follows: a decrease in behavioral problems was reported in 10 patients (32.2%), an increase in expressive language was reported in 7 patients (22.5%), improved cognition was reported in 4 patients (12,9%), an increase in social interaction was reported in 3 patients (9.6%), and a decrease in stereotypes was reported in 1 patient (3.2%). The parents reported improvement in cognition among patients who adhered to CBD-enriched cannabis treatment for over two years. The antipsychotic drug could be stopped only in one patient who showed mild ASD symptoms. No change could be made in other drug use and doses. Additionally, this study includes an extensive review of the literature regarding CBD treatment in autism spectrum disorder. According to recent studies, the average dose of CBD was 3.8±2.6 mg/kg/day. The ratio of CBD to THC in the used preparations was 20:1. The most significant improvements
were seen in the behavioral problems reported in 20–70% of the patients.
Conclusion: Using lower doses of CBD and trace THC seems to be promising in managing behavioral problems associated with autism. In addition, this treatment could be effective in managing the core symptoms and cognitive

CBD: How It Works
Martin A. Lee
O’Shaughnessy’s • Autumn 2011
https://beyondthc.com/wp-content/upl.../CBDiary21.pdf

CBG, CBD, Δ9-THC, CBN, CBGA, CBDA and Δ9-THCA as antioxidant agents and their intervention abilities in antioxidant action
Andrzej L.Dawidowicz , Małgorzata Olszowy-Tomczyk Rafał Typek
Fitoterapia 152 (2021)
DOI: 10.1016/j.fitote.2021.104915
https://www.sciencedirect.com/scienc...0d9f127aca88db
Positive effect of some cannabinoids in the treatment and prophylaxis of a wide variety of oxidation-associated diseases and growing popularity of supplements containing cannabinoids, mainly cannabinoid oils (e.g. CBD oil, CBG oil), in the self-medication of humans cause a growing interest in the antioxidant properties of these compounds, especially those not showing psychotropic effects. Herein, we report the antioxidant activity of cannabigerol (CBG), cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol (CBN), cannabigerolic acid (CBGA), cannabinolic acid (CBDA) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) estimated by spectrophotometric methods: ABTS, DPPH, ORAC, beta-carotene CUPRAC and FRAP. The presented data prove that all the examined cannabinoids exhibit antioxidant activity manifested in their ability to scavenge free radicals, to prevent the oxidation process and to reduce metal ions. Although the intensity of these activities is not the same for the individual cannabinoids it is comparable for all of them with that of E vitamin. As results from the research, the significance of the two types of electron sources presenting in examined cannabinoids, phenolic groups and double bonds transferring electrons, depends on the type of electronaccepting species - radicals/metal ions.

Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression
Fabrı´cio A. Moreira, Maximilian Grieb
Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 133–144
DOI: 10.1016/j.beem.2008.09.003
Both agonists (e.g. D9 -tetrahydrocannabinol, nabilone) and antagonists (e.g. rimonabant, taranabant) of the cannabinoid type-1 (CB1) receptor have been explored as therapeutic agents in diverse fields of medicine such as pain management and obesity with associated metabolic dysregulation, respectively. CB1 receptors are widely distributed in the central nervous system and are involved in the modulation of emotion, stress and habituation responses, behaviours that are thought to be dysregulated in human psychiatric disorders. Accordingly, CB1 receptor activation may, in some cases, precipitate episodes of psychosis and panic, while its inhibition may lead to behaviours reminiscent of depression and anxiety-related disorders. The present review discusses these sideeffects, which have to be taken into account in the therapeutic exploitation of the endocannabinoid system.

Challenges for Clinical Cannabis and Cannabinoid Research in the United States
Ziva D. Cooper, Donald I. Abrams, Steven Gust, Alejandro Salicrup, Douglas C. Throckmorton
J Natl Cancer Inst Monogr (2021) 2021(58): lgab009
doi: 10.1093/jncimonographs/lgab009
https://academic.oup.com/jncimono/ar...58/114/6446199
Significant changes have occurred in the policy landscape surrounding cannabis legalization, production, and use around the globe and across the United States. With widespread availability of novel cannabis and cannabis-based products, there is an urgent need to understand their safety and effectiveness for medical indications. Three primary barriers contribute to the dif- ficulty in initiating research geared toward answering the most pressing public health questions: the US regulatory status of cannabis and cannabinoids, sources for cannabis and cannabinoid study medications, and limited funding and resources to support studies. Despite these hurdles, research is rapidly increasing, and recent changes in the United States have paved the way for exciting new work. Here, challenges and barriers to cannabis and cannabinoid research are described from the perspectives of the National Institute on Drug Abuse, National Institutes of Health; the US Food and Drug Administration; and 2 clinical researchers. Barriers specifically to studying cannabis, cannabinoids, and cancer are emphasized.

Changes in California cannabis exposures following recreational legalization and the COVID-19 pandemic
Winter Roth, Mitchell Tam, Carrie Bi, June Kim, Justin Lewis, Raymond Ho & Dorie E. Apollonio
Clinical Toxicology 2021
https://www.tandfonline.com/doi/full...06212?needAcce ss=true
Doi: 10.1080/15563650.2021.2006212
Introduction
Since 2012, eighteen states and the District of Columbia have legalized recreational cannabis. Past research suggests this policy change is associated with increased cannabis exposures however this has not yet been studied in California, despite its status as the world’s largest legal cannabis market.
Methods
This observational, retrospective study analyzed trends in cannabis exposures reported to the California Poison Control System (CPCS) from 2010 to 2020. We assessed shifts in exposures before and after the legalization of recreational cannabis in November 2016, the establishment of recreational retail sales in January 2018, and the institution of a statewide shelter-in-place order due to the COVID-19 pandemic in March 2020 using interrupted time-series analysis and reviewed all records to identify specific products associated with exposures.
Results
Between 2010 and 2020 edible exposures increased from near zero to 79% of exposures in 2020. Cannabis exposures significantly increased following recreational legalization in 2016 (by an estimated 2.07 exposures per month [CI: 0.60, 3.55]; p < 0.01) and initiation of retail sales in 2018 (0.85 [CI: 0.12, 1.58]; p < 0.05). There was no significant change in cannabis exposures following the first shelter-in-place order of the COVID-19 pandemic (1.59 [CI: −1.61, 3.68]; p = 0.43). Cannabis exposures for those thirteen and under increased significantly both after recreational legalization (1.04 [CI: 0.38, 1.70]) and after the opening of the retail sales market (0.73 [CI: 0.34, 1.12]), but not following the shelter-in-place order (1.59 [CI: −1.61, 3.68]), nor was there a significant change for those older than thirteen.
Conclusions
Our findings suggest that cannabis legalization is linked to increased exposures, particularly for products such as gummies and candy edibles among children under the age of thirteen. Clinicians should be aware of these risks and communicate them to patients, and policymakers should consider stronger regulations on packaging to reduce these exposures.

Changes in Hepatic Phospholipid Metabolism in Rats under UV Irradiation and Topically Treated with Cannabidiol
Michał Biernacki , Anna Jastrząb , Elżbieta Skrzydlewska
Antioxidants (Basel). 2021 Jul 21;10(8):1157.
doi: 10.3390/antiox10081157
The liver is a key metabolic organ that is particularly sensitive to environmental factors, including UV radiation. As UV radiation induces oxidative stress and inflammation, natural compounds are under investigation as one method to counteract these consequences. The aim of this study was to assess the effect of topical application of phytocannabinoid-cannabidiol (CBD) on the skin of nude rats chronically irradiated with UVA/UVB, paying particular attention to its impact on the liver antioxidants and phospholipid metabolism. The results of this study indicate that CBD reaches the rat liver where it is then metabolized into decarbonylated cannabidiol, 7-hydroxy-cannabidiol and cannabidiol-glucuronide. CBD increased the levels of GSH and vitamin A after UVB radiation. Moreover, CBD prevents the increase of 4-hydroxynonenal and 8-iso-prostaglandin-F2α levels in UVA-irradiated rats. As a consequence of reductions in phospholipase A2 and cyclooxygenases activity following UV irradiation, CBD upregulates the level of 2-arachidonoylglycerol and downregulates prostaglandin E2 and leukotriene B4. Finally, CBD enhances decreased level of 15-deoxy-Δ-12,14-prostaglandin J2 after UVB radiation and 15-hydroxyeicosatetraenoic acid after UVA radiation. These data show that CBD applied to the skin prevents ROS- and enzyme-dependent phospholipid metabolism in the liver of UV-irradiated rats, suggesting that it may be used as an internal organ protector


CHARACTERISTICS OF MEDICAL CANNABIS USAGE AMONG PATIENTS WITH FIBROMYALGIA
Harefuah 159(5):343-348*May 2020
George Habib, Uriel Levinger
https://www.researchgate.net/publica...H_FIBROMYALGIA
Introduction: Medical cannabis (MC) is becoming more and more popular among patients with chronic pain syndromes. In this study we evaluated the characteristics of MC use among patients with fibromyalgia.
Methods: All patients with fibromyalgia who were followed up at Laniado Hospital in Netanya and at the Nazareth Hospital in Nazareth, in addition to all patients followed at the different health service organizations by the first author were located and contacted regarding a large number of parameters. The data included demographic information, duration of fibromyalgia symptoms and diagnosis, duration of MC use, monthly consumption of MC, frequency of daily use, number of species of MC currently used, number of species previously used, types and features of MC supplied, methods of MC consumption, delay in MC supply, symptoms of cannabis withdrawal during delay in supply or shortage of MC, familiarity with the content of (-)- trans-??-tetrahydrocannabinol (THC) and Cannabidiol (CBD) of the current species used. Furthermore, information was retrieved on current medications for fibromyalgia and medications for fibromyalgia stopped after starting MC consumption, discontinuation of MC treatment, companies growing and supplying the currently consumed MC, names of current MC species, dominance of Sativa or Indica of the current MC species, and the impact of MC on clinical parameters such as pain, sleep, anxiety, depression, memory, concentration and weight. In addition, questions were presented regarding work, outdoor leisure time, driving, sharing their own MC with other people, opinion on the reform of MC in Israel and adverse effects of MC.
Results: One-hundred and one patients completed the study; 73% of the participants were female with a mean age of 45±11.8 years. The mean duration of fibromyalgia symptoms and diagnosis were 8.39±6 and 4.69±3.9 years, respectively. The mean duration of MC consumption was 15.3±12.6 months and the mean monthly consumption amount was 28.6±10.2 g. 54% smoked pure cannabis, 18% used vaporized cannabis only and 3 participants only used MC oil. The rest used a variety of combinations. The mean minimal daily frequency of MC consumption was 4.11±2.9 times and the mean maximal daily frequency was 7.9±5.6 times. The mean number of current daily MC species was 2.11±1 and the mean number of tried species was 6.7±5.2 for each participant; 47% of all the participants stopped any other treatment for fibromyalgia and 51% reduced the dose or the number of other medications for fibromyalgia. One patient only stopped MC treatment. Tikun Olam was the manufacturing company with the largest number of clients and its most popular species for daytime was "Alaska" and "Erez" for night-time. Mean improvement in sleep and pain was slightly more than 77% with less improvement in other parameters; 36% of the patients reported weight gain, while 16% reported weight loss; 51% reported having more leisure time outdoors. Nearly all patients refused sharing any amount of their MC with friends or family members, and all patients recommended MC treatment for their loved ones once they develop severe fibromyalgia; 61% of the participants were against the reform of MC and 11% were in favor of it. Nearly one quarter of the patients reported mild adverse effects and one patient developed a psychotic attack (was consuming 70 gram of MC monthly).
Conclusions: MC is an effective treatment for fibromyalgia, with nearly zero % withdrawal from this treatment. The mean daily amount consumed was relatively low, less than 1 gram, and the main method of consumption was smoking with a huge variety in the frequency of smoking during the day and night among the participants. MC treatment enabled nearly half of the patients to discontinue any treatment for fibromyalgia and all participants recommended MC treatment for their loved ones in case they develop severe fibromyalgia. Most participants were against the reform of MC in Israel. Mild adverse effects were reported in nearly a quarter of the patients but did not result in discontinuing its consumption.

Characterization of bioactive compounds in defatted hempseed (Cannabis sativaL.) by UHPLC-HRMS/MS and anti-inflammatory activity in primary human monocytes.
Rea Martinez, J., Montserrat-de la Paz, S., De la Puerta, R., Garcia-Gimenez, M. D., & Fernandez-Anche, A.
Food & Function. (2020).
doi:10.1039/d0fo00066c
Hempseed (Cannabis sativa L.) has beneficial impact on human health mainly because of its wide variability of bioactive compounds. However, many of them are not fully characterized yet. In this work, hempseed was defatted and through a bio-guided studied, two fractions (F03 and F05) with the highest content of phenols, flavonoids and antioxidant capacity were selected. Fractions were chemically analyzed by UHPLC HRMS/MS. The anti-inflammatory capacities of these compounds were evaluated on human monocytes using flow cytometry, RT-qPCR and Elisa procedures. A high amount of phenolic compounds were identified, with the major compound being: N-trans-caffeoyltyramine (6.36 mg/g in F05 and 1.28 mg/g in F03). Both, F03 and F05 significantly reduced the inflammatory competence of LPS- treated human primary monocytes, decreasing TNF-? and IL-6 gene expression and secretion. These findings indicate that in the defatted fraction of the hempseed there are a wide number of compounds with beneficial potential to prevent and treat inflammatory disorders, as well as other processes caused by oxidative stress.

Characterization of Cannabidiol in Alternative Biological Specimens and Urine, After Consumption of an Oral Capsule.
Ameline, A., Raul, J.-S., & Kintz, P.
Journal of Analytical Toxicology. (2020).
doi:10.1093/jat/bkaa191
Among the hundred cannabinoids present in cannabis sativa indica, cannabidiol is a phytocannabinoid discovered in 1940, which can account for up to 40 % of the plant’s extract. Medically, it has been proposed to treat convulsions, inflammation, anxiety and nausea. Contrary to the hallucinogenic ingredient of the plant, delta-9-tetrahydrocannabinol, cannabidiol does not seem to have a sedative effect, which can increase its popularity among users. The identification of cannabidiol in blood and urine has been widely described in the scientific literature for several years. Only few data after cannabis use has been reported regarding cannabidiol identification in alternative specimens, such as oral fluid, sweat, exhaled breath and hair. Cannabidiol capsules were purchased in the United States, from a grocery store and a green capsule containing 22 mg of cannabidiol was orally administered to a 59-year-old healthy man. Oral fluid was collected over 8 hours using the NeoSal™ device. Sweat was collected with PharmCheck™ sweat patch technology
over 7 days. Exhaled breath was collected with the ExaBreath® DrugTrap device over 8 hours. Beard hair was collected 7 and 14 days after administration. Finally, urine specimens were collected over 48 hours in plastic tubes without preservative. Cannabidiol was only detected in oral fluid at 15 minutes, at 20 pg/mL. Increasing concentrations, up to 96 pg/patch of cannabidiol, were detected in the sweat patches. Cannabidiol was detected during 45 minutes in exhaled breath (Cmax 302 pg/filter at 30 minutes). Cannabidiol produced a very low but significant chromatographic signal in beard hair, with concentrations lower than 1 pg/mg. Finally, cannabidiol tested positive in urine after enzymatic hydrolysis with a Cmax at 70 ng/mL, after 6 hours.


Characterization of Cannabis sativa allergens.
Nayak, A. P., Green, B. J., Sussman, G., Berlin, N., Lata, H., Chandra, S., … Beezhold, D. H.
Annals of Allergy, Asthma & Immunology, 111(1), 32–37.e4. (2013).
doi:10.1016/j.anai.2013.04.018
Background: Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa. Objective: To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients. Methods: Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified. Results: Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity. Conclusion: Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species.

Characterization of the peripheral FAAH inhibitor, URB937, in animal models of acute and chronic migraine.
Greco, R., Demartini, C., Zanaboni, A., Casini, I., De Icco, R., Reggiani, A., … Tassorelli, C.
Neurobiology of Disease,*147, 105157. (2021). doi:10.1016/j.nbd.2020.105157
Inhibiting the activity of fatty-acid amide hydrolase (FAAH), the enzyme that deactivates the endocannabinoid anandamide, enhances anandamide-mediated signaling and holds promise as a molecular target for the treatment of human pathologies such as anxiety and pain. We have previously shown that the peripherally restricted
FAAH inhibitor, URB937, prevents nitroglycerin-induced hyperalgesia – an animal model of migraine – and attenuates the activation of brain areas that are relevant for migraine pain, e.g. trigeminal nucleus caudalis and locus coeruleus. The current study is aimed at profiling the behavioral and biochemical effects of URB937 in animal models of acute and chronic migraine. We evaluated the effects of URB937 in two rat models that capture aspects of acute and chronic migraine, and are based on single or repeated administration of the vasodilating
drug, nitroglycerin (NTG). In addition to nocifensive behavior, in trigeminal ganglia and medulla, we measured mRNA levels of neuropeptides and pro-inflammatory cytokines along with tissue levels of anandamide and palmitoylethanolamide (PEA), an endogenous agonist of peroxisome proliferator-activated receptor type-a (PPAR-a), which is also a FAAH substrate. In the acute migraine model, we also investigated the effect of subtype-selective antagonist for cannabinoid receptors 1 and 2 (AM251 and AM630, respectively) on nocifensive
behavior and on levels of neuropeptides and pro-inflammatory cytokines. In the acute migraine paradigm, URB937 significantly reduced hyperalgesia in the orofacial formalin test when administered either before or after NTG. This effect was accompanied by an increase in anandamide and PEA levels in target neural tissue, depended upon CB1 receptor activation, and was associated with a decrease in calcitonin gene-related peptide (CGRP), substance P and cytokines TNF-alpha and IL-6 mRNA. Similar effects were observed in the chronic migraine paradigm, where URB937 counteracted NTG-induced trigeminal hyperalgesia and prevented the increase in
neuropeptide and cytokine transcription. The results show that peripheral FAAH inhibition by URB937 effectively reduces both acute and chronic NTGinduced trigeminal hyperalgesia, likely via augmented anandamide-mediated CB1 receptor activation. These effects are associated with inhibition of neuropeptidergic and inflammatory pathways.

Chemical and biological studies of ? -carotene after exposure to Cannabis sativa smoke.
Do Monte, D. S., Bezerra Tenório, J. A., Bastos, I. V. G. A., de S. Mendonça, F., Neto, J. E., da Silva, T. G., & Ramos, C. S.
Toxicology Reports, 3, 516–522. (2016).
doi:10.1016/j.toxrep.2016.06.001
Considering the increase in consumption of Cannabis sativa and the use of the compound ?-carotene (BC) as supplement, we investigated potential changes in the chemical and biological proprieties of BC after exposure to C. sativa smoke (CSS). Our results showed that the BC exposed to CSS underwent 98.8% degradation and suffered loss ofits antiradical activity. The major degradation products identified were 3- hydroxy-2,4,4-trimethylpentyl)2-methylpropanoate and (2-ethyl-3-hydroxyhexyl)2-methylpropanoate compounds. These are found in higher levels in the exhalations of colorectal cancer patients and are similar to the toxic products associated with lipid peroxidation of polyunsaturated fatty acids. In toxicological assays using micro-crustacean Artemia salina the BC was non-toxic, while the BC degraded by CSS had a toxicity of LC50 = 397.35 g/mL. In Wistar rats, females treated with BC degraded by CSS (BCCSS) showed whitish liver spots, alterations in liver weight and in bilirubin and alkaline phosphatase levels, and decrease in the number of leukocytes associated with atypical lymphocytosis. In male rats, there was an increase in the number of leukocytes when compared to the control group. In the histopathological analysis, the cortical region of the kidneys showed the presence of discrete amorphous eosinophilic material (cylinders) in the lumen of the proximate and distal convoluted tubules. In general, the BC in contact with CSS undergoes chemical changes and exhibits toxicity to rats and Artemia salina.

Chemical profiles of cannabis sativa medicinal oil using different extraction and concentration methods
Cesar N. Pegoraro, Diego Nutter, Mario Thevenon & Cristina L. Ramirez
NATURAL PRODUCT RESEARCH 2019 Sep 12:1-4.
DOI: 10.1080/14786419.2019.1663515 (NOT Full article)
The present study shows the variability in chemical profiles of three different varieties of Cannabis sativa extracts used in medicinal cannabis oil when using two different extraction and evaporation methods. Procedures were compared by analyzing rate of total terpenoids to the principal cannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD), using gas chromatography coupled to mass spectrometry (GC-MS). The extraction of inflorescences using soxhlet method showed the highest mass of final extract. Also, the use of heating and the presence of oxygen when evaporating the solvents, greatly modified the final profiles due to evaporation or chemical reactions. These variations in chemical profiles must be carefully taken into account and standardized in the elaboration of medicinal oils.

Chemistry, Metabolism, and Toxicology of Cannabis: Clinical Implications
Priyamvada Sharma, Pratima Murthy, M.M. Srinivas Bharath
Iran J Psychiatry 2012; 7:4: 149-156
https://www.ncbi.nlm.nih.gov/pmc/art...IJPS-7-149.pdf
Cannabis is one of the most widely abused substances throughout the world. The primary psychoactive constituent of cannabis, delta 9- tetrahydrocannabinol (?9_THC), produces a myriad of pharmacological effects in animals and humans. Although it is used as a recreational drug, it can potentially lead to dependence and behavioral disturbances and its heavy use may increase the risk for psychotic disorders. Many studies that endeavor to understand the mechanism of action of cannabis concentrate on pharmacokinetics and pharmacodynamics of cannabinoids in humans. However, there is limited research on the chronic adverse effects and retention of cannabinoids in human subjects. Cannabis can be detected in body fluids following exposure through active/passive inhalation and exposure through breastfeeding. Cannabis detection is directly dependent on accurate analytical procedures for detection of metabolites and verification of recent use. In this review, an attempt has been made to summarize the properties of cannabis and its derivatives, and to discuss the implications of its use with emphasis on bioavailability, limit of detection, carry over period and passive inhalation, important factors for detection and diagnosis

Chinese Herbal Medicine (MaZiRenWan) Improves Bowel Movement in Functional Constipation Through Down-Regulating Oleamide
Tao Huang, Ling Zhao, Cheng-Yuan Lin, Lin Lu, Zi-Wan Ning, Dong-Dong Hu, Linda L. D. Zhong, Zhi-Jun Yang and Zhao-Xiang Bian
(2020) Chinese Herbal Medicine (MaZiRenWan)
Front. Pharmacol. 10:1570.
doi: 10.3389/fphar.2019.01570
In a prospective, randomized, three-arms, controlled clinical study, Chinese Herbal Medicine MaZiRenWan (MZRW, also known as Hemp Seed Pill) demonstrates comparable efficacy with Senna for functional constipation (FC) during an 8-week treatment period. Both MZRW and Senna are better than a placebo; relative to Senna and a placebo, MZRW displayed a more sustained effect during the 8-week follow-up period. The characteristic pharmacological mechanism responsible for this observation is still unclear. To explore this, we collected pre- and post-treatment serum samples of 85 FC patients from MZRW/Senna/placebo treatment groups for pharmacometabolomic analysis. An ultrahigh-performance liquid chromatography-mass spectrometer (UPLCMS) was used for metabolic profiling and quantification. In vivo studies were conducted in constipated C57BL/6J mice to verify the effects and corresponding mechanism(s) of the action of MZRW. Pearson correlation analysis, paired t-test, one-way ANOVA analysis, c2 test, and Student t-test were used to interpret the clinical and preclinical data. Changes in levels of circulating oleamide and its derivatives negatively correlate with improvement in complete spontaneous bowel movement (CSBM) in the MZRW group (Pearson r = -0.59, p = 0.00057). The same did not hold true for either Senna or placebo groups. Oleamide is a known regulator of intestinal motility. MZRW treatment resulted in reduced levels of circulating oleamide in FC patients. Experimental verification showed that MZRW attenuated oleamide-induced slow intestinal motility in mice. MZRW decreased oleamide levels in serum, ileum, and colon in normal mice, but increased expression of colonic fatty acid amide hydrolase (FAAH). In conclusion, MZRW improved bowel movement in FC by down-regulating oleamide, possibly by enhancing FAAH-mediated degradation. Our findings suggest a novel therapeutic strategy for FC.

Chronic Administration of Cannabidiol to Healthy Volunteers and Epileptic Patients
Cunha JM, Carlini EA, Pereira AE, Ramos OL, Pimentel C, Gagliardi R, Sanvito WL, Lander N, Mechoulam R.
Pharmacology · ;21(3):175-85. February 1980
DOI: 10.1159/000137430
In phase 1 of the study, 3 mg/kg daily of cannabidiol (CBD) was given for 30 days to 8 health human volunteers. Another 8 volunteers received the same number of identical capsules containing glucose as placebo in a double-blind setting. Neurological and physical examinations, blood and urine analysis, ECG and EEG were performed at weekly intervals. In phase 2 of the study, 15 patients suffering from secondary generalized epilepsy with temporal focus were randomly divided into two groups. Each patient received, in a double-blind procedure, 200-300 mg daily of CBD or placebo. The drugs were administered for along as 4 1/2 months. Clinical and laboratory examinations, EEG and ECG were performed at 15- or 30-day intervals. Throughout the experiment the patients continued to take the antiepileptic drugs prescribed before the experiment, although these drugs no longer controlled the signs of the disease. All patients and volunteers tolerated CBD very well and no signs of toxicity or serious side effects were detected on examination. 4 of the 8 CBD subjects remained almost free of convulsive crises throughout the experiment and 3 other patients demonstrated partial improvement in their clinical condition. CBD was ineffective in 1 patient. The clinical condition of 7 placebo patients remained unchanged whereas the condition of 1 patient clearly improved. The potential use of CBD as an antiepileptic drug and its possible potentiating effect on other antiepileptic drugs are discussed.

Clinical and Pre-Clinical Evidence for Functional Interactions of Cannabidiol and Δ9-Tetrahydrocannabinol.
Boggs, Douglas L; Nguyen, Jacques D; Morgenson, Daralyn; Taffe, Michael A; Ranganathan, Mohini
Neuropsychopharmacology, (), –. (2017).
doi:10.1038/npp.2017.209
The plant Cannabis sativa, commonly called cannabis or marijuana, has been used for its psychotropic and mind-altering side effects for millennia. There has been growing attention in recent years on its potential therapeutic efficacy as municipalities and legislative bodies in the United States, Canada, and other countries grapple with enacting policy to facilitate the use of cannabis or its constituents for medical purposes. There are over 550 chemical compounds and over 100 phytocannabinoids isolated from cannabis, including ∆ 9 -tetrahydrocannabinol (THC) and Cannabidiol (CBD). THC is thought to produce the main psychoactive effects of cannabis, while CBD does not appear to have similar effects. Studies conflict as to whether CBD attenuates or exacerbates the behavioral and cognitive effects of THC. This includes effects of CBD on THC induced anxiety, psychosis and cognitive deficits. In this article, we review the available evidence on the pharmacology and behavioral interactions of THC and CBD from pre-clinical and human studies particularly with reference to anxiety and psychosis like symptoms. Both THC and CBD, as well as other cannabinoid molecules, are currently being evaluated for medicinal purposes, separately and in combination. Future cannabisrelated policy decisions should include consideration of scientific findings including the individual and interactive effects of CBD and THC.

Clinical effects of a single dose of cannabinoids to patients with chronic lymphocytic leukemia
Christopher M. Melén, Magali Merrien, Agata M. Wasik, Georgios Panagiotidis, Olof Beck, Kristina Sonnevi, Henna-Riikka Junlén, Birger
Christensson, Birgitta Sander & Björn Engelbrekt Wahlin Leukemia & Lymphoma, 2022
DOI: 10.1080/10428194.2021.2020776
This phase II clinical trial investigates a one-time oromucosal dose of tetrahydrocannabinol/cannabidiol (THC/CBD) in 23 patients with indolent leukemic B cell lymphomas. Primary endpoint was a significant reduction in leukemic B cells. Grade 1_2 adverse events were seen in 91% of the patients; most common were dry mouth (78%), vertigo (70%), and somnolence (43%). After THC/CBD a significant reduction in leukemic B cells (median, 11%) occurred within two hours (p..014), and remained for 6 h without induction of apoptosis or proliferation. Normal B cells and T cells were also reduced. CXCR4 expression increased on leukemic cells and T cells. All effects were gone by 24 h. Our results show that a single dose of THC/CBD affects a wide variety of leukocytes and only transiently reduce malignant cells in blood. Based on this study, THC/ CBD shows no therapeutic potential for indolent B cell lymphomas (EudraCT trial no. 2014- 005553-39).

Clinical Outcome Data of First Cohort of Chronic Pain Patients Treated With Cannabis-Based Sublingual Oils in the United Kingdom: Analysis From the UK Medical Cannabis Registry
Michal Kawka, Simon Erridge, Carl Holvey, Azfer Usmani, Mohammad Sajad, Michael W. Platt, James J. Rucker, Mikael H. Sodergren,
The Journal of Clinical Pharmacology 2021, 61(12) 1545–1554
DOI: 10.1002/jcph.1961
https://accp1.onlinelibrary.wiley.co.. .1002/jcph.1961
Cannabis-based medicinal products (CBMPs) are an emerging therapeutic option in the management of primary chronic pain, using the role of the endocannabinoid system in modulating central and peripheral pain processes. Despite promising preclinical data, there is a paucity of high-quality evidence to support the use of CBMPs for chronic pain. This study aimed to investigate the health-related quality-of-life outcomes of patients with chronic pain who were prescribed CBMP oil preparations (Adven, Curaleaf International, Guernsey, UK). This study is a case series of patients from the UK Medical Cannabis Registry, who were treated with CBMP oils for an indication of chronic pain. The primary outcomes were the changes in Brief Pain Inventory short form, Short-Form McGill Pain Questionnaire-2, Visual Analog Scale Pain, General Anxiety Disorder-7, Sleep Quality Scale, and EQ-5D-5L, at 1, 3, and 6 months. One hundred ten patients were included. Significant improvements in Sleep Quality Scale, EQ-5D-5L pain and discomfort subscale, and Brief Pain Inventory Interference Subscale (P < .05) at 1, 3, and 6 months were demonstrated. There were no notable differences between cannabis-naïve and previous cannabis users in quality-of-life outcomes. The adverse event incidence was 30.0%, with most (n = 58; 92.1%) adverse events being mild or moderate in intensity. Treatment of chronic pain with Adven CBMP oils was associated with an improvement in pain-specific outcomes, health-related quality of life, and self-reported sleep quality. Relative safety was demonstrated over medium-term prescribed use. While these findings must be treated with caution considering the limitations of study design, they can inform future clinical trials.

Clinician Beliefs and Practices Related to Cannabis
Gillian L. Schauer, Rashid Njai, and Althea M. Grant
Cannabis and Cannabinoid Research 2021
DOI: 10.1089/can.2020.0165
https://www.liebertpub.com/doi/pdf/1.../can.2020.0165
Introduction: Medical cannabis (marijuana) use is legal in 33 U.S. states and the District of Columbia. Clinicians can play an important role in helping patients access and weigh potential benefits and risks of medicinal cannabis. Accordingly, this study aimed to assess clinician beliefs and practices related to cannabis.
Methods: Data are from 1506 family practice doctors, internists, nurse practitioners, and oncologists who responded to the 2018 DocStyles, a web-based panel survey of clinicians. Questions assessed medicinal uses for and practices related to cannabis and assessed clinicians' knowledge of cannabis legality in their state. Logistic regression was used to assess
multivariable correlates of asking about, assessing, and recommending cannabis.
Results: Over two-thirds (68.9%) of clinicians surveyed believe that cannabis has medicinal uses and just over a quarter (26.6%) had ever recommended cannabis to a patient. Clinicians who believed cannabis had medicinal uses had 5.9 times the adjusted odds (95% con dence interval 3.9–8.9) of recommending cannabis to patients. Beliefs about conditions for medical cannabis use did not necessarily align with the current scienti c evidence. Nearly two-thirds (60.0%) of clinicians surveyed incorrectly reported the legal status of cannabis in their state.
Discussion: Findings suggest that while clinicians believe that cannabis has medicinal uses, they may not have a full understanding of the scientific evidence and may not accurately understand their state-based policies for cannabis legalization and use. Given that clinicians are responsible for recommending medicinal cannabis in most states that have legalized it, ongoing education about the health effects of cannabis is warranted.

CMCR Center For Medicinal Cannabis Research
Check out the studies links current and inactive studies, and the Publications links, Scientific Publications, CMCR Reports, Other Reports, Presentations, CMCR Symposium for addition articles
https://www.cmcr.ucsd.edu/
Short-Term Effects of Cannabis Therapy on Spasticity in MS
Sleep and Medicinal Cannabis
Placebo-controlled, Double Blind Trial of Medicinal Cannabis in Painful HIV Neuropathy
Impact of Repeated Cannabis Treatments on Driving Abilities
Mechanisms of Cannabinoid Analgesia
Effects of Cannabis Therapy on Endogenous Cannabinoids
Effects of Medicinal Cannabis on CD4 immunity in AIDS
Analgesic Efficacy of Smoked Cannabis
Efficacy of Inhaled Cannabis in Diabetic Peripheral Neuropathy
A Double Blind, Active Placebo Controlled Crossover Trial of the Antinociceptive Effects of Smoked Marijuana on Subjects with Neuropathic Pain; Correlation with Changes in Mood, Cognition, and Psychomotor Performance
The Analgesic Effect of Vaporized Cannabis on Neuropathic Pain in Spinal Cord Injury
Vaporization as a "Smokeless" Cannabis Delivery System
Cannabis for Treatment of HIV-Related Peripheral Neuropathy

Collagen Hydrogels Loaded with Silver Nanoparticles and Cannabis Sativa Oil
Pablo Edmundo Antezana, Sofia Municoy, Claudio Javier Pérez and Martin Federico Desimon
Antibiotics 2021, 10, 1420.
DOI: 10.3390/ antibiotics10111420
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-01420.pdf
Wounds represent a major healthcare problem especially in hospital-associated infections where multi-drug resistant strains are often involved. Nowadays, biomaterials with therapeutic molecules play an active role in wound healing and infection prevention. In this work, the development of collagen hydrogels loaded with silver nanoparticles and Cannabis sativa oil extract is described. The presence of the silver nanoparticles gives interesting feature to the biomaterial such as improved mechanical properties or resistance to collagenase degradation but most important is the long-lasting antimicrobial effect. Cannabis sativa oil, which is known for its anti-inflammatory and analgesic effects, possesses antioxidant activity and successfully improved the biocompatibility and also enhances the antimicrobial activity of the nanocomposite. Altogether, these results suggest that this novel nanocomposite biomaterial is a promising alternative to common treatments of wound infections and wound healing.

Colon carcinogenesis is inhibited by the TRPM8 antagonist cannabigerol, a Cannabis-derived non-psychotropic cannabinoid.
Borrelli, F., Pagano, E., Romano, B., Panzera, S., Maiello, F., Coppola, D., … Izzo, A. A.
Carcinogenesis, 35(12), 2787–2797.(2014).
doi:10.1093/carcin/bgu205
Cannabigerol (CBG) is a safe non-psychotropic Cannabis-derived cannabinoid which interacts with specific targets involved in carcinogenesis. Specifically, CBG potently blocks transient receptor
potential (TRP) M8 (TRPM8), activates TRPA1, TRPV1 and TRPV2 channels, blocks 5-HT1A receptors and inhibits the reuptake of endocannabinoids. Here, we investigated whether CBG protects against colon tumorigenesis. Cell growth was evaluated in colorectal cancer cells using the MTT and NR assays; apoptosis was examined by histology and by assessing caspase 3/7 activity; ROS production by a fluorescent probe; cannabinoid (CB) receptors, TRP and CHOP mRNA expression were quantified by RT-PCR; shRNA-vector silencing of TRPM8 was performed by electroporation. The in vivo antineoplastic effect of CBG was assessed using mouse models of colon cancer. Colorectal cancer cells expressed TRPM8, CB 1, CB 2, 5HT1A receptors, TRPA1, TRPV1 and TRPV2 mRNA. CBG promoted apoptosis, stimulated ROS production, up-regulated CHOP mRNA and reduced cell growth in colorectal cancer cells. CBG effect on cell growth was independent from TRPA1, TRPV1 and TRPV2 channels activation, was further increased by a CB 2 receptor antagonist, and mimicked by other TRPM8 channel blockers but not by a 5-HT1A antagonist. Furthermore, the effect of CBG on cell growth and on CHOP mRNA expression was reduced in TRPM8 silenced cells. In vivo, CBG inhibited the growth of xenograft tumors as well as chemically-induced colon carcinogenesis. CBG hampers colon cancer progression in vivo and selectively inhibits the growth of colorectal cancer cells, an effect shared by other TRPM8 antagonists. CBG should be considered translationally in colorectal cancer prevention and cure.
Summary The non-psychotropic Cannabis ingredient cannabigerol – as well as other TRPM8 antagonists - inhibits the growth of colorectal cancer cells mainly via a pro-apoptotic mechanism and hinders the development and the growth of colon carcinogenesis in vivo.

Comparative assessment of antimicrobial, antiradical and cytotoxic activities of cannabidiol and its propyl analogue cannabidivarin
Chiara Russo, Margherita Lavorgna, Roberta Nugnes, Elena Orlo & Marina
Nature scientific reports (2021)
DOI:10.1038/s41598-021-01975-z
https://www.nature.com/articles/s41598-021-01975-z.pdf
Cannabidiol and cannabidivarin are phytocannabinoids produced by Cannabis indica and Cannabis sativa. Cannabidiol has been studied more extensively than its propyl analogue cannabidivarin. Therefore, we performed a battery of in vitro biological assays to compare the cytotoxic, antiradical and antibacterial activities of both cannabinoids. Potential mitochondrial metabolism alterations, DNA synthesis inhibition, and plasma membrane damage were studied by MTT assay, BrdU-ELISA and LDH assay of cancer and normal human cells exposed to cannabinoids. ABTS and DPPH assays were performed to observe the efects of the cannabinoids on free radicals. Microbial susceptibility tests were performed to study the activity of the cannabinoids in two bacterial species implicated in human infections, Escherichia coli and Staphylococcus aureus. The results showed that the cannabinoids induced medium levels of cytotoxicity in cancer and normal cells at concentrations ranging from 15.80 to 48.63 and from 31.89 to 151.70 µM, respectively, after 72 h of exposure. Cannabinoids did not exhibit a strong antioxidant capacity in scavenging ABTS or DPPH radicals. No evident diferences were observed between the two cannabinoids in antimicrobial activity, except with respect to S. aureus, which showed greater susceptibility to cannabidiol than to cannabidivarin after 72 h of exposure.

Comparison of Efficacy of Cannabinoids versus Commercial Oral Care Products in Reducing Bacterial Content from Dental Plaque: A Preliminary Observation
Stahl et al.
Cureus 12(1): e6809. 2020
DOI 10.7759/cureus.6809
Background Dental plaque is a complex biofilm that gets formed on the teeth and acts as a reservoir of different microbes. It is the root cause for the occurrence of several dental problems and diseases, including cavities, bad breath, bleeding gums, tooth decay, and tooth loss. Therefore, it should be regularly removed using suitable oral care aids. Objectives The present study compared the efficacy of oral care products and cannabinoids in reducing the bacterial content of dental plaques. Methods Sixty adults aged 18 to 45 years were categorized into six groups based on the Dutch periodontal screening index. Dental plaques of the adults were collected using paro-toothpick sticks and spread on two Petri dishes, each with four divisions. On Petri dish-A, cannabidiol (CBD), cannabichromene (CBC), cannabinol (CBN), and cannabigerol (CBG) were used, and on Petri dish-B, cannabigerolic acid (CBGA), Oral B, Colgate, and Cannabite F (a toothpaste formulation of pomegranate and algae) were used. The Petri dishes were sealed and incubated, followed by counting the number of colonies. Results By evaluating the colony count of the dental bacteria isolated from six groups, it was found that cannabinoids were more effective in reducing the bacterial colony count in dental plaques as compared to the well-established synthetic oral care products such as Oral B and Colgate. Conclusion Cannabinoids have the potential to be used as an effective antibacterial agent against dental plaque-associated bacteria. Moreover, it provides a safer alternative for synthetic antibiotics to reduce the development of drug resistance

Component of Cannabis, Cannabidiol, as a Possible Drug against the Cytotoxicity of A?(31–35) and A?(25–35) Peptides: An Investigation by Molecular Dynamics and Well-Tempered Metadynamics Simulations
Wojciech Chrobak, Dawid Wojciech Pacut, Fredrik Blomgren, Alexander Rodin, Jan Swenson, and Inna Ermilova
ACS Chem. Neurosci. 2021
DOI: 10.1021/acschemneuro.0c00692
In this work cannabidiol (CBD) was investigated as a possible drug against the cytotoxicity of A?(31–35) and A?(25–35) peptides with the help of atomistic molecular dynamics (MD) and well-tempered metadynamics simulations. Four interrelated mechanisms of possible actions of CBD are proposed from our computations. This implies that one mechanism can be a cause or/and a consequence of another. CBD is able to decrease the aggregation of peptides at certain concentrations of compounds in water. This particular action is more prominent for A?(25–35), since originally A?(31–35) did not exhibit aggregation properties in aqueous solutions. Interactions of CBD with the peptides affect secondary structures of the latter ones. Clusters of CBD are seen as possible adsorbents of A?(31–35) and A?(25–35) since peptides are tending to aggregate around them. And last but not least, CBD exhibits binding to MET35. All four mechanisms of actions can possibly inhibit the A?-cytotoxicity as discussed in this paper. Moreover, the amount of water also played a role in peptide clustering: with a growing concentration of peptides in water without a drug, the aggregation of both A?(31–35) and A?(25–35) increased. The number of hydrogen bonds between peptides and water was significantly higher for simulations with A?(25–35) at the higher concentration of peptides, while for A?(31–35) that difference was rather insignificant. The presence of CBD did not substantially affect the number of hydrogen bonds in the simulated systems.

Computational characterization and epitope prediction for Bet-v1 like protein of Cannabis sativa
Zarrin Basharat
PeerJ Preprints (2016)
DOI: 10.7287/peerj.preprints.2305v1
Cannabis sativa encodes a Bet-v1 like protein is an allergen and a causuative agent of pollen allergy. Multiple sequence alignment of this protein revealed conserved residues in Betv1 domain. Identification of linear epitopes of this protein was done after preliminary bioinformatics characterization and structure prediction. Structure prediction was done using Modeller software and minimized using Swiss PDBViewer. Six linear epitopes were then, predicted using EMBOSS antigenic program. Phylogenetic analysis of Bet-v1 with other sequences demonstrated divergence patterns with allergens of other species but revealed conserved residues in allergenic epitopes. This study can serve as an informational aid in the development of hypoallergenic vaccine for Cannabis sativa allergy.

CONCEPTUAL REVIEW ON VIJAYA (CANNABIS SATIVA LINN.): A FORGOTTEN AMBROSIA
G.Siva Ram, C.Murali Krishna, D.Panchajanya Kumar, K.Midhuna Mohan, G. Babu
Int. J. Res. Ayurveda Pharm. 9 (2), 2018
DOI: 10.7897/2277-4343.09228
Vijaya (Cannabis sativa Linn.) is associated with spiritual and medicinal aspect of Indian cultural heritage. Currently it is a controlled substance placed under Schedule-1 in the United Nations Convention on Psychotropic Substances due to its abusive & narcotic nature. Ayurveda, the science of life has details regarding Vijaya identification, cultivation techniques, collection method, purification process, formulations, indications, antidotes, specific dietetics, etc. According to Ayurveda Vijaya, an analeptic herb is originated from amrita (ambrosia) & having the properties of Visha (poison) requires Sodhana (purification) to be utilized as a medicament. Purification helps to detoxify the unwanted qualities & also improves the therapeutic value. Approximately fifty diseases have been cited in Ayurvedic classics which can be treated with single or compound preparations of Vijaya. Bioavailability & efficacy is very quick as it is an ushna virya (hot potency) herb having Tikshna (sharp), Vyavayi (bioavailability even before digestion) & Yogavahi (synergistic action) properties. In this twenty first century research in the therapeutic aspects of Cannabis sativa lead to an increase in the awareness & knowledge of the ‘medical Cannabis’ among the scientific community. Revalidation of the medicinal evidence of Vijaya present in the ancient Ayurvedic literature provides scope for more refined research.

Consumer Experiences with Delta-8-THC: Medical Use, Pharmaceutical Substitution, and Comparisons with Delta-9-THC
Daniel J. Kruger, Jessica S. Kruger
Cannabis and Cannabinoid Research Nov 2021
DOI: 10.1089/can.2021.0124
Introduction: Cannabis products containing delta-8-THC became widely available in most of the United States in late 2020 and rapidly became a significant source of revenue for hemp processing companies, especially in states where use of delta-9-THC remains illegal or requires professional authorization for medical use. Scientific research on the use of delta-8-THC is scarce, previous clinical studies included a combined total of 14 participants, leading some state governments to prohibit it until its properties and effects are better understood.
Methods: Researchers developed an online survey for delta-8-THC consumers addressing a broad range of issues regarding delta-8-THC, including use for the treatment of health and medical conditions. Previous survey studies on the medical use of cannabis and cannabis products informed survey components.
Results: Patterns of delta-8-THC use had both similarities with and differences from the use of delta-9-THC cannabis and products. Administration methods were primarily edibles (64%) and vaping concentrates (48%). About half of the participants (51%) used delta-8-THC to treat a range of health and medical conditions, primarily anxiety or panic attacks (69%), stress (52%), depression or bipolar disorder (46%), and chronic pain (41%). Participants compared delta-8-THC very favorably with both delta-9-THC and pharmaceutical drugs and reported substantial levels of substitution for both. Most participants did not inform their primary care provider of their delta-8-THC use (78%) and were not confident of their primary care provider's ability to integrate medical cannabis into their treatment (70%). Knowledge of effective dosages was low, and participants' knowledge of delta-8-THC was primarily from the Internet and their own experiences.
Conclusion: Harm reduction is a central component of public health. Although the legal environment is becoming more restrictive for delta-8-THC in comparison to delta-9-THC, results suggest that delta-8-THC may be equally effective for desired purposes of cannabis use and lower in undesirable or adverse effects. All policies and practices should be informed by empirical evidence. Considerable research will be needed to systematically verify the patterns reported by participants, and collaborations among academic researchers, government, and the cannabis industry may be valuable in developing the knowledge base for delta-8-THC and other cannabinoids.
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Contaminants of Concern in Cannabis: Microbes, Heavy Metals and Pesticides
May 2017
In book: Cannabis sativa L. - Botany and Biotechnology
John McPartland, Kevin McKernan
DOI: 10.1007/978-3-319-54564-6_22
Microbiological contaminants pose a potential threat to cannabis consumers. Bacteria and fungi may cause opportunistic infections in immunocompromized individuals. Even dead organisms may trigger allergies and asthma. Toxins from microbial overloads, such as Shigla toxin and aflatoxins, may pose a problem—unlikely, but possible. The Cannabis plant hosts a robust microbiome; the identification of these organisms is underway. Cannabis bioaccumulates heavy metals in its tissues, so avidly that hemp crops have been used for bioremediation. Heavy metals cause myriad human diseases, so their presence in crops destined for human consumption must be minimized. Pesticide residues in cannabis pose a unique situation among crop plants—the Environmental Protection Agency (EPA) will not propose pesticides guidelines, because Cannabis is illegal on the federal level. The use of illegal pesticides is a rising crisis, and a breakdown in ethics. Testing for pesticide residues and maximal limits are proposed.

Conversion of cannabidiol to ?9-tetrahydrocannabinol and related cannabinoids in artificial gastric juice, and their pharmacological effects in mice
Kazuhito Watanabe · Yuka Itokawa · Satoshi Yamaori · Tatsuya Funahashi · Toshiyuki Kimura · Toshiyuki Kaji · Noriyuki Usami · Ikuo Yamamoto
Forensic Toxicol (2007) 25:16–21
DOI 10.1007/s11419-007-0021-y
Cannabidiol (CBD), a nonpsychoactive cannabinoid, was found to be converted to 9? –hydroxyhexahydrocannabinol (9? -OH-HHC) and 8-hydroxyiso - hexahydrocannabinol (8-OH-iso -HHC) together with ?9 -tetrahydrocannabinol (?9 -THC), a psychoactive cannabinoid, and cannabinol in artificial gastric juice. These cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS) by comparison with the spectral data of the authentic compounds. Pharmacological effects of 9? -OH-HHC and 8-OH-iso -HHC in mice were examined using catalepsy, hypothermia, pentobarbital- induced sleep prolongation, and antinociception against acetic acid-induced writhing as indices. The ED50 values (effective dose producing a 50% reduction of control; mg/kg, i.v.) of 9? -OH-HHC and 8-OH-iso -
HHC for the cataleptogenic effect were 8.0 and 30.4, respectively. 8-OH-iso -HHC (10 mg/kg, i.v.) produced a significant hypothermia from 15 to 90 min after administration, although 9? -OH-HHC failed to induce such an effect at the same dose. However, both HHCs (10 mg/ kg, i.v.) significantly prolonged pentobarbital-induced sleeping time by 1.8 to 8.0 times as compared with the control solution with 1% Tween 80-saline. The ED50 values (mg/kg, i.v.) of 9? -OH-HHC and 8-OH-iso –HHC for the antinociceptive effect were 14.1 and 39.4, respectively. The present study demonstrated that CBD can be converted to ?9 -THC and its related cannabinoids, 9? - OH-HHC and 8-OH-iso -HHC, in artifi cial gastric juice, and that these HHCs show ?9 -THC-like effects in mice, although their pharmacological effects were less potent than those of ?9 -THC.

Correlation of Breath and Blood ?9-Tetrahydrocannabinol Concentrations and Release Kinetics Following Controlled Administration of Smoked Cannabis.
Lynch, K. L., Luo, Y. R., Hooshfar, S., & Yun, C.
Clinical Chemistry, clinchem.2019.304501. (2019).
doi:10.1373/clinchem.2019.304501
BACKGROUND: Cannabis use results in impaired driving and an increased risk of motor vehicle crashes. Cannabinoid concentrations in blood and other matrices can remain high long after use, prohibiting the differentiation between acute and chronic exposure. Exhaled breath has been proposed as an alternative matrix in which concentrations may more closely correspond to the window of impairment; however, efficient capture and analytically sensitive detection methods are required for measurement.
METHODS: Timed blood and breath samples were collected from 20 volunteers before and after controlled administration of smoked cannabis. Cannabinoid concentrations were measured using LC-MS/MS to determine release kinetics and correlation between the 2 matrices.
RESULTS: 9-Tetrahydrocannabinol (THC) was detected in exhaled breath for all individuals at baseline through 3 h after cannabis use. THC concentrations in breath were highest at the 15-min timepoint (median 17.8 pg/L) and declined to 5% of this concentration in all participants 3 h after smoking. The decay curve kinetics observed for blood and breath were highly correlated within individuals and across the population.
CONCLUSIONS: THC can be reliably detected throughout the presumed 3-h impairment window following controlled administration of smoked cannabis. The findings support breath THC concentrations as representing a physiological process and are correlated to blood concentrations, albeit with a shorter window of detection.

Craving and emotional responses to trauma and cannabis cues in trauma-exposed cannabis users: Influence of PTSD symptom severity
Pablo Romero-Sanchiz , Ioan T Mahu , Sean P Barrett, Joshua P Salmon, Mohammed Al-Hamdani, Jennifer E Swansburg, Sherry H Stewart
Addict Behav . 2022 Feb;125:107126.
doi: 10.1016/j.addbeh.2021.107126
Conditioned craving to trauma cues and avoidance learning have both been implicated in the high concurrence of trauma-related distress and substance misuse. Using a cue-exposure paradigm involving personalized trauma, cannabis, and neutral cues, we examined if conditioned craving and/or elevated negative affect to trauma cues are mechanisms linking PTSD and cannabis use disorder. Fifty-one trauma-exposed cannabis users were randomly presented the three cue types. Craving and emotional responses were evaluated after each cue using the Marijuana Craving Questionnaire-Short Form (Heishman et al., 2001) and the Positive and Negative Affect Schedule (Watson et al., 1988). Relief cannabis craving (compulsivity and emotionality) was significantly higher after trauma than cannabis and neutral cues (p's < 0.001) and was also higher among those with more severe PTSD symptoms (p's < 0.05). The relationship between PTSD symptom severity and cannabis craving was stronger after trauma than cannabis cues for the compulsivity component of craving (p < .05). Relief craving was also higher after the cannabis cue than after the neutral cue (expectancy and purposefulness; p < .001). Negative affect was significantly higher: after trauma than cannabis and neutral cues (p's < 0.001); and among those with more severe PTSD symptoms (p < . 005). Positive affect was significantly lower after trauma than cannabis cues (p < .05). Trauma cue exposure might promote cannabis misuse through conditioned craving as well as the desire to relieve negative affect. Conditioned cannabis craving involving an uncontrollable compulsion to use cannabis in response to trauma reminders appears particularly likely among cannabis users with more severe PTSD symptoms.
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Cue-induced craving for marijuana in cannabis-dependent adults.
Lundahl, L. H., & Johanson, C.-E.
Experimental and Clinical Psychopharmacology, 19(3), 224–230. (2011).
doi:10.1037/a0023030
Recent interest in the development of medications for treatment of cannabis-use disorders indicates the need for laboratory models to evaluate potential compounds prior to undertaking clinical trials. To investigate whether a cue-reactivity paradigm could induce marijuana craving in cannabis-dependent adults, 16 (eight female) cannabis-dependent and 16 (eight female) cannabis-na?¨ve participants were exposed to neutral and marijuana-related cues, and subsequent changes in mood, self-reported craving, and physiologic function were assessed. Significant Group X cue interactions were found on all three VAS craving indices as well as on the Compulsivity scale of the Marijuana Craving Questionnaire-Brief Form (MCQ-BF). Cannabis-dependent individuals responded to marijuana-related cues with significantly increased reports of marijuana craving compared to neutral cue exposure, although there were no cue-induced changes in any of the physiological measures. There were no significant gender differences on any of the measures. These results indicate that marijuana craving can be induced and assessed in cannabis-dependent, healthy adults within a laboratory setting, and support the need for further research of the cue reactivity paradigm in the development of medications to treat cannabis-use disorders

Current application of cannabidiol (CBD) in the management and treatment of neurological disorders.
Fiani, B., Sarhadi, K. J., Soula, M., Zafar, A., & Quadri, S. A.
Neurological Sciences. (2020).
doi:10.1007/s10072-020-04514-2
Cannabidiol (CBD), which is nonintoxicating pharmacologically relevant constituents of Cannabis, demonstrates several beneficial effects. It has been found to have antioxidative, anti-inflammatory, and neuroprotective effects. As the medicinal use of CBD is gaining popularity for treatment of various disorders, the recent flare-up of largely unproven and unregulated cannabis based preparations on medical therapeutics may have its greatest impact in the field of neurology. Currently, as lot of clinical trials are underway, CBD demonstrates remarkable potential to become a supplemental therapy in various neurological conditions. It has shown promise in the treatment of neurological disorders such as anxiety, chronic pain, trigeminal neuralgia, epilepsy, and essential tremors as well as psychiatric disorders. While recent FDA-approved prescription drugs have demonstrated safety, efficacy, and consistency enough for regulatory approval in spasticity in multiple sclerosis (MS) and in Dravet and Lennox-Gastaut Syndromes (LGS), many therapeutic challenges still remain. In the current review, the authors have shed light on the application of CBD in the management and treatment of various neurological disorders.

Current Status and Prospects for Cannabidiol Preparations as New Therapeutic Agents
Fasinu PS, Phillips S, ElSohly MA, Walker LA
Pharmacotherapy
DOI: 10.1002/phar.1780
There is growing pressure for states and the federal government to legalize the use of cannabis products for medical purposes in the United States. Sixteen states have legalized (or possession of) products high in cannabidiol (CBD) and with restricted ?9 -tetrahydrocannabinol (?9 -THC) content. In most of these states, the intent is for use in refractory epileptic seizures in children, but in a few states, the indications are broader. The objectives of this review are to provide an overview of the pharmacology and toxicology of CBD; to summarize some of the regulatory, safety, and cultural issues relevant to the further exploitation of its antiepileptic or other pharmacologic activities; and to assess the current status and prospects for clinical development of CBD and CBD-rich preparations for medical use in the United States. Unlike ?9 -THC, CBD elicits its pharmacologic effects without exerting any significant intrinsic activity on the cannabinoid receptors (CB1 and CB2 ), whose activation results in the psychotropic effects characteristic of ?9 -THC, and CBD possesses several pharmacologic activities that give it a high potential for therapeutic use. CBD exhibits neuroprotective, antiepileptic, anxiolytic, antipsychotic, and antiinflammatory properties. In combination with ?9 -THC, CBD has received regulatory approvals in several European countries and is currently under study in U.S. Food and Drug Administration-registered trials in the United States. A number of states have passed legislation to allow for the use of CBD-rich, limited ?9 -THC-content preparations of cannabis for certain pathologic conditions. CBD is currently being studied in several clinical trials and is at different stages of clinical development for various medical indications. Judging from clinical findings reported so far, CBD and CBD-enriched preparations have great potential utility, but uncertainties regarding sourcing, long-term safety, abuse potential, and regulatory dilemmas remain.

CYP2C-catalyzed delta(9)-tetrahydrocannabinol metabolism: Kinetics, pharmacogenetics and interaction with phenytoin
Tina M. Bland , Robert L. Haining , Timothy S. Tracy , Patrick S. Callery
Biochemical Pharmacology 70 (2005) 1096–1103
doi:10.1016/j.bcp.2005.07.007
delta(9)-Tetrahydrocannabinol (D9-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-D9-THC, which is oxidized further to 11-nor-9-carboxy-D9-THC. The hydroxylation is thought to be mediated by CYP2C9. The present study was designed to address the kinetics and pharmacogenetics of CYP2C-mediated metabolism of (D9)-THC by studying its metabolism in human liver microsomes and expressed enzymes. Expressed CYP2C9.1 exhibited high affinity for the hydroxylation of D9-THC (apparent Km, 2 mM), similar to that observed in human liver microsomes (apparent Km, 0.8 mM). In contrast, the calculated intrinsic clearance (apparent Vm/Km) for CYP2C9.2 and CYP2C9.3 was approximately 30% that of the wild type, CYP2C9.1. Given the high affinity of CYP2C9 for the hydroxylation of D9-THC, we evaluated the potential for an interaction between D9- THC, 11-hydroxy-D9-THC, or 11-nor-9-carboxy-D9-THC and the CYP2C9 substrate, phenytoin. Surprisingly, D9-THC increased the rate of phenytoin hydroxylation in human liver microsomes and expressed CYP2C9 enzyme. Similar increases in rate were observed with coincubation of 11-hydroxy-D9-THC and 11-nor-9-carboxy-D9-THC with phenytoin. These in vitro data suggest the potential for an interaction from the concomitant administration of D9-THC and phenytoin that could result in decreased phenytoin concentrations in vivo.

Daily cannabis use is associated with lower CNS inflammation in people with HIV.
C. Wei-Ming Watson, Laura M. Campbell, Ni Sun-Suslow, Suzi Hong, Anya Umlauf, Ronald J. Ellis, Jenny Iudicello, Scott Letendre, Thomas D. Marcotte, Robert K. Heaton, Erin E. Morgan, Igor Grant
J Int Neuropsychol Soc. 2021 July ; 27(6): 661–672.
https://www.ncbi.nlm.nih.gov/labs/pm...ms-1659467.pdf
doi:10.1017/S1355617720001447.
Objective: Recent cannabis exposure has been associated with lower rates of neurocognitive impairment in people with HIV (PWH). Cannabis’ anti-inflammatory properties may underlie this relationship by reducing chronic neuroinflammation in PWH. This study examined relations between cannabis use and inflammatory biomarkers in cerebrospinal fluid (CSF) and plasma, and cognitive correlates of these biomarkers within a community-based sample of PWH. Methods: 263 individuals were categorized into four groups: HIV− non-cannabis users (n = 65), HIV+ non-cannabis users (n = 105), HIV+ moderate cannabis users (n = 62), and HIV+ daily cannabis users (n = 31). Differences in pro-inflammatory biomarkers (IL-6, MCP-1/CCL2, IP-10/ CXCL10, sCD14, sTNFR-II, TNF-α) by study group were determined by Kruskal-Wallis tests. Multivariable linear regressions examined relationships between biomarkers and seven cognitive domains, adjusting for age, sex/gender, race/ethnicity, education, current CD4 count, and current tobacco use.
Results: HIV+ daily cannabis users showed lower MCP-1 and IP-10 levels in CSF compared to HIV+ non-cannabis users (p = 0.015; p = 0.039), and were similar to HIV− non-cannabis users. Plasma biomarkers showed no differences by cannabis use. Among PWH, lower CSF MCP-1 and lower CSF IP-10 were associated with better learning performance (p = 0.016; p = 0.036).
Conclusions: Current daily cannabis use was associated with lower levels of pro-inflammatory chemokines implicated in HIV pathogenesis and these chemokines were linked to the cognitive domain of learning which is commonly impaired in PWH. Cannabinoid-related reductions of MCP-1 and IP-10, if confirmed, suggest a role for medicinal cannabis in the mitigation of persistent inflammation and cognitive impacts of HIV


Daily use of high-potency cannabis is associated with more positive symptoms in first-episode psychosis patients: the EU-GEI case–control study.
Quattrone, D., Ferraro, L., Tripoli, G., La Cascia, C., Quigley, H., … Quattrone, A.
Psychological Medicine, 1–9. (2020).
doi:10.1017/s0033291720000082
Background. Daily use of high-potency cannabis has been reported to carry a high risk for developing a psychotic disorder. However, the evidence is mixed on whether any pattern of cannabis use is associated with a particular symptomatology in firstepisode psychosis (FEP) patients. Method. We analysed data from 901 FEP patients and 1235 controls recruited across six countries, as part of the European Network of National Schizophrenia Networks Studying GeneEnvironment Interactions (EU-GEI) study. We used item response modelling to estimate two bifactor models, which included general and specific dimensions of psychotic symptoms in patients and psychotic experiences in controls. The associations between these dimensions and cannabis use were evaluated using linear mixed-effects models analyses. Results. In patients, there was a linear relationship between the positive symptom dimension and the extent of lifetime exposure to cannabis, with daily users of high-potency cannabis having the highest score (B = 0.35; 95% CI 0.14–0.56). Moreover, negative symptoms were more common among patients who never used cannabis compared with those with any pattern of use (B = ?0.22; 95% CI ?0.37 to ?0.07). In controls, psychotic experiences were associated with current use of cannabis but not with the extent of lifetime use. Neither patients nor controls presented differences in depressive dimension related to cannabis use. Conclusions. Our findings provide the first large-scale evidence that FEP patients with a history of daily use of high-potency cannabis present with more positive and less negative symptoms, compared with those who never used cannabis or used lowpotency types.

Decreased sensitivity in adolescent versus adult rats to the antidepressant-like effects of cannabidiol.
Bis-Humbert, C., García-Cabrerizo, R., & García-Fuster, M. J.
Psychopharmacology. (2020).
doi:10.1007/s00213-020-05481-4
Rationale Cannabidiol is a non-psychoactive phytocannabinoid with great therapeutic potential in diverse psychiatric disorders; however, its antidepressant potential has been mainly ascertained in adult rats. Objectives To compare the antidepressant-like response induced by cannabidiol in adolescent and adult rats and the possible parallel modulation of hippocampal neurogenesis. Methods Male Sprague-Dawley rats were repeatedly treated with cannabidiol (3, 10, and 30 mg/kg) or vehicle (1 mL/kg) during adolescence (postnatal days, PND 27-33) or adulthood (PND 141-147) and exposed to 3 consecutive tests (forced swim, open field, two-bottle choice) that quantified behavioral despair, anxiety, and sucrose intake respectively. Results Cannabidiol induced differential effects depending on the age and dose administered, with a decreased sensitivity observed in adolescent rats: (1) cannabidiol (30 mg/kg) decreased body weight only in adult rats; (2) cannabidiol ameliorated behavioral despair in adolescent and adult rats, but with a different dose sensitivity (10 vs. 30 mg/kg), and with a different extent (2 vs. 21 days post-treatment); (3) cannabidiol did not modulate anxiety-like behavior at any dose tested in adolescent or adult rats; and (4) cannabidiol increased sucrose intake in adult rats. Conclusions Our findings support the notion that cannabidiol exerts antidepressant- and anorexigenic-like effects in adult rats and demonstrate a decreased potential when administered in adolescent rats. Moreover, since cannabidiol did not modulate hippocampal neurogenesis (cell proliferation and early neuronal survival) in adolescent or adult rats, the results revealed potential antidepressant-like effects induced by cannabidiol without the need of regulating hippocampal neurogenesis.

?9?TETRAHYDROCANNABINOLIC ACID ALLEVIATES COLLAGEN?INDUCED ARTHRITIS: ROLE OF PPAR? AND CB 1 RECEPTORS.
Palomares, B., Garrido?Rodriguez, M., Gonzalo?Consuegra, C., Gómez?Cañas, M., Saen?oon, S., Soliva, R., … Muñoz, E.
British Journal of Pharmacology. (2020).
doi:10.1111/bph.15155
Background and Purpose: ? 9 -THCA-A, the precursor of ? 9 -THC, is a non-psychotropic phytocannabinoid that shows PPAR? agonistic activity. Herein, we investigated ? 9 -THCA ability to modulate classic cannabinoid receptors (CB1 and CB2) and evaluated its anti-arthritis activity. Experimental Approach: Cannabinoid receptors binding and intrinsic activity, as well as their downstream signaling were analyzed in vitro and in silico. The anti-arthritis properties of ? 9 - THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasmatic disease biomarkers were identified by liquid chromatographytandem mass spectrometry (LC-MS/MS) based on proteomic and ELISA assays. Key Results: Functional and docking analyses showed that ? 9 -THCA-A can act as an orthosteric CB1 agonist and also as a positive allosteric modulator in the presence of CP- 55,940. In addition, ? 9 -THCA-A seemed to be an inverse agonist for CB2. In vivo experiments showed that ? 9 -THCA-A reduced arthritis in CIA mice. ? 9 -THCA-A prevented the infiltration of inflammatory cells; synovium hyperplasia and cartilage damage. Furthermore, ? 9 -THCA-A inhibited the expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of ? 9 -THCA-A was ablated by either SR141716 or T0070907. Analysis of plasmatic biomarkers as well as determination of cytokines and anti-collagen antibodies confirmed that ? 9 -THCA-A mediates its activity mainly through PPAR? and CB1 pathways. Conclusion and Implications: ? 9 -THCA-A modulates CB1 receptor through the orthosteric and allosteric binding sites. In addition, our studies document that ? 9 -THCA-A exerts antiarthritis activity through CB1/PPAR? pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as Rheumatoid Arthritis (RA).

?9-Tetrahydrocannabinolic Acid markedly alleviates liver fibrosis and inflammation in mice
Beatriz Carmona-Hidalgo , Isabel González-Mariscal , Adela García-Martín , María E Prados , Francisco Ruiz-Pino , Giovanni Appendino , Manuel Tena-Sempere , Eduardo Muñoz
Phytomedicine (2020)*Nov 30;81:153426.
*doi: 10.1016/j.phymed.2020.153426.
Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, and it is closely associated to obesity, type 2 diabetes mellitus, and dyslipidemia. Medicinal cannabis and some neutral cannabinoids have been suggested as a potential therapy for liver diseases.
Hypothesis: ?9-tetrahydrocannabinolic acid (?9-THCA), the non-psychotropic precursor of ?9-THC, is one of the most abundant cannabinoids presents in Cannabis Sativa. However, its biological activities have been poorly investigated. Herein, we studied the antifibrotic and antiinflammatory activities of ?9-THCA in two different animal models of liver injury, providing a rationale for additional studies on the medicinal use of this cannabinoid in the treatment of liver fibrosis and the management of NAFLD.
Study design: The antifibrotic activity of ?9-THCA in vitro was investigated in the cell lines LX-2 and NIH-3T3-Col1A2-luc. Non-alcoholic liver fibrosis was induced in mice by CCl4treatment or, alternatively, by 23-week high fat diet (HFD) feeding. ?9-THCA was administered daily intraperitoneally during the CCl4 treatment or during the last 3 weeks in HFD-fed mice.
Methods: TGF?-induced profibrotic gene expression was analyzed by luciferase and qPCR assays. Liver fibrosis and inflammation were assessed by immunochemistry and qPCR. Blood glucose, insulin, leptin and triglyceride levels were measured in HFD mice.
Results: ?9-THCA inhibited the expression of Tenascin C (TNC) and Col3A1 induced by TGF? in LX-2 cells and the transcriptional activity of the Col1A2 promoter in fibroblasts. ?9-THCA significantly attenuated CCl4-induced liver fibrosis and inflammation and reduced T cell and macrophage infiltration. Mice fed HFD for 23 weeks developed severe obesity (DIO), fatty liver and marked liver fibrosis, accompanied by immune cell infiltration. ?9-THCA, significantly reduced body weight and adiposity, improved glucose tolerance, and drastically attenuated DIO-induced liver fibrosis and immune cell infiltration.
Conclusions: ?9-THCA prevents TGF?-induced fibrotic markers in vitro and liver inflammation and fibrogenesis in vivo, providing a rationale for additional studies on the medicinal use of this cannabinoid, as well as cannabis preparations containing it, for the treatment of liver fibrosis and the management of NAFLD.

Delta-9-Tetrahydrocannabinol, Cannabidiol, and Acute Psychotomimetic States: A Balancing Act of the Principal Phyto-Cannabinoids on Human Brain and Behavior
Suhas Ganesh, Jose Cortes-Briones, Ashley M. Schnakenberg Martin, Patrick D. Skosnik, Deepak C. D’Souza, and Mohini Ranganathan,
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0166
Background: THC and CBD are the principal phyto-cannabinoids in the cannabis plant. The differential and possibly antagonistic effects of these compounds on specific brain and behavioral responses, and the mechanisms
underlying their effects have generated extensive interest in pre-clinical and clinical neuroscience investigations.
Methods: In this double-blind randomized placebo-controlled counterbalanced Human Laboratory Study, we examined the effects of three different dose ratios of CBD:THC (1:1, 2:1, and 3:1) on ‘‘neural noise,’’ an electrophysiological biomarker of psychosis known to be sensitive to cannabinoids as well as subjective and psychotomimetic effects. Healthy volunteers (n = 28, 12 women) with at least one prior exposure to cannabis participated in the study.
Outcomes: The lowest CBD (2.5 mg):THC (0.035 mg/kg) ratio (1:1) resulted in maximal attenuation of both THC induced psychotomimetic effects (Positive and Negative Syndrome Scale [PANSS] positive: Anova Type Statistic [ATS] = 7.83, pcorrected = 0.015) and neural noise (ATS = 8.83, pcorrected = 0.009). Further addition of CBD did not reduce the subjective experience of THC-induced ‘‘high’’ ( p > 0.05 for all CBD doses).
Interpretation: These novel results demonstrate that CBD attenuates specific THC-induced subjective and objective effects relevant to psychosis in a dose/ratio-dependent manner. Given the increasing global trend of cannabis liberalization and application for medical indications, these results assume considerable significance given the potential dose-related interactions of these key phyto-cannabinoids. Trial registration: The trial was registered in clinicaltrials.gov ID: NCT01180374.
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D9-Tetrahydrocannabinol and Cannabidiol Differentially Regulate Intraocular Pressure
Sally Miller, Laura Daily, Emma Leishman, Heather Bradshaw, and Alex Straiker
Invest Ophthalmol Vis Sci.
2018;59:5904–5911. https://doi.org/
10.1167/iovs.18-24838
PURPOSE. It has been known for nearly 50 years that cannabis and the psychoactive constituent D9-tetrahydrocannabinol (THC) reduce intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a major cause of blindness. THC likely acts via one of the known cannabinoid-related receptors (CB1, CB2, GPR18, GPR119, GPR55) but this has never been determined explicitly. Cannabidiol (CBD) is a second major constituent of cannabis that has been found to be without effect on IOP in most studies.
METHODS. Effects of topically applied THC and CBD were tested in living mice by using tonometry and measurements of mRNA levels. In addition the lipidomic consequences of CBD treatment were tested by using lipid analysis.
RESULTS. We now report that a single topical application of THC lowered IOP substantially (~28%) for 8 hours in male mice. This effect is due to combined activation of CB1 and GPR18 receptors each of which has been shown to lower ocular pressure when activated. We also found that the effect was sex-dependent, being stronger in male mice, and that mRNA levels of CB1 and GPR18 were higher in males. Far from inactive, CBD was found to have two opposing effects on ocular pressure, one of which involved antagonism of tonic signaling. CBD prevents THC from lowering ocular pressure.
CONCLUSIONS. We conclude that THC lowers IOP by activating two receptors—CB1 and GPR18—but in a sex-dependent manner. CBD, contrary to expectation, has two opposing effects on IOP and can interfere with the effects of THC.

Delta-9-Tetrahydrocannabinol and Cannabidiol Drug-Drug Interactions
Paul T. Kocisa, *Kent E. Vrana
Med Cannabis Cannabinoids
DOI: 10.1159/000507998
Although prescribing information (PI) is often the initial source of information when identifying potential drug-drug interactions, it may only provide a limited number of exemplars or only reference a class of medications without providing any specific medication examples. In the case of medical cannabis and medicinal cannabinoids, this is further complicated by the fact that the increased therapeutic use of marijuana extracts and cannabidiol oil will not have regulatory agency approved PI. The objective of this study was to provide a detailed and comprehensive drug-drug interaction list that is aligned with cannabinoid manufacturer PI. The cannabinoid drug-drug interaction information is listed in this article and online supplementary material as a PRECIPITANT (cannabinoid) medication that either INHIBITS/INDUCES the metabolism or competes for the same SUBSTRATE target (metabolic enzyme) of an OBJECT (OTHER) medication. In addition to a comprehensive list of drug-drug interactions, we also provide a list of 57 prescription medications displaying a narrow therapeutic index that are potentially impacted by concomitant cannabinoid use (whether through prescription use of cannabinoid medications or therapeutic/recreational use of cannabis and its extracts).

?9-tetrahydrocannabinol exposure during rat pregnancy leads to symmetrical fetal growth restriction and labyrinth-specific vascular defects in the placenta.
Natale, B. V., Gustin, K. N., Lee, K., Holloway, A. C., Laviolette, S. R., Natale, D. R. C., & Hardy, D. B.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-019-57318-6
1 in 5 women report cannabis use during pregnancy, with nausea cited as their primary motivation. Studies show that (-)-?9–tetrahydrocannabinol (?9-THC), the major psychoactive ingredient in cannabis, causes fetal growth restriction, though the mechanisms are not well understood. Given the critical role of the placenta to transfer oxygen and nutrients from mother, to the fetus, any compromise in the development of fetal-placental circulation signifcantly afects maternal-fetal exchange and thereby, fetal growth. The goal of this study was to examine, in rats, the impact of maternal ?9-THC exposure on fetal development, neonatal outcomes, and placental development. Dams received a daily intraperitoneal injection (i.p.) of vehicle control or ?9-THC (3mg/kg) from embryonic (E)6.5 through 22. Dams were allowed to deliver normally to measure pregnancy and neonatal outcomes, with a subset sacrifced at E19.5 for placenta assessment via immunohistochemistry and qPCR. Gestational ?9-THC exposure resulted in pups born with symmetrical fetal growth restriction, with catch up growth by postnatal day (PND)21. During pregnancy there were no changes to maternal food intake, maternal weight gain, litter size, or gestational length. E19.5 placentas from ?9-THC-exposed pregnancies exhibited a phenotype characterized by increased labyrinth area, reduced Epcam expression (marker of labyrinth trophoblast progenitors), altered maternal blood space, decreased fetal capillary area and an increased recruitment of pericytes with greater collagen deposition, when compared to vehicle controls. Further, at E19.5 labyrinth trophoblast had reduced glucose transporter 1 (GLUT1) and glucocorticoid receptor (GR) expression in response to ?9-THC exposure. In conclusion, maternal exposure to ?9-THC efectively compromised fetal growth, which may be a result of the adversely afected labyrinth zone development. These fndings implicate GLUT1 as a ?9-THC target and provide a potential mechanism for the fetal growth restriction observed in wwomen who use cannabis during pregnancy


?9-tetrahydrocannabinol (?9-THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson’s disease.
Carroll, C. B., Zeissler, M.-L., Hanemann, C. O., & Zajicek, J. P.
Neuropathology and Applied Neurobiology, 38(6), 535–547.(2012).
doi:10.1111/j.1365-2990.2011.01248.x
Aims: D9 -tetrahydrocannabinol (D9 -THC) is neuroprotective in models of Parkinson’s disease (PD). Although CB1 receptors are increased within the basal ganglia of PD patients and animal models, current evidence suggests a role for CB1 receptor-independent mechanisms. Here, we utilized a human neuronal cell culture PD model to further investigate the protective properties of D9 - THC. Methods: Differentiated SH-SY5Y neuroblastoma cells were exposed to PD-relevant toxins: 1-methyl-4- phenylpyridinium (MPP+ ), lactacystin and paraquat. Changes in CB1 receptor level were determined by quantitative polymerase chain reaction and Western blotting. Cannabinoids and modulatory compounds were co-administered with toxins for 48 h and the effects on cell death, viability, apoptosis and oxidative stress assessed. Results: We found CB1 receptor up-regulation in response to MPP+ , lactacystin and paraquat and a protective effect of D9 -THC against all three toxins. This neuroprotective effect was not reproduced by the CB1 receptor agonist WIN55,212-2 or blocked by the CB1 antagonist AM251. Furthermore, the antioxidants a-tocopherol and butylhydroxytoluene as well as the antioxidant cannabinoids, nabilone and cannabidiol were unable to elicit the same neuroprotection as D9 -THC. However, the peroxisome proliferator-activated receptor-gamma (PPARg) antagonist T0070907 dose-dependently blocked the neuroprotective, antioxidant and anti-apoptotic effects of D9 -THC, while the PPARg agonist pioglitazone resulted in protection from MPP+ -induced neurotoxicity. Furthermore, D9 -THC increased PPARg expression in MPP+ -treated SH-SY5Y cells, another indicator of PPARg activation. Conclusions: We have demonstrated up-regulation of the CB1 receptor in direct response to neuronal injury in a human PD cell culture model, and a direct neuronal protective effect of D9 -THC that may be mediated through PPARg activation

Delta-9-THC based monotherapy in fibromyalgia patients on experimentally induced pain, axon reflex flare, and pain relief.
Schley M1, Legler A, Skopp G, Schmelz M, Konrad C, Rukwied R.
Curr Med Res Opin. 2006 Jul;22(7):1269-76.
DOI:10.1185/030079906X112651
OBJECTIVE: Fibromyalgia (FM) is a chronic pain syndrome characterized by a distinct mechanical hyperalgesia and chronic pain. Recently, cannabinoids have been demonstrated as providing anti-nociceptive and anti-hyperalgesic effects in animal and human studies. Here, we explored in nine FM patients the efficacy of orally administered delta-9-tetrahydrocannabinol (THC) on electrically induced pain, axon reflex flare, and psychometric variables.
RESEARCH DESIGN AND METHODS: Patients received a daily dose of 2.5-15 mg of delta-9-THC, with a weekly increase of 2.5 mg, as long as no side effects were reported. Psychometric variables were assessed each week by means of the West Haven-Yale Multidimensional Pain Inventory (MPI), Pittsburgh Sleep Quality Index (PSQI), Medical outcome survey-short form (MOS SF-36), the Pain Disability Index (PDI), and the Fibromyalgia Impact Questionnaire (FIQ). In addition, patients recorded daily, in a diary, their overall pain intensity on a numeric scale. Each week, pain and axon reflex flare was evoked experimentally by administration of high intensity constant current pulses (1 Hz, pulse width 0.2 ms, current increase stepwise from 2.5-12.5 mA every 3 minutes) delivered via small surface electrodes, attached to the volar forearm skin.
MAIN OUTCOME MEASURES: Daily pain recordings by the patient, experimentally induced pain, and axon reflex flare recorded by a laser Doppler scanner.
RESULTS: Five of nine FM patients withdrew during the study due to adverse side effects. Delta-9-THC had no effect on the axon reflex flare, whereas electrically induced pain was significantly attenuated after doses of 10-15 mg delta-9-THC (p < 0.05). Daily-recorded pain of the FM patients was significantly reduced (p < 0.01).
CONCLUSIONS: This pilot study demonstrated that a generalized statement that delta-9-THC is an analgetic drug cannot be made. However, a sub-population of FM patients reported significant benefit from the delta-9-THC monotherapy. The unaffected electrically induced axon reflex flare, but decreased pain perception, suggests a central mode of action of the cannabinoid.

?9-Tetrahydrocannabinol Increases Dopamine D1-D2 Receptor Heteromer and Elicits Phenotypic Reprogramming in Adult Primate Striatal Neurons.
Hasbi, A., Madras, B. K., Bergman, J., Kohut, S., Lin, Z., Withey, S. L., & George, S. R.
iScience, 100794. (2019).
doi:10.1016/j.isci.2019.100794
Long-term cannabis users manifest deficits in dopaminergic functions, reflecting THCinduced neuroadaptive dysfunctional dopamine signaling, similar to those observed upon dopamine D1-D2 heteromer activation. The molecular mechanisms remain largely unknown. We show evolutionary and regional differences in D1-D2 heteromer abundance in mammalian striatum. Importantly, chronic THC increased the number of D1-D2 heteromerexpressing neurons, and the number of heteromers within individual neurons in adult monkey striatum. The majority of neurons displayed a phenotype co-expressing the characteristic markers of both striatonigral and striatopallidal neurons. Furthermore, THC increased D1-D2-
linked calcium signaling markers (pCaMKII?, pThr75-DARPP-32, BDNF/pTrkB) and inhibited cAMP signaling (pThr34-DARPP-32, pERK1/2, pS845-GluA1, pGSK3). Cannabidiol attenuated most but not all of these THC-induced neuroadaptations. Targeted pathway analyses linked these changes to neurological and psychological disorders. These data underline the importance of the D1-D2 receptor heteromer in cannabis use-related disorders, with THC-induced changes likely responsible for the reported adverse effects observed in heavy long-term users.

Delta‑9‑tetrahydrocannabinol reduces willingness to exert effort in women
Margaret C. Wardle · Elisa Pabon · Heather E. Webber · Harriet de Wit
Psychopharmacology
DOI: 10.1007/s00213-021-06032-1
https://link.springer.com/content/pdf/10.1007/s00213-021-06032-1.pdf
Background The use of cannabis has been clinically associated with decreased motivation to engage in normally rewarding activities. However, evidence from previous controlled studies is mixed. Method In this study, we examined the effects of acute delta-9-tetrahydrocannabinol (THC) versus placebo on a task measuring willingness to exert effort for rewards. This is a secondary analysis of a larger study examining interactions between ovarian hormones and THC. In this within-subjects study, oral THC and placebo were administered under double-blind conditions in counterbalanced order to healthy young adult (M age=24 years) women with previous cannabis experience who were not regular users. Forty subjects completed three 4-h sessions with PL, 7.5 and 15 mg THC, while an additional 18 completed only PL and 15 mg THC sessions (design abridged due to pandemic). At each session, they completed a task consisting of making repeated choices between a hard and an easy task, which were worth varying amounts of money at varying probabilities. Results THC dose-dependently decreased hard task choices (drug effect, b= −0.79, SE=0.29, z= −2.67, p<0.01), especially at moderate to high expected values of reward (drug×probability×amount interaction, b=0.77, SE=0.38, z=1.99, p=0.04). THC also slowed task performance (drug efect, b=0.01, SE=0.005, t(5.24)=2.11, p=0.04), but the effect of THC on choice was still significant after controlling for this psychomotor slowing. Conclusions These fndings support the idea that cannabis acutely reduces motivation to earn non-drug rewards. Still to be determined are the neurochemical mechanisms underlying this efect.

9-Tetrahydrocannabinol (THC), 11-Hydroxy-THC, and 11-Nor-9-carboxy-THC Plasma Pharmacokinetics during and after Continuous High-Dose Oral THC.
Schwilke, E. W., Schwope, D. M., Karschner, E. L., Lowe, R. H., Darwin, W. D., Kelly, D. L., … Huestis, M. A.
Clinical Chemistry, 55(12), 2180–2189.(2009).
doi:10.1373/clinchem.2008.122119
BACKGROUND: 9 -Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis and an active cannabinoid pharmacotherapy component. No plasma pharmacokinetic data after repeated oral THC administration are available. METHODS: Six adult male daily cannabis smokers resided on a closed clinical research unit. Oral THC capsules (20 mg) were administered every 4 –8 h in escalating total daily doses (40 –120 mg) for 7 days. Free and glucuronidated plasma THC, 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THC COOH) were quantified by 2-dimensional GC-MS during and after dosing. RESULTS: Free plasma THC, 11-OH-THC, and THCCOOH concentrations 19.5 h after admission (before controlled oral THC dosing) were mean 4.3 (SE 1.1), 1.3 (0.5), and 34.0 (8.4) g/L, respectively. During oral dosing, free 11-OH-THC and THCCOOH increased steadily, whereas THC did not. Mean peak plasma free THC, 11-OH-THC, and THCCOOH concentrations were 3.8 (0.5), 3.0 (0.7), and 196.9 (39.9) g/L, respectively, 22.5 h after the last dose. Escherichia coli -glucuronidase hydrolysis of 264 cannabinoid specimens yielded statistically significant increases in THC, 11-OH-THC, and THCCOOH concentrations (P 0.001), but conjugated concentrations were underestimated owing to incomplete enzymatic hydrolysis. CONCLUSIONS: Plasma THC concentrations remained 1 g/L for at least 1 day after daily cannabis smoking and also after cessation of multiple oral THC doses. We report for the first time free plasma THC concentrations after multiple high-dose oral THC throughout the day and night, and after Escherichia coli -glucuronidase hydrolysis. These data will aid in the interpretation of plasma THC concentrations after multiple oral doses.

?9-Tetrahydrocannabivarin (THCV): a commentary on potential therapeutic benefit for the management of obesity and diabetes
Amos Abioye, Oladapo Ayodele, Aleksandra Marinkovic, Risha Patidar, Adeola Akinwekomi and Adekunle Sanyaolu
Journal of Cannabis Research (2020) 2:6
doi 10.1186/s42238-020-0016-7
?9-Tetrahydrocannabivarin (THCV) is a cannabis-derived compound with unique properties that set it apart from the more common cannabinoids, such as ?9-tetrahydrocannabinol (THC). The main advantage of THCV over THC is the lack of psychoactive effects. In rodent studies, THCV decreases appetite, increases satiety, and up-regulates energy metabolism, making it a clinically useful remedy for weight loss and management of obesity and type 2 diabetic patients. The distinctions between THCV and THC in terms of glycemic control, glucose metabolism, and energy regulation have been demonstrated in previous studies. Also, the effect of THCV on dyslipidemia and glycemic control in type 2 diabetics showed reduced fasting plasma glucose concentration when compared to a placebo group. In contrast, THC is indicated in individuals with cachexia. However, the uniquely diverse properties of THCV provide neuroprotection, appetite suppression, glycemic control, and reduced side effects, etc.; therefore, making it a potential priority candidate for the development of clinically useful therapies in the future. Hopefully, THCV could provide an optional platform for the treatment of life-threatening diseases

Dendritic Cell Regulation by Cannabinoid-Based Drugs.
Mattias Svensson, Puran Chen and Oscar Hammarfjord
Pharmaceuticals 2010, 3, 2733-2750;
doi:10.3390/ph3082733
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4033947/pdf/pharmaceuticals-03-02733.pdf
Cannabinoid pharmacology has made important advances in recent years after the cannabinoid system was discovered. Studies in experimental models and in humans have produced promising results using cannabinoid-based drugs for the treatment of obesity and cancer, as well as neuroinflammatory and chronic inflammatory diseases. Moreover, as we discuss here, additional studies also indicates that these drugs have immunosuppressive and anti-inflammatory properties including modulation of immune cell function. Thus, manipulation of the endocannabinoid system in vivo may provide novel therapeutic strategies against inflammatory disorders. At least two types of cannabinoid receptors, cannabinoid 1 and cannabinoid 2 receptors are expressed on immune cells such as dendritic cells (DC). Dendritic cells are recognized for their critical role in initiating and maintaining immune responses. Therefore, DC are potential targets for cannabinoidmediated modulation. Here, we review the effects of cannabinoids on DC and provide some perspective concerning the therapeutic potential of cannabinoids for the treatment of human diseases involving aberrant inflammatory processes.

Determining factors for Cannabis use among Moroccans Schizophrenic Patients: A cross sectional study
Avicenna Journal of Phytomedicine 10(1):89-100 January 2020
Sara Bouri, Hanane Hanane, Khadija El Ayoubi Idrissi, Sanae Achour
https://www.researchgate.net/publica...ectional_study
Objective: Cannabis use is considered a major clinical problem associated with a poorer outcome in patients with schizophrenia. The objective of the present study was to assess the prevalence of cannabis us among patients with schizophrenia. The assessment consists in comparing some factors related to substance use in a population of schizophrenic patients between cannabis users and non. Materials and methods: Four hundred and three participants who were examined prospectively during their hospitalization answered the PANNS scale of schizophrenia, GAF, BIS-10, CDSS, and MARS. The consumption of cannabis was investigated using urine toxicological analysis. Sociodemographic, clinical and therapeutic data were also recorded. Results: The prevalence of cannabis use among schizophrenic inpatients was 49%. Patients with cannabis use were younger (31.7 vs 34.9 years old, p<0.001), more often male (52 vs 20% female, p<0.001), and they presented more often a history of imprisonment (68.8% vs 31.2%, p<0.001). Patients who were users of cannabis had a lower age at onset of the disease than non-users (23.6 vs 24.8 years, p=0.029), and more often with poor medication adherence (p=0.001). Logistic regression revealed that factors associated with cannabis use among schizophrenics were the age, gender, history of imprisonment and poor medication adherence. Conclusion: The study showed that a high prevalence of cannabis use among patients with schizophrenia which was associated with negative overall outcomes. Determining comorbid substance use disorders among schizophrenic patients is crucial as it may contribute to establish a better therapeutic strategy.

Deveiopment of Indication-specific Cannabis chemovars
Ethan B. Russo, Mark A. Lewis
Napro poster at ICRS 2018
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Development of intestinal bioavailability orediction (IBP) and phytochemical relative antitoxidant potential prediction (PRAPP) models for optimizing functional food value of Cannabis sativa (hemp).
Kimber Wise, Sophie N.B. Selby-Pham, Jamie Selby-Pham and Harsharn Gill
INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2020, VOL. 23, NO. 1, 1287–1295
DOI: 10.1080/10942912.2020.1797783
Oxidative stress and inflammation (OSI) occurs naturally during many biological processes including digestion, metabolism, and exercise. While small, transient amounts of OSI are considered normal, unregulated, or chronic OSI can damage the vascular-circulatory system, which can result in chronic illnesses such as cardiovascular disease (CVD), atherosclerosis, and cancer. Antioxidant phytochemicals have the capacity to mitigate OSI through radical scavenging activity or the induction of endogenous mechanisms, but to achieve optimal reductions in OSI, the timing of antioxidant effects must occur during the onset of OSI – a concept known as ‘bio-matching.’ Additionally, the bioavailability and antioxidant capacity of active phytochemicals should be accounted for during pharmacokinetic assessments to guide bio-matching. Herein two quantitative structure–activity relationship (QSAR) predictive models are presented: the intestinal absorption prediction (IBP) model for predicting compound bioavailability (r2 = 0.93), and the phytochemical relative antioxidant potential prediction (PRAPP) model for predicting antioxidant capacity (r2 = 0.89). Application of these models to a characterized hemp meal phytochemical profile, along with established models for predicting Tmax and Tó, generated a composite antioxidant fingerprint, which predicted a peak in antioxidant activity 36 min after ingestion in liquid form. Accordingly, hemp meal-based protein powders (a common exercise supplement) should be consumed 26 min prior to completion of exercise to achieve bio-matching with the onset of exercise-induced OSI 10 min after exercise. The IBP and PRAPP models presented herein could be useful tools in understanding phytochemical complex antioxidant pharmacodynamics and in optimizing the consumption of hemp meal and other functional foods to achieve bio-matching of composite antioxidant activity with OSI profiles. .

Developmental regulation of fear learning and anxiety behavior by endocannabinoids
T. T. Lee, M. Hill, F. Lee January 2016
Genes, Brain and Behavior (2016) 15: 108–124
DOI:10.1111/gbb.12253
The developing brain undergoes substantial maturation into adulthood and the development of specific neural structures occurs on differing timelines. Transient imbalances between developmental trajectories of corticolimbic structures, which are known to contribute to regulation over fear learning and anxiety, can leave an individual susceptible to mental illness, particularly anxiety disorders. There is a substantial body of literature indicating that the endocannabinoid (eCB) system critically regulates stress responsivity and emotional behavior throughout the life span, making this system a novel therapeutic target for stress- and anxiety-related disorders. During early life and adolescence, corticolimbic eCB signaling changes dynamically and coincides with different sensitive periods of fear learning, suggesting that eCB signaling underlies age-specific fear learning responses. Moreover, perturbations to these normative fluctuations in corticolimbic eCB signaling, such as stress or cannabinoid exposure, could serve as a neural substrate contributing to alterations to the normative developmental trajectory of neural structures governing emotional behavior and fear learning. In this review, we first introduce the components of the eCB system and discuss clinical and rodent models showing eCB regulation of fear learning and anxiety in adulthood. Next, we highlight distinct fear learning and regulation pro- files throughout development and discuss the ontogeny of the eCB system in the central nervous system, and models of pharmacological augmentation of eCB signaling during development in the context of fear learning and anxiety.

Diabetes and Cannabinoid CB1 receptor deficiency promote similar early onset aging-like changes in the skin
Ermelindo C Leal , Liane I F Moura , Roksana M Pirzgalska , Dorinda Marques-da-Silva , Catherine Ledent , Attila Köfalvi,, Eugenia Carvalho
Exp Gerontol. 2021 Aug 24;154:111528.
DOI: 10.1016/j.exger.2021.111528
Background: The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model.
Methods: We evaluated several signals of skin aging in wild-type control (WT), WT streptozotocin-induced type 1 diabetic mice (WT DM), CB1R knockout (CB1RKO) and CB1RKO DM mice. We quantified markers of inflammation, angiogenesis, antioxidant enzymes and collagen content. Moreover, we evaluate reactive oxygen species (ROS) levels and macrophage phenotype, M1 and M2.
Results: CB1R expression is decreased in the skin of WT DM mice and collagen levels are decreased in the skin of WT DM, CB1RKO and CB1RKO DM mice. Additionally, the absence of CB1R correlated with higher expression of pro-inflammatory markers, also evident in WT DM or CB1RKO DM mice. Moreover, the M1/M2 macrophage ratio and ROS levels were significantly elevated but in the diabetic WT and the CB1RKO mice, consistent with a significant decrease in the antioxidant capacity of the skin.
Conclusions: Our results indicate that CB1R deficiency in the skin may lead to accelerated skin aging due to the increased production of ROS, a decrease in the antioxidant defenses and a higher pro-inflammatory environment. A significant decrease in the CB1R expression may be a significant contributing factor to the early aging-like changes in diabetes.
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Not Cannabis specific
Dietary favanols improve cerebral cortical oxygenation and cognition in healthy adults
GabrieleGratton, Samuel R. Weaver, ClaireV. Burley, KathyA. Low, Edward L. Maclin, Paul W. Johns, Quang S. Pham, Samuel J. E. Lucas, Monica Fabiani, & Catarina Rendeiro
Nature Scientific Reports
10.1038/s41598-020-76160-9
Cocoa favanols protect humans against vascular disease, as evidenced by improvements in peripheral endothelial function, likely through nitric oxide signalling. Emerging evidence also suggests that favanol-rich diets protect against cognitive aging, but mechanisms remain elusive. In a randomized double-blind within-subject acute study in healthy young adults, we link these two lines of research by showing, for the frst time, that favanol intake leads to faster and greater brain oxygenation responses to hypercapnia, as well as higher performance only when cognitive demand is high. Individual diference analyses further show that participants who beneft from favanols intake during hypercapnia are also those who do so in the cognitive challenge. These data support the hypothesis that similar vascular mechanisms underlie both the peripheral and cerebral efects of favanols. They further show the importance of studies combining physiological and graded cognitive challenges in young adults to investigate the actions of dietary favanols on brain function.

Differential Effects of Cannabidiol and a Novel Cannabidiol Analog on Oxycodone Place Preference and Analgesia in Mice: an Opioid Abuse Deterrent with Analgesic Properties
Hannah M. Harris, Waseem Gul, Mahmoud A. ElSohly, and Kenneth J. Sufka
Cannabis and Cannabinoid Research 24 Dec 2021
DOI: 10.1089/can.2021.0050
https://www.liebertpub.com/doi/10.10...rid:crossref.o rg&rfr_dat=cr_pub%20%200pubme d
https://www.liebertpub.com/doi/epub/.../can.2021.0050
Background and Purpose: This study sought to determine whether cannabidiol (CBD) or a CBD derivative, CBD monovalinate monohemisuccinate (CBD-val-HS), could attenuate the development of oxycodone reward while retaining its analgesic effects.
Experimental Approach: To determine the effect on oxycodone reward, animals were enrolled in the conditioned place preference paradigm and received either saline or oxycodone (3.0 mg/kg) in combination with either CBD or CBD-val-HS utilizing three sets of drug-/no drug-conditioning trials. To determine if the doses of CBD or CBD-val-HS that blocked opioid reward would affect nociceptive processes, animals were enrolled in the hot plate and abdominal writhing assays when administered alone or in combination with a subanalgesic (1.0 mg/kg) or analgesic (3.0 mg/kg) dose of oxycodone.
Key Results: Results from condition place preference demonstrated CBD was not able attenuate oxycodone place preference while CBD-val-HS attenuated these rewarding effects at 8.0 mg/kg and was void of rewarding or aversive properties. In contrast to CBD, CBD-val-HS alone produced analgesic effects in both nociceptive assays but was most effective compared with oxycodone against thermal nociception. Of interest, there was a differential interaction of CBD and CBD-val-HS×oxycodone across the two nociceptive assays producing subadditive responses on the hot plate assay, whereas additive responses were observed in the abdominal writhing assay.
Conclusion: These findings suggest CBD-val-HS alone, a nonrewarding analgesic compound, could be useful in pain management and addiction treatment settings.

Differential sensitivity to the acute psychotomimetic effects of delta-9-tetrahydrocannabinol associated with its differential acute effects on glial function and cortisol
Marco Colizzi , Nathalie Weltens , David J Lythgoe , Steve Cr Williams , Lukas Van Oudenhove , Sagnik Bhattacharyya
Psychol Med 2020 Oct 27;1-8.
doi: 10.1017/S0033291720003827.
Background: Cannabis use has been associated with psychosis through exposure to delta-9-tetrahydrocannabinol (?9-THC), its key psychoactive ingredient. Although preclinical and human evidence suggests that ?9-THC acutely modulates glial function and hypothalamic-pituitary-adrenal (HPA) axis activity, whether differential sensitivity to the acute psychotomimetic effects of ?9-THC is associated with differential effects of ?9-THC on glial function and HPA-axis response has never been tested.
Methods: A double-blind, randomized, placebo-controlled, crossover study investigated whether sensitivity to the psychotomimetic effects of ?9-THC moderates the acute effects of a single ?9-THC dose (1.19 mg/2 ml) on myo-inositol levels, a surrogate marker of glia, in the Anterior Cingulate Cortex (ACC), and circadian cortisol levels, the key neuroendocrine marker of the HPA-axis, in a set of 16 healthy participants (seven males) with modest previous cannabis exposure.
Results: The ?9-THC-induced change in ACC myo-inositol levels differed significantly between those sensitive to (?9-THC minus placebo; M = -0.251, s.d. = 1.242) and those not sensitive (M = 1.615, s.d. = 1.753) to the psychotomimetic effects of the drug (t(14) = 2.459, p = 0.028). Further, the ?9-THC-induced change in cortisol levels over the study period (baseline minus 2.5 h post-drug injection) differed significantly between those sensitive to (?9-THC minus placebo; M = -275.4, s.d. = 207.519) and those not sensitive (M = 74.2, s.d. = 209.281) to the psychotomimetic effects of the drug (t(13) = 3.068, p = 0.009). Specifically, ?9-THC exposure lowered ACC myo-inositol levels and disrupted the physiological diurnal cortisol decrease only in those subjects developing transient psychosis-like symptoms.
Conclusions: The interindividual differences in transient psychosis-like effects of ?9-THC are the result of its differential impact on glial function and stress response.

Discovery and Isolation of Anandamide and Other Endocannabinoids
Lumr O. Hanus
November 2007 Chemistry & Biodiversity 4(8):1828-41
DOI: 10.1002/cbdv.200790154
1. Introduction
2. Cannabinoids in Plant and Body
3. Cannabinoid Receptors
4. Isolation of Anandamide
5. Other Endocannabinoids
6. The Endocannabinoid Congeners
7. Conclusions
1. Introduction. –Cannabis had many uses as a cultivated plant already in Neolithic China. The first known record of the use of cannabis as a medicine was published in China 5000 years ago in the reign of the Emperor Chen Nung. However, it was not until the 19th century that cannabis became a common medicine in Europe, and its analgesic, anticonvulsant, anti-spasmodic, anti-emetic, and hypnotic uses were recognized. The Assyrians, who ruled large parts of the Middle East for nearly a millennium, ca. 3000 years ago, have left us a pharmaceutical legacy on hundreds of clay tablets. Cannabis was one of the major drugs of their pharmacopoeia. They named this plant according to its use. Campbell identified the Sumerian term a-zal-la and the Akkadian term azulla as cannabis on the basis of their similarities to the Syrian azal, meaning >to spin?. He also took the Assyrian word gurgurangu as another reference to cannabis because of its similarity to garganinj, the Persian word for cannabis. Building on these similarities, Campbell then identified the Sumerian drug gan-zi-gun-nu as hashish (a robber (gan) who spins away (gun-nu) the soul (zi)), and this word has been translated as >the drug that takes away the mind? (from today point of viewwe can translate it as >cannabimimetic?). A letter written around 680 B.C. by an unknown woman to the mother of the Assyrian king, Esarhaddon, mentions a substance called qu-nu-bu which also may have been cannabis, but again there is no certainty for this identification. Pliny the Elder, an ancient Roman nobleman, scientist, and historian, author of Naturalis Historia, >Pliny¬s Natural History? (79 a.d.) wrote that >The roots boiled in water ease cramped joints, gout too and similar violent pain?. 1828 CHEMISTRY & BIODIVERSITY – Vol. 4 (2007) C 2007 Verlag Helvetica Chimica Acta AG, ZFrich Pedanius Dioscorides (ca. 40 in Anazarbus, Cilicia – ca. 90), an ancient Greek physician, pharmacologist, and botanist who practiced in Rome at the times of Nero (90 a.d.) mentions that >The sodden root when placed on inflammations sooths them, eliminates edema and disperses obdurate matter above inflamed joints? [5]. French physician Jacques Joseph Moreau remains the most-cited connection between cannabis and the art community. Moreau first used hashish while traveling through the Middle East in the 1830s. He hypothesized that cannabis-induced sensations might model the hallucinations and delusions common in psychotic individuals. He had hoped this research might help treatment of the mentally ill. The outspoken hedonist and popular novelist Pierre Jules Theophile Gautier assisted Moreau in this research. He not only participated himself, but he also recruited other members of France?s artistic community (Charles Pierre Baudelaire, one of the greatest poets of the 19th century; Honore´ de Balzac, Alexandre Dumas, and Gustave Flaubert). This crewof experimenters donned around 1835 the name the Club des Hashichins (Hashish Club), and met monthly in an old mansion in Paris [6]. The single most complete and authoritative work on the history of the genus Cannabis published E. L. Abel, Marijuana – The First Twelve Thousand Years, Plenum Press, New York, 1980

Disease-modifying effects of natural ?9-tetrahydrocannabinol in endometriosis-associated pain
Alejandra Escudero-Lara1, Josep Argerich1, David Cabañero1* and Rafael Maldonado
Doi: 10.1101/715938.
Endometriosis is a chronic painful disease highly prevalent in women that is defined by growth of endometrial tissue outside the uterine cavity and lacks adequate treatment. Medical use of cannabis derivatives is a current hot topic and it is unknown whether phytocannabinoids may modify endometriosis symptoms and development. Here we evaluate the effects of repeated exposure to ?9-tetrahydrocannabinol (THC) in a mouse model of surgically-induced endometriosis. In this model, female mice develop pelvic mechanical hypersensitivity, anxiety-like behavior and sharp memory deficits associated
to the presence of extrauterine endometrial cysts. Interestingly, chronic THC (2 mg/kg/day) provides sustained alleviation of pelvic hypersensitivity without altering the anxiogenic phenotype, modifies uterine innervation and restores cognitive function, an effect correlated with neuroinflammatory changes in prefrontal cortex. Strikingly, THC also inhibits the development of endometrial cysts. These data highlight the interest of scheduled clinical trials designed to investigate possible benefits of THC for women with endometriosis.

Distinct Functions of Endogenous Cannabinoid System in Alcohol Abuse Disorders.
Basavarajappa, B. S., Joshi, V., Shivakumar, M., & Subbanna, S.
British Journal of Pharmacology. (2019).
doi:10.1111/bph.14780
?9-tetrahydrocannabinol (?9-THC), the principal active component in Cannabis sativa extracts such as marijuana, participates in cell signaling by binding to cell surface receptors. CB1 receptors (CB1s) are present in both inhibitory and excitatory presynaptic terminals. CB2 receptors (CB2s) found in neuronal subpopulations in addition to microglial cells and astrocytes and are present in both pre- and postsynaptic terminals. Subsequent to endocannabinoid (eCB) system discoveries, studies have suggested that alcohol alters the eCB system and that the eCB system plays a major role in the motivation to abuse alcohol. Preclinical studies have provided evidence that chronic alcohol consumption modulates eCBs and CB1 expression in brain addiction circuits. In addition, studies have further established the distinct function of the eCB system in the development of fetal alcohol spectrum disorders. This review provides a recent and comprehensive assessment of the literature related to the function of the eCB system in alcohol abuse disorders.

Distribution of cannabinoid synthase genes in non-Cannabis organisms
Niranjan Aryal* , Debbie Figueroa Orellana and Jamal Bouie
Journal of Cannabis Research 2019, 1(2019):8
Doi: .1186/s42238-019-0008-7
The discovery of phytocannabinoid synthesizing enzymes, tetrahydrocannabinolic acid synthase (THCAs) and cannabidiolic acid synthase (CBDAs) was a breakthrough in Cannabis research. However, their evolutionary aspects and distribution across organisms has not been adequately studied. We searched for THCAs and CBDAs genes in organisms other than Cannabis plants using the database available in NCBI. Both cannabinoid synthases seem to be widely distributed in the plant kingdom. Of several complete or partial sequences of cannabinoid synthases-likes, CBDAs-like from Morus notabilis matched closely to CsCBDAs and CsTHCAs. When amino acid sequences of CsTHCAs, CsCBDAs and MnCBDAs-like were compared to each other, and to the motif database stored in Expasy, all three proteins contained the FAD_PCMH (PCMH-type FAD-binding) domain indicating the conservation of this domain in cannabinoid synthases. Apart from FAD binding, Berberine Bridge Enzyme (BBE-likes), which catalyzes the synthesis of isoquinoline alkaloids in many plants such as mulberry, poplas and citrus, were the other most closely related enzymes to CsTHCAs and CsCBDAs. We also searched for THCAs and CBDAs in fungal and bacterial kingdom but could not find any notably similar sequence. However, partial mRNA from FAD binding enzyme from Trametes versicolor and 6-hydroxy D nicotine oxidase from Aspergillus saccharolyticus matched the CsTHCA sequence and a partial mRNA from a hypothetical protein in Pneumocystis carinii was the most closely matched fungal enzyme to the CsCBDA. Our database search showed that Morus notabilis from mulberry family could be the candidate plant for further studies. Comparative transcriptomic and metabolomic studies for mulberry and Cannabis plants could provide a much clear concepts on the co-evolution of these syanthases. Moreover, the understanding of cannabinoid synthesis pathway is still evolving, in-depth bioinformatics and functional analysis of the enzymes involved are required for pharmaceutical research and industrial advancement.

Does cannabidiol reduce severe behavioural problems in children with intellectual disability? Study protocol for a pilot single-site phase I/II randomised placebo controlled trial
Efron D, Taylor K, Payne JM, et al
BMJ Open 2020;10:e034362.
doi:10.1136/ bmjopen-2019-034362
Abstract Introduction Severe behavioural problems (SBPs) are a common contributor to morbidity and reduced quality of life in children with intellectual disability (ID). Current medication treatment for SBP is associated with a high risk of side effects. Innovative and safe interventions are urgently needed. Anecdotal reports and preliminary research suggest that medicinal cannabis may be effective in managing SBP in children with developmental disabilities. In particular, cannabidiol (CBD) may be a plausible and safe alternative to current medications. Families who are in urgent need of solutions are seeking cannabis for their ID children with SBP. However there is no evidence from randomised controlled trials to support the use of CBD for SBP. This pilot study aims to investigate the feasibility of conducting a randomised placebocontrolled trial of CBD to improve SBP in children with ID. Methods and analysis This is a single-site, double-blind, parallel-group, randomised, placebo-controlled pilot study of 10 participants comparing 98% CBD oil with placebo in reducing SBP in children aged 8–16 years with ID. Eligible participants will be randomised 1:1 to receive either CBD 20mg/kg/day or placebo for 8 weeks. Data will be collected regarding the feasibility and acceptability of all study components, including recruitment, drop-out rate, study visit attendance, protocol adherence and the time burden of parent questionnaires. Safety outcomes and adverse events will be recorded. All data will be reported using descriptive statistics. These data will inform the design of a full scale randomised controlled trial to evaluate the efficacy of CBD in this patient group. Ethics and dissemination This protocol has received ethics approval from the Royal Children’s Hospital ethics committee (Human Research Ethics Committee no. 38236). Results will be disseminated through peer-reviewed journals

Does Cannabis Extract Obtained From Cannabis Flowers With Maximum Allowed Residual Level of Aflatoxins and Ochratoxin a Have an Impact on Human Safety and Health?
Tijana Serafimovska, Sasho Stefanovski, Joachim Erler, Zlatko Keskovski, Gjoshe Stefkov, Marija Mitevska, Marija Darkovska Serafimovska, Trajan Balkanov and Jasmina Tonic Ribarska
Front. Med. 8:759856.
doi: 10.3389/fmed.2021.759856
https://www.frontiersin.org/articles...21.759856/full
Aim: The aim of this study was to investigate whether the cannabis extract obtained from cannabis flowers that contain the maximum allowed level of mycotoxins affects human safety and health. For that purpose, a novel liquid chromatography with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of aflatoxins and ochratoxin A (OchA) in cannabis extracts to demonstrate that this analytical method is suitable for the intended experimental design.
Methods: Experimental design was done by adding maximum allowed concentration of aflatoxins (B1, B2, G1, G2) and OchA according to the European Pharmacopeia related to cannabis flowers. The concentration of aflatoxins and OchA was determined using the same LC/MS/MS analytical method in the starting material (dry flower) before preparing the spiked sample and after obtaining decarboxylated extract with ethanol 96%.
Results: The results obtained indicate that aflatoxins and OchA, primarily added to the cannabis dried flowers, were also determined into the obtained final extract in amounts much higher (m/m) than in the starting plant material.
Conclusion: With this experiment, we have shown that mycotoxins, especially aflatoxins, which are extremely toxic secondary metabolites, can reach critical values in cannabis extracts obtained from dry cannabis flowers with the maximum allowed quantity of mycotoxins. This can pose a great risk to consumers and their health especially to those with compromised immune systems.

Dose-dependent cannabis use, depressive symptoms, and FAAH genotype predict sleep quality in emerging adults: a pilot study
Kristin E Maple, Kymberly A McDaniel, Skyler G Shollenbarger, Krista M Lisdah
The American Journal of Drug and Alcohol Abuse 42(4):1-10 (2016)
DOI: 10.3109/00952990.2016.1141913
Background: Cannabis has been shown to affect sleep in humans. Findings from animal studies indicate that higher endocannabinoid levels promote sleep, suggesting that chronic use of cannabis, which downregulates endocannabinoid activity, may disrupt sleep. Objectives: This study sought to determine if past-year cannabis use and genes that regulate endocannabinoid signaling, FAAH rs324420 and CNR1 rs2180619, predicted sleep quality. As depression has been previously associated with both cannabis and sleep, the secondary aim was to determine if depressive symptoms moderated or mediated these relationships. Methods: Data were collected from 41 emerging adult (ages 18-25) cannabis users. Exclusion criteria included Axis I disorders (besides SUD) and medical and neurologic disorders. Relationships were tested using multiple regressions, controlling for demographic variables, past-year substance use, and length of cannabis abstinence. Results: Greater past-year cannabis use and FAAH C/C genotype were associated with poorer sleep quality. CNR1 genotype did not significantly predict sleep quality. Depressive symptoms moderated the relationship between cannabis use and sleep at a nonsignificant trend level, such that participants with the higher cannabis use and depressive symptoms reported the more impaired sleep. Depressive symptoms mediated the relationship between FAAH genotype and sleep quality. Conclusions: This study demonstrates a dose-dependent relationship between chronic cannabis use and reported sleep quality, independent of abstinence length. Furthermore, it provides novel evidence that depressive symptoms mediate the relationship between FAAH genotype and sleep quality in humans. These findings suggest potential targets to impact sleep disruptions in cannabis users.

Dose, Multiple Dose, and Food Effect Trial of the Safety, Tolerability and Pharmacokinetics of Highly Purifed Cannabidiol in Healthy Subjects
Lesley Taylor, Barry Gidal, Graham Blakey, Bola Tayo, Gilmour Morrison
CNS Drugs (2018) 32:1053–1067
doiL 10.1007/s40263-018-0578-5
Background A formal single ascending and multiple dose pharmacokinetic (PK) trial of cannabidiol (CBD) oral solution was required to determine the safety and tolerability of CBD, the maximum tolerated dose, and to examine the efect of food on CBD PK parameters.
Objective This trial assessed the safety, tolerability and PK of CBD oral solution in healthy adult volunteers, as well as the efect of food on CBD PK parameters.
Methods The study consisted of three*arms: single ascending dose (1500, 3000, 4500 or 6000*mg CBD [n=6 per group]/ placebo [n=8; 2 per CBD dose group]), multiple dose (750*or 1500*mg CBD [n=9 per group]/placebo [n=6; 3 per CBD dose group] twice daily), and food efect (1500*mg CBD single dose [n=12]). All subjects completed all trial arms and were analyzed as planned.
Results CBD was generally well tolerated. Diarrhea, nausea, headache, and somnolence were the most common adverse events (AEs) across all trial arms, with an increased incidence of some gastrointestinal and nervous system disorder AEs (most notably diarrhea and headache) apparent in subjects taking CBD compared with placebo. All AEs were of mild or moderate severity; none were severe or serious. There were no deaths or discontinuations in the trial. After single oral doses, CBD appeared rapidly in plasma; time to maximum plasma concentration (tmax) was approximately 4–5*h. The major circulating metabolite was 7-carboxy-CBD, then parent CBD, 7-hydroxy-CBD (active metabolite), and 6-hydroxy-CBD (a relatively minor metabolite). Plasma exposure to CBD [maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from time zero to time t (AUCt)] increased in a less than dose-proportional manner (Cmax slope 0.73; AUCt slope 0.64). Oral clearance of CBD was high (1111–1909*L/h) and apparent volume of distribution was large (20,963–42,849*L). CBD reached steady state after approximately 2*days, with moderate accumulation (1.8- to 2.6-fold) after 750*and 1500*mg CBD twice daily. After 7*days, a twofold increase in CBD dose resulted in 1.6- and 1.9-fold increases in geometric mean Cmax and area under the plasma concentration-time curve over a dosing interval (AUC?), respectively. CBD elimination was multiphasic; the terminal elimination half-life was approximately 60*h after 750 and 1500*mg CBD twice daily; and efective half-life estimates ranged from 10 to 17*h. Cmax was 541.2*ng/mL and AUC? was 3236*ng·h/mL after 1500*mg CBD twice daily. A high-fat meal increased CBD plasma exposure (Cmax and AUCt ) by 4.85- and 4.2-fold, respectively; there was no efect of food on tmax or terminal half-life.

Dosage Related Efficacy and Tolerability of Cannabidiol in Children With Treatment-Resistant Epileptic Encephalopathy: Preliminary Results of the CARE-E Study
Richard J. Huntsman, Richard Tang-Wai, Jane Alcorn, Stephanie Vuong, Bryan Acton, Scott Corley, Robert Laprairie, Andrew W. Lyon, Simona Meier, Darrell D. Mousseau, Doris Newmeyer, Erin Prosser-Loose, Blair Seifert, Jose Tellez-Zenteno, Linda Huh, Edward Leung and Philippe Major Front. Neurol. 10:716. (2019)
doi: 10.3389/fneur.2019.00716 https://www.frontiersin.org/articles...019.00716/full Purpose: There is uncertainty regarding the appropriate dose of Cannabidiol (CBD) for childhood epilepsy. We present the preliminary data of seven participants from the Cannabidiol in Children with Refractory Epileptic Encephalopathy (CARE-E) study.
Methods: The study is an open-label, prospective, dose-escalation trial. Participants received escalating doses of a Cannabis Herbal Extract (CHE) preparation of 1:20 19-tetrahydrocannabinol (THC): CBD up to 10–12mg CBD/kg/day. Seizure frequency was monitored in daily logs, participants underwent regular electroencephalograms, and parents filled out modified Quality of Life in Childhood Epilepsy (QOLCE) and Side Effect rating scale questionnaires. Steady-state trough levels (Css, Min) of selected cannabinoids were quantified.
Results: All seven participants tolerated the CHE up to 10–12mg CBD/kg/day and had improvements in seizure frequency and QOLCE scores. CSS,Min plasma levels for CBD, THC, and cannabichromene (CBC) showed dose-independent pharmacokinetics in all but one participant. CSS,Min CBD levels associated with a >50% reduction in seizures and seizure freedom were lower than those reported previously with purified CBD. In most patients, CSS,Min levels of THC remained lower than what would be expected to cause intoxication. Conclusion: The preliminary data suggest an initial CBD target dose of 5–6 mg/kg/day when a 1:20 THC:CBD CHE is used. Possible non-linear pharmacokinetics of CBD and CBC needs investigation. The reduction in seizure frequency seen suggests improved seizure control when a whole plant CHE is used. Plasma THC levels suggest a low risk of THC intoxication when a 1:20 THC:CBD CHE is used in doses up to 12 mg/kg CBD/kg/day.

Dose-Related Inhibition of Capsaicin Responses by Cannabinoids CBG, CBD, THC and their Combination in Cultured Sensory Neurons
Uma Anand Christian Oldfield, Barbara Pacchetti, Praveen Anand, Mikael H Sodergren
DOI: 10.2147/JPR.S336773
Journal of Pain Research 2021:14
https://www.dovepress.com/getfile.php?fileID=76128
Background: The analgesic effects of Cannabis sativa are mediated by ∆9 tetrahydrocannabinol (THC), but the contributions of other bioactive complex components, including cannabigerol (CBG) and cannabidiol (CBD), are unclear. We describe the individual and combined effects of CBG, CBD and THC, on blocking capsaicin responses in dorsal root ganglion (DRG) neurons, in an in vitro model of nociceptor hypersensitivity.
Materials and Methods: Adult rat DRG were dissected and enzyme digested to obtain a neuronal suspension in BSF2 medium containing 2% fetal calf serum, and the neurotrophic factors NGF and GDNF. After 48 h, cultured neurons were loaded with Fura-2 AM, to determine the effects of cannabinoids on capsaicin responses using calcium imaging. In control experiments, neurons were treated with vehicle, followed by 1 µM capsaicin. In cannabinoid treated cultures, CBG, CBD or THC were applied individually, or combined (1:1:1 ratio), followed by 1 µM capsaicin. Data from n = 6 experiments were analysed with Student’s t-test and Pearson’s correlation coefficient.
Results: CBG, CBD and THC, applied individually, elicited dose-related calcium influx in a subset of DRG neurons, and a corresponding dose-related reduction of subsequent responses to capsaicin. Maximum inhibition of capsaicin responses was observed at 30 µM CBG, 100 µM CBD, and 100 µM THC individually, and with combined CBD+CBG +THC (1:1:1) at 90 µM. THC+CBD+CBG combined in a 1:1:1 proportion has the potential to enhance the potency of these compounds applied individually. There was a high correlation between cannabinoid-mediated calcium influx and reduction of capsaicin responses: CBG = −0.88, THC = −0.97, CBD = −0.99 and combined CBG + THC + CBD = −1.00.
Conclusion: CBG, CBD and THC demonstrated potent dose-related inhibition of capsaicin responses in DRG neurons when applied individually in vitro, and enhanced when applied in combination, being most effective at 90 μM. Thus, efficacy and tolerability of THC could be improved in combination with CBG and CBD at optimal concentrations, which deserve further studies in vivo

Drug interactions with cannabinoids
Tony Antoniou, Jack Bodkin, Joanne M.-W. Ho
CMAJ 2020 March 2;192:E206.
doi: 10.1503/cmaj.191097
https://www.cmaj.ca/content/cmaj/192/9/E206.full.pdf
1. Cannabinoid levels can be increased by other medications
Delta-9-tetrahydrocannabinol (THC) and cannabidiol are pharmacologically active cannabinoids in marijuana that are metabolized by cytochrome P450 (CYP)3A4; THC is also metabolized by CYP2C9, a liver enzyme.1 A pharmacokinetic study found that the CYP3A4 inhibitor ketoconazole nearly doubled THC and cannabidiol concentrations,2 and similar interactions could occur with other CYP3A4 inhibitors, including macrolides and verapamil, augmenting the psychoactive effects of THC and dose-related adverse effects of cannabidiol (e.g., somnolence, transaminase elevation).1,2 CYP2C9 inhibitors such as cotrimoxazole, fluoxetine and amiodarone would also be expected to increase THC exposure and psychoactive effects.
2. Cannabinoids can affect levels of other drugs
Cannabidiol inhibits CYP2C19, increasing levels of the active metabolite of clobazam threefold.1,3 Interactions with other drugs metabolized by CYP2C19 (Appendix 1, available at www.cmaj.ca/lookup/suppl/ doi:10.1503/cmaj.191097/-/DC1) should be anticipated. Very high international normalized ratio levels and bleeding have been reported with combined used of warfarin and marijuana.1 A case reporting a threefold increase in tacrolimus levels following the addition of cannabidiol shows that CYP3A4/5 inhibition can also occur.
3. Smoking marijuana can increase clearance of some drugs
Smoked marijuana increases the clearance of theophylline 40%.1 Similar findings would be expected for other drugs metabolized by CYP1A2, such as olanzapine. Increased drug clearance occurs with regular marijuana use (> 2 marijuana cigarettes per week); no effect of occasional use has been reported. 4. Additive effects can occur with other drugs
Additive effects can occur when marijuana is combined with sympathomimetics (e.g., tachycardia, hypertension), central nervous system depressants such as alcohol and opioids (e.g., drowsiness, ataxia), and anticholinergics (e.g., tachycardia, confusion).
5. There are potential “red flag” interactions
Though further research is needed, marijuana may have serious interactions with drugs including warfarin (increased international normalized ratio and risk of bleeding); clobazam (increased risk of benzodiazepine toxicity); central nervous sytem depressants and sympathomimetics (additive effects); and theophylline, clozapine and olanzapine (reduced efficacy). Patients should be advised about possible increased cannabinoid effects with concomitant CYP3A4 and 2C9 inhibitors (Appendix 1).1,5 Alternatives that do not interact with marijuana should be selected when clinically feasible
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Medical Cannabis/Endocannabinoids part 2 E-Z. RE-ALPHABETIZED

Early evidence of the impact of cannabis legalization on cannabis use, cannabis use disorder, and the use of other substances: Findings from state policy evaluations

Rosanna Smart & Rosalie Liccardo Pacula
The American Journal of Drug and Alcohol Abuse
DOI: 10.1080/00952990.2019.1669626
Background: The past decade has seen unprecedented shifts in the cannabis policy environment, and the public health impacts of these changes will hinge on how they affect patterns of cannabis use and the use and harms associated with other substances.
Objectives: To review existing research on how state cannabis policy impacts substance use, emphasizing studies using methods for causal inference and highlighting gaps in our understanding of policy impacts on evolving cannabis markets. Methods: Narrative review of quasi-experimental studies for how medical cannabis laws (MCLs) and recreational cannabis laws (RCLs) affect cannabis use and use disorders, as well as the use of or harms from alcohol, opioids, and tobacco.
Results: Research suggests MCLs increase adult but not adolescent cannabis use, and provisions of the laws associated with less regulated supply may increase adult cannabis use disorders. These laws may reduce some opioid-related harms, while their impacts on alcohol and tobacco use remain uncertain. Research on RCLs is just emerging, but findings suggest little impact on the prevalence of adolescent cannabis use, potential increases in college student use, and unknown effects on other substance use.
Conclusions: Research on how MCLs influence cannabis use has advanced our understanding of the importance of heterogeneity in policies, populations, and market dynamics, but studies of how MCLs relate to other substance use often ignore these factors. Understanding effects of cannabis laws requires greater attention to differences in short- versus long-term effects of the laws, nuances of policies and patterns of consumption, and careful consideration of appropriate control groups.

Effect of baseline cannabis use and working-memory network function on changes in cannabis use in heavy cannabis users: a prospective fMRI study
Cousijn, J.; Wiers, R.W.; Ridderinkhof, K.Richard.; van den Brink, W.; Veltman, D.J.; Goudriaan, A.E.
Human Brain Mapping 35(5): 2470-2482. 2014.
DOI: 10.1002/hbm.22342
Theoretical models of addiction suggest that a substance use disorder represents an imbalance between hypersensitive motivational processes and deficient regulatory executive functions. Working-memory (a central executive function) may be a powerful predictor of the course of drug use and drug-related problems. Goal of the current functional magnetic resonance imaging study was to assess the predictive power of working-memory network function for future cannabis use and cannabis-related problem severity in heavy cannabis users. Tensor independent component analysis was used to investigate differences in working-memory network function between 32 heavy cannabis users and 41 nonusing controls during an N-back working-memory task. In addition, associations were examined between working-memory network function and cannabis use and problem severity at baseline and at 6-month follow-up. Behavioral performance and working-memory network function did not significantly differ between heavy cannabis users and controls. However, among heavy cannabis users, individual differences in working-memory network response had an independent effect on change in weekly cannabis use 6 months later (?R(2) = 0.11, P = 0.006, f(2) = 0.37) beyond baseline cannabis use (?R(2) = 0.41) and a behavioral measure of approach bias (?R(2) = 0.18): a stronger network response during the N-back task was related to an increase in weekly cannabis use. These findings imply that heavy cannabis users requiring greater effort to accurately complete an N-back working-memory task have a higher probability of escalating cannabis use. Working-memory network function may be a biomarker for the prediction of course and treatment outcome in cannabis users.

Effect of Cannabinoid 2 Receptor Modulation on the Peripheral Immune Response in Central Nervous System Injury-Induced Immunodeficiency Syndrome
Saki Sultana, Ian Burkovskiy, Juan Zhou, Melanie M. Kelly, and Christian Lehmann
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0130
https://www.researchgate.net/publica...iency_Syndrome
Acute central nervous system (CNS) injury, such as stroke, spinal cord injury, or traumatic brain injury can result in dysregulated immune response, and the condition is known as CNS injury-induced immunodeficiency syndrome (CIDS). The endocannabinoid system is an important homeostatic regulator in the CNS and immune system. Activation of cannabinoid 2 receptors (CB2R) on immune cells has been reported to dampen inflammation, suggesting a potential role of CB2R in the peripheral immune response following CNS injury. In this study, we have investigated the effect of CB2R modulation on the peripheral immune response during CIDS. Materials and Methods: Experimental CNS injury was induced in C57BL/6 mice through intracerebral injection of the vasopressor peptide, endothelin-1. A selective CB2R agonist (HU308) was used as an early treatment before the onset of CIDS and AM630, a selective CB2R antagonist, was administered as a later-phase therapy to combat the systemic immunodeficiency following the CNS injury. The peripheral immune response to endotoxin was studied 24 h after the CNS injury using intravital microscopy to examine leukocyte activation within the intestinal microcirculation in mice. Brain infarct size, and plasma levels of cytokines and soluble adhesion molecules were measured as additional parameters for the assessment of treatment outcomes. Results: Our results showed that early CB2R activation with HU308 reduced brain injury size and restored leukocyte response to endotoxin in the peripheral microcirculation. Late CB2R inhibition with AM630 also improved the peripheral leukocyte response to endotoxin and did not exacerbate the extent of brain injury. Discussion: CB2R activation has the potential to mitigate CNS injury as an early treatment by limiting neuroinflammation and preventing the development of CIDS. At the later stage with already-established CIDS, treatment may require dampening CB2R activation to improve the patient's outcome.

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on nocturnal sleep and early-morning behavior in young adults.
Nicholson AN1, Turner C, Stone BM, Robson PJ.
J Clin Psychopharmacol. 2004 Jun;24(3):305-13.
DOI: 10.1097/01.jcp.0000125688.05091.8f
The effects of cannabis extracts on nocturnal sleep, early-morning performance, memory, and sleepiness were studied in 8 healthy volunteers (4 males, 4 females; 21 to 34 years). The study was double-blind and placebo-controlled with a 4-way crossover design. The 4 treatments were placebo, 15 mg Delta-9-tetrahydrocannabinol (THC), 5 mg THC combined with 5 mg cannabidiol (CBD), and 15 mg THC combined with 15 mg CBD. These were formulated in 50:50 ethanol to propylene glycol and administered using an oromucosal spray during a 30-minute period from 10 pm. The electroencephalogram was recorded during the sleep period (11 pm to 7 am). Performance, sleep latency, and subjective assessments of sleepiness and mood were measured from 8:30 am (10 hours after drug administration). There were no effects of 15 mg THC on nocturnal sleep. With the concomitant administration of the drugs (5 mg THC and 5 mg CBD to 15 mg THC and 15 mg CBD), there was a decrease in stage 3 sleep, and with the higher dose combination, wakefulness was increased. The next day, with 15 mg THC, memory was impaired, sleep latency was reduced, and the subjects reported increased sleepiness and changes in mood. With the lower dose combination, reaction time was faster on the digit recall task, and with the higher dose combination, subjects reported increased sleepiness and changes in mood. Fifteen milligrams THC would appear to be sedative, while 15 mg CBD appears to have alerting properties as it increased awake activity during sleep and counteracted the residual sedative activity of 15 mg THC.

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system
Grégoire Chevalier, Eleni Siopi, Laure Guenin-Macé, Maud Pascal, Thomas Laval, Aline Rifflet, Ivo Gomperts Boneca, Caroline Demangel, Benoit Colsch, Alain Pruvost, Emeline Chu-Van, Aurélie Messager, François Leulier, Gabriel Lepousez, Gérard Eberl, & Pierre-Marie Lledo
Nature Communications Dec 2020
10.1038/s41467-020-19931-2
Depression is the leading cause of disability worldwide. Recent observations have revealed an association between mood disorders and alterations of the intestinal microbiota. Here, using unpredictable chronic mild stress (UCMS) as a mouse model of depression, we show that UCMS mice display phenotypic alterations, which could be transferred from UCMS donors to naïve recipient mice by fecal microbiota transplantation. The cellular and behavioral alterations observed in recipient mice were accompanied by a decrease in the endocannabinoid (eCB) signaling due to lower peripheral levels of fatty acid precursors of eCB ligands. The adverse effects of UCMS-transferred microbiota were alleviated by selectively enhancing the central eCB or by complementation with a strain of the Lactobacilli genus. Our findings provide a mechanistic scenario for how chronic stress, diet and gut microbiota generate a pathological feed-forward loop that contributes to despair behavior via the central eCB system.

Effect of medical cannabis on thermal quantitative measurements of pain in patients with Parkinson’s disease
A. Shohet, A. Khlebtovsky, N. Roizen, Y. Roditi, R. Djaldetti
2016 EJP
DOI: 10.1002/ejp.942
Background: Cannabis can alleviate pain of various etiologies. This study assessed the effect of cannabis on motor symptoms and pain parameters in patients with Parkinson’s disease (PD).
Methods: Twenty patients with PD who were licensed to use cannabis underwent evaluation before and 30 min after cannabis consumption and again after long-term use. Motor function was assessed with the Unified PD Rating scale (UPDRS) by two raters, one blinded. Pain was assessed with the Pain Rating Index (PRI) and Visual Analogue Scale
(VAS) of the short-form McGill Pain Questionnaire. Thermal quantitative sensory testing (QST) was performed in 18 patients. The two consecutive QST measurements were validated in 12 cannabis-na?ve patients with PD.
Results: There was a significant decrease from baseline to 30 min after cannabis consumption in mean motor UPDRS score (38.1 18 to 30.4 15.6, p < 0.0001), total PRI (27 13.5 to 9.7 11, p = 0.001), and VAS score (6.4 2.8 to 3.6 3.1, p = 0.0005). Mean cold pain threshold decreased significantly in the more affected limb, but only after exclusion of two patients who consumed cannabis by vaporizer rather than smoking (19.5 5.2 to 15.6 8.7 °C, p = 0.02). After longterm (median 14 weeks) exposure, mean heat pain threshold decreased significantly in the more affected limb in all treated patients (43.6 3.5 to 40.9 3.3 °C, p = 0.05) and in cannabis smokers (43.7 3.6 to 40.3 2.5 °C, p = 0.008).
Conclusions: Cannabis improved motor scores and pain symptoms in PD patients, together with a dissociate effect on heat and cold pain thresholds. Peripheral and central pathways are probably modulated by cannabis.
Significance: Quantitative sensory test results are significantly altered following cannabis consumption in patients with PD. Cannabis probably acts on pain in PD via peripheral and central pathways.

Effect of preoperative cannabis use on perioperative outcomes_ a retrospective cohort study. 46th Annual Regional Anesthesiology and Acute Pain Medicine Meeting
Betty Huiyu Zhang, Haris Saud, Neil Sengupta, Max Chen, Devyani Bakshi, Elizabeth Richardson, Li Wang, Harsha Shanthanna
POSTER
DOI: 10.1136/rapm-2020-101812
Potential Mechanisms of Interactions Relevant to Surgery:
• Cannabinoid receptors interact with gamma aminobutyric acid, norepinephrine and acetylcholine release, and modulates opioid and N-methyl-D-aspartate receptors.
• Concerns around anesthetic dosing of propofol, opioids, ketamine, and benzodiazepine
Design: Retrospective chart review with propensity-score matched pairs analysis.
Clinical Question: Is cannabis use associated with increased risk of postoperative complications or increased use of analgesics?
Primary Outcome: Composite of a) respiratory arrest or cardiac arrest, b) intensive care admission, c) stroke, d) myocardial infarction, and e) mortality during this hospital stay
Secondary Outcomes: pain scores, intra/postoperative opioid use, induction dose of propofol, nausea/vomiting in the post-anesthesia care unit.
Statistical Analysis: Confounders were controlled for using a propensity score-matched analysis to assess the adjusted association between cannabis use and study outcomes.
Find Pdf

Effect of surfactant concentration and sterilization process on intraocular pressure–lowering activity of ?9-tetrahydrocannabinol-valine-hemisuccinate (NB1111) nanoemulsions.
Drug Delivery and Translational Research. (2020).
doi:10.1007/s13346-020-00871-9
The use of ?9 -tetrahydrocannabinol (THC) and ?9 -tetrahydrocannabinol-valine-hemisuccinate (THC-VHS; NB1111) has recently been investigated in the management of intraocular pressure (IOP). The current study was undertaken to develop an optimized THC-VHS-loaded nanoemulsion formulation (NE; THC-VHS-NE) that could improve the drug load and duration of activity. THC-VHS-NE formulation was prepared by homogenization followed by ultrasonication. Sesame oil, Tween®80, and Poloxamer®188 were used as the oil, surfactant, and cosurfactant, respectively. Stability of the optimized THC-VHS-NE formulation was observed at 4 °C. The IOP lowering efect of the lead formulations, commercial timolol, and latanoprost ophthalmic solutions, as well as an emulsion in Tocrisolve™ (THC-VHS-TOC), was studied in New Zealand White rabbits following topical administration. The efect of surfactant concentration and sterilization process on IOP-lowering activity was also studied. THC-VHS-NE formulations (0.5, 1.0, and 2.0% w/v) showed dose dependent duration of action. The 1.0%w/v THC-VHS-NE formulation was selected for further evaluation because of its desirable physical and chemical characteristics. THC-VHS-NE formulation prepared with 2% w/v Tween®80 exhibited a higher drop in IOP than the 0.75 and 4.0% w/v of Tween®80 containing formulations. The IOP-lowering duration was, however, similar for the formulations with 0.75 and 2.0% Tween®80, while that with 4.0% Tween®80 was shorter. THC-VHS-NE formulation produced a greater drop in IOP (p <0.05) and a longer duration of activity compared to THC-VHS-TOC, latanoprost, and timolol. The formulation could be sterilized by fltration without impacting product attributes. Overall, the optimized THC-VHS-NE formulation demonstrated a signifcantly better IOP reduction profle in the test model compared to the commercial ophthalmic solutions evaluated.

COLOR="Red"]Effectiveness and tolerability of THC:CBD oromucosal spray as add-on measure in patients with severe chronic pain: analysis of 12-week open-label real-world data provided by the German Pain e-Registry[/COLOR]
Ueberall, M., Essner, U., & Mueller-Schwefe, G. H. H.
Journal of Pain Research, Volume 12, 1577–1604. (2019). doi:10.2147/jpr.s192174
Objective: To evaluate effectiveness, tolerability and safety of an oromucosal spray containing ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD), as add-on treatment in patients with severe chronic pain (SCP).
Methods: Exploratory analysis of anonymized 12-week routine/open-label data provided by the German Pain e-Registry (GPR) on adult SCP patients treated with THC:CBD oromucosal spray in 2017.
Results: Among those 30.228 cases documented in the GPR in 2017, 800 (2.6%; 57% female, mean ± SD age: 46.3±9.7 years) received a treatment with THC:CBD. All patients fulfilled the legislative preconditions for a treatment with cannabis as medicine as defined by the German Act Amending Narcotics and Other Regulations. THC:CBD-treatment was followed by an aggregated nine-factor symptom relief (ASR-9) improvement at end of week 12 vs baseline of 39.0±26.5% (95%-CI: 36.9–41.1, median: 42, range ?41 to 85). A full ASR-9 response (ie, a 50%-improvement in all 9 factors) was found for 123 patients (15.4%), while 488 patients (56.0%) presented with an ?50% improvement in at least 5 of 9 ASR factors. With a 54.9±17.2% (median: 56%, range: ?6 to 85) improvement was significantly superior in the neuropathic pain subgroup (n=497, 62.1%) vs those with mixed (n=249, 31.1%; ASR-9: 18.2±12.0, median: 19, range: ?12 to 42%) or nociceptive pain (n=54, 6.8%; ASR-9: ?11.9±10.5, median: ?11, range: ?41% to 12%; p<0.001 for each). 159 patients (19.9%) reported at least one of 206 TEAEs, most of them of mild intensity (n=81.6%). Most frequently reported TEAEs were increased appetite (n=50, 6.3%) and dysgeusia (n=23, 2.9%). TEAE-related discontinuations were reported for 32 patients (4.0%). 113 (14.1%) patients discontinued due to inadequate pain relief, most of them with nociceptive pain (n=40, 74.1%), least with neuropathic pain (n=1, 0.2%; p<0.001)
Conclusion: THC:CBD oromucosal spray proved to be an effective and well-tolerated add-on treatment for patients with elsewhere refractory chronic pain – especially of neuropathic origin.

Effects of acute cannabidiol administration on anxiety and tremors induced by a Simulated Public Speaking Test in patients with Parkinson’s disease.
De Faria, S. M., de Morais Fabrício, D., Tumas, V., Castro, P. C., Ponti, M. A., Hallak, J. E., … Chagas, M. H. N.
Journal of Psychopharmacology, (2020).
doi:10.1177/0269881119895536
Background: Cannabidiol (CBD) is one of the main components of Cannabis sativa and has anxiolytic properties, but no study has been conducted to evaluate the effects of CBD on anxiety signs and symptoms in patients with Parkinson’s disease (PD). This study aimed to evaluate the impacts of acute CBD administration at a dose of 300 mg on anxiety measures and tremors induced by a Simulated Public Speaking Test (SPST) in individuals with PD. Methods: A randomised, double-blinded, placebo-controlled, crossover clinical trial was conducted. A total of 24 individuals with PD were included and underwent two experimental sessions within a 15-day interval. After taking CBD or a placebo, participants underwent the SPST. During the test, the following data were collected: heart rate, systemic blood pressure and tremor frequency and amplitude. In addition, the Visual Analog Mood Scales (VAMS) and Self-Statements during Public Speaking Scale were applied. Statistical analysis was performed by repeated-measures analysis of variance (ANOVA) while considering the drug, SPST phase and interactions between these variables. Results: There were statistically significant differences in the VAMS anxiety factor for the drug; CBD attenuated the anxiety experimentally induced by the SPST. Repeated-measures ANOVA showed significant differences in the drug for the variable related to tremor amplitude as recorded by the accelerometer. Conclusion: Acute CBD administration at a dose of 300 mg decreased anxiety in patients with PD, and there was also decreased tremor amplitude in an anxiogenic situation.

Effects of Cannabidiol and Delta-9-Tetrahydrocannabinol on Emotion, Cognition, and Attention: A Double-Blind, Placebo-Controlled, Randomized Experimental Trial in Healthy Volunteers.
Woelfl, T., Rohleder, C., Mueller, J. K., Lange, B., Reuter, A., Schmidt, A. M., … Leweke, F. M.
Frontiers in Psychiatry, 11.(2020).
doi:10.3389/fpsyt.2020.576877
The two main phytocannabinoids—delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD)—have been extensively studied, and it has been shown that THC can induce transient psychosis. At the same time, CBD appears to have no psychotomimetic potential. On the contrary, emerging evidence for CBD’s antipsychotic properties suggests that it may attenuate effects induced by THC. Thus, we investigated and compared the effects of THC and CBD administration on emotion, cognition, and attention as well as the impact of CBD pre-treatment on THC effects in healthy volunteers. We performed a placebo-controlled, double-blind, experimental trial (GEI-TCP II; ClinicalTrials.gov identifier: NCT02487381) with 60 healthy volunteers randomly allocated to four parallel intervention groups, receiving either placebo, 800 mg CBD, 20 mg THC, or both cannabinoids. Subjects underwent neuropsychological tests assessing working memory (Letter Number Sequencing test), cognitive processing speed (Digit Symbol Coding task), attention (d2 Test of Attention), and emotional state (adjective mood rating scale [EWL]). Administration of CBD alone did not influence the emotional state, cognitive performance, and attention. At the same time, THC affected two of six emotional categories—more precisely, the performance-related activity and extraversion—, reduced the cognitive processing speed and impaired the performance on the d2 Test of Attention. Interestingly, pre-treatment with CBD did not attenuate the effects induced by THC. These findings show that the acute intake of CBD itself has no effect per se in healthy volunteers and that a single dose of CBD prior to THC administration was insufficient to mitigate the detrimental impact of THC in the given setting. This is in support of a complex interaction between CBD and THC whose effects are not counterbalanced by CBD under all circumstances.

Effects of Cannabidiol Chewing Gum on Perceived Pain and Well-Being of Irritable Bowel Syndrome Patients: A Placebo-Controlled Crossover Exploratory Intervention Study with Symptom-Driven Dosing
Anne-Claire B, van Orten-Luiten, Nicole M. de Roos, Soumia Majait, Ben J.M. Witteman, and Renger F. Witkamp
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0087
https://www.liebertpub.com/doi/pdf/1.../can.2020.0087
Background: Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders. Its pathophysiology is diverse and variable, involving disturbed gut–brain interactions, altered motility and secretion, visceral hypersensitivity, increased intestinal permeability, immune activation, and changes in gut microbiota. Complaints experienced by patients suffering from IBS and its co-morbidities strongly impair quality of life (QoL), and available treatments are often unsatisfactory. Anecdotal reports and preclinical data suggest that the endocannabinoid system and functionally related mechanisms could offer treatment targets. Cannabidiol (CBD) is a candidate agent of interest with a broad molecular target profile and the absence of psychoactive properties.
Materials and Methods: In 32 female IBS patients, we explored the effect of a chewing gum formulation containing 50 mg CBD on abdominal pain and perceived well-being in a randomized, double-blinded, placebocontrolled cross-over trial. Chewing gums were used on-demand guided by pain symptoms with a maximum of six per day. Pain intensity was assessed by a visual analogue scale (scale 0.0–10.0), and QoL was evaluated with the IBS-36 questionnaire.
Results: There was no statistically significant difference in pain scores between CBD and placebo at a group level. Subgroup and individual analyses showed a highly variable picture. No indications were found for symptomdriven intake, which also remained lower than expected overall.
Conclusions: With the current design, based on the assumption that IBS patients would adjust their intake to their perceived symptom relief, no differences at the group level were found between CBD and placebo gum in pain scores and the number of gums used. The low use of the gums also indicates that the benefits experienced by these patients generally did not outweigh practical disadvantages such as prolonged chewing throughout the day. The very high intra- and inter-individual variation in IBS symptoms warrant future trials that are more personalized, for example by applying an N-of-1 (rotating) design with individualized dose titration.

Effects of cannabidiol in cannabis flower: Implications for harm reduction
Laurel P Gibson, Hollis C Karoly, Jarrod M Ellingson, Jost Klawitter, Cristina Sempio, Julia E Squeri, Angela D Bryan, L Cinnamon Bidwell , Kent E Hutchison
Addict Biol. 2021 Aug 31;e13092.
doi: 10.1111/adb.13092
Using a federally compatible, naturalistic at-home administration procedure, the present study examined the acute effects of three cannabis flower chemovars with different tetrahydrocannabinol (THC) to cannabidiol (CBD) ratios, in order to test whether chemovars with a higher CBD content produce different effects. Participants were randomly assigned to ad libitum administration of one of three chemovars (THC-dominant: 24% THC, 1% CBD; THC+CBD: 9% THC, 10% CBD; CBD-dominant: 1% THC, 23% CBD); 159 regular cannabis users (male = 94, female = 65) were assessed in a mobile pharmacology lab before, immediately after, and 1 h after ad libitum administration of their assigned chemovar. Plasma cannabinoids as well as positive (e.g., high, elation) and negative (e.g., paranoia and anxiety) subjective effects were assessed at each time points. Participants who used the CBD-dominant and THC + CBD chemovars had significantly less THC and more CBD in plasma samples compared to participants who used the THC-dominant chemovar. Further, the THC + CBD chemovar was associated with similar levels of positive subjective effects, but significantly less paranoia and anxiety, as compared to the THC-dominant chemovar. This is one of the first studies to examine the differential effects of various THC to CBD ratios using chemovars that are widely available in state-regulated markets. Individuals using a THC + CBD chemovar had significantly lower plasma THC concentrations and reported less paranoia and anxiety while also reporting similar positive mood effects as compared to individuals using THC only, which is intriguing from a harm reduction perspective. Further research is needed to clarify the harm reduction potential of CBD in cannabis products.
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Effects of cannabidiol on simulated driving and cognitive performance: A dose-ranging randomised controlled trial
Danielle McCartney, Anastasia S Suraev, Peter T Doohan, Christopher Irwin, Richard C Kevin, Ronald R Grunstein, Camilla M Hoyos, and Iain S McGregor
Journal of Psychopharmacology 1–12
DOI: 10.1117/02698811221095356
Background: Cannabidiol (CBD), a major cannabinoid of Cannabis sativa, is widely consumed in prescription and non-prescription products. While CBD is generally considered ‘non-intoxicating’, its effects on safety-sensitive tasks are still under scrutiny.
Aim: We investigated the effects of CBD on driving performance.
Methods: Healthy adults (n=17) completed four treatment sessions involving the oral administration of a placebo, or 15, 300 or 1500mg CBD in a randomised, double-blind, crossover design. Simulated driving performance was assessed between ~45–75 and ~210–240min post-treatment (Drives 1 and 2) using a two-part scenario with ‘standard’ and ‘car following’ (CF) components. The primary outcome was standard deviation of lateral position (SDLP), a well-established measure of vehicular control. Cognitive function, subjective experiences and plasma CBD concentrations were also measured. Non-inferiority analyses tested the hypothesis that CBD would not increase SDLP by more than a margin equivalent to a 0.05% blood alcohol concentration (Cohen’s dz =0.50).
Results: Non-inferiority was established during the standard component of Drive 1 and CF component of Drive 2 on all CBD treatments and during the standard component of Drive 2 on the 15 and 1500mg treatments (95% CIs<0.5). The remaining comparisons to placebo were inconclusive (the 95% CIs included 0 and 0.50). No dose of CBD impaired cognition or induced feelings of intoxication (ps>0.05). CBD was unexpectedly found to persist in plasma for prolonged periods of time (e.g. >4weeks at 1500mg).
Conclusion: Acute, oral CBD treatment does not appear to induce feelings of intoxication and is unlikely to impair cognitive function or driving performance

Effects of cannabinoid receptor type 2 in respiratory syncytial virus infection in human subjects and mice.
Tahamtan, A., Samieipoor, Y., Nayeri, F. S., Rahbarimanesh, A. A., Izadi, A., Rashidi-Nezhad, A., … Salimi, V.
Virulence, 9(1), 217–230.(2017).
doi:10.1080/21505594.2017.1389369
An accumulating body of evidence suggests that the endocannabinoid system plays a significant role in pathophysiological processes and impacts disease severity. Here we investigate the possible role of a cannabinoid receptor type 2 (CB2) functional variant in determining disease severity and the potential pharmacological therapeutic effects of CB2 activation in viral respiratory infection. The common missense variant (CAA/CGG; Q63R) of the gene-encoding CB2 receptor (CNR2) was evaluated in 90 inpatient and 90 outpatient children with acute respiratory tract infection (ARTI). The frequency distribution of respiratory syncytial virus (RSV)-the main cause of severe cases of bronchiolitis and pneumonia in children-was studied in all collected samples. The mechanism through which CB2 affects clinical outcomes in case of RSV infection was studied in Balb/c mice model using AM630 as a CB2 antagonist. The potential therapeutic effect of CB2 activation during RSV infection was studied using a selective agonist, JWH133. The CB2 Q63R variation was associated with increased risk of hospitalization in children with ARTI. Children carrying the QQ genotype were more prone to developing severe ARTI (OR=3.275, 95% CI: 1.221-8.705; p=0.019). Of all the children enrolled in the study, 83 patients (46.1%) were found positive for RSV infection. The associated risk of developing severe ARTI following RSV infection increased more than two-fold in children carrying the Q allele (OR=2.148, 95% CI: 1.092-4.224; p=0.026). In mice, the blockade of CB2 by AM630 during RSV infection enhanced the influx of BAL cells and production of cytokines/chemokines while exaggerating lung pathology. CB2 activation by JWH133 reduces the influx of BAL cells and production of cytokines/chemokines while alleviating lung pathology. Collectively, CB2 is associated with RSV severity during infancy and may serve as a therapeutic target in RSV infection through the alleviation of virus-associated immunopathology.

Effects of cannabidivarin (CBDV) on brain excitation and inhibition systems in adults with and without Autism Spectrum Disorder (ASD): a single dose trial during magnetic resonance spectroscopy
Charlotte M Pretzsch, Bogdan Voinescu, David Lythgoe, Jamie Horder, Maria Andreina Mendez, Robert Wichers, Laura Ajram, Glynis Ivin, Martin Heasman, Richard A. E. Edden, Steven Williams, Declan G. M. Murphy, Eileen Daly and Gráinne M. McAlonan
Translational Psychiatry ( 2019) 9:313
DOI: 10.1038/s41398-019-0654-8
https://www.nature.com/articles/s41398-019-0654-8.pdf
Autism spectrum disorder (ASD) is a high cost neurodevelopmental condition; and there are currently no effective pharmacological treatments for its core symptoms. This has led some families and researchers to trial alternative remedies – including the non-intoxicating Cannabis sativa-derived compound cannabidivarin (CBDV). However, how CBDV affects the human brain is unknown. Previous (pre)clinical evidence suggests that CBDV may modulate brain excitatory-inhibitory systems, which are implicated in ASD. Hence, our main aim was to test, for the fi rst time, if CBDV shifts glutamate and/or GABA metabolites – markers of the brain’ s primary excitatory and inhibitory system - in both the ‘ typical’ and autistic brain. Our subsidiary aim was to determine whether, within ASD, brain responsivity to CBDV
challenge is related to baseline biological phenotype. We tested this using a repeated-measures, double-blind, randomized-order, cross-over design. We used magnetic resonance spectroscopy (MRS) to compare glutamate (Glx =
glutamate + glutamine) and GABA + (GABA + macromolecules) levels following placebo (baseline) and 600 mg CBDV in 34 healthy men with (n = 17) and without (n = 17) ASD. Data acquisition from regions previously reliably linked to ASD (dorsomedial prefrontal cortex, DMPFC; left basal ganglia, BG) commenced 2 h (peak plasma levels) after placebo/CBDV administration. Where CBDV signifi cantly shifted metabolite levels, we examined the relationship of this change with baseline metabolite levels. Test sessions were at least 13 days apart to ensure CBDV wash-out. CBDV signifi cantly increased Glx in the BG of both groups. However, this impact was not uniform across individuals. In the ASD group, and not in the typically developing controls, the ‘ shift’ in Glx correlated negatively with baseline Glx concentration. In contrast, CBDV had no signifi cant impact on Glx in the DMPFC, or on GABA+ in either voxel in either group. Our findings suggest that, as measured by MRS, CBDV modulates the glutamate-GABA system in the BG but not in frontal regions. Moreover, there is individual variation in response depending on baseline biochemistry. Future studies should examine the effect of CBDV on behaviour and if the response to an acute dose of CBDV could predict a potential clinical treatment response in ASD.

Effects of cold plasma, gamma and e-beam irradiations on reduction of fungal colony forming unit levels in medical cannabis inflorescences
Shachar Jerushalmi, Marcel Maymon, Aviv Dombrovsky and Stanley Freeman
Journal of Cannabis Research (2020) 2:12
doi: 10.1186/s42238-020-00020-6
Background: The use of medical cannabis (MC) in the medical field has been expanding over the last decade, as more therapeutic beneficial properties of MC are discovered, ranging from general analgesics to anti-inflammatory and anti-bacterial treatments. Together with the intensified utilization of MC, concerns regarding the safety of usage, especially in immunocompromised patients, have arisen. Similar to other plants, MC may be infected by fungal plant pathogens (molds) that sporulate in the tissues while other fungal spores (nonpathogenic) may be present at high concentrations in MC inflorescences, causing a health hazard when inhaled. Since MC is not grown under sterile conditions, it is crucial to evaluate current available methods for reduction of molds in inflorescences that will not damage the active compounds. Three different sterilization methods of inflorescences were examined in this research; gamma irradiation, beta irradiation (e-beam) and cold plasma to determine their efficacy in reduction of fungal colony forming units (CFUs) in vivo.
Methods: The examined methods were evaluated for decontamination of both uninoculated and artificially inoculated Botrytis cinerea MC inflorescences, by assessing total yeast and mold (TYM) CFU levels per g plant tissue. In addition, e-beam treatment was also tested on naturally infected commercial MC inflorescences.
Results: All tested methods significantly reduced TYM CFUs at the tested dosages. Gamma irradiation reduced CFU levels by approximately 6- and 4.5-log fold, in uninoculated and artificially inoculated B. cinerea MC inflorescences, respectively. The effective dosage for elimination of 50% (ED50)TYM CFU of uninoculated MC inflorescence treated with e-beam was calculated as 3.6 KGy. In naturally infected commercial MC inflorescences, e-beam treatments reduced TYM CFU levels by approximately 5-log-fold. A 10 min exposure to cold plasma treatment resulted in 5- log-fold reduction in TYM CFU levels in both uninoculated and artificially inoculated B. cinerea MC inflorescences.
Conclusions: Although gamma irradiation was very effective in reducing TYM CFU levels, it is the most expensive and complicated method for MC sterilization. Both e-beam and cold plasma treatments have greater potential since they are cheaper and simpler to apply, and are equally effective for MC sterilization.

Effects of dietary hemp seed oil to sows on fatty acid profiles, nutritional and immune status of piglets.
Vodolazska, D., & Lauridsen, C.
Journal of Animal Science and Biotechnology, 11(1). (2020).
Effects of dietary hemp seed oil to sows doi:10.1186/s40104-020-0429-3
Background: The oil from industrial hemp seeds (Cannabis sativa) is an ideal source of stearidonic acid, which is a precursor fatty acid for the long-chained n-3 polyunsaturated fatty acids. These fatty acids are important for neonatal development, health and immunity. Hemp seed oil has been investigated for the influence on human health, but research on the impact in pig nutrition is scarce. The aim of our research was to study the effect of dietary hemp seed oil relative to soybean oil to lactating sows on the transfer of fatty acids to the off-spring and the effect on piglets’ immune and nutritional status. Results: The fatty acid composition of the hemp seed and the soybean oil influenced the fatty acid composition of sow plasma, colostrum and mature milk. The highest proportion of C18:3n-3, C18:4n-3 and C20:4n-6 was obtained in mature milk fat of sows fed 5% hemp seed oil diet when compared to the other dietary fat sources (5% soybean oil or a 50:50 mix of hemp and soybean oil at 5%). The effect of dietary oil supplementation to sows was reflected in the plasma fatty acids profile of piglets. Notably the proportion of C20:5n-3 and C22:5n-3 was the highest in plasma of piglets suckling sows fed hemp seed oil-containing diets, whereas no C18:4n-3 could be detected hence indicating conversion of ?-linolenic acid (ALA) and stearidonic acid (SDA) to the longer chained n-3 polyunsaturated fatty acids. Dietary fat source also influenced number of born piglets, their weight gain during first week, plasma concentration of glucose and IgG, and haematological profile. Conclusions: The hemp seed oil resulted in direct maternal supply with n-3 long-chain polyunsaturated fatty acids (LCPUFA), especially ALA and SDA, and piglets were able to convert these fatty acids obtained via the sow milk intake to C20:5n-3 and C22:5n-3. Furthermore, some interesting effects of the 5% hemp seed oil was obtained with regard to piglet initial body weight gain and glucose, which could be of interest for further research, i.e., the capability of hemp seed oil to benefit piglets during early life

Effects of High-Potency Cannabis on Psychomotor Performance in Frequent Cannabis Users.
Karoly, H. C., Milburn, M. A., Brooks-Russell, A., Brown, M., Streufert, J., Bryan, A. D., … Bidwell, L. C.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2020.0048
Background: Recently increased access to cannabis products in the United States has been associated with increased rates of driving after cannabis use. Although numerous studies indicate that cannabis impairs psychomotor and neurocognitive functions that can affect driving ability, the determination of cannabis-impaired driving risk is complicated by the extent to which frequent cannabis users develop tolerance to THC’s subjective, cognitive, and psychomotor effects, and by the fact that there is no validated behavioral or biological marker of recent cannabis use or cannabis-related impairment. This study examined the psychomotor impairment-related effects experienced by frequent cannabis users in Colorado after naturalistic consumption of smoked cannabis, both immediately and 1 h postuse.
Results were then validated in a smaller replication sample from Washington state.
Methods: In the primary Colorado study, participants (n = 70) used the DRUID mobile app, a brief measure of psychomotor and cognitive domains that are sensitive to the effects of cannabis. First, participants used DRUID to establish a sober baseline impairment score. During a second appointment, they used DRUID at three time points: preuse, immediately after acutely using cannabis, and 1 h postuse. In the Washington replication sample, participants (n = 39) used DRUID before acute cannabis consumption and then every half hour for 2.5 h. Results: In both studies, peak DRUID impairment effects were seen immediately after cannabis use, with recovery of performance at 1 h postuse. Specifically, significant quadratic effects of time emerged for both studies (Colorado study: (b = 0.935, SE = 0.204, p < 0.001); Washington study: b = 3.0299, SE = 1.3085, p < 0.01). Domain-specific effects were tested in the larger Colorado study and were observed for reaction time within a complex divided attention task and a postural-stability balance task. Conclusions: These findings demonstrate that psychomotor impairment emerges immediately after acute cannabis use even in regular users, but decreases significantly 1 h postuse. These results underscore the potential utility of the DRUID app for assessing acute cannabis-related psychomotor impairment. Further research is needed to explore whether the DRUID app and/or the specific psychomotor functions it assesses might serve as a tool for measuring cannabis-related driving impairment. Clinical trials registration number for the Colorado Study: NCT03522103.

Effects of long term storage on secondary metabolite profiles of cannabis resin.
Grafström K, Andersson K, Pettersson N, Dalgaard J, Dunne SJ
Forensic Sci Int. 2019 Aug;301:331-340.
doi: 10.1016/j.forsciint.2019.05.035. Epub 2019 May 25.
The structural identification and the monitoring of the relative concentrations of a wide range of major (3) and minor secondary (16) metabolites used as marker substances for profiling of cannabis resin using GC-FID at the Swedish National Forensic Centre (NFC) has facilitated the mapping of their chemical and physical behaviors over a period of 48months whilst stored under different conditions (exposure to light, exposure to air, temperature). In all cases the behavior of this group of sesquiterpenes, sesquiterpenoids, cannabinoids and waxes could be directly related to their chemical lability/functionality. In particular, the identification of homologue triads for both ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD) together with a group of seemingly chemically inert substances (for example, cannabicyclol(CBL) and the waxes (n-alkanes)) has created new tools for the establishment of common origins between samples of cannabisresins aged under different conditions. Since sampling of the resin blocks in NFC's method for profiling of cannabis resin is made below the surface, the effects of light incursion were found to be negligible. The effects of exposure to air (and indirectly temperature) were found to be more significant, not unexpectedly as many of the observed transformations were based on oxidation or rearrangement processes.

EFFECTS OF MARIHUANA IN LABORATORY ANIMALS AND IN MAN.
CARLINI, E. A., KARNIOL, I. G., RENAULT, P. F., & SCHUSTER, C. R.
British Journal of Pharmacology, 50(2), 299–309. (1974).
doi:10.1111/j.1476-5381.1974.tb08576.x
1 The pharmacological potencies of the resins from three different samples of Brazilian marihuana (A, B and C) were determined through corneal areflexia in rabbits, decrease of spontaneous motor activity and induction of catatonia in mice, and decrease of rope climbing performance of rats. 2 The A9 -tetrahydrocannabinol (A9 THC) content of the marihuanas, measured by gas chromatography, was 0.82, 2.02 and 0.52%, respectively, for samples A, B and C. Approximately 2% cannabinol was present in samples A and B whereas the content of cannabidiol was approximately 0.1%. 3 The petroleum ether extraction of the samples A, B and C yielded, respectively, 12.06, 14.56 and 4.26% of resin. 4 In all animal tests resin B was nearly twice as active as resin A, whereas C was the weakest. 5 The smoke of the marihuana samples was inhaled by 33 human subjects, under a double-blind standardized procedure. Pulse rate, a time production task and an evaluation of psychological effects were recorded. 6 The smoke of 250 mg of sample B provoked disruption of the time production task, increased pulse rate, and induced strong psychological reactions in four of the six subjects who received it. Similar effects, although slightly smaller, were obtained with 500 mg of sample A. On the other hand, 500 mg of sample C did not differ from placebo. 7 It is suggested that it is possible by means of animal tests to predict the potency of a marihuana sample in man. 8 In parallel experiments, A9-THC was administered to other human subjects and to laboratory animals in a manner similar to that in which the marihuana samples were administered. 9 Comparison of the results between the marihuanas and A9 -THC showed that in man and in the laboratory animals marihuanas A and B induced effects two to four times greater than expected from their A9 -THC content. 10 It is suggested that there may be potentiation of the effects of A9 -THC by other substances

Effects of non-euphoric plant cannabinoids on muscle quality and performance of dystrophic mdx mice
Fabio Arturo Iannotti ,Ester Pagano, Aniello Schiano Moriello, Filomena Gra zia Alvino, Nicolina Cristina Sorrentino, LucaD’Orsi, Elisabetta Gazzerro, Raffaele Capasso, Elvira De Leonibus, Luciano De Petrocellis and Vincenzo Di Marzo
British Journal of Pharmacology 176(10) August 2018
DOI: 10.1111/bph.14460
https://www.researchgate.net/publica...ophic_mdx_mice
Background and purpose: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy leads to the accumulation of damaged intracellular organelles that greatly contribute to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, including cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV) to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo. Key results: We found that CBD and CBDV promote the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. CBD and CBDV also promoted the differentiation of myoblasts from DMD donors. In primary cultures prepared from satellite cells isolated from healthy donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case mostly via TRPA1 activation. In mdx mice, CBD (60 mg Kg-1 ), CBDV (60 mg Kg-1 ) prevented the loss of locomotor activity at two distinct ages (from 5 to 7 and 32 to 34 weeks of age). This effect was associated with a reduction in tissue and plasma pro-inflammatory markers, together with the restoration of autophagy. Conclusion and implications: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients.

Effects of oral, smoked, and vaporized cannabis on endocrine pathways related to appetite and metabolism: a randomized, double-blind, placebo-controlled, human laboratory study.
Farokhnia, M., McDiarmid, G. R., Newmeyer, M. N., Munjal, V., Abulseoud, O. A., Huestis, M. A., & Leggio, L.
Translational Psychiatry, 10(1). (2020).
doi:10.1038/s41398-020-0756-3
As perspectives on cannabis continue to shift, understanding the physiological and behavioral effects of cannabis use is of paramount importance. Previous data suggest that cannabis use influences food intake, appetite, and metabolism, yet human research in this regard remains scant. The present study investigated the effects of cannabis administration, via different routes, on peripheral concentrations of appetitive and metabolic hormones in a sample of cannabis users. This was a randomized, crossover, double-blind, placebo-controlled study. Twenty participants underwent four experimental sessions during which oral cannabis, smoked cannabis, vaporized cannabis, or placebo was administered. Active compounds contained 6.9 ± 0.95% (~50.6 mg) ?9-tetrahydrocannabinol (THC). Repeated blood samples were obtained, and the following endocrine markers were measured: total ghrelin, acyl-ghrelin, leptin, glucagon-like peptide-1 (GLP-1), and insulin. Results showed a significant drug main effect (p = 0.001), as well as a significant drug × time-point interaction effect (p = 0.01) on insulin. The spike in blood insulin concentrations observed under the placebo condition (probably due to the intake of brownie) was blunted by cannabis administration. A significant drug main effect (p = 0.001), as well as a trend-level drug × time-point interaction effect (p = 0.08) was also detected for GLP-1, suggesting that GLP-1 concentrations were lower under cannabis, compared to the placebo condition. Finally, a significant drug main effect (p = 0.01) was found for total ghrelin, suggesting that total ghrelin concentrations during the oral cannabis session were higher than the smoked and vaporized cannabis sessions. In conclusion, cannabis administration in this study modulated blood concentrations of some appetitive and metabolic hormones, chiefly insulin, in cannabis users. Understanding the mechanisms underpinning these effects may provide additional information on the cross-talk between cannabinoids and physiological pathways related to appetite and metabolism.

Effects of Smoked Cannabis and Oral ?9-Tetrahydrocannabinol on Nausea and Emesis After Cancer Chemotherapy: A Review of State Clinical Trials
Richard E. Musty Rita Rossi
Journal of Cannabis Therapeutics, Vol. 1(1) 2001
https://www.cannabis-med.org/data/pdf/2001-01-2.pdf
Background. In 1999 the Institute of Medicine (IOM) issued a report entitled Marijuana and Medicine (Joy, Watson and Benson, 1999). It recommended the development of cannabinoid drug delivery systems which might be effective for nausea, vomiting and AIDS wasting syndrome, among other chronic disorders. The report went on to recognize that patients should be allowed to smoke marijuana if they failed to achieve relief from approved symptoms that could be relieved by cannabinoid drugs with rapid onset. Recommended criteria of the report included: access to marijuana within 24 hours of submission by a physician, supervision that allows for assessment of treatment effectiveness, and an oversight strategy comparable to an institutional review board. In this context a review of previously unpublished state-run clinical trials with Cannabis sativa (marijuana and/ or ?9-tetrahydrocannabinol capsules) to test efficacy in reducing nausea and vomiting following cancer chemotherapy is warranted. The impetus for these studies came from individual state legislatures responding to constituents’ claims that smoking marijuana reduced or blocked nausea and vomiting. Methods. Technical reports were obtained from 6 states which had conducted clinical trials. Each protocol was examined for the procedure used, the experimental design of the clinical trial and the results obtained. Data were available on 748 patients who smoked marijuana prior to and/or after cancer chemotherapy and 345 patients who used the oral THC capsule.
Results. Patients who smoked marijuana experienced 70-100% relief from nausea and vomiting, while those who used the THC capsule
experienced 76-88% relief.
Conclusions. On the basis of these studies, it appears that smoked marijuana can be a very successful treatment for nausea and vomitingfollowing cancer chemotherapy. The development of smokeless inhalation devices could certainly reduce yhe potential harm from smoking marijuana.

Effects of standardized Cannabis sativa extract and ionizing radiation in melanoma cells in vitro
Jamal Naderi1, Nasim Dana1, Shaghayegh Haghjooy Javanmard1, Alireza Amooheidari2, Maryam Yahay1, Golnaz Vasegh
Journal of Cancer Research and Therapeutics 2020 Vol 16 Issue 6
https://www.cancerjournal.net/articl...;aulast=Naderi
Context: Melanoma causes the highest number of skin cancer-related deaths worldwide. New treatment methods are essential for the management of this life-threatening disease.
Aims: In this study, we investigated the efficacy of a standardized Cannabis sativa extract alone or in combination with single radiation dose (6 Gy) in B16F10 mouse melanoma cells in an extract dose-dependent manner.
Materials and Methods: C. sativa extract at three concentrations (25, 12.5, and 6.25 ?g/mL) alone for 72 h or in combination with radiation (24 h incubation after the extract treatment + 48 h incubation after exposure to radiation) were evaluated for cell viability of melanoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells were also treated with 6.25 ?g/mL extract alone for 72 h before analyzing C. sativa-induced cell death by flow cytometry.
Results: Administration of the extract alone or alongside radiation substantially inhibited melanoma cell viability and proliferation in the extract dose response-dependent manner. The inhibition of melanoma cell viability was paralleled by an increase in necrosis but not apoptosis when melanoma cells were treated with the extract alone. Radiation alone did not have any antiproliferative effects, and radiation also did not synergize antiproliferative effects of the extract when the extract and radiation were combined.
Conclusion: Our data suggest that C. sativa extract may have significant health and physiological implications for the treatment of melanoma. The results of this study also indicate that B16F10 mouse melanoma cells are radioresistant. Taken together, these findings may lead to the identification of new therapeutic strategy for the management of melanoma.

Effects of systemic endocannabinoid manipulation on social and exploratory behavior in prairie voles (Microtus ochrogaster).
Simmons, T. C., Singh, A. L. K., & Bales, K. L.
Psychopharmacology. (2020).
doi:10.1007/s00213-020-05683-w
Rationale Anandamide is an endocannabinoid that contributes to certain aspects of social behavior, like play and reward, by binding to cannabinoid receptor type 1 (CB1). Most interesting is the recent discovery that anandamide may be mobilized by oxytocin receptor activation under certain contexts, particularly in the nucleus accumbens. Objectives Given the established role of oxytocin and the nucleus accumbens in the neurobiology of pair-bonding, we investigated whether systemic administration of brain-permeable modulators of the endocannabinoid system could alter preferential partner contact in both male and female prairie voles. Methods Specifically, we tested whether intraperitoneal administration of the neutral CB1 antagonist AM4113 (4.0–16.0 mg/kg) or the anandamide hydrolysis inhibitor URB597 (5.0–20.0 mg/kg) could prevent or facilitate partner preference formation, respectively. To further investigate the specificity of effects on partner preference, we repeated our URB597 dosing regimen on an additional group of females and tested their anxiety-related behavior in both an elevated-plus maze and a light/dark test. Results AM4113 administration had no effect on partner preference. But while URB597 also had no effect on partner preference, low-dose females did increase absolute preferential contact with either the partner or the stranger; individual females spent significant contact time with either the partner or the stranger. None of our outcome measures in either anxiety test showed significant effects of treatment. Conclusions Our results reveal that experimentally increasing anandamide levels in female prairie voles can increase social contact with both a familiar and novel male via unknown mechanisms that are likely separate from anxiety reduction.

Effects of tetrahydrocannabinols on human oral cancer cell proliferation, apoptosis, autophagy, oxidative stress, and DNA damage
Abdelhabib Semlali , Sarra Beji, , Ikram Ajala , Mahmoud Rouabhia
Arch Oral Biol. Volume 129, September 2021
DOI: 10.1016/j.archoralbio.2021.105200
Objective: Cannabinoids, including delta-8- and delta-9-tetrahydrocannabinol (THC) have a palliative care impact and may therefore be beneficial against cancer. The aim of this study was to investigate the effect of Δ9-THC and Δ8-THC on oral cancer cell behaviors.
Design: The Ca9-22 oral cancer cells were cultured in the presence or not of various concentrations of Δ9-THC and Δ8-THC for different times. The cultures were then used to measure cell viability/proliferation, apoptosis, autophagy, oxidative stress, antioxidant activity, and inhibition of signaling pathways MAP-Kinase, NF-κB, and β-catenin.
Results: Both cannabinoids were found to decrease cell viability/proliferation by blocking the cell cycle progression from the S to the G2/M phase and enhancing their apoptosis and autophagy. Δ9-THC and Δ8-THC also suppressed the migration/invasion by inhibiting epithelial-mesenchymal transition markers, such as E-cadherin, in addition to decreasing reactive oxygen species (ROS) production and increasing glutathione (GSH) and the expression of mtMP. Δ9-THC and Δ8-THC also downregulated cyclin D1, p53, NOXA, PUMAα, and DRAM expressions but increased p21 and H2AX expression.
Conclusion: We demonstrated that cannabinoids (Δ9-THC and Δ8-THC) were able to decrease oral cancer cell growth through various mechanisms, including apoptosis, autophagy, and oxidative stress. These results suggest a potential use of these molecules as a therapy against oral cancer.

Effects of therapeutic cannabis on simulated driving: A pilot study
Patricia Di Ciano, Ana Matamoros, Hayley A Hamilton, Robert E Mann, Patrick A Byrne, Andrew Fares, Christine M Wickens, Tara Marie Watson, Bernard Le Foll, Bruna Brands, Justin Matheson, Ana Matamoros, Patricia Di Ciano
Journal of Concurrent Disorders Vol. (TBD) No. (TBD), 2020 (pp. TBD)
FIND DOI:
Background: Although medical cannabis has been available to Canadians since 2001, there is little research on the effects of cannabis on driving in individuals who use cannabis medically. This pilot study sought to determine the effects of therapeutic cannabis use on simulated driving.
Methods: Eligible participants reported daily use of cannabis for therapeutic purposes, with a medical authorization. Prior to the test session, participants were asked not to smoke their regular dose. Participants (n=14) completed self-report questionnaires, including subjective effects questionnaires (visual analog scales), the Addiction Research Centre Inventory (ARCI), and Profile of Mood States (POMS), and provided blood (for determination of THC and metabolites). They also drove a simulator both before and after smoking their usual daily dose of cannabis. Outcome measures on simulated driving consisted of overall mean speed, straightaway mean speed, straightaway lateral control, and brake latency. Speed and lateral control were also measured under cognitive load.
Results: After smoking cannabis, overall mean speed was reduced. No effects of therapeutic cannabis were found on straightaway mean speed or straightaway lateral control for either condition (standard or cognitive load) or on brake latency. After smoking therapeutic cannabis in the lab, changes in speed and lateral control were negatively correlated with the amount of cannabis smoked per day. Prior to smoking therapeutic cannabis in the lab, under baseline conditions, speed and lateral control under cognitive load were also correlated with the amount of cannabis used per day. Therapeutic cannabis use increased subjective reports and blood levels of THC and metabolites. Conclusions: The present study suggests that, even with repeated daily use, cannabis consumption among therapeutic users may alter driving behavior. This has implications for road safety and use of cannabis for therapeutic purposes

Efficacy and Safety of Cannabidiol and Tetrahydrocannabivarin on Glycemic and Lipid Parameters in Patients With Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled, Parallel Group Pilot Study.
Jadoon, K. A., Ratcliffe, S. H., Barrett, D. A., Thomas, E. L., Stott, C., Bell, J. D., … Tan, G. D.
Diabetes Care, 39(10), 1777–1786.(2016).
doi:10.2337/dc16-0650
OBJECTIVE Cannabidiol (CBD) and D9 -tetrahydrocannabivarin (THCV) are nonpsychoactive phytocannabinoids affecting lipid and glucose metabolism in animal models. This study set out to examine the effects of these compounds in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS In this randomized, double-blind, placebo-controlled study, 62 subjects with noninsulin-treated type 2 diabetes were randomized to five treatment arms: CBD (100 mg twice daily), THCV (5 mg twice daily), 1:1 ratio of CBD and THCV (5 mg/5 mg, twice daily), 20:1 ratio of CBD and THCV (100 mg/5 mg, twice daily), or matched placebo for 13 weeks. The primary end point was a change in HDLcholesterol concentrations from baseline. Secondary/tertiary end points included changes in glycemic control, lipid profile, insulin sensitivity, body weight, liver triglyceride content, adipose tissue distribution, appetite, markers of inflammation, markers of vascular function, gut hormones, circulating endocannabinoids, and adipokine concentrations. Safety and tolerability end points were also evaluated. RESULTS Compared with placebo, THCV significantly decreased fasting plasma glucose (estimated treatment difference [ETD] = 21.2 mmol/L; P < 0.05) and improved pancreatic b-cell function (HOMA2 b-cell function [ETD = 244.51 points; P < 0.01]), adiponectin (ETD = 25.9 3 106 pg/mL; P < 0.01), and apolipoprotein A (ETD = 26.02 mmol/L; P < 0.05), although plasma HDL was unaffected. Compared with baseline (but not placebo), CBD decreased resistin (2898 pg/ml; P < 0.05) and increased glucose-dependent insulinotropic peptide (21.9 pg/ml; P < 0.05). None of the combination treatments had a significant impact on end points. CBD and THCV were well tolerated. CONCLUSIONS THCV could represent a new therapeutic agent in glycemic control in subjects with type 2 diabetes.

Efficacy and safety of medical cannabinoids in children: a systematic review and meta analysis
NirTreves, Noa Mor, KarelAllegaert, Hely Bassalov, Matitiahu Berkovitch, Orit E. Stolar & Ilan Matok
Nature Scientific Reports (2021) 11:23462
DOI:10.1038/s41598-021-02770-6
https://www.nature.com/articles/s41598-021-02770-6.pdf
Despite the increased use of medical cannabinoids, the efcacy and safety of the treatment among children remain uncertain. The objective was to study the efcacy and safety of medical cannabinoids in children. The search included studies through 11-May-2020. Selection criteria included studies evaluating efcacy and safety outcomes of medical cannabinoids (tetrahydrocannabinol, cannabidiol and other cannabis derivatives) versus control in children, independently assessed by two reviewers. Eight studies were included, all of which are randomized controlled trials. Cannabidiol is associated with 50% reduction in seizures rate (Relative Risk (RR)= 1.69, 95% CI [1.20–2.36]) and caregiver global impression of change (Median Estimated diference= (− 1), 95%CI [− 1.39–(− 0.60)]) in Dravet syndrome, compared to placebo. While cannabidiol was associated with a reduction in reported seizure events (RR= 0.59, 95% CI [0.36–0.97]), no association was found in products contained also tetrahydrocannabinol (RR= 1.35, 95% CI [0.46–4.03]). Higher dose of cannabidiol was associated with decreased appetite (RR= 2.40, 95% CI [1.39–4.15]). A qualitative assessment suggests that medical cannabinoids might be associated with adverse mental events. In conclusion, cannabidiol is associated with clinical improvement in Dravet syndrome. However, cannabidiol is also associated with decreased appetite. Adverse mental events were reported as well, however, more research should be performed to assess well this outcome

Efficacy of Inhaled Cannabis on Painful Diabetic Neuropathy
Mark S. Wallace, Thomas D. Marcotte, Anya Umlauf, Ben Gouaux, J.H. Atkinson,
Journal of Pain (2015),16(7), 616–627.
DOI: 10.1016/j.jpain.2015.03.008
A randomized, double-blinded, placebo controlled crossover study was conducted in 16 patients with painful diabetic peripheral neuropathy to assess the short-term efficacy and tolerability of inhaled cannabis. In a cross-over design, each participant was exposed to a single dosing session of placebo, low (1% tetrahydrocannabinol, THC), medium (4% THC), or high (7% THC) doses of cannabis. Baseline spontaneous pain, evoked pain and cognitive testing were performed. Subjects were then administered aerosolized cannabis or placebo and the pain intensity and subjective highness score was measured at 5, 15, 30, 45, and 60 minutes and then every 30 minutes for an additional 3 hours. Cognitive testing was performed at 5 and 30 minutes and then every 30 minutes for an additional 3 hours. The primary analysis compared differences in spontaneous pain over time between doses using linear mixed effects models. There was a significant difference in spontaneous pain scores between doses (p<0.001). Specific significant comparisons were placebo versus low, medium, high dose (p = 0.031, 0.04 and <0.001 respectively) and high versus low, medium (both p<0.001). There was a significant effect of the high dose on foam brush and von Frey evoked pain (both p<0.001). There was a significant negative effect (impaired performance) of the high dose on two of the three neuropsychological tests (Paced Auditory Serial Addition Test, Trail Making Test B.

EHP-101, an oral formulation of the cannabidiol aminoquinone VCE-004.8, alleviates bleomycin-induced skin and lung fibrosis.
García-Martín, A., Garrido-Rodríguez, M., Navarrete, C., del Río, C., Bellido, M. L., Appendino, G., … Muñoz, E.
Biochemical Pharmacology.(2018).
doi:10.1016/j.bcp.2018.07.047*
Systemic sclerosis (SSc) or scleroderma is a chronic multi-organ autoimmune disease characterized by vascular, immunological, and fibrotic abnormalities. The etiology of SSc is unknown, but there is growing evidence that dysfunction of the endocannabinoid system (ECS) plays a critical role in its development. Since the semi-synthetic cannabinoquinoid VCE-004.8 could alleviate bleomycin (BLM)-induced skin fibrosis, we have investigated an oral lipid formulation (EHP-101) of this dual PPAR?/CB2 receptors activator for the prevention of skin- and lung fibrosis and of collagen accumulation in BLM challenged mice. Immunohistochemistry analysis of the skin showed that EHP-101 could prevent macrophage infiltration as well as the expression of Tenascin C (TNC), vascular cell adhesion molecule 1 (VCAM1), and the ?-smooth muscle actin (SMA). EHP-101 could also prevent the reduced expression of vascular CD31 typical of skin fibrosis. RNAseq analysis of skin biopsies showed a clear effect of EHP-101 in the inflammatory and epithelial-mesenchymal transition transcriptomic signatures. TGF-?-regulated genes [matrix metalloproteinase-3 (Mmp3), cytochrome b- 245 heavy chain (Cybb), lymphocyte antigen 6E (Ly6e), vascular cell adhesion molecule-1 (Vcam1) and Integrin alpha-5 (Itga5)] were induced in BLM mice and repressed by EHP-101 treatment. By intersecting differentially expressed genes in EHP- 101-treated mice with a dataset of human scleroderma intrinsic genes, 53 overlapped genes were discovered, including biomarkers of SSc like the C-C motif chemokine 2 (Ccl2) and the interleukin 13 receptor subunit alpha 1 (IL-13Ra1) genes. Taken together, these data provide a rationale for further developing VCE-004.8 as an orally active agent to alleviate scleroderma and, possibly, other fibrotic diseases as well.

Elephant at Warsaw Zoo to test cannabis-extract oil
By Joshua Nevett BBC News
https://www.bbc.com/news/world-europe-53907689
This year has been a difficult time for Fredzia, a young female African elephant at Warsaw Zoo in the Polish capital.
The zoo's four elephants became three in March, following the death of Erna, the largest female and elder of the herd.
Erna's death left Fredzia in mourning, putting her under stress.
Zoo keepers noticed an immediate change in Frezia's behaviour, as she attempted to make sense of life without Erna.
"Fredzia reacted strangely when she saw Erna's body. She was really excited," Dr Agnieszka Czujkowska, head of the zoo's Animal Rehabilitation Department, told the BBC. "But you could see that she was also grieving actually, she was also depressed.”

Endocannabinoid functions controlling neuronal specification during brain development
Tibor Harkany, Erik Keimpema, Klaudia Barabás, Jan Mulder
Molecular and Cellular Endocrinology 2008, 286 (1-2),
DOI: 10.1016/j.mce.2008.02.011
Endocannabinoids (eCBs) regulate a broad range of physiological functions in the postnatal brain and are implicated in the neuropathogenesis of psychiatric and metabolic diseases. Accumulating evidence indicates that eCB signaling also serves key functions during neurodevelopment; and is inherently involved in the control of neurogenesis, neural progenitor proliferation, lineage segregation, and the migration and phenotypic specification of immature neurons. Recent advances in developmental biology define fundamental eCB-driven cellular mechanisms that also contribute to our understanding of the molecular substrates of prenatal drug, in particular cannabis, actions. Here, we summarize known organizing principles of eCB signaling systems in the developing telencephalon, and outline the sequence of decision points and underlying signaling pathways upon CB1 cannabinoid receptor activation that contribute to neuronal diversification in the developing brain. Finally, we discuss how these novel principles affect the formation of complex neuronal networks.

Endocannabinoid-Mediated Neuromodulation in the Olfactory Bulb: Functional and Therapeutic Significance.
Bhatia-Dey, N., & Heinbockel, T.
International Journal of Molecular Sciences, 21(8), (2020). doi:10.3390/ijms21082850
Endocannabinoid synthesis in the human body is naturally occurring and on-demand. It occurs in response to physiological and environmental stimuli, such as stress, anxiety, hunger, other factors negatively disrupting homeostasis, as well as the therapeutic use of the phytocannabinoid cannabidiol and recreational use of exogenous cannabis, which can lead to cannabis use disorder. Together with their specific receptors CB1R and CB2R, endocannabinoids are major components of endocannabinoid-mediated neuromodulation in a rapid and sustained manner. Extensive research on endocannabinoid function and expression includes studies in limbic system structures such as the hippocampus and amygdala. The wide distribution of endocannabinoids, their on-demand synthesis at widely different sites, their co-existence in specific regions of the body, their quantitative differences in tissue type, and different pathological conditions indicate their diverse biological functions that utilize specific and overlapping pathways in multiple organ systems. Here, we review emerging evidence of these pathways with a special emphasis on the role of endocannabinoids in decelerating neurodegenerative pathology through neural networks initiated by cells in the main olfactory bulb.

Endocannabinoid Receptor-1 and Sympathetic Nervous System Mediate the Beneficial Metabolic Effects of Gastric Bypass.
Ye, Y., Abu El Haija, M., Morgan, D. A., Guo, D., Song, Y., Frank, A., … Mokadem, M.
Cell Reports, 33(4), 108270. (2020).
doi:10.1016/j.celrep.2020.108270
The exact mechanisms underlying the metabolic effects of bariatric surgery remain unclear. Here, we demonstrate, using a combination of direct and indirect calorimetry, an increase in total resting metabolic rate (RMR) and specifically anaerobic RMR after Roux-en-Y gastric bypass (RYGB), but not sleeve gastrectomy (SG). We also show an RYGB-specific increase in splanchnic sympathetic nerve activity and ‘‘browning’’ of visceral mesenteric fat. Consequently, selective splanchnic denervation abolishes all beneficial metabolic outcomes of gastric bypass that involve changes in the endocannabinoid signaling within the small intestine. Furthermore, we demonstrate that administration of rimonabant, an endocannabinoid receptor-1 (CB1) inverse agonist, to obese mice mimics RYGB-specific effects on energy balance and splanchnic nerve activity. On the other hand, arachidonoylethanolamide (AEA), a CB1 agonist, attenuates the weight loss and metabolic signature of this procedure. These findings identify CB1 as a key player in energy regulation post-RYGB via a pathway involving the sympathetic nervous system.

Endocannabinoid signaling enhances visual responses through modulation of intracellular chloride levels in retinal ganglion cells.
Miraucourt LS, Tsui J, Gobert D, Desjardins JF, Schohl A, Sild M, Spratt P, Castonguay A, De Koninck Y, Marsh-Armstrong N, Wiseman PW,Ruthazer ES
Elife. 2016 Aug 8;5. pii: e15932.
doi: 10.7554/eLife.15932.
Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl(-) levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl(-) regulation.

Endocannabinoid signaling in Alzheimers disease: current knowledge and future directions
C D' Addario , A Di Francesco , L Trabace , A Finazzi Agro , V Cuomo , M Maccarrone
Biol Regul Homeost Agents. 27(2 Suppl):61-73 Apr-Jun 2013.
https://www.researchgate.net/publica...g_in_Alzheimer's_disease_Current_knowledge_a nd_future_directions
The importance of the endocannabinoid system (ECS) in the modulation functions of the central nervous system has been extensively investigated during the last few years. In particular, accumulated evidence has implicated ECS in the pathophysiology of Alzheimer’s disease (AD), that is a progressive, degenerative, and irreversible disorder characterized by the accumulation in the brain of beta-amyloid fragments forming insoluble plaques, and of intracellular neurofibrillary tangles (NTFs) associated with synaptic and neuronal loss. In all the processes involved in the formation of both plaques and NFTs, the key-role played by the ECS has been documented. Here, we review current knowledge and future directions of ECS modulation both in animal models of AD and in human tissues, underlying the role of endocannabinoid signaling in the development of AD hallmarks. Overall, the available data suggest that next generation therapeutics might target distinct ECS elements, for instance CB2 receptor or fatty acid amide hydrolase, as a promising approach to halt or at least to slow down disease progression.

Endocannabinoid signaling in brain diseases: Emerging revelance of glial cells
Ana Bernal-Chico, Vanja Tepavcevic, Andrea Manterola, Carmen Utrilla, Carlos Matute, Susana Mato Glia.
Glia. 2022;1–24.
DOI: 10.1002/glia.24172
https://onlinelibrary.wiley.com/doi/epdf/10.1002/glia.24172
The discovery of cannabinoid receptors as the primary molecular targets of psychotropic cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) in late 1980s paved the way for investigations on the effects of cannabis-based therapeutics in brain pathology. Ever since, a wealth of results obtained from studies on human tissue samples and animal models have highlighted a promising therapeutic potential of cannabinoids and endocannabinoids in a variety of neurological disorders. However, clinical success has been limited and major questions concerning endocannabinoid signaling need to be satisfactorily addressed, particularly with regard to their role as modulators of glial cells in neurodegenerative diseases. Indeed, recent studies have brought into the limelight diverse, often unexpected functions of astrocytes, oligodendrocytes, and microglia in brain injury and disease, thus providing scientific basis for targeting glial cells to treat brain disorders. This Review summarizes the current knowledge on the molecular and cellular hallmarks of endocannabinoid signaling in glial cells and its clinical relevance in neurodegenerative and chronic inflammatory disorders.

Endocannabinoid System: An overview of its potential in current medical practice
Zadalla Mouslech, Vasiliki Valla
Neuroendocrinology Letters Volume 30 No. 2 2009
https://www.academia.edu/13907210/En...dical_practice
The endocannabinoid system (ECS) is a lipid signalling system, comprising of the endogenous cannabis-like ligands (endocannabinoids) anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which derive from arachidonic acid. These bind to a family of G-protein-coupled receptors, called CB1 and CB2. The cannabinoid receptor 1 (CB1R) is distributed in brain areas associated with motor control, emotional responses, motivated behaviour and energy homeostasis. In the periphery, the same receptor is expressed in the adipose tissue, pancreas, liver, GI tract, skeletal muscles, heart and the reproduction system. The CB2R is mainly expressed in the immune system regulating its functions. Endocannabinoids are synthesized and released upon demand in a receptor-dependent way. They act as retrograde signalling messengers in GABAergic and glutamatergic synapses and as modulators of postsynaptic transmission, interacting with other neurotransmitters. Endocannabinoids are transported into cells by a specific uptake system and degraded by the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The ECS is involved in various pathophysiological conditions in central and peripheral tissues. It is implicated in the hormonal regulation of food intake, cardiovascular, gastrointestinal, immune, behavioral, antiproliferative and mammalian reproduction functions. Recent advances have correlated the ECS with drug addiction and alcoholism. The growing number of preclinical and clinical data on ECS modulators is bound to result in novel therapeutic approaches for a number of diseases currently treated inadequately. The ECS dysregulation has been correlated to obesity and metabolic syndrome pathogenesis. Rimonabant is the first CB1 blocker launched to treat cardiometabolic risk factors in obese and overweight patients. Phase III clinical trials showed the drug’s ability to regulate intra-abdominal fat tissue levels, lipidemic, glycemic and inflammatory parameters. However, safety conerns have led to its withdrawal. The role of endocannabinoids in mammalian reproduction is an emerging research area given their implication in fertilization, preimplantation embryo and spermatogenesis. The relevant preclinical data on endocannabinoid signalling open up new perspectives as a target to improve infertility and reproductive health in humans

Endocannabinoid System and Fear Conditioning (Chapter 16)
Leonardo B.M.Resstel*Fabrício A.Moreira†Francisco S.Guimarães
Vitamins & Hormones Volume 81, 2009, Pages 421-440
doi: 10.1016/S0083-6729(09)81016-9
The endocannabinoid system has been proposed to modulate neuronal functions involved in distinct types of defensive reactions, possibly counteracting the harmful consequences of stressful stimuli. However, the precise brain sites for this action remain to be further explored. This chapter summarizes the data about the role of the endocannabinoid system in the processing of conditioned fear as well as the potential neural subtract for its actions.

Endocannabinoid system and mood disorders: priming a target for new therapies
Vincenzo Micale , Vincenzo Di Marzo, Alexandra Sulcova, Carsten T Wotjak, Filippo Drago
Pharmacol Ther. 2013 Apr;138(1):18-37.
doi: 10.1016/j.pharmthera.2012.12.002
The endocannabinoid system (ECS), comprising two G protein-coupled receptors (the cannabinoid receptors 1 and 2 [CB1 and CB2] for marijuana's psychoactive principle ∆(9)-tetrahydrocannabinol [∆(9)-THC]), their endogenous small lipid ligands (namely anandamide [AEA] and 2-arachidonoylglycerol [2-AG], also known as endocannabinoids), and the proteins for endocannabinoid biosynthesis and degradation, has been suggested as a pro-homeostatic and pleiotropic signaling system activated in a time- and tissue-specific way during physiopathological conditions. In the brain activation of this system modulates the release of excitatory and inhibitory neurotransmitters and of cytokines from glial cells. As such, the ECS is strongly involved in neuropsychiatric disorders, particularly in affective disturbances such as anxiety and depression. It has been proposed that synthetic molecules that inhibit endocannabinoid degradation can exploit the selectivity of endocannabinoid action, thus activating cannabinoid receptors only in those tissues where there is perturbed endocannabinoid turnover due to the disorder, and avoiding the potential side effects of direct CB1 and CB2 activation. However, the realization that endocannabinoids, and AEA in particular, also act at other molecular targets, and that these mediators can be deactivated by redundant pathways, has recently led to question the efficacy of such approach, thus opening the way to new multi-target therapeutic strategies, and to the use of non-psychotropic cannabinoids, such as cannabidiol (CBD), which act via several parallel mechanisms, including indirect interactions with the ECS. The state of the art of the possible therapeutic use of endocannabinoid deactivation inhibitors and phytocannabinoids in mood disorders is discussed in this review article.

Endocannabinoid System Components: Overview and Tissue Distribution.
Joshi, N., & Onaivi, E. S. (2019).
Recent Advances in Cannabinoid Physiology and Pathology, 1–12.
doi:10.1007/978-3-030-21737-2_1
Marijuana/cannabinoid research has been transformed into mainstream science during the last half-century. Evidence based research and remarkable biotechnological advances demonstrate that phytocannabinoids and endocannabinoid (eCBs) acting on cannabinoid receptors (CBRs) regulate various aspects of human physiological, behavioral, immunological and metabolic functions. The distribution and function of the components of the endocannabinoid system (ECS) in the central nervous system (CNS) and immune processes have garnished significant research focus with major milestones. With these advances in biotechnology, rapid extension of the ECS research in the periphery has gained momentum. In this chapter, we review the components and tissue distribution of this previously unknown but ubiquitous and complex ECS that is involved in almost all aspects of mammalian physiology and pathology

Endocannabinoids and immune regulation
Rupal Pandey, Khalida Mousawy, Mitzi Nagarkatti, and Prakash Nagarkatti
Pharmacol Res. 2009 August ; 60(2): 85–92.
doi: 10.1016/j.phrs.2009.03.019
Cannabinoid pharmacology has made important advances in recent years after the discovery of the cannabinoid receptors. These discoveries have added to our understanding of exogenous and endogenous cannabinoid signaling along with exploring the various pathways of their biosynthesis, molecular structure, inactivation, and anatomical distribution of their receptors throughout the body. The endocannabinoid system is involved in immunoregulation and neuroprotection. In this article, we have reviewed the possible mechanisms of the regulation of the immune response by endocannabinoids which include modulation of immune response in different cell types, effect on cytokine network, induction of apoptosis in immune cells and downregulation of innate and adaptive immune response. Studies from our laboratory have suggested that administration of endocannabinoids or use of inhibitors of enzymes that breakdown the endocannabinoids, leads to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Thus, manipulation of endocannabinoids in vivo may constitute a novel treatment modality against inflammatory disorders.

Endocannabinoids and neurodegenerative diseases
Vincenzo Micale, Carmen Mazzola, Filippo Drago
Pharmacological Research 56 (2007) 382–392
doi:10.1016/j.phrs.2007.09.008
The cannabinoid CB1 and CB2 receptors, the endogenous endocannabinoid (EC) ligands anandamide (AEA) and 2-arachidonylethanolamide, and the degradative enzymes fatty acid amide hydrolase (FAAH) and monoglyceride lipase (ML) are key elements of the EC system implicated in different physiological functions including cognition, motor activity and immune responses. Thus, both the possible neuroprotective role of ECs and their modulating action on neurotransmitter systems affected in several neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and multiple sclerosis (MS) are currently under investigation. Accumulating data show an unbalance in the EC system (i.e. decrease of neuronal cannabinoid CB1 receptors, increase of glial cannabinoid CB2 receptors and over-expression of FAAH in astrocytes) in experimental models of AD as well as in post-mortem brain tissue of AD patients, suggesting its possible role in inflammatory processes and in neuroprotection. However, the mechanisms of the EC modulation of immune response are not fully understood. By contrast, in HD a reduced EC signaling, given both by the loss of cannabinoid CB1 receptors and decrease of ECs in brain structures involved in movement control as basal ganglia, has been well documented in preclinical and clinical studies. Thus, in the present review we discuss recent data concerning the role of the EC system in the pathophysiology of AD and HD, two neurodegenerative diseases characterized by cognitive deficit and motor impairment, respectively. We focus on the effects of compounds modulating the EC system (agonists/antagonists of cannabin

Endocannabinoids and related lipids in serum from patients with amyotrophic lateral sclerosis.
Carter, G. T., McLaughlin, R. J., Cuttler, C., Sauber, G. J., Weeks, D. L., Hillard, C. J., & Weiss, M. D
Muscle & Nerve. . (2020).
doi:10.1002/mus.27096
Introduction The goals of this study were to determine whether serum concentrations of endocannabinoids (eCB) and related lipids predict disease status in patients with amyotrophic lateral sclerosis (ALS) relative to healthy controls, and whether concentrations correlate with disease duration and severity.
Methods Serum concentrations of the eCBs 2-arachidonoylglycerol (2-AG) and Narachidonoylethanolamine (AEA), and related lipids palmitoylethanolamine (PEA), oleoylethanolamine (OEA), and 2-oleoylglycerol (2-OG), were measured in samples from 47 patients with ALS and 19 healthy adults. Hierarchical binary logistic and linear regression
analyses assessed whether lipid concentrations predicted disease status (ALS or healthy control), duration, or severity.
Results Binary logistic regression revealed that, after controlling for age and gender, 2-AG, 2-OG and AEA concentrations were unique predictors of the presence of ALS, demonstrating odds ratios of 0.86 (p=.039), 1.03 (p=.023), and 42.17 (p=.026), respectively. When all 5 lipids and covariates (age, sex, race, ethnicity, BMI, presence of a feeding tube) were included, the resulting model had an overall classification accuracy of 92.9%. Hierarchical linear regression analyses indicated that in patients with ALS, AEA and OEA inversely correlated with disease duration (p=.030 and .031 respectively), while PEA demonstrated a positive relationship with disease duration (p=.013). None of the lipids examined predicted disease severity.
Discussion These findings support previous studies indicating significant alterations in concentrations of circulating lipids in patients with ALS. They suggest that arachidonic and oleic acid containing small lipids may serve as biomarkers for identifying the presence and duration of this disease.

*Endocannabinoids and Their Pharmacological Actions
Roger G Pertwee
Handbook of experimental pharmacology 231:1-37 September 2015
DOI: 10.1007/978-3-319-20825-1_1
The endocannabinoid system consists of G protein-coupled cannabinoid CB1 and CB2 receptors, of endogenous compounds known as endocannabinoids that can target these receptors, of enzymes that catalyse endocannabinoid biosynthesis and metabolism, and of processes responsible for the cellular uptake of some endocannabinoids. This review presents in vitro evidence that most or all of the following 13 compounds are probably orthosteric endocannabinoids since they have all been detected in mammalian tissues in one or more investigation, and all been found to bind to cannabinoid receptors, probably to an orthosteric site: anandamide, 2-arachidonoylglycerol, noladin ether, dihomo-?-linolenoylethanolamide, virodhamine, oleamide, docosahexaenoylethanolamide, eicosapentaenoylethanolamide, sphingosine, docosatetraenoylethanolamide, N-arachidonoyldopamine, N-oleoyldopamine and haemopressin. In addition, this review describes in vitro findings that suggest that the first eight of these compounds can activate CB1 and sometimes also CB2 receptors and that another two of these compounds are CB1 receptor antagonists (sphingosine) or antagonists/inverse agonists (haemopressin). Evidence for the existence of at least three allosteric endocannabinoids is also presented. These endogenous compounds appear to target allosteric sites on cannabinoid receptors in vitro, either as negative allosteric modulators of the CB1 receptor (pepcan-12 and pregnenolone) or as positive allosteric modulators of this receptor (lipoxin A4) or of the CB2 receptor (pepcan-12). Also discussed are current in vitro data that indicate the extent to which some established or putative orthosteric endocannabinoids seem to target non-cannabinoid receptors and ion channels, particularly at concentrations at which they have been found to interact with CB1 or CB2 receptors.

Endocannabinoids and traumatic brain injury
Esther Shohami, Ayelet Cohen?Yeshurun, Lital Magid, Merav Algali, Raphael Mechoulam
British Journal of Pharmacology (2011) 163 1402–1410
doi: 10.1111/j.1476-5381.2011.01343.x
Traumatic brain injury (TBI) represents the leading cause of death in young individuals. It triggers the accumulation of harmful mediators, leading to secondary damage, yet protective mechanisms are also set in motion. The endocannabinoid (eCB) system consists of ligands, such as anandamide and 2?arachidonoyl?glycerol (2?AG), receptors (e.g. CB1, CB2), transporters and enzymes, which are responsible for the ‘on?demand’ synthesis and degradation of these lipid mediators. There is a large body of evidence showing that eCB are markedly increased in response to pathogenic events. This fact, as well as numerous studies on experimental models of brain toxicity, neuroinflammation and trauma supports the notion that the eCB are part of the brain's compensatory or repair mechanisms. These are mediated via CB receptors signalling pathways that are linked to neuronal survival and repair. The levels of 2?AG, the most highly abundant eCB, are significantly elevated after TBI and when administered to TBI mice, 2?AG decreases brain oedema, inflammation and infarct volume and improves clinical recovery. The role of CB1 in mediating these effects was demonstrated using selective antagonists or CB1 knockout mice. CB2 were shown in other models of brain insults to reduce white blood cell rolling and adhesion, to reduce infarct size and to improve motor function. This review is focused on the role the eCB system plays as a self?neuroprotective mechanism and its potential as a basis for the development of novel therapeutic modality for the treatment of CNS pathologies with special emphasis on TBI.

Endocannabinoids in Appetite Control and the Treatment of Obesity
Tim C Kirkham, Sonia A Tucci
CNS & Neurological Disorders - Drug Targets, 2006, 5, 275-292
DOI: 10,2174/187152706777452272
https://www.researchgate.net/publica...ent_of_Obesity
Abstract: Research into the endocannabinoid ‘system’ has grown exponentially in recent years, with the discovery of cannabinoid receptors and their endogenous ligands, such as anandamide and 2-arachidonoylglycerol (2-AG). Important advances have been made in our understanding of endocannabinoid transduction mechanisms, their metabolic pathways, and of the biological processes in which they are implicated. A decade of endocannabinoid studies has promoted new insights into neural regulation and mammalian physiology that are as revolutionary as those arising from the discovery of the endogenous opioid peptides in the 1970s. Thus, endocannabinoids have been found to act as retrograde signals: released by postsynaptic neurons, they bind to presynaptic heteroceptors to modulate the release of inhibitory and excitatory neurotransmitters through multiple G-protein-coupled receptor (GPCR)-linked effector mechanisms. The metabolic pathways of anandamide and 2-AG have now been been characterised in great detail, and we can anticipate that these pathways – together with endocannabinoid uptake mechanisms – will complement cannabinoid receptors as targets for the pharmacological analysis of the physiological functions of these substances. Specific insights into the potential role
of endocannabinoid-CB1 receptor systems in central appetite control, peripheral metabolism and body weight regulation herald the clinical application of CB1 receptor antagonists in the management of obesity and its associated disorders.

Endocannabinoids in Body Weight Control
Henrike Horn, Beatrice Böhme, Laura Dietrich and Marco Koch
Pharmaceuticals 2018, 11, 55;
doi:10.3390/ph11020055
https://www.ncbi.nlm.nih.gov/pmc/art...s-11-00055.pdf
Maintenance of body weight is fundamental to maintain one’s health and to promote longevity. Nevertheless, it appears that the global obesity epidemic is still constantly increasing. Endocannabinoids (eCBs) are lipid messengers that are involved in overall body weight control by interfering with manifold central and peripheral regulatory circuits that orchestrate energy homeostasis. Initially, blocking of eCB signaling by first generation cannabinoid type 1 receptor (CB1) inverse agonists such as rimonabant revealed body weight-reducing effects in laboratory animals and men. Unfortunately, rimonabant also induced severe psychiatric side effects. At this point, it became clear that future cannabinoid research has to decipher more precisely the underlying central and peripheral mechanisms behind eCB-driven control of feeding behavior and whole body energy metabolism. Here, we will summarize the most recent advances in understanding how central eCBs interfere with circuits in the brain that control food intake and energy expenditure. Next, we will focus on how peripheral eCBs affect food digestion, nutrient transformation and energy expenditure by interfering with signaling cascades in the gastrointestinal tract, liver, pancreas, fat depots and endocrine glands. To finally outline the safe future potential of cannabinoids as medicines, our overall goal is to address the molecular, cellular and pharmacological logic behind central and peripheral eCB-mediated body weight control, and to figure out how these precise mechanistic insights are currently transferred into the development of next generation cannabinoid medicines displaying clearly improved safety profiles, such as significantly reduced side effects.

Endocannabinoids in the regulation of appetite and body weight
T. C. Kirkham
Behavioural Pharmacology 2005, 16:297–313
DOI: 10.1097/00008877-200509000-00004
https://pages.ucsd.edu/~mboyle/COGS1...%20in%20the%20 regulation%20of%20appetite%20a nd%20body%20weight-2005.pdf
The discovery of cannabinoid receptors, together with the development of selective cannabinoid receptor antagonists, has encouraged a resurgence of cannabinoid pharmacology. With the identification of endogenous agonists, such as anandamide, scientists have sought to uncover the biological role of endocannabinoid systems; initially guided by the long-established actions of cannabis and exogenous cannabinoids such as D9 -tetrahydrocannabinol (THC). In particular, considerable research has examined endocannabinoid involvement in appetite, eating behaviour and body weight regulation. It is now confirmed that endocannabinoids, acting at brain CB1 cannabinoid receptors, stimulate appetite and ingestive behaviours, partly through interactions with more established orexigenic and anorexigenic signals. Key structures such as the nucleus accumbens and hypothalamic nuclei are sensitive sites for the hyperphagic actions of these substances, and endocannabinoid activity in these regions varies in relation to nutritional status and feeding expression. Behavioural studies indicate that endocannabinoids increase eating motivation by enhancing the incentive salience and hedonic evaluation of ingesta. Moreover, there is strong evidence of an endocannabinoid role in energy metabolism and fuel storage. Recent developments point to potential clinical benefits of cannabinoid receptor antagonists in the management of obesity, and of agonists in the treatment of other disorders of eating and body weight regulation.

Endocannabinoids in the Treatment of Mood Disorders: Evidence from Animal Models
Francis Rodriguez Bambico, Andrea Duranti, Andrea Tontini, Giorgio Tarzia and Gabriella Gobbi Current Pharmaceutical Design, 2009, 15, DOI: 10.2174/138161209788168029 https://www.researchgate.net/publica...nabinoids_in_t he_Treatment_of_Mood_Disorders _Evidence_from_Animal_Models Among all mental disorders, major depression has the highest rate of prevalence and incidence of morbidity. Currently available antidepressant therapies have limited efficacies; consequently, research on new drugs for the treatment of mood disorders has become increasingly critical. Recent preclinical evidences that cannabinoid agonists and endocannabinoid enhancers, such as the fatty acid amide hydrolase (FAAH) inhibitors, can impact mood regulation have opened a new line of research in antidepressant drug discovery. However, the neurobiological mechanisms linking the endocannabinoid system with the pathophysiology of mood disorders and antidepressant action remain unclarified. In this review, we have presented an update on preclinical data indicating the antidepressant potential of cannabinoid agonists and endocannabinoid enhancers in comparison to standard antidepressants. Data obtained from CB1 knockout (CB1-/-) and FAAH knockout (FAAH-/-) mice have also been examined within this context. We have illustrated how the various classes of antidepressants exert their therapeutic action. In particular, all antidepressants increase the neurotransmission of serotonin after long-term treatment, enhance the tonic activity of hippocampal 5-HT1A receptors, promote neurogenesis, and modulate (decrease or increase) the firing activity of noradrenergic neurons. Interestingly, cannabinoid agonists and endocannabinoid enhancers increase serotonin and noradrenergic neuronal firing activity, increase serotonin release in the hippocampus, as well as promote neurogenesis. Since cannabinoid-derived drugs potentiate monoaminergic neurotransmission and hippocampal neurogenesis through distinct pathways compared to classical antidepressants, they may represent an alternative drug class in the pharmacotherapy of mood and other neuropsychiatric disorders.

Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.
Ellermann, M., Pacheco, A. R., Jimenez, A. G., Russell, R. M., Cuesta, S., Kumar, A., … Sperandio, V.
Cell. (2020)
doi:10.1016/j.cell.2020.09.022
Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems.

Endogenous cannabinoid system as a modulator of food intake
D Cota, G Marsicano, B Lutz, V Vicennati, GK Stalla, R Pasquali and U Pagotto
International Journal of Obesity (2003) 27, 289–301
doi: 10.1038/sj.ijo.802250
The ability of Cannabis sativa (marijuana) to increase hunger has been noticed for centuries, although intensive research on its
molecular mode of action started only after the characterization of its main psychoactive component D9-tetrahydrocannabinol in
the late 1960s. Despite the public concern related to the abuse of marijuana and its derivatives, scientific studies have pointed to
the therapeutic potentials of cannabinoid compounds and have highlighted their ability to stimulate appetite, especially for sweet and palatable food. Later, the discovery of specific receptors and their endogenous ligands (endocannabinoids) suggested the existence of an endogenous cannabinoid system, providing a physiological basis for biological effects induced by marijuana and other cannabinoids. Epidemiological reports describing the appetite-stimulating properties of cannabinoids and the recent insights into the molecular mechanisms underlying cannabinoid action have proposed a central role of the cannabinoid system in obesity. The aim of this review is to provide an extensive overview on the role of this neuromodulatory system in feeding behavior by summarizing the most relevant data obtained from human and animal studies and by elucidating the interactions of the cannabinoid system with the most important neuronal networks and metabolic pathways involved in the control of food intake. Finally, a critical evaluation of future potential therapeutical applications of cannabinoid antagonists in the therapy of obesity and eating disorders will be discussed.

Endogenous cannabinoids and appetite
Tim C. Kirkham* and Claire M. Williams
Nutrition Research Reviews (2001), 14, 65–86
DOI: 10.1079/NRR200118
Since pre-history, Cannabis sativa has been exploited for its potent and manifold pharmacological actions. Amongst the most renowned of these actions is a tendency to provoke ravenous eating. The characterization of the psychoactive principals in cannabis (exogenous cannabinoids) and, more recently, the discovery of specific brain cannabinoid receptors and their endogenous ligands (endocannabinoids) has stimulated research into the physiological roles of endocannabinoid systems. In this review, we critically discuss evidence from the literature that describe studies on animals and human subjects to support endocannabinoid involvement in the control of appetite. We describe the hyperphagic actions of the exogenous cannabinoid, 9-tetrahydrocannabinol, and the endogenous CB1 ligands, anandamide and 2-arachidonylglycerol, and present evidence to support a specific role of endocannabinoid systems in appetitive processes related to the incentive and reward properties of food. A case is made for more comprehensive and systematic analyses of cannabinoid actions on eating, in the anticipation of improved therapies for disorders of appetite and body weight, and a better understanding of the biopsychological processes underlying hunger.

Endometriosis and Cannabis Consumption During the COVID-19 Pandemic: An International Cross-Sectional Survey
Mike Armour, Justin Sinclair, Junipearl Cheng, Preston Davis, Aaish Hameed, Harini Meegahapola, Krithika Rajashekar, Sunethra Suresh, Andrew Proudfoot, and Mathew Leonardi
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0162
Introduction: Endometriosis affects 1 in 10 women worldwide, with most experiencing difficulties achieving adequate symptom control. These difficulties have been compounded by the onset of the COVID-19 pandemic
due to worldwide shifts in health care resource allocation. As cannabis is a relatively common form of selfmanagement in endometriosis, this study aims to explore the impact of the COVID-19 pandemic on cannabis consumption in those with endometriosis.
Methods: An anonymous, cross-sectional online international survey was developed and promoted by endometriosis advocacy/support organizations worldwide. Respondents needed to have a diagnosis of endometriosis and be aged between 18 and 55.
Results: A total of 1634 responses were received from 46 different countries. The average age of respondents was 30, with a mean diagnosis age of 25. Eight hundred forty-six respondents (51%) reported consuming cannabis
in the past 3 months, with 55% of these reporting use for symptom management only. One in five respondents (20%) reported having consumed cannabis previously, the most common reason for discontinuation (65%) was access difficulties during COVID. Those who had legal access were more likely to consume cannabis than those without ( p < 0.0001) and were more likely to disclose usage to health care professionals ( p < 0.0001). The most common reasons for consuming cannabis during COVID was increased stress/anxiety (59%) and lack of access to normal medical care (48%). Pre-pandemic, cannabis was mostly consumed at least once a day (61%) and in inhaled forms (51.6%). Consumption increased for most people (57%) during the pandemic. During the pandemic just under a quarter (23%) of respondents changed their mode of consumption, with a reduction in inhaled forms (39.5%) and an increase in consumption of edibles (40%) or oil (25.2%).
Conclusions: Cannabis consumption, especially for symptom relief, was relatively common among those with endometriosis, with some people starting their consumption of cannabis due to health care restrictions that occurred due to the COVID-19 pandemic. Difficulties accessing cannabis and unpleasant/unwanted side effects were the most common reasons for lack of current cannabis consumption in those who had previously consumed it. Cannabis consumption may form an important part of endometriosis management especially when access to routine medical care is restricted.
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Enhanced Brain Disposition and Effects of D9-Tetrahydrocannabinol in P-Glycoprotein and Breast Cancer Resistance Protein Knockout Mice
PLoS ONE 7(4): e35937.
doi:10.1371/journal.pone.0035937
Adena S. Spiro , Alexander Wong , Aurelie A. Boucher , Jonathon C. Arnold
The ABC transporters P-glycoprotein (P-gp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis D9-tetrahydrocannabinol (THC) has affinity for Pgp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that mice devoid of Abcb1 and Abcg2 retain higher brain THC levels and are more sensitive to cannabinoid-induced hypothermia than wild-type (WT) mice. Abcb1a/b (2/2), Abcg2 (2/2) and wild-type (WT) mice were injected with THC before brain and blood were collected and THC concentrations determined. Another cohort of mice was examined for THC-induced hypothermia by measuring rectal body temperature. Brain THC concentrations were higher in both Abcb1a/b (2/2) and Abcg2 (2/2) mice than WT mice. ABC transporter knockout mice exhibited delayed elimination of THC from the brain with the effect being more prominent in Abcg2 (2/2) mice. ABC transporter knockout mice were more sensitive to THC-induced hypothermia compared to WT mice. These results show P-gp and Bcrp prolong. the brain disposition and hypothermic effects of THC and offer a novel mechanism for both genetic vulnerability to the psychoactive effects of cannabis and drug interactions between CNS therapies and cannabis

Enhancing Brain Pregnenolone May Protect Cannabis Intoxication but Should Not Be Considered as an Anti-addiction Therapeutic: Hypothesizing Dopaminergic Blockade and Promoting Anti-Reward
Kenneth Blum, Marlene Oscar-Berman, Eric R. Braverman, Marcelo Febo, Mona Li and Mark S. Gold
J Reward Defic Syndr1(1): 20-23.
doi: 10.17756/jrds.2015-005
Many US states now embrace the medical and recreational use of Cannabis. Changes in the laws have heightened interest and encouraged research into both cannabinoid products and the potential harms of Cannabis use, addiction, and intoxication. Some research into those harms will be reviewed here and misgivings about the use of Pregnenolone, to treat cannabis addiction and intoxication explained. Pregnenolone considered the inactive precursor of all steroid hormones, has recently been shown to protect the brain from Cannabis intoxication. The major active ingredient of Cannabis sativa (marijuana), ?9-tetrahydrocannabinol (THC) enhances Pregnenolone synthesis in the brain via stimulation of the type-1 cannabinoid (CB1) receptor. This steroid has been shown to inhibit the activity of the CB1 receptor thereby reducing many of the effects of THC. While this mechanism seems correct, in our opinion, Vallee et al., incorrectly suggest that blocking CB1 receptors could open unforeseen approaches to the treatment of cannabis intoxication and addiction. In this hypothesis, we caution the scientific community that, other CB1 receptor blockers, such as, Rimonabant (SR141718) have been pulled off the market in Europe. In addition, CB1 receptor blockers were rejected by the FDA due to mood changes including suicide ideation. Blocking CB1 receptors would result in reduced neuronal release of Dopamine by disinhibition of GABA signaling. Longterm blockade of cannabinoid receptors could occur with raising Pregnenolone brain levels, may induce a hypodopaminergic state, and lead to aberrant substance and nonsubstance (behavioral) addictions.

Enhancing effects of acute exposure to cannabis smoke on working memory performance.
Blaes, S. L., Orsini, C. A., Holik, H. M., Stubbs, T. D., Ferguson, S. N., Heshmati, S. C., … Setlow, B.
Neurobiology of Learning and Memory (2018).
doi:10.1016/j.nlm.2018.12.001
Numerous preclinical studies show that acute cannabinoid administration impairs cognitive performance. Almost all of this research has employed cannabinoid injections, however, whereas smoking is the preferred route of cannabis administration in humans. The goal of these experiments was to systematically determine how acute exposure to cannabis smoke affects
working memory performance in a rat model. Adult male (n = 15) and female (n = 16) LongEvans rats were trained in a food-motivated delayed response working memory task. Prior to test sessions, rats were exposed to smoke generated by burning different numbers of cannabis or placebo cigarettes, using a within-subjects design. Exposure to cannabis smoke had no effect on male rats’ performance, but surprisingly, enhanced working memory accuracy in females, which tended to perform less accurately than males under baseline conditions. In addition, cannabis smoke enhanced working memory accuracy in a subgroup of male rats that performed comparably to the worst-performing females. Exposure to placebo smoke had no effect on performance, suggesting that the cannabinoid content of cannabis smoke was critical for its effects on working memory. Follow-up experiments showed that acute administration of
either ?9-tetrahydrocannabinol (0.0, 0.3, 1.0, 3.0 mg/kg) or the cannabinoid receptor type 1 antagonist rimonabant (0.0, 0.2, 0.6, 2.0 mg/kg) impaired working memory performance. These results indicate that differences in the route, timing, or dose of cannabinoid administration can yield distinct cognitive outcomes, and highlight the need for further investigation of this topic.

Epidemiology of Cannabis Use Disorder
Marsha Lopez, Carlos Blanco
In book: Cannabis Use Disorders January 2019
DOI: 10.1007/978-3-319-90365-1_2
The field of epidemiology provides information about the occurrence and distribution of cannabis use disorders (CUD) over the years, giving clinicians a greater understanding of who may be at increased or decreased risk of cannabis-related problems and why, with the ultimate goal of helping to help guide interventions to reduce public health burden. This chapter describes the goals of epidemiology, defines what constitutes cannabis use and disorder, and summarizes national CUD trends at a population level. Included are discussions of risk factors, subgroup differences, and comorbidity as integral components of a comprehensive picture of CUD, as well as a necessary and timely discussion of cannabis policy and how the continuing evolution of cannabis legislation across US states could impact cannabis use and disorder, and how we treat them, moving forward.

Epidiolex (Cannabidiol): A New Hope for Patients With Dravet or Lennox-Gastaut Syndromes.
Chen, Jeffrey W.; Borgelt, Laura M.; Blackmer, Allison B.
Annals of Pharmacotherapy, (), 106002801882212–(2019).
doi:10.1177/1060028018822124
Objective: To review the efficacy, safety, pharmacology and pharmacokinetics of pure, plant-derived cannabidiol (CBD; Epidiolex) in the treatment of Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS). Data Sources: Relevant information was identified through EMBASE and Ovid MEDLINE (1946 to October 2018). Product labeling and https://www.clinicaltrials.gov were also reviewed. Study Selection/Data Extraction: English language articles evaluating efficacy and safety in humans with treatment-resistant epilepsies were reviewed; additional pharmacology and pharmacokinetic studies in humans, animals, and in vitro were also included. Data Synthesis: Pure, plant-based CBD is a pharmaceutical grade extract that exhibits clinically significant antiseizure properties, with a hypothesized multimodal mechanism of action. In the GWPCARE trial series, CBD displayed superior efficacy in reducing key seizure frequencies (convulsive seizures in DS; drop seizures in LGS) by 17% to 23% compared with placebo as adjunctive therapy to standard antiepileptic drugs in patients 2 years of age and older. Common adverse effects were somnolence, diarrhea, and elevated hepatic transaminases. Noteworthy drug-drug interactions included clobazam, valproates, and significant inducers/inhibitors of CYP2C19 and 3A4 enzymes. Relevance to Patient Care and Clinical Practice: A discussion regarding CBD dosing, administration, adverse effects, monitoring parameters, and interactions is provided to guide clinicians. CBD offers patients with DS and LGS a new treatment option for refractory seizures. Conclusion: This is the first cannabis-derived medication with approval from the US Food and Drug Administration. This CBD formulation significantly reduces seizures as an adjunct to standard antiepileptic therapies in patients ≥2 years old with DS and LGS and is well tolerated.

Epidiolex (Cannabidiol) Primer: Frequently Asked Questions for Patients and Caregivers.
Renad Abu-Sawwa and Caitlin Stehling
The Journal of Pediatric Pharmacology and Therapeutics:
January-February 2020, Vol. 25, No. 1, pp. 75-77.
https://doi.org/10.5863/1551-6776-25.1.75
Volume 25, Issue 1 (January-February 2020)
https://www.jppt.org/doi/pdf/10.5863/1551-6776-25.1.75

Epigenetic regulation of the cannabinoid receptor CB1 in an activity-based rat model of anorexia nervosa.
D’Addario, C., Zaplatic, E., Giunti, E., Pucci, M., Micioni Di Bonaventura, M. V., Scherma, M., … Fadda, P.
International Journal of Eating Disorders. (2020).
doi:10.1002/eat.23271
Objective: Both environmental and genetic factors are known to contribute to the development of anorexia nervosa (AN), but the exact etiology remains poorly understood. Herein, we studied the transcriptional regulation of the endocannabinoid system, an interesting target for body weight maintenance and the control of food intake and energy balance. Method: We used two well-characterized animal models of AN: (a) the activity-based anorexia (ABA) model in which rats, housed with running wheels and subjected to daily food restriction, show reductions in body weight and increase in physical activity; (b) the genetic anx/anx mouse displaying the core features of AN: low food intake and emaciation. Results: Among the evaluated endocannabinoid system components, we observed a selective and significant down-regulation of the gene encoding for the type 1 cannabinoid receptor (Cnr1) in ABA rats' hypothalamus and nucleus accumbens and, in the latter area, a consistent, significant and correlated increase in DNA methylation at the gene promoter. No changes were evident in the anx/anx mice except for a downregulation of Cnr1, in the prefrontal cortex. Discussion: Our findings support a possible role for Cnr1 in the ABA animal model of AN. In particular, its regulation in the nucleus accumbens appears to be triggered by environmental cues due to the consistent epigenetic modulation of the promoter. These data warrant further studies on Cnr1 regulation as a possible target for treatment of AN

ESCAPE PAIN - Cannamedical® starts nationwide pain therapy study
Press release
https://www.apotheke-adhoc.de/branch...&mc_eid=UNIQID
Cologne -
Optimal patient care requires medical progress and professional information. As the market leader, Cannamedical makes a significant contribution to research into therapeutic success with medicinal cannabis through the nationwide pain therapy study ESCAPE PAIN.
In cooperation with leading experts, Cannamedical is working to advance the clinical evidence for the use and effectiveness of cannabis-based drugs. The evaluation of the data sets of 500 patients, which are to be collected as part of the non-interventional study ESCAPE PAIN, support both patient safety and the continuous improvement and standardization of an individual therapy with cannabinoids.

Essential Fatty Acids and Lipid mediators
G. Caramia
Ped. Med. Chir. (Med. Surg. Ped.), 2012, 34: 65-72
DOI: 10.4081/pmc.2012.2 (In Italian)
In 1929 Burr and Burr discovered the essential fatty acids omega-6 and omega-3. Since then, researchers have shown a growing interest in polyunsaturated fatty acids (PUFA) as precursors of "lipid mediator" molecules, often with opposing effects, prostaglandins, prostacyclins, thromboxanes, leukotrienes, lipossines, resolvines, protectines, maresins that regulate immunity, platelet aggregation, inflammation, etc. They showed that the balance between omega-3 and omega-6 acids has a profound influence on all the body's inflammatory responses and a raised level of PUFA omega-3 in tissue correlate with a reduced incidence of degenerative cardiovascular disease, some mental illnesses such as depression, and neuro-degenerative diseases such as Alzheimer's. The CYP-catalyzed epoxidation and hydroxylation of arachidonic acid (AA) were established recently as the so-called third branch of AGE cascade. Cytochrome P450 (CYP) epoxygenases convert AA to four epoxyeicosatrienoic acid (EET) regioisomers, that produce vascular relaxation anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are accessible to CYP enzymes in the same way as AA. Metabolites derived from EPA include epoxye-icosatetraenoic acids (EETR) and hydroxyeicosapentaenoic acids (19- and 20-HEPE), whereas DHA include epoxydocosapentaenoic acids (EDPs) hydroxydocosahexaenoic acids (21- and 22-HDoHE). For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates and the available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA. From AA derives also endocannabinoids like anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol, capable of mimicking the pharmacological actions of the active principle of Cannabis sativa preparations such as hashish and marijuana (-)-Delta9-tetrahydrocannabinol. They act as true 'endogenous cannabinoids' by binding and functionally activating one or both cannabinoid receptor present on nervous and peripheral cell membranes. Enzymes that carry out anandamide oxidation are the same fatty acid oxygenases that are known to act on endogenous arachidonic acid namely, the members of the COX, LOX, and P450 families of enzymes. Recent advances in the biochemistry and pharmacology of the endocannabinoid system, also for its central and peripheral roles in regulating food intake, will offer the development of novel therapeutic agents.

Ethnomedicinal ....antiinflammatory and analgesics
Anil Kumar
In book: Ethnomedicine: A Source of Complementary Therapeutics March 2021
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Evaluation of pharmacokinetics and acute anti?inflammatory potential of two oral cannabidiol preparations in healthy adults.
Hobbs, J. M., Vazquez, A. R., Remijan, N. D., Trotter, R. E., McMillan, T. V., Freedman, K. E., … Weir, T. L.
Phytotherapy Research.(2020).
doi:10.1002/ptr.6651
Cannabidiol (CBD) is a dietary supplement with numerous purported health benefits and an expanding commercial market. Commercially available CBD preparations range from tinctures, oils, and powders, to foods and beverages. Despite widespread use, information regarding bioavailability of these formulations is limited. The purpose of this study was to test the bioavailability of two oral formulations of CBD in humans and explore their potential acute anti-inflammatory activity. We conducted a pilot randomized, parallel arm, double-blind study in 10 healthy adults to determine differences in pharmacokinetics of commercially available water and lipid-soluble CBD powders. Participants consumed a single 30 mg dose, which is within the range of typical commercial supplement doses, and blood samples were collected over 6 hr and analyzed for CBD concentrations. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and T = 90 min, cultured and stimulated with bacterial lipopolysaccharide (LPS) to induce an inflammatory response. Cell supernatants were assayed for IL-10 and TNF, markers of inflammation, using enzyme-linked immunosorbent assays. The water-soluble powder had Cmax = 2.82 ng/ml, Tmax = 90 min, and was approximately ×4.5 more bioavailable than the lipid-soluble form. TNF was decreased in LPS-stimulated PBMCs collected 90 min after CBD exposure relative to cells collected at baseline. This study provides pilot data for designing and powering future studies to establish the anti-inflammatory potential and bioavailability of a larger variety of commercial CBD products consumed by humans.

Evaluation of prevalent phytocannabinoids in the acetic acid model of visceral nociception.
Booker, L., Naidu, P. S., Razdan, R. K., Mahadevan, A., & Lichtman, A. H.
Drug and Alcohol Dependence, 105(1-2), 42–47(2009).
doi:10.1016/j.drugalcdep.2009.06.00
Considerable preclinical research has demonstrated the efficacy of 9-tetrahydrocannabinol (9-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of 9-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, 9-THC and CBN bound to the CB1 receptor and produced antinociceptive effects. The CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB1 receptor with similar affinity as 9-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of 9-THC when both drugs were given in combination. Importantly, the antinociceptive effects of 9-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of 9-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of 9-THC.

Evaluation of THC-Related Neuropsychiatric Symptoms Among Adults Aged 50 Years and Older: A Systematic Review and Metaregression Analysis
Latha Velayudhan, Katie Louise McGoohan, Sagnik Bhattacharyya
JAMA Netw Open. 2021 Feb 1;4(2)
DOI: 10.1001/jamanetworkopen.2020.35913
This systematic review and metaregression analysis estimates the association between the delta-9-tetrahydrocannabinol (THC) dose of cannabinoid-based medicines and neuropsychiatric adverse events among adults aged 50 years and older.

Evaluation of the Possible Anticonvulsant Effect of ?9-Tetrahydrocannabinolic Acid in Murine Seizure Models.
Benson, M. J., Anderson, L. L., Low, I. K., Luo, J. L., Kevin, R. C., Zhou, C., … Arnold, J. C.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2020.0073
Introduction: The cannabinoid D9 -tetrahydrocannabinolic acid (D9 -THCA) has long been suggested in review articles and anecdotal reports to be anticonvulsant; yet, there is scant evidence supporting this notion. The objective of this study was to interrogate the anticonvulsant potential of D9 -THCA in various seizure models—the Scn1a + / mouse model of Dravet syndrome, the 6-Hz model of psychomotor seizures and the maximal electroshock (MES) model of generalized tonic-clonic seizures.
Materials and Methods: We examined the effect of acute D9 -THCA treatment against hyperthermia-induced seizures, and subchronic treatment on spontaneous seizures and survival in the Scn1a + / mice. We also studied the effect of acute D9 -THCA treatment on the critical current thresholds in the 6-Hz and MES tests using outbred Swiss mice. Highly purified D9 -THCA was used in the studies or a mixture of D9 -THCA and D9 -THC.
Results: We observed mixed anticonvulsant and proconvulsant effects of D9 -THCA across the seizure models. Highly pure D9 -THCA did not affect hyperthermia-induced seizures in Scn1a + / mice. A D9 -THCA/D9 -THC mixture was anticonvulsant in the 6-Hz threshold test, but purified D9 -THCA and D9 -THC had no effect. Conversely, both D9 -THCA and D9 -THC administered individually were proconvulsant in the MES threshold test but had no effect when administered as a D9 -THCA/D9 -THC mixture. The D9 -THCA/D9 -THC mixture, however, increased spontaneous seizure severity and increased mortality of Scn1a + / mice.
Discussion: The anticonvulsant profile of D9 -THCA was variable depending on the seizure model used and presence of D9 -THC. Because of the unstable nature of D9 -THCA, further exploration of D9 -THCA through formal anticonvulsant drug development is problematic without stabilization. Future studies may better focus on determining the mechanisms by which combined D9 -THCA and D9 -THC alters seizure thresholds, as this may uncover novel targets for the control of refractory partial seizures.

Evaluation of the potential of the phytocannabinoids, cannabidivarin (CBDV) and Δ9 -tetrahydrocannabivarin (THCV), to produce CB1 receptor inverse agonism symptoms of nausea in rats
Erin M Rock, Martin A Sticht, Marnie Duncan, Colin Stott and Linda A Parker
British Journal of Pharmacology (2013) 170 671–678
DOI:10.1111/bph.12322
https://www.ncbi.nlm.nih.gov/pmc/art...h0170-0671.pdf
BACKGROUND AND PURPOSE The cannabinoid 1 (CB1) receptor inverse agonists/antagonists, rimonabant (SR141716, SR) and AM251, produce nausea and potentiate toxin-induced nausea by inverse agonism (rather than antagonism) of the CB1 receptor. Here, we evaluated two phytocannabinoids, cannabidivarin (CBDV) and Δ9 -tetrahydrocannabivarin (THCV), for their ability to produce these behavioural effect characteristics of CB1 receptor inverse agonism in rats.
EXPERIMENTAL APPROACH In experiment 1, we investigated the potential of THCV and CBDV to produce conditioned gaping (measure of nausea-induced behaviour) in the same manner as SR and AM251. In experiment 2, we investigated the potential of THCV and CBDV to enhance conditioned gaping produced by a toxin in the same manner as CB1 receptor inverse agonists.
KEY RESULTS SR (10 and 20 mg·kg−1 ) and AM251 (10 mg·kg−1 ) produced conditioned gaping; however, THCV (10 or 20 mg·kg−1 ) and CBDV (10 or 200 mg·kg−1 ) did not. At a subthreshold dose for producing nausea, SR (2.5 mg·kg−1 ) enhanced lithium chloride (LiCl)-induced conditioned gaping, whereas Δ9 -tetrahydrocannabinol (THC, 2.5 and 10 mg·kg−1 ), THCV (2.5 or 10 mg·kg−1 ) and CBDV (2.5 or 200 mg·kg−1 ) did not; in fact, THC (2.5 and 10 mg·kg−1 ), THCV (10 mg·kg−1 ) and CBDV (200 mg·kg−1 ) suppressed LiCl-induced conditioned gaping, suggesting anti-nausea potential.
CONCLUSIONS AND IMPLICATIONS The pattern of findings indicates that neither THCV nor CBDV produced a behavioural profile characteristic of CB1 receptor inverse agonists. As well, these compounds may have therapeutic potential in reducing nausea.


Evaluating the effects of gamma-irradiation for decontamination of medicinal cannabis
Arno Hazekamp
Frontiers in Pharmacology
Frontiers in Pharmacology 7 April 2016
DOI: 10.3389/fphar.2016.00108
In several countries with a National medicinal cannabis program, pharmaceutical regulations specify that herbal cannabis products must adhere to strict safety standards regarding microbial contamination. Treatment by gamma irradiation currently seems the only method available to meet these requirements. We evaluated the effects of irradiation treatment of four different cannabis varieties covering different chemical compositions. Samples were compared before and after standard gamma-irradiation treatment by performing quantitative HPLC analysis of major cannabinoids, as well as qualitative GC analysis of full cannabinoid and terpene profiles. In addition, water content and microscopic appearance of the cannabis flowers was evaluated. This study found that treatment did not cause changes in the content of THC and CBD, generally considered as the most important therapeutically active components of medicinal cannabis. Likewise, the water content and the microscopic structure of the dried cannabis flowers were not altered by standard irradiation protocol in the cannabis varieties studied. The effect of gamma-irradiation was limited to a reduction of some terpenes present in the cannabis, but keeping the terpene profile qualitatively the same. Based on the results presented in this report, gamma irradiation of herbal cannabis remains the recommended method of decontamination, at least until other more generally accepted methods have been developed and validated.

Evaluating the Suitability and Potential Efficiency of Cannabis sativa Oil for Patients with Primary Burning Mouth Syndrome: A Prospective, Open-Label, Single-Arm Pilot Study.
Gambino, A., Cabras, M., Panagiotakos, E., Calvo, F., Macciotta, A., Cafaro, A., … Arduino, P. G.
Pain Medicine.(2020).
doi:10.1093/pm/pnaa318
Objective. To evaluate the use of a Cannabis sativa oil in the management of patients diagnosed with primary burning mouth syndrome (BMS).
Design. Prospective, open-label, single-arm pilot study. Setting. University hospital. Subjects. Seventeen patients with diagnosed BMS were included.
Methods. Subjects were treated for 4 weeks with a full cannabis plant extract, which was prepared from standardized plant material (cannabis flos) in specialized pharmacies by means of Romano-Hazekamp extraction and was diluted in oil (1 g of cannabis in 10 g of olive oil). The primary outcome was the change in pain intensity (assessed by the visual analog scale, Present Pain Intensity scale, McGill Pain Questionnaire, and Oral Health Impact Profiles) at the end of the protocol and during the succeeding 24 weeks; the neuropathic pain was also investigated with a specific interview questionnaire (DN4-interview [Douleur Neuropathique en 4 Questions]). Levels of anxiety and depression were considered as secondary outcomes, together with reported adverse events due to the speci- fied treatment.
Results. Subjects showed a statistically significant improvement over time in terms of a clinical remission of the oral symptoms. Levels of anxiety and depression also changed statistically, displaying a favorable improvement. No serious reactions were detailed. None of the patients had to stop the treatment due to adverse events.
Conclusions. In this pilot evaluation, the C. sativa oil provided was effective and well tolerated in patients with primary BMS. Further bigger and properly defined randomized controlled trials, with different therapeutic approaches or placebo control, are needed, however.

Even High Doses of Oral Cannabidiol Do Not Cause THC-Like Effects in Humans: Comment on Merrick et al.
Grotenhermen, F., Russo, E., & Zuardi, A. W.
Cannabis and Cannabinoid Research 2016;1(1):102–112;
DOI: 10.1089/can.2015.0004. Cannabis and Cannabinoid Research, 2(1), 1–4.doi:10.1089/can.2016.0036
This short communication examines the question whether the experimental data presented in a study by Merrick et al. are of clinical relevance. These authors found that cannabidiol (CBD), a major cannabinoid of the cannabis plant devoid of psychotropic effects and of great interest for therapeutic use in several medical conditions, may be converted in gastric fluid into the psychoactive cannabinoids delta-8-THC and delta-9-THC to a relevant degree. They concluded that ‘‘the acidic environment during normal gastrointestinal transit can expose orally CBD-treated patients to levels of THC and other psychoactive cannabinoids that may exceed the threshold for a positive physiological response.’’ They issued a warning concerning oral use of CBD and recommend the development of other delivery methods. However, the available clinical data do not support this conclusion and recommendation, since even high doses of oral CBD do not cause psychological, psychomotor, cognitive, or physical effects that are characteristic for THC or cannabis rich in THC. On the contrary, in the past decades and by several groups, high doses of oral CBD were consistently shown to cause opposite effects to those of THC in clinical studies. In addition, administration of CBD did not result in detectable THC blood concentrations. Thus, there is no reason to avoid oral use of CBD, which has been demonstrated to be a safe means of administration of CBD, even at very high doses

Exercise-induced euphoria and anxiolysis do not depend on endogenous opioids in humans
Michael Siebers, Sarah V. Biedermann, Laura Bindila, Beat Lutz, Johannes Fuss
Psychoneuroendocrinology Volume 126, April 2021, 105173
DOI: 10.1016/j.psyneuen.2021.105173
A runner's high describes a sense of well-being during endurance exercise characterized by euphoria and anxiolysis. It has been a widespread belief that the release of endogenous opioids, such as endorphins, underlie a runner's high. However, exercise leads to the release of two classes of rewarding molecules, endocannabinoids (eCBs) and opioids. In mice, we have shown that core features of a runner's high depend on cannabinoid receptors but not opioid receptors. In the present study, we aimed to corroborate in humans that endorphins do not play a significant role in the underlying mechanism of a runner's high. Thus, we investigated whether the development of two core features of a runner's high, euphoria and reduced anxiety levels, depend on opioid signaling by using the opioid receptor antagonist naltrexone (NAL) in a double-blind, randomized, placebo (PLA)-controlled experiment. Participants (N = 63) exhibited increased euphoria and decreased anxiety after 45 min of running (RUN) on a treadmill in a moderate-intensity range compared to walking (WALK). RUN led to higher plasma levels of the eCBs anandamide (AEA) and 2-arachidonoglycerol (2-AG). Opioid blockade did not prevent the development of euphoria and reduced anxiety as well as elevation of eCB levels following exercise. Moreover, the fraction of participants reporting a subjective runner's high was comparable in the NAL and PLA-treated group. Therefore, this study indicates that the development of a runner's high does not depend on opioid signaling in humans, but makes eCBs strong candidates in humans, as previously shown in mice.
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Study Questions:
Do endorphins play a significant role in humans in the underlying mechanism of runner’s high?
Methods:
In a double-blind, randomized, placebo-controlled experiment, the opioid-receptor antagonist naltrexone was used to assess whether euphoria and reduced anxiety levels, two core features of runners high, were dependent on opioid signaling. A cohort of 63 healthy adults (32 women, 31 men, 18-50 years of age) who regularly perform endurance exercise were included. Participants were randomized to receive either naloxone 50 mg or placebo before exercise; on two occasions separated by at least 30 days, participants performed moderate-intensity running or regular walking for 45 minutes on a treadmill. Standardized questionnaires and subjective grading were used to assess emotional state and the presence of runner’s high after exercise. Blood samples were collected and analyzed for endocannabinoids (eCBs).
Results:
Participants experienced increased euphoria and decreased anxiety after 45 minutes of running on a treadmill in a moderate-intensity range compared to walking. Running led to higher plasma levels of the eCBs anandamide and 2-arachidonoglycerol. Opioid blockade with naloxone did not prevent the development of euphoria and reduced anxiety or elevation of eCB levels following exercise. The fraction of participants reporting a subjective runner’s high was comparable in the naloxone (7 of 32) and the placebo-treated groups (5 of 31).
Conclusions:
The authors concluded that this study indicates that the development of runner’s high in humans does not depend on opioid signaling; and makes eCBs strong candidates for agents responsible for runner’s high in humans, as previously demonstrated in mice.
Perspective:
Runner’s high describes a sense of well-being during endurance exercise, characterized by euphoria and anxiolysis. The release of endogenous opioids, such as endorphins, has been widely believed to be responsible for runner’s high. However, exercise is associated with the release of both opioids and eCBs, both classes of molecules potentially associated with reward processing. This double-blind, randomized, placebo-controlled study revealed that running for 45 minutes compared to walking resulted in higher eCB levels, and that opioid-blockade with naloxone did not interfere with the development of subjective runner’s high. Although the scenario of a 45-minute treadmill run might not be the best scenario to induce runner’s high, the study provides provocative data suggesting that eCBs, not endogenous opioids, are responsible for runner’s high.

Expanding Research on Cannabis-Based Medicines for Liver Steatosis: A Low-Risk High-Reward Way Out of the Present Deadlock?
Tangui Barré , Vincenzo Di Marzo, Fabienne Marcellin, Patrizia Burra, and Patrizia Carrieri
13 Apr 2022 DOI: 10.1089/can.2022.0014
https://www.liebertpub.com/doi/epdf/10.1089/can.2022.0014
Obesity and nonalcoholic fatty liver disease (NAFLD) constitute global and growing epidemics that result in therapeutic dead ends. There is an urgent need for new and accessible treatments to improve and widen both preventive and curative approaches against NAFLD. The endocannabinoid system (ECS) is recognized as a complex signaling apparatus closely related to metabolic disorders and is a key target for treating NAFLD. Despite a lack of conclusive clinical trials, observational and pre-clinical studies highlight putative benefits of phytocannabinoids on liver steatosis through multiple pathways. Owing to both its safety profile and its diversity of active compounds acting primarily
(although not exclusively) on the ECS—and its expanded version, the endocannabinoidome, the Cannabis plant should be considered a major prospect in the treatment of NAFLD. However, seizing this opportunity, and intensifying clinical research in this direction, will require overcoming both scientific and nonscientific barriers. .

Experimental methods to study the role of the peripheral cannabinoid receptor in immune function
Nancy E Buckley , Diep Burbridge, Manop Buranapramest, Tanya Ferguson, Renee Y Paau
Methods Mol Med. 2006;123:19-40.
doi: 10.1385/1-59259-999-0:19
https://www.researchgate.net/publica...mmune_function
Marijuana components, such as delta-9-tetrahydrocannabinol, and endogenous cannabinoids, such as anandamide and 2-arachydonoylglycerol, alter diverse immune functions. Two cannabinoid receptors have been discovered to date, the central cannabinoid receptor (CB1R) and the peripheral cannabinoid receptor (CB2R). The CB1R is expressed predominantly in the central nervous system. The CB2R is expressed mainly in cells of the immune system, suggesting that the CB2R is involved in immunoregulatory events. Cannabinoids have been shown to modulate diverse immune functions including cytokine production, lymphocyte proliferation, and humoral and cell-mediated immune responses. In addition, cannabinoids have been shown to induce different signal transduction pathways. However, the role of cannabinoids and their receptors in the immune system remains unclear. The objective of the experimental methods described herein is to investigate the role of CB2R activation in specific splenocyte and macrophage functions using a mouse lacking the CB2R. Interestingly, our findings,thus far suggest that basal CB2R activation modulates lymphocyte proliferation and cytokine secretion and macrophage phagocytic activity. Therefore, data obtained using the methodology described in this chapter will help us elucidate the role of cannabinoids and the CB2R in the immune system

Expert Panel on Understanding Cannabis: Medicine, Society, Government
Kyle Kazan, Daniele Piomelli, Dana Rohrabacher, and Lori Ajax
Cannabis and Cannabinoid Research 2017, 2.1
Doi: 10.1089/can.2017.29010.kka
On May 5, 2017, the Department of Anatomy and Neurobiology of the University of California Irvine convened an expert panel to discuss issues related to cannabis at the interface between medical science, society,
and government. The three invited speakers—Dr. Daniele Piomelli (University of California, Irvine), U.S. Representative Dana Rohrabacher (California’s 48th District), and Lori Ajax (Bureau of Medical Marijuana
Regulation California Department of Consumer Affairs)—briefly introduced each one of the topics to an audience of more than 200 people, and a lively discussion ensued. What follows are excerpts from the discussion.

Exploring cannabis concentrates on the legal market: User profiles, product strength, and health-related outcomes.
Cinnamon Bidwelll, L., YorkWilliams, S. L., Mueller, R., Bryan, A. D., & Hutchison, K. E.
Addictive Behaviors Reports.(2018).
doi:10.1016/j.abrep.2018.08.004
Background: Concentrated cannabis products are increasingly available and used, particularly in states with legal cannabis, but little is known about the profiles and characteristics of concentrate users. We aimed to characterize user profiles of cannabis users living in states with legal medical or recreational cannabis who reported using concentrates to those who do not use concentrates.
Methods: An anonymous online survey was advertised in California, Colorado, Nevada, Oregon, and Washington. We compared respondents who endorsed frequent concentrate use (FC; N=67) (i.e. 4 days/week) with cannabis users who never use concentrates (NC; N=64), and with those who smoke/vaporize cannabis flower frequently but never or very rarely use concentrates (FF; N=60), on measures related to cannabis use patterns and cannabinoid product strength, other substance use, and occupational functioning and health.
Results: FC endorsed more symptoms of cannabis use disorder as compared to nonconcentrate users

Exploring the diagnosis and profile of cannabis allergy.
Decuyper, I. I., Van Gasse, A. L., Faber, M. A., Elst, J., Mertens, C., Rihs, H.-P., … Ebo, D. G. .
The Journal of Allergy and Clinical Immunology: In Practice. (2018)
doi:10.1016/j.jaip.2018.09.017
Background: Cannabis allergy (CA) has mainly been attributed to Can s 3, the nonspecific lipid transfer protein (nsLTP) of Cannabis sativa. Nevertheless, standardized diagnostic tests are lacking and research on CA is scarce.
Objective: To explore the performance of 5 cannabis diagnostic tests and the phenotypic profile of CA.
Methods: A total of 120 patients with CA were included and stratified according to the nature of their cannabis-related symptoms; 62 healthy and 189 atopic controls were included.
Specific IgE (sIgE) hemp, sIgE and basophil activation test (BAT) with a recombinant Can s 3 protein from Cannabis sativa (rCan s 3), BAT with a crude cannabis extract, and a skin prick test (SPT) with an nCan s 3-rich cannabis extract were performed. Clinical information was based on patient history and a standardized questionnaire.
Results:*First, up to 72% of CA reporting likely-anaphylaxis (CA-A) are Can s 3 sensitized. Actually, the Can s 3-based diagnostic tests show the best combination of positive and negative predictive values, 80% and 60%, respectively. sIgE hemp displays 82% sensitivity but only 32% specificity. Secondly, Can s 3+CA reported significantly more cofactor-mediated reactions and displayed significantly more sensitizations to other nsLTPs than Can s 3-CA. Finally, the highest prevalence of systemic reactions to plant-derived foods was seen in CA-A, namely 72%.
Conclusions:*The most effective and practical tests to confirm CA are the SPT with an nCan s 3-rich extract and the sIgE rCan s 3. Can s 3 sensitization entails a risk of systemic reactions to plant-derived foods and cofactor-mediated reactions. However, as Can s 3 sensitization is not absolute, other cannabis allergens probably play a role.

Exploring the munchies: An online survey of users’ experiences of cannabis effects on appetite and the development of a Cannabinoid Eating Experience Questionnaire.
Roberts, C. A., Jager, G., Christiansen, P., & Kirkham, T. C.
Journal of Psychopharmacology, 026988111986252.(2019).
doi:10.1177/0269881119862526
Background: Cannabis intoxication is commonly reported to increase appetite and enhance appreciation of food (the ‘munchies’). These effects are attributed to activation of the endocannabinoid system. However, the psychological changes that underlie these phenomena are under-researched. We report here the results of an extensive online survey of cannabis users with an exploratory Cannabinoid Eating Experience Questionnaire (CEEQ). Method: Frequent cannabis users completed a 46-item questionnaire about their eating behaviour under the influence of cannabis. An Englishspeaking sample (n=591) provided data for the initial exploratory validation of the scale. A second Dutch-language survey (n=163) was used for confirmatory factor analysis. Test-retest reliability was based on a third English-speaking sample (n=40) who completed the revised, 28-item CEEQ twice across 2 weeks.
Results: Principal components analysis provided a two-factor solution. Factor 1 (hedonic) comprised 14 items that related primarily to the enjoyment and altered sensory aspects of eating. Factor 2 (appetitive) comprised a further 14 items related to motivational factors that instigate or promote eating. The two-factor structure was supported by confirmatory factor analysis. Both the hedonic and appetitive subscales had good internal reliability (?=0.92 for each subscale, in two independent samples). Good test-retest reliability was obtained for the revised 28-item questionnaire

Exploring the relationship between marijuana smoking and COVID-19 outcomes.
FASIH SAMI SIDDIQUI, SAUD BIN ABDUL SATTAR, CHAPMAN WEI, NAWAL MUSTAFA, RADU GROVU, AHMAD MUSTAFA
CRITICAL CARE| VOLUME 164, ISSUE 4, SUPPLEMENT , A1691, OCTOBER 2023
DOI:https://doi.org/10.1016/j.chest.2023.07.1163
Marijuana use is becoming increasingly prevalent worldwide, yet the full spectrum of its effects largely remains unknown. Although cannabinoids have immunomodulatory properties, there remains a significant gap in our understanding of the potential impact of marijuana use on COVID-19 outcomes. The purpose of the study is to compare the outcomes of COVID-19 infection on individuals who use marijuana and those who do not.

Expression of endocannabinoid system components in human airway epithelial cells: impact of sex and chronic respiratory disease status
Matthew F. Fantauzzi, Jennifer A. Aguiar, Benjamin J.-M. Tremblay, Michael J. Mansfield, Toyoshi Yanagihara, Abiram Chandiramohan, Spencer Revill, Min Hyung Ryu, Chris Carlsten, Kjetil Ask, Martin Stämpfli,Andrew C. Doxey, Jeremy A. Hirota
ERJ Open Research 2020 6: 00128-2020;
DOI: 10.1183/23120541.00128-2020
https://sci-hub.se/https://openres.e...6/4/00128-2020
Cannabis smoking is the dominant route of delivery, with the airway epithelium functioning as the site of first contact. The endocannabinoid system is responsible for mediating the physiological effects of inhaled phytocannabinoids. The expression of the endocannabinoid system in the airway epithelium and contribution to normal physiological responses remains to be defined.
To begin to address this knowledge gap, a curated dataset of 1090 unique human bronchial brushing gene expression profiles was created. The dataset included 616 healthy subjects, 136 subjects with asthma, and 338 subjects with COPD. A 32-gene endocannabinoid signature was analysed across all samples with sex and disease-specific analyses performed. Immunohistochemistry and immunoblots were performed to probe in situ and in vitro protein expression.
CB1, CB2, and TRPV1 protein signal is detectable in human airway epithelial cells in situ and in vitro, justifying examining the downstream endocannabinoid pathway. Sex status was associated with differential expression of 7 of 32 genes. In contrast, disease status was associated with differential expression of 21 of 32 genes in people with asthma and 26 of 32 genes in people with COPD. We confirm at the protein level that TRPV1, the most differentially expressed candidate in our analyses, was upregulated in airway epithelial cells from people with asthma relative to healthy subjects. Our data demonstrate that the endocannabinoid system is expressed in human airway epithelial cells with expression impacted by disease status and minimally by sex. The data suggest that cannabis consumers may have differential physiological responses in the respiratory mucosa

FAAH inhibition for treatment of problematic cannabis use
Tony P George
Lancet Psychiatry Published online December 6, 2018
doi: 10.1016/S2215-0366(18)30462-0
In their Article in The Lancet Psychiatry, Deepak Cyril D’Souza and colleagues from Yale University describe the results from a randomised clinical trial of a fatty acid amide hydrolase (FAAH) inhibitor in 70 men seeking treatment for cannabis dependence (according to DSM-IV criteria). Participants were randomly assigned to an oral FAAH Inhibitor (PF-04457845 [provided by Pfizer, Groton, VT, USA]; figure; 4 mg per day) or matching placebo and underwent a period of initial abstinence from cannabis for about 1 week during an inpatient stay on a research unit; they were then were discharged to outpatient care for an additional 3-week treatment period on study medications. The primary outcomes were treatmentrelated differences in cannabis withdrawal symptoms during admission, and self-reported cannabis use by timeline follow-back methods, and urine THC-COOH concentrations at end of treatment. Other outcome measures included craving symptoms and sleep measures. The results of this study suggest positive effects of PF-04457845 versus placebo on cannabis use, withdrawal symptoms, and sleep, with mediumto-large effect sizes suggesting that these effects are clinically meaningful. PF-04457845 reduced cannabis withdrawal symptoms (difference in mean symptom score on first day of treatment 4·96 [95% CI 0·71–9·21], padj=0·048), self-reported cannabis use at 4 weeks (difference in joints per day 0·88 [95% CI 0·29–1·46]; p<0·0003), and urinary THC-COOH concentrations (difference 392·37 ng/mL [95% CI 17·55–767·18]; p=0·009).

Fatal Aspergillosis Associated with Smoking Contaminated Marijuana, in A Marrow Transplant Recipient.
Hamadeh, R., Ardehali, A., Locksley, R. M., & York, M. K.
Chest, 94(2), 432–433. (1988).
doi:10.1378/chest.94.2.432
A 34-year-old man presented with pulmonary aspergillosis on the 75th day after marrow transplant for chronic myelogenous leukemia. The patient had smoked marijuana heavily for several weeks prior to admission. Cultures of the marijuana revealed AspergiUus fumigatus with morphology and growth characteristics identical to the organism grown from open lung biopsy specimen. Despite aggressive antifungal therapy, the patient died with disseminated disease. Physicians should be aware of this potentially lethal complication of marijuana use in compromised hosts.

FDA Issues Warning Letters to Companies Claiming Their CBD Products Can Treat COVID-19
April 1, 2022 – Cannabis Business Times News
https://www.cannabisbusinesstimes.c...ies-claiming-cbd-products-can-treat-covid-19/.
The federal agency is warning seven companies that they are falsely advertising their wares.
Several companies have been claiming that their CBD products can cure, treat, or prevent COVID-19, but the U.S. Food and Drug Administration (FDA) says they are wrong.
Seven companies—Cureganics, Heaven’s Organics LLC, Functional Remedies LLC, Greenway Herbal Products LLC, CBD Social, UPSY LLC and Nature’s Highway—received warning letters from the FDA in conjunction with the Federal Trade Commission (FTC) on March 28, according to a Miami Herald report.
The FDA and FTC are issuing warning letters to businesses that they say are “selling fraudulent products with claims to prevent, treat, mitigate, diagnose, or cure coronavirus disease 2019 (COVID-19),” according to the FDA’s website.
For example, in its letter to CBD Social, the FDA claims it “has observed that your website offers cannabidiol (CBD) products for sale in the United States and that these products are intended to mitigate, prevent, treat, diagnose, or cure COVID-19 in people. Based on our review, these products are unapproved new drugs sold in violation of section 505(a) of the Federal Food, Drug, and Cosmetic Act (FD&C Act), 21 U.S.C. § 355(a). Furthermore, these products are misbranded drugs under section 502 of the FD&C Act, 21 U.S.C. § 352. The introduction or delivery for introduction of these products into interstate commerce is prohibited under sections 301(a) and (d) of the FD&C Act, 21 U.S.C. § 331(a) and (d).”
The warning letter says that CBD Social cited studies on its website to “misleadingly represent” its products “as safe and/or effective for the treatment or prevention of COVID-19,” including a 2021 National Institute of Health (NIH) study, a 2022 University of Chicago study, and a 2022 study from the American Chemical Society and American Society of Pharmacognosy. .

Fetal Exposure to Cannabis and Childhood Metabolic Outcomes: The Healthy Start Study
Brianna F. Moore, Katherine A. Sauder, Allison L. B. Shapiro, Tessa Crume, Gregory L. Kinney, and Dana Dabelea
The Journal of Clinical Endocrinology & Metabolism, 2022, XX, 1–8
DOI: 10.1210/clinem/dgac101
https://watermark.silverchair.com/dgac101.pdf?token=AQECAHi208BE 49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfK Ac485ysgAAAtswggLXBgkqhkiG9w0B BwagggLIMIICxAIBADCCAr0GCSqGSI b3DQEHATAeBglghkgBZQMEAS4wEQQM osS-Ac166NuJprghAgEQgIICjrXEbeMpY7 wlKwa-nHCx7tX1jjUSycrnvdHVl0rAYa2eLh vlbTQOk75T61fSct2bsZJNfpqXOtSY YoSXAgFJxUoiczKwCZP5wOrZ1KT4tJ tfWo7iIVH3mfvjoZ73AJeWZmbebdF1 E1R-ZSgLAIczIVVvh4ZoAi_dmFvXesoBji HOlVrDksVF--isiyrlcmoaoBxPpy22ky0wPMLw60WN Tjlx9SpWzjceAGD2_xtBfiG8_rLucA QwTix3Cu0PRno89dqNOhZ0Qf-lwgRDrrLkQ6czVaO8L_j21wXJtArsN WhGVvrP0DuCRim6ENgl6S63em1J2N6 akajz5cT2u1cP1mVXKFQ7sPMpwOHdu OcrX5tk8Ts7Hl25U-0MM1fgQiJzPvAdfIBgihoTiV2SSU7Q ZhNHjvqoTikVDZaBZ4ZfZ25F6rdqmH YDg4PSrOXEcu0fCpsiTrjQ4CeDPgtB GeYZDrYcgbVsPhmDEeG9egFBDmoWki ugAD2GrAssOGPi4ZRwdOj-8wSka6CrBm9EgdpkyUD0UGXFSkGxnN fG7yyksVsuCQBIMmYj09VtTP3LRuyC H2hzax6pmpwRSQozGvkjGJ9PIdkyEX 7Ulmv2pdfWYG9M_gI97_zWd-Git-QTAKbgeUpfsm2_1fkaCGI9e3-09AHnDC5BiWAkHQWEW0CwyTcjoCCan-_YmzD6DEc-c827D_JJ70ou5JpVoaaAKN1wI7VBSL knHZv2NWGOPTcqohB889nI0THZinRH jx7OlBFfhjaAO8__nNX1hc4-oipZYQz_DmVRE3KawjT_eqJOylikjY 2a-binjtJ5w0SQNY9YXhh49m_qTupmBcv Ghae-ow8AsO4dmRpZH2lJ0yX-pw
Objective: To assess the impact of fetal exposure to cannabis on adiposity and glucose-insulin traits in early life.
Research Design and Methods: We leveraged a subsample of 103 mother-child pairs from Healthy Start, an ethnically diverse Colorado-based cohort. Twelve cannabinoids/metabolites of cannabis (including Δ9-tetrahydrocannabinol and cannabidiol) were measured in maternal urine collected at ~27 weeks’ gestation. Fetal exposure to cannabis was dichotomized as exposed (any cannabinoid > limit of detection [LOD]) and not exposed (all cannabinoids < LOD). Fat mass and fat-free mass were measured via air displacement plethysmography at follow-up (mean age: 4.7 years). Glucose and insulin were obtained after an overnight fast. Generalized linear models estimated the associations between fetal exposure to cannabis with adiposity measures (fat mass [kg], fat-free mass [kg], adiposity [fat mass percentage], body mass index [BMI], and BMI z-scores) and metabolic measures (glucose [mg/dL], insulin [uIU/mL], and homeostatic model assessment of insulin resistance [HOMA-IR]).
Results: Approximately 15% of the women had detectable levels of any cannabinoid, indicating fetal exposure to cannabis. Exposed offspring had higher fat mass (1.0 kg; 95% CI, 0.3-1.7), fat-free mass (1.2 kg; 95% CI, 0.4-2.0), adiposity (2.6%; 95% CI, 0.1-5.2), and fasting glucose (5.6 mg/dL; 95% CI, 0.8-10.3) compared with nonexposed offspring. No associations were found with fasting insulin (in the fully adjusted model), HOMA-IR, BMI, or BMI z-scores.
Conclusions: We provide novel evidence to suggest an association between fetal exposure to cannabis with increased adiposity and fasting glucose in childhood, a finding that should be validated in other cohorts.

Fine Particulate Matter Exposure From Secondhand Cannabis Bong Smoking
Patton Khuu Nguyen, S. Katharine Hammond
JAMA Network Open. 2022;5(3):e224744.
doi:10.1001/jamanetworkopen.2022.4744
https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2790510?xnpe_tifc=b.YdOfQNOfoX bDY8xIndxMpZhfEWVjQsVuU_O.VjOI olhkUstIYDhIbA4FP_hNl8bdhNtIHp bDPNxfb7bDHdb9puOIUNhkoJhkUL4k nD4dhN&utm_source=eNews&utm_ca mpaign=TCS%20Newsletter%20Week %2014%202022&utm_medium=email
Introduction
Secondhand cannabis smoke (SHCS) is a novel exposure source uncharacterized in homes but containing known health risk factors.1 Although 27%of young adults believe SHCS exposure is safe,2 cannabis smoke has several hundred toxic chemicals, carcinogens, and fine particulate matter (PM2.5), many at higher concentrations than tobacco smoke.1 Decades of secondhand tobacco smoke (SHTS) research demonstrate causal links to cancer, respiratory and cardiovascular diseases, preterm birth, and decreased immune function.3 These concerns have not translated to cannabis bong smoking, a popular consumption method in social settings among young adults, wherein smoke is drawn through water. However, like SHTS, 1 minute of SHCS caused significant endothelial dysfunction in rats.4 This cohort study measured PM2.5 levels from social bong smoking; it is the first, to our knowledge, to quantify SHCS levels from social cannabis smoking in the home.
Methods
Levels of PM2.5 were measured before, during, and after 8 cannabis social-smoking sessions in one 20-m2 household living room (eMethods in the Supplement). An aerosol monitor (SidePak AM510; TSI Inc) measured PM2.5 concentrations where a nonsmoker might sit. The University of California, Berkeley, Office for the Protection of Human Subjects deemed this study not human participants research and waived review. This study followed the STROBE reporting guideline. The Wilcoxon rank sum 2-sided test assessed statistical differences between PM2.5 concentrations before and during smoking. Analysis was performed using RStudio, version 1.4 (RStudio). Two-sided P < .05 indicated statistical significance.
Results
Home cannabis bong smoking significantly increased PM2.5 from background levels (conditions existing before the smoking began) in all sessions by 100-fold to 1000-fold for 6 of 8 sessions; the other 2 sessions had high background and significantly increased PM2.5 more than 20-fold (P < .001 for all 8 sessions). During the first 10 minutes of smoking, mean (SD) PM2.5 concentrations increased to 410 (220) μg/m3, after 15 minutes to 570 (290) μg/m3, after 30 minutes to 1000 (320) μg/m3, and went as high as 2500 μg/m3 in 1 session (Figure). The concentration during smoking increased to a mean (SD) of 1300 (280) μg/m3 (Table). During 2-hour smoking sessions, mean (SD) 5-minute peak PM2.5 concentration was 1700 (460) μg/m3 and remained half that 90 minutes after smoking ceased. Each half hour after smoking ceased, mean concentration declined to 78%of peak value, then 60%, then 40%, and, after 110 minutes, 31%. In the 1 session monitored for 12 hours after smoking stopped, PM2.5 remained elevated at 50 μg/m3, more than 10 times the background concentration. Cannabis bong smoking in the home generated 4 times greater PM2.5 concentrations than cigarette or tobacco hookah (waterpipe) smoking (Table).
Discussion
The PM2.5 concentrations generated in a home during social cannabis bong smoking to which a nonsmoking resident might be exposed were greatly increased compared with background levels, and PM2.5 decayed only gradually after smoking ceased. After 15 minutes of smoking, mean PM2.5 (570 μg/m3) (Figure)was more than twice the US Environmental Protection Agency (EPA) hazardous air quality threshold (>250 μg/m3). If one assumes the exposure concentrations were at the mean levels observed, a single home smoking session with no other exposures would generate an estimated mean daily concentration (200 μg/m3) that greatly exceeds the average in cigarette smoking homes (44 μg/m3), nonsmoking homes (15 μg/m3), and the US EPA daily standard (35 μg/m3).3 A strength of this study is that measurements were made during actual social bong smoking sessions without artificial constraints. Limitations include that cannabis smoking was not directly
observed. .

Flavonoid Derivative of Cannabis Demonstrates Therapeutic Potential in Preclinical Models of Metastatic Pancreatic Cancer.
Moreau, M., Ibeh, U., Decosmo, K., Bih, N., Yasmin-Karim, S., Toyang, N., … Ngwa, W.
Frontiers in Oncology, 9. (2019).
doi:10.3389/fonc.2019.00660
Pancreatic cancer is particularly refractory to modern therapies, with a 5-year survival rate for patients at a dismal 8%. One of the significant barriers to effective treatment is the immunosuppressive pancreatic tumor microenvironment and development of resistance to treatment. New treatment options to increase both the survival and quality of life of patients are urgently needed. This study reports on a new non-cannabinoid, non-psychoactive derivative of cannabis, termed FBL-03G, with the potential to treat pancreatic cancer. In vitro results show major increase in apoptosis and consequential decrease in survival for two pancreatic cancer modelsPanc-02 and KPC pancreatic cancer cells treated with varying concentrations of FBL-03G and radiotherapy. Meanwhile, in vivo results demonstrate therapeutic efficacy in delaying both local and metastatic tumor progression in animal models with pancreatic cancer when using FBL-03G sustainably delivered from smart radiotherapy biomaterials. Repeated experiments also showed significant (P < 0.0001) increase in survival for animals with pancreatic cancer compared to control cohorts. The findings demonstrate the potential for this new cannabis derivative in the treatment of both localized and advanced pancreatic cancer, providing impetus for further studies toward clinical translation

Food effect on pharmacokinetics of cannabidiol oral capsules in adult patients with refractory epilepsy.
Birnbaum, A. K., Karanam, A., Marino, S. E., Barkley, C. M., Remmel, R. P., Roslawski, M., … Leppik, I. E.
Epilepsia. (2019).
doi:10.1111/epi.16093
Objective: To evaluate the pharmacokinetics of a purified oral cannabidiol (CBD) capsule administered with and without food in adults with refractory epilepsy.
Methods: Adult patients who were prescribed CBD for seizures, had localization? related intractable epilepsy with ?4 seizures per month, and qualified for Minnesotacannabis were enrolled. A single dose of 99% pure CBD capsules was taken under both fasting (no breakfast) and fed (high fat 840?860 calorie) conditions. Blood sampling for CBD plasma concentrations was performed under each condition between 0 and 72*hours post?dose and measured by a validated liquid chormatography?mass spectometry assay. CBD pharmacokinetic profiles including maximum concentration (Cmax), area?under?the?curve from zero to infinity (AUC0??), and time?to? maximum concentration (Tmax) were calculated. The confidence intervals (CIs) for log?transformed Cmax and AUC0?? ratios between fed and fasting states were calculated. Seizure and adverse events information was collected.
Results: Eight patients completed the study. On average Cmax was 14 times and AUC0?? 4 times higher in the fed state. The 90% CI for the ratio of fed versus fast conditions for Cmax and AUC0?? were 7.47?31.86 and 3.42?7.82, respectively. No sequence or period effect for Cmax and AUC0?? was observed. No adverse events were reported.
Significance: Administering CBD as a capsule rather than a liquid allows for more precise determination of pharmacokinetics parameters and is more representative of CBD swallowed products. The fat content of a meal can lead to significant increases in Cmax and AUC0?? and can account for variability in bioavailability and overall drug exposure within patients with oral products.

For whom the endocannabinoid tolls: Modulation of innate immune function and implications for psychiatric disorders
Rebecca J Henry , Daniel M Kerr , David P Finn , Michelle Roche
Prog Neuropsychopharmacol Biol Psychiatry. 2016.
doi: 10.1016/j.pnpbp.2015.03.006
Toll-like receptors (TLRs) mediate the innate immune response to pathogens and are critical in the host defence, homeostasis and response to injury. However, uncontrolled and aberrant TLR activation can elicit potent effects on neurotransmission and neurodegenerative cascades and has been proposed to trigger the onset of certain neurodegenerative disorders and elicit detrimental effects on the progression and outcome of established disease. Over the past decade, there has been increasing evidence demonstrating that the endocannabinoid system can elicit potent modulatory effects on inflammatory processes, with clinical and preclinical evidence demonstrating beneficial effects on disease severity and symptoms in several inflammatory conditions. This review examines the evidence supporting a modulatory effect of endocannabinoids on TLR-mediated immune responses both peripherally and centrally, and the implications for psychiatric disorders such as depression and schizophrenia.

Forbidden herbs: Alzate’s defense of pipiltzintzintlis.
Dierksmeier, L.
Colonial Latin American Review, 29(2), 292–315.(2020).
doi:10.1080/10609164.2020.1755941
In a 1772 newspaper article, José Antonio Alzate y Ramírez (1737–1799) defended the medicinal benefits of cannabis against the prohibition of the Inquisition. Alzate concluded that the indigenous herb pipiltzintzintlis1 was ‘nothing else but cannabis.’ 2 As Spanish physician Juan de Cárdenas contended almost two centuries earlier, Alzate argued for a disassociation of the plant’s narcotic or hallucinogenic properties and effects from its potential demonic influence.3 In the following pages, I investigate the Church’s prohibition of the herb pipiltzintzintlis. I analyze Alzate’s evidence in support of medicinal cannabis use, ranging from firsthand experience to second-hand accounts to medical encyclopedias, thereby exposing the range of eighteenth-century sources supporting medicinal marijuana use. I end with conclusions on the epistemic value bestowed on different sources of scientific evidence in eighteenth-century Mexico and on the role that censorship played in the circulation of scientific knowledge

Fool’s gold: diseased marijuana and cannabis hyperemesis syndrome
Oscar Armando Dorantes
J Investig Med 2021;69:1063–1064.
doi:10.1136/jim-2021-001980
https://jim.bmj.com/content/jim/69/5/1063.full.pdf
I am writing in response to the article by Gajendran et al1 and the editorial by Ron Shay, 'Cannabis hyperemesis syndrome: the conundrum
is here to stay'.2 As an emic cultural investigator (anthropologist) and from my vantage point, the problem of cannabis hyperemesis syndrome could easily go away just as quickly as it has appeared. There is a large disconnect between the professional medical community and the marijuana subculture. The majority of the latter are somewhat in denial that cannabis is the cause of this condition and, on the flip side, doctors feel strongly about their conclusions. Perhaps both can be right, because in the marijuana subculture community there are two types of marijuana: 'the sticky, skunky weed' or the 'the bunk weed', 'the dank or the regs' or otherwise viewed as the 'good stuff versus not good stuff'. Were any of the samples of consumed marijuana analyzed during the marijuana hyperemesis diagnosis, as presented in Gajendran et al's paper?

REMOVE?
Forbidden herbs? The effects of cannabis were a controversial topic 250 years ago
University of Tübingen
https://phys.org/news/2020-07-forbid...d0a4-365750754
Should cannabis be legalized for medicinal purposes or will it remain an illegal drug? This has been discussed in many countries for years—and has been a point of contention for much longer than expected: Already in Mexico in the 18th century, priest and scientist José Antonio Alzate y Ramírez campaigned for the healing effects of the controversial plant—against the position of the Spanish Crown and the Inquisition. The historian Dr. Laura Dierksmeier from the Collaborative Research Center Resource Cultures at the University of Tübingen examines the public debate at that time in Mexico. Her study "Forbidden herbs: Alzate's defense of pipiltzintzintlis" was published on July 7th in the journal Colonial Latin American Review.

Free and Glucuronide Urine Cannabinoids after Controlled Smoked, Vaporized, and Oral Cannabis Administration in Frequent and Occasional Cannabis Users.
Huestis, M. A., Sempio, C., Newmeyer, M. N., Andersson, M., Barnes, A. J., Abulseoud, O. A., … Smith, M. L.
Journal of Analytical Toxicology.(2020).
doi:10.1093/jat/bkaa046
BACKGROUND: Total urinary 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) concentrations are generally reported following cannabis administration. Few data are available for glucuronide and minor cannabinoid metabolite concentrations.
METHODS: All urine specimens from 11 frequent and 9 occasional cannabis users were analyzed for 11 cannabinoids for up to 85 h by LC–MS-MS following controlled smoked, vaporized or oral 50.6 mg ? 9 - tetrahydrocannabinol (THC) in a randomized, placebo-controlled, within-subject dosing design.
RESULTS: No cannabidiol, cannabinol, cannabigerol, tetrahydrocannabivarin (THCV), THC, 11-OH-THC, ? 9 -tetrahydrocannabinolic acid were detected in urine. Median THCCOOH-glucuronide maximum concentrations (Cmax) following smoked, vaporized and oral routes were 68.0, 26.7 and 360 ?g/L for occasional and 378, 248 and 485 ?g/L for frequent users, respectively. Median time to specific-gravity normalized Cmax (Tmax) was 5.1–7.9 h for all routes and all users. Median Cmax for THCCOOH, THCglucuronide and 11-nor-9-carboxy-? 9 -THCV (THCVCOOH) were less than 7.5% of THCCOOHglucuronide Cmax concentrations. Only THC-glucuronide mean Tmax differed between routes and groups, and was often present only in occasional users’ first urine void. Multiple THCCOOH-glucuronide and THCCOOH peaks were observed. We also evaluated these urinary data with published models for determining recency of cannabis use.
CONCLUSIONS: These urinary cannabinoid marker concentrations from occasional and frequent cannabis users following three routes of administration provide a scientific database to assess single urine concentrations in cannabis monitoring programs. New target analytes (CBD, CBN, CBG, THCV and phase II metabolites) were not found in urine. The results are important to officials in drug treatment, workplace and criminal justice drug monitoring programs, as well as policy makers with responsibility for cannabis regulations.

From Cannabis sativa to Cannabidiol: Promising Therapeutic Candidate for the Treatment of Neurodegenerative Diseases.
Cassano, T., Villani, R., Pace, L., Carbone, A., Bukke, V. N., Orkisz, S., … Serviddio, G.
Frontiers in Pharmacology, 11.(2020).
doi:10.3389/fphar.2020.00124
Cannabis sativa, commonly known as marijuana, contains a pool of secondary plant metabolites with therapeutic effects. Besides D9-tetrahydrocannabinol that is the principal psychoactive constituent of Cannabis, cannabidiol (CBD) is the most abundant nonpsychoactive phytocannabinoid and may represent a prototype for antiinflammatory drug development for human pathologies where both the inflammation and oxidative stress (OS) play an important role to their etiology and progression. To this regard, Alzheimer's disease (AD), Parkinson's disease (PD), the most common neurodegenerative disorders, are characterized by extensive oxidative damage to different biological substrates that can cause cell death by different pathways. Most cases of neurodegenerative diseases have a complex etiology with a variety of factors contributing to the progression of the neurodegenerative processes; therefore, promising treatment strategies should simultaneously target multiple substrates in order to stop and/ or slow down the neurodegeneration. In this context, CBD, which interacts with the eCB system, but has also cannabinoid receptor-independent mechanism, might be a good candidate as a prototype for anti-oxidant drug development for the major neurodegenerative disorders, such as PD and AD. This review summarizes the multiple molecular pathways that underlie the positive effects of CBD, which may have a considerable impact on the progression of the major neurodegenerative disorders.

Functional Crosstalk between CB and TRPV1 Receptors Protects Nigrostriatal Dopaminergic Neurons in the MPTP Model of Parkinson’s Disease
Rayul Wi, Young Cheul Chung, and Byung Kwan Jin
Hindawi Journal of Immunology Research Volume 2020, Article ID 5093493,
doi: 10.1155/2020/5093493
The present study examined whether crosstalk between cannabinoid (CB) and transient potential receptor vanilloid type 1 (TRPV1) could contribute to the survival of nigrostriatal dopamine neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD). MPTP induced a significant loss of nigrostriatal dopamine neurons and glial activation in the substantia nigra (SN) and striatum (STR) as visualized by tyrosine hydroxylase (TH) or macrophage antigen complex-1 (MAC-1) or glial fibrillary acidic protein (GFAP) immunocytochemistry, respectively. RT-PCR analysis shows the upregulation of inducible nitric oxide synthase, interleukin-1?, and tumor necrosis factor-? in microglia in the SN in vivo, indicating the activation of the inflammatory system. By contrast, treatment with capsaicin (a specific TRPV1 agonist) increased the survival of dopamine neurons in the SN and their fibers and dopamine levels in the STR in MPTP mice. Capsaicin neuroprotection is accompanied by inhibiting MPTP-induced glial activation and production of inflammatory cytokines. Treatment with AM251 and AM630 (CB1/2 antagonists) abolished capsaicin-induced beneficial effects, indicating the existence of a functional crosstalk between CB and TRPV1. Moreover, treatment with anandamide (an endogenous agonist for both CB and TRVP1) rescued nigrostriatal dopamine neurons and reduced gliosis-derived neuroinflammatory responses in MPTP mice. These results suggest that the cannabinoid and vanilloid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with neuroinflammation.

General risks of harm with cannabinoids, cannabis, and cannabis-based medicine possibly relevant to patients receiving these for pain management: an overview of systematic reviews
Mohammed Mohiuddin, Fiona M Blyth, Louisa Degenhardt, Ian Gilron
Pain July 2020
DOI: 10.1097/j.pain.0000000000002000
The growing demand for improved pain treatments together with expanding legalization of, and access to, cannabinoids, cannabis, and
cannabis-based medicines has intensified the focus on risk benefit considerations in pain management. Given limited harms data from analgesic clinical trials, we conducted an overview of systematic reviews focused on all harms possibly relevant to patients receiving cannabinoids for pain management. This PROSPERO-registered, PRISMA-compliant systematic overview identified 79 reviews, encompassing over 2200 individual reports about psychiatric and psychosocial harms, cognitive/behavioral effects, motor vehicle accidents, cardiovascular respiratory, cancer-related, maternal/fetal, and general harms. Reviews,and their included studies, were of variable quality. Available evidence suggests variable associations between cannabis exposure (ranging from monthly to daily use based largely on self-report) and psychosis, motor vehicle accidents,respiratory problems, and other harms. Most evidence comes from settings other than that of pain
management (eg, nonmedicinal and experimental) but does signal a need for caution and more robust harms evaluation in future studies.
Given partial overlap between patients receiving cannabinoids for pain management and individuals using cannabinoids for other reasons,
lessons from the crisis of oversupply and overuse of opioids in some parts of the world emphasize the need to broadly consider harm evidence from real-world settings. The advancement of research on cannabinoidharms will serve to guideoptimalapproaches to the use of cannabinoids for painmanagement. In the meantime, this evidence should be carefully examined when making risk-benefit considerations about the use of cannabinoids, cannabis, and cannabis-based medicine for chronic pain.

Genetic basis of cannabis use: a systematic review
Alannah Hillmer , Caroul Chawar , Stephanie Sanger , Alessia D’Elia , Mehreen Butt , Raveena Kapoor , Flavio Kapczinski , Lehana Thabane and Zainab Samaan
BMC Med Genomics (2021) 14:203
DOI: 10.1186/s12920-021-01035-5
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_1035.pdf
Background: With the increase in cannabis use rates, cannabis use disorder is being reported as one of the most common drug use disorders globally. Cannabis use has several known physical, psychological, and social adverse events, such as altered judgement, poor educational outcomes, and respiratory symptoms. The propensity for taking cannabis and the development of a cannabis use disorder may be genetically infuenced for some individuals. Heritability estimates suggest a genetic basis for cannabis use, and several genome-wide association studies (GWASs) have identifed possible regions of association, albeit with inconsistent fndings. This systematic review aims to summarize the fndings from GWASs investigating cannabis use and cannabis use disorder.
Methods: This systematic review incorporates articles that have performed a GWAS investigating cannabis use or cannabis use disorder. MEDLINE, Web of Science, EMBASE, CINAHL, GWAS Catalog, GWAS Central, and NIH Database of Genotype and Phenotype were searched using a comprehensive search strategy. All studies were screened in duplicate, and the quality of evidence was assessed using the quality of genetic association studies (Q-Genie) tool. All studies underwent qualitative synthesis; however, quantitative analysis was not feasible.
Results: Our search identifed 5984 articles. Six studies met our eligibility criteria and were included in this review. All six studies reported results that met our signifcance threshold of p≤1.0× 10–7. In total 96 genetic variants were identifed. While meta-analysis was not possible, this review identifed the following genes, ANKFN1, INTS7, PI4K2B, CSMD1, CST7, ACSS1, and SCN9A, to be associated with cannabis use. These regions were previously reported in diferent mental health conditions, however not in relation to cannabis use.
Conclusion: This systematic review summarized GWAS fndings within the feld of cannabis research. While a metaanalysis was not possible, the summary of fndings serves to inform future candidate gene studies and replication efforts.

Genetic predisposition to schizophrenia associated with increased use of cannabis
Robert A Power, Karin Verweij, Moha Zuhairmed, Nicholas G. Martin
June 2014
Molecular Psychiatry 19(11)
DOI: 10.1038/mp.2014.51
Cannabis is the most commonly used illicit drug worldwide. With debate surrounding the legalization and control of use, investigating its health risks has become a pressing area of research. One established association is that between cannabis use and schizophrenia, a debilitating psychiatric disorder affecting ~1% of the population over their lifetime. Although considerable evidence implicates cannabis use as a component cause of schizophrenia, it remains unclear whether this is entirely due to cannabis directly raising risk of psychosis, or whether the same genes that increases psychosis risk may also increase risk of cannabis use. In a sample of 2082 healthy individuals, we show an association between an individual's burden of schizophrenia risk alleles and use of cannabis. This was significant both for comparing those who have ever versus never used cannabis (P=2.6 × 10(-)(4)), and for quantity of use within users (P=3.0 × 10(-3)). Although directly predicting only a small amount of the variance in cannabis use, these findings suggest that part of the association between schizophrenia and cannabis is due to a shared genetic aetiology. This form of gene-environment correlation is an important consideration when calculating the impact of environmental risk factors, including cannabis use

Genital Warts Do Not Respond to Systemic Recombinant Interferon Alfa-2a Treatment during Cannabis Consumption
February 1991 Dermatologica 183(3):203-7
Gerd Gross, Angeliki Roussaki-Schulze, Hans Ikenberg, N Drees
DOI: 10.1159/000247670
The case of a 22-year-old man suffering from genital warts is described. The lesions responded completely to recombinant interferon alfa-2a only after discontinuation of cannabis consumption. Cannabis was detected using the enzyme immunoassay/1-trans-tetrahydrocannabinoid method in urine. Southern blotting of frozen genital wart biopsy material revealed papillomavirus type 11 DNA, the amount of which increased significantly during interferon treatment. The final clearing of lesions after discontinuation of cannabis consumption implicates that the drug-induced impairment of cellular immunity was reversible. It is concluded that drug abuse and especially cannabis consumption may play some role in the world-wide increase in genital papillomavirus disease and in the high number of recalcitrant courses of genital warts.

Genome-wide association study implicates CHRNA2 in cannabis use disorder
Ditte Demontis?, Veera Manikandan Rajagopal?, Thorgeir E. Thorgeirsson?, Thomas D. Als?, Jakob Grove, Kalle Leppälä, Daniel F. Gudbjartsson?, Jonatan Pallesen, Carsten Hjorthøj?, Gunnar W. Reginsson, Thorarinn Tyrfingsson, Valgerdur Runarsdottir, Per Qvist?, Jane Hvarregaard Christensen, Jonas Bybjerg-Grauholm?, Marie Bækvad-Hansen, Laura M. Huckins?, Eli A. Stahl?, Allan Timmermann, Esben Agerbo, David M. Hougaard?, Thomas Werge, Ole Mors, Preben Bo Mortensen, Merete Nordentoft, Mark J. Daly, Hreinn Stefansson?, Kari Stefansson?, Mette Nyegaard, and Anders D. Børglum?
Nat Neurosci. 2019 Jun 17.
doi: 10.1038/s41593-019-0416-1. [Epub ahead of print]
Cannabis is the most frequently used illicit psychoactive substance worldwide; around one in ten users become dependent. The risk for cannabis use disorder (CUD) has a strong genetic component, with twin heritability estimates ranging from 51 to 70%. Here we performed a genome-wide association study of CUD in 2,387 cases and 48,985 controls, followed by replication in 5,501 cases and 301,041 controls. We report a genome-wide significant risk locus for CUD (P=?9.31?×?10?12) that replicates in an independent population (Preplication=?3.27?×?10?3, Pmeta-analysis=?9.09?×?10?12). The index variant (rs56372821) is a strong expression quantitative trait locus for cholinergic receptor nicotinic ?2 subunit (CHRNA2); analyses of the genetically regulated gene expression identified a significant association of CHRNA2 expression with CUD in brain tissue. At the polygenic level, analyses revealed a significant decrease in the risk of CUD with increased load of variants associated with cognitive performance. The results provide biological insights and inform on the genetic architecture of CUD.
https://sci-hub.tw/10.1038/s41593-019-0416-1
DOI: 10.1038/s41593-019-0416-1

Genome-wide Association Study of Cannabis Dependence Severity, Novel Risk Variants, and Shared Genetic Risks
Richard Sherva, QianWang, Henry Kranzler, Hongyu Zhao, Ryan Koesterer,
Aryeh Herman, Lindsay A. Farrer, Joel Gelernter
JAMA Psychiatry. 73(5) March 2016
DOI:10.1001/jamapsychiatry.2016.0036
IMPORTANCE Cannabis dependence (CAD) is a serious problem worldwide and is of growing importance in the United States because cannabis is increasingly available legally. Although genetic factors contribute substantially to CAD risk, at present no well-established specific genetic risk factors for CAD have been elucidated.
OBJECTIVE To report findings for DSM-IV CAD criteria from association analyses performed in large cohorts of African American and European American participants from 3 studies of substance use disorder genetics.
DESIGN, SETTING, AND PARTICIPANTS This genome-wide association study for DSM-IV CAD criterion count was performed in 3 independent substance dependence cohorts (the Yale-Penn Study, Study of Addiction: Genetics and Environment [SAGE], and International Consortium on the Genetics of Heroin Dependence [ICGHD]). A referral sample and volunteers recruited in the community and from substance abuse treatment centers included 6000 African American and 8754 European American participants, including some from small families. Participants from the Yale-Penn Study were recruited from 2000 to 2013. Data were collected for the SAGE trial from 1990 to 2007 and for the ICGHD from 2004 to 2009. Data were analyzed from January 2, 2013, to November 9, 2015.
MAIN OUTCOMES AND MEASURES Criterion count for DSM-IV CAD.
RESULTS Among the 14 754 participants, 7879 were male, 6875 were female, and the mean (SD) age was 39.2 (10.2) years. Three independent regions with genome-wide significant single-nucleotide polymorphism associations were identified, considering the largest possible sample. These included rs143244591 (? = 0.54, P = 4.32 Å~ 10?10 for the meta-analysis) in novel antisense transcript RP11-206M11.7; rs146091982 (? = 0.54, P = 1.33 Å~ 10?9 for the meta-analysis) in the solute carrier family 35 member G1 gene (SLC35G1); and rs77378271 (? = 0.29, P = 2.13 Å~ 10?8 for the meta-analysis) in the CUB and Sushi multiple domains 1 gene (CSMD1). Also noted was evidence of genome-level pleiotropy between CAD and major depressive disorder and for an association with single-nucleotide polymorphisms in genes associated with schizophrenia risk. Several of the genes identified have functions related to neuronal calcium homeostasis or central nervous system development.
CONCLUSIONS AND RELEVANCE These results are the first, to our knowledge, to identify specific CAD risk alleles and potential genetic factors contributing to the comorbidity of CAD with major depression and schizophrenia.

Genome-wide DNA methylation analysis of heavy cannabis exposure in a New Zealand longitudinal cohort.
Osborne, A. J., Pearson, J. F., Noble, A. J., Gemmell, N. J., Horwood, L. J., Boden, J. M., … Kennedy, M. A.
Translational Psychiatry, 10(1). (2020).
doi:10.1038/s41398-020-0800-3
Cannabis use is of increasing public health interest globally. Here we examined the effect of heavy cannabis use, with and without tobacco, on genome-wide DNA methylation in a longitudinal birth cohort (Christchurch Health and Development Study, CHDS). A total of 48 heavy cannabis users were selected from the CHDS cohort, on the basis of their adult exposure to cannabis and tobacco, and DNA methylation assessed from whole blood samples, collected at approximately age 28. Methylation in heavy cannabis users was assessed, relative to non-users (n = 48 controls) via the Illumina Infinium® MethylationEPIC BeadChip. We found the most differentially methylated sites in cannabis with tobacco users were in the AHRR and F2RL3 genes, replicating previous studies on the effects of tobacco. Cannabis-only users had no evidence of differential methylation in these genes, or at any other loci at the epigenome-wide significance level (P < 10?7 ). However, there were 521 sites differentially methylated at P < 0.001 which were enriched for genes involved in neuronal signalling (glutamatergic synapse and long-term potentiation) and cardiomyopathy. Further, the most differentially methylated loci were associated with genes with reported roles in brain function (e.g. TMEM190, MUC3L, CDC20 and SP9). We conclude that the effects of cannabis use on the mature human blood methylome differ from, and are less pronounced than, the effects of tobacco use, and that larger sample sizes are required to investigate this further.

Not Cannabis specific
Geraniol Restores Antibiotic Activities against Multidrug-Resistant Isolates from Gram-Negative Species.
Lorenzi, V., Muselli, A., Bernardini, A. F., Berti, L., Pages, J.-M., Amaral, L., & Bolla, J.-M.
Antimicrobial Agents and Chemotherapy, 53(5), 2209–2211.(2009).
doi:10.1128/aac.00919-08
The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine -naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of -lactams, quinolones, and chloramphenicol.

Glucose metabolism links astroglial mitochondria to cannabinoid effects.
Jimenez-Blasco, D., Busquets-Garcia, A., Hebert-Chatelain, E., Serrat, R., Vicente-Gutierrez, C., Ioannidou, C., … Marsicano, G.
Nature.(2020).
doi:10.1038/s41586-020-2470-y
Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1–5 . By contrast, astrocytes are under neuronal control through specifc neurotransmitter receptors5–7 . However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifcally, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and afects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these efects abolishes the efect of cannabinoid treatment on the observed behaviour. These fndings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fne-tune neuronal activity and behaviour in mice.

Not Cannabis specific
Glucagon and a glucagon-GLP-1 dual-agonist increases cardiac performance with different metabolic effects in insulin-resistant hearts
LN Axelsen, W Keung, HD Pedersen, E Meier, D Riber, AL Kjølbye, JS Petersen, SD Proctor, N-H Holstein-Rathlou and GD Lopaschuk
British Journal of Pharmacology (2012) 165 2736–2748
DOI:10.1111/j.1476-5381.2011.01714.x
BACKGROUND AND PURPOSE The prevalence of heart disease continues to rise, particularly in subjects with insulin resistance (IR), and improved therapies for these patients is an important challenge. In this study we evaluated cardiac function and energy metabolism in IR JCR:LA-cp rat hearts before and after treatment with an inotropic compound (glucagon), a glucagon-like peptide-1 (GLP-1) receptor agonist (ZP131) or a glucagon-GLP-1 dual-agonist (ZP2495).
EXPERIMENTAL APPROACH Hearts from IR and lean JCR:LA rats were isolated and perfused in the working heart mode for measurement of cardiac function and metabolism before and after addition of vehicle, glucagon, ZP131 or ZP2495. Subsequently, cardiac levels of nucleotides and short-chain CoA esters were measured by HPLC.
KEY RESULTS Hearts from IR rats showed decreased rates of glycolysis and glucose oxidation, plus increased palmitate oxidation rates, although cardiac function and energy state (measured by ATP/AMP ratios) was normal compared with control rats. Glucagon increased glucose oxidation and glycolytic rates in control and IR hearts, but the increase was not enough to avoid AMP and ADP accumulation in IR hearts. ZP131 had no significant metabolic or functional effects in either IR or control hearts. In contrast, ZP2495 increased glucose oxidation and glycolytic rates in IR hearts to a similar extent to that of glucagon but with no concomitant accumulation of AMP or ADP.
CONCLUSION AND IMPLICATIONS Whereas glucagon compromised the energetic state of IR hearts, glucagon-GLP-1 dual-agonist ZP2495 appeared to preserve it. Therefore, a glucagon-GLP-1 dual-agonist may be beneficial compared with glucagon alone in the treatment of severe heart failure or cardiogenic shock in subjects with IR.

Glucocorticoids shift arachidonic acid metabolism toward endocannabinoid synthesis: A non-genomic anti-inflammatory switch
Renato Malcher-Lopes, Alier Franco, Jeffrey G. Tasker
European Journal of Pharmacology 583 (2008) 322–339
doi:10.1016/j.ejphar.2007.12.033
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2367369/
Glucocorticoids are capable of exerting both genomic and non-genomic actions in target cells of multiple tissues, including the brain, which trigger an array of electrophysiological, metabolic, secretory and inflammatory regulatory responses. Here, we have attempted to show how glucocorticoids may generate a rapid anti-inflammatory response by promoting arachidonic acid-containing endocannabinoids biosynthesis. According to our hypothesized model, non-genomic action of glucocorticoids results in the global shift of membrane lipid metabolism, subverting metabolic pathways toward the synthesis of the anti-inflammatory endocannabinoids, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), and away from arachidonic acid production. Post-transcriptional inhibition of cyclooxygenase-2 (COX2) synthesis by glucocorticoids assists this mechanism by suppressing the synthesis of pro-inflammatory prostaglandins as well as endocannabinoid-derived prostanoids. In the central nervous system (CNS) this may represent a major neuroprotective system, which may cross-talk with leptin signaling in the hypothalamus allowing for the coordination between energy homeostasis and the inflammatory response. .

Guided web-based treatment program for reducing cannabis use: a randomized controlled trial.
Sinadinovic, K., Johansson, M., Johansson, A.-S., Lundqvist, T., Lindner, P., & Hermansson, U.
Addiction Science & Clinical Practice, 15(1).(2020).*
doi:10.1186/s13722-020-00185-8*
Background: The aim of this study was to investigate the efects of a web-based treatment program with therapist guidance for adults and adolescents with regular cannabis use from the general population. Methods: A double blinded randomized controlled trial with a parallel group design was conducted (intervention group n=151, wait-list control group n=152). Follow-up 12 weeks from treatment commencement of a 13-module intervention. The primary outcome was frequency of cannabis use. Time by group interaction efects were modeled using generalized estimated equations and the instrumental variable approach was used to estimate the efect of intervention adherence. Results: At follow-up, the intention to treat (ITT) analyses did not show any signifcant time by group efects. A signifcant association between intervention adherence and scores on the cannabis abuse screening test (CAST) was found. Secondary analysis excluding participants who had received other professional help revealed time by group efects for secondary outcomes gram cannabis consumed past week, number of dependency criteria and CAST score. Due to methodological limitations, these latter results should be interpreted with caution. Conclusions: In this study we did not fnd a web-based treatment program with therapist guidance to be more efective than a waiting-list in reducing frequency of cannabis use.

Hair endocannabinoid concentrations in individuals with acute and weight-recovered anorexia nervosa
Friederike I Tam , Julius Steding , Jonas L Steinhäuser, Franziska Ritschel , Wei Gao,, Kerstin Weidner , Veit Roessner , Clemens Kirschbaum , Stefan Ehrlich
Prog Neuropsychopharmacol Biol Psychiatry 2021 Jan 11;110243.
doi: 10.1016/j.pnpbp.2021.110243
Background: The endocannabinoid system has been suggested to modulate energy metabolism and stress response and could be an important factor in the pathophysiology of anorexia nervosa (AN). In the context of AN, excessive physical activity may influence endocannabinoid concentrations. The objective of this study was to investigate hair endocannabinoid concentrations at different stages of the disorder. Measurement in hair allows for a cumulative assessment of endocannabinoid concentrations independent of circadian rhythms.
Methods: In a combined cross-sectional and longitudinal design, we measured hair concentrations of the endocannabinoids anandamide and 2-arachidonoylglycerol and the endocannabinoid-related compounds palmitoylethanolamide, oleoylethanolamide, and stearoylethanolamide in female underweight patients with acute AN (n = 67, reassessment of n = 47 after short-term weight restoration with a body mass index increase of at least 14%), individuals long-term recovered from AN (n = 27), and healthy control participants (n = 84).
Results: Hair concentrations of anandamide and all endocannabinoid-related compounds were elevated in acute AN and decreased over the course of short-term weight restoration. Anandamide concentrations remained elevated in long-term recovered AN patients. In long-term recovered patients, physical activity correlated positively with the concentrations of all endocannabinoid-related compounds.
Conclusion: The current study provides evidence for a significant alteration of the endocannabinoid system in acute AN, which may partly persist into long-term recovery. The endocannabinoid system may be a possible target for pharmaceutical interventions in AN, which should be explored in further preclinical and subsequently clinical randomized controlled trials.

Hair Regrowth with Cannabidiol (CBD)-rich Hemp Extract A Case Series
Gregory Smith, John Satino
Cannabis: Vol. 4 No. 1 (2021)
https://publications.sciences.ucf.ed.. .rticle/view/78
https://www.sciencegate.app/app/docu...is/2021.01.003
Androgenetic alopecia (AGA) is the most common cause of hair loss. Several FDA approved medications are available but offer limited results. Studies have shown that the endocannabinoid system (ECS) is a key player in hair follicle cell growth. The ECS cannabinoid type one (CB1) receptors are well expressed in the hair follicle cells. Cannabidiol (CBD) is a negative allosteric modulator of the CB1 receptor and has been shown to result in hair shaft elongation. In addition, the hair follicle cycle phases are controlled by the ECS vanilloid receptor-1 (TRPV1). CBD has also been shown to increase Wnt signaling pathways that are involved in the differentiation of dermal progenitor cells into new hair follicles and maintaining the anagen phase of the hair cycle. The effects of CBD on hair growth are dose dependent and higher doses may result in premature entry into the catagen phase via a receptor known as vanilloid receptor-4 (TRPV4). Topical application of CBD reaches hair follicles where it is a CB1 negative modulator, and TRPV1, and TRPV4 agonist. A study was done of 35 subjects with AGA using a once daily topical hemp oil formulation, averaging about 3-4 mg per day of CBD and minimal amounts of other cannabinoids for six months. A hair count of the greatest area of alopecia was carried out before treatment and again after six months. The results revealed that men did slightly better than women, and the vertex area did better than the temporal areas. On average there was statistically significant 93.5% increase in hair after 6 months. All subjects had some regrowth. There were no reported adverse effects. Since the CBD works through novel mechanisms different from finasteride and minoxidil it can be used in conjunction with these current drugs and would be expected to have synergistic effects.

Handbook of Cannabis Therapeutics From Bench to Bedside
Ethan Russo and Franjo Grotenhermen
https://saltonverde.com/wp-content/u...to_bedside.pdf
The human race prides itself as the only animal through evolution to become a rational being and therefore able to make decisions and take action based on logic and analysis of available data, rather than simply by instinct, other genetic factors, and experience, accumulated mostly through conditioning, as found in other animals. But is this human pride justified? My involvement within the limited field of cannabis therapeutics certainly shows that humans still value instincts and antiquated ideas, some based on a total lack of rationality much more than on reasoned facts. In June 2005 the U.S. Supreme Court declared medical use of cannabis illegal on the federal level, although some states have allowed its use in certain medical conditions. Was there scientific and medical logic behind this decision? Cannabis sativa is a part of the medical-cultural heritage of many lands. It is mentioned in Assyrian clay tablets as a drug for treating neurological conditions, and it was used for a variety of medical problems in China and India when Northern Europe was still governed by barbarian tribes. Even as late as the end of the nineteenth century cannabis was described by Queen Victoria’s physician as “by far the most useful of drugs . . . in some painful conditions,” but during most of the twentieth century it was regarded as a nuisance—an illegal drug which did not quite fit the official view of a dangerous narcotic. Yet during the past forty years, science has accumulated a vastly increasing bulk of knowledge of this drug. Its chemistry was clarified during the 1960s, and its psychoactive principle, ?9-tetrahydrocannabinol (?9-THC), was identified. In spite of its mild psychotropic effects, in the United States in 1985 THC was approved as a prescription drug against vomiting and nausea caused by cancer chemotherapy, and in 1992 as an appetite enhancer, most often used by AIDS patients. Over the past twenty years, the mechanism of action of THC was shown to be quite unique. This plant molecule activates two receptors, CB1 (found predominantly in the central nervous system) and CB2 (found mostly in the immune system). These receptors are part of the endocannabinoid system, which is involved in myriad physiological processes. It is activated by specific ligands called endocannabinoids, the best known being anandamide and 2-arachidonoyl glycerol, both derivatives of a fatty acid, arachidonic acid. Although completely different in chemical structure from the endocannabinoids, THC binds to the same receptors and elicits the same physiological responses. The endocannabinoids and THC enhance appetite, reduce pain, and affect many of the symptoms of diseases, in particular those associated with the central nervous and the immune systems, as well as impacting a long list of additional body processes. But THC is not the only cannabis constituent with physiological activity. Cannabidiol (CBD), a nonpsychotropic cannabinoid, has been found to be anxiolytic, antiepileptic, and antischizophrenic in animals and humans. We know very little about the activity of the dozens of additional but mostly minor cannabinoids that are present in the plant. Many sufferers of various diseases claim that the effect of using the total plant is better than the effect of THC alone. In part, this may be due to the different modes of administration— marijuana is, of course, usually smoked, while THC is administered mostly orally. However, I believe that the combination of CBD with THC may represent a better drug than THC alone. As these are the main plant cannabinoids, a cannabis plant with a standardized content of CBD and THC may represent a valuable therapeutic drug. Unfortunately, the present irrational bias against the plant may block the introduction of this type of medicine in many countries. The use of neurotransmitters and neuromodulators as medicinal agents is certainly not new. Many modern drugs are based on them. There is every reason to expect that cannabinoid-based drugs will eventually also be introduced. So why block a valuable medicine? The only reason I can think of is lack of reason in the face of compelling scientific evidence. This book is a compilation of many articles published originally in the now defunct and difficult-to-obtain Journal of Cannabis Therapeutics. The chapters deal with the history of the medical use of cannabis, the pharmacology and pharmacokinetics of THC and other cannabinoids, the endocannabinoid system, and with the various medicinal uses (including the side effects) of the drug. It is, in my view, an excellent overall presentation of the expanding topic of cannabis therapeutics.
-Raphael Mechoulam

Health Claims About Cannabidiol Products: A Retrospective Analysis of U.S. Food and Drug Administration Warning Letters from 2015 to 2019
Kimberly G. Wagoner , Allison J. Lazard , E. Alfonso Romero-Sandoval , and Beth A. Reboussin
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0166
https://www.liebertpub.com/doi/pdfpl.../can.2020.0166
Background: Cannabidiol (CBD) products are increasingly available to consumers in the United States and are subject to regulation by the U.S. Food and Drug Administration (FDA). CBD products cannot be marketed as unapproved new drugs with claims of therapeutic benefit. In addition, because CBD is the active ingredient in a FDA-approved CBD product, Epidiolex, CBD cannot be marketed as, or in, food products or dietary supplements. The FDA has issued Warning Letters to promote voluntary regulatory compliance. These letters provide insights as to the types of violations for CBD products detected in the U.S. market.
Objective: The goal of this retrospective study was to content analyze Warning Letters issued by the FDA to identify illicit marketing of CBD products.
Design: Warning Letters issued by the FDA between 2015 and 2019 were content analyzed using a deductive approach. We extracted year of issuance, issuing offce, and claim types that are currently prohibited by the FDA, including (i) unapproved new drug, (ii) misbranded drug, (iii) false and/or misleading, (iv) FDA-approved/endorsed, (v) dietary supplement, and (vi) adulterated food product. In addition, we documented the disease or conditions the product claimed to affect, pharmacological effects, and location of violation.
Results: Of the 39 Warning Letters issued, 97% were for violations made on company websites and 56% were for social media accounts. Almost all letters (97%) cited violations of marketing CBD as an unapproved new drug. These illicit therapeutic claims were made for >125 unique health problems, including cancer (87.2%), diabetes (71.8%), inflammation (66.7%), pain (66.7%), and arthritis (66.7%). The majority of letters (79.5%) also cited illicit marketing of CBD as a dietary supplement or food product. CBD was promoted as having 16 unique pharmacological effects, including anti-inflammatory (53.8%), anticancer (43.6%), and antipsychotic (30.8%).
Conclusions: CBD products have been unlawfully advertised online as unauthorized drugs with health claims that promote therapeutic benefits and as dietary supplements. Efforts are needed to regulate and monitor illicit advertising so consumers are not misled about the risks and benefits of CBD use.
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HEMP AS A MEDICAMENT
ACTA UNIVERSITATIS PALACKIANAE OLOMUCENSIS - TOM. VI.
Prof. Jan Kabelik
1955
https://www.chanvre-info.ch/info/en/..._kabelikEN.pdf
History of the medicinal use of hemp.......................... .............................. ......4
Properties of isolated substances.................... .............................. .................11
Methods and results of the bacteriological experiments................... ..................23
Survey of clinical experiences................... .............................. .......................38
Therapeutic results in stomatology .............................. .............................. ....48
Cannabis indica in oto-rhino-laryngology .............................. ..........................50
Importance of hemp seeds in the tuberculosis therapy....................... ...............51
References of the Study......................... .............................. ........................ 75

Hempseed (Cannabis sativa) Peptides WVSPLAGRT and IGFLIIWV Exert Anti-inflammatory Activity in the LPS-Stimulated Human Hepatic Cell Line
Ivan Cruz-Chamorro, Guillermo Santos-Sánchez, Carlotta Bollati, Martina Bartolomei, Jianqiang Li, Anna Arnoldi, and Carmen Lammi J. Agric. Food Chem. 2022, 70, 577−583 DOI: 10.1021/acs.jafc.1c07520 https://www.ncbi.nlm.nih.gov/labs/pm...es/PMC8778635/
WVSPLAGRT (H2) and IGFLIIWV (H3) are two transepithelial transported intestinal peptides obtained from the hydrolysis of hempseed protein with pepsin, which exert antioxidant activity in HepG2 cells. Notably, both peptides reduce the H2 O2 -induced reactive oxygen species, lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells via the modulation of the nuclear factor erythroid 2-related factor 2 and the inducible nitric oxide synthase (iNOS) pathways, respectively. Due to the close link between inflammation and oxidative stress and with the objective of fostering the multifunctional behavior of bioactive peptides, in this study, the molecular characterization of the anti-inflammatory and immunomodulatory properties of H2 and H3 was carried out in HepG2 cells. In fact, both peptides were shown to modulate the production of pro (IFN-γ : − 33.0 ± 6.7% H2, p = 0.011; − 13.1 ± 2.0% H3, p = <0.0001; TNF: − 17.6 ± 1.7% H2, p = 0.0004; − 20.3 ± 1.7% H3, p = <0.0001; and IL-6: − 15.1 ± 6.5% H3, p = 0.010)- and anti (IL-10: +9.6 ± 3.1% H2, p = 0.010; +26.0 ± 2.3% H3, p = < 0.0001)-inflammatory cytokines and NO (− 9.0 ± 0.7% H2, p = <0.0001; − 7.2 ± 1.8% H3, p = <0.0001) through regulation of the NF-κ B and iNOS pathways, respectively, in HepG2 cells stimulated by lipopolysaccharides.

Heterogeneous Ozonolysis of Tetrahydrocannabinol: Implications for Third hand Cannabis Smoke
Aaron D. L. Wylie and Jonathan P. D. Abbatt
Environmental Science & Technology
DOI: 10.1021/acs.est.0c03728
Third hand smoke (THS) deposits to surfaces following smoking events and is a source of chemical exposure to humans. However, the evolution of THS in indoor environments is not well understood. Cannabis THS is a chemically distinct and prevalent form of THS, which has not been studied. The heterogeneous reaction of ?9 -tetrahydrocannabinol (THC), a major component of cannabis smoke, with ozone was examined as a pure compound and within cannabis smoke. Oxidative decay via ozonolysis and product formation were monitored by liquid chromatography?tandem mass spectrometry. Epoxide, dicarbonyl, and secondary ozonide THC reaction products were detected from both pure THC and cannabis experiments, with the product ratios dependent on relative humidity. The observed reaction kinetics for loss of THC on glass and cotton surfaces are consistent with a relatively short loss lifetime, which will be strongly dependent on the film thickness, ozone mixing ratio, and ozone reactivity of the surface substrate. The low volatility of THC and its oxidation products suggest that their contributions to thirdhand cannabis smoke will be less significant than the role that nicotine plays in thirdhand tobacco smoke.

High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs
Miran Aswad, Haya Hamza, Antonina Pechkovsky, Anastasiia Zikrach, Tania Popov, Yaniv Zohar, Eduardo Shahar and Igal Louria-Hayon
Front. Immunol. 13:875546.
doi: 10.3389/fimmu.2022.875546
https://www.frontiersin.org/articles/10.3389/fimmu.2022.875546/full
Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinfl ammation. They are often detrimental, and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have antiinfl amatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects
of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed signifi cant reductions in pro-inflammatory cytokines in human-derived PBMCs, neutrophils and T cells. In vivo mouse studies, using a systemically infl amed mouse model, showed reductions in pro-inflammatory cytokines TNFa and IL-1b and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation, as in severe COVID-19 disease, is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition, the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were signifi cantly reduced, demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung infl amed mouse model, we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1b , MCP-1, IL-6 and TNFa , in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions, in which the secretion of cytokines is dysregulated, as it is in severe COVID-19 disease or other infectious or inflammatory diseases. .

Highs and lows of cannabinoid-dopamine interactions: effects of genetic variability and pharmacological modulation of catechol-O-methyl transferase on the acute response to delta-9-tetrahydrocannabinol in humans.
Ranganathan, M., De Aquino, J. P., Cortes-Briones, J. A., Radhakrishnan, R., Pittman, B., Bhakta, S., & D’Souza, D. C.
Psychopharmacology. (2019).
doi:10.1007/s00213-019-05273-5
Rationale The catechol-O-methyl transferase (COMT) enzyme has been implicated in determining dopaminergic tone and the effects of delta-9-tetrahydrocannabinol (THC) in the human brain. Objective This study was designed to evaluate the effect of (1) a functional polymorphism and (2) acute pharmacological inhibition of COMT on the acute response to THC in humans. Methods Sub-study I: The effect of intravenous (IV) THC (0.05 mg/kg) was investigated in 74 healthy subjects genotyped for the COMT rs4680 (Val/Met) polymorphism in a 2-test-day double-blind, randomized, placebo-controlled study. Sub-study II: COMT rs4680 homozygous subjects (Val/Val and Met/Met) from sub-study I received the COMT enzyme inhibitor tolcapone (200 mg) followed by IV THC or placebo on two additional test days. Subjective, behavioral, and cognitive data were obtained periodically on each test day. Results Sub-study I: Val/Val individuals were most sensitive to THC-induced attention and working memory deficits. In contrast, the psychotomimetic and subjective effects of THC were not influenced by COMT genotype. Sub-study II: Tolcapone reduced THC-induced working memory deficits, but not THC’s psychotomimetic effects. Tolcapone and COMT genotype (met/met) were associated with an increased report of feeling Bmellow.^ Conclusions The interaction between COMT rs4680 polymorphisms and tolcapone on the cognitive, but not on the psychotomimetic and overall subjective effects of THC, suggests that modulation of dopaminergic signaling may selectively influence specific cannabinoid effects in healthy individuals. The role of dopaminergic signaling in the cognitive effects of cannabinoids should be considered in drug development efforts targeting these effects.

History of cannabis as a medicine: a review
Antonio Waldo Zuardi
Rev Bras Psiquiatr. 2006;28(2):153-7
https://www.scielo.br/pdf/rbp/v28n2/29785.pdf
Cannabis as a medicine was used before the Christian era in Asia, mainly in India. The introduction of cannabis in the Western medicine occurred in the midst of the 19th century, reaching the climax in the last decade of that century, with the availability and usage of cannabis extracts or tinctures. In the first decades of the 20th century, the Western medical use of cannabis significantly decreased largely due to difficulties to obtain consistent results from batches of plant material of different potencies. The identification of the chemical structure of cannabis components and the possibility of obtaining its pure constituents were related to a significant increase in scientific interest in such plant, since 1965. This interest was renewed in the 1990’s with the description of cannabinoid receptors and the identification of an endogenous cannabinoid system in the brain. A new and more consistent cycle of the use of cannabis derivatives as medication begins, since treatment effectiveness and safety started to be scientifically proven.

Hit the chronic… physical activity: are cannabis associated mental health changes in adolescents attenuated by remaining active?
Duncan, M. J., Patte, K. A., & Leatherdale, S. T.
Social Psychiatry and Psychiatric Epidemiology. (2020).
doi:10.1007/s00127-020-01900-1
Purpose High-frequency cannabis use in adolescents has been associated with adult mental illness. In contrast, physical activity has been demonstrated to beneft mental health status. The purpose of this study was to examine whether, within a 1-year prospective study design, changes in cannabis use frequency are associated with changes in mental health, and whether meeting physical activity guidelines moderates these associations.
Methods COMPASS (2012–2021) is a hierarchical longitudinal health data survey from a rolling cohort of secondary school students across Canada; student-level mental health data linked from Years 5 (2016/17) and 6 (2017/18) were analysed (n=3173, 12 schools). Multilevel conditional change regression models were used to assess associations between mental health scores change, cannabis use change and physical activity guideline adherence change after adjusting for covariates.
Results Adopting at least weekly cannabis use was associated with increases in depressive and anxiety symptoms and decreases in psychosocial well-being. Maintaining physical activity guidelines across both years improved psychosocial well-being regardless of cannabis use frequency, and ofset increases in depressive symptoms among individuals who adopted high frequency cannabis use. Physical activity adherence had no apparent relationship with anxiety symptoms.
Conclusion Regardless of the sequence of events, adopting high frequency cannabis use may be a useful behavioural marker of current or future emotional distress, and the need for interventions to address mental health. Physical activity adherence may be one approach to minimizing potential changes in mental health associated with increasing cannabis use.

How does CBD regulate gene expression?
Adrian Devitt-Lee and Martin A Lee
O’Shaughnessy’s• Winter2015/16
https://www.projectcbd.org/science/c...ene-expression
There is growing interest among medical scientists in the gene-regulating properties of cannabidiol (CBD), the non-psychoactive plant cannabinoid. Researchers at the California Pacific Medical Center have shown that CBD reduces brain cancer and breast cancer cell proliferation and metastasis by inhibiting the expression of the ID-1 gene. ID-1 expression is implicated in several kinds of ag¬gressive cancer.
In 2012, Israeli scientists identified more than 1,200 genes affected by CBD. Some 680 “gene transcripts” were upregulated (“turned on”) by CBD and 524 were down¬regulated (“turned off”). The probe focused on CBD’s role in maintaining the right amount of zinc within cells (zinc homeostasis).
In the same study, THC was found to regulate 94 genes. “The results show that CBD, but much less so THC, af¬fects the expression of genes involved in zinc homeostasis and suggest that the regulation of zinc levels could have an important role through which CBD may exert its anti-oxidant and anti-inflammatory effects,” the researchers concluded.

How to prevent cannabis-induced psychological distress . . . in politicians
Franjo Grotenhermen
THE LANCET • Vol 363 • May 15, 2004
DOI:10.1016/S0140-6736(04)16235-1

Human Cannabinoid Pharmacokinetics
Marilyn A. Huestis
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
A multitude of roles for the endogenous cannabinoid system has been proposed by recent research efforts. A large number of endogenous cannabinoid neurotransmitters or endocannabinoids have been identified, and the CB-1 and CB-2 cannabinoid receptors have been characterized. The presence of other receptors, transporters, and enzymes responsible for the synthesis or metabolism of endocannabinoids are becoming known at an extraordinary pace. The complex functions of this novel system have created multiple new targets for pharmacotherapies. Research has focused on separating the behavioral psychoactive effects of cannabinoid agonists from
therapeutic effects. These efforts have been largely unsuccessful. Another strategy centers on changing the pharmacokinetics of drug delivery to maximize therapeutic effect and minimize cognitive and subjective drug effects. Development of oral, rectal, and transdermal medications of synthetic D9-tetrahydrocannabinol (THC)1) are examples of this type of approach. Additionally, the potential therapeutic benefits of administering unique combinations of cannabinoids and other chemicals present in the plant Cannabis sativa is being investigated by the oromucosal route. There also is strong interest in medications based on antagonizing endocannabinoid action.

Human Metabolites of Cannabidiol: A Review on Their Formation, Biological Activity, and Relevance in Therapy
Istvan Ujvary, and Lum?r Hanus
Cannabis and Cannabinoid Research, 1(1), 90–101.
DOI: 10.1089/can.2015.0012
Cannabidiol (CBD), the main nonpsychoactive constituent of Cannabis sativa, has shown a wide range of therapeutically promising pharmacological effects either as a sole drug or in combination with other drugs in adjunctive therapy. However, the targets involved in the therapeutic effects of CBD appear to be elusive. Furthermore, scarce information is available on the biological activity of its human metabolites which, when formed in pharmacologically relevant concentration, might contribute to or even account for the observed therapeutic effects. The present overview summarizes our current knowledge on the pharmacokinetics and metabolic fate of CBD in humans, reviews studies on the biological activity of CBD metabolites either in vitro or in vivo, and discusses relevant drug–drug interactions. To facilitate further research in the area, the reported syntheses of CBD metabolites are also catalogued.

Human Pharmacokinetic Parameters of Orally Administered ?9-Tetrahydrocannabinol Capsules Are Altered by Fed Versus Fasted Conditions and Sex Differences.
Lunn, S., Diaz, P., O’Hearn, S., Cahill, S. P., Blake, A., Narine, K., & Dyck, J. R. B.
Cannabis and Cannabinoid Research, 4(4), 255–264. (2019). doi:10.1089/can.2019.0037
Background: There is variability in thereportedD9 -tetrahydrocannabinol (THC) and 11-hydroxy-tetrahydrocannabinol (11-OH-THC) pharmacokinetic (PK) and pharmacodynamic (PD) parameters between studies and there is limited investigation into how the presence of food or sex affect these parameters. In this study, we examined the PK and PD parameters of an encapsulated THC extract and its major active metabolite, 11-OH-THC, under different fed states. Methods: The study was a single-dose, randomized, double-blinded, four-way crossover investigation. THC capsules (1 or 2 · 5 mg) were administered to 28 healthy adults (13 females: 15 males) under a fasted condition or after a high-fat meal. Blood samples were collected and PK parameters were determined through noncompartmental analysis. Adverse events (AEs), cognitive function (through completion of digit symbol substitution tests), blood pressure, and heart rate were also recorded. Results: The presence of high-fat food significantly enhanced time to peak plasma concentration (Tmax) and area under the curve (AUC0–24) for both THC and 11-OH-THC and reduced THC’s apparent volume of distribution (Vz/F) and apparent clearance (Cl/F). Females had a significantly greater peak plasma concentration (Cmax) compared with males after 5 mg THC in a fasted state. No cardiovascular or cognitive effects and only mild AEs (somnolence, fatigue, and euphoric mood) were reported. Conclusion: These findings may help to inform the guidelines provided by governing health bodies on the effects of cannabis, such as time to onset and duration of action, and aid health care practitioners in their prescribing practices. Furthermore, the doses used in this study are safe to consider for future interventional studies in disease conditions where THC has been shown to have therapeutic efficacy.

NOT CANNABIS SPRCIFIC
Human tumor cell growth inhibition by nontoxic anthocyanidins, the pigments in fruits and vegetables.
Zhang, Y., Vareed, S. K., & Nair, M. G.
Life Sciences, 76(13), 1465–1472. (2005).
doi:10.1016/j.lfs.2004.08.025
Anthocyanidins, the aglycones of anthocyanins, impart brilliant colors in many fruits and vegetables. The widespread consumption of diets rich in anthocyanin and anthocyanidins prompted us to determine their inhibitory effects on human cancer cell proliferation. Five anthocyanidins, cyanidin (1), delphinidin (2), pelargonidin (3), petunidin (4) and malvidin (5), and four anthocyanins, cyanidin-3-glucoside, cyanidin-3-galactoside, delphinidin- 3-galactoside and pelargonidin-3-galactoside were tested for cell proliferation inhibitory activity against human cancer cell lines, AGS (stomach), HCT-116 (colon), MCF-7 (breast), NCI H460 (lung), and SF-268 (Central Nervous System, CNS) at 12.5–200 Ag/mL concentrations. The viability of cells after exposure to anthocyanins and anthocyanidins was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric methods. The anthocyanins assayed did not inhibit cell proliferation of cell lines tested at 200 Ag/mL. However, anthocyanidins showed cell proliferation inhibitory activity. Malvidin inhibited AGS, HCT-116, NCIH460, MCF-7 and SF-268 cell growth by 69, 75.7, 67.7, 74.7 and 40.5%, respectively, at 200 Ag/mL. Similarly, pelargonidin inhibited AGS, HCT-116, NCI H460, MCF-7 and SF-268 cell growth by 64, 63, 62, 63 and 34%, respectively, at 200 Ag/mL. At 200 Ag/mL, cyanidin, delphinidin and petunidin inhibited the breast cancer cell growth by 47, 66 and 53%, respectively. This is the first report of tumor cell proliferation inhibitory activity by anthocyanidins.

Hypnotic and Antiepileptic Effects of Cannabidiol
ELISALDO A. CARLINI, and JOMAR M. CUNHA
J Clin Pharmacol. 1981; 21:417S-427S.
DOI:10.1002/j.1552-4604.1981.tb02622.x
Clinical trials with cannabidiol (CBD) in healthy volunteers, insomniacs, and
epileptic patients conducted in the authors’ laboratory from 1972 up to the present are reviewed. Acute doses of cannabidiol ranging from 10 to 600 mg and chronic administration of 10 mg for 20 days or 3 mg/kg/day for 30 days did not induce psychologic or physical symptoms suggestive of psychotropic or toxic effects; however, several volunteers complained of somnolence. Complementary laboratory tests (EKG, blood pressure, and blood and urine analysis) revealed no sign of toxicity. Doses of 40,80, and 160 mg cannabidiol were compared to placebo and 5 mg nitrazepam in 15 insomniac volunteers. Subjects receiving 160 mg cannabidiol reported having slept significantly more than those receiving placebo; the volunteers also reported significantly less dream recall with the three doses of cannabidiol than with placebo. Fifteen patients suffering from secondary generalized epilepsy refractory to
known antiepileptic drugs received either 200 to 300 mg cannabidiol daily or placebo for as long as 4.5 months. Seven out of the eight epileptics receiving cannabidiol had improvement of their disease state, whereas only one placebo patient improved.

IAMC Bulletin June 6 2021
http://www.cannabis-med.org/english/...l.php?id=618#1

IASP Presidential Task Force on Cannabis and Cannabinoid Analgesia research agenda on the use of cannabinoids, cannabis, and cannabis-based medicines for pain management
Haroutounian, Simon; Arendt-Nielsen, Lars; Belton, Joletta; Blyth, Fiona M.; Degenhardt, Louisa; Di Forti, Marta; Eccleston, Christopher; Finn, David P; Finnerup, Nanna B; Fisher, Emma; Fogarty, Alexandra E.; Gilron, Ian; Hohmann, Andrea G; Kalso, Eija; Krane, Elliot; Mohiuddin, Mohammed; Moore, R Andrew; Rowbotham, Michael; Soliman, Nadia; Wallace, Mark; Zinboonyahgoon, Nantthasorn; Rice, Andrew SC
PAIN: March 12, 2021 - Volume
doi: 10.1097/j.pain.0000000000002266
https://journals.lww.com/pain/_layou...V1cm9uZXQubmw=
The President of the International Association for the Study of Pain (IASP) established a Taskforce on Cannabis and Cannabinoid Analgesia, to systematically examine the evidence on (i) analgesic pharmacology of cannabinoids and preclinical evidence on their efficacy in animal models of injury-related or pathological persistent pain, (ii) the clinical efficacy of cannabis, cannabinoids and cannabis-based medicines (CBM) for pain, (iii) harms related to long-term use of cannabinoids, as well as (iv) societal issues and policy implications related to the use of these compounds for pain management. Here, we summarize key knowledge gaps identified in the Taskforce outputs and propose a research agenda for generating high-quality evidence on the topic.
The systematic assessment of preclinical and clinical literature identified gaps in rigor of study design and reporting across the translational spectrum. We provide recommendations to improve the quality, rigor, transparency, and reproducibility of preclinical and clinical research on cannabis and cannabinoids for pain, as well as for the conduct of systematic reviews on the topic. Gaps related to comprehensive understanding of the endocannabinoid system and cannabinoid pharmacology, including pharmacokinetics and drug formulation aspects, are discussed. We outline key areas where high quality clinical trials with cannabinoids are needed. Important remaining questions about long-term and short-term safety of cannabis and cannabinoids are emphasized. Finally, regulatory, societal and policy challenges associated with medicinal and non-medicinal use of cannabis are highlighted, with recommendations for improving patient safety and reducing societal harms in the context of pain management.

Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol.
Linciano, P., Citti, C., Russo, F., Tolomeo, F., Laganà, A., Capriotti, A. L., … Cannazza, G.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-020-79042-2
The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and ?9 -tetrahydrocannabinol (?9 -THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and ?9 -THCB) and a heptyl-(in CBDP and ?9 -THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that flls the gap between the pentyl and heptyl homologs of CBD and ?9 -THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and ?9 -tetrahydrocannabihexol (?9 -THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and ?9 -THCH have been already identifed and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and ?9 -tetrahydrocannabinol monomethyl ether (?9-THCM). The unambiguously identifcation in cannabis extract of the n-hexyl homologues of CBD and ?9 -THC diferent from the corresponding methylated isomers (CBDM, CBGM and ?9 -THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantifcation of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of ?9 -THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confrmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and ?9 -THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

Identification of Cannabis pollens using an allergic patient’s immunoglobulin E and purification and characterization of allergens in Cannabis pollens
Hiroyuki Tanaka , Mie Degawa , Etsuo Kawata , Jun Hayashi ,
Yukihiro Shoyama
Forensic Science International 97 (1998) 139–153
doi.org/10.1016/S0379-0738(98)00152-2
Cannabis pollen allergens were detected using the serum of an allergic patient. The allergens were then purified by sequential column chromatography (including DE52 cellulose and phenyl- Sepharose CL-4B) and preparative HPLC. The molecular weight of the allergens were determined as 10 050 and 13 706 by matrix-assisted laser desorption / ionization time of flight mass
spectrometry.We utilised Western blotting and development of an enzyme-linked immunosorbent assay for the detection of Cannabis pollen allergens.

Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes
Rongrong Jiang, Satoshi Yamaori, Shuso Takeda, Ikuo Yamamoto, Kazuhito Watanabe
Life Sciences 89 (5-6), (2011) 165–170
doi:10.1016/j.lfs.2011.05.018
Aims: Cannabidiol (CBD), one of the major constituents in marijuana, has been shown to be extensively metabolized by experimental animals and humans. However, human hepatic enzymes responsible for the CBD metabolism remain to be elucidated. In this study, we examined in vitro metabolism of CBD with human liver microsomes (HLMs) to clarify cytochrome P450 (CYP) isoforms involved in the CBD oxidations.
Main methods: Oxidations of CBD in HLMs and recombinant human CYP enzymes were analyzed by gas chromatography/mass spectrometry.
Key findings: CBD was metabolized by pooled HLMs to eight monohydroxylated metabolites (6?-OH-, 6?-OH-, 7-OH-, 1?-OH-, 2?-OH-, 3?-OH-, 4?-OH-, and 5?-OH-CBDs). Among these metabolites, 6?-OH-, 6?-OH-, 7-OH-, and 4?-OH-CBDs were the major ones as estimated from the relative abundance of m/z 478, which was a predominant fragment ion of trimethylsilyl derivatives of the metabolites. Seven of 14 recombinant human CYP enzymes examined (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) were capable of metabolizing CBD. The correlations between CYP isoform-specific activities and CBD oxidative activities in 16 individual HLMs indicated that 6?-OH- and 4?-OH-CBDs were mainly formed by CYP3A4, which was supported by inhibition studies using ketoconazole and an anti-CYP3A4 antibody. The correlation and inhibition studies also showed that CBD 6?-hydroxylation was mainly catalyzed by CYP3A4 and CYP2C19, whereas CBD 7-hydroxylation was predominantly catalyzed by CYP2C19.
Significance: This study indicated that CBD was extensively metabolized by HLMs. These results suggest that CYP3A4 and CYP2C19 may be major isoforms responsible for 6?-, 6?-, 7-, and/or 4?-hydroxylations of CBD in HLMs.

Identification of Psychoactive Degradants of Cannabidiol in Simulated Gastric and Physiological Fluid.
Merrick, J., Lane, B., Sebree, T., Yaksh, T., O’Neill, C., & Banks, S. L.
Cannabis and Cannabinoid Research, 1(1), 102–112. (2016).
doi:10.1089/can.2015.0004
Introduction: In recent research, orally administered cannabidiol (CBD) showed a relatively high incidence of somnolence in a pediatric population. Previous work has suggested that when CBD is exposed to an acidic environment, it degrades to D9 -tetrahydrocannabinol (THC) and other psychoactive cannabinoids. To gain a better understanding of quantitative exposure, we completed an in vitro study by evaluating the formation of psychoactive cannabinoids when CBD is exposed to simulated gastric fluid (SGF).
Methods: Materials included synthetic CBD, D8 -THC, and D9 -THC. Linearity was demonstrated for each component over the concentration range used in this study. CBD was spiked into media containing 1% sodium dodecyl sulfate (SDS). Samples were analyzed using chromatography with UV and mass spectrometry detection. An assessment time of 3 h was chosen as representative of the maximal duration of exposure to gastric fluid.
Results: CBD in SGF with 1% SDS was degraded about 85% after 60 min and more than 98% at 120 min. The degradation followed first-order kinetics at a rate constant of 0.031 min1 (R2 = 0.9933). The major products formed were D9 -THC and D8 -THC with less significant levels of other related cannabinoids. CBD in physiological buffer performed as a control did not convert to THC. Confirmation of THC formation was demonstrated by comparison of mass spectral analysis, mass identification, and retention time of D9 -THC and D8 -THC in the SGF samples against authentic reference standards.
Conclusions: SGF converts CBD into the psychoactive components D9 -THC and D8 -THC. The first-order kinetics observed in this study allowed estimated levels to be calculated and indicated that the acidic environment during normal gastrointestinal transit can expose orally CBD-treated patients to levels of THC and other psychoactive cannabinoids that may exceed the threshold for a physiological response. Delivery methods that decrease the potential for formation of psychoactive cannabinoids should be explored.

Illicit Cannabis Usage as a Management Strategy in New Zealand Women with Endometriosis: An Online Survey
JOURNAL OF WOMEN’S HEALTH 2020
DOI: 10.1089/jwh.2020.8668
Mike Armour, Justin Sinclair, Geoff Noller, Jane Girling, Maria Larcombe, Mahmoud A. Al-Dabbas, Erika Hollow, Deborah Bush, Dip Tchg, and Neil Johnson
Background: Endometriosis affects around 10% of women worldwide. Many women with endometriosis struggle with finding adequate pain management, and data from other countries suggest that women use cannabis, either legal or illicit, to help manage their endometriosis symptoms. The aim of this study was to determine use of cannabis where endometriosis was self-identified as a condition that was being treated with cannabis, as well as the impact of cannabis use on the usage on other pharmaceuticals.
Materials and Methods: A cross-sectional online survey of those using cannabis for health-related conditions run between May and July 2019. This article reports on the subset of this larger data set for those reporting they had a diagnosis of endometriosis and/or polycystic ovary syndrome. Data were collected on demographics, modes of cannabis administration, symptoms treated, changes in pharmaceutical usage, and adverse events.
Results: Two hundred thirteen valid responses were analyzed. Mean age of respondents was 32 years and 79.8% were current cannabis users. The most common outcomes that cannabis was used for were to improve pain relief (95.5%) and to improve sleep (95.5%). Respondents reported that their symptom was ‘‘much better’’ for pain (81%), sleep (79%), and nausea or vomiting (61%). Over three-quarters (81.4%) indicated cannabis had reduced their normal medication usage. Over half (59%) were able to completely stop a medication, most commonly (66%) analgesics. Opioids (40%) were the most common class of analgesic stopped.
Conclusions: Cannabis is reported as an effective intervention for pain and other endometriosis symptoms with potential substitution effects on opioid usage.

Immune Responses Regulated by Cannabidiol.
Nichols, J. M., & Kaplan, B. L. F.
Cannabis and Cannabinoid Research. (2019).
doi:10.1089/can.2018.0073
Introduction: Cannabidiol (CBD) as Epidiolex (GW Pharmaceuticals) was recently approved by the U.S. Food and Drug Administration (FDA) to treat rare forms of epilepsy in patients 2 years of age and older. Together with the increased societal acceptance of recreational cannabis and CBD oil for putative medical use in many states, the exposure to CBD is increasing, even though all of its biological effects are not understood. Once such example is the ability of CBD to be anti-inflammatory and immune suppressive, so the purpose of this review is to summarize effects and mechanisms of CBD in the immune system. It includes a consideration of reports identifying receptors through which CBD acts, since the ‘‘CBD receptor,’’ if a single one exists, has not been definitively identified for the myriad immune system effects. The review then provides a summary of in vivo and in vitro effects in the immune system, in autoimmune models, with a focus on experimental autoimmune encephalomyelitis, and ends with identification of knowledge gaps. Conclusion: Overall, the data overwhelmingly support the notion that CBD is immune suppressive and that the mechanisms involve direct suppression of activation of various immune cell types, induction of apoptosis, and promotion of regulatory cells, which, in turn, control other immune cell targets.

Impact of circadian rhythmicity and sleep restriction on circulating endocannabinoid (eCB) N-arachidonoylethanolamine (anandamide)
Erin C. Hanlon, PhD
Psychoneuroendocrinology 2019 Oct 4;111:104471.
doi: 10.1016/j.psyneuen.2019.104471.
Objective: The endocannabinoid (eCB) system is involved in diverse aspects of human physiology and behavior but little is known about the impact of circadian rhythmicity on the system. The two most studied endocannabinoids, AEA (ananamide) and 2-AG, can be measured in peripheral blood however
the functional relevance of peripheral eCB levels is not clear. Having previously detailed the 24-hr profile of serum 2-AG, here we report the 24-hr serum profile of AEA to determine if these two endocannabinoids vary in parallel across the biological day including a nocturnal 8.5-h sleep period.
Further, we assessed and compared the effect of a physiological challenge, in the form of sleep restriction to 4.5-h, on these two profiles.
Methods: In this randomized crossover study, we examined serum concentrations of AEA across a 24-hr period in fourteen young adults. Congeners of AEA, the structural analogs oleoylethanolamide (OEA)
and palmitoylethanolamide (PEA) were simultaneously assayed. Prior to 24-hr blood sampling, each participant was exposed to two nights of normal (8.5 hrs) or restricted sleep (4.5 hrs). The two sleep conditions were separated by at least one month. In both sleep conditions, during the period of blood sampling, each individual ate the same high-carbohydrate meal at 0900, 1400, and 1900.
Results: Mean 24-hr concentrations of AEA were 0.697 ± 0.11 pmol/ml. A reproducible biphasic 24-h profile of AEA was observed with a first peak occurring during early sleep (0200) and a second peak in the mid-afternoon (1500) while a nadir was detected in the mid-morning (1000). The 24-hr profiles for both OEA and PEA followed a similar pattern to that observed for AEA. AEA, OEA, and PEA levels were not affected by sleep restriction at any time of day, contrasting with the elevation of early afternoon levels previously observed for 2-AG.
Conclusions: The 24-hr rhythm of AEA is markedly different from that of 2-AG, being of lesser amplitude and biphasic, rather than monophasic. These observations suggest distinct regulatory pathways of the two eCB and indicate that time of day needs to be carefully controlled in studies attempting to delineate their relative roles. Moreover, unlike 2-AG, AEA is not altered by sleep restriction, suggesting that physiological perturbations may affect AEA and 2-AG differently. Similar 24-hr profiles were observed for OEA and PEA following normal and restricted sleep, further corroborating the validity of the wave-shape and lack of response to sleep loss observed for the AEA profile. Therapeutic approaches involving agonism or antagonism of peripheral eCB signaling will likely need to be tailored according to time of day.

Impact of D9-Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells
Torsten Lowin, Christina Kok, Sophie Smutny and Georg Pongratz
Biomedicines 2022, 10, 1118.
DOI: 10.3390/biomedicines10051118
9-Tetrahydrocannabinol (THC) has demonstrated anti-inflammatory effects in animal models of arthritis, but its mechanism of action and cellular targets are still unclear. The purpose of this study is to elucidate the effects of THC (0.1–25 _M) on synovial fibroblasts from patients with rheumatoid arthritis (RASF) and peripheral blood mononuclear cells (PBMC) from healthy donors in respect to proliferation, calcium mobilization, drug uptake, cytokine and immunoglobulin production. Intracellular calcium and drug uptake were determined by fluorescent dyes Cal-520 and PoPo3, respectively. Cytokine and immunoglobulin production were evaluated by ELISA. Cannabinoid receptors 1 and 2 (CB1 and CB2) were detected by flow cytometry. RASF express CB1 and CB2 and the latter was increased by tumor necrosis factor (TNF). In RASF, THC (_5 _M) increased intracellular calcium levels/PoPo3 uptake in a TRPA1-dependent manner and reduced interleukin-8 (IL-8) and matrixmetalloprotease 3 (MMP-3) production at high concentrations (25 _M). Proliferation was slightly enhanced at intermediate THC concentrations (1–10 _M) but was completely abrogated at 25 _M. In PBMC alone, THC decreased interleukin-10 (IL-10) production and increased immunoglobulin G (IgG). In PBMC/RASF co-culture, THC decreased TNF production when cells were stimulated with interferon- (IFN-) or CpG. THC provides pro- and anti-inflammatory effects in RASF and PBMC. This is dependent on the activating stimulus and concentration of THC. Therefore, THC might be used to treat inflammation in RA but it might need titrating to determine the effective concentration.

Impact of smoking cannabidiol (CBD)-rich marijuana on driving ability
Tim J. Gelmia, Wolfgang Weinmanna and Matthias Pfäfflic
Forensic Sciences Research 2021, VOL. 6, NO. 3, 195–207
DOI: 10.1080/20961790.2021.1946924
https://www.tandfonline.com/doi/full...0.2021.1946924
https://www.tandfonline.com/doi/epub...46924?needAcce ss=true
To investigate effects of smoking cannabidiol (CBD)-rich marijuana on driving ability and determine free CBD and Δ9-tetrahydrocannabinol (THC) concentrations in capillary blood samples, a randomised, double-blind, placebo-controlled, two-way crossover pilot study was conducted with 33 participants. Participants smoked a joint containing 500 mg of tobacco and either 500 mg of CBD-rich marijuana (16.6% total CBD; 0.9% total THC) or 500 mg of a placebo substance, then performed three different dimensions of the Vienna Test System TRAFFIC examining reaction time, behaviour under stress, and concentration performance. For further assessment of participants’ fitness to drive, three tests of balance and coordination were evaluated and vital signs (blood pressure and pulse) were measured. Dried blood spot samples of capillary blood were taken after smoking and after completion of the tests to determine the cannabinoid concentrations (CBD, THC and THC-metabolites). The results revealed no significant differences between the effects of smoking CBD-rich marijuana and placebo on reaction time, motor time, behaviour under stress, or concentration performance. Maximum free CBD and THC concentrations in capillary blood were detected shortly after smoking, ranging between 2.6–440.0 ng/mL and 6.7–102.0 ng/mL, respectively. After 45 min, capillary blood concentrations had already
declined and were in the range of 1.9–135.0 ng/mL (free CBD) and 0.9–38.0 ng/mL (free THC). Although the observed levels of free THC concentrations have been reported to cause symptoms of impairment in previous studies in which THC-rich marijuana was smoked, no signs of impairment were found in the current study. This finding suggests that higher CBD concentrations cause a negative allosteric effect in the endocannabinoid system, preventing the formation of such symptoms. Nevertheless, it is recommended that consumers refrain from driving for several hours after smoking CBD-rich marijuana, as legal THC concentration limits may be exceeded.

Impacts of cannabinoid epigenetics on human development:
Reece, A. S., & Hulse, G. K.
reflections on Murphy et. al. “cannabinoid exposure and altered DNA methylation in rat and human sperm” epigenetics 2018; 13: 1208-1221.
Epigenetics, 1–16. (2019)
doi:10.1080/15592294.2019.1633868
Recent data from the Kollins lab (‘Cannabinoid exposure and altered DNA methylation in rat and human sperm’ Epigenetics 2018; 13: 1208–1221) indicated epigenetic effects of cannabis use on sperm in man parallel those in rats and showed substantial shifts in both hypo- and hyper-DNA methylation with the latter predominating. This provides one likely mechanism for the transgenerational transmission of epigenomic instability with sperm as the vector. It therefore contributes important pathophysiological insights into the probable mechanisms underlying the epidemiology of prenatal cannabis exposure potentially explaining diverse features of cannabis-related teratology including effects on the neuraxis, cardiovasculature, immune stimulation, secondary genomic instability and carcinogenesis related to both adult and pediatric cancers. The potentially inheritable and therefore multigenerational nature of these defects needs to be carefully considered in the light of recent teratological and neurobehavioural trends in diverse jurisdictions such as the USA nationally, Hawaii, Colorado, Canada, France and Australia, particularly relating to mental retardation, age-related morbidity and oncogenesis including inheritable cancerogenesis. Increasing demonstrations that the epigenome can respond directly and in real time and retain memories of environmental exposures of many kinds implies that the genome-epigenome is much more sensitive to environmental toxicants than has been generally realized. Issues of long-term multigenerational inheritance amplify these concerns. Further research particularly on the epigenomic toxicology of many cannabinoids is also required.

Importance of Hemp seeds in the tuberculosis therapy
Jan Kabelik
1955 - ACTA UNIVERSITATIS PALACKIANAE OLOMUCENSIS - TOM. VI.
https://www.bushka.cz/KabelikEN/hempseed.html
IMPORTANCE OF HEMP SEEDS IN THE THERAPY OF TUBERCULOSIS
JOSEF SIREK
Tuberculosis Hospital at Jince 1954
If we are discussing the use of hemp seed in the therapy of tuberculosis, we don't think about chemotherapy in today's sense of the word, not even the treatment specifically directed against the Koch bacillus and not a treatment oriented towards diseases that cause changes in the lungs. We think of a total conservative treatment with the emphases on its dietetic or therapeutic nutrition. Therapeutic nutrition becomes then a branch of therapy, namely as a part of the overall effort to "treat the whole human being". For this reason we, according to the Soviet example, begin to understand the importance of the treatment through nutrition in therapy, not excluding physiotherapy. These are, however, the beginnings, lacking direction and therefore up to this point, not too fruitful. One should not be, however, surprised. Even in the Soviet Union the whole question of treatment through nutrition is still controversial, and even the most important nutritional work of the Pevzner school has been subjected to a very critical assessment. Although it was primarily M.I. Pevzner and out of other Soviet authors namely L. M. Model who also in conjunction with tuberculosis rightfully stressed the most important elements of the treatment through nutrition. Another Soviet author, O. L. Gordon, in his presumation entitled "Justification of treatment through nutrition in the clinic and prospects for its expansion" said the following: "Treatment through nutrition is a therapeutic method. As a part of complex therapy it has two goals: To satisfy the physiological needs of the sick organism and to actively influence the pathological process. In this aspect the treatment through nutrition differs from the ordinary nutrition of a sick person". F.K. Mensikov coworker of the Clinic for treatment through nutrition, added that during complex therapy of all diseases proper nutrition is a primary need, while a nutrition which does not take into account the status of the patient could have a negative effect. "Particularly, the treatment through nutrition has an important effect in situations where we need to support regeneration of the tissues, namely when we don't have a medication which could actively support such process". (Presented on December 10, 1954, at the occasion of a scientific conference at Palacky University at Olomouc, as a part of the topic "Hemp seed as a treatment”).

In an exploratory randomized, double-blind, placebo-controlled, cross-over study, psychoactive doses of intravenous delta-9-tetrahydrocannabinol fail to produce antinociceptive effects in healthy human volunteers.
Schindler, E. A. D., Schnakenberg Martin, A. M., Sewell, R. A., Ranganathan, M., DeForest, A., Pittman, B. P., … D’Souza, D. C
Psychopharmacology.(2020).
doi:10.1007/s00213-020-05595-9
Rationale Animal studies and anecdotal human reports suggest that cannabinoids have antinociceptive effects. Controlled human studies have produced mixed results.
Objectives We sought to reduce existing variability by investigating the effects of intravenous delta-9-tetrahydrocannabinol (THC) in several pain paradigms within the same human subjects, addressing some of the limitations to the published literature.
Methods In this exploratory randomized, double-blind, placebo-controlled, cross-over study, healthy human subjects received 0.01 mg/kg or 0.03 mg/kg intravenous THC or placebo (ethanol vehicle) infused over 10 min on three test days, each separated by at least 72 h. Capsaicin (250 μg) was injected intradermally to induce chemical pain and hyperalgesia. Four other forms of acute pain were induced: mechanical (von Frey filament), hot and cold (thermode), and electrical (pulse generator). Pain ratings were obtained before drug administration, at peak drug effects, and 2 h after drug administration and included both objective and subjective measures. THC drug effects and vital signs were also collected during experimental sessions. Nonparametric analysis with repeated measures was performed.
Results THC induced euphoria, perceptual and cognitive alterations, and tachycardia in a dose-related manner, but failed to have significant effects in experimentally induced acute chemical, mechanical, thermal, or electrical pain and capsaicin-induced hyperalgesia.
Conclusions In this exploratory controlled study, intravenous THC lacked significant antinociceptive properties in experimental models of acute pain and capsaicin-induced hyperalgesia in healthy human subjects. Continued study of THC and other cannabinoids through high-quality, controlled studies in both healthy volunteers and patients with pain conditions is warranted to inform the growing demand for the clinical application of cannabinoids in pain management.

In Search of Preventative Strategies: Novel Anti-Inflammatory High-CBD Cannabis Sativa Extracts Modulate ACE2 Expression in COVID-19 Gateway Tissues.
Wang, B.; Kovalchuk, A.; Li, D.; Ilnytskyy, Y.; Kovalchuk, I.; Kovalchuk, O.
Preprints 2020,
https://www.preprints.org/manuscript/202004.0315/v1
doi: 10.20944/preprints202004.0315.v1
With the rapidly growing pandemic of COVID-19 caused by the new and challenging to treat zoonotic SARS-CoV2 coronavirus, there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. Inhibition of viral entry and thereby spread constitute plausible therapeutic avenues. Similar to other respiratory pathogens, SARS-CoV2 is transmitted through respiratory droplets, with potential for aerosol and contact spread. It uses receptor-mediated entry into the human host via angiotensin-converting enzyme II (ACE2) that is expressed in lung tissue, as well as oral and nasal mucosa, kidney, testes, and the gastrointestinal tract. Modulation of ACE2 levels in these gateway tissues may prove a plausible strategy for decreasing disease susceptibility. Cannabis sativa, especially one high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been proposed to modulate gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. Working under the Health Canada research license, we have developed over 800 new Cannabis sativa lines and extracts and hypothesized that high-CBD C. sativa extracts may be used to modulate ACE2 expression in COVID-19 target tissues. Screening C. sativa extracts using artificial human 3D models of oral, airway, and intestinal tissues, we identified 13 high CBD C. sativa extracts that modulate ACE2 gene expression and ACE2 protein levels. Our initial data suggest that some C. sativa extract down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV2 entry into host cells. While our most effective extracts require further large-scale validation, our study is crucial for the future analysis of the effects of medical cannabis on COVID-19. The extracts of our most successful and novel high CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the treatment of COVID-19 as an adjunct therapy. They can be used to develop easy-to-use preventative treatments in the form of mouthwash and throat gargle products for both clinical and at-home use. Such products ought to be tested for their potential to decrease viral entry via the oral mucosa. Given the current dire and rapidly evolving epidemiological situation, every possible therapeutic opportunity and avenue must be considered.

In Vitro Screening of Three Commercial Cannabis-Based Products on ATP-Binding Cassette and Solute-Carrier Transporter Function.
Anderson, L. L., Etchart, M. G., MacNair, L., Land, M. H., Mosesova, I. A., Bonn-Miller, M. O., & Arnold, J. C.
Cannabis and Cannabinoid Research.(2020).
doi:10.1089/can.2020.0053
Introduction: Legalization of medicinal cannabis around the world has led to an increase in the use of commercial cannabis-based products in the community. These cannabis-based products are being used in combination with conventional drugs to treat a variety of health conditions. Moreover, recreational cannabis-based products may be used in combination with other drugs. In this setting, there is increased potential for drug–drug interactions (DDIs) involving commercial cannabis-based products. Since DDIs can lead to serious adverse events, drug regulatory bodies require that every investigational drug be evaluated for DDI potential at metabolic enzymes and transporters. However, this seldom occurs for cannabis-based products due to legislation in many jurisdictions allowing a direct pathway to market. This study aimed to examine the inhibitory potential of three commercially available cannabis-based products at human ATP-binding cassette (ABC) and solute-carrier (SLC) transporters.
Materials and Methods: Three commercial cannabis-based products (Spectrum Yellow, Tweed Argyle, and Spectrum Red) that contain differing concentrations of cannabidiol (CBD) and D9 -tetrahydrocannabinol (D9 -THC) were evaluated for DDI potential at 12 drug transporters. HEK293 cells or vesicles expressing human ABC transporters (ABCB1, ABCC2, ABCG2, or ABCB11) and SLC transporters (SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLCO1B1, SLCO1B3, SLC47A1, and SLC47A2) were used to measure transporter function.
Results: Spectrum Yellow and Tweed Argyle inhibited ABCG2 transporter function. The IC50 value of Spectrum Yellow based on CBD and D9 -THC content was 4.5 lM for CBD and 0.20 lM for D9 -THC, and the IC50 value of Tweed Argyle was 9.3 lM for CBD and 6.0 lM for D9 -THC. Tweed Argyle also inhibited ABCB11 transporter function with an IC50 value of 11.9 lM for CBD and 7.7 lM for D9 -THC. SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 transporter functions were modestly inhibited by high concentrations of the cannabis-based products. The three cannabis-based products did not inhibit ABCB1, ABCC2, SLC47A1, SLC47A2, or SLC22A8 transporters.
Discussion: Novel findings were that the cannabis-based products inhibited ABCB11, SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 (although modestly in most instances). Spectrum Yellow and Tweed Argyle potently inhibited ABCG2, and future in vivo DDI studies could be conducted to assess whether cannabis products affect the pharmacokinetics of medications that are ABCG2 substrates.

In vivo Evidence for Therapeutic Properties of Cannabidiol (CBD) for Alzheimer's Disease.
Watt G, Karl T
Front Pharmacol. 2017 Feb 3;8:20. eCollection 2017.
doi: 10.3389/fphar.2017.00020.
Alzheimer's disease (AD) is a debilitating neurodegenerative disease that is affecting an increasing number of people. It is characterized by the accumulation of amyloid-? and tau hyperphosphorylation as well as neuroinflammation and oxidative stress. Current AD treatments do not stop or reverse the disease progression, highlighting the need for new, more effective therapeutics. Cannabidiol (CBD) is a non-psychoactivephytocannabinoid that has demonstrated neuroprotective, anti-inflammatory and antioxidant properties in vitro. Thus, it is investigated as a potential multifunctional treatment option for AD. Here, we summarize the current status quo of in vivo effects of CBD in established pharmacological and transgenic animal models for AD. The studies demonstrate the ability of CBD to reduce reactive gliosis and the neuroinflammatory response as well as to promote neurogenesis. Importantly, CBD also reverses and prevents the development of cognitive deficits in AD rodent models. Interestingly, combination therapies of CBD and ?9-tetrahydrocannabinol (THC), the main active ingredient of cannabis sativa, show that CBD can antagonize the psychoactive effects associated with THC and possibly mediate greater therapeutic benefits than either phytocannabinoid alone. The studies provide "proof of principle" that CBD and possibly CBD-THC combinations are valid candidates for novel AD therapies. Further investigations should address the long-term potential of CBD and evaluate mechanisms involved in the therapeutic effects describe

Increased Anandamide and Decreased Pain and Depression after Exercise in Fibromyalgia.
Stensson N, Gerdle B, Ernberg M, Mannerkorpi K3, Kosek E, Ghafouri B
Med Sci Sports Exerc. 2020 Mar 10.
doi: 10.1249/MSS.0000000000002293.
https://www.ncbi.nlm.nih.gov/pubmed/32168104
PURPOSE:
Physical exercise is increasingly being promoted by healthcare for chronic pain conditions with beneficial outcomes such as: pain and fatigue reduction, and increased quality of life. Nevertheless, knowledge about biochemical consequences of physical exercise in chronic pain is still relatively poor.The endocannabinoid system has been suggested to play a role for acute exercise-induced reward and pain inhibition. The aim of this study is to investigate the chronic outcomes of resistance exercise on levels of endocannabinoids and related lipids in fibromyalgia (FM).
METHODS:
This study examine the outcomes of a 15-week person-centered resistance exercise program on plasma levels of the lipid mediators; anandamide, 2-arachidonoylglycerol (2-AG), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and stearoylethanolamide (SEA) sampled from 37 women with FM and 33 healthy controls. The associations between clinical scorings of pain, depression, anxiety, fatigue, and muscle strength with levels of these lipid mediators before and after the exercise program are also analysed.
RESULTS:
After the 15 weeks exercise program anandamide levels were significantly increased and SEA levels significantly decreased in FM. Pain intensity and depression scorings decreased and muscle strength increased, and in a multivariate context muscle strength was positively associated with 2-AG levels after the resistance exercise program in FM.
CONCLUSIONS:
The increased anandamide and decreased SEA in women with fibromyalgia after the 15 weeks program might point to a chronic effect of resistance exercise. Pain and depression scorings decreased in the fibromyalgia group after the program but no associations between pain, depression and lipid level changes were assured.

INDIAN HEMP DRUGS AND INSANITY.
G. F. W. Ewens,
THE Indian Medical Gazette NOVEMBER, 1904.
https://www.ncbi.nlm.nih.gov/pmc/art...71518-0021.pdf
the Superintendent of the Central Lunatic Asylum, Lahore, raises again the question which ten years ago was very much debated among medical men in India.
It will be observed that, as a result of his experience while in charge of the large asylum at Lahore, Capt. Evvens is of opinion (1) that there is a form of mental disease which seems to have a direct relation to the excessive use of hemp drugs, as "a definite effect following a definite cause (2) it has a " definite train of symptoms of a fairly regular character."
Those of us who were in India ten years ago
will remember the Report of the Indian Hemp
Drugs Commission.

Influence of Plasticizers on the Stability and Release of a Prodrug of Δ9-Tetrahydrocannabinol Incorporated in Poly (Ethylene Oxide) Matrices,
Sridhar Thumma, Mahmoud A. ElSohly, Shuang-Qing Zhang, Waseem Gul and Michael A. Repka,
European Journal of Pharmaceutics and Biopharmaceutics, and 70(2):605-614, 2008.
DOI: 10.1016/j.ejpb.2008.06.009
The objective of this research was to stabilize a heat-labile novel prodrug of Δ9-tetrahydrocannabinol (THC), THC-hemiglutarate (THC-HG), in polyethylene oxide (PEO) [PolyOx® WSR N-80 (PEO N-80), MW 200,000 Daltons] polymeric matrix systems produced by hot-melt fabrication for systemic delivery of THC through the oral transmucosal route. For this purpose, the effects of processing conditions (processing temperature and heating duration), plasticizer type and concentration and storage conditions on the stability of the prodrug were investigated. The selected plasticizers studied included vitamin E succinate (VES), acetyltributyl citrate (ATBC), triethyl citrate (TEC), triacetin and polyethylene glycol 8000 (PEG 8000). Furthermore, the influence of plasticizer concentration on drug release was also studied. The stability of THC-HG in PEO matrices was influenced by all the aforementioned variables. Films processed at 110 °C for 7 min were found to be favorable for hot-melt processing with a post-processing drug content of 95%, while significant degradation of THC-HG (∼42%) was observed in those processed at 200 °C for 15 min. The degradation of the prodrug during hot-melt fabrication and also upon storage was considerably reduced in the presence of the plasticizers investigated, VES being the most effective. Modulation of the microenvironmental pH to an acidic range via incorporation of citric acid in PEO-plasticizer matrices significantly improved the stability of the prodrug, with almost 90% of the theoretical drug remaining as opposed to only 15% remaining in PEO-only matrices when stored at 40 °C for up to 3 months. The release of drug from PEO matrices was influenced both by the plasticizer type and concentration. A faster release resulted from water-soluble plasticizers, PEG 8000 and triacetin, and with increasing concentration. However, a slower release was observed with an increase in concentration of water-insoluble plasticizers, VES and ATBC.
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Influence of Treatment of Tourette Syndrome with D9 Tetrahydrocannabinol (D9-THC) on Neuropsychological Performance
K. R. Mu?ller-Vahl, A. Koblenz, M. Jöbges, H. Kolbe, H. M. Emrich, U. Schneider
Pharmacopsychiatry 2001; 34: 19±24
DOI:10.1055/s-2001-15191
Previous studies have suggested that marijuana (cannabis sativa) and delta-9-tetrahydrocannabinol (D9-THC), the major psychoactive ingredient of marijuana, are effective in the therapy of tics and associated behavioral disorders in Tourette Syndrome (TS). Because there is also evidence that
cannabis sativa may cause cognitive impairment in healthy users, we performed a randomized double-blind placebo controlled crossover trial for D9-THC in 12 adult TS patients to investigate whether treatment of TS with a single dose of D9-THC at 5.0 to 10.0 mg causes significant side effects on
neuropsychological performance. Using a variety of neuropsychological tests, we found no significant differences after treatment with D9-THC compared to placebo treatment in verbal and visual memory, reaction time, intelligence, sustained attention, divided attention, vigilance, or mood. Only when using the Symptom Checklist 90-R (SCL-90-R) did our data provide evidence for a deterioration of obsessive-compulsive behavior (OCB) and a trend towards an increase in phobic anxiety. However, these results should be interpreted with caution as SCL-90-R has known limitations on measuring OCB. We suggest that the increase in phobic anxiety is mainly due to the fact that a single-dose treatment rules out the possibility of administering the dosage slowly. In contrast to results obtained from healthy marijuana users, a single dose treatment with D9-THC in patients suffering from TS does not cause cognitive impairment. We therefore suggest that further investigations should concentrate on the effects of a longer-term therapy of TS with D9-THC.

Ingestion of a THC-Rich Cannabis Oil in People with Fibromyalgia: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.
Chaves, C., Bittencourt, P. C. T., & Pelegrini, A.
Pain Medicine, 21(10), 2212–2218.(2020).
doi:10.1093/pm/pnaa303
Objective. To determine the benefit of a tetrahydrocannabinol (THC)-rich cannabis oil on symptoms and quality of life of fibromyalgia patients. Methods. A double-blind, randomized, placebo-controlled clinical trial was conducted for eight weeks to determine the benefit of a THC-rich cannabis oil (24.44 mg/mL of THC and 0.51 mg/mL of cannabidiol [CBD]) on symptoms and quality of life of 17 women with fibromyalgia, residents of a neighborhood with a low socioeconomic profile and a high incidence of violence in the city of Florianopolis, Brazil. The initial dose was one drop (1.22 mg of THC and 0.02 mg of CBD) a day with subsequent increases according to symptoms. The Fibromyalgia Impact Questionnaire (FIQ) was applied at pre- and postintervention moments and in five visits over eight weeks.
Results. There were no significant differences on baseline FIQ score between groups. However, after the intervention, the cannabis group presented a significant decrease in FIQ score in comparison with the placebo group (P¼ 0.005) and in comparison with cannabis group baseline score. (P< 0.001). Analyzing isolated items on the FIQ, the cannabis group presented significant improvement on the “feel good,” “pain,” “do work,” and “fatigue” scores. The placebo group presented significant improvement on the “depression” score after intervention. There were no intolerable adverse effects.
Conclusions. Phytocannabinoids can be a low-cost and well-tolerated therapy to reduce symptoms and increase the quality of life of patients with fibromyalgia. Future studies are still needed to assess long-term benefits, and studies with different varieties of cannabinoids associated with a washout period must be done to enhance our knowledge of cannabis action in this health condition.

Inhalation Absorption Prediction (IAP) Model for Predicting Medicinal Cannabis Phytochemical Pharmacokinetics

Kimber Wise, Harsharn Gill, Jamie Selby-Pham

Pharmacogn. Commn. 2019; 9(3): 85-90

https://phcogcommn.org/wp-content/uploads/2021/08/PharmaComm-9-3-85.pdf

Introduction: The medicinal benefits from inhalation of Cannabis sativa phytochemicals have been extensively reported. Whilst in-silico models are available for prediction of phytochemical pharmacokinetics post-ingestion, no models are available to accurately predict inhalation pharmacokinetics. Therefore, the aim of this study was to explore the relationship between phytochemical physicochemical properties and inhalation pharmacokinetics and to develop an in-silico model for predicting the time of maximal compound concentration in plasma (Tmax) and compound elimination half-life (T.), following inhalation. Methods: A training set of compound pharmacokinetic data was collated from previous publications and compared to physicochemical parameters using regression analyses. Physicochemical parameters that correlated with Tmax and T. were combined to develop a statistical model, which constructs functional fingerprints predicting compound concentrations in plasma post inhalation. Predicted functional fingerprints for three cannabis bioactive compounds were constructed and biomatched against previously reported physiological effects. Results: Inhalation Tmax was predicted (r2 = 0.84) by compound volume (Vol), topological surface area (TPSA) and molecular weight (MW), whilst T. was predicted (r2 = 0.87) by molecular weight, volume and number of rotatable bonds (nrot). The resulting inhalation absorption prediction (IAP) model was achieved by combining Tmax and T. predictions. The IAP model was applied to cannabis metabolites which accurately predicted decay functions in-vivo and biomatching with associated physiological effects. Conclusion: The IAP model was applied successfully to cannabis phytochemicals to explore the pharmacokinetics underpinning their medicinal effects. This study demonstrates the utility of the IAP model and highlights its applicability during the investigation of medicinal plants and their modes of action.


Inhaled Cannabis Suppresses Chemotherapy-Induced Neuropathic Nociception by Decoupling the Raphe Nucleus: A Functional Imaging Study in Rats
Ilayda Alkislar, Alison R. Miller, Andrea G. Hohmann, Aymen H. Sadaka, Xuezhu Cai, Praveen Kulkarni, Craig Ferris
Biological Psychiatry: Cognitive Neuroscience and Neuroimaging 2020
DOI: 10.1016/j.bpsc.2020.11.015
Background Efficacy of inhaled cannabis for treating pain is controversial. Effective treatment for chemotherapy-induced neuropathy represents an unmet medical need. We hypothesized that cannabis reduces neuropathic pain by reducing functional coupling in the raphe nuclei. Methods We assessed the impact of inhalation of vaporized cannabis plant (containing 10.3% ??-tetrahydrocannabinol (THC)/0.05% cannabidiol (CBD)) or placebo cannabis on brain resting state BOLD functional connectivity and pain behavior induced by paclitaxel in rats. Rats received paclitaxel to produce chemotherapy-induced peripheral neuropathy (CIPN) or its vehicle. Behavioral and imaging studies were performed after neuropathy was established and stable. Images were registered to, and analyzed, using a 3D MRI rat atlas providing site-specific data on over 168 different brain areas. Results Prior to vaporization, paclitaxel produced cold allodynia. Inhaled vaporized cannabis increased cold withdrawal latencies relative to pre-vaporization or placebo cannabis, consistent with THC-induced antinociception. In paclitaxel-treated rats, the midbrain serotonergic system, comprising the dorsal and median raphe, showed hyperconnectivity to cortical, brainstem, and hippocampal areas, consistent with nociceptive processing. Inhalation of vaporized cannabis uncoupled paclitaxel-induced hyperconnectivity patterns. No such changes in connectivity or cold responsiveness were observed following placebo cannabis vaporization. Conclusion Inhaled vaporized cannabis plant uncoupled brain resting state connectivity in the raphe nuclei, normalizing paclitaxel-induced hyperconnectivity to levels observed in vehicle-treated rats. Inhaled vaporized cannabis produced antinociception in both paclitaxel- and vehicle-treated rats. Our studies elucidate neural circuitry implicated in the therapeutic effects of THC and support a role for functional imaging studies in animals in guiding indications for future clinical trials.

Inhaled medicinal cannabis and the imunocompromised patient.
Ruchlemer, R., Amit-Kohn, M., Raveh, D., & Hanuš, L.
Supportive Care in Cancer, 23(3), 819–822. (2014).
doi:10.1007/s00520-014-2429-3
Medicinal cannabis is an invaluable adjunct therapy for pain relief, nausea, anorexia, and mood modification in cancer patients and is available as cookies or cakes, as sublingual drops, as a vaporized mist, or for smoking. However, as with every herb, various microorganisms are carried on its leaves and flowers which when inhaled could expose the user, in particular immunocompromised patients, to the risk of opportunistic lung infections, primarily from inhaled molds. The objective of this study was to identify the safest way of using medicinal cannabis in immunosuppressed patients by finding the optimal method of sterilization with minimal loss of activity of cannabis. We describe the results of culturing the cannabis herb, three methods of sterilization, and the measured loss of a main cannabinoid compound activity. Systematic sterilization of medicinal cannabis can eliminate the risk of fatal opportunistic infections associated with cannabis among patients at risk.

Inhibition of Carboxylesterase 1 by Major Cannabinoids and Selected Metabolites.
Qian, Y., Wang, X., & Markowitz, J. S.
Drug Metabolism and Disposition, dmd.(2019).
doi:10.1124/dmd.118.086074
The escalating use of medical cannabis and significant recreational use of cannabis in recent years has led to higher potential for metabolic interactions between cannabis or one or more of its components and concurrently used medications. Although there have been a significant number of in vitro and in vivo assessments of the effects of cannabis on cytochrome P450 and UDP-glucuronosyltransferase enzyme systems, there is limited information regarding the effects of cannabis on the major hepatic esterase, carboxylesterase 1 (CES1). In this study, we investigated the in vitro inhibitory effects of the individual major cannabinoids and metabolites ?9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-nor-?9-tetrahydrocannabinol-carboxylic acid (THC-COOH), and 11-Hydroxy-?9-tetrahydrocannabinol (11-OH-THC) on CES1 activity. S9 fractions from human embryonic kidney (HEK 293) cells stably expressing CES1 were utilized in the assessment of cannabinoid inhibitory effects. THC, CBD, and
CBN each exhibited substantial inhibitory potency, and were further studied to determine their mechanism of inhibition and kinetic parameters. The inhibition of CES1 by THC, CBD, and CBN was reversible and appears to proceed through a mixed competitive-noncompetitive mechanism. The Ki values for THC, CBD, and CBN inhibition were 0.541, 0.974, and 0.263 µM (0.170, 0.306, and 0.0817 µg/ml), respectively. Inhibition potency was increased when THC, CBD, and CBN were combined. Compared to the potential unbound plasma concentrations attainable clinically, the Ki values suggest a potential for clinically significant inhibition of CES1 by THC and CBD. CBN however, is expected to have limited impact on CES1. Carefully designed clinical studies are warranted to establish the clinical significance of these in vitro findings.

Inhibition of endocannabinoid neuronal uptake and hydrolysis as strategies for developing anxiolytic drugs
Luara A. Batistaa, Pedro H. Gobirab, Thercia G. Vianab, Daniele C. Aguiarc and Fabricio A. Moreira Behavioural Pharmacology 2014, 25:425–433
https://d1wqtxts1xzle7.cloudfront.ne.. .A.Review.Behav. Pharmacol.2014.pdf DOI: 10.1097/FBP.0000000000000073 The endocannabinoid system comprises the CB1 and CB2 receptors (the targets of the Cannabis sativa compound delta-9-tetrahydrocannabinol), the endogenous ligands (endocannabinoids) arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, their synthesizing machinery and membrane transport system, and the hydrolyzing enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The endocannabinoids may act on demand to confer protection against aversive stimuli, which suggests that increasing their brain levels may represent an approach for treatment of anxiety-related disorders. Thus, this article reviews the profile of endocannabinoid reuptake and hydrolysis inhibitors in experimental tests predictive of anxiolytic activity. The FAAH inhibitors and the blockers of anandamide transport, in contrast to direct CB1 receptor agonists, induce anxiolytic effects at doses that do not interfere with motor activity. MAGL inhibitors also reduce anxiety-like behavior, although they are more likely to impair motor activity. Regarding their mechanisms, increasing anandamide levels induce responses mediated by the CB1 receptor and occluded by the transient receptor potential vanilloid type-1 channels, whereas the effects of increasing 2-arachidonoyl glycerol depend on both CB1 and CB2 receptors. Their neuroanatomical targets include various structures related to anxiety and fear responses. Understanding the pharmacological properties of FAAH and MAGL inhibitors may contribute toward the development of new anxiolytic interventions based on the endocannabinoid system.

Inhibition of sodium conductance by cannabigerol contributes to a reduction of dorsal root ganglion neuron excitability
Mohammad-Reza Ghovanloo, Mark Estacion, Grant P Higerd, Peng Zhao, Sulayman Dib-Hajj, Stephen G Waxman
Br J Pharmacol . 2022 Mar 16.
DOI: 10.1111/bph.15833https://bpspubs.onlinelibrary.wiley... .1111/bph.15833
Background and purpose:
Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor for ∆9-tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10 μM) blocks voltage-gated sodium (Nav) currents in CNS neurons; however, the underlying mechanism is not well-understood. Genetic and functional studies have validated Nav1.7 as an opportune target for analgesic drug development. The effects of CBG on Nav1.7 channels, which may contribute to its analgesic properties, have not been previously investigated.
Experimental approach: To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling.
Key results: We found that CBG is a ~10-fold state-dependent Nav inhibitor (KI -KR : ~2-20 μM) with an average Hill-slope of ~2. We determined that at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation; however, as the concentration is increased, CBG also induces a hyperpolarizing shift in half-voltage of inactivation. Our modeling and multielectrode array recordings suggest that CBG attenuates DRG excitability.
Conclusions and implications: Inhibition of Nav1.7 in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav1.7 channels are inactivated at DRG resting membrane potential, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav1.7 compared to other Navs (via Gmax block).
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Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites
Shamema Nasrin, Christy J. W. Watson, Keti Bardhi, Gabriela Fort, Gang Chen, and Philip Lazarus
Drug Metab Dispos 49:1081–1089, December 2021
https://doi.org/10.1124/dmd.121.000530
https://dmd.aspetjournals.org/conten.../1081.full.pdf
The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids D9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 mM, 0.22 ± 0.045 mM, 0.40 ± 0.10 mM, and 0.82 ± 0.15 mM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 lM and 0.51 ± 0.063 lM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function.

Innovative Clinical-Organizational Model to Ensure Appropriateness and Quality in the Management of Medical Cannabis: An Italian Regional Case
Eleonora Russo, Clara Cannas, Maria Susanna Rivetti, Carla Villa and Barbara Rebesco
Healthcare 2021, 9(11), 1425;
DOI: 10.3390/healthcare9111425
https://www.ncbi.nlm.nih.gov/pmc/art...e-09-01425.pdf
This work focuses on the clinical-organizational model implemented in an Italian region (Liguria) to streamline the access procedures to galenic cannabis preparations. The competent local health care authority that takes care of tracing a virtuous path to obtain common, uniform and shared protocols and ensure high standards of care is A.Li.Sa. (Azienda Ligure Sanitaria), a public organization with the function of coordination, direction and governance of the health care in the regional hospitals and health facilities. To this purpose, different working groups and a board meeting have been set up with the main role to define and develop technical standards to be applied to the prescription, preparation and dispensing of pharmaceutical forms based on therapeutic cannabis. In particular, the galenic preparations provided by the Italian Ministry of Health, described in detail in the regional standard operating protocols, are described and discussed. Moreover, the most significant data monitored from 2018 to 2020 and collected by hospitals and the evaluation of those derived from local pharmacies and health facilities are presented, discussed and compared in regards to their adherence and coherence with the Italian Institute of Health (ISS) data.

Innovative methods for the preparation of medical Cannabis oils with a high content in both cannabinoids and terpenes
Ternelli, M., Brighenti, V., Anceschi, L., Poto, M., Bertelli, D., Licata, M., & Pellati, F.
Journal of Pharmaceutical and Biomedical Analysis, 2020).
doi:10.1016/j.jpba.2020.113296
Cannabis-based medications are being increasingly used for the treatment of different clinical conditions. Among all galenic products, olive oil extracts from medical Cannabis are the most prescribed ones for their ease of preparation and usage. A great variety of methods have been
described so far for the preparation of medical Cannabis oils to reach a high yield of ?9-tetrahydrocannabinol (?9-THC), but poor attention has been paid to the preservation of the terpene fraction from the plant, which may contribute to the overall bioactivity of the extracts. In this context, the present study was aimed at the chemical characterization of different medical Cannabis oils prepared by following both innovative and existing extraction protocols, with particular attention to both cannabinoids and terpenes, in order to set up a suitable method to obtain an extract rich in both chemical classes. In particular, six different extraction procedures were followed, based on different techniques, of which all but one included a decarboxylation of the plant material. The profile of cannabinoids was studied in detail by means of HPLC-ESI-MS/MS, while terpenes were characterized by means both GC-MS and GC-FID techniques coupled with solid-phase microextraction operated in the head-space mode (HS-SPME). An innovative method that is based on the extraction of the oil by dynamic maceration at room temperature from plant inflorescences, which were partially decarboxylated in a closed system at a moderate temperature and partially pre-extracted with ethanol, produced similar yields of bioactive compounds as that obtained by using a microwave-assisted distillation of the essential oil from the plant material, in combination with a maceration extraction of the oil from the residue. Both these new methods provided a higher efficiency over already existing extraction procedures of medical Cannabis oils and they can be applied to obtain a product with a high therapeutic value.

Innovative Strategies for Enhancing Topical and Transdermal Drug Delivery
D.I.J. Morrow, P.A. McCarron, A.D. Woolfson and R.F. Donnelly*
The Open Drug Delivery Journal, 2007, 1, 36-59
1874-1266/07 2007 Bentham Science Publishers Ltd.
DOI: 10.2174/1874126600701010036
School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
Abstact: Historically, the skin was thought to be a simple homogenous barrier. However, it is now known to be a highly
specialised organ, and plays a key role in homeostasis. The protective properties of the skin are provided by the outermost
layer, the epidermis, which safeguards against chemical, microbial, and physical attack. The exceptional barrier properties
of the skin result in it being a challenging route for the delivery of therapeutic agents. This article reviews strategies developed
to enhance the skin penetration of drugs, ranging from conventional approaches, for example the use of chemical
penetration enhancers to those in early-stage development, such as microscissioning.

Insomnia treatment: a new multitasking natural compound based on melatonin and cannabis extracts
G Palmieri, M Vadalà, V Corazzari, B Palmieri
Clin Ter 7;173(1):91-96
doi: 10.7417/CT.2022.2399
https://pubmed.ncbi.nlm.nih.gov/35147654/
Objectives. Our main aim was to investigate the short-term thera- peutic effects, safety/tolerability of natural compound, composed of melatonin (1,5 mg) and cannabis extracts (2.5 mg CBD) in patients with sleep disorders
Methods. In this spontaneous, anecdotal, retrospective, “compas- sionate-use,” observational, open-label study, 20 patients (age 43-96 years) were appealed to our “Second Opinion Medical Consulting Net- work” (Modena, Italy), because of a variable pattern of sleep disorders and anxiety and were instructed to take sublingually the compound (20 drops) overnight for 3 months of treatment. Tolerability and adverse effects were assessed monthly during the treatment period through direct contact (email or telephone) or visit if required.
Results. PSQUI and HAM-A scores evidenced reduction in mood alterations, including anxiety, panic, paranoia, depression (P < 0.03), in pain (P < 0.02) and good general health perceptions.

Conclusions. These data suggest that the formula CBD-melatonin could be competitive with the classic hypnotic synthetic drugs, the antioxidant activity of melatonin offers a further benefit to the brain network, restoring the biological clock functions, while CBD, redu- cing chronic pain perception, helps to complete the neuromuscular relaxation and to relieve anxiety fulfilling a very balanced sensation of wellbeing during the sleep

https://www.imc-nederland.nl/
Institute of Medicinal Cannabis Netherlands was founded in 2021 with the following 3 goals
Information and Education To promote that the treatment of patients who (could) benefit from the use of medicinal cannabis takes place in a responsible manner and under medical supervision.
Coordination Research Promoting coordination and collaboration between partners in scientific research with and into medicinal cannabis and cannabinoids.
Advocacy Representing the interests of persons and parties in the Netherlands who are involved in the pharmaceutical chain with medicinal cannabis and cannabinoids intended for use by patients and research into this.

Interindividual Variation in the Pharmacokinetics
of ?9-Tetrahydrocannabinol as Related to Genetic Polymorphisms in CYP2C9
C Sachse-Seeboth, J Pfeil, D Sehrt, I Meineke, M Tzvetkov, E Bruns, W Poser, SV Vormfelde and J Brockmöller
Clinical pharmacology & Therapeutics | VOLUME 85 NUMBER 3 | MARCH 2009
doi:10.1038/clpt.2008.213
The impact of the CYP2C9 polymorphism on the pharmacokinetics of orally administered ?9-tetrahydrocannabinol (THC) was studied in 43 healthy volunteers. THC pharmacokinetics did not differ by CYP2C9*2 allele status.
However, the median area under the curve of THC was threefold higher and that of 11-nor-9-carboxy-9-tetrahydrocannabinol was 70% lower in CYP2C9*3/*3 homozygotes than in CYP2C9*1/*1 homozygotes. CYP2C9*3 carriers also showed a trend toward increased sedation following administration of THC. Therefore, the CYP2C9*3 variant may influence both the therapeutic and adverse effects of THC

Interplay between synaptic endocannabinoid signaling and metaplasticity in neuronal circuit function and dysfunction
Miriam Melis, Barbara Greco and Raffaella Tonini
European Journal of Neuroscience, Vol. 39, pp. 1189–1201, 2014
doi:10.1111/ejn.12501
Synaptic neuromodulation acts across different functional domains to regulate cognitive processing and behavior. Recent challenges are related to elucidating the molecular and cellular mechanisms through which neuromodulatory pathways act on multiple time scales to signal state-dependent contingencies at the synaptic level or to stabilise synaptic connections during behavior. Here, we present a framework with the synaptic neuromodulators endocannabinoids (eCBs) as key players in dynamic synaptic changes. Modulation of various molecular components of the eCB pathway yields interconnected functional activation states of eCB signaling (prior, tonic, and persistent), which may contribute to metaplastic control of synaptic and behavioral functions in health and disease. The emerging picture supports aberrant metaplasticity as a contributor to cognitive dysfunction associated with several pathological states in which eCB signaling, or other neuromodulatory pathways, are deregulated.

Interventions for Cannabis Use Disorder
Ken C. Winters, Joel Mader, Alan J. Budney, Catherine Stanger, Ashley A. Knapp, Denise D. Walker
Current Opinion in Psychology (Dec 2020)
DOI: 10.1016/j.copsyc.2020.11.002
How to treat adults and adolescents with a Cannabis Use Disorder is a burgeoning research area. This article reviews the empirical literature pertaining to the several psychosocial approaches (cognitive-behavior therapy, motivational enhancement and contingency management), all of which are associated with favorable outcomes. We also review the emerging research on the use of pharmacotherapy, brief interventions
and technology-delivered interventions, and conclude with an overview of future research needs.

Introduction to Cannabis Use Disorders
Ivan D. Montoya, Susan R. B. Weiss
In book: Cannabis Use Disorders January 2019
DOI: 10.1007/978-3-319-90365-1_1
Cannabis is a natural product that, when used chronically, has been associated with the development of cannabis use disorder. This disorder, as described in the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) of the American Psychiatric Association (2013. Diagnostic and statistical manual of mental disorders, 5th edn. American Psychiatric Association, Washington, DC), is clinically characterized by the inability to stop using cannabis despite psychosocial/medical problems, the presence of craving, the need to use larger amounts to obtain the same effect (tolerance), and/or the onset of symptoms when its use is stopped (withdrawal). The current increase in the frequency of cannabis use, the availability and use of cannabis products with high concentration of the psychoactive ingredient tetrahydrocannabinol (THC), and the presence in the markets of highly potent synthetic cannabinoids may result in an increased risk of CUD. Recent studies have provided a wealth of information about the endocannabinoid system, the effects of cannabinoids, and the brain mechanisms of CUD. These new discoveries are allowing the identification of novel pharmacological targets and medications that may affect the endocannabinoid system and may be useful for the treatment of CUD and other medical conditions. The purpose of this book is to provide an overview of the epidemiology, neurobiological basis, developmental aspects, and clinical characteristics of CUD, including its pharmacological and non-pharmacological therapeutic approaches.

Investigating the Causal Effect of Cannabis Use on Cognitive Function with a Quasi-Experimental Co-Twin Design.
Ross, J. M., Ellingson, J. M., Rhee, S. H., Hewitt, J. K., Corley, R. P., Lessem, J. M., & Friedman, N. P.
Drug and Alcohol Dependence, 107712.(2019).
doi:10.1016/j.drugalcdep.2019.107712
Background: It is unclear whether cannabis use causes cognitive decline; several studies show an association between cannabis use and cognitive decline, but quasi-experimental twin studies have found little support for a causal effect. Here, we evaluate the association of cannabis use with general cognitive ability and executive functions (EFs) while controlling for genetic and shared environmental confounds in a longitudinal twin study.
Methods: We first examined the phenotypic associations between cannabis initiation, frequency, and use disorder with cognitive abilities, while also controlling for pre-use general cognitive ability and other substance involvement. We tested the concurrent association between the cannabis use variables and cognitive abilities in late adolescence and young adulthood and the longitudinal association between cannabis use variables during adolescence and young adulthood cognitive abilities. Next, we used multilevel models to test whether these relations reflect between- and/or withintwin pair associations.
Results: Phenotypically, cannabis use was related to poorer cognitive functioning, although most associations were negligible after accounting for other substance use. Nevertheless, there were few significant within-family twin-specific associations, except that age 17 cannabis frequency was associated with worse age 23 Common EF and general cognitive ability. Conclusions: We found little support for a potential causal effect of cannabis use on cognition, consistent with previous twin studies. Results suggest that cannabis use may not cause decline in cognitive ability among a normative sample of cannabis users.

Involvement of Cannabinoids in Cellular Proliferation
María L López-Rodríguez, Alma Viso, Silvia Ortega-Gutierrez, Inés Díaz-Laviada
Mini Reviews in Medicinal Chemistry 5(1):97-106 February 2005
DOI: 10.2174/1389557053402819
https://www.researchgate.net/publication/8092132_Involvement_of_Cannabi noids_in_Cellular_Proliferatio n
The endogenous canabinoid system (ECS) is involved in the regulation of an important number of central and peripheral physiological effects. Among all these functions, the control of the cellular proliferation has become a focus of major attention as opening new therapeutic possibilities for the use of cannabinoids as potential antitumor agents. The capacity of endogenous and synthetic cannabinoids to induce apoptosis of different tumoral cells in culture and in vivo, the mechanism underlying and the potential therapeutic applications are discussed in this review.

Investigating the Therapeutic Mechanism of Cannabidiol in a Human Induced Pluripotent Stem Call (iPSC)-Based Cellular Model of Dravet Syndrome.
Sun, Y., & Dolmetsch, R. E. (2019).
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXIII
doi:10.1101/sqb.2018.83.038174
Dravet syndrome is an infantile epileptic encephalopathy primarily caused by loss-of-function variants of the gene SCN1A. Standard treatment regimens have very limited efficacy to combat the life-threatening seizures in Dravet syndrome or the behavioral-cognitive comorbidities of the disease. Recently there has been encouraging progress in developing new treatments for this disorder. One of the clinical advances is cannabidiol (CBD), a compound naturally found in cannabis and shown to further reduce convulsive seizures in patients when used together with existing drug regimens. Like many other natural products, the exact therapeutic mechanism of CBD remains undefined. Previously we have established a human cellular model of Dravet syndrome by differentiating patient-derived induced pluripotent stem cells (iPSCs) into telencephalic inhibitory and excitatory neurons. Here we have applied this model to investigate the antiepileptic mechanism(s) of CBD at the cellular level. We first determined the effect of escalating the concentrations of CBD on neuronal excitability, using primary culture of rat cortical neurons. We found modulatory effects on excitability at submicromolar concentrations and toxic effects at high concentrations (15 µM). We then tested CBD at 50 nM, a concentration that corresponds to the estimated human clinical exposure, in telencephalic neurons derived from a patient iPSC line and control cell line H9. This 50 nM of CBD increased the excitability of inhibitory neurons but decreased the excitability of excitatory neurons, without changing the amplitude of sodium currents in either cell type. Our findings suggest a cell type–dependent mechanism for the therapeutic action of CBD in Dravet syndrome that is independent of sodium channel activity

Investigation of hand functions in individuals with cannabis and its derivatives use disorder

Bekir Güçlü, Baki Umut Tuğay, Fatih Özden, Esra Erğun Keşli & Cemal Onur Noyan

Irish Journal of Medical Science (1971 -) (2022)

https://link.springer.com/article/10.1007/s11845-022-03006-w

Background​

The present study was aimed to compare the grip/pinch strengths and manual dexterity of individuals with and without the use of cannabis and its derivatives.

Methods​

A cross-sectional prospective study was conducted with 66 individuals, including 33 cases with the use of cannabis (and its derivatives) and 33 age- and sex-matched controls. Grip and pinch strengths were evaluated with a dynamometer. The Nine-Hole Peg Test (9HPT), Minnesota Manual Dexterity Test (MMDT), and Michigan Hand Outcomes Questionnaire (MHQ) were used to assess the hand function.

Results​

The hand grip strength and dominant hand 2-point pinch (2PP) grip strength were less in individuals with substance use disorder (SUD) (p < 0.05). The 9HPT duration of the SUD patients was higher (p < 0.05). On the other hand, the MMDT insertion and rotation test results were different between the groups (p < 0.05). Grip strength was related with the MMDT insertion and rotation tests (r =  −0.411 to −0.480). There was significant correlation between grip strength with dominant hand 9HPT (r =  −0.370) and between dominant hand 3-point pinch (3PP) strength with MMDT insertion (r =  −0.378). In addition, dominant hand 2PP strength was correlated with overall hand function of MHQ (r = 0.382).

Conclusion​

The individuals with cannabis use disorder showed reduced grip strength on both sides and decreased 2PP strength on the dominant side compared to healthy individuals. In addition, there is a decrease in the hand skills of individuals with cannabis use disorder. Decreased grip strength of individuals with cannabis use disorder affected their hand skills negatively.
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Involvement of dopamine receptor in the actions of non-psychoactive phytocannabinoids
Sarah H. Shrader, Yong-Guang Tong, Maxwell B. Duff, Jonathan H. Freedman, Zhao-Hui Song
Biochemical and Biophysical Research Communications (2020)
DOI: 10.1016/j.bbrc.2020.10.021
Nematode Caenorhabditis elegans (C. elegans) exhibited a vigorous swimming behavior in liquid medium. Addition of dopamine inhibited the swimming behavior, causing paralysis in 65% of wild-type nematodes. Interestingly, phytocannabinoids cannabidiol (CBD) or cannabidivarin (CBDV), caused paralysis in 40% of the animals. Knockout of DOP-3, the dopamine D2-like receptor critical for locomotor behavior, eliminated the paralysis induced by dopamine, CBD, and CBDV. In contrast, both CBD and CBDV caused paralysis in animals lacking CAT-2, an enzyme necessary for dopamine synthesis. Co-administration of dopamine with either CBD or CBDV caused paralysis similar to that of either phytocannabinoid treatment alone. These data support the notion that CBD and CBDV act as functional partial agonists on dopamine D2-like receptors in vivo. The discovery that dopamine receptor is involved in the actions of phytocannabinoids moves a significant step toward our understanding of the mechanisms for medical uses of cannabis in the treatment of neurological and psychiatric disorders.

Irritable Bowel Syndrome: Manipulating the Endocannabinoid System as First-Line Treatment .
Viola Brugnatelli, Fabio Turco, Ulderico Freo and Gastone Zanette
Front. Neurosci. 14:371.
doi: 10.3389/fnins.2020.00371
Irritable Bowel Syndrome (IBS) is a functional disorder characterized by abdominal pain, spasms, and altered bowel movements, either predominantly diarrhea (IBS-D), or predominantly constipation (IBS-C), or alternating between those states (Saha, 2014). In the Western world it affects the 10–15% of the population (Canavan et al., 2014). IBS represents a visceral
hypersensitivity, with features of gastrointestinal (GI) allodynia and hyperalgesia. Considered a life-long condition, it is clear that significant gastrointestinal insults, such as food poisoning or antibiotic administration, may generate attacks that persist, often indefinitely. Attacks are associated with anxiety and depression, but controversy carries on to which incites the other (Saha, 2014). It is possible that some patients may develop a vicious cycle of worsening physical and psychological symptoms (Jones et al., 2013, 2017). Currently, IBS sufferers are prescribed opioids, anticholinergics, and antidepressants, however with quite suboptimal results. Other compounds have been formulated to interact with serotoninergic circuitry, nevertheless these have been withdrawn from certain markets due to association with ischemic colitis (alosetron, cilansetron) and cardiovascular events (tegaserod),
leaving, de facto, an urgent clinical need (Ford et al., 2014; Lexicomp Online, 2017). The Endocannabinoid System (ECS) is known to modulate several functions, including mood, anxiety, and memory retrieval of traumatic events and it directly coordinates GI propulsion, secretion, inflammation, and nociception, providing a rationale for agents capable of interacting with the ECS as treatment candidates for IBS (Russo, 2016).

Is Early Exposure to Cannabis Associated with Bipolar disorder? Results from a Finnish Birth-Cohort Study
Alexander Denissoff, Antti Mustonen, Anni-Emilia Alakokkare, James G Scott, Musa B Sami, Jouko Miettunen, Solja Niemelä
Addiction. 2022 Mar 21.
doi: 10.1111/add.15881.
Background and aims: There are few longitudinal studies assessing the association of cannabis use and subsequent onset of bipolar disorder. We aimed to measure the association between early cannabis exposure and subsequent bipolar disorder.
Design, setting, and participants: Observational study linking a sample from the Northern Finland Birth Cohort 1986 (n=6,325) to nationwide register data to examine the association of life-time cannabis exposure at age 15/16 years and subsequent bipolar disorder until age 33 (until the end of 2018). 6,325 individuals (48.8% males) were included in the analysis.
Measurements: Cannabis exposure was measured via self-report. Bipolar disorder was measured via bipolar disorder-related diagnostic codes (ICD-10: F30.xx, F31.xx) collected from the Care Register for Health Care 2001-2018, the Register of Primary Health Care Visits 2011 - 2018, the medication reimbursement register of the Social Insurance Institution of Finland 2001 - 2005 and the disability pensions of the Finnish Center for Pensions 2001 - 2016. Potential confounders included demographic characteristics, parental psychiatric disorders, emotional and behavioral problems and other substance use.
Findings: 352 adolescents (5.6 %) reported any cannabis use until the age of 15-16 years. Of the whole sample, 66 (1.0 %) were diagnosed with bipolar disorder. Adolescent cannabis use was associated with bipolar disorder (hazard ratio

=3.46; 95% confidence interval [CI] 1.81-6.61). This association remained statistically significant after adjusting for sex, family structure, and parental psychiatric disorders (HR =3.00; 95% CI 1.47-6.13) and after further adjusting for adolescent emotional and behavioral problems (HR =2.34; 95% CI 1.11-4.94). Further adjustments for frequent alcohol intoxications, daily smoking and lifetime illicit drug use attenuated the associations to statistically non-significant.
Conclusions: In Finland, the positive association between early cannabis exposure and subsequent development of bipolar disorder appears to be confounded by other substance use.
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Is marijuana use associated with decreased use of prescription opioids? Toxicological findings from two US national samples of drivers
Guohua Li and Stanford Chihuri
Substance Abuse Treatment, Prevention, and Policy (2020) 15:12
doi: 10.1186/s13011-020-00257-7
Background: State governments in the United States are increasingly viewing marijuana legalization as a policy option for controlling the opioid epidemic under the premise that marijuana is a less harmful substitute for opioids. The purpose of this study is to assess whether marijuana use is associated with decreased odds of prescription opioid use. Methods: A cross-sectional study design was applied to toxicological testing data from two national samples of drivers: 1) the 2011–2016 Fatality Analysis Reporting System (FARS) and 2) the 2013–2014 National Roadside Survey of Alcohol and Drug Use by Drivers (NRS). Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) estimated from multivariable logistic regression models were used to assess the associations of marijuana use with prescription opioid use and alcohol use. Results: Among the 47,602 drivers from the FARS, 15.7% tested positive for marijuana and 6.9% positive for prescription opioids. Compared with drivers testing negative for marijuana, those testing positive for marijuana were 28% more likely to test positive for prescription opioids (adjusted OR = 1.28, 95% CI = 1.15–1.42). Among the 7881 drivers from the NRS, 7.9% tested positive for marijuana and 4.5% positive for prescription opioids. Relative to drivers testing negative for marijuana, those testing positive for marijuana were twice as likely to test positive for prescription opioids (adjusted OR = 2.03, 95% CI = 1.29–3.20). In both study samples, marijuana use was associated with significantly increased odds of alcohol positivity. Conclusions: Drivers who test positive for marijuana are significantly more likely to test positive for prescription opioids. Longitudinal studies with rigorous designs and toxicological testing data are needed to further address the substitution hypothesis between marijuana and prescription opioids

Is Recovery from Cannabis Dependence Possible? Factors that Help or Hinder Recovery in a National Sample of Canadians with a History of Cannabis Dependence.
Fuller-Thomson, E., Jayanthikumar, J., Redmond, M. L. & Agbeyaka, S.
Advances in Preventive Medicine, 2020, 1–9.(2020).
doi:10.1155/2020/9618398
Objectives. To identify among Canadian adults who have ever been dependent upon cannabis, the prevalence of risk and protective factorsassociated with (1) cannabis remission, (2) the absence of psychiatric disorders or addictions in the past year (APD), and (3) positive mental health (PMH). Method. Data from Statistics Canada’s nationally representative 2012 Canadian Community Health Survey-Mental Health ( = 20, 777, of whom 336 have a history of cannabis dependence) was used. Chi-square tests and logistic regression analyses were conducted. ¬e World Health Organization Composite International Diagnostic Interview (WHO-CIDI) measures were used to determine lifetime cannabis dependence, past-year remission from cannabis depression, and the absence of psychiatric disorders in the past year (APD) (i.e., no suicidal ideation, depressive episodes, anxiety disorders, bipolar disorders, or any substance dependence). PMH is comprised of three factors: APD, happiness or life satisfaction and social and psychological well-being. Results. Among those with a history of cannabis dependence, 72% were in remission from cannabis dependence. Although 53% were free of major psychiatric
disorders and any substance dependence and 43% of respondents were in PMH, these percentages were dramatically lower than those without a history of cannabis dependence (92% and 74%, respectively). Positive outcomes were more common among women, older respondents, those with higher levels of social support, and those who had never had major depressive disorder or generalized anxiety disorder. Conclusion. Although many Canadians with a history of cannabis dependence achieve remission and a large minority are truly resilient and achieve PMH, many are failing to thrive. Targeted outreach is warranted for the most vulnerable individuals with a history of cannabis dependence (e.g., men, younger respondents, those with low social support and a history of mental illness).

Is the Adolescent Brain at Greater Vulnerability to the Effects of Cannabis?
Blest-Hopley, G., Colizzi, M., Giampietro, V., & Bhattacharyya, S.
A Narrative Review of the Evidence. Frontiers in Psychiatry, 11.(2020).
doi:10.3389/fpsyt.2020.00859
Cannabis use during the critical neurodevelopmental period of adolescence, may lead to brain structural, functional, and histological alterations that may underpin some of the longer-term behavioral and psychological harms associated with it. The endocannabinoid system performs a key regulatory and homeostatic role, that undergoes developmental changes during adolescence making it potentially more susceptible to the effects of exposure to cannabis during adolescence. Here, we synthesize evidence from human studies of adolescent cannabis users showing alterations in cognitive performance as well as in brain structure and function with relevant preclinical evidence to summarize the current state of knowledge. We also focus on the limited evidence that speaks to the hypothesis that cannabis use during adolescence, may pose a greater risk than use during adulthood, identify gaps in current evidence and suggest directions for new research. Existing literature is consistent with the association of cannabis use during adolescence and neurological changes. Adolescence cannabis users show altered functional connectivity within known functional circuits, that may underlie inefficient recruitment of brain regions, as largely increased functional activation has been observed compared to controls. This disruption in some cases may contribute to the development of adverse mental health conditions; increasing the chances or accelerating the onset, of their development. Preclinical evidence, further supports disruption from cannabis use being specific to the developmental period. Future studies are required to better investigate adolescent cannabis use with more accuracy using better defined groups or longitudinal studies and examine the permanency of these changes following caseation of use. Furthermore, research is required to identify heritable risk factors to cannabis use. There is a need for caution when considering the therapeutic potential of cannabis for adolescence and particularly in public discourse leading to potential trivialization of possible harm from cannabis use in adolescence. Current evidence indicates that adolescence is a sensitive period during which cannabis use may result in adverse neurocognitive effects that appear to show a level of permanency into adulthood.

Is the Urine Cannabinoid Level Measured via a Commercial Point-of-Care Semiquantitative Immunoassay a Cannabis Withdrawal Syndrome Severity Predictor?
Claus, B. B., Specka, M., McAnally, H., Scherbaum, N., Schifano, F., & Bonnet, U.
Frontiers in Psychiatry, 11.(2020).
doi:10.3389/fpsyt.2020.598150
Background: For cannabis-dependent subjects, the relationship between cannabis withdrawal syndrome (CWS) severity and the urine cannabinoid concentrations are unclear; we investigated this using a commercial point-of-care (POC) enzyme immunoassay detecting 11-nor-9-carboxy-Delta-9-tetrahydrocannabinol (THC-COOH). Methods: Observational study of 78 adult chronic cannabis-dependent subjects assessed over a 24-day inpatient detoxification treatment, with 13 serial measurement days. Repeated Measures Correlation and Multilevel Linear Models were employed. Results: Absolute urinary THC-COOH levels significantly correlated with Marijuana Withdrawal Checklist (MWC) scores across the entire study duration (r = 0.248; p < 0.001). Correlation between serial creatinine-adjusted THC-COOH ratios and serial MWC scores emerged as significant only in the sample with higher MWC scores (>11 points) at admission (n = 21; r = 0.247; p = 0.002). The aforementioned significant relationships have persisted when replacing the absolute THC-COOH-levels with the (relative) dayto-day change in urinary THC-COOH levels. MWC scores were significantly correlated with the Clinical Global Impression-Severity (CGI-S; r = 0.812; p < 0.001). Females showed a significantly slower decline in urine THC-COOH levels and prolonged CWS course characterized by substantial illness severity (per CGI-S), occurring in nearly 30% of cases
Conclusion: Urine cannabinoid levels (THC-COOH) determined by POC assay significantly predicted CWS severity (moderate correlation), guiding detoxification treatment duration. In patients with MWC > 11 points upon admission, creatinine-adjusted THC-COOH ratios also significantly predicted CWS severity—again with moderate effect size. Females showed prolonged urinary THC-COOH elimination and cannabis withdrawal.

Is there a specific ‘cannabis psychosis’?
Wayne Hall and Louisa Degenhard
in book: Marijuana and MadnessPsychiatry and Neurobiology , Chapter 6, pp. 89 – 100 (2004)
DOI: 10.1017/CBO9780511543630.008
There are good reasons to be concerned about the possibility that cannabis use may be a cause of psychotic disorders. Psychoses are serious and disabling disorders (Bromet et al., 1995). Cannabis is widely used during late adolescence in many developed societies (Hall et al., 1999), and high doses of tetrahydrocannabinol (THC) – the psychoactive substance in cannabis – have been reported to produce psychotic symptoms such as visual and auditory hallucinations, delusional ideas and thought disorder in normal volunteers (Hall et al., 2001; and see Chapter 5). There are a number of hypotheses about the relationship between cannabis use and psychosis that need to be distinguished (Thornicroft, 1990). The strongest hypothesis in causal terms is that heavy cannabis use causes a specific ‘cannabis psychosis’. It assumes that these psychoses would not occur in the absence of cannabis use, and that the causal role of cannabis can be inferred from the symptoms and their relationship to cannabis use; that is, they are preceded by heavy cannabis use and remit after abstinence. It also assumes that cannabis psychoses are qualitatively different fromother psychotic disorders. This hypothesis is the subject of this chapter. The potential role of cannabis as a causal agent for schizophrenia per se is the subject of Chapter 7.

Isolated Pauci-Immune Pulmonary Capillaritis Associated with Hydrocarbon Inhalation and Marijuana Smoking: An Unusual Case of Severe Hypoxemia
Jason S. Oh, Uni Wong, Divyansh Bajaj, and Stella E. Hines
Hindawi Case Reports in Pulmonology Volume 2020, h
DOI:10.1155/2020/1264859
We present a case report of a patient with Isolated pauci-immune pulmonary capillaritis (IPIPC). A 40-year-old male presented with acute onset severe hypoxemic respiratory failure. He had just returned home from work as a cabinetmaker, where he experienced inhalational exposure to hydrocarbons and solvents, and had smoked a marijuana cigarette. He was hypotensive, and his chest imaging showed bilateral dependent in‘ltrates. His hypoxemia made little improvement a’er conventional ventilator support and broad-spectrum antibacterial therapy and he was considered too unstable to tolerate diagnostic bronchoscopy with bronchoalveolar lavage. His laboratory evaluation initially showed microscopic hematuria which later cleared, but other tests including serologic autoimmune assessment were negative, and he did not have any traditional risk factors for vasculitis. A video-assisted thoracoscopic lung biopsy revealed diuse alveolar hemorrhage with pulmonary capillaritis on histopathology. He was diagnosed with IPIPC and initiated on immunosuppressive therapy. He was soon liberated from mechanical ventilation and improved to hospital discharge. Diuse alveolar hemorrhage from Goodpasture’s Syndrome has manifested following inhalation of hydrocarbons and following smoking. is has not previously been reported with IPIPC. Given the lack of other ‘ndings and risk factors, his IPIPC was likely associated with occupational exposures to hydrocarbons as a cabinetmaker compounded by marijuana smoking.

Isolation, Purification, and Antimicrobial Characterization of Cannabidiolic Acid and Cannabidiol from Cannabis sativa L.
Martinenghi, L. D., Jønsson, R., Lund, T., & Jenssen, H.
Biomolecules, 10(6), 900.(2020).
doi:10.3390/biom10060900
The emergence of multi?drug resistant bacteria such as methicillin?resistant Staphylococcus aureus (MRSA) causes a major threat to public health due to its limited therapeutic options. There is an urgent need for the development of new effective antimicrobial agents and alternative strategies that are effective against resistant bacteria. The parallel legalization of cannabis and its products has fueled research into its many therapeutic avenues in many countries around the world. This study aimed at the development of a reliable method for the extraction, purification, characterization, and quantification of cannabidiolic acid (CBDA) and its decarboxylated form cannabidiol (CBD) present in the fiber type Cannabis sativa L. The two compounds were extracted by ethanol, purified on a C18 sep?pack column, and the extracts were analyzed by high performance liquid chromatography coupled with ultraviolet (UV)–vis and ESI?MS (electrospray ionization mass spectrometry) detection. The antimicrobial effect of CBDA and CBD was also evaluated. CBD displayed a substantial inhibitory effect on Gram?positive bacteria with minimal inhibitory concentrations ranging from 1 to 2 ?g/mL. Time kill analysis and minimal bactericidal concentration revealed potential bactericidal activity of CBDA and CBD. While cannabinoids showed a significant antimicrobial effect on the Gram?positive S. aureus and Staphylococcus epidermidis, no activity was noticed on Gram?negative Escherichia coli and Pseudomonas aeruginosa. CBDA presented a two?fold lower antimicrobial activity than its decarboxylated form, suggesting that the antimicrobial pharmacophore of the analyzed cannabinoids falls in the ability for permeabilizing the bacterial cell membrane and acting as a detergent?like agent. A synergy test performed on MRSA with CBD and a range of antibiotics did not indicate a synergetic effect, but noteworthy no antagonist influence either. CBD and CBDA manifested low hemolytic activity on human red blood cells. Likewise, the safety of CBD toward human keratinocyte cells presents no toxicity at a concentration of up to seven?fold higher than the antibacterial minimal inhibitory concentration. Similarly, both CBD and CBDA are well tolerated by mammals, including humans, and conserve a safe value limits for blood? contacting drug development. Overall, CBD exhibited a strong antimicrobial effect against Gram? positive strains and could serve as an alternative drug for tackling MRSA

Knowledge and perspectives about the use of cannabis as a medicine: a mixed methods observational study in a cohort of New Zealand general practice patients
Oldfield K, Eathorne A, Maijers I, Beasley R, Semprini A, Braithwaite I.
N Z Med J. 2020 Sep 25;133(1522):96-111.
DOI:??????FIND
Aim: To determine what patients presenting to general practice (GP) understand about the use of cannabis as a medicine, beliefs of how this may impact their medical conditions and interactions with doctors.
Method: An in-person survey of 134 GP patients from four GP practices throughout the North Island of New Zealand undertaken from November 2018 to October 2019.
Results: Fifty-five percent of the sample were female, with 40% of all participants aged 60 years plus. Ninety-one percent of participants indicated they would use a prescribed medicinal cannabis product while 45% reported they believed it may be of some benefit to their medical condition. Of those who believed it beneficial, 71% indicated they thought it useful for pain relief. Participants indicated comfort discussing medicinal cannabis use with GPs and specialists (92% respectively); however, less than 10% had done this.
Conclusions: Just under half of patients surveyed believe that medicinal cannabis products may be helpful to their condition, and while the majority report willingness, few have discussed this with their GP or specialist. There is need for accessible, accurate information regarding the use of cannabis-based medicine for patients and doctors alike to guide the patient-doctor consultation and decrease barriers to open discussion.

Knowledge of Tetrahydrocannabinol and Cannabidiol Levels Among Cannabis Consumers in the United States and Canada
David Hammond and Samantha Goodman
Cannabis and Cannabinoid Research Volume X, Number X, 2020
https://www.liebertpub.com/doi/pdf/1.../can.2020.0092
DOI: 10.1089/can.2020.0092
Objective: This study aimed to examine consumer knowledge of tetrahydrocannabinol (THC) and cannabidiol (CBD) levels for usual cannabis products.
Methods: Data are from the International Cannabis Policy Study conducted online in August–September 2018. Respondents included 6471 past 12-month cannabis users, aged 16–65 years, recruited from the Nielsen Global Insights Consumer Panel using nonprobability methods. Respondents were recruited from Canada, which had not yet legalized nonmedical cannabis (n = 2354), and US states that had (n = 2160) and had not (n = 1957) legalized nonmedical cannabis.
Results: Participants reported descriptive THC:CBD ratios (e.g., high THC, low CBD) and numeric THC and CBD levels (mg or %) for products they usually use in each of nine product categories. Few consumers knew and were able to report the numeric THC or CBD levels of their usual cannabis products. For example, only 10% of dried herb consumers reported the THC level, approximately 30% of whom reported implausible values. A greater proportion of consumers reported a descriptive THC:CBD ratio of their usual product, ranging from 50.9% of edible users to 78.2% of orally ingested oil users. Consumers were substantially more likely to report products high in THC versus low in THC for all products except topicals and tinctures, whereas similar proportions reported using products high and low in CBD. Despite some evidence of greater knowledge in legal jurisdictions, knowledge was still low in states with legal cannabis markets.
Conclusions: Consumer knowledge of THC and CBD levels was low, with only modest differences between consumers living in jurisdictions that had and had not legalized nonmedical cannabis. The findings cast doubt on the validity of self-reported cannabinoid levels

Knowledge, Perception, and Use of Cannabis Therapy in Patients with Inflammatory Bowel Disease.
Luis A Muñiz-Camacho, Frances I Negrón-Quintana, Luis A Ramos-Burgos
P R Health Sci J 2021 Sep;40(3):110-114
doi: 10.14309/01.ajg.0000592344.36842.0d
https://journals.lww.com/ajg/Abstrac...erception,_and _Use_of_Cannabis.702.aspx
https://journals.lww.com/ajg/pages/a...px?year=2019&i ssue=10001&article=00702&type= Abstract
Objective: In 2017, the government of Puerto Rico legalized medical cannabis for several conditions including Crohn's disease (CD). There is little information about cannabis use in this population. This study aimed to develop a demographic characterization and evaluate patient perception on cannabis use for Inflammatory Bowel Disease (IBD) at the University of Puerto Rico Center for Inflammatory Bowel Diseases.
Methods: One hundred patients of ages 21 or older with a confirmed diagnosis of IBD were recruited to complete a voluntary anonymous questionnaire.
Results: 27% of the surveyed participants reported use of cannabis. Of these, 39% reported moderate knowledge and 53% reported little to no knowledge of medical cannabis. The majority did not discuss cannabis use with their physician (78%), and most saw improvement of their symptoms (68%).
Conclusion: Cannabis is frequently considered by patients as a treatment option for IBD but most have limited knowledge about its use. The low number of patients that discuss cannabis use with their physician suggests the need for physician awareness of unreported use. It should also lead to the development of strategies for patient orientation regarding the uses, properties, and expectations of cannabis as a therapy.

Labeling of Cannabidiol Products: A Public Health Perspective.
Corroon, J., MacKay, D., & Dolphin, W.
Cannabis and Cannabinoid Research. (2020). doi:10.1089/can.2019.0101
Introduction: Interest in the therapeutic use of cannabidiol (CBD) has reached a fever-pitch in recent months, as CBD-containing products appear everywhere from online retailers to grocery stores and gas stations. The widespread availability of hemp-derived CBD products is confounding given that CBD is a U.S. Food and Drug Administration (FDA)-approved drug, and thus precluded from being added to food and beverages, or included in dietary supplements. The use by manufacturers of disease-related claims on marketing materials and product labels, along with the federal legalization of hemp in December 2018, has created political pressure on FDA to promulgate regulations. Conclusions: Accurate and informative labeling of hemp and hemp-derived CBD products is an important public health issue. FDA-regulated product labels are considered an essential tool for protecting consumers and enabling informed decision-making. Untruthful or unsubstantiated health-related claims, and unallowed Drug Claims, in marketing materials and on labels of CBD products may create harm by enticing consumers to forgo more evidence-based medical interventions. Furthermore, missing or inaccurate labeling of the amount of CBD, delta-9 tetrahydrocannabinol (THC), and potentially harmful contaminants such as pesticides, naturallyoccurring yeast and mold or heavy metals may result in harm and/or lack of efficacy. Manufacturers of these products may reasonably be expected to understand and adhere to FDA regulations for labeling and marketing of food, dietary supplements and drugs, both over-the-counter (OTC) and prescription, even though FDA has interpreted federal law as excluding them from these categories. As manufacturers prepare for forthcoming regulations, a better understanding of the basic framework for FDA labeling and marketing regulations for food, dietary supplements and drugs is warranted.

Leading Companies in Cannabis Market
Project: Cannabis Market Report and Patent Landscape Analysis May 2019
Twinkle Pandagre
https://www.researchgate.net/publica...annabis_Market
Leading Companies in Cannabis Market present the comprehensive market, patent information and analysis for the existing players to diversify and new players to invest in this technology and it also presents the exponentially growing Cannabis market for medical and recreational purposes.

Letter to the Editor: Possible Drug–Drug Interactions Between Cannabinoids and Candidate COVID-19 Drugs
M. Hunter Land Laura MacNair Brian F. Thomas, Erica N. Peters, Marcel O. Bonn-Miller
Cannabis and Cannabinoid ResearchVol. 5, No. 4, (2020) Letters to the Editor
DOI: 10.1089/can.2020.0054
Cannabinoid preparations are available across the globe as regulatory agency-approved medicines, through medical cannabis programs, and as hemp-derived products. Many regions, including most provinces in Canada, have designated cannabis businesses as “essential” services during the coronavirus disease 2019 (COVID-19) pandemic, and sales of cannabis remain strong in an otherwise economically challenging time. In light of the potential increased use of cannabis and a recent surge in research to rapidly identify medications to treat COVID-19, it is critical to delineate possible pharmacokinetic (PK) and pharmacodynamic (PD) drug–drug interactions (DDIs) between cannabinoids and such experimental medications. Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are lipophilic, highly protein bound, have a large volume of distribution, a long half-life, bioaccumulate, and share common metabolic pathways within the cytochrome P450 (CYP450) family (e.g., 3A4, 2C9, and 2C19), drug transporters (e.g., breast cancer resistance protein), and plasma protein-binding substrates. Both THC and CBD have been shown to have clinically significant PK (e.g., warfarin and clobazam)1,2 and PD interactions (e.g., valproic acid).2 The severity of some cannabinoid DDIs, such as potential hepatocellular injury and sedation,2,3 further highlights the importance of early identification of possible interactions between cannabinoids and medications that may be used to treat COVID-19.

License heritable gene editing like medical cannabis.
Julian H.
Nature. 2019 Jun;570(7762):446.
DOI:10.1038/d41586-019-01971-4
https://www.nature.com/articles/d41586-019-01971-4
In my view, a moratorium on the technology would be regulatory theatre (see E. Lander et al. Nature 567, 165–168; 2019). CRISPR gene editing is cheap and easily accessible, and its practice and products are hard to detect. Like cannabis prohibition, a moratorium risks fostering a black market in unregulated and potentially harmful ‘products’, in this case heritable gene variants.

Lifetime cannabis exposure and small airway function in a population-based cohort study
Hua Shin Tan, Helena M. McAnally, Jack Dummer and Robert J. Hancox
ERJ Open Research 2022 8: 00688-2021;
DOI: 10.1183/23120541.00688-2021
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9062301/pdf/00688-2021.pdf
Background and objective The long-term effects of cannabis on small airway function remain unclear. We investigated associations between cannabis use and small airway function in a general population sample. Methods Cannabis use was ascertained at multiple ages from age 18 to 45 years and quantified as jointyears among 895 participants in the Dunedin Multidisciplinary Health and Development Study. Small airway function at ages 38 and 45 years was measured using impulse oscillometry (IOS) before and after inhalation of salbutamol. Analyses used multiple linear regression adjusting for tobacco use, body mass index and height. Longitudinal analyses of cannabis use between 38 and 45 years also adjusted for IOS at age 38 years. Results Associations between lifetime cannabis joint-years and IOS differed between men and women: in women, cannabis use was associated with pre-bronchodilator resistance at 5 Hz (R5) and 20 Hz (R20), reactance at 5 Hz, area of reactance and resonant frequency, and marginally associated with the difference between R5 and R20. Cannabis use was only statistically significantly associated with pre-bronchodilator resonant frequency in men. Cannabis use between the ages of 38 and 45 years was associated with a similar pattern of changes in IOS measures. After salbutamol, cannabis use was only statistically significantly associated with R5 and R20 among women and none of the IOS measures among men. Conclusions Cannabis use is associated with small airway dysfunction at age 45 years, indicating an increase in peripheral airway resistance and reactance. These associations were greater and mostly only statistically significant among women. Associations were weaker and mostly nonsignificant after bronchodilator use, suggesting that cannabis-induced changes in small airways may be at least partially reversible.

Living with a Hope of Survival Is Challenged by a Lack of Clinical Evidence: An Interview Study among Cancer Patients Using Cannabis-Based Medicine
Dorte Buchwald , Dorte Brønnum , Dorte Melgaard , Peter D C Leutscher
J Palliat Med ( Aug 2020)
doi: 10.1089/jpm.2019.0298
Background: There is an increasing focus among cancer patients on the use of cannabis-based medicine (CBM) as a supplement to conventional palliative care. However, physicians are reluctant to engage in dialog with the patients as clinical evidence is lacking. As a result, the patients are often left alone to rely on their own judgment in purchasing CBM products on the illegal market.
Objective: Our study aimed to collect information from CBM treatment-experienced cancer patients receiving palliative care regarding treatment decision rationale and outcome.
Design: A qualitative interview study using thematic analysis was performed.
Setting/Participants: A total of 20 informants took part in individual interviews.
Results: To the question addressing the main rationale for starting CBM treatment, all 20 patients responded that they carried a hope that cannabis would have a curative effect on the cancer disease. Most patients reported relief of symptoms, such as insomnia, anxiety, nausea, and pain, after initiation of CBM treatment, but this outcome was perceived as less of a focus in comparison to hope of a cure. Conclusion: This study contributes with knowledge from the perspective of the cancer patient in palliative care regarding the decision behind the use of CBM. There seems to be striving for surviving cancer based on the rationale that cannabis may constitute curative properties. Relief of symptoms is perceived as a secondary reason for treatment. This knowledge is essential in the dialog between the health professional and the cancer patient about the use of CBM products for treatment.

Long-Term Aberrations To Cerebellar Endocannabinoids Induced By Early-Life Stress.
Moussa-Tooks, A. B., Larson, E. R., Gimeno, A. F., Leishman, E., Bartolomeo, L. A., Bradshaw, H. B., … Hetrick, W. P.
Scientific Reports, (2020).
10(1). doi:10.1038/s41598-020-64075-4
Emerging evidence points to the role of the endocannabinoid system in long-term stress-induced neural remodeling with studies on stress-induced endocannabinoid dysregulation focusing on cerebral changes that are temporally proximal to stressors. Little is known about temporally distal and sexspecifc efects, especially in cerebellum, which is vulnerable to early developmental stress and is dense with cannabinoid receptors. Following limited bedding at postnatal days 2–9, adult (postnatal day 70) cerebellar and hippocampal endocannabinoids, related lipids, and mRNA were assessed, and behavioral performance evaluated. Regional and sex-specifc efects were present at baseline and following earlylife stress. Limited bedding impaired peripherally-measured basal corticosterone in adult males only. In the CNS, early-life stress (1) decreased 2-arachidonoyl glycerol and arachidonic acid in the cerebellar interpositus nucleus in males only; (2) decreased 2-arachidonoyl glycerol in females only in cerebellar Crus I; and (3) increased dorsal hippocampus prostaglandins in males only. Cerebellar interpositus transcriptomics revealed substantial sex efects, with minimal stress efects. Stress did impair novel object recognition in both sexes and social preference in females. Accordingly, the cerebellar endocannabinoid system exhibits robust sex-specifc diferences, malleable through early-life stress, suggesting the role of endocannabinoids and stress to sexual diferentiation of the brain and cerebellarrelated dysfunctions.

Long-term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open-label extension trial
Elizabeth A. Thiele | E. Martina Bebin | Francis Filloux | Patrick Kwan | Rachael Loftus | Farhad Sahebkar | Steven Sparagana | James Wheless
Epilepsia. 2021;00:1–14.
DOI: 10.1111/epi.17150
https://onlinelibrary.wiley.com/doi/...1111/epi.17150
Objective: To evaluate the long-term safety and efficacy of add-on cannabidiol (CBD) in patients with seizures associated with tuberous sclerosis complex (TSC) in the open-label extension (OLE) of the randomized, placebo-controlled phase 3 trial GWPCARE6 (NCT02544763). Results of an interim (February 2019 data cut) analysis are reported.
Methods: Patients who completed the randomized trial enrolled to receive CBD (Epidiolex® in the United States; Epidyolex® in the EU; 100 mg/mL oral solution). The initial target dose was 25 mg/kg/day, which, based on response and tolerability, could be decreased or increased up to 50 mg/kg/day. The primary end point was safety. Key secondary end points included percentage reduction in TSC-associated (countable focal and generalized) seizures, responder rates, and Subject/Caregiver Global Impression of Change (S/CGIC).
Results: Of 201 patients who completed the randomized phase, 199 (99%) entered the OLE. Mean age was 13 years (range, 1–57). At the time of analysis, 5% of patients had completed treatment, 20% had withdrawn, and 75% were ongoing. One-year retention rate was 79%. Median treatment time was 267 days (range,18–910) at a 27 mg/kg/day mean modal dose. Most patients (92%) had an adverse event (AE). Most common AEs were diarrhea (42%), seizure (22%), and decreased appetite (20%). AEs led to permanent discontinuation in 6% of patients. There was one death that was deemed treatment unrelated by the investigator. Elevated liver transaminases occurred in 17 patients (9%) patients; 12 were taking valproate. Median percentage reductions in seizure frequency (12-week windows across 48 weeks) were 54%–68%. Seizure responder rates (≥50%, ≥75%, 100% reduction) were 53%–61%, 29%–45%, and 6%–11% across 12-week windows for 48 weeks. Improvement on the S/CGIC scale was reported by 87% of patients/ caregivers at 26 weeks.

Long-term consequences of URB597 administration during adolescence on cannabinoid CB1 receptor binding in brain areas
Eva María Marco, Tiziana Rubino, Walter Adriani, María-Paz Viveros, Daniela Parolaro, Giovanni Laviol
BRAIN RESEARCH 1257 (2009) 25 – 3 1
DOI: 10.1016/j.brainres.2008.12.037
Despite the alarming increment in the use and abuse of cannabis preparations among young people, little is known about possible long-term consequences of targeting the endocannabinoid system during the critical developmental period of adolescence. Therefore, we aimed to analyze possible long-lasting neurobiological consequences of enhancing endocannabinoid signalling during adolescence, by means of blocking anandamide (AEA) hydrolysis. Adolescent Wistar male rats were administered an inhibitor of AEA hydrolysis, i.e. URB597 (0, 0.1 or 0.5 mg/kg/day from postnatal days 38 to 43). The expression of brain cannabinoid receptor type 1 (CB1R) was then analyzed by [(3)H]CP-55,940 auto-radiographic binding at adulthood. Repeated URB597 administration during adolescence persistently modified CB1R binding in a region-dependent manner. A long-lasting decrease of CB1R binding levels was found in caudate-putamen, nucleus accumbens, ventral tegmental area and hippocampus, while an opposite increment was observed in the locus coeruleus. Present results provide evidence for long-lasting effects of adolescent URB597 administration. Activation of endocannabinoid transmission during the still plastic phase of adolescence may have implications for the maturational end-point of the endocannabinoid system itself, which could lead to permanent alterations in neuronal brain circuits and behavioural responses. Insights into the developmental trajectories of this neuromodulatory system may help us to better understand and prevent outcomes of neonatal and adolescent cannabis exposure.

Long-term safety and treatment effects of cannabidiol in children and adults with treatment-resistant epilepsies: Expanded access program results.
Szaflarski, J. P., Bebin, E. M., Comi, A. M., Patel, A. D., Joshi, C., … Checketts, D.
Epilepsia, 59(8), 1540–1548. (2018).
doi:10.1111/epi.14477
Objective: Since 2014, cannabidiol (CBD) has been administered to patients with treatment?resistant epilepsies (TREs) in an ongoing expanded?access program (EAP). We report interim results on the safety and efficacy of CBD in EAP patients treated through December 2016. Methods: Twenty?five US?based EAP sites enrolling patients with TRE taking stable doses of antiepileptic drugs (AEDs) at baseline were included. During the 4?week baseline period, parents/caregivers kept diaries of all countable seizure types. Patients received oral CBD starting at 2?10 mg/kg/d, titrated to a maximum dose of 25?50 mg/kg/d. Patient visits were every 2?4 weeks through 16 weeks and every 2?12 weeks thereafter. Efficacy endpoints included the percentage change from baseline in median monthly convulsive and total seizure frequency, and percentage of patients with ?50%, ?75%, and 100% reductions in seizures vs baseline. Data were analyzed descriptively for the efficacy analysis set and using the last?observation?carried?forwa rd method to account for missing data. Adverse events (AEs) were documented at each visit. Results: Of 607 patients in the safety dataset, 146 (24%) withdrew; the most common reasons were lack of efficacy (89 [15%]) and AEs (32 [5%]). Mean age was 13 years (range, 0.4?62). Median number of concomitant AEDs was 3 (range, 0?10). Median CBD dose was 25 mg/kg/d; median treatment duration was 48 weeks. Add? on CBD reduced median monthly convulsive seizures by 51% and total seizures by 48% at 12 weeks; reductions were similar through 96 weeks. Proportion of patients with ?50%, ?75%, and 100% reductions in convulsive seizures were 52%, 31%, and 11%, respectively, at 12 weeks, with similar rates through 96 weeks. CBD was generally well tolerated; most common AEs were diarrhea (29%) and somnolence (22%).

Lost in the Haze: The Physician’s Role in Cannabinoid Prescribing and Advising.
Johnston, C. B., & Vanderah, T.
The American Journal of Medicine. (2019).
doi:10.1016/j.amjmed.2019.05.049
Ten states and the District of Columbia have legalized cannabis for recreational use and 33 states have legalized cannabis for medicinal use. Although the use of cannabis remains federally illegal,
some of its derivative compounds have been approved by the Food and Drug Administration for prescription use (e.g., dronabinol, nabilone, Epidiolex®). According to a 2017 Gallup poll, 64% of
Americans favored marijuana legalization. An increasing number of people are using cannabinoids to treat a variety of ailments. Extracts of cannabidiol (CBD) are proliferating in dispensaries and other outlets across the United States. Individuals and internet websites provide medical guidance - often based on minimal or no data - on what indications should warrant use of cannabinoids. But where are physicians in this rapidly changing landscape? Increasingly, providers are left behind as dispensaries become the preferred experts on cannabis strains for specific conditions. Many institutions issue prohibitions or confusing policies on whether providers can recommend cannabis, leaving providers bewildered. Cannabis remains a Schedule 1 substance that is still illegal under federal law, and the science of cannabinoid effectiveness lags way behind anecdotal evidence, which understandably makes many providers uneasy in making recommendations regarding cannabinoids for their patients.

Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice
Sabine Steffens, Niels R. Veillard, Claire Arnaud, Graziano Pelli, Fabienne Burger, Christian Staub, Andreas Zimmer, Jean-Louis Frossard & François Mach
Nature volume 434, pages782–786 (2005)
DOI: 10.1038/nature03389
Atherosclerosis is a chronic inflammatory disease, and is the primary cause of heart disease and stroke in Western countries1. Derivatives of cannabinoids such as delta-9-tetrahydrocannabinol (THC) modulate immune functions2 and therefore have potential for the treatment of inflammatory diseases. We investigated the effects of THC in a murine model of established atherosclerosis. Oral administration of THC (1?mg?kg-1 per day) resulted in significant inhibition of disease progression. This effective dose is lower than the dose usually associated with psychotropic effects of THC. Furthermore, we detected the CB2 receptor (the main cannabinoid receptor expressed on immune cells2,3) in both human and mouse atherosclerotic plaques. Lymphoid cells isolated from THC-treated mice showed diminished proliferation capacity and decreased interferon-? secretion. Macrophage chemotaxis, which is a crucial step for the development of atherosclerosis1, was also inhibited in vitro by THC. All these effects were completely blocked by a specific CB2 receptor antagonist4. Our data demonstrate that oral treatment with a low dose of THC inhibits atherosclerosis progression in the apolipoprotein E knockout mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, THC or cannabinoids with activity at the CB2 receptor may be valuable targets for treating atherosclerosis.

Low-Dose Vaporized Cannabis Significantly Improves Neuropathic Pain
Barth Wilsey, Thomas Marcotte, Reena Deutsch, Ben Gouaux, Staci Sakai, and Haylee Donaghe
The Journal of Pain, Vol 14, No 2 (February), 2013: pp 136-148
Doi: 10.1016/j.jpain.2012.10.009
We conducted a double-blind, placebo-controlled, crossover study evaluating the analgesic efficacy of vaporized cannabis in subjects, the majority of whom were experiencing neuropathic pain despite traditional treatment. Thirty-nine patients with central and peripheral neuropathic pain underwent a standardized procedure for inhaling medium-dose (3.53%), low-dose (1.29%), or placebo cannabis with the primary outcome being visual analog scale pain intensity. Psychoactive side effects and neuropsychological performance were also evaluated. Mixed-effects regression models demonstrated an analgesic response to vaporized cannabis. There was no significant difference between the 2 active dose groups’ results (P > .7). The number needed to treat (NNT) to achieve 30% pain reduction was 3.2 for placebo versus low-dose, 2.9 for placebo versus medium-dose, and 25 for medium- versus low-dose. As these NNTs are comparable to those of traditional neuropathic pain medications, cannabis has analgesic efficacy with the low dose being as effective a pain reliever as the medium dose. Psychoactive effects were minimal and well tolerated, and neuropsychological effects were of limited duration and readily reversible within 1 to 2 hours. Vaporized cannabis, even at low doses, may present an effective option for patients with treatment-resistant neuropathic pain.
Perspective: The analgesia obtained from a low dose of delta-9-tetrahydrocannabinol (1.29%) in patients, most of whom were experiencing neuropathic pain despite conventional treatments, is a clinically significant outcome. In general, the effect sizes on cognitive testing were consistent
with this minimal dose. As a result, one might not anticipate a significant impact on daily functioning.

Low doses of widely consumed cannabinoids (cannabidiol and cannabidivarin) cause DNA damage and chromosomal aberrations in human-derived cells
Chiara Russo · Franziska Ferk · Miroslav Mišík · Nathalie Ropek · Armen Nersesyan · Doris Mejri ·Klaus Holzmann · Margherita Lavorgna · Marina Isidori · Siegfried Knasmu?ller
Archives of Toxicology (2019) 93:179–188
doi: 10.1007/s00204-018-2322-9
Cannabidiol (CBD) and cannabidivarin (CBDV) are natural cannabinoids which are consumed in increasing amounts worldwide in cannabis extracts, as they prevent epilepsy, anxiety, and seizures. It was claimed that they may be useful in cancer therapy and have anti-inflammatory properties. Adverse long-term effects of these drugs (induction of cancer and infertility) which are related to damage of the genetic material have not been investigated. Therefore, we studied their DNA-damaging properties in human-derived cell lines under conditions which reflect the exposure of consumers. Both compounds induced DNA damage in single cell gel electrophoresis (SCGE) experiments in a human liver cell line (HepG2) and in buccal-derived cells (TR146) at low levels (? 0.2 ?M). Results of micronucleus (MN) cytome assays showed that the damage leads to formation of MNi which reflect chromosomal aberrations and leads to nuclear buds and bridges which are a consequence of gene amplifications and dicentric chromosomes. Additional experiments indicate that these effects are caused by oxidative base damage and that liver enzymes (S9) increase the genotoxic activity of both compounds. Our findings show that low concentrations of CBD and CBDV cause damage of the genetic material in human-derived cells. Furthermore, earlier studies showed that they cause chromosomal aberrations and MN in bone marrow of mice. Fixation of damage of the DNA in the form of chromosomal damage is generally considered to be essential in the multistep process of malignancy, therefore the currently available data are indicative for potential carcinogenic properties of the cannabinoids.

L-Theanine Prevents Long-Term Affective and Cognitive Side-Effects of Adolescent ?-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.
De Felice, M., Renard, J., Hudson, R., Szkudlarek, H. J., Pereira, B. J., Schmid, S., … Laviolette, S. R.
The Journal of Neuroscience, JN–RM–1050–20. (2020). doi:10.1523/jneurosci.1050-20.2020
Chronic adolescent exposure to '-9-Tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioural abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathological alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. L-theanine is an amino acid analogue of L-glutamate and L-glutamine derived from various plant sources, including green tea leaves. L-
theanine has previously been shown to modulate levels of GABA, DA and glutamate in various neural regions and to possess neuroprotective properties. Using a pre-clinical model of adolescent THC exposure in male rats, we report that L-theanine pre-treatment prior to adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect which persists into adulthood. In addition, pre-treatment with L-theanine blocked THC-induced downregulation of local GSK-3 and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and sub-cortical DAergic dysregulation. Finally, L-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long- term neuroprotective effects of L-theanine in the mesocorticolimbic system.

Mandatory pharmacist-led education session for patients seeking medical cannabis
Vikas Parihar, Laura Katz, Anna Rogers, Lisa Patterson, Ramesh Zacharias
Pharmacy Practice Vol 18 No 4 (2020): Oct-Dec
DOI: 10.18549/PharmPract.2020.4.2088
Objective: The primary objectives of this pre-post session study, was to evaluate the impact of a pharmacist-led education session on the perceived benefits and safety of cannabis among patients with chronic pain, as well as determine the influence of pharmacist education on the selection of safer cannabis products and dosage forms for medical use among patients.
Methods: A retrospective analysis of completed pre-post session questionnaires was conducted among chronic pain patients attending a mandatory education session led by a pharmacist, prior to being authorized cannabis in clinic. All questionnaire data was analyzed using SPSS v. 25. Demographic and sample characteristics were reviewed using univariate analyses. Chi-Square tests were employed to determine if the group-based education significantly affected knowledge, perception of efficacy and safety of cannabis.
Results: Of the 260 session participants, 203 completed pre-post session questionnaires. After the session, a majority of current cannabis users (33.8%) and cannabis naïve/past users (56.9%) reported they would use a low THC product in the future, and a majority of current users (54.5%) would use a high CBD product in the future. After education, participants were more likely to report cannabis as having the potential for addiction (chi-square =42.6, p <0.0001) and harm (chi-square =34.0, p <0.0001).
Conclusions: Pharmacist counselling and education has the potential to influence patient selection and use of cannabis, from more harmful to safer products, as well as moderate the potential perceived benefits of use

Maintenance of Blinding in Clinical Trials and the Implications for Studying Analgesia Using Cannabinoids
Barth Wilsey, Reena Deutsch, and Thomas D. Marcotte
Cannabis and Cannabinoid Research Volume 1.1, 2016
DOI: 10.1089/can.2016.0016
The design of analgesic clinical trials invariably involves a comparison between placebo and active study medication. An assumption is made that treatment effects can be approximated by subtracting the response to placebo from that attained with the use of active study medication. However, the psychoactivity of cannabinoids may unmask their presence and lead to an expectation and/or conditioning of pain relief. For example, study participants biased toward the belief that cannabis is beneficial for their condition might be more inclined to report positive effects if they were to accurately identify the active treatment because of its psychoactivity. This may lead to incorrect assumptions regarding the efficacy of a cannabinoid. Methodologies designed to counteract unmasking need to be implemented in the design phase of a study. During the clinical trial, it is also important to query participants as to which treatment they believe they have received. Blinding can be considered to be preserved when the accuracy of treatment guesses is not considerably different than random guessing, which is estimated to be correct 50% of the time. After a study has been completed, the use of statistical methodologies such as regression and mediation analysis are worthy of consideration to see whether psychoactive effects biased the results.

Management of chronic pain with Jalaprakshalana (water-wash) Shodhita (processed) Bhanga(Cannabis sativa L.) in cancer patients with deprived quality of life: An open-label single arm clinical trial.
Tavhare SD1, Acharya R2, Reddy RG3, Dhiman KS
Ayu. 2019 Jan-Mar;40(1):34-43.
doi: 10.4103/ayu.AYU_43_19.
https://www.ayujournal.org/article.a...aulast=Tavhare
INTRODUCTION:
Pain is a common and complex symptom of cancer having physical, social, spiritual and psychological aspects. Approximately 70%-80% of cancer patients experiences pain, as reported in India. Ayurveda recommends use of Shodhita (Processed)Bhanga (Cannabis) for the management of pain but no research yet carried out on its clinical effectiveness.
OBJECTIVE:
To assess the analgesic potential of Jala-Prakshalana (Water-wash) processed Cannabis sativa L. leaves powder in cancer patients with deprived quality of life (QOL) through openlabel single arm clinical trial.
MATERIALS AND METHODS:
Waterwash processed Cannabis leaves powder filled in capsule, was administered in 24 cancer patients with deprived QOL presenting complaints of pain, anxiety or depression; for a period of 4 weeks; in a dose of 250 mg thrice a day; along with 50 ml of cow's milk and 4 g of crystal sugar. Primary outcome i.e. pain was measured by Wong-Bakers FACES Pain Scale (FACES), Objective Pain Assessment (OPA) scale and Neuropathic Pain Scale (NPS). Secondary outcome namely anxiety was quantified by Hospital Anxiety and Depression Scale (HADS), QOL by FACT-G scale, performance score by Eastern Cooperative Oncology Group (ECOG) and Karnofsky score.
RESULTS:
Significant reduction in pain was found on FACES Pain Scale (P < 0.05), OPA (P < 0.05), NPS (P < 0.001), HADS (P < 0.001), FACT-G scale (P < 0.001), performance status score like ECOG (P < 0.05) and Karnofsky score (P < 0.01).
CONCLUSION:
Jalaprakshalana Shodhita Bhanga powder in a dose of 250 mg thrice per day; relieves cancerinduced pain, anxiety and depression significantly and does not cause any major adverse effect and withdrawal symptoms during trial period.

Marijuana and Myocardial Infarction in the UK Biobank Cohort
Steven Lehrer, Peter H. Rheinstein
Cureus (2022)
https://assets.cureus.com/uploads/or...113-j6ohwy.pdf
DOI: 10.7759/cureus.22054
Background: Atrial fibrillation, ventricular tachycardia, acute coronary syndromes, and cardiac arrest have been attributed to marijuana. But the National Academy of Science’s 2017 Report, The Health Effects of Cannabis and Cannabinoids, found limited evidence that acute marijuana smoking is positively associated with an increased risk of acute myocardial infarction, and uncovered no evidence to support or refute associations between any chronic effects of marijuana use and increased risk of myocardial infarct (MI).
Aims: We sought to determine the association of marijuana smoking with MI in the UK Biobank cohort. Because red wine is a mood-altering substance, we compared the effect of marijuana with red wine on MI incidence.
Methods: Our analysis included all subjects with MI. The diagnosis was ascertained using the 10th Revision of the International Classification of Diseases (ICD10 I21). Marijuana was recorded in UKB Category 143, medical conditions, marijuana use. Cigarette smoking information was from UKB Category 100058, smoking. To compare marijuana smoking with the effect of wine drinking we used data from UKB Category 10051, alcohol.
Results: With marijuana use, MI incidence decreased (p < 0.001, two tail Fisher exact test). Red wine was associated with lower MI incidence, although the incidence begins to rise at 11 or more glasses per week (p < 0.001, two tail Fisher exact test). Multivariate analysis was done with logistic regression, MI dependent variable, cigarette pack-years, diabetes type 2, sex, BMI, hypertension, marijuana use, age, red wine consumption, independent variables. Odds ratio (O.R.) 0.844 associated with marijuana use indicates that MI was less likely in marijuana users and was comparable to the effect of red wine (O.R. 0.847).
Conclusion: Marijuana, which has not been shown to have the favorable physiologic effects of red wine on the heart, does reduce MI risk to an extent comparable to red wine. Perhaps both affect the heart by reducing stress.

Marijuana compounds a non conventional therapudic approach to epilepsy in children
Mariana Babayeva, Malka Fuzailov, Paulina Rozenfeld and Paramita Basu

J Addict Addictv Disord 2014, 1: 002.
DOI: 10.24966/AAD-7276/100002
http://www.heraldopenaccess.us/artic...n-children.pdf
Epilepsy in children is a complex disease, with a variety of distinct syndromes and many alternative treatment options. Even with a plethora of available treatment options, childhood epilepsies are commonly associated with seizures that are resistant to existing treatment methods. Treatment of pediatric epilepsy is challenging and requires more effective therapy to avoid short-term and long-term neurological disorders. Marijuana has been used to treat disease since ancient times. Marijuana ingredients Cannabidiol (CBD) and D9-Tetrahydrocannabinol (THC) have created a significant research interest as potential therapy options in epilepsy treatment. THC is the major psychoactive component of marijuana that aids in reducing epileptic seizures. CBD has proven to have anticonvulsant effect not only in experimental models but also in clinical studies. Research studies have provided strong evidence for safety and anticonvulsant properties of medical marijuana. Principal concerns regarding the use of medical marijuana in children include lack of standardization and regulation, imprecise dosing, possible adverse side effects and medication interactions.


Marijuana Dependence: Not Just Smoke and Mirrors
Divya Ramesh, Joel E. Schlosburg, Jason M. Wiebelhaus, Aron H. Lichtman
ILAR J. 2011 January ; 52(3): 295–308. doi:10.1093/ilar.52.3.295
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606907/pdf/nihms380778.pdf
Marijuana (Cannabis sativa) is the most commonly used illicit drug worldwide as well as in the Unites States. Prolonged use of marijuana or repeated administration of its primary psychoactive constituent, Δ9 -tetrahydrocannabinol (THC), can lead to physical dependence in humans and laboratory animals. The changes that occur with repeated cannabis use include alterations in behavioral, physiological, and biochemical responses. A variety of withdrawal responses occur in cannabis-dependent individuals: anger, aggression, irritability, anxiety and nervousness, decreased appetite or weight loss, restlessness, and sleep difficulties with strange dreams. But the long halflife and other pharmacokinetic properties of THC result in delayed expression of withdrawal symptoms, and because of the lack of contiguity between drug cessation and withdrawal responses the latter are not readily recognized as a clinically relevant syndrome. Over the past 30 years, a substantial body of clinical and laboratory animal research has emerged supporting the assertion that chronic exposure to cannabinoids produces physical dependence and may contribute to drug maintenance in cannabis-dependent individuals. However, no medications are approved to treat cannabis dependence and withdrawal. In this review, we describe preclinical and clinical research that supports the existence of a cannabinoid withdrawal syndrome. In addition, we review research evaluating potential pharmacotherapies (e.g., THC, a variety of antidepressant drugs, and lithium) to reduce cannabis withdrawal responses and examine how expanded knowledge about the regulatory mechanisms in the endocannabinoid system may lead to promising new therapeutic targets. .

Marijuana Equivalency in Portion and Dosage
An assessment of physical and pharmacokinetic relationships in marijuana production and consumption in Colorado
Prepared for the Colorado Department of Revenue
DOI: 10.13140/RG.2.2.30577.28004
An assessment of physical and pharmacokinetic relationships in marijuana production and consumption in Colorado
Prepared for the Colorado Department of Revenue (2015)
The original legislation to legalize and regulate marijuana in Colorado does not explicitly restrict marijuana concentrates and infused edibles. Over time, these marijuana products have become more popular, prompting new legislation to remedy the omission. House Bill 14-1361 now stipulates limits upon marijuana flower portions, “or their equivalent.”
This study provides scientific and data driven evidence in order to understand these equivalencies. The results provide comparisons between marijuana flower, concen- trates and infused products specifically for Colorado’s marijuana market.
Equivalency can be viewed from three perspectives: production, dosing, and market price. The first perspective relates to physical production, where infused edibles or concentrates are traced back into their corresponding weight of flower or trim inputs. This enables the conversion from non-flower products into a common flower-based denominator, so that aggregate use can be measured across different marijuana product types.

Marijuana Issues: Bibliography
ANRF (2020)
https://no-smoke.org/wp-content/uplo...bliography.pdf
This partial bibliography of studies is provided for informational purposes only. It should not be considered to replace medical advice from your physician, nor should it be considered as a list of every existing study on this topic. The ANR Foundation is unable to provide copies of any of the studies. Links to either the study’s abstract or full text are provided when available. The citations referenced below are presented without comment regarding the source, including any potential conflict of interest. Due diligence is recommended in researching the source of any given study before relying on its conclusions.

Marijuana, Ondansetron, and Promethazine Are Perceived as Most Effective Treatments for Gastrointestinal Nausea.
Zikos, T. A., Nguyen, L., Kamal, A., Fernandez-Becker, N., Regalia, K., Nandwani, M., Clarke, J. O.
Digestive Diseases and Sciences. (2020).
doi:10.1007/s10620-020-06195-5
Background Many anti-nausea treatments are available for chronic gastrointestinal syndromes, but data on efcacy and comparative efectiveness are sparse. Aims To conduct a sectional survey study of patients with chronic nausea to assess comparative efectiveness of commonly used anti-nausea treatments. Methods Outpatients at a single center presenting for gastroenterology evaluation were asked to rate anti-nausea efcacy on a scale of 0 (no efcacy) to 5 (very efective) of 29 commonly used anti-nausea treatments and provide other information about their symptoms. Additional information was collected from the patients’ chart. The primary outcome was to determine which treatments were better or worse than average using a t test. The secondary outcome was to assess diferential response by individual patient characteristics using multiple linear regression. Results One hundred and ffty-three patients completed the survey. The mean efcacy score of all anti-nausea treatments evaluated was 1.73. After adjustment, three treatments had scores statically higher than the mean, including marijuana (2.75, p

Marijuana, Spice ‘herbal high’, and early neural development: implications for rescheduling and legalization
Delphine Psychoyosa, and K. Yaragudri Vinod
Drug Test Anal. 2013 January ; 5(1): 27–45.
doi: 10.1002/dta.1390
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147784/
Marijuana is the most widely used illicit drug by pregnant women in the world. In utero exposure to Δ9 -tetrahydrocannabinol (Δ9 -THC), a major psychoactive component of marijuana, is associated with an increased risk for anencephaly and neurobehavioural deficiencies in the offspring, including attention deficit hyperactivity disorder (ADHD), learning disabilities, and memory impairment. Recent studies demonstrate that the developing central nervous system (CNS) is susceptible to the effects of Δ9 -THC and other cannabimimetics, including the psychoactive ingredients of the branded product ‘Spice ’ branded products. These exocannabinoids interfere with the function of an endocannabinoid (eCB) system, present in the developing CNS from E12.5 (week 5 of gestation in humans), and required for proliferation, migration, and differentiation of neurons. Until recently, it was not known whether the eCB system is also present in the developing CNS during the initial stages of its ontogeny, i.e. from E7.0 onwards (week 2 of gestation in humans), and if so, whether this system is also susceptible to the action of exocannabinoids. Here, we review current data, in which the presence of an eCB system during the initial stage of development of the CNS is demonstrated. Furthermore, we focus on recent advances on the effect of canabimimetics on early gestation. The relevance of these findings and potential adverse developmental consequences of in utero exposure to ‘high potency’ marijuana, Spice branded products and/or cannabinoid research chemicals during this period is discussed. Finally, we address the implication of these findings in terms of the potential dangers of synthetic cannabinoid use during pregnancy, and the ongoing debate over legalization of marijuana.

Marijuana Use Among Women of Reproductive Age With Disabilities
Panagiota Kitsantas, Salman M. Aljoudi, Edward J. Booth, Melanie L. Kornides
Am J Prev Med 2021;61(4):554−562.
https://doi.org/10.1016/j.amepre.2021.04.008
Introduction: Despite the increasing prevalence and potential adverse health outcomes associated with marijuana use, limited research exists related to its use in women of reproductive age with disabilities. The purpose of this study is to examine the past-month marijuana use in women of reproductive age with disabilities.
Methods: Data from the 2015−2019 National Survey on Drug Use and Health included 76,765 women of reproductive age (18−44 years). Descriptive statistics and adjusted logistic regression analyses were conducted to assess the associations between past-month marijuana use and overall disability, including the type of disability.
Results: In this sample, 12.6% of women reported past-month marijuana use. Approximately, 21% of women with disabilities reported past-month marijuana use, compared with only 11.1% of women without a disability. Marijuana use was more prevalent in women with disabilities who were younger (aged ≤25 years), who were non-Hispanic White, who were nonmarried, who had at least some college education, and who used alcohol or tobacco. Women with disabilities had 1.68 (95% CI=1.57, 1.80) higher odds of reporting past-month marijuana use than those with no disabilities. The odds of past-month marijuana use were higher among those with cognitive (AOR=1.78, 95% CI=1.64, 1.94), sensory (AOR=1.30, 95% CI=1.12, 1.51), and daily activities−related (AOR=1.64, 95% CI=1.49, 1.80) disabilities than among their counterparts without disabilities.
Conclusions: This study found an increased prevalence of past-month marijuana use among women of reproductive age with disabilities. Enhanced screening and counseling using evidence- based practices during routine care for women with disabilities may be necessary to mitigate marijuana use

Marijuana Use by Breastfeeding Mothers and Cannabinoid Concentrations in Breast Milk
Pediatrics 142(3):e20181076 August 2018
DOI: 10.1542/peds.2018-1076
Kerri A. Bertrand, Nathan J Hanan, Gordon Honerkamp-Smith, Christina D Chambers
BACKGROUND AND OBJECTIVE: Marijuana is the most commonly used recreational drug among breastfeeding women. With legalization of marijuana in several US states and a 1990 study in which authors documented psychomotor deficits in infants breastfed by mothers using marijuana, there is a need for information on potential exposure to the breastfed infant. Our objective with this study was to quantify cannabinoids in human milk after maternal marijuana use. Methods: Between 2014 and 2017, 50 breastfeeding women who reported marijuana use provided 54 breast milk samples to a research repository, Mommy's Milk. Concentrations of ?-9-tetrahydrocannabinol (?9-THC), 11-hydroxy-?-9-tetrahydrocannabinol, cannabidiol, and cannabinol were measured by using liquid chromatography mass spectrometry electrospray ionization. Results: ?9-THC was detectable in 34 (63%) of the 54 samples up to ?6 days after last reported use; the median concentration of ?9-THC was 9.47 ng/mL (range: 1.01-323.00). Five samples had detectable levels of 11-hydroxy-?-9-tetrahydrocannabinol (range: 1.33-12.80 ng/mL) or cannabidiol (range: 1.32-8.56 ng/mL). The sample with the highest concentration of cannabidiol (8.56 ng/mL) did not have measurable ?9-THC. Cannabinol was not detected in any samples. The number of hours since last use was a significant predictor of log ?9-THC concentrations (-0.03; 95% confidence interval [CI] -0.04 to -0.01; P = .005). Adjusted for time since last use, the number of daily uses and time from sample collection to analysis were also significant predictors of log ?9-THC concentrations (0.51; 95% CI 0.03 to 0.99; P = .039; 0.08; 95% CI 0.00 to 0.15; P = .038, respectively). Conclusions: ?9-THC was measurable in a majority of breast milk samples up to ?6 days after maternal marijuana use.

Marijuana use does not affect the outcomes of bariatric surgery.
Shockcor, N., Adnan, S. M., Siegel, A., Wise, E., Zafar, S. N., & Kligman, M.
Surgical Endoscopy.(2020).
doi:10.1007/s00464-020-07497-5
Background The decriminalization of marijuana and legalization of derived products requires investigation of their efect on healthcare-related outcomes. Unfortunately, little data are available on the impact of marijuana use on surgical outcomes. We aimed to determine the efect of marijuana use on 30-day complications and 1-year weight loss following laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic sleeve gastrectomy (LSG). Methods At a large academic center, 1176 consecutive patients undergoing primary bariatric surgery from 2012 to 2017 were identifed and separated into cohorts according to marijuana use. The only exclusions were 19 patients lost to follow-up. Propensity score matching, using logistic regression according to preoperative age, gender, BMI, and comorbid conditions, yielded 73 patient pairs for the control and study arms. All patients were followed two years postoperatively. Results Excess BMI lost did not difer between marijuana users and controls at 3 weeks (23.0% vs 18.9%, p=0.095), 3 months (42.0% vs 38.1%, p=0.416), 6 months (60.6% vs 63.1%, p=0.631), 1 year (78.2% vs 77.3%, p=0.789), or 2 years (89.1% vs 74.5%, p=0.604). No diferences in the rate of major 30-day postoperative complications, including readmission, infection, thromboembolic events, bleeding events and reoperation rates, were found between groups. Follow-up rate at two years was lower in marijuana users (12.3% vs 27.4%, p=0.023). Conclusion This study suggests marijuana use has no impact on 30-day complications or weight loss following bariatric surgery, and should not be a contraindication to bariatric surgery

Marijuana Use During Pregnancy and Breastfeeding: Implications for Neonatal and Childhood Outcomes
Sheryl A. Ryan, Seth D. Ammerman, Mary E. O’Connor,
Pediatrics. 2018;142(3):
doi: 10.1542/peds.2018-1889
Marijuana is one of the most widely used substances during pregnancy in
the United States. Emerging data on the ability of cannabinoids to cross
the placenta and affect the development of the fetus raise concerns about
both pregnancy outcomes and long-term consequences for the infant or
child. Social media is used to tout the use of marijuana for severe nausea
associated with pregnancy. Concerns have also been raised about marijuana
use by breastfeeding mothers. With this clinical report, we provide data on
the current rates of marijuana use among pregnant and lactating women,
discuss what is known about the effects of marijuana on fetal development
and later neurodevelopmental and behavioral outcomes, and address
implications for education and policy.

Marijuana Use in Inflammatory Bowel Disease: Understanding the Prevalence and the Potential Pitfalls
Frank I. Scott
Crohn’s & Colitis 360 • Volume 2, Number 2, April 2020
https://www.ncbi.nlm.nih.gov/pmc/art...df/otaa016.pdf
doi: 10.1093/crocol/otaa016
Many patients with inflammatory bowel disease experiment with marijuana for symptom control. Marijuana may help reduce abdominal pain but does not control inflammatory bowel disease related inflammation. Patients should discuss their marijuana use with their providers and continue appropriate medications if considering marijuana.

Marijuana Use in Transplantation: A Call for Clarity
Levi ME, Montague BT, Thurstone C, Kumar D, Huprikar SS, Kotton CN
Clin Transplant. 2019 Feb;33(2):e13456.
doi: 10.1111/ctr.13456
Transplant centers have varying policies for marijuana (MJ) use in donors, transplant candidates and recipients. Rationales for these differences range from concerns for fungal complications, impaired adherence and drug interactions. This paper reviews the current status of MJ policies and practices in transplant centers and results of a survey sent to the American Society of Transplantation (AST) membership by the Executive Committee of the
AST Infectious Diseases Community of Practice. The purpose of the survey was to compare policies and concerns of MJ use to actual observed complications. Of the 3,321 surveys sent, 225 members (8%) responded. Transplant centers varied in their approval processes, differing even in organ types within the same institutions. Furthermore, there was discordance among transplant centers in their perceived risks of marijuana use as opposed to complications actually observed.
An increasing number of states continue to legalize medical and recreational MJ resulting in widespread availability. Further research is needed to assess the validity of concerns for complications of MJ use in potential donors and recipients. Ultimately, standardized guidelines should be established based on studies and evidence based criteria to assist transplant programs in their policies around the use of cannabis in their donors and recipients.

Maternal cannabis use is associated with suppression of immune gene networks in placenta and increased anxiety phenotypes in offspring
Gregory Rompala, Yoko Nomura, and Yasmin L. Hurd
PNAS November 23, 2021 118 (47) e2106115118;
https://doi.org/10.1073/pnas.2106115118
https://www.pnas.org/content/118/47/e2106115118
While cannabis is among the most used recreational drugs during pregnancy, the impact of maternal cannabis use (mCB) on fetal and child development remains unclear. Here, we assessed the effects of mCB on psychosocial and physiological measures in young children along with the potential relevance of the in utero environment reflected in the placental transcriptome. Children (∼3 to 6 y) were assessed for hair hormone levels, neurobehavioral traits on the Behavioral Assessment System for Children (BASC-2) survey, and heart rate variability (HRV) at rest and during auditory startle. For a subset of children with behavioral assessments, placental specimens collected at birth were processed for RNA sequencing. Hair hormone analysis revealed increased cortisol levels in mCB children. In addition, mCB was associated with greater anxiety, aggression, and hyperactivity. Children with mCB also showed a reduction in the high-frequency component of HRV at baseline, reflecting reduced vagal tone. In the placenta, there was reduced expression of many genes involved in immune system function including type I interferon, neutrophil, and cytokine-signaling pathways. Finally, several of these mCB-linked immune genes organized into coexpression networks that correlated with child anxiety and hyperactivity. Overall, our findings reveal a relationship between mCB and immune response gene networks in the placenta as a potential mediator of risk for anxiety-related problems in early childhood.
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Medical and Health Consequences of Marijuana
Jag H Khalsa
In book: Marijuana and the Cannabinoids November 2007
DOI: 10.1007/978-1-59259-947-9_10
Marijuana is the most frequently used illegal drug in the world today. Some 146 million people, or 3.7% of the population 15–64 years of age, consumed Cannabis in 2001–2003 (1). In the United States, 95 million Americans over the age of 12 have tried marijuana at least once. In 2002, an estimated 15 million Americans had used the drug in the month before a survey (2), representing 6.2% of the population age 12 years and older. Marijuana was used either alone or in combination with other drugs by 75% of the current illicit drug users. Approximately 2-3 million new users of marijuana are added each year, with about 1.1% becoming clinically dependent on it (3). In the case of young people, according to a recent survey of high school students known as Monitoring the Future, supported by the US National Institute on Drug Abuse (NIDA) and conducted yearly, at least 19% of 8th graders had tried marijuana at least once and 18% of 10th graders were “current” drug users (i.e., had used the drug within the past month before the survey). Among 12th graders, nearly 48% had tried marijuana at least once, and approx 21% were “current”marijuana users (4). Marijuana use by young people has increased or decreased at various times during the last decade, possibly as a result of its potency, which has been on the rise, although nonsignificantly from a 3% concentration of ?9-tetrahyrocannabinol (THC; marijuana’s active chemical constituent) in 1991 to 4.4% in 1997—possibly because of changes in the perceptions of youths about marijuana’s dangers or other unknown factors. Research suggests that marijuana use usually peaks in the late teens to early 20s, and then declines in later years (5).

Medical cannabis and insomnia in older adults with chronic pain: a cross-sectional study.
Sznitman, S. R., Vulfsons, S., Meiri, D., & Weinstein, G.
BMJ Supportive & Palliative Care, bmjspcare–2019–001938.(2020).
doi:10.1136/bmjspcare-2019-001938
Objectives Medical cannabis (MC) is increasingly being used for treatment of chronic pain symptoms. Among patients there is also a growing preference for the use of MC to manage sleep problems. The aim of the current study was to examine the associations between use of whole plant cannabis and sleep problems among chronic pain patients.
Methods A total of 128 individuals with chronic pain over the age of 50 years were recruited from the Rambam Institute for Pain Medicine in Haifa, Israel. Of them, 66 were MC users and 62 were non-users. Regression models tested the differences in sleep problems between the two groups. Furthermore, Pearson correlations between MC use measures (dose, length and frequency of use, number of strains used, tetrahydrocannabinol/cannabidiol levels) and sleep problems were assessed among MC users.
Results After adjustment for age, sex, pain level and use of sleep and anti-depressant medications, MC use was associated with less problems with waking up at night compared with non-MC use. No group differences were found for problems with falling asleep or waking up early without managing to fall back asleep. Frequent MC use was associated with more problems waking up at night and falling asleep.
Conclusions MC use may have an overall positive effect on maintaining sleep throughout the night in chronic pain patients. At the same time, tolerance towards potential sleep-inducing properties of MC may occur with frequent use. More research based on randomised control trials and other longitudinal designs is warranted.

Medical cannabis as an alternative for opioids for chronic pain: A case report.
Caldera, F. E.
SAGE Open Medical Case Reports, 8, 2050313X2090701.(2020).
doi:10.1177/2050313x20907015
Opioid medication–related deaths have increased to epidemic proportions in the last decade. This report describes a case of 43-year-old female with a traumatic brain injury who developed chronic pain and opioid dependence. The patient expressed concerns and wanted weaning off opioids. Recent legalization of medical marijuana in Pennsylvania allows us to try it as an alternative to opioids for chronic pain. Medical cannibus has risks associated with administration but is safer than opioids. Our patient was successfully weaned off her opioid medications with the help of medical cannibus and pain remained well controlled. More studies need to be done on using medical cannibus as an alternative to opioids.

Medical Cannabis Certification in a Large Pediatric Oncology Center.
Skrypek, M. M., Bostrom, B. C., & Bendel, A. E.
Children, 6(6), 79.(2019).
doi:10.3390/children6060079
In Minnesota, medical cannabis was approved for use in 2014. From July 2015 to February 2019, our center certified 103 pediatric and young adult patients for the use of medical cannabis under the qualifying conditions of cancer and treatment-related symptoms. Here, we provide a review of the literature on medical cannabis use in pediatric and young adult cancer patients. We also provide demographic data on our patients certified for medical cannabis. The most common diagnoses were leukemia/lymphoma (36%), brain tumors (37%), and malignant solid tumors (26%). The most common indications were chemotherapy-related nausea, pain, and cancer cachexia. The age range at certification was 1.4–28.7 years (median 15.3 years). The time from cancer diagnosis to certification ranged from 0.5–197 months (median 8.9 months). The majority (94%) were certified during their first line of treatment. In the 32 patients who died from recurrent or progressive cancer, the time from certification to death was 1.3–30.3 months (median 4.4 years). Despite requesting certification, a subset (24%) never had medical cannabis dispensed. In our experience, pediatric and young adult oncology patients are interested in medical cannabis to help manage treatment-related symptoms. Ongoing analysis of this data will identify the therapeutic efficacy of medical cannabis.

Medical cannabis for inflammatory bowel disease: real-life experience of mode of consumption and assessment of side-effects.
Naftali T, Bar-Lev Schleider L, Sklerovsky Benjaminov F, Lish I, Konikoff FM, Ringel Y
Eur J Gastroenterol Hepatol. 2019 Nov;31(11):1376-1381.
doi: 10.1097/MEG.0000000000001565.
OBJECTIVE:
Use of medical cannabis for improving symptoms of inflammatory bowel disease is increasing. However, reports on long-term outcomes are lacking. This prospective, observational study assessed the effects of licensed cannabis use among patients with inflammatory bowel disease.
METHODS:
Dose and mode of consumption, adverse events, use of other medications, and long-term effects were evaluated among 127 patients with inflammatory bowel disease using legalized medical cannabis. Blood count, albumin, and C-reactive protein were assessed before, 1 month, and at least 1 year after medical cannabis therapy was initiated. Questionnaires on disease activity, patient function, and signs of addiction were completed by patients and by a significant family member to assess its effects.
RESULTS:
The average dose used was 31 ± 15 g/month. The average Harvey-Bradshaw index improved from 14 ± 6.7 to 7 ± 4.7 (P < 0.001) during a median follow-up of 44 months (interquartile range, 24-56 months). There was a slight, but statistically significant, average weight gain of 2 kg within 1 year of cannabis use. The need for other medications was significantly reduced. Employment among patients increased from 65 to 74% (P < 0.05). We conclude that the majority of inflammatory bowel disease patients using cannabis are satisfied with a dose of 30 g/month. We did not observe negative effects of cannabis use on the patients' social or occupational status.
CONCLUSIONS:
Cannabis use by inflammatory bowel disease patients can induce clinical improvement and is associated with reduced use of medication and slight weight gain. Most patients respond well to a dose of 30 g/month, or 21 mg ?9-tetra- hydrocannabinol (THC) and 170 mg Cannabidiol (CBD) per day.

B]Medical Cannabis in Cancer Patients: A Survey of a Community Hematology Oncology Population[/B]
David M Macari 1, Bolanle Gbadamosi, Ishmael Jaiyesimi, Susanna Gaikazian
Am J Clin Oncol (2020) June 2
doi: 10.1097/COC.0000000000000718
Objectives: Cancer patients are using medical cannabis (MC) to address symptoms; however, little data exist to guide clinicians when counseling patients. We seek to define the patterns of MC use among cancer patients, as well as efficacy and safety of MC.
Materials and methods: Cancer patients attending oncology office visits at Beaumont Hospital, Michigan from July to December 2018 were anonymously surveyed. The survey included data regarding demographics, diagnosis, treatment, symptom burden, and MC use. Patients who reported MC use since their cancer diagnosis completed a section on patterns of use, efficacy, and safety.
Results: The response rate was 188 of 327 (57.5%). MC use was reported by 46 of 188 (24.5%). A median composite baseline symptom score ranging from 8 (best) to 32 (worst) was higher in patients using MC versus nonusers; 17.5 versus 14.4 (P<0.001). Pain was the symptom with the highest frequency of improvement 34/42 (81%), followed by appetite 34/44 (77.3%), and anxiety 32/44 (73%). MC improved the ability to tolerate treatment in 24/44 (54.5%). Cloudy thinking is the symptom that worsened the most 7/42 (16.7%), with decreased energy being experienced by 4/41 (9.8%) of the users.
Conclusions: MC was utilized by a significant portion of cancer patients in this sample, across age, diagnosis, stage, and treatment. Patients with a higher severity of baseline symptoms were more likely to use MC and report a favorable efficacy profile of MC. Minimal toxicity was reported in this cohort. Prospective studies are needed to define the efficacy and safety of MC.

Medical Cannabis for Intractable Epilepsy in Childhood: A Review
Bruria Ben-Zeev
Rambam Maimonides Med J. 2020 Jan; 11(1):
doi:*10.5041/RMMJ.10387
In recent years, cannabis has been gaining increasing interest in both the medical research and clinical fields, with regard to its therapeutic effects in various disorders. One of the major fields of interest is its role as an anticonvulsant for refractory epilepsy, especially in the pediatric population. This paper presents and discusses the current accumulated knowledge regarding artisanal cannabis and Epidiolex®, a United States Food and Drug Administration (FDA)-approved pure cannabidiol (CBD), in epilepsy management in pediatrics, by reviewing the literature and raising debate regarding further research directions.

Medical cannabis for severe treatment resistant epilepsy in children: a case-series of 10 patients
Rayyan Zafar, Anne Schlag, Lawrence Phillips, David J Nutt
BMJ Paediatrics Open 2021;5:e001234.
doi: 10.1136/bmjpo-2021-001234
https://bmjpaedsopen.bmj.com/content...01234.full.pdf
Objectives To report the findings of a case-series of 10 children suffering with intractable epilepsies in the UK to determine the feasibility for using whole-plant cannabis medicines to treat seizures in children.
Setting This study was conducted retrospectively through collecting clinical data from caretakers and clinicians on study outcome variables. Participants were recruited through the MedCann Support and End our Pain charity groups which are patient representative groups that support children who are using medical cannabis to treat their epilepsies. Medicines were prescribed to patients by clinicians in both National Health Service and private medical practices. Follow-up calls were conducted throughout the period January 2021 to May 2021 to keep data recorded up to date.
Participants Ten children, 18 years old or under, with intractable epilepsies were recruited from two charities. There were no limitations on diagnosis, sex or ethnic origin.
Interventions Participants were treated with a range of whole-plant medical cannabis oils. Individual dosing regimens were determined by clinicians.
Primary outcome measure The primary outcome measure was seizure frequency.
Results Seizure frequency across all 10 participants reduced by 86% with no significant adverse events. Participants reduced use of antiepileptic drugs from an average of seven to one following treatment with medical cannabis. We also noted significant financial costs of £874 per month to obtain these medicines through private prescriptions.
Conclusions This study establishes the feasibility of whole-plant medical cannabis as an effective and well-tolerated medicine for reducing seizure frequency in children suffering with intractable epilepsies. These findings justify the potential value of further research into the reported therapeutic benefit of whole-plant Medicinal cannabis products.

Medical Cannabis in Children
Adi Aran, and Dalit Cayam-Rand
Rambam Maimonides Med J January 2020 Volume 11 Issue 1
doi:*10.5041/RMMJ.10386
https://www.ncbi.nlm.nih.gov/pmc/art...11-1-e0003.pdf
The use of medical cannabis in children is rapidly growing. While robust evidence currently exists only for pure cannabidiol (CBD) to treat specific types of refractory epilepsy, in most cases, artisanal strains of CBDrich medical cannabis are being used to treat children with various types of refractory epilepsy or irritability associated with autism spectrum disorder (ASD). Other common pediatric disorders that are being considered for cannabis treatment are Tourette syndrome and spasticity. As recreational cannabis use during youth is associated with serious adverse events and medical cannabis use is believed to have a relatively high placebo effect, decisions to use medical cannabis during childhood and adolescence should be made with caution and based on evidence. This review summarizes the current evidence for safety, tolerability, and efficacy of medical cannabis in children with epilepsy and in children with ASD. The main risks associated with use of ?9-tetrahydrocannabinol (THC) and CBD in the pediatric population are described, as well as the debate regarding the use of whole-plant extract to retain a possible “entourage effect” as opposed to pure cannabinoids that are more standardized and reproducible.

Medical Cannabis Used as an Alternative Treatment for Chronic Pain Demonstrates Reduction in Chronic Opioid Use – A Prospective Study Gregory Benedict, Annas Sabbagh, and Till Conermann Pain Physician 2022; 25:E113-E119 www.painphysicianjournal.com
https://www.painphysicianjournal.com.. .e=NzQwOA%3D%3D Background: Chronic opioid therapy (COT) has been used to treat many chronic pain conditions even with poor evidence for its long-term effectiveness. Medical cannabis has emerged with certain pain-relieving properties, which has led to questions as to its’ potential application, especially in relation to its effect on opioid use.
Objectives: This study investigates a proposed clinical context in offering medical cannabis as a treatment for chronic pain for those already using chronic opioid therapy. It then details patients’ daily morphine milligram equivalent (MME) usage.
Study Design: This single-center prospective study follows a group of patients trialing medical cannabis treatment for chronic pain that is already using COT in order to determine individual efficacy. Continued medical cannabis treatment was a decision made by the patient, after trialing medical cannabis, to either continue medical cannabis along with COT at a reduced daily MME, or to revert back to their previous COT regimen.
Setting: This study was performed at the Allegheny Health Network Institute for Pain Medicine in Pittsburgh, Pennsylvania. The state of Pennsylvania legalized medical cannabis in April of 2016, and it became available to patients in February of 2018 through medical dispensaries.
Methods: One hundred and fifteen patients met the inclusion criteria, with the majority of those excluded due to not being treated with COT. Of the 115 who chose to undergo a medical cannabis trial in addition to their COT, 75 chose to remain certified for medical cannabis as they had significant pain relief and subsequently weaned down on opioids. Additionally, of the 115 choosing to undergo a medical cannabis trial, 30 chose to be decertified due to ineffectiveness or side effects, and those were placed back on their COT regimen. The other 10 were not included for other denoted reasons. Compliance was monitored through urine drug screens (UDS).
Results: There was a 67.1% average decrease in daily MME/patient from 49.9 to 16.4 MME at the first follow-up. There was a 73.3% decrease in MME at second follow-up from 49.9 to 13.3 MME with an ANOVA analysis denoting a significant difference of P < 0.0001.
Limitations: The period of follow-up presented at this point includes their first 6 months of treatment with medical cannabis and COT concomitantly.
Conclusions: Presenting medical cannabis to chronic pain patients on COT should be done in the context of a patient choice between medical cannabis WITH decrement of COT or continued current dose of COT in order to maximize effectiveness in opioid reduction as well as to limit polypharmacy concerns regarding medical cannabis. Allowing for a temporary short-term period where patients may trial medical cannabis, while concomitantly gradually weaning their COT, is also essential in determining medical cannabis’ individual effectiveness for that patient’s specific type of chronic pain, which should serve to maximize long-term opioid reduction results and hence decrease opioid-related overdose deaths. .

Medical Marijuana: Clearing Away the Smoke
Igor Grant, J. Hampton Atkinson, Ben Gouaux and Barth Wilsey
The Open Neurology Journal, 2012, 6, 18-25
Recent advances in understanding of the mode of action of tetrahydrocannabinol and related cannabinoid in-gredients of marijuana, plus the accumulating anecdotal reports on potential medical benefits have spurred increasing re-search into possible medicinal uses of cannabis. Recent clinical trials with smoked and vaporized marijuana, as well as other botanical extracts indicate the likelihood that the cannabinoids can be useful in the management of neuropathic pain, spasticity due to multiple sclerosis, and possibly other indications. As with all medications, benefits and risks need to be weighed in recommending cannabis to patients. We present an algorithm that may be useful to physicians in determining whether cannabis might be recommended as a treatment in jurisdictions where such use is permitted

Medical Marijuana Efficacy: A Survey
Ngugi M. Kinyungu, Josiah Gikungi, Ariel Davson
Open Journal of Anesthesiology, 2022, 12, 91-98
https://www.scirp.org/pdf/ojanes_2022030716163914.pdf
Background: Medical marijuana use has seen a rise and acceptance in the last several years and while its efficacy has been spoken and discussed in lore, data on efficacy still remains scarce.
Aim: We conducted a survey on chronic pain patients certified for medical marijuana to assess pain relief and efficacy.
Findings: Our survey found that the pain score with the use of medical marijuana was halved in our 20 respondents and sleep was improved. Overall function improved.
Conclusion: The survey shows that medical marijuana is efficacious in chronic pain patients and further studies need to be done and access to the medication needs to be improved so as to benefit more patients.

Medical Marijuana for Digestive Disorders: High Time to Prescribe?
Mark E. Gerich, MD, Robert W. Isfort, Bryan Brimhall, and Corey A. Siegel,
The American Journal of GASTROENTEROLOGY VOL. 110 | FEB 2015
ttps://static1.squarespace.com/static/57faf399cd0f68a69b4c670c/t/5839e6a2be6594d45acf9e82/1480189604172/Digestive+disease+review+natur e.pdf
doi: 10.1038/ajg.2014.245.
The use of recreational and medical marijuana is increasingly accepted by the general public in the United States. Along with growing interest in marijuana use has come an understanding of marijuana’s effects on normal physiology and disease, primarily through elucidation of the human endocannabinoid system. Scientifi c inquiry into this system has indicated potential roles for marijuana in the modulation of gastrointestinal symptoms and disease. Some patients with gastrointestinal disorders already turn to marijuana for symptomatic relief, often without a clear understanding of the risks and benefi ts of marijuana for their condition. Unfortunately, that lack of understanding is shared by healthcare providers. Marijuana’s federal legal status as a Schedule I controlled substance has limited clinical investigation of its effects. There are also potential legal ramifi cations for physicians who provide recommendations for marijuana for their patients. Despite these constraints, as an increasing number of patients consider marijuana as a potential therapy for their digestive disorders, health-care providers will be asked to discuss the issues surrounding medical marijuana with their patients.

Medical Marijuana Is the Cart Before the Horse?
Deepak Cyril D'Souza, Mohini Ranganathan
JAMA June 23/30, 2015 Volume 313, Number 24
DOI: 10.1001/jama.2015.6407
This Editorial discusses some of the medical and legal considerations surrounding use of medical marijuana and cannabinoid drugs.
There is a pressing need to develop newmedications formany debilitating conditions. Novel approaches based on marijuana or its constituent cannabinoids, if proven, could be added to the armamentarium of available treatments. In this issue of JAMA, reviews by Whiting et al1 and Hill2 provide detailed assessment of the pharmacology, indications, benefits, adverse effects, and laws related tomedicalmarijuana and the cannabinoids, and the results and conclusions are consistent. There is some evidence to support the use of marijuana for nausea and vomiting related to chemotherapy, specific pain syndromes, and spasticity from multiple sclerosis. However, for most other indications that qualify by state law for use ofmedicalmarijuana, such as hepatitis C, Crohn disease, Parkinson disease, or Tourette syndrome, the evidence supporting its use is of poor quality. State laws vary widely regarding conditions for which marijuana is approved and the dispensable legal limit. Both reviews raise important issues worthy of further discussion

Medical use of cannabis and cannabinoids
Prepared by Wayne Hall
European Monitoring Centre for Drugs and Drug Addiction
Questions and answers for policymaking December 2018
doi:10.2810/979004
The medical use of preparations derived from the Cannabis sativa plant has a long history. However, by the twentieth century, medical use of cannabis had largely declined, and its consumption for medical purposes was already very limited when in 1961 cannabis was included in the United Nations Single Convention on Narcotic Drugs and classified as a drug that had no medical uses (see ‘A brief history of the medical use of cannabis and cannabinoids’, on page 7). In the past 20 years, however, there has been a resurgence of patient interest in using cannabis and cannabinoids to treat a variety of conditions, including chronic pain, cancer pain, depression, anxiety disorders, sleep disturbances and neurological disorders, the symptoms of which are reportedly improved by using cannabis (NASEM, 2017).
Increased patient interest in the medical use of cannabis has been accompanied by renewed scientific interest in the medical use of substances found in the cannabis plant, namely cannabinoids. This followed the discovery, in the early 1990s, of a cannabinoid system in the human brain and body that was implicated in the control of important biological functions, such as cognition, memory, pain, sleep and immune functioning. However, the classification of cannabis as a drug without medical uses made it difficult to conduct clinical research (NASEM, 2017).
In the mid-1990s, citizens in several US states responded to patient demand for cannabis by passing referenda that legalised the medical use of cannabis for people with a variety of illnesses, such as chronic pain, terminal cancer and multiple sclerosis. A similar approach was later adopted in many other US states. In 1999, Canada introduced a medical cannabis programme that expanded over the subsequent decades in response to court decisions. In the early 2000s, Israel (2001) and the Netherlands (2003), and later other countries, such as Switzerland (2011), Czechia (2013), Australia (2016) and Germany (2017), legislated to allow the medical use of cannabis under specified conditions. Over a similar period, clinical trials have provided the basis for granting an authorisation for marketing in many EU Member States of a medicinal product, primarily based on cannabis extracts, that has proven effective in the treatment of muscle spasticity due to multiple sclerosis.
Most EU countries now allow, or are considering allowing, the medical use of cannabis or cannabinoids in some form. However, the approaches taken vary widely in terms of both the products allowed and the regulatory frameworks governing their provision. In this context, this report aims to provide a brief overview of current knowledge and the latest developments relating to medical use of cannabis and cannabinoids.
The report is intended to help a broad audience of interested readers, such as policymakers, practitioners, potential patients and the public, to understand the scientific, clinical and regulatory issues that arise when consideration is given to making cannabis or cannabinoids available to treat the symptoms of medical illnesses.

Medical use of cannabis for glaucoma
Paul Rafuse, Yvonne M. Buys
Canadian Journal of Ophthalmology 54(1):7-8 February 2019
DOI: 10.1016/j.jcjo.2018.11.001
The clinical utility of cannabis (sometimes referred to as marijuana or marihuana) for the treatment of glaucoma is limited by the inability to separate the potential clinical action from the undesirable neuropsychological and behavioural effects. The Canadian Ophthalmological Society does not support the medical use of cannabis for the treatment of glaucoma due to the short duration of action, the incidence of undesirable psychotropic and other systemic side effects, and the absence of scientific evidence showing a beneficial effect on the course of the disease. This is in contrast to other more effective and less harmful medical, laser, and surgical modalities for the treatment of glaucoma.
(I like how the latter suggests the elderly (and of course younger people with alzheimer) can use hash or flower or oil or edibles as they like rather than needing a specific extract from some pharma co. It’s like vegetables and fruits, a diverse diet of cannabis can help keep you healthy, don’t need supplements in pill form.)

Medicinal cannabis
Geke A H van den Elsen, Florentien E M In 't Hout, Marjan de Vries, Robbert-Jan Verkes
Nederlands tijdschrift voor geneeskunde 163 May 2019
https://www.researchgate.net/publica...cinal_cannabis
Medicinal cannabis The use of cannabis products for medical purposes is rapidly increasing in the Netherlands. Studies suggest that these products have positive effects in the treatment of chronic neuropathic pain, multiple-sclerosis-related spasticity, certain epilepsy syndromes and chemotherapy-related nausea and vomiting. The interpretation of these findings is impeded by methodological shortcomings, such as a small number of participants. Differences in product composition and dosage form mean that study resultsare often not directly comparable. Responsible prescribing requires that the patient be very well informed about the goal of treatment, alternative forms of treatment and the side effects. A history of psychosis, relevant cardiac co-morbidity, recurrent falls, addiction problems, pregnancy and breastfeeding are all contra-indications to the use of medical cannabis.

Medicinal cannabis
Geke A H van den Elsen, Florentien E M In 't Hout, Marjan de Vries, Robbert-Jan Verkes
Nederlands tijdschrift voor geneeskunde 163 May 2019
https://www.researchgate.net/publica...cinal_cannabis
Medicinal cannabis The use of cannabis products for medical purposes is rapidly increasing in the Netherlands. Studies suggest that these products have positive effects in the treatment of chronic neuropathic pain, multiple-sclerosis-related spasticity, certain epilepsy syndromes and chemotherapy-related nausea and vomiting. The interpretation of these findings is impeded by methodological shortcomings, such as a small number of participants. Differences in product composition and dosage form mean that study resultsare often not directly comparable. Responsible prescribing requires that the patient be very well informed about the goal of treatment, alternative forms of treatment and the side effects. A history of psychosis, relevant cardiac co-morbidity, recurrent falls, addiction problems, pregnancy and breastfeeding are all contra-indications to the use of medical cannabis.

Medical Cannabis for Gilles de la Tourette Syndrome: An Open- Label Prospective Study
Saar Anis , Corinne Zalomek, Amos D. Korczyn, Alina Rosenberg, Nir Giladi, and Tanya Gurevich
Behavioural Neurology Volume 2022, 10 pages
DOI: 10.1155/2022/5141773
https://downloads.hindawi.com/journa...22/5141773.pdf
Objectives. Assessing the effectiveness and tolerability of medical cannabis (MC) treatment on Gilles de la Tourette syndrome (GTS) patients. Methods. We report on an open-label, prospective study on the effect of MC on adult GTS patients. MC mode of use was decided by the treating neurologist and the patient. Δ9-Tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) content within MC product and monthly dose were titrated during the study. Following treatment initiation, patients were assessed after 4 and 12 weeks for efficacy, tolerability, and side effects. Results. Eighteen patients entered the study. Baseline Yale Global Tic Severity Scale- (YGTSS) Total (range 0-100) was 60:3 ± 17:1. Three patients did not reach the end of follow-up period. The most common mode of administration was smoking (80%). Following twelve weeks of treatment, a significant 38% average reduction (p = 0:002) of YGTSS-Total and a 20% reduction (p = 0:043) of Premonitory Urge for Tic Scale (PUTS) were observed. Common side effects were dry mouth (66.7%), fatigue (53.3%), and dizziness (46.7%). Three patients suffered from psychiatric side effects including worsening of obsessive compulsive disorder (stopped treatment), panic attack, and anxiety (resolved with treatment modification). Six patients (40%) reported cognitive side effects regarding time perception, visuospatial disorientation, confusion, slow processing speed, and attention. Conclusions. MC treatment demonstrates good efficacy and tolerability in adult GTS patients. Predilection for smoking rather than using oil drops requires further comparative studies to evaluate the efficacy of each. Cognitive and psychiatric side effects have to be monitored and addressed.

Medicinal Cannabis: History, Pharmacology, And Implications for the Acute Care Setting
Mary Barna Bridgeman, Daniel T. Abazia
https://www.ncbi.nlm.nih.gov/pmc/art...ptj4203180.pdf
Medicinal cannabis, or medicinal marijuana, is a therapy that has garnered much national attention in recent years. Controversies surrounding legal, ethical, and societal implications associated with use; safe administration, packaging, and dispensing; adverse health consequences and deaths attributed to marijuana intoxication; and therapeutic indications based on limited clinical data represent some of the complexities associated with this treatment. Marijuana is currently recognized by the U.S. Drug Enforcement Agency’s (DEA’s) Comprehensive Drug Abuse Prevention and Control Act (Controlled Substances Act) of 1970 as a Schedule I controlled substance, defined as
having a high potential for abuse, no currently accepted medicinal use in treatment in the United States, and a lack of accepted safety data for use of the treatment under medical supervision. Cannabis is the most commonly cultivated, trafficked, and abused illicit drug worldwide; according to the World Health Organization (WHO), marijuana consumption has an annual prevalence rate of approximately 147 million individuals or nearly 2.5% of the global population.
In 2014, approximately 22.2 million Americans 12 years of age or older reported current cannabis use, with 8.4% of this population reporting use within the previous month.3General cannabis use, both for recreational and medicinal purposes, has garnered increasing acceptance across the country as evidenced by legislative actions, ballot measures, and public opinion polls; an October 2016 Gallup poll on American’s views on legalizing cannabis indicated that 60% of the population surveyed believed the substance should be legalized.
Further, a recent Quinnipiac University poll concluded 54% of American voters surveyed would favor the legalization of cannabis without additional constraints, while 81% of respondents favored legalization of cannabis for medicinal purposes.
Limited data suggest that health care providers also may consider this therapy in certain circumstances. In the United States, cannabis is approved for medicinal use in 28 states, the District of Columbia, Guam, and Puerto Rico as of January 2017. The use and acceptance of medicinal cannabis continues to evolve, as shown by the growing number of states now permitting use for specific medical indications. The Food and Drug Administration (FDA) has considered how it might support the scientific rigor of medicinal cannabis claims, and the review of public data regarding safety and abuse potential is ongoing. The purpose of this article is to review the historical significance of the use of medicinal cannabis and to discuss its pharmacology, pharmacokinetics, and select evidence on medicinal uses, as well as to describe the implications of evolving medicinal cannabis regulations and their effects on the acute care hospital setting.

Medicinal Cannabis: In Vitro Validation of Vaporizers for the Smoke-Free Inhalation of Cannabis.
Lanz, C., Mattsson, J., Soydaner, U., & Brenneisen, R.
PLOS ONE, 11(1), (2016). e0147286
doi:10.1371/journal.pone.0147286
Inhalation by vaporization is a promising application mode for cannabis in medicine. An in vitro validation of 5 commercial vaporizers was performed with THC-type and CBD-type cannabis. Gas chromatography/mass spectrometry was used to determine recoveries of total THC (THCtot) and total CBD (CBDtot) in the vapor. High-performance liquid chromatography with photodiode array detection was used for the quantitation of acidic cannabinoids in the residue and to calculate decarboxylation efficiencies. Recoveries of THCtot and CBDtot in the vapor of 4 electrically-driven vaporizers were 58.4 and 51.4%, 66.8 and 56.1%, 82.7 and 70.0% and 54.6 and 56.7% for Volcano Medic1, Plenty Vaporizer1, Arizer Solo1 and DaVinci Vaporizer1, respectively. Decarboxylation efficiency was excellent for THC ( 97.3%) and CBD ( 94.6%). The gas-powered Vape-or-Smoke™ showed recoveries of THCtot and CBDtot in the vapor of 55.9 and 45.9%, respectively, and a decarboxylation efficiency of 87.7 for both cannabinoids. However, combustion of cannabis was observed with this device. Temperature-controlled, electrically-driven vaporizers efficiently decarboxylate inactive acidic cannabinoids and reliably release their corresponding neutral, active cannabinoids. Thus, they offer a promising application mode for the safe and efficient administration of medicinal cannabis.

Medicinal cannabis pilot programme (Denmark)
https://laegemiddelstyrelsen.dk/en/special/medicinal-cannabis-/medicinal-cannabis-pilot-programme/
On 1 January 2018, a medicinal cannabis pilot programme entered into force. The programme allows doctors to prescribe new types of cannabis products that were not legal in Denmark before. The pilot programme runs until 31 December 2025.
The purpose of the pilot programme is to offer patients a lawful way of testing treatment with medicinal cannabis if they have experienced no benefits from authorised medicines. That is the intention with the programme.
The products available in the pilot programme depend on the manufacturers of cannabis products. They apply for admission of cannabis products to the programme with the aim of making these products available for prescribers. The available cannabis products are therefore likely to change during the course of the pilot programme.
The currently available cannabis products can be seen on Medicine Prices, www.medicinpriser.dk.
The cannabis products included in the pilot programme are not authorised medicines – not in Denmark or any other country. Usually, the products are not tested in clinical trials like authorised medicines. So compared to authorised medicines, doctors have limited evidence of the effects and side effects. One of the implications thereof is that doctors must accept full responsibility for the prescription of a product, e.g. by determining the dose for the individual patient. They can neither consult a package leaflet nor a summary of product characteristics to assess what beneficial effects or side effects the individual patient is likely to expect

Medicinal cannabis use in palliative care
Anthony Herbert, Janet Hardy
AJGP Vol. 50, No. 6, June 2021
https://www1.racgp.org.au/getattachm...tive-care.aspx
Background
Legislative change in recent years allows the prescription of medicinal cannabis for patients receiving palliative care. There appears to be widespread community support of its use in this context.
Objective
The aim of this article is to provide an overview of current research on the use of medicinal cannabis within the palliative care context for both children and adults.
Discussion
The evidence needed to support the use of medicinal cannabis for symptom control is limited and still emerging. It is unlikely that medicinal cannabis will be used as a first-line agent for the management of pain, nausea and other specific symptoms, but it may have an adjuvant or complementary role in symptom management within palliative care. Consistent with the Therapeutic Goods Administration advice, these authors recommend that general practitioners encourage patients to participate in trials or programs that objectively monitor benefit and toxicity of medicinal cannabis.

Medical Cannabis Use in Palliative Care: Review of Clinical Effectiveness and Guidelines – An Update
Erika MacDonald, Kelly Farrah
CADTH; 2019 Oct. (CADTH rapid response report: summary with critical appraisal).
https://www.ncbi.nlm.nih.gov/books/N..._NBK551867.pdf
Research Questions
1. What is the clinical effectiveness of medical cannabis products for symptom control in adult palliative care patients?
2. What are the evidence-based guidelines regarding medical cannabis products for symptom control in adult palliative care patients?
Key Findings The clinical effectiveness of medical cannabis for symptom control in adult palliative care patients is unclear, due to a lack of quality and quantity of evidence; this lack of evidence applies to the cannabis plant, its extracts and synthetic cannabinoids. From a systematic review of nine randomized controlled trials, low quality evidence suggests that in patients with HIV, dronabinol (a synthetic cannabinoid) may be more effective than placebo for appetite and weight gain, at the expense of increased risk of psychiatric adverse effects. In patients with cancer, dronabinol may be less effective than megestrol for improvement in appetite, weight gain and health-related quality of life, and may increase risk of withdrawal due to adverse events as compared to megestrol. Similarly, in patients with HIV, dronabinol may be less effective than megestrol for weight gain. Two evidence-based guidelines address the use of medical cannabis in a palliative care setting. The first evidence-based guideline explicitly recommends against the use of medical cannabis as a first or second line option for palliative cancer pain. The guideline suggests that it could be considered in the case of refractory symptoms and with careful consideration of potential risks. The second evidence-based guideline similarly recommends that medical cannabis only be used in the palliative care setting when other treatments have failed, and after consideration of the potential for adverse events and drug interactions.
Methods Literature Search Methods This report updates and expands on a previous CADTH summary of abstracts report.9 For the current report, a limited literature search was conducted by an information specialist on key resources including PubMed, the Cochrane Library, the University of York Centre for Reviews and Dissemination (CRD) databases, the websites of Canadian and major international health technology agencies, as well as a focused Internet search. The search strategy was comprised of both controlled vocabulary, such as the National Library of Medicine’s MeSH (Medical Subject Headings), and keywords. The main search concepts were cannabis and palliative care. No filters were applied to limit the retrieval by study type. Where possible, retrieval was limited to the human population. The search was also limited to English language documents published between January 1, 2018 and September 24, 2019. The literature search for the previous summary of abstracts report was limited to English language documents published between January 1, 2013 and August 17, 2018

Medical cannabis or cannabinoids for chronic non-cancer and cancer related pain: a systematic review and meta-analysis of randomised clinical trials Li Wang,Patrick J Hong, Curtis May, Yasir Rehman, Yvgeniy Oparin, Chris J Hong, Brian Y Hong, Mahmood AminiLari, Lucas Gallo, Alka Kaushal, Samantha Craigie, Rachel J Couban, Elena Kum, Harsha Shanthanna, Ira Price, Suneel Upadhye, Mark A Ware, Fiona Campbell, Rachelle Buchbinder, Thomas Agoritsas, Jason W Busse BMJ 2021;373:n1034 DOI: 10.1136/bmj.n1034
https://www.bmj.com/content/bmj/374/bmj.n1034.full.pdf
OBJECTIVE To determine the benefits and harms of medical cannabis and cannabinoids for chronic pain.
DESIGN Systematic review and meta-analysis.
DATA SOURCES MEDLINE, EMBASE, AMED, PsycInfo, CENTRAL, CINAHL, PubMed, Web of Science, Cannabis-Med, Epistemonikos, and trial registries up to January 2021.
STUDY SELECTION Randomised clinical trials of medical cannabis or cannabinoids versus any non-cannabis control for chronic pain at ≥1 month follow-up. DATA EXTRACTION AND SYNTHESIS Paired reviewers independently assessed risk of bias and extracted data. We performed random-effects models meta-analyses and used GRADE to assess the certainty of evidence.
RESULTS A total of 32 trials with 5174 adult patients were included, 29 of which compared medical cannabis or cannabinoids with placebo. Medical cannabis was administered orally (n=30) or topically (n=2). Clinical populations included chronic non-cancer pain (n=28) and cancer related pain (n=4). Length of follow-up ranged from 1 to 5.5 months. Compared with placebo, non-inhaled medical cannabis probably results in a small increase in the proportion of patients experiencing at least the minimally important difference (MID) of 1 cm (on a 10 cm visual analogue scale (VAS)) in pain relief (modelled risk difference (RD) of 10% (95% confidence interval 5% to 15%), based on a weighted mean difference (WMD) of −0.50 cm (95% CI −0.75 to −0.25 cm, moderate certainty)). Medical cannabis taken orally results in a very small improvement in physical functioning (4% modelled RD (0.1% to 8%) for achieving at least the MID of 10 points on the 100-point SF-36 physical functioning scale, WMD of 1.67 points (0.03 to 3.31, high certainty)), and a small improvement in sleep quality (6% modelled RD (2% to 9%) for achieving at least the MID of 1 cm on a 10 cm VAS, WMD of −0.35 cm (−0.55 to −0.14 cm, high certainty)). Medical cannabis taken orally does not improve emotional, role, or social functioning (high certainty). Moderate certainty evidence shows that medical cannabis taken orally probably results in a small increased risk of transient cognitive impairment (RD 2% (0.1% to 6%)), vomiting (RD 3% (0.4% to 6%)), drowsiness (RD 5% (2% to 8%)), impaired attention (RD 3% (1% to 8%)), and nausea (RD 5% (2% to 8%)), but not diarrhoea; while high certainty evidence shows greater
increased risk of dizziness (RD 9% (5% to 14%)) for trials with <3 months follow-up versus RD 28% (18% to 43%) for trials with ≥3 months followup; interaction test P=0.003; moderate credibility of subgroup effect). CONCLUSIONS Moderate to high certainty evidence shows that non-inhaled medical cannabis or cannabinoids results in a small to very small improvement in pain relief, physical functioning, and sleep quality among patients with chronic pain, along with several transient adverse side effects, compared with placebo. The accompanying BMJ Rapid Recommendation provides contextualised guidance based on this body of evidence.

Medical Marijuana Efficacy: A Survey
Ngugi M. Kinyungu, Josiah Gikungi, Ariel Davson
Open Journal of Anesthesiology, 2022, 12, 91-98
https://www.scirp.org/pdf/ojanes_2022030716163914.pdf
Background: Medical marijuana use has seen a rise and acceptance in the last several years and while its efficacy has been spoken and discussed in lore, data on efficacy still remains scarce.
Aim: We conducted a survey on chronic pain patients certified for medical marijuana to assess pain relief and efficacy.
Findings: Our survey found that the pain score with the use of medical marijuana was halved in our 20 respondents and sleep was improved. Overall function improved.
Conclusion: The survey shows that medical marijuana is efficacious in chronic pain patients and further studies need to be done and access to the medication needs to be improved so as to benefit more patients. .

Medicinal Properties of Cannabinoids, Terpenes, and Flavonoids in Cannabis, and Benefits in Migraine, Headache, and Pain: An Update on Current Evidence and Cannabis Science
Eric P Baron
Headache The Journal of Head and Face Pain 58(7):1139-1186 August 2018
DOI: 10.1111/head.13345
Background.—Comprehensive literature reviews of historical perspectives and evidence supporting cannabis/ cannabinoids in the treatment of pain, including migraine and headache, with associated neurobiological mechanisms of pain modulation have been well described. Most of the existing literature reports on the cannabinoids ?9 -tetrahydrocannabinol (THC) and cannabidiol (CBD), or cannabis in general. There are many cannabis strains that vary widely in the composition of cannabinoids, terpenes, flavonoids, and other compounds. These components work synergistically to produce wide variations in benefits, side effects, and strain characteristics. Knowledge of the individual medicinal properties of the cannabinoids, terpenes, and flavonoids is necessary to cross-breed strains to obtain optimal standardized synergistic compositions. This will enable targeting individual symptoms and/or diseases, including migraine, headache, and pain. Objective.—Review the medical literature for the use of cannabis/cannabinoids in the treatment of migraine, headache, facial pain, and other chronic pain syndromes, and for supporting evidence of a potential role in combatting the opioid epidemic. Review the medical literature involving major and minor cannabinoids, primary and secondary terpenes, and flavonoids that underlie the synergistic entourage effects of cannabis. Summarize the individual medicinal benefits of these substances, including analgesic and anti-inflammatory properties. Conclusion.—There is accumulating evidence for various therapeutic benefits of cannabis/cannabinoids, especially in the treatment of pain, which may also apply to the treatment of migraine and headache. There is also supporting evidence that cannabis may assist in opioid detoxification and weaning, thus making it a potential weapon in battling the opioid epidemic. Cannabis science is a rapidly evolving medical sector and industry with increasingly regulated production standards. Further research is anticipated to optimize breeding of strain-specific synergistic ratios of cannabinoids, terpenes, and other phytochemicals for predictable user effects, characteristics, and improved symptom and diseasetargeted therapies.

Medicinal Use of Cannabis in Children and Pregnant Women
Koren G, Cohen R
Rambam Maimonides Med J 2020;11 (1)
doi:*10.5041/RMMJ.10382
https://www.ncbi.nlm.nih.gov/pmc/art...11-1-e0005.pdf
The increasing medicinal use of cannabis during recent years has largely overlooked children and pregnant women due to litigious and ethical concerns. However, over the last few years medicine has observed increasing numbers of children treated with cannabis for autism spectrum disorder (ASD) and fetal alcohol spectrum disorder (FASD), and pregnant women treated for hyperemesis gravidarum (HG). This review provides an account of major findings discovered through this research. Specifically, cannabis may offer therapeutic advantages to behavioral symptoms of autism spectrum disorder and fetal alcohol spectrum disorder, and to the severe nausea and vomiting in hyperemesis gravidarum. The use of medical cannabis in children and pregnant women should be further discussed and researched in this patient population.

Medicinal Uses of Marijuana and Cannabinoids
Franjo Grotenhermen Dr. med. & Kirsten Mu?ller-Vahl
Critical Reviews in Plant Sciences, Pages 1-27 Published online: 17 Feb 2017
DOI:10.1080/07352689.2016.1265360
In the past two decades, there has been increasing interest in the therapeutic potential of cannabis and single cannabinoids, mainly cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC). THC and cannabis products rich in THC exert their effects mainly through the activation of cannabinoid receptors (CB1 and CB2). Since 1975, 140 controlled clinical trials using different cannabinoids or whole-plant preparations for the treatment of a large number of disorders and symptoms have been conducted. Results have led to the approval of cannabis-based medicines [dronabinol, nabilone, and the cannabis extract nabiximols (Sativex®, THC:CBD = 1:1)] as well as cannabis flowers in several countries. Controlled clinical studies provide substantial evidence for the use of cannabinoid receptor agonists in cancer chemotherapy induced nausea and vomiting, appetite loss and cachexia in cancer and HIV patients, neuropathic and chronic pain, and in spasticity in multiple sclerosis. In addition, there is also some evidence suggesting a therapeutic potential of cannabis-based medicines in other indications including Tourette syndrome, spinal cord injury, Crohn's disease, irritable bowel syndrome, and glaucoma. In several other indications, small uncontrolled and single-case studies reporting beneficial effects are available, for example in posttraumatic stress disorder, attention deficit hyperactivity disorder, and migraine. The most common side effects of THC and cannabis-based medicines rich in THC are sedation and dizziness (in more than 10% of patients), psychological effects, and dry mouth. Tolerance to these side effects nearly always develops within a short time. Withdrawal symptoms are hardly ever a problem in the therapeutic setting. In recent years there is an increasing interest in the medical use of CBD, which exerts no intoxicating side effects and is usually well-tolerated. Preliminary data suggest promising effects in the treatment of anxiety disorders, schizophrenia, dystonia, and some forms of epilepsy. This review gives an overview on clinical studies which have been published over the past 40 years.


Medicinal cannabis in Thailand
1-year experience after legalization
Zinboonyahgoon, Nantthasorn; Srisuma, Sahaphume; Limsawart, Wirun; Rice, Andrew S.C.; Suthisisang, Chuthamanee
PAIN: September 30, 2020 -
doi: 10.1097/j.pain.0000000000001936
No Abstract available

Medicinal Properties of Cannabinoids, Terpenes, and Flavonoids in Cannabis, and Benefits in Migraine, Headache, and Pain: An Update on Current Evidence and Cannabis Science.
Baron, E. P.
Heaache: The Journal of Head and Face Pain, 58(7), 1139–1186.(2018).
doi:10.1111/head.13345
Background.—Comprehensive literature reviews of historical perspectives and evidence supporting cannabis/ cannabinoids in the treatment of pain, including migraine and headache, with associated neurobiological mechanisms of pain modulation have been well described. Most of the existing literature reports on the cannabinoids ?9 - tetrahydrocannabinol (THC) and cannabidiol (CBD), or cannabis in general. There are many cannabis strains that vary widely in the composition of cannabinoids, terpenes, flavonoids, and other compounds. These components work synergistically to produce wide variations in benefits, side effects, and strain characteristics. Knowledge of the individual medicinal properties of the cannabinoids, terpenes, and flavonoids is necessary to cross-breed strains to obtain optimal standardized synergistic compositions. This will enable targeting individual symptoms and/or diseases, including migraine, headache, and pain. Objective.—Review the medical literature for the use of cannabis/cannabinoids in the treatment of migraine, headache, facial pain, and other chronic pain syndromes, and for supporting evidence of a potential role in combatting the opioid epidemic. Review the medical literature involving major and minor cannabinoids, primary and secondary terpenes, and flavonoids that underlie the synergistic entourage effects of cannabis. Summarize the individual medicinal benefits of these substances, including analgesic and anti-inflammatory properties.
Conclusion.—There is accumulating evidence for various therapeutic benefits of cannabis/cannabinoids, especially in the treatment of pain, which may also apply to the treatment of migraine and headache. There is also supporting evidence that cannabis may assist in opioid detoxification and weaning, thus making it a potential weapon in battling the opioid epidemic. Cannabis science is a rapidly evolving medical sector and industry with increasingly regulated production standards. Further research is anticipated to optimize breeding of strain-specific synergistic ratios of cannabinoids, terpenes, and other phytochemicals for predictable user effects, characteristics, and improved symptom and diseasetargeted therapies

Medicinal Uses of Marijuana and Cannabinoids.
Grotenhermen, F., & Müller-Vahl, K. (2016).
Critical Reviews in Plant Sciences, 35(5-6), 378–405.
doi:10.1080/07352689.2016.126536
In the past two decades, there has been increasing interest in the therapeutic potential of cannabis and single cannabinoids, mainly cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC). THC and cannabis products rich in THC exert their effects mainly through the activation of cannabinoid receptors (CB1 and CB2). Since 1975, 140 controlled clinical trials using different cannabinoids or whole-plant preparations for the treatment of a large number of disorders and symptoms have been conducted. Results have led to the approval of cannabis-based medicines [dronabinol, nabilone, and the cannabis extract nabiximols (Sativex, THC:CBD D 1:1)] as well as cannabis flowers in several countries. Controlled clinical studies provide substantial evidence for the use of cannabinoid receptor agonists in cancer chemotherapy induced nausea and vomiting, appetite loss and cachexia in cancer and HIV patients, neuropathic and chronic pain, and in spasticity in multiple sclerosis. In addition, there is also some evidence suggesting a therapeutic potential of cannabisbased medicines in other indications including Tourette syndrome, spinal cord injury, Crohn’s disease, irritable bowel syndrome, and glaucoma. In several other indications, small uncontrolled and single-case studies reporting beneficial effects are available, for example in posttraumatic stress disorder, attention deficit hyperactivity disorder, and migraine. The most common side effects of THC and cannabis-based medicines rich in THC are sedation and dizziness (in more than 10% of patients), psychological effects, and dry mouth. Tolerance to these side effects nearly always develops within a short time. Withdrawal symptoms are hardly ever a problem in the therapeutic setting. In recent years there is an increasing interest in the medical use of CBD, which exerts no intoxicating side effects and is usually well-tolerated. Preliminary data suggest promising effects in the treatment of anxiety disorders, schizophrenia, dystonia, and some forms of epilepsy. This review gives an overview on clinical studies which have been published over the past 40 years.

Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates
Joseph Molnar, D Szabó, Rozalia Pusztai Yukihiro Shoyama
Anticancer research 20(2A):861-7 March 2000
FIND DOI OR LINK
in the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Metabolites of cannabidiol identified in human urine.
Harvey DJ1, Mechoulam R.
Xenobiotica. 1990 Mar;20(3):303-20.
DOI: 10.3109/00498259009046849
1. Urine from a dystonic patient treated with cannabidiol (CBD) was examined by g.l.c.-mass spectrometry for CBD metabolites. Metabolites were identified as their trimethylsilyl (TMS), [2H9]TMS, and methyl ester/TMS derivatives and as the TMS derivatives of the product of lithium aluminium deuteride reduction. 2. Thirty-three metabolites were identified in addition to unmetabolized CBD, and a further four metabolites were partially characterized. 3. The major metabolic route was hydroxylation and oxidation at C-7 followed by further hydroxylation in the pentyl and propenyl groups to give 1"-, 2"-, 3"-, 4"- and 10-hydroxy derivatives of CBD-7-oic acid. Other metabolites, mainly acids, were formed by beta-oxidation and related biotransformations from the pentyl side-chain and these were also hydroxylated at C-6 or C-7. The major oxidized metabolite was CBD-7-oic acid containing a hydroxyethyl side-chain. 4. Two 8,9-dihydroxy compounds, presumably derived from the corresponding epoxide were identified. 5. Also present were several cyclized cannabinoids including delta-6- and delta-1-tetrahydrocannabinol and cannabinol. 6. This is the first metabolic study of CBD in humans; most observed metabolic routes were typical of those found for CBD and related cannabinoids in other species.

MGC kicks off Perth medicinal cannabis trial
Matt Birney
The West Australian
ASX-listed MGC Pharmaceuticals has teamed up with the University of Notre Dame in Perth to commence a new clinical trial for its “CogniCann” phytocannabinoid-derived medicine, after the first bulk shipment of the medicine arrived in Australia.
The company said CogniCann was developed with the specific aim to treat symptoms associated with dementia and Alzheimer’s disease.
The trial will involve a total of 50 patients with dementia or Alzheimer’s disease over the age of 65 at various aged care facilities across Perth. The specific aim is to evaluate the potential behavioural benefits of CogniCann on those patients as the company said the number of patients with various behavioural difficulties is growing globally.
MGC Pharma said that this Phase two clinical trial was looking to confirm the clinical effectiveness of the CogniCann drug and determine the therapeutic individual dose response. Results of the trial are expected in the third quarter of 2021.

Microbiological contaminants of marijuana
J. M. McPartland
JIHA Journal International Hemp Association Vol. 1 No. 2 December 1994
https://www.internationalhempassocia.. ./iha01205.html
https://www.*********.com/threads/mi...rijuana.23271/
If you go the the IHA WWW site, I just type in IHA hemp in my browser and I get a link for the IHA, click on the JIHA archives then next page "click here" then on the next page click on Microbiological contaminants of marijuana, the 5th article and you can get this for free.
Do not know why if I put the direct http link it shows up on IC as https instead of a http address, or why https does not work some hyyps links work fine. I just type in IHA hemp in my browser and I get a link for the IHA.
https://www.internationalhempassocia.. ./iha01205.html
Use of marijuana as a medicament is on the rise. Many medical marijuana users have a suppressed immune system, owing to their disease or treatment. Herbal marijuana, whether field grown or hydroponically cultivated, contains many microorganisms. Many of these organisms may pose a threat to immunosuppressed individuals. The microflora of marijuana is well described in the literature. Similarly, the microflora that cause opportunistic infections in AIDS patients is well documented. These separate literatures are correlated with commentary, and methods for detecting and eliminating microbial contaminants are discussed.
Marijuana is classified as a Schedule I drug "having no medical use, with a high potential for abuse" by the US Drug Enforcement Administration. Nevertheless, a handful of US citizens are permitted use of government-supplied "medical marijuana" for a variety of ailments (Bowersox 1992). Additionally, a large clinical trial utilizing marijuana in AIDS patients has been approved in San Francisco (Doblin 1994). Doblin and Kleiman (1991) document the widespread illicit use of marijuana for medical purposes. Nearly half of the oncologists polled by Doblin and Kleiman recommended that their patients experiment with marijuana as a medicine. Many of these patients, however, are immuno-suppressed, making them very susceptible to opportunistic bacteria and fungi. Since hundreds of bacteria and fungi are associated with the cultivation of marijuana (McPartland 1991), possible microbiological hazards need to be assessed.
A majority of the organisms associated with marijuana are strictly plant pathogens. They cannot infect healthy humans. But a subset of opportunistic plant pathogens are associated with "post-harvest" or "storage" decay of marijuana. These organisms may infect immuno-compromised individuals and become opportunistic human pathogens. Additionally, many opportunist organisms on plants produce dangerous toxins and/or elicit allergenic reactions when inhaled.
Lastly, a handful of strictly human pathogens have been isolated from marijuana, as contaminants. These agents are highly infectious, and some also exude toxins.

Another interesting handbook for testing Cannabis Microbiologicals
Microbiological Safety Testing Of Cannabis
https://cannabissafetyinstitute.org/...f-Cannabis.pdf
Cannabis is increasingly becoming legal at the state level in the U.S., for either medical or recreational use. Each of these states has had to wrestle with the question of how to ensure the safety of a new product that is not covered under any existing safety guidelines. Safety testing
in other agricultural industries is regulated by the FDA, the USDA, or by other federal agencies, all of which have been unable to assist the states in this case. The few states that have produced safety testing guidelines for Cannabis were forced to develop them from scratch, without the regulatory and scientific support that federal agencies typically provide. In the absence of this federal guidance, regulators in each state have turned to different sources for information, and each state has produced a unique set of rules and regulations (if they have produced one at all). Many of these are in outright conflict with each other, and they are largely not grounded on scientific research. This whitepaper is focused entirely on the question of microbiological safety, and has been written in order to promote the adoption of regulatory guidelines for the Cannabis industry that are rational, consistent, and safe. We have gathered what data there are on this issue and related ones, and assembled a broad collection of experts on the general subjects of plant microbiology, medical microbiology, and safety-testing of agricultural and food products.

Microglial Cannabinoid Type 1 Receptor Regulates Brain Inflammation in a Sex-Specific Manner
Julia De Meij, Zain Alfanek, Lydie Morel, Fanny Decoeur, Quentin Leyrolle, Katherine Picard, Micael Carrier,
Agnes Aubert, Alexandra Se´re´ , Ce´line Lucas, Gerald Laforest, Jean-Christophe Helbling, Marie-Eve Tremblay,
Daniela Cota, Marie-Pierre Moisan, Giovanni Marsicano, Sophie Laye´, and Agne`s Nadjar
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2020.0170
https://www.liebertpub.com/doi/abs/1...ournalCode=can
Background: Neuroinflammation is a key feature shared by most, if not all, neuropathologies. It involves complex biological processes that act as a protective mechanism to fight against the injurious stimuli, but it can lead to
tissue damage if self-perpetuating. In this context, microglia, the main cellular actor of neuroinflammation in the brain, are seen as a double-edged sword. By phagocyting neuronal debris, these cells can not only provide tissue repair but can also contribute to neuronal damage by releasing harmful substances, including inflammatory cytokines. The mechanisms guiding these apparent opposing actions are poorly known. The endocannabinoid system modulates the release of inflammatory factors such as cytokines and could represent a functional link between microglia and neuroinflammatory processes. According to transcriptomic databases and in vitro studies, microglia, the main source of cytokines in pathological conditions, express the cannabinoid type 1 receptor (CB1R).
Methods: We thus developed a conditional mouse model of CB1R deletion specifically in microglia, which was subjected to an immune challenge (peripheral lipopolysaccharide injection).
Results: Our results reveal that microglial CB1R differentially controls sickness behavior in males and females.
Conclusion: These findings add to the comprehension of neuroinflammatory processes and might be of great interest for future studies aimed at developing therapeutic strategies for brain disorders with higher prevalence in men.
Find PDF I have

Microglial Phenotypes and Their Relationship to the Cannabinoid System: Therapeutic Implications for Parkinson’s Disease
Rachel Kelly, Valerie Joers, Malú G. Tansey, Declan P. McKernan and Eilís Dowd
Molecules 2020, 25, 453;
doi:10.3390/molecules25030453
Parkinson’s disease is a neurodegenerative disorder, the motor symptoms of which are associated classically with Lewy body formation and nigrostriatal degeneration. Neuroinflammation has been implicated in the progression of this disease, by which microglia become chronically activated in response to α-synuclein pathology and dying neurons, thereby acquiring dishomeostatic phenotypes that are cytotoxic and can cause further neuronal death. Microglia have a functional endocannabinoid signaling system, expressing the cannabinoid receptors in addition to being capable of synthesizing and degrading endocannabinoids. Alterations in the cannabinoid system—particularly an upregulation in the immunomodulatory CB2 receptor—have been demonstrated to be related to the microglial activation state and hence the microglial phenotype. This paper will review studies that examine the relationship between the cannabinoid system and microglial activation, and how this association could be manipulated for therapeutic benefit in Parkinson’s disease.

Mind Blown: Exploding Head Syndrome as a Side Effect of Marijuana
Christopher Missak, Jenie George, Indira Gurubhagavatula
Sleep 43(Supplement_1):A472-A473 may 2020
DOI: 10.1093/sleep/zsaa056.1233
Marijuana use is increasing the United States and has been associated with increased sensory perception, euphoria, and altered cognition. Exploding head syndrome is a parasomnia characterized by loud explosion-like noises occurring prior to or during sleep. This the first report to link marijuana use with the occurrence of EHS. Report of Case A 45-year-old man with depression, anxiety and multisubstance abuse reported two years of neuropsychiatric symptoms including: “fireworks going off in my brain,” visual hallucinations, and sudden arousals characterized by panic and vertigo. He reported bouts of occupational stress and severe anxiety previously treated with clonazepam for six months, but discontinued it due to poor clinical response. Normal findings were reported on a previous work-up including: MRI, EEG, and head CT Scan. During his visit, he reported that he had been smoking marijuana three times a day for 24 years. One month prior to his visit he discontinued marijuana, stopped clonazepam, and started using mirtazapine with improvement in all aforementioned symptoms. During the same month, he relapsed and smoked marijuana once with a sudden return of all previously described symptoms on the night following its use. These symptoms then resolved and did not recur again until 9 months later, after another episode of weekend marijuana use. An in-lab polysomnogram revealed an apnea-hypopnea index 2.2 per hour, increased alpha intrusion throughout the study, and no epileptiform activity. No parasomnias were observed during rapid-eye-movement (REM) or non-REM sleep. Conclusion The etiology of EHS remains elusive. A review of the research has proposed five major theories and case reports suggest a complex etiology. Given the temporal correlation with marijuana use and symptom resolution with its cessation, we presume that EHS resulted from a complex interplay between neurons susceptible to cannabinoids and their derivatives.

Mini-Review Cannabis in palliative care: current challenges and practical recommendations
Claude Cyr, Maria Fernanda Arboleda, Sunil Kumar Aggarwal, Lynda G. Balneaves, Paul Daeninck, Andrée Néron, Erin Prosk, Antonio Vigano
Ann Palliat Med 2018
doi: 10.21037/apm.2018.06.04
https://www.researchgate.net/publica...cationCoverPdf
Pain and symptom control challenges are common in palliative care, and the search for other therapeutic strategies is ongoing. Unfortunately, patients and their caregivers are receiving little information or support from healthcare providers regarding the increasingly popular cannabinoid-based medicines (CBM). Clinicians, meanwhile, feel understandably perplexed by the discrepancy between the available evidence and the rapid interest in which patients and their families have demonstrated for CBM. There is an urgent need to address the many challenges that are delaying the appropriate integration of CBM into clinical practice, notwithstanding the obvious need for a solid general knowledge of pharmacology, mechanism of action and available clinical evidence supporting its use. The authors will address these challenges and provide practical recommendations regarding patient assessment for the use of CBM. The authors will also make suggestions regarding patient expectations in order to define clear objectives, review the necessary precautions prior to initiating treatment, aid in selecting the appropriate strain and route of administration as well as establishing proper titration and monitoring protocols. The authors will also discuss the lesser known but potentially therapeutic psychoactive effects of cannabis. As this class of therapeutic agents are likely to play a major role in palliative medicine in the near future, clinicians would benefit from familiarizing themselves with CBM and we can expect that patients and their caregivers will appreciate receiving support in their search for safe and effective therapeutic alternatives.

Mitochondria: a possible nexus for the regulation of energy homeostasis by the endocannabinoid system?
Christopher Lipina, Andrew J. Irving, and Harinder S. Hundal
American Journal of Physiology-Endocrinology and MetabolismVol. 307, No. 1,July 14
DOI: 10.1152/ajpendo.00100.2014
The endocannabinoid system (ECS) regulates numerous cellular and physiological processes through the activation of receptors targeted by endogenously produced ligands called endocannabinoids. Importantly, this signaling system is known to play an important role in modulating energy balance and glucose homeostasis. For example, current evidence indicates that the ECS becomes overactive during obesity whereby its central and peripheral stimulation drives metabolic processes that mimic the metabolic syndrome. Herein, we examine the role of the ECS in modulating the function of mitochondria, which play a pivotal role in maintaining cellular and systemic energy homeostasis, in large part due to their ability to tightly coordinate glucose and lipid utilization. Because of this, mitochondrial dysfunction is often associated with peripheral insulin resistance and glucose intolerance as well as the manifestation of excess lipid accumulation in the obese state. This review aims to highlight the different ways through which the ECS may impact upon mitochondrial abundance and/or oxidative capacity and, where possible, relate these findings to obesity-induced perturbations in metabolic function. Furthermore, we explore the potential implications of these findings in terms of the pathogenesis of metabolic disorders and how these may be used to strategically develop therapies targeting the ECS.

Mode of Cannabis Use and Factors Related to Frequency of Cannabis Use Among Breastfeeding Mothers: Results from an Online Survey
Christine D. Garner, Kathleen Kendall-Tackett, Christina Young, Teresa Baker, and Thomas W. Hale
Breastfeeding Medicine Dec 2021
DOI: 10.1089/bfm.2021.0151
Background: In the United States, 5% of breastfeeding mothers report using cannabis. Frequent cannabis use results in higher delta-9-tetrahydrocannabinol (THC) in breast milk, and mode of cannabis use may also impact risk to the infant. The aim of this study was to understand how breastfeeding mothers use cannabis and factors related to frequency of its use.
Methods: An anonymous online survey was conducted among mothers who used cannabis while breastfeeding. Frequency of cannabis use was ascertained along with modes of and reasons for cannabis use. Respondents were grouped by frequency of use: less-than-daily (n = 686), low-daily (1–3 times/day; n = 423), and high-daily (≥4 times/day; n = 218). Chi-square and analysis of variance tested between-group differences, and ordinal logistic regression examined factors associated with cannabis use frequency.
Results: Smoking (88%) was the most common mode of cannabis consumption, followed by vaping (48%) and oral/edibles (36%). Smoking and vaping differed by cannabis use frequency. Only 54% used cannabis to get high, but was reported more among frequent users. In contrast, 89% of mothers used cannabis for mental or physical health symptoms, including anxiety, depression, gastrointestinal symptoms, chronic pain, and posttraumatic stress disorder. These symptoms differed by cannabis use frequency. Reporting more symptoms was associated with higher frequency of use. The odds of increasing cannabis use frequency was 2.7 for those reporting 1–2 health reasons, 5.6 for those reporting 3–4 health reasons, and 13.1 for reporting ≥5 health reasons.
Conclusions: Strategies are needed to address maternal mental and physical health, which may be key to reducing cannabis use among breastfeeding mothers.
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Model based analysis on systemic availability of coadministered cannabinoids after controlled vaporised administration.
]Liu, Z., Galettis, P., Broyd, S. J., Hell, H., Greenwood, L., Krey, P., Martin, J. H.
Internal Medicine Journal. (2019)
doi:10.1111/imj.14415
Aims The most important two medicinal cannabinoids are ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Vaporised administration is superior due to its higher systemic availability, lower individual variability and faster drug delivery. Although it is common THC is coadministered with CBD, the influence of CBD on the pharmacokinetics, especially the systemic availability of THC after vaporised administration, is unknown. This study aimed to investigate the influence of different doses of coadministered CBD on the systemic availability of THC, and to compare the availability of THC and CBD in a sample of frequent and infrequent cannabis users.
Methods The study used a randomised, double-blind, crossover placebo-controlled design. THC and/or CBD in ethanol was vaporised and inhaled. Plasma concentrations of THC and CBD were analysed. The THC data created in this study were pooled together with published THC pharmacokinetic data in order to cover all the phases of THC disposition. Population pharmacokinetic model of THC was developed based on the pooled data. The model of CBD was developed based on the data created in this study.
Results Population pharmacokinetic models of THC and CBD were developed. With concomitant inhalation of high dose CBD, the systemic availability of THC decreased
significantly. Frequent cannabis users appeared to have higher systemic availability of THC and CBD when high dose CBD was administered.
Conclusions The results observed in this study are useful for guiding future pharmacokinetic studies of medicinal cannabinoids, and for development of dosing guidelines for medical use of cannabis in the real world‘ setting.

Modification of the hemodynamic and molecular features of phosphine, a potent mitochondrial toxicant in the heart, by cannabidiol
Mohammad Reza Hooshangi Shayesteh, Hamed Haghi-Aminjan, Maryam Baeeri, Mahban Rahimifard, Shokoufeh Hassani, Mehdi Gholami, Saeideh Momtaz, Seyed Alireza Salami, Maryam Armandeh, Behnaz Bameri, Mahedeh Samadi, Taraneh Mousavi, Seyed Nasser Ostad & Mohammad Abdollahi
Toxicology Mechanisms and Methods Volume 32, 2022 - Issue 4
DOI: 10.1080/15376516.2021.1998851
https://www.tandfonline.com/doi/abs/10.1080/15376516.2021.1998851?journalCode=itxm20
Aluminum phosphide (AlP) poisoning is common in many countries responsible for high mortality. The heart is the main target organ in AlP poisoning. Several studies have reported the beneficial effects of cannabidiol (CBD) in reducing heart injuries. This study aimed to investigate the possible protective effect of CBD on cardiac toxicity caused by AlP poisoning. Study groups included almond oil, normal saline, sole CBD (100 µg/kg), AlP (11.5 mg/kg), and four groups of AlP + CBD (following AlP gavage, CBD administrated at doses of 5, 25, 50, and 100 μg/kg via intravenous (iv) injection). Thirty minutes after AlP treatment, an electronic cardiovascular device (PowerLab) was used to record electrocardiographic (ECG) changes, heart rate (HR), and blood pressure (BP) for three hours. Cardiac tissue was examined for the activities of mitochondrial complexes, ADP/ATP ratio, the release of cytochrome C, mitochondrial membrane potential (MMP), apoptosis, oxidative stress parameter, and cardiac biomarkers at 12 and 24 hours time points. AlP administration caused abnormal ECG, decreased HR, and BP. AlP also significantly reduced mitochondrial complex I and IV activity and ADP/ATP ratio. The level of cytochrome C release, apoptosis, oxidative stress, and cardiac biomarkers was considerably increased by AlP, which was compensated following CBD administration. CBD was able to improve hemodynamic function to some extent in AlP poisoned rats. CBD restored ATP levels and mitochondrial function and decreased oxidative damage and thus, prevented the heart cells from entering the apoptotic stage. Further clinical trials are needed to explore any possible benefits of CBD in AlP-poisoned patients.
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Modulation of striatal functional connectivity differences in adults with and without autism spectrum disorder in a single-dose randomized trial of cannabidivarin
Charlotte M. Pretzsch, Dorothea L. Floris, Bogdan Voinescu, Malka Elsahib, Maria A. Mendez, Robert Wichers, Laura Ajram, Glynis Ivin, Martin Heasman, Elise Pretzsch, Steven Williams, Declan G. M. Murphy, Eileen Daly & Gráinne M. McAlonan
Molecular Autism (2021) 12:49
DOI: 10.1186/s13229-021-00454-6
https://molecularautism.biomedcentra.. .21-00454-6.pdf
Background: Autism spectrum disorder (ASD) has a high cost to afected individuals and society, but treatments for core symptoms are lacking. To expand intervention options, it is crucial to gain a better understanding of potential treatment targets, and their engagement, in the brain. For instance, the striatum (caudate, putamen, and nucleus accumbens) plays a central role during development and its (atypical) functional connectivity (FC) may contribute to multiple ASD symptoms. We have previously shown, in the adult autistic and neurotypical brain, the non-intoxicating cannabinoid cannabidivarin (CBDV) alters the balance of striatal ‘excitatory–inhibitory’ metabolites, which help regulate FC, but the efects of CBDV on (atypical) striatal FC are unknown.
Methods: To examine this in a small pilot study, we acquired resting state functional magnetic resonance imaging data from 28 men (15 neurotypicals, 13 ASD) on two occasions in a repeated-measures, double-blind, placebocontrolled study. We then used a seed-based approach to (1) compare striatal FC between groups and (2) examine the efect of pharmacological probing (600 mg CBDV/matched placebo) on atypical striatal FC in ASD. Visits were separated by at least 13 days to allow for drug washout.
Results: Compared to the neurotypicals, ASD individuals had lower FC between the ventral striatum and frontal and pericentral regions (which have been associated with emotion, motor, and vision processing). Further, they had higher intra-striatal FC and higher putamenal FC with temporal regions involved in speech and language. In ASD, CBDV reduced hyperconnectivity to the neurotypical level.
Limitations: Our fndings should be considered in light of several methodological aspects, in particular our participant group (restricted to male adults), which limits the generalizability of our fndings to the wider and heterogeneous ASD population.

Modulation Of The Endo-Cannabinoid System: Therapeutic Potential Against Cocaine Dependence Gianluigi Tanda
Pharmacol Res. 2007 Nov; 56(5): 406–417.
doi: 10.1016/j.phrs.2007.09.001
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2134985/ Dependence on cocaine is still a main unresolved medical and social concern, and in spite of research efforts, no pharmacological therapy against cocaine dependence is yet available. Recent studies have shown that the endocannabinoid system participates in specific stages and aspects of drug dependence in general, and some of this evidence suggests an involvement of the cannabinoid system in cocaine effects. For example, cocaine administration has been shown to alter brain endocannabinoid levels, and the endocannabinoid system has been involved in long-term modifications of brain processes that might play a role in neuro/behavioral effects of psychostimulant drugs like cocaine. Human studies show that marijuana dependence is frequently associated with cocaine dependence, and that the cannabinoid receptor CNR1 gene polymorphism might be related to cocaine addiction. This article will review the main papers in the field showing how a modulation of different components of the cannabinoid system might interact with some of the neurobiological/behavioral effects of cocaine related to its reinforcing effects, evaluated in preclinical models or in clinical settings. The goal of this review will be to provide insights into the complex picture of cocaine abuse and addiction, and to extrapolate from such endocannabinoid-cocaine interactions useful information to test the therapeutic potential of cannabinoid ligands and endocannabinoid-level enhancers against cocaine dependence for future preclinical/clinical trials.

Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: novel anti-tumor mechanisms of . Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex
T. Rubino, N. Realini, C. Castiglioni, C. Guidali, D. Vigano´, E. Marras, S. Petrosino, G. Perletti, M. Maccarrone, V. Di Marzo and D. Parolaro
Cerebral Cortex June 2008;18:1292--1301
doi:10.1093/cercor/bhm161
In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due toTRPV1 stimulation or the lack of CB1 activation, respectively.

Molecular and Biochemical Mechanism of Cannabidiol in the Management of the Inflammatory and Oxidative Processes Associated with Endometriosis
Tiziana Genovese, Marika Cordaro, Rosalba Siracusa, Daniela Impellizzeri, Sebastiano Caudullo, Emanuela Raffone, Francesco Macrí, Livia Interdonato, Enrico Gugliandolo, Claudia Interlandi, Rosalia Crupi, Ramona D’Amico, Roberta Fusco, Salvatore Cuzzocrea and Rosanna Di Paola
Int. J. Mol. Sci. 2022, 23,5427.
DOI: 0.3390/ ijms23105427
Endometriosis is usually associated with inflammation and chronic pelvic pain. This paper focuses the attention on the anti-inflammatory, anti-oxidant and analgesic effects of cannabidiol (CBD) and on its potential role in endometriosis. We employed an in vivo model of endometriosis and administered CBD daily by gavage. CBD administration strongly reduced lesions diameter, volume and area. In particular, it was able to modify lesion morphology, reducing epithelial glands and stroma. CBD showed anti-oxidant effects reducing lipid peroxidation, the expression of Nox-1 and Nox-4 enzymes. CBD restored the oxidative equilibrium of the endogenous cellular defense as showed by the SOD activity and the GSH levels in the lesions. CBD also showed important antifibrotic effects as showed by the Masson trichrome staining and by downregulated expression of MMP-9, iNOS and TGF-β. CBD was able to reduce inflammation both in the harvested lesions, as showed by the increased Ikb-α and reduced COX2 cytosolic expressions and reduced NFkB nuclear localization, and in the peritoneal fluids as showed by the decreased TNF-α, PGE2 and IL-1α levels. CBD has important analgesic effects as showed by the reduced mast cells recruitment in the spinal cord and the reduced release of neuro-sensitizing and pro-inflammatory mediators. In conclusion, the collected data showed that CBD has an effective and coordinated effects in endometriosis suppression.

Molecular diagnosis in cannabis allergy.
Armentia, A., Herrero, M., Martín-Armentia, B., Rihs, H.-P., Postigo, I., & Martínez-Quesada, J.
The Journal of Allergy and Clinical Immunology: In Practice, 2(3), 351–352. (2014)
doi:10.1016/j.jaip.2014.01.015
Studies have described isolated cases of Cannabis sativa sensitization as unusual cases of lipid transfer protein (LTP) allergy. Serologic investigations that used ImmunoCAP (Thermofisher Scientific, Uppsala, Sweden) and ISAC (Thermofisher Scientific, Uppsala, Sweden) microarraye confirmed IgE positivity for peach LTP (nPrup3), hazelnut lipid transfer protein (rCor a 8), and mugwort LTP (nArt v3). Reports suggest that C sativa sensitization may be mediated by 2 mechanisms, cross-reactivity (mainly with LTPs and thaumatinlike protein), and exposure-related de novo sensitization. LTPs
sensitize primarily through the airways. We characterized the sensitization profile of patients diagnosed with primary cannabis allergy who experienced new-onset asthma after cannabis handling or smoking. The protocol was approved by the clinical research ethics committees, and all participants gave written informed consent
Clinical Implications
* Cannabis may be an important allergen in young people.
* The most important allergens implicated were lipid transfer proteins, which may be responsible for primary sensitization and might induce further lipid transfer protein food allergies.
* Patients sensitized to tobacco or tomatoes are at risk

Molecular docking analysis of phyto-constituents from Cannabis sativa with pfDHFR
Temitope . David1, Niyi S. Adelakun, Olaposi . Omotuyi, Damilohun S. Metibemu,Oluwafemi E. Ekun Gabriel O. eniafe, Olumide K. Inyang, Bamidele Adewumi, Ojochenemi A. Enejoh, Raymond T. Owolabi, Eunice I. Oribamise
Bioinformation 14(9): 574-579 (2018)
doi: 10.6026/97320630014574.
Available antimalarial drugs have been associated with numerous side effects, which include skin rashes and myelo-suppression. Therefore, it is of interest to explore compounds from natural source having drug-like properties without side effect. This study focuses on the screening of compounds from Cannabis sativa against malaria Plasmodium falciparum dihydrofolate reductase for antimalarial properties using Glide (Schrodinger maestro 2018-1). The result showed that phytochemicals from Cannabis sativa binds with a higher affinity and lower free energy than the standard ligand with isovitexin and vitexin having a glide score of -11.485 and -10.601 respectively, sophoroside has a glide score of -9.711 which is lower than the cycloguanil (co-crystallized ligand) having a glide score of -6.908. This result gives new perception to the use of Cannabis sativa as antimicrobial agent.

Modification of the hemodynamic and molecular features of phosphine, a potent mitochondrial toxicant in the heart, by cannabidiol
Mohammad Reza Hooshangi Shayesteh, Hamed Haghi-Aminjan, Maryam Baeeri, Mahban Rahimifard, Shokoufeh Hassani, Mehdi Gholami, Saeideh Momtaz, Seyed Alireza Salami, Maryam Armandeh, Behnaz Bameri, Mahedeh Samadi, Taraneh Mousavi, Seyed Nasser Ostad & Mohammad Abdollahi
Toxicology Mechanisms and Methods Volume 32, 2022 - Issue 4
DOI: 10.1080/15376516.2021.1998851
https://www.tandfonline.com/doi/abs/10.1080/15376516.2021.1998851?journalCode=itxm20
Aluminum phosphide (AlP) poisoning is common in many countries responsible for high mortality. The heart is the main target organ in AlP poisoning. Several studies have reported the beneficial effects of cannabidiol (CBD) in reducing heart injuries. This study aimed to investigate the possible protective effect of CBD on cardiac toxicity caused by AlP poisoning. Study groups included almond oil, normal saline, sole CBD (100 µg/kg), AlP (11.5 mg/kg), and four groups of AlP + CBD (following AlP gavage, CBD administrated at doses of 5, 25, 50, and 100 μg/kg via intravenous (iv) injection). Thirty minutes after AlP treatment, an electronic cardiovascular device (PowerLab) was used to record electrocardiographic (ECG) changes, heart rate (HR), and blood pressure (BP) for three hours. Cardiac tissue was examined for the activities of mitochondrial complexes, ADP/ATP ratio, the release of cytochrome C, mitochondrial membrane potential (MMP), apoptosis, oxidative stress parameter, and cardiac biomarkers at 12 and 24 hours time points. AlP administration caused abnormal ECG, decreased HR, and BP. AlP also significantly reduced mitochondrial complex I and IV activity and ADP/ATP ratio. The level of cytochrome C release, apoptosis, oxidative stress, and cardiac biomarkers was considerably increased by AlP, which was compensated following CBD administration. CBD was able to improve hemodynamic function to some extent in AlP poisoned rats. CBD restored ATP levels and mitochondrial function and decreased oxidative damage and thus, prevented the heart cells from entering the apoptotic stage. Further clinical trials are needed to explore any possible benefits of CBD in AlP-poisoned patients.
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Mood, sleep and pain comorbidity outcomes in cannabis dependent patients: Findings from a nabiximols versus placebo randomised controlled trial

Mark Montebello, Meryem Jefferies, Llewellyn Mills, Raimondo Bruno, Jan Copeland, Iain McGregori Consuelo Rivas, Melissa A.Jackson, Catherine Silsbury, Adrian Dunlop, Nicholas Lintzeris
Drug and Alcohol Dependence Volume 234, 1 May 2022, 109388
DOI: 10.1016/j.drugalcdep.2022.109388
https://www.sciencedirect.com/science/article/abs/pii/S0376871622001259?via=ihub
Highlights Depression, anxiety, stress, stressand sleep disturbance symptoms decreased with cannabis dependence treatment.
• Pain symptoms improved only in the treatment period for cannabis dependence.
• Participants in this trial who qualified as cases at baseline had elevated comorbidity symptoms.
• There was no evidence that nabiximols treatment is a barrier to achieving reductions in the comorbid symptoms examined.
• Abstinence is not required to achieve improvements in comorbid mood, sleep and pain symptoms.

Background​

Mood, sleep and pain problems are common comorbidities among treatment-seeking cannabis-dependent patients. There is limited evidence suggesting treatment for cannabis dependence is associated with their improvement. This study explored the impact of cannabis dependence treatment on these comorbidities.

Methods​

This is a secondary analysis from a 12-week double-blind placebo-controlled trial testing the efficacy of a cannabis agonist (nabiximols) against placebo in reducing illicit cannabis use in 128 cannabis-dependent participants. Outcome measurements including DASS-21 (Depression, Anxiety, and Stress subscales); Insomnia Severity Index (ISI); and Brief Pain Inventory (BPI), were performed at weeks 0, 4, 8, 12 and 24. Each was analysed as continuous outcomes and as binary cases based on validated clinical cut-offs.

Results​

Among those whose DASS and ISI scores were in the moderate to severe range at baseline, after controlling for cannabis use, there was a gradual decrease in severity of symptoms over the course of the trial. BPI decreased significantly until week 12 and then rose again in the post-treatment period during weeks 12–24. Neither pharmacotherapy type (nabiximols vs placebo)
nor number of counselling sessions contributed significant explanatory power to any of the models and were excluded from the final analyses for both continuous and categorical outcomes.

Conclusions​

Participants in this trial who qualified as cases at baseline had elevated comorbidity symptoms. There was no evidence that nabiximols treatment is a barrier to achieving reductions in the comorbid symptoms examined. Cannabis dependence treatment reduced illicit cannabis use and improved comorbidity symptoms, even when complete abstinence was not achieved.

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Morphometric Study on Cytoskeletal Components of Neuronal and Astroglial Cells After Chronic CB1 Agonist Treatment
Patricia Tagliaferro, Alberto J. Ramos, Emmanuel S. Onaivi, Sergio G. Evrard, Maite Duhalde Vega, and Alicia Brusco
Chapter 5 in book Marijuana and Cannabinoid Research Edited by Emmanuel S. Onaivi, Methods and Protocols Marijuana and Cannabinoid Research https://www.researchgate.net/profile...ntal_methods_t o_study_the_role_of_the_periph eral_cannabinoid_receptor_in_i mmune_function/links/09e4150994ee2087a2000000/Experimental-methods-to-study-the-role-of-the-peripheral-cannabinoid-receptor-in-immune-function.pdf#page=108
One of the major goals for the use of digital image analysis systems in neuroanatomy is to visualize structures, cells, or other tissue components in order to compare various populations. In addition, digital image analysis allows semi-quantification of cell labeling because it is capable of measuring simultaneously the staining intensity, location, size, and shape of labeled profiles. In the present work, the morphological changes in the CB1 hippocampal area and corpus striatum induced by chronic treatment with the synthetic CB1-receptor agonist WIN55,212-2 were analyzed as an example of digital image analysis application. Twice-daily treatment for 14 d with the CB1-receptor agonist demonstrated significant changes in the expression of neuronal cytoskeletal proteins and in neuronal morphology, as evidenced by immunocytochemical and digital analysis studies. However, changes in the expression of astroglial cytoskeletal proteins were not found

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders
Alline Cristina Campos, Fabricio Araújo Moreira, Felipe Villela Gomes, Elaine Aparecida Del Bel, and Francisco Silveira Guimarães
Philos Trans R Soc Lond B Biol Sci. 2012 Dec 5; 367(1607): 3364–3378.
doi: 10.1098/rstb.2011.0389
https://www.ncbi.nlm.nih.gov/labs/pm...tb20110389.pdf
Cannabidiol (CBD) is amajor phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound D9 –tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1Amediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamidemediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed withCBD.Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARg receptors agonism, intracellular (Ca2fl ) increase, etc.), on CBD behavioural effects.

Nabilone administration in refractory chronic diarrhea: a case series.
Pellesi, L., Verga, M. C., De Maria, N., Villa, E., Pini, L. A., & Guerzoni, S.
BMC Gastroenterology, 19(1). (2019).
doi:10.1186/s12876-019-1024-y
Background: Daily cannabis assumption is currently associated with several physical and mental health problems, however in the past it was prescribed for a multitude of symptoms, including vomiting, abdominal pain and diarrhea. Through the years, the endocannabinoid system has been recognized in the homeostatic mechanisms of the gut, as well as in the physiological control of intestinal motility and secretion. Accordingly, cannabinoids may be a promising therapy against several gastrointestinal conditions, such as abdominal pain and motility-related disorders. Case presentation: We retrospectively analysed the efficacy and safety of a CB1-receptor agonist administered in six patients with refractory chronic diarrhea, between April 2008 and July 2016. After three months of therapy, oral nabilone improved the health of nearly all patients, with visible improvements in reducing diarrheal symptoms and weight gain. Most of the benefits persisted through the three-month follow-up. Only one patient interrupted the treatment after one month, due to severe fatigue and mental confusion; the symptoms disappeared in the follow-up period. Conclusions: These findings encourage the study of cannabinoids acting on CB1 receptors in chronic gastrointestinal disorders, especially in refractory chronic diarrhea, offering a chance for a substantial improvement in the quality of life of selected patients, with a reasonable safety profile.

Nabiximols combined with motivational enhancement/cognitive behavioral therapy for the treatment of cannabis dependence: A pilot randomized clinical trial.
Trigo, J. M., Soliman, A., Quilty, L. C., Fischer, B., Rehm, J., Selby, P., … Le Foll, B.
PLOS ONE, 13(1), e0190768.(2018).
doi:10.1371/journal.pone.0190768
Background The current lack of pharmacological treatments for cannabis use disorder (CUD) warrants novel approaches and further investigation of promising pharmacotherapy. We previously showed that nabiximols (27 mg/ml ?9 -tetrahydrocannabinol (THC)/ 25 mg/ml cannabidiol (CBD), Sativex®) can decrease cannabis withdrawal symptoms. Here, we assessed in a pilot study the tolerability and safety of self-titrated nabiximols vs. placebo among 40 treatment-seeking cannabis-dependent participants.
Methods Subjects participated in a double blind randomized clinical trial, with as-needed nabiximols up to 113.4 mg THC/105 mg CBD or placebo daily for 12 weeks, concurrently with Motivational Enhancement Therapy and Cognitive Behavioral Therapy (MET/CBT). Primary outcome measures were tolerability and abstinence, secondary outcome measures were days and amount of cannabis use, withdrawal, and craving scores. Participants received up to CDN$ 855 in compensation for their time.
Results Medication was well tolerated and no serious adverse events (SAEs) were observed. Rates of adverse events did not differ between treatment arms (F1,39 = 0.205, NS). There was no significant change in abstinence rates at trial end. Participants were not able to differentiate between subjective effects associated with nabiximols or placebo treatments (F1,40 = 0.585, NS). Cannabis use was reduced in the nabiximols (70.5%) and placebo groups (42.6%). Nabiximols reduced cannabis craving but no significant differences between the nabiximols and placebo groups were observed on withdrawal scores.
Conclusions Nabiximols in combination with MET/CBT was well tolerated and allowed for reduction of cannabis use. Future clinical trials should explore the potential of high doses of nabiximols for cannabis dependence.

Nabiximols for the Treatment of Cannabis Dependence.
Lintzeris, N., Bhardwaj, A., Mills, L., Dunlop, A., Copeland, J., … McGregor, I.
JAMA Internal Medicine. (2019).
doi:10.1001/jamainternmed.2019.1993
IMPORTANCE There are no effective medications for treating dependence on cannabis.
OBJECTIVE To examine the safety and efficacy of nabiximols in the treatment of patients with cannabis dependence.
DESIGN, SETTING, AND PARTICIPANTS This parallel double-blind randomized clinical trial comparing nabiximols with placebo in a 12-week, multisite outpatient study recruited participants from February 3, 2016, to June 14, 2017, at 4 outpatient specialist alcohol and drug treatment services in New South Wales, Australia. Participants had cannabis dependence (as defined by the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision) and were seeking treatment, were nonresponsive to prior treatment attempts, were 18 to 64 years of age, had no other substance use disorder, had no severe medical or psychiatric conditions, were not pregnant, were not mandated by a court to undergo treatment, and provided informed consent. Results for primary efficacy measures and all secondary outcomes were obtained using a modified intention-to-treat data set.
INTERVENTIONS Participants received 12-week treatment involving weekly clinical reviews, structured counseling, and flexible medication doses—up to 32 sprays daily (tetrahydrocannabinol, 86.4 mg, and cannabidiol, 80 mg), dispensed weekly.
MAIN OUTCOMES AND MEASURES Primary outcome was self-reported number of days using illicit cannabis during the 12-week period. Other outcomes included alternate cannabis use parameters (periods of abstinence, withdrawal, cravings, and problems), safety parameters (adverse events and aberrant medication use), health status, other substance use, and treatment retention.
RESULTS A total of 128 participants (30 women and 98 men; mean [SD] age, 35.0 [10.9] years) were randomized and received at least 1 dose of study medication. Participants had used a mean (SD) of 2.3 (2.1) g of cannabis on a mean (SD) of 25.7 (4.5) days in the past 28 days. Treatment retention was comparable for the 2 groups (placebo, 30 of 67 participants [44.8%]; nabiximols, 30 of 61 participants [49.2%]), and both groups used similar mean (SD) doses (placebo, 18.5 [9.5] sprays daily; nabiximols, 17.6 [9.5] sprays daily, equivalent to a mean [SD] of 47.5 [25.7] mg of tetrahydrocannabinol and 44.0 [23.8] mg of cannabidiol). For the primary end point, the placebo group reported significantly more days using cannabis during the 12 weeks (mean [SD], 53.1 [33.0] days) than the nabiximols group (mean [SD], 35.0 [32.4] days; estimated difference, 18.6 days; 95% CI, 3.5-33.7 days; P = .02). Both groups showed comparable improvements in health status, with no substantial changes in other substance use. Medication was well tolerated with few adverse events.
CONCLUSIONS AND RELEVANCE This study demonstrates that cannabinoid agonist treatment, in this case using nabiximols, in combination with psychosocial interventions is a safe approach for reducing cannabis use among individuals with cannabis dependence who are seeking treatment.

B]Natural (?9-THC) and synthetic (JWH-018) cannabinoids induce seizures by acting through the cannabinoid CB1 receptor.[/B]
Malyshevskaya O, Aritake K, Kaushik MK, Uchiyama N, Cherasse Y, Kikura-Hanajiri R, Urade Y.
Sci Rep. 2017 Sep 5;7(1):10516.
doi: 10.1038/s41598-017-10447-2.
Natural cannabinoids and their synthetic substitutes are the most widely used recreational drugs. Numerous clinical cases describe acute toxic symptoms and neurological consequences following inhalation of the mixture of synthetic cannabinoids known as "Spice." Here we report that an intraperitoneal administration of the natural cannabinoid ?9-tetrahydrocannabinol (10?mg/kg), one of the main constituent of marijuana, or the synthetic cannabinoid JWH-018 (2.5?mg/kg) triggered electrographic seizures in mice, recorded by electroencephalography and videography. Administration of JWH-018 (1.5, 2.5 and 5?mg/kg) increased seizure spikes dose-dependently. Pretreatment of mice with AM-251 (5?mg/kg), a cannabinoid receptor 1-selective antagonist, completely prevented cannabinoid-induced seizures. These data imply that abuse of cannabinoids can be dangerous and represents an emerging public health threat. Additionally, our data strongly suggest that AM-251 could be used as a crucial prophylactic therapy for cannabinoid-induced seizures or similar life-threatening conditions.

Naturally occurring and related synthetic cannabinoids and their potential therapeutic applications
Ahmed M. Galal, Desmond Slade, Waseem Gul, Abir T. El-Alfy, Daneel Ferreira, and Mahmoud A. Elsohly
Recent Patents on CNS Drug Discovery, 2009, 4, 112-136
https://www.researchgate.net/publica...iscov_4112-136
Naturally occurring cannabinoids (phytocannabinoids) are biosynthetically related terpenophenolic compounds
uniquely produced by the highly variable plant, Cannabis sativa L. Natural and synthetic cannabinoids have been
extensively studied since the discovery that the psychotropic effects of cannabis are mainly due to!9-THC. However,
cannabinoids exert pharmacological actions on other biological systems such as the cardiovascular, immune and
endocrine systems. Most of these effects have been attributed to the ability of these compounds to interact with the
cannabinoid CB1 and CB2 receptors. The FDA approval of Marinol®, a product containing synthetic 9-THC (dronabinol), in 1985 for the control of nausea and vomiting in cancer patients receiving chemotherapy, and in 1992 as an appetite stimulant for AIDS patients, has further intensified the research interest in these compounds. This article reviews patents (2003-2007) that describe methods for isolation of cannabinoids from cannabis, chemical and chromatographic methods for their purification, synthesis, and potential therapeutic applications of these compounds.

Neural responses to reward anticipation and feedback in adult and adolescent cannabis users and controls
Martine Skumlien, Claire Mokrysz, Tom P. Freeman, Matthew B. Wall, Michael Bloomfield, Rachel Lees, Anna Borissova, Kat Petrilli, James Carson, Tiernan Coughlan, Shelan Ofori, Christelle Langley, Barbara J. Sahakian , H. Valerie Curran and Will Lawn
Neuropsychopharmacology Nature
https://www.nature.com/articles/s41386-022-01316-2.pdf .
Chronic use of drugs may alter the brain’s reward system, though the extant literature concerning long-term cannabis use and neural correlates of reward processing has shown mixed results. Adolescents may be more vulnerable to the adverse effects of cannabis than adults; however, this has not been investigated for reward processing. As part of the ‘CannTeen’ study, in the largest functional magnetic resonance imaging study of reward processing and cannabis use to date, we investigated reward anticipation and feedback in 125 adult (26–29 years) and adolescent (16–17 years) cannabis users (1–7 days/week cannabis use) and genderand age-matched controls, using the Monetary Incentive Delay task. Blood-oxygen-level-dependent responses were examined using region of interest (ROI) analyses in the bilateral ventral striatum for reward anticipation and right ventral striatum and left ventromedial prefrontal cortex for feedback, and exploratory whole-brain analyses. Results showed no User-Group or User-Group × Age-Group effects during reward anticipation or feedback in pre-defined ROIs. These null findings were supported by post hoc Bayesian analyses. However, in the whole-brain analysis, cannabis users had greater feedback activity in the prefrontal and inferior parietal cortex compared to controls. In conclusion, cannabis users and controls had similar neural responses during reward anticipation and in hypothesised reward-related regions during reward feedback. The whole-brain analysis revealed tentative evidence of greater fronto-parietal activity in cannabis users during feedback. Adolescents showed no increased vulnerability compared with adults. Overall, reward anticipation and feedback processing appear spared in adolescent and adult cannabis users, but future longitudinal studies are needed to corroborate this.

Nausea-Induced Conditioned Gaping Reactions in Rats Produced by High-Dose Synthetic Cannabinoid, JWH-018.
DeVuono, M. V., Hrelja, K. M., Petrie, G. N., Limebeer, C. L., Rock, E. M., Hill, M. N., & Parker, L. A.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2019.0103
Introduction: Cannabinoid hyperemesis syndrome is becoming a more prominently reported side effect of cannabis containing high-dose D9 -tetrahydrocannabinol (THC) and designer cannabinoid drugs such as ‘‘Spice.’’ One active ingredient that has been found in ‘‘Spice’’ is 1-pentyl-3-(1-naphthoyl)indole ( JWH-018), a synthetic full agonist of the cannabinoid 1 (CB1) receptor. In this study, we evaluated the potential of different doses of JWH-018 to produce conditioned gaping in rats, an index of nausea. Materials and Methods: Rats received 3 daily conditioning trials in which saccharin was paired with JWH-018 (0.0, 0.1, 1, and 3 mg/kg, intraperitoneal [i.p.]). Then the potential of pretreatment with the CB1 antagonist, rimonabant (SR), to prevent JWH-018-induced conditioned gaping was determined. To begin to understand the potential mechanism underlying JWH-018-induced nausea, serum collected from trunk blood was subjected to a corticosterone (CORT) analysis in rats receiving three daily injections with vehicle (VEH) or JWH-018 (3 mg/kg). Results: At doses of 1 and 3 mg/kg (i.p.), JWH-018 produced nausea-like conditioned gaping reactions. The conditioned gaping produced by 3 mg/kg JWH-018 was reversed by pretreatment with rimonabant, which did not modify gaping on its own. Treatment with JWH-018 elevated serum CORT levels compared to vehicle-treated rats. Conclusions: As we have previously reported with high-dose THC, JWH-018 produced conditioned gaping in rats, reflective of a nausea effect mediated by its action on CB1 receptors and accompanied by elevated CORT, reflective of hypothalamic-pituitary-adrenal (HPA) activation

Neuroinflammation and the Glial Endocannabinoid System
Maria Pazos in book Cannabinoida and the Brain (2008) Chapter 16 pg 331
Attila Köfalvi Editor
https://www.academia.edu/4790006/Neu...card=thumbnail

The remarkable density and wide distribution of cannabinoid CB1 receptors in the central nervous system served to explain many of the well-known pharmacological effects of natural, synthetic and endogenous cannabinoids. This receptor type is one of the most abundant cerebral receptors so far described. Its presynaptic location in neurons allows its participation in a myriad of cerebral functions, such as those controlling motor activity or memory and to mediate cannabinoid-induced neuroprotection. At the same time, the psychoactive effects derived from CB1 activation limited the development of novel therapeutic approaches on the use of cannabinoids. However, recent data have raised the possible interest of the endocannabinoid system in neuroinflammation. These new perspectives can be summarized mostly at two levels: (1) the participation of other components of the endocannabinoid system, mainly CB2 receptors and fatty acid amide hydrolase (FAAH), in neuroinflammatory processes; and (2) the predominance of the glial endocannabinoid system over the neuronal endocannabinoid system under pathological conditions. We now know that dramatic changes take place in the endocannabinoid system in the human brain, suggesting its possible involvement in several prevalent diseases, such as Alzheimer's disease, multiple sclerosis or viral encephalitis. This is the subject of the present review.


Neuropharmacological Effects of the Main Phytocannabinoids: A Narrative Review
Rafael G Dos Santos , Jaime E C Hallak , José Alexandre S Crippa
Adv Exp Med Biol. 2021;1264:29-45.
doi: 10.1007/978-3-030-57369-0_3.
Cannabis can synthetize more than 400 compounds, including terpenes, flavonoids, and more than 100 phytocannabinoids. The main phytocannabinoids are ?-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Cannabis-based products are used as medicines in several countries. In this text, we present an overview of the main neurochemical mechanisms of action of the phytocannabinoids, especially THC and CBD. We also reviewed the indications and adverse effects of the main cannabis-based medicinal products. THC acts as a partial agonist at cannabinoid 1/2 receptors (CB1/2). It is responsible for the characteristic effects of cannabis, such as euphoria, relaxation, and changes in perceptions. THC can also produce dysphoria, anxiety, and psychotic symptoms. THC is used therapeutically in nausea and vomiting due to chemotherapy, as an appetite stimulant, and in chronic pain. CBD acts as a noncompetitive negative allosteric modulator of the CB1 receptor, as an inverse agonist of the CB2 receptor, and as an inhibitor of the reuptake of the endocannabinoid anandamide. Moreover, CBD also activates 5-HT1Aserotonergic receptors and vanilloid receptors. Its use in treatment-resistant epilepsy syndromes is approved in some countries. CBD does not produce the typical effects associated with THC and has anxiolytic and antipsychotic effects. Some of the most common adverse effects of CBD are diarrhea, somnolence, nausea, and transaminase elevations (with concomitant use of antiepileptics). The mechanisms of action involved in both the therapeutic and adverse effects of the phytocannabinoids are not fully understood, involving not only the endocannabinoid system. This "promiscuous" pharmacology could be responsible for their wide therapeutic spectrum.

Neuroprotection by Cannabinoids in Neurodegenerative Diseases
Walter Milano, Anna Capasso
Alzheimer's, Dementia & Cognitive Neurology
DOI: 10.15761/ADCN.1000120 jan 2018
https://www.oatext.com/neuroprotecti...e-diseases.php
The cannabinoids are found to have particular application as neuroprotectants for mental and motor dysfuction in neurodegenerative diseases. The neuroprotective properties of cannabinoids suggest their therapeutic use for limiting neurological damage. The cannabinoids treatments should not only aim to alleviate specific symptoms but also attempt to delay/arrest disease progression and to repair the damaged structures. The author conducted a review of studies published between 1974 and 2011. The search was performed using the following PubMed search terms: “Cannabinoids” and “Neurodegenerative Diseases” and 287 papers were detected. The articles were examined and the overlapping or insufficiently clear works were excluded. Finally we chose 117 articles regarding the latest international guidelines, the pathophysiology of neurodegenerative diseases and the various therapeutic choices. The studies reported in the present review support the view that the cannabinoid signalling system is a key modulatory element in the activity of the basal ganglia. This idea is supported by different anatomical, electrophysiological, pharmacological and biochemical data. Furthermore, these studies indicate that the cannabinoid system is impaired in different neurological disorders that directly or indirectly affect the basal ganglia, which supports the idea of developing novel pharmacotherapies with compounds that selectively target specific elements of the cannabinoid system.
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Neuroprotective and Symptomatic Effects of Cannabidiol in an Animal Model of Parkinson's Disease
Claudio Giuliano, Miriam Francavilla, Gerardo Ongari, Alessandro Petese, Cristina Ghezzi, Nora Rossini , Fabio Blandini , Silvia Cerri
Int J Mol Sci. 2021 Aug 18;22(16):8920.
doi: 10.3390/ijms22168920
https://www.ncbi.nlm.nih.gov/pmc/art...s-22-08920.pdf
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta, leading to classical PD motor symptoms. Current therapies are purely symptomatic and do not modify disease progression. Cannabidiol (CBD), one of the main phytocannabinoids identified in Cannabis Sativa, which exhibits a large spectrum of therapeutic properties, including anti-inflammatory and antioxidant effects, suggesting its potential as disease-modifying agent for PD. The aim of this study was to evaluate the effects of chronic treatment with CBD (10 mg/kg, i.p.) on PD-associated neurodegenerative and neuroinflammatory processes, and motor deficits in the 6-hydroxydopamine model. Moreover, we investigated the potential mechanisms by which CBD exerted its effects in this model. CBD-treated animals showed a reduction of nigrostriatal degeneration accompanied by a damping of the neuroinflammatory response and an improvement of motor performance. In particular, CBD exhibits a preferential action on astrocytes and activates the astrocytic transient receptor potential vanilloid 1 (TRPV1), thus, enhancing the endogenous neuroprotective response of ciliary neurotrophic factor (CNTF). These results overall support the potential therapeutic utility of CBD in PD, as both neuroprotective and symptomatic agent.

Neuroprotective effect of cannabinoids in neurodegenerative diseases
Carlos Suero-Garcia, Lucia Martin-Banderas, M.A. Holgado
DOI: 10.1016/j.jddst.2017.04.023
https://www.researchgate.net/publica...rotective_effe ct_of_cannabinoids_in_neurodeg enerative_diseases
Objectives: It is analysed the actual situation of the investigations related to cannabinoids substances, as well as their interaction with the organism, classification, therapeutics effects and their use in neurodegenerative diseases. Methods: This study is based on the review of multiples scientific articles directly related with the cannabinoides substance and its synthetic derivates, with a special attention on the way the organism interacts and the effects the substances cause in it. Specifcally, the study will get deep into the neuroantiinflammatory and analgesic effects of these substances, related with the neuroprotector effect in neurodegenerative diseases such as Alzheimer, Parkinson, Multiple Sclerosis and Amyotrophic Lateral Sclerosis. Results: From thousands years, the Cannabis Sativa has been used by multiples cultures with different purposes such as joy, textile, analgesics etc. But is not till ends of the XX century when there are diverse scientifc studies related with the Cannabis encouraged. The plant has 400 components, 60 of them belong to the cannabinoides group. The main ones are cannabinol, cannabidiol and tetrahidrocannabinol. With the discoverment of the cannabinoids substances, its derivates, and the receptors which interact with them, it is increased the therapeutic possibilities and remarking the neuroprotective effect which these substances contain. Conclusions: It has been demostraded the huge potential of the cannabinoids as therapeutic substances apart from its analgesic and antiemetic uses, that is, neurodegenaritve diseases in which they can not only decrease its symptons but stop disease process. Another possible application could be in the oncologic area, being particularly intense the investigation's activity realised the last 15 years.

Neuroprotective Effects of Phytocannabinoid-Based Medicines in Experimental Models of Huntington’s Disease
Onintza Sagredo, M. Ruth Pazos, Valentina Satta, Jose ́ A. Ramos, Roger G. Pertwee, and Javier Ferna ́ndez-Ruiz
Journal of Neuroscience Research 89:1509–1518 (2011)
DOI: 10.1002/jnr.22682
We studied whether combinations of botanical extracts enriched in either D9-tetrahydrocannabinol (D9-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, provide neuroprotec- tion in rat models of Huntington’s disease (HD). We used rats intoxicated with 3-nitropropionate (3NP) that were given combinations of D9-THC- and CBD-enriched bo- tanical extracts. The issue was also studied in malonate- lesioned rats. The administration of D9-THC- and CBD- enriched botanical extracts combined in a ratio of 1:1 as in Sativex attenuated 3NP-induced GABA deficiency, loss of Nissl-stained neurons, down-regulation of Crecep- tor and IGF-1 expression, and up-regulation of calpain expression, whereas it completely reversed the reduction in superoxide dismutase-1 expression. Similar responses were generally found with other combinations of D9-THC- and CBD-enriched botanical extracts, suggesting that these effects are probably related to the antioxidant and CB and CB receptor-independent properties of both phytocannabinoids. In fact, selective antagonists for both receptor types, i.e., SR141716 and AM630, respectively, were unable to prevent the positive effects on calpain expression caused in 3NP-intoxicated rats by the 1:1 combination of D9-THC and CBD. Finally, this combina- tion also reversed the up-regulation of proinflammatory markers such as inducible nitric oxide synthase observed in malonate-lesioned rats. In conclusion, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying disease pro- gression in HD, a disorder that is currently poorly man- aged in the clinic, prompting an urgent need for clinical trials with agents showing positive results in preclinical studies.

Neuroprotective Properties of Cannabigerol in Huntington’s Disease: Studies in R6/2 Mice and 3-Nitropropionate-lesioned Mice.
Valdeolivas, S., Navarrete, C., Cantarero, I., Bellido, M. L., Muñoz, E., & Sagredo, O.
Neurotherapeutics, 12(1), 185–199.(2014).
doi:10.1007/s13311-014-0304-z
Different plant-derived and synthetic cannabinoids have shown to be neuroprotective in experimental models of Huntington’s disease (HD) through cannabinoid receptordependent and/or independent mechanisms. Herein, we studied the effects of cannabigerol (CBG), a nonpsychotropic phytocannabinoid, in 2 different in vivo models of HD. CBG was extremely active as neuroprotectant in mice intoxicated with 3-nitropropionate (3NP), improving motor deficits and preserving striatal neurons against 3NP toxicity. In addition, CBG attenuated the reactive microgliosis and the upregulation of proinflammatory markers induced by 3NP, and improved the levels of antioxidant defenses that were also significantly reduced by 3NP. We also investigated the neuroprotective properties of CBG in R6/2 mice. Treatment with this phytocannabinoid produced a much lower, but significant, recovery in the deteriorated rotarod performance typical of R6/2 mice. Using HD array analysis, we were able to identify a series of genes linked to this disease (e.g., symplekin, Sin3a, Rcor1, histone deacetylase 2, huntingtin-associated protein 1, ? subunit of the gamma-aminobutyric acid-A receptor (GABAA), and hippocalcin), whose expression was altered in R6/2 mice but partially normalized by CBG treatment. We also observed a modest improvement in the gene expression for brain-derived neurotrophic factor (BDNF), insulinlike growth factor-1 (IGF-1), and peroxisome proliferatoractivated receptor-? (PPAR?), which is altered in these mice, as well as a small, but significant, reduction in the aggregation of mutant huntingtin in the striatal parenchyma in CBGtreated animals. In conclusion, our results open new research avenues for the use of CBG, alone or in combination with other phytocannabinoids or therapies, for the treatment of neurodegenerative diseases such as HD.

New Perspectives on the Use of Cannabis in the Treatment of Psychiatric Disorders.
Scherma M., Masia, P., Deidda M., Fratta W., Tanda G., & Fadda P.
Medicines, 5(4), 107. (2018)
doi:10.3390/medicines5040107
Following the discovery of the endocannabinoid system and its potential as a therapeutic target for various pathological conditions, growing interest led researchers to investigate the role of cannabis and its derivatives for medical purposes. The compounds ∆9-tetrahydrocannabinol and cannabidiol are the most abundant phytocannabinoids found in cannabis extracts, as well as the most studied. The present review aims to provide an overview of the current evidence for their beneficial effects in treating psychiatric disorders, including schizophrenia, anxiety, and depression. Nevertheless, further investigations are required to clarify many pending issues, especially those relative to the assessment of benefits and risks when using cannabis for therapeutic purposes, thereby also helping national and federal jurisdictions to remain updated.

Non-Cannabinoid Metabolites of Cannabis sativa L. with Therapeutic Potential
Henry Lowe, Blair Steele, Joseph Bryant, Ngeh Toyang, Wilfred Ngwa
Plants 2021, 10, 400.
DOI: 10.3390/ plants10020400
https://www.ncbi.nlm.nih.gov/pmc/art...s-10-00400.pdf
The cannabis plant (Cannabis sativa L.) produces an estimated 545 chemical compounds of different biogenetic classes. In addition to economic value, many of these phytochemicals have medicinal and physiological activity. The plant is most popularly known for its two most-prominent and most-studied secondary metabolites—∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and cannabidiol (CBD). Both ∆ 9 -THC and CBD have a wide therapeutic window across many ailments and form part of a class of secondary metabolites called cannabinoids—of which approximately over 104 exist. This review will focus on non-cannabinoid metabolites of Cannabis sativa that also have therapeutic potential, some of which share medicinal properties similar to those of cannabinoids. The most notable of these non-cannabinoid phytochemicals are flavonoids and terpenes. We will also discuss future directions in cannabis research and development of cannabis-based pharmaceuticals. Caflanone, a flavonoid molecule with selective activity against the human viruses including the coronavirus OC43 (HCov-OC43) that is responsible for COVID-19, and certain cancers, is one of the most promising non-cannabinoid molecules that is being advanced into clinical trials. As validated by thousands of years of the use of cannabis for medicinal purposes, vast anecdotal evidence abounds on the medicinal benefits of the plant. These benefits are attributed to the many phytochemicals in this plant, including non-cannabinoids. The most promising non-cannabinoids with potential to alleviate global disease burdens are discussed.

Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-kB-mediated signaling
Vittoria Borgonetti, Cristina Benatti, Paolo Governa, Giovanni Isoldi, Federica Pellati, Silvia Alboni, Fabio Tascedda, Monica Montopoli, Nicoletta Galeotti, Fabrizio Manetti, Elisabetta Miraldi, Marco Biagi, Giovanna Rigillo
DOI: 10.1002/ptr.7458
https://onlinelibrary.wiley.com/doi/epdf/10.1002/ptr.7458
Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and β-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms
Luciano De Petrocellis, Alessia Ligresti, Aniello Schiano Moriello,
Mariagrazia Iappelli, Roberta Verde, Colin G Stott, Luigia Cristino,
Pierangelo Orlando and Vincenzo Di Marzo
British Journal of Pharmacology (2013) 168 79–102
DOI:10.1111/j.1476-5381.2012.02027.x
BACKGROUND AND PURPOSE Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than
D9-tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival.
EXPERIMENTAL APPROACH We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells.
KEY RESULTS Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h.
Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1–10 mM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and
intracellular Ca2+). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis.
CONCLUSIONS AND IMPLICATIONS These data support the clinical testing of CBD against prostate carcinoma.

Normalization of mediotemporal and prefrontal activity, and mediotemporal-striatal connectivity, may underlie antipsychotic effects of cannabidiol in psychosis.
O’Neill, A., Wilson, R., Blest-Hopley, G., Annibale, L., Colizzi, M., Brammer, M., Bhattacharyya, S.
Psychological Medicine, 1–11. (2020).
doi:10.1017/s0033291719003519
Background. Recent evidence suggests that cannabidiol (CBD), a non-intoxicating ingredient present in cannabis extract, has an antipsychotic effect in people with established psychosis. However, the effect of CBD on the neurocognitive mechanisms underlying psychosis is unknown. Methods. Patients with established psychosis on standard antipsychotic treatment were studied on separate days at least one week apart, to investigate the effects of a single dose of orally administered CBD (600 mg) compared to a matched placebo (PLB), using a double-blind, randomized, PLB-controlled, repeated-measures, within-subject cross-over design. Three hours after taking the study drug participants were scanned using a block design functional magnetic resonance imaging (fMRI) paradigm, while performing a verbal paired associate learning task. Fifteen psychosis patients completed both study days, 13 completed both scanning sessions. Nineteen healthy controls (HC) were also scanned using the same fMRI paradigm under identical conditions, but without any drug administration. Effects of CBD on brain activation measured using the blood oxygen level-dependent hemodynamic response fMRI signal were studied in the mediotemporal, prefrontal, and striatal regions of interest. Results. Compared to HC, psychosis patients under PLB had altered prefrontal activation during verbal encoding, as well as altered mediotemporal and prefrontal activation and greater mediotemporal-striatal functional connectivity during verbal recall. CBD attenuated dysfunction in these regions such that activation under its influence was intermediate between the PLB condition and HC. CBD also attenuated hippocampal-striatal functional connectivity and caused trend-level symptom reduction in psychosis patients. Conclusions. This suggests that normalization of mediotemporal and prefrontal dysfunction and mediotemporal-striatal functional connectivity may underlie the antipsychotic effects of CBD.

Novel Δ8‑Tetrahydrocannabinol Vaporizers Contain Unlabeled Adulterants, Unintended Byproducts of Chemical Synthesis, and Heavy Metals
Jiries Meehan-Atrash and Irfan Rahman Chem. Res. Toxicol. (2022) DOI: 10.1021/acs.chemrestox.1c00388 Supporting Information: separate Pdf Cannabis e-cigarettes containing Δ8 –tetrahydrocannabinol (Δ8 -THC) produced synthetically from hemp-derived cannabidiol (CBD) have recently risen in popularity as a legal means of cannabis consumption, but questions surrounding purity and unlabeled additives have created doubts of their safety. Herein, NMR, GC-MS, and ICP-MS were used to analyze major componentsof 27 products from 10 brands, and it was determined none of these had accurate Δ8 -THC labeling, 11 had unlabeled cutting agents, and all contained reaction side-products including olivetol, Δ4(8) -iso tetrahydrocannabinol, 9-ethoxyhexahydrocannabinol, Δ9 –tetrahydrocannabinol (Δ9 -THC), heavy metals, and a novel previously undescribed cannabinoid, iso -tetrahydrocannabifuran.

Novel Δ9-tetrahydrocannabinol formulation Namisol® has beneficial pharmacokinetics and promising pharmacodynamic effects.
Klumpers, L. E., Beumer, T. L., van Hasselt, J. G. C., Lipplaa, A., Karger, L. B., Kleinloog, H. D., … van Gerven, J. M. A.
British Journal of Clinical Pharmacology, 74(1), 42–53.(2012).
doi:10.1111/j.1365-2125.2012.04164.x
AIMS Among the main disadvantages of currently available D9 -tetrahydrocannabinol (THC) formulations are dosing difficulties due to poor pharmacokinetic characteristics. Namisol® is a novel THC formulation, designed to improve THC absorption. The study objectives were to investigate the optimal administration route, pharmacokinetics (PK), pharmacodynamics (PD) and tolerability of Namisol®. METHODS This first in human study consisted of two parts. Panel I included healthy males and females (n = 6/6) in a double-blind, double-dummy, randomized, crossover study with sublingual (crushed tablet) and oral administration of Namisol® (5 mg THC). Based on these results, male and female (n = 4/5) participants from panel I received oral THC 6.5 and 8.0 mg or matching placebo in a randomized, crossover, rising dose study during panel II. PD measurements were body sway; visual analogue scales (VAS) mood, psychedelic and heart rate. THC and 11-OH-THC population PK analysis was performed. RESULTS Sublingual administration showed a flat concentration profile compared with oral administration. Oral THC apparent t1/2 was 72–80 min, tmax was 39–56 min and Cmax 2.92–4.69 ng ml-1 . THC affected body sway (60.8%, 95% CI 29.5, 99.8), external perception (0.078 log mm, 95% CI 0.019, 0.137), alertness (-2.7 mm, 95% CI -4.5, -0.9) feeling high (0.256 log mm, 95% CI 0.093, 0.418) and heart rate (5.6 beats min–1, 95% CI 2.7, 6.5). Namisol® was well tolerated. CONCLUSIONS Oral Namisol® showed promising PK and PD characteristics. Variability and tmax of THC plasma concentrations were smaller for Namisol® than reported for studies using oral dronabinol and nabilone. This study was performed in a limited number of healthy volunteers. Therefore, future research on Namisol® should study clinical effects in patient populations.

Novel Pharmacologic Approaches to Treating Cannabis Use Disorder
Rebecca E. Balter & Ziva D. Cooper & Margaret Haney
Curr Addict Rep (2014) 1:137–143
DOI 10.1007/s40429-014-0011-1
With large and increasing numbers of people using cannabis, the development of cannabis use disorder (CUD) is a growing public health concern. Despite the success of evidence-based psychosocial therapies, the low rates of initial abstinence and high rates of relapse during and following treatment for CUD suggest a need for adjunct pharmacotherapies. Here we review the literature on medication development for the treatment of CUD, with a particular focus on studies published within the last three years (2010–2013). Studies in both the human laboratory and in the clinic have tested medications with a wide variety of mechanisms. In the laboratory, the following medication strategies have been shown to decrease cannabis withdrawal and selfadministration following a period of abstinence (a model of relapse): the cannabinoid receptor agonist, nabilone, and the adrenergic agonist, lofexidine, alone and in combination with dronabinol (synthetic THC), supporting clinical testing of these medication strategies. Antidepressant, anxiolytic and antipsychotic drugs targeting monoamines (norepinephrine, dopamine, and serotonin) have generally failed to decrease withdrawal symptoms or laboratory measures of relapse. In terms of clinical trials, dronabinol and multiple antidepressants
(fluoxetine, venlafaxine and buspirone) have failed to decrease cannabis use. Preliminary results from controlled clinical trials with gabapentin and N-acetylcysteine (NAC) support further research on these medication strategies. Data from open label and laboratory studies suggest that lithium and oxytocin also warrant further testing. Overall, it is likely that different medications will be needed to target distinct aspects of problematic cannabis use: craving, ongoing use, withdrawal and relapse. Continued research is needed in preclinical, laboratory and clinical settings. .

ON THE PREPARATIONS OF THE INDIAN HEMP, OR GUJNJAH,* (Cannabis Indica)
W. B. O'SIAUGHNESSY,
https://www.ncbi.nlm.nih.gov/pmc/art...00864-0001.pdf
The narcotic effects of hemp are popularly known in the South of Africa, South America, Turkey, Egypt, Asia Minor, India, and the adjacent territories of the Malays, Burmese, and Siamese. In all these countries hemp is used in various forms, by the dissipated and depraved, as the ready agent of a pleasing intoxication. In the popular medicine of these nations, we find it extensively employed for a multitude of affections, especially those in which spasm or neuralgic pain are the prominent symptoms. But in Western Europe its use, either as a stimulant or as a remedy, is equally unknown. With the exception of the trial, as a frolic, of the Egyptian " hasheesh," by a few youths in Marseilles, and of the clinical use of the wine of hemp by Hahnemann, as shown in a sub. sequent extract, I have been unable to trace any notice of the employment of this drug in Europe. Much difference of opinion exists on the question, whether the hemp so abundant in Europe, even in high northern latitudes, is identical in specific characters with the hemp of Asia Minor and India. The extraordinary symptoms produced by the latter depend on a resinous secretion with which it abounds, and which seems totally absent in the European kind. The closest physical resemblance or even identity exists between both plants; difference of climate seems to me more than sufficient to account for the absence of the resinous secretion, and consequent want of narcotic power in that indigenous in colder countries.

Observational study on role of crude cannabis in pain control and quality of life in terminally ill cancer patients: An Indian perspective.
Dusi, V., Attili, S. V. S., & Singaraju, M.
Annals of Oncology, 30, ix119. (2019).
doi:10.1093/annonc/mdz430.005
In India, most of these patients do shift to alternative medicine in desperation, with active ingredients, such as steroids/cannabinoids, and few unclassified anti-tumor substances. After listing and analyzing these the two most frequently observed ones are steroids and cannabinoids wherever patients have satisfactory pain control. Methods: An interview based study was conducted as a part of QOL Data collection, which included other symptoms (such as fatigue, cachexia, well-being etc.) in the period of 2016-2018. One of key inclusions was - patients with advanced cancer progressed on multiple lines for which there was no standard of care. After data collection, a subset analysis was conducted with reference to pain control and use of alternative medication. For ease of analysis subjects were grouped into 4 categories based on pain control with opioids and concurrent use of cannabinoids. Subjects whose nature of the medication was not known were excluded. All four groups were analyzed for the pain control with help of visual analogy scale (VAS). Results: The baseline demographic characters in all four groups were well balanced and depicted in Table -1. Overall there were no statistically significant differences in the duration of symptoms, average dose of opioid analgesia, performance status and the stage of disease. Pain relief was better in the cannabis group, when it was used independently or in combination with opioids. No significant additional side effects pertaining to cannabis were reported by the patients. The overall qualities of life, as well as weight gain and nausea control were better in the cannabis group. Conclusions: We could infer that Bhang [cannabis crude form], is an effective analgesic independently having synergy with opioids. It also improved overall QOL, especially in cachexia, without adverse effects. If scientifically proven with pharmaceutical grade, it will be a significant addition to the symptomatic care of terminally ill cancer patients. Though available in US, India still does not have regulatory approval for medical cannabis

Observed Impact of Long-term Consumption of Oral Cannabidiol on Liver Function in Healthy Adults
Robert Kaufmann, Keith Aqua, Jeff Lombardo, and Martin Lee
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0114
https://www.liebertpub.com/doi/epdf/.../can.2021.0114
https://pubmed.ncbi.nlm.nih.gov/34918948/
Introduction: Previous studies have suggested that prescribed cannabidiol (CBD) products may cause elevations in liver tests (LT). This study compared the prevalence of elevated LT in an adult population self-administering CBD with the normal and general adult population prevalences.
Materials and Methods: Adults 18–75 years of age across the United States taking CBD orally for a minimum of 30 days were recruited from 12 individual CBD product companies in this decentralized, observational study and sent their standard CBD regimen from the company of their choice. An app-based, 21CFR Part 11 decentralized clinical study platform (ValidCare Study) was used to securely automate consent inclusion/exclusion criteria and
collect all the data for this study, including: demographic information, medical history, reasons for taking, dosage, current medications dosage, adverse effects, and efficacy. At the end of 30 days, LTs were obtained. Follow-up
LTs were offered to all individuals with elevated alanine transaminase (ALT) values.
Results: A total of 28,121 individuals were invited to participate in this study, 1475 enrolled, and 839 (female: 65.3%, male: 34.7%) completed the study. Full-spectrum hemp oil was used by 55.7%, CBD-isolate by 40.5%, and broad spectrum by 3.8%. The mean – SD daily dose of CBD was 50.3 + 40.7 mg. The prevalence of elevated ALT was 9.1%, aspartate aminotransferase (AST) 4.0%, alkaline phosphatase 1.9%, total bilirubin 1.7%, with 85.5% of the ALT elevations < 2 · the upper limit of normal (ULN) with only 0.3% having ALT levels > 3 · ULN. The prevalence of ALT and AST elevations (9.1% and 4.0%) were not significantly different from known adult general population prevalences (8.9% and 4.9%). There was no significant association between CBD dosage and LT values. Thirty-three individuals with elevated ALT levels had follow-up LT performed with 21 having normal LT, 8 having the same severity of ALT elevation, and 4 having an increase in severity, 1 of which ultimately became normal.
Conclusions: Self-medication of CBD does not appear to be associated with an increased prevalence of LT elevation and most of the LT elevations are likely due to the conditions/medications for which the individuals are taking CBD.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.
Kobayashi, Y., Watanabe, N., Kitakaze, T., Sugimoto, K., Izawa, T., Kai, K., … Yamaji, R.
British Journal of Nutrition, 1–35. (2020).
doi:10.1017/s0007114520004304
Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nM in the male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nM after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg/day) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area (CSA) of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nM in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt, mTOR, and p70S6K in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of LC3-II and p62, but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II, and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ? 100 nM. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the PI3K/Akt/mTOR signalling pathway and restoring autophagy flux.

Olivetolic acid, a cannabinoid precursor in Cannabis sativa, but not CBGA methyl ester exhibits a modest anticonvulsant effect in a mouse model of Dravet syndrome
Lyndsey L Anderson, Michael Udoh, Declan Everett-Morgan, Marika Heblinski, Iain S McGregor, Samuel D Banister, Jonathon C Arnold
J Cannabis Res . 2022 Jan 4;4(1):2.
doi: 10.1186/s42238-021-00113-w.
https://www.ncbi.nlm.nih.gov/labs/pm...rticle_113.pdf
Objective: Cannabigerolic acid (CBGA), a precursor cannabinoid in Cannabis sativa, has recently been found to have anticonvulsant properties in the Scn1a+/- mouse model of Dravet syndrome. Poor brain penetration and chemical instability of CBGA limits its potential as an anticonvulsant therapy. Here, we examined whether CBGA methyl ester, a more stable analogue of CBGA, might have superior pharmacokinetic and anticonvulsant properties. In addition, we examined whether olivetolic acid, the biosynthetic precursor to CBGA with a truncated (des-geranyl) form, might possess minimum structural requirements for anticonvulsant activity. We also examined whether olivetolic acid and CBGA methyl ester retain activity at the epilepsy-relevant drug targets of CBGA: G-protein-coupled receptor 55 (GPR55) and T-type calcium channels.
Methods: The brain and plasma pharmacokinetic profiles of CBGA methyl ester and olivetolic acid were examined following 10 mg/kg intraperitoneal (i.p.) administration in mice (n = 4). The anticonvulsant potential of each was examined in male and female Scn1a+/- mice (n = 17-19) against hyperthermia-induced seizures (10-100 mg/kg, i.p.). CBGA methyl ester and olivetolic acid were also screened in vitro against T-type calcium channels and GPR55 using intracellular calcium and ERK phosphorylation assays, respectively.
Results: CBGA methyl ester exhibited relatively limited brain penetration (13%), although somewhat superior to that of 2% for CBGA. No anticonvulsant effects were observed against thermally induced seizures in Scn1a+/- mice. Olivetolic acid also showed poor brain penetration (1%) but had a modest anticonvulsant effect in Scn1a+/- mice increasing the thermally induced seizure temperature threshold by approximately 0.4°C at a dose of 100 mg/kg. Neither CBGA methyl ester nor olivetolic acid displayed pharmacological activity at GPR55 or T-type calcium channels.
Conclusions: Olivetolic acid displayed modest anticonvulsant activity against hyperthermia-induced seizures in the Scn1a+/- mouse model of Dravet syndrome despite poor brain penetration. The effect was, however, comparable to the known anticonvulsant cannabinoid cannabidiol in this model. Future studies could explore the anticonvulsant mechanism(s) of action of olivetolic acid and examine whether its anticonvulsant effect extends to other seizure types.

One in Five Autism Caregivers Give Their Child CBD Products
Emily Ansell Elfer
Autism Parenting Magazine Oct 6, 2021
https://www.autismparentingmagazine.co m/autism-cbd-products/
In a survey sent out by Autism Parenting Magazine (APM) to more than 160,000 email subscribers around the world, 18.6% of respondents confirmed they use CBD for a child on the spectrum to help relieve a variety of autism symptoms. Breaking the data down further, 22.16% of USA-based caregivers use CBD versus 14.29% of UK caregivers.
A total of 72.4% of respondents identified themselves as autism parents, while the remaining participants were grandparents, full-time carers, teachers, therapists, doctors, or individuals on the spectrum.
The data revealed 76.3% participants use CBD only (less than 0.3% THC), 13.7% use CBD/THC combination, while the remaining 10% use “Other” forms including Hemp, CBD/THC & Epidiolex, and CBD with Terpenes, among others.
When asked to identify the primary reason for using CBD with their child, 42.9% responded with anxiety relief, 36.9% stated challenging behavior, 5.1% said pain relief and inflammation, 8.1% said sleep and relaxation, 4.3% said seizures, while the remaining respondents identified “Other” reasons such as increasing speech and supporting potty training.
Oils (oral drops and topical sprays) were the preferred CBD format, with 60.8% of respondents opting to use these. Meanwhile, 21.5% use gummies and topicals, 7.5% use capsules or tablets, 5.1% use lotions or balms, and 1.9% use vape. The remaining respondents selected “Other” options including honey sticks and patches.
A large number of respondents use CBD for a teenager on the spectrum with 21.39% of CBD users confirming their child is aged 13 to 18. When asked if they started using CBD during the COVID-19 pandemic, a whopping 31.3% said yes. Meanwhile, 16.6% have increased the amount of CBD they give their child since the pandemic began.
Another interesting find was that only 20.1% have a doctor’s prescription for CBD usage. Despite this, 21.7% reported it was their doctor who recommended trying CBD.
Meanwhile, 27.4% were recommended by a friend or family member and 23.6% were recommended by another autism parent. The remaining respondents selected “Other” with many saying usage stemmed from their own research.
When asked if they would recommend CBD products to other parents on the spectrum, a huge majority of 82.9% said they would.

Online patient-provider cannabis consultations.
Gali, K., Narode, R., Young-Wolff, K. C., Rubinstein, M. L., Rutledge, G., & Prochaska, J. J.
Preventive Medicine, (2020).
doi:10.1016/j.ypmed.2020.105987
Cannabis has been legalized, decriminalized, or medicalized in over half the U.S. states. With restrictions on cannabis research, accepted standards to guide clinical practice are lacking. Analyzing online communications through a digital health platform, we characterized patient questions about cannabis use and provider responses. Coded for content were 4,579 questions posted anonymously online between March 2011 through January 2017, and the responses from 1,439 U.S. licensed clinicians. Provider responses to medical cannabis use questions were coded for sentiment: ?negative?, ?positive?, and ?mixed.? Responses could be ?thanked? by patients and receive ?agrees? from providers. The most frequent themes were detection of cannabis use (25.3%), health harms (19.9%), co -use with other substances (9.1%), and medical use (8.2%). The 425 medical cannabis use questions most frequently related to treatment of mental illness (20.3%), pain (20.0%), and cancer care (6.7%). The 762 provider responses regarding medical cannabis use were coded for sentiment as 59.5% negative, 28.6% mixed, and 11.8% positive. Provider sentiment was most positive regarding cannabis use for palliative care and most negative for treating respiratory conditions, poor appetite, and mental illness. The proportion of positive sentiment responses increased from 17.6% to 32.4%. Provider responses coded as negative sentiment received more provider ?Agrees? (mean rank=280) than those coded as positive (mean rank=215), beta coefficient=0.33; 95% CI: 0.05, 0.62; p=.02. Cannabis use is a health topic of public interest. Variability in provider responses reflects the need for more research and consensus building to inform evidence -based clinical guidelines for cannabis use in medicine

Onset of Regular Cannabis Use & Young Adult Insomnia: An Analysis of Shared Genetic Liability
Evan A. Winiger, Spencer B. Huggett, Alexander S. Hatoum, Naomi P. Friedman, Christopher L. Drake, Kenneth P. Wright Jr., John K. Hewitt
Oxford University Press on behalf of the Sleep Research Society.
DOI: 10.1093/sleep/zsz293
Study Objectives: Estimate the genetic and environmental influences on the relationship between onset of regular cannabis use and young adult insomnia. Methods: In a population-based twin cohort of 1882 twins (56% female, mean age = 22.99, SD = 2.97) we explored the genetic/environmental etiology of the relationship between onset of regular cannabis use and insomnia-related outcomes via multivariate twin models. Results: Controlling for sex, current depression symptoms, and prior diagnosis of an anxiety or depression disorder, adult twins who reported early onset for regular cannabis use (age 17 or younger) were more likely to have insomnia (? = 0.07, p = 0.024) and insomnia with short sleep on weekdays (? = 0.08, p = 0.003) as young adults. We found significant genetic contributions for the onset of regular cannabis use (a 2 = 76%, p < 0.001), insomnia (a 2 = 44%, p < 0.001) and insomnia with short sleep on weekdays (a 2 = 37%, p < 0.001). We found significant genetic correlations between onset of regular use and both insomnia (rA = 0.20, p = 0.047) and insomnia with short sleep on weekdays (rA = 0.25, p = 0.008) but no significant environmental associations between these traits. Conclusion: We found common genetic liabilities for early onset of regular cannabis use and insomnia, implying pleiotropic influences of genes on both traits.

Opioid and healthcare service use in medical cannabis patients with chronic pain: a prospective study
Sharon Sznitman, Carolyn Mabouk, Zahi Said and Simon Vulfsons
BMJ Support Palliat Care. 2021 Sep 14;
DOI: 10.1136/bmjspcare-2020-002661
https://spcare.bmj.com/content/early...20-002661.long
Background Various jurisdictions have legalised medical cannabis (MC) for use in chronic pain treatment. The objective of this study was to determine if the use of MC is related to a reduction in the use of prescription opioids and other prescription medications and healthcare services.
Methods A retrospective cohort study was conducted using the medical files of 68 Israeli patients with chronic pain using MC. Number of prescription medications filled and healthcare services used were recorded separately for the baseline period (6 months prior to the start of MC treatment) and 6 months’ follow-up. Paired t-tests were used to compare each individual to himself/herself from baseline to follow-up.
Results Patients filled less opioid prescription medication at follow-up compared with baseline, and the reduction was of small effect size. There were no significant changes in the use of other medications or use of healthcare services from pre-MC treatment to follow-up.
Conclusions MC may be related to a significant yet small reduction in opioid prescription medication. Further prospective studies with representative samples are warranted to confirm the potential small opioid-sparing effects of MC treatment, its clinical importance, if any, and potential lack of association with other healthcare-related services and medication use. Due to methodological limitations of the data used in this study, results may be regarded as preliminary and causal inferences cannot be made.
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Opposite Effects of Δ-9-Tetrahydrocannabinol and Cannabidiol on Human Brain Function and Psychopathology.

Bhattacharyya, S., Morrison, P. D., Fusar-Poli, P., Martin-Santos, R., Borgwardt, S., Winton-Brown, T., … McGuire, P. K.
Neuropsychopharmacology, 35(3), 764–774.(2009).
doi:10.1038/npp.2009.184
D-9-tetrahydrocannabinol (D-9-THC) and Cannabidiol (CBD), the two main ingredients of the Cannabis sativa plant have distinct symptomatic and behavioral effects. We used functional magnetic resonance imaging (fMRI) in healthy volunteers to examine whether D-9-THC and CBD had opposite effects on regional brain function. We then assessed whether pretreatment with CBD can prevent the acute psychotic symptoms induced by D-9-THC. Fifteen healthy men with minimal earlier exposure to cannabis were scanned while performing a verbal memory task, a response inhibition task, a sensory processing task, and when viewing fearful faces. Subjects were scanned on three occasions, each preceded by oral administration of D-9-THC, CBD, or placebo. BOLD responses were measured using fMRI. In a second experiment, six healthy volunteers were administered D-9-THC intravenously on two occasions, after placebo or CBD pretreatment to examine whether CBD could block the psychotic symptoms induced by D-9-THC. D-9-THC and CBD had opposite effects on activation relative to placebo in the striatum during verbal recall, in the hippocampus during the response inhibition task, in the amygdala when subjects viewed fearful faces, in the superior temporal cortex when subjects listened to speech, and in the occipital cortex during visual processing. In the second experiment, pretreatment with CBD prevented the acute induction of psychotic symptoms by D-9-tetrahydrocannabinol. D-9-THC and CBD can have opposite effects on regional brain function, which may underlie their different symptomatic and behavioral effects, and CBD’s ability to block the psychotogenic effects of D-9-THC

Opposite Roles for Cannabidiol and δ-9-Tetrahydrocannabinol in Psychotomimetic Effects of Cannabis Extracts A Naturalistic Controlled Study
Alberto Sainz-Cort , Daniel Jimenez-Garrido, Elena Muñoz-Marron, Raquel Viejo-Sobera, Joost Heeroma, Jose Carlos Bouso
J Clin Psychopharmacol. 2021 Aug 19.
doi: 10.1097/JCP.0000000000001457
Background: Although δ-9-tetrahydrocannabinol (THC), the main cannabinoid from the cannabis plant, is responsible for the psychotomimetic effects of cannabis, cannabidiol (CBD), the second most abundant cannabinoid in the cannabis plant, does not show any psychotomimetic effect. Cannabidiol has even been proposed to be antipsychotic and to counteract some of the psychotomimetic effects of THC. The aim of this study was to test the potential antipsychotomimetic effects of CBD.
Method: Eighteen members from a cannabis social club were tested for subjective and psychotomimetic effects under the effects of different full-spectrum cannabis extracts containing either THC, CBD, THC + CBD, or placebo in a naturalistic, randomized, double-blind, crossover, placebo-controlled study.
Results: Results showed that participants under the effects of THC + CBD showed lower psychotomimetic scores in subjective scales when compared with THC alone. Subjective scores were lower under the effects of CBD and placebo when compared with THC + CBD. Cannabidiol and placebo did not show any psychotomimetic effect.
Conclusions: This study provides evidence for both the psychotomimetic effects of THC and the antipsychotomimetic effects of CBD when it is coadministered with THC in real-world situations, which can be very relevant for the clinical practice of medical cannabis. Ultimately, this study substantiates the link between the endocannabinoid system and psychotic-like symptoms and has important implications for the understanding of schizophrenia and the therapeutic potential of CBD as an antipsychotic. Lastly, we demonstrate how reliable methodologies can be implemented in real situations to collect valid ecological evidence outside classic laboratory settings.
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Oral Cannabidiol does not Alter the Subjective, Reinforcing or Cardiovascular Effects of Smoked Cannabis.
Haney M1, Malcolm RJ2, Babalonis S3, Nuzzo PA3, Cooper ZD1, Bedi G1, Gray KM2, McRae-Clark A2, Lofwall MR3, Sparenborg S4, Walsh SL3.
Neuropsychopharmacology.
DOI: 10.1038/npp.2015.367
Cannabidiol (CBD), a constituent of cannabis with few psychoactive effects, has been reported in some studies to attenuate certain aspects of ?9-tetrahydrocannabinol (THC) intoxication. However, most studies have tested only one dose of CBD in combination with one dose of oral THC making it difficult to assess the nature of this interaction. Further, the effect of oral CBD on smoked cannabis administration is unknown. The objective of this multi-site, randomized, double-blind, within-subject laboratory study was to assess the influence of CBD (0, 200, 400, 800?mg, p.o.) pretreatment on the reinforcing, subjective, cognitive, and physiological effects of smoked cannabis [0.01 (inactive), 5.30-5.80% THC]. Non-treatment-seeking, healthy cannabis smokers (n=31; 17M,14F) completed 8 outpatient sessions. CBD was administered 90?min prior to cannabis administration. The behavioral and cardiovascular effects of cannabis were measured at baseline and repeatedly throughout the session. A subset of participants (n=8) completed an additional session to measure plasma CBD concentrations after administration of the highest CBD dose (800?mg). Under placebo CBD conditions, active cannabis (1) was self-administered by significantly more participants than placebo cannabis, and (2) produced significant, time-dependent increases in ratings of 'High,' 'Good Effect,' ratings of the cannabis cigarette (eg, strength, liking) and heart rate relative to inactive cannabis. CBD, which alone produced no significant psychoactive or cardiovascular effects, did not significantly alter any of these outcomes. Cannabis self-administration, subjective effects, and cannabis ratings did not vary as a function of CBDdose relative to placebo capsules. These findings suggest that oral CBD does not reduce the reinforcing, physiological or positive subjective effects of smoked cannabis.

Oral Ingestion of Cannabis sativa: Risks, Benefits, and Effects on Malaria-Infected Hosts
Olugbenga Akinola, Elizabeth O. Ogbeche, Hidayah A. Olumoh-Abdul, Abdulmusawwir O. Alli-Oluwafuyi, Aboyeji L. Oyewole, Abdulbasit Amin, Wahab Imam AbdulMajeed, Olayemi Joseph Olajide, Abdurrazaq B. Nafiu, Anoka A. Njan, Olufunke E. Olorundare, and Grace O. Gbotosho. Cannabis and Cannabinoid Research
Volume 3.1, 2018
DOI: 10.1089/can.2018.0043 https://www.researchgate.net/publica...Infected_Hosts
Background: The emergence of a multidrug-resistant strain of Plasmodium falciparum (Pf Pailin) raises concern about malaria control strategies. Unfortunately, the role(s) of natural plants/remedies in curtailing malaria catastrophe remains uncertain. The claims of potential antimalarial activity of Cannabis sativa in vivo have not been well established nor the consequences defined. This study was, therefore, designed to evaluate the effects of whole cannabis consumption on malaria-infected host.
Methods: Thirty mice were inoculated with dose of 1 · 107 chloroquine-resistant Plasmodium berghei ANKAinfected erythrocyte and divided into six treatment groups. Cannabis diet formulations were prepared based on weighted percentages of dried cannabis and standard mice diet and the study animals were fed ad libitum. Chemosuppression of parasitemia, survival rates, parasite clearance, and recrudescence time were evaluated. Histopathological studies were performed on the prefrontal cortex (PFC) and hippocampus of the animals after 14 days’ consumption of cannabis diet formulation by naive mice.
Results: There was a significant difference ( p < 0.05) in the day-4 chemosuppression of parasitemia between the animals that were fed C. sativa and chloroquine relative to the untreated controls. There was also a significant difference in the survival rate ( p < 0.05) of animals fed C. sativa diet (40%, 20%, 10%, and 1%) in contrast to control animals on standard mice diet. A parasite clearance time of 2.18 – 0.4 was recorded in the chloroquine treatment group, whereas recrudescence in chloroquine group occurred on day 7. There were slight histomorphological changes in the PFC and cell densities of the dentate gyrus of the hippocampus of animals that were fed C. sativa.
Conclusions: C. sativa displayed mild antimalarial activity in vivo. There was evident reduction in symptomatic manifestation of malaria disease, though unrelated to levels of parasitemia. This disease tolerance status may be beneficial, butmay also constitute a transmission burden through asymptomatic carriage of parasites by habitual cannabis users.

Overlapping patterns of recreational and medical cannabis use in a large community sample of cannabis users
Jasmine Turna. Iris Balodis, Catharine Munn, Michael Van Ameringen, Jason Busse, James MacKillop
Comprehensive Psychiatry (2020)
DOI: 10.1016/j.comppsych.2020.152188
Background: Regulatory changes are increasing access to both medical cannabis and cannabis in general. As such, understanding patterns of recreational and medical cannabis use is a high public health priority. Objectives: Patterns of cannabis use (recreational and medical), other substance use , and psychiatric symptoms were characterized in a large sample of community adult cannabis users in Canada, prior to federal cannabis legalization. Methods: This was a self-report assessment of 709 cannabis users (Mean age = 30.19 (11.82) years; 55.01% female). Patterns of overall substance use and psychiatric symptomatology were compared based on recreational/medical cannabis status were examined.
Results: Overall, 61.4% of participants endorsed exclusively recreational use , while 38.6% reported some level of medical use. Of all medical users, only 23.4% reported authorization from a health professional . Recreational cannabis users typically reported infrequent use (less than weekly), whereas medical users modally reported daily use. Compared to recreational users, medical users reported more problematic cannabis use in addition to greater psychiatric symptomatology (anxiety, depression and trauma). Interestingly, a large majority of medical users also reported using recreationally (80.6%), while exclusive medical use was less common (19.3%) . This dual motives group reported more daily cannabis use and more alcohol and tobacco use. Compared to medical -only users, individuals using cannabis for both medical and recreational purposes more often used cannabis to treat psychiatric conditions.
Conclusions: These findings reveal the differences in cannabis use patterns and preferences between recreational and medical users, and even within medical users. In particular, dual motives individuals, who use cannabis for both positively and negatively reinforcing purposes, may warrant special attention as a subpopulation.

Overview of Cannabis including Kampo Medicine and Therapy for Treatment of Dementia: A Review
Wenger Tibor, Kazuhito Watanabe, Yui Sasaki, Yukihiro Shoyama
Frontiers in Pharmacology 12 March 2022
DOI: 10.3389/fphar.2021.713228
https://www.frontiersin.org/articles/10.3389/fphar.2021.713228/full
Cannabis sativa L. is an annual herb oldest cultivated plants as a source of fiber since about 5000 B.C. On the other hand, the cannabis flower and seed are listed in Shennong’s classic Materia Medica approximately 2000 years ago. The formulas prescribed with cannabis in Kampo medicine have been summarized. Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the major neurological and psychiatric cannabinoids, and develop to drugs. It becomes evident that the therapeutic CBD and/or THC are the important candidate of anti-dementia drugs having different mechanism for Alzheimer’s patients. Two receptors and endocannabinoids are also discussed for underlying mechanism of action. In order to promote the breeding of cannabis plant containing higher concentration of target cannabinoid the biosynthetic enzymes were isolated, cloning and the tertiary structure of THCA synthase determined by x-ray analysis resulting in the possibility of molecular breeding for cannabinoids.


P.0357 Effect of a combination therapy with cannabidiol and β-caryophyllene in SCN1A-A1783V mice, an experimental model of dravet syndrome
Cristina Alonso Gómez, Valentina Satta, I Hernández Fisac, O. Sagredo Ezkioga
European neuropsychopharmacology Dec 2021
10.1016/j.euroneuro.21.10.338
Abstract?
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Palmitoylethanolamide: A Potential Alternative to Cannabidiol

Paul Clayton, Silma Subah, Ruchitha Venkatesh, Mariko Hill, Nathasha Bogoda
Journal of Dietary Supplements 2021
DOI: 10.1080/19390211.2021.2005733
https://www.tandfonline.com/doi/full...05733?scroll=t op&needAccess=true
The endocannabinoid system (ECS) is a widespread cell signaling network that maintains homeostasis in response to endogenous and exogenous stressors. This has made the ECS an attractive therapeutic target for various disease states. The ECS is a well-known target of exogenous phytocannabinoids derived from cannabis plants, the most well characterized being Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). However, the therapeutic efficacy of cannabis products comes with a risk of toxicity and high abuse potential due to the psychoactivity of THC. CBD, on the other hand, is reported to have beneficial medicinal properties including analgesic, neuroprotective, anxiolytic, anticonvulsant, and antipsychotic activities, while apparently lacking the toxicity of THC. Nevertheless, not only is the currently available scientific data concerning CBD’s efficacy insufficient, there is also ambiguity surrounding its regulatory status and safety in humans that brings inherent risks to manufacturers. There is a demand for alternative compounds combining similar effects with a robust safety profile and regulatory approval. Palmitoylethanolamide (PEA) is an endocannabinoid-like lipid mediator, primarily known for its anti-inflammatory, analgesic and neuroprotective properties. It appears to have a multi-modal mechanism of action, by primarily activating the nuclear receptor PPAR-α while also potentially working through the ECS, thus targeting similar pathways as CBD.
With proven efficacy in several therapeutic areas, its safety and tolerability profile and the development of formulations that
maximize its bioavailability, PEA is a promising alternative to CBD.
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Palmitoylethanolamide dampens neuroinflammation and anxiety-like behavior in obese mice
Adriano Lama, Claudio Pirozzi, Ilenia Severi, Maria Grazia Morgese, Martina Senzacqua, Chiara Annunziata, Federica Comella, Filomena Del Piano, Stefania Schiavone, Stefania Petrosino, Maria Pina Mollica, Sabrina Diano, Luigia Trabace, Antonio Calignano, Antonio Giordano, Giuseppina Mattace Raso, Rosaria Meli
Brain, Behavior, and Immunity 102 (2022) 110-123
doi: 10.1016/j.bbi.2022.02.008
https://reader.elsevier.com/reader/...egion=us-east-1&originCreation=20220424173633
High-fat diet (HFD) consumption leads to obesity and a chronic state of low-grade inflammation, named met-ainflammation. Notably, metainflammation contributes to neuroinflammation due to the increased levels of circulating free fatty acids and cytokines. It indicates a strict interplay between peripheral and central counterparts in the pathogenic mechanisms of obesity-related mood disorders. In this context, the impairment of internal hypothalamic circuitry runs in tandem with the alteration of other brain areas associated with emotional processing (i.e., hippocampus and amygdala). Palmitoylethanolamide (PEA), an endogenous lipid mediator belonging to the N-acylethanolamines family, has been extensively studied for its pleiotropic effects both at central and peripheral level.
Our study aimed to elucidate PEA capability in limiting obesity-induced anxiety-like behavior and neuroinflammation-related features in an experimental model of HFD-fed obese mice. PEA treatment promoted an improvement in anxiety-like behavior of obese mice and the systemic inflammation, reducing serum pro-inflammatory mediators (i.e., TNF-α, IL-1β, MCP-1, LPS). In the amygdala, PEA increased dopamine turnover, as well as GABA levels. PEA also counteracted the overactivation of HPA axis, reducing the expression of hypothalamic corticotropin-releasing hormone and its type 1 receptor. Moreover, PEA attenuated the immunoreactivity of Iba-1 and GFAP and reduced pro-inflammatory pathways and cytokine production in both the hypothalamus and hippocampus. This finding, together with the reduced transcription of mast cell markers (chymase 1 and tryptase β2) in the hippocampus, indicated the weakening of immune cell activation underlying the neuroprotective effect of PEA. Obesity-driven neuroinflammation was also associated with the disruption of blood–brain barrier (BBB) in the hippocampus. PEA limited the albumin extravasation and restored tight junction transcription modified by HFD. To gain mechanistic insight, we designed an in vitro model of metabolic injury using human neuroblastoma SH-SY5Y cells insulted by a mix of glucosamine and glucose.
Here, PEA directly counteracted inflammation and mitochondrial dysfunction in a PPAR-α-dependent manner since the pharmacological blockade of the receptor reverted its effects.
Our results strengthen the therapeutic potential of PEA in obesity-related neuropsychiatric comorbidities, controlling neuroinflammation, BBB disruption, and neurotransmitter imbalance involved in behavioral Dysfunction

Peering Through the Haze of Smoked vs Vaporized Cannabis—To Vape or Not to Vape?
Solowij, N.
JAMA Network Open, 1(7), e184838. (2018).
doi:10.1001/jamanetworkopen.2018.4838
We are living in an era of unprecedented rapid change to cannabis use legislation across jurisdictions worldwide. Increased availability of cannabis, and inherent perceived endorsement of its apparent harmlessness, will inevitably lead to some increase in uptake by novice or infrequent users.1,2 Missives around potential safety of delivery by 1 method vs another circulate, yet like much of the anecdotal information that abounds, there is little scientific examination of concomitant consequences. In a placebo-controlled crossover trial conducted in healthy adults who infrequently use cannabis, Spindle and colleagues3 report a comparison of acute effects of smoked vs vaporized cannabis at 2 different doses. Dose-orderly subjective drug effects, cardiovascular effects, and impaired cognitive and psychomotor function were observed, with greater effects and higher blood cannabinoid concentrations achieved from vaporization. The authors warn that even relatively low-potency cannabis can adversely affect inexperienced users.

Perceived Efficacy, Reduced Prescription Drug Use, and Minimal Side Effects of Cannabis in Patients with Chronic Orthopedic Pain
Ari Greis, Eric Larsen, Conan Liu, Bryan Renslo, Anjithaa Radakrishnan, and Adrianne R. Wilson-Poe
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0088
https://www.liebertpub.com/doi/epdf/.../can.2021.0088
Introduction: Although cannabis is widely used for the treatment of chronic pain, most research relies on patient self-report and few studies have objectively quantified its efficacy and side effects. Extant inventories for measuring cannabis use were not designed to capture the medically relevant features of cannabis use, but rather were designed to detect problematic use or cannabis use disorder. Thus, we sought to capture the medically relevant features of cannabis use in a population of patients with orthopedic pain and pair these data with objective measures of pain and prescription drug use.
Materials and Methods: In this prospective observational study, orthopedic pain patients were enrolled in Pennsylvania’s medical cannabis program by their treating pain management physician, received cannabis education from their physician at the time of certification, and purchased products from state-licensed cannabis retailers.
Results: Medical cannabis use was associated with clinical improvements in pain, function, and quality of life with reductions in prescription drug use; 73% either ceased or decreased opioid consumption and 31% discontinued benzodiazepines. Importantly, 52% of patients did not experience intoxication as a side effect of cannabis therapy. Significant clinical benefits of cannabis occurred within 3 months of initiating cannabis therapy and plateaued at the subsequent follow-ups.
Conclusions: This work provides a direct relationship between the initiation of cannabis therapy and objectively fewer opioid and benzodiazepine prescriptions. Our work also identifies specific subpopulations of patients for whom cannabis may be most efficacious in reducing opioid consumption, and it highlights the importance of both physician involvement and patient self-titration in symptom management with cannabis.

Personal Account of Medical Use of Cannabis
Hodges, Clare. (2002). Personal Account of Medical Use of Cannabis. Journal of Cannabis Therapeutics. 2(3-4).
DOI: 10.1300/J175v02n03_11.
The author provides a personal account of her sojourn with multiple sclerosis and its treatment with smoked and oral preparations of cannabis. Additional information is provided as to the effects, dosing and delivery of cannabis employed by 250 members of the Alliance for Cannabis Therapeutics.

Perspectives on formation of medical cannabis market in Ukraine based on holistic approach
Nataliia Aliekperova, ?ostyantyn Kosyachenko and Oleksandr Kaniura
Journal of Cannabis Research (2020) 2:33
Doi 10.1186/s42238-020-00044-y
Background: Nowadays, medical cannabis still remains inaccesible for patients in the former Union of Soviet Socialist Republics (USSR). Even registered medicinal products based on herbal or synthetic cannabinoids, like Sativex, are practically unavailable due to their high cost and narrow scope of application (for example, in Lithuania). However, before the absolute prohibition of medical cannabis in the USSR, in line with Single Convention of 1961, the State Pharmacopoeia of the USSR (eighth edition) published monographs on such medicinal products as “Herba Cannabis indicae”, “Extractum Cannabis indicae spissum” and “Fructus Cannabis”, which could be prescribed by physicians with precaution.
Objectives: Formation of a holistic approach aimed at the creation of appropriate conditions for the development of medical cannabis market and the improvement of life quality and health of Ukrainian patients.
Methods: We analyzed legislation and regulation mechanisms for medical cannabis in the USSR, and the present availability of these products for patients in the former USSR, such as Lithuania, Georgia, Estonia, Russian Federation, and Ukraine. Four hundred thirty-five Ukrainian pharmacy students participated in the quantitative analysis (a survey) that took place on April–May, 2019 at Bogomolets National Medical University (Kyiv, Ukraine). They were surveyed about legalization of medical cannabis in Ukraine, advisability of including cannabis and cannabinoids related data into educational programs, and other issues. Qualitative analysis we applied consisted of the stakeholder analysis and Strength, Weakness, Opportunity, and Threat (SWOT) analysis. We classified the key stakeholders into the patients, healthcare professionals, legislative and regulatory bodies, pharmaceutical companies, pharmacies, etc., and ranked them based on their power and interest in the development of potential medical cannabis market in Ukraine. We also identified their expectations and goals. SWOT analysis allows us to evaluate predictable risks and opportunities, as well as strong and weak aspects of the effective development of medical cannabis industry in Ukraine.

Pharmacists and the future of cannabis medicine.
Schmitz, N., & Richert, L.
Journal of the American Pharmacists Association (2019)..
doi:10.1016/j.japh.2019.11.007
Objectives: To summarize the history and evolution of cannabis use and policies and to review current therapeutic uses, safety, and the central role pharmacists can play. Summary: Cannabis regulation and use have evolved over the centuries and are becoming more widely accepted, with over two-thirds of states in the United States having an approved cannabis program. However, changing policy and a paucity of controlled clinical trials has led to questions on the safety and effectiveness of cannabinoid therapies. Although there are conditions for which cannabinoids may be helpful, potential contraindications, adverse effects, and drug-drug interactions should be taken into account. Conclusion: Pharmacists are in a unique position based on their accessibility, knowledge, and skills to guide product selection, dosing, and discuss drug interactions and adverse effects to educate patients on safe cannabis use, whether it be delta-9-tetrahydrocannabinol, cannabidiol, or a combination thereof. Pharmacists and pharmacy organizations, moreover, should advocate for an integral role in the medical cannabis movement to ensure patient safety and evaluate cannabinoid pharmacology, pharmacokinetics, drug-drug interactions, safety, and efficacy through rigorous investigations.

Pharmacodynamic effects of vaporized and oral cannabidiol (CBD) and vaporized CBD-dominant cannabis in infrequent cannabis users
Tory Spindle, Edward J Cone, Elia Goffi, Elise M Weerts, John Mitchell, Ruth E. Winecker, George E. Bigelow, Ron Flegel, Ryan Vandrey,
Drug and Alcohol Dependence April 2020
DOI: 10.1016/j.drugalcdep.2020.107937
The use and availability of oral and inhalable products containing cannabidiol (CBD) as the principal constituent has increased with expanded cannabis/hemp legalization. However, few controlled clinical laboratory studies have evaluated the pharmacodynamic effects of oral or vaporized CBD or CBD-dominant cannabis. Methods Eighteen healthy adults (9 men; 9 women) completed four, double-blind, double-dummy, drug administration sessions. Sessions were separated by ?1 week and included self-administration of 100 mg oral CBD, 100 mg vaporized CBD, vaporized CBD-dominant cannabis (100 mg CBD; 3.7 mg THC), and placebo. Study outcomes included: subjective drug effects, vital signs, cognitive/psychomotor performance, and whole blood THC and CBD concentrations. Results Vaporized CBD and CBD-dominant cannabis increased ratings on several subjective items (e.g., Like Drug Effect) relative to placebo. Subjective effects did not differ between oral CBD and placebo and were generally higher for CBD-dominant cannabis compared to vaporized CBD. CBD did not increase ratings for several items typically associated with acute cannabis/THC exposure (e.g., Paranoid). Women reported qualitatively higher ratings for Pleasant Drug Effect than men after vaporized CBD and CBD-dominant cannabis use. CBD-dominant cannabis increased heart rate compared to placebo. Cognitive/psychomotor impairment was not observed in any drug condition. Conclusions Vaporized CBD and CBD-dominant cannabis produced discriminable subjective drug effects, which were sometimes stronger in women, but did not produce cognitive/psychomotor impairment. Subjective effects of oral CBD did not differ from placebo. Future research should further elucidate the subjective effects of various types of CBD products (e.g., inhaled, oral, topical), which appear to be distinct from THC-dominant products.

Pharmacokinetic and Safety Evaluation of Various Oral Doses of a Novel 1:20 THC:CBD Cannabis Herbal Extract in Dogs.
Chicoine, A., Illing, K., Vuong, S., Pinto, K. R., Alcorn, J., & Cosford, K.
Frontiers in Veterinary Science, 7.(2020).
doi:10.3389/fvets.2020.583404
Objective: To determine the pharmacokinetics (PK) and safety of various oral doses of a Cannabis herbal extract (CHE) containing a 1:20 ratio of 19-tetrahydrocannabinol (THC):cannabidiol (CBD) in 13 healthy Beagle-cross dogs.
Methods: Single-dose PK was assessed after oral administration of CHE at low, medium, or high doses [2, 5, or 10 mg CBD and 0.1, 0.25, or 0.5 mg THC per kg of body weight (bw), respectively; n = 6 per group]. Dogs were monitored for adverse events for up to 48 h post-dose. Evaluations of neurological signs, clinical laboratory abnormalities, and other adverse events were performed in two separate study phases: a multiple-dose phase with 12 dogs receiving five medium doses (5 mg CBD/kg bw) at 12 h intervals, and a single low-dose (2 mg CBD/kg bw), randomized, blinded, negative controlled study with 13 dogs.
Results: Cannabinoids CBD, THC, CBC, and metabolites 6-OH-CBD, 7-OH-CBD, 11-OH-THC, and THC-COOH were quantified in plasma. CBD and THC were rapidly absorbed (mean Tmax of 1.9–2.3 h) and initially depleted rapidly (mean CBD T1/2? of 2.3–2.6 h). A prolonged elimination phase (mean CBD T1/2? of 13.3–24.4 h) was observed. CBD and THC concentrations increased in a dose-dependent (non-linear) manner, with disproportionally greater cannabinoid exposure relative to the dose increase. Neurological signs (hyperesthesia or proprioceptive deficits) were noted in five of six dogs in the high-dose group, but only occasionally or rarely in the medium- and low-dose groups, respectively. Presence and severity of clinical signs correlated with plasma cannabinoid concentrations. Dogs appeared to develop a tolerance to cannabinoid effects after multiple CHE doses, with fewer neurological signs noted after the final (fifth) vs. first dose. No clinically meaningful changes in blood count or chemistry values occurred after multiple CHE doses.
Clinical Significance: Dogs tolerated the 1:20 THC:CBD formulation well at low and medium doses, but clinically meaningful neurological signs were observed at high doses. Because of non-proportional increases in plasma cannabinoid concentrations with increasing doses, as well as potential differences in CHE product composition and bioavailability, the possibility of adverse events and dose regimen consistency should be discussed with dog owners.

Pharmacokinetic Investigation of Commercially Available Edible Marijuana Products in Humans: Potential Influence of Body Composition and Influence on Glucose Control
Taylor Russell Ewell, Kieran Shay Struebin Abbotts, Natasha N. Bondareva Williams, Hannah Michelle Butterklee, Matthew Charles Bomar, Kole Jerel Harms, Jordan Douglas Rebik, Sarah Margaret Mast, Natalie Akagi, Gregory P. Dooley and Christopher Bell
Pharmaceuticals 2021, 14, 817.
DOI: 10.3390/ph14080817
https://www.ncbi.nlm.nih.gov/pmc/art...s-14-00817.pdf
The purpose of the study was to describe and compare the pharmacokinetics of five commercial edible marijuana products, determine the influence of body composition on pharmacokinetics, and, in light of epidemiology suggesting marijuana may offer diabetes protection, explore the influence of edible marijuana on glucose tolerance. Seven regular users of marijuana self-administered five edible products in a randomized crossover design; each product contained 10 mg of delta-9- tetrahydrocannabinol (THC). Thirty minutes following marijuana ingestion, participants imbibed a 75 g glucose beverage. Time-to-peak plasma THC concentration ranged between 35 and 90 min; maximal plasma THC concentration (Cmax) ranged between 3.2 and 5.5 ng/mL. Differences between products in plasma THC concentration during the first 20–30 min were detected (p = 0.019). Relations were identified between body composition and pharmacokinetic parameters for some products; however, none of these body composition characteristics were consistently related to pharmacokinetics across all five of the products. Edible marijuana had no effect on oral glucose tolerance compared with a marijuana-free control (Matsuda Index; p > 0.395). Commercially available edible marijuana products evoke different plasma THC concentrations shortly after ingestion, but do not appear to influence acute glucose regulation. These data may allow recreational marijuana users to make informed decisions pertaining to rates of edible marijuana ingestion and avoid overdose.

Pharmacokinetics and metabolism of the plant cannabinoids, delta9-tetrahydrocannabinol, cannabidiol and cannabinol.
Huestis M.A.
Handb Exp Pharmacol. 2005;(168):657-90.
DOI: 10.1007/3-540-26573-2_23
Increasing interest in the biology, chemistry, pharmacology, and toxicology of cannabinoids and in the development of cannabinoid medications necessitates an understanding of cannabinoid pharmacokinetics and disposition into biological fluids and tissues. A drug's pharmacokinetics determines the onset, magnitude, and duration of its pharmacodynamic effects. This review of cannabinoid pharmacokinetics encompasses absorption following diverse routes of administration and from different drug formulations, distribution of analytes throughout the body, metabolism by different tissues and organs, elimination from the body in the feces, urine, sweat, oral fluid, and hair, and how these processes change over time. Cannabinoid pharmacokinetic research has been especially challenging due to low analyte concentrations, rapid and extensive metabolism, and physicochemical characteristics that hinder the separation of drugs of interest from biological matrices--and from each other--and lower drug recovery due to adsorption of compounds of interest to multiple surfaces. delta9-Tetrahydrocannabinol, the primary psychoactive component of Cannabis sativa, and its metabolites 11-hydroxy-delta9-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol are the focus of this chapter, although cannabidiol and cannabinol, two other cannabinoids with an interesting array of activities, will also be reviewed. Additional material will be presented on the interpretation of cannabinoid concentrations in human biological tissues and fluids following controlled drug administration.

Pharmacokinetics of cannabichromene in a medical cannabis product also containing cannabidiol and Δ9 tetrahydrocannabinol: a pilot study Erica N. Peters · Laura MacNair · Irina Mosesova · Uwe Christians · Cristina Sempio · Jost Klawitter · M. Hunter Land · Mark A. Ware · Cynthia Turcotte · Marcel O. Bonn Miller
European Journal of Clinical Pharmacology (2022) 78:259–265
DOI: 10.1007/s00228-021-03232-8
https://link.springer.com/content/pd...21-03232-8.pdf
Purpose Cannabichromene (CBC) is a phytocannabinoid commonly found in cannabis, yet its acute post-dose pharmacokinetics (PK) have not been examined in humans. This is a secondary data analysis from a trial investigating Spectrum Yellow oil, an oral cannabis product used for medical purposes that contained 20 mg cannabidiol (CBD), 0.9 mg Δ9 - tetrahydrocannabinol (THC), and 1.1 mg CBC, per 1 mL of oil. Methods Participants (N=43) were randomized to one of 5 groups: 120 mg CBD, 5.4 mg THC, and 6.6 mg CBC daily; 240 mg CBD, 10.8 mg THC, and 13.2 mg CBC daily; 360 mg CBD, 16.2 mg THC, and 19.8 mg CBC daily; 480 mg CBD, 21.6 mg THC, and 26.4 mg CBC daily; or placebo. Study medication was administered every 12 h for 7 days. Plasma CBC concentrations were analyzed by a validated two-dimensional high-performance liquid chromatography–tandem mass spectrometry assay. Results After a single dose and after the final dose, the Cmax of CBC increased by 1.3–1.8-fold for each twofold increase in dose; the tmax range was 1.6–4.3 h. Based on the ratio of administered CBD, THC, and CBC to the plasma concentration, the dose of CBD was 18 times higher than the dose of CBC, yet the AUC0–t of CBD was only 6.6–9.8-fold higher than the AUC0–t of CBC; the dose of THC was similar to the dose of CBC, yet THC was quantifiable in fewer plasma samples than was CBC. Conclusions CBC may have preferential absorption over CBD and THC when administered together

Pharmacokinetics of Sativex® in Dogs: Towards a Potential Cannabinoid-Based Therapy for Canine Disorders.
Fernández-Trapero, Pérez-Díaz, Espejo-Porras, de Lago, & Fernández-Ruiz.
Biomolecules, 10(2), 279.(2020).
doi:10.3390/biom10020279
The phytocannabinoid?based medicine Sativex® is currently marketed for the treatment of spasticity and pain in multiple sclerosis patients and is being investigated for other central and peripheral pathological conditions. It may also serve in Veterinary Medicine for the treatment of domestic animals, in particular for dogs affected by different pathologies, including human?like pathological conditions. With the purpose of assessing different dosing paradigms for using Sativex in Veterinary Medicine, we investigated its pharmacokinetics when administered to naïve dogs via sublingual delivery. In the single dose arm of the study, adult Beagle dogs were treated with 3 consecutive sprays of Sativex, and blood samples were collected at 12 intervals up to 24 h later. In the multiple dose arm of the study, Beagle dogs received 3 sprays daily for 14 days, and blood samples were collected for 24 h post final dose. Blood was used to obtain plasma samples and to determine the levels of cannabidiol (CBD), ?9?tetrahydrocannabinol (?9?THC) and its metabolite 11? hydroxy??9?THC. Maximal plasma concentrations of both ?9?THC (Cmax = 18.5 ng/mL) and CBD (Cmax = 10.5 ng/mL) were achieved 2 h after administration in the single dose condition and at 1 h in the multiple dose treatment (?9?THC: Cmax = 24.5 ng/mL; CBD: Cmax = 15.2 ng/mL). 11?hydroxy??9? THC, which is mainly formed in the liver from ?9?THC, was almost undetected, which is consistent with the use of sublingual delivery. A potential progressive accumulation of both CBD and ?9?THC was detected following repeated exposure, with maximum plasma concentrations for both cannabinoids being achieved following multiple dose. Neurological status, body temperature, respiratory rate and some hemodynamic parameters were also recorded in both conditions, but in general, no changes were observed. In conclusion, this study demonstrates that single or multiple dose sublingual administration of Sativex to naïve dogs results in the expected pharmacokinetic profile, with maximal levels of phytocannabinoids detected at 1–2 h and suggested progressive accumulation after the multiple dose treatment.

Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation
Patricia Rivera, Laura Bindila,Antoni Pastor,Margarita Pérez-Martín,Francisco J. Pavón,Antonia Serrano,Rafael de la Torre,Beat Lutz,Fernando Rodríguez de Fonseca andJuan Suárez
Front. Cell. Neurosci., 27 March 2015
DOI: 10.3389/fncel.2015.00098
https://www.frontiersin.org/articles/10.3389/fncel.2015.00098/full
Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3+ or BrdU+ cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3+), astroglia (GFAP+), and microglia (Iba1+ cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3+ and BrdU+ subgranular cells as well as GFAP+, Iba1+ and cleaved caspase-3+ cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3+, GFAP+ and 3-weeks-old BrdU+ cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context


Pharmacologic Treatment of Cannabinoid Hyperemesis Syndrome: A Systematic Review
John R. Richards Brent K. Gordon Aaron R. Danielson Aimee K. Moulin
harmacotherapy: The Journal of Human Pharmacology and Drug Therapy Volume 37, Issue 6 June 2017 Pages 725-734
DOI: 10.1002/phar.1931
Cannabinoid hyperemesis syndrome (CHS) has become more prevalent with increasing cannabis use. CHS is often resistant to standard antiemetics. The objective of this study is to review the current evidence for pharmacologic treatment of CHS. Medline, PsycINFO, DARE, OpenGrey, Google Scholar, and the Cochrane Library were searched from inception to February 2017. Articles were selected and reviewed independently. Evidence was graded using Oxford Center for Evidence?Based Medicine guidelines. The search resulted in 1262 articles with 63 of them eligible for inclusion (205 human subjects). There were 4 prospective level?2, 3 retrospective level?3 studies, 12 level?4 case series, and 44 level?5 case reports. Among level?2 studies (64 subjects), tricyclic antidepressants (TCAs) and lorazepam were discussed as effective long? and short?term treatments, respectively, in two studies. Ondansetron, promethazine, diphenhydramine, and opioids were also mentioned, but the authors did not comment on their efficacy. Among level?3 studies (43 subjects), one reported effective treatment with antiepileptics zonisamide and levetiracetam, but not TCAs. Another reported favorable response to morphine, ondansetron, and lorazepam but did not specify the actual number of patients receiving specific treatment. Among the level?4 case series (54 subjects), benzodiazepines, haloperidol, and capsaicin were reported as helpful. For level?5 case reports (44 subjects), benzodiazepines, metoclopramide, haloperidol, ondansetron, morphine, and capsaicin were reported as effective. Effective treatments mentioned only once included fentanyl, diazepam, promethazine, methadone, nabilone, levomepromazine, piritramide, and pantoprazole. Hot showers and baths were cited in all level?4 and ?5 articles as universally effective. High?quality evidence for pharmacologic treatment of CHS is limited. Benzodiazepines, followed by haloperidol and capsaicin, were most frequently reported as effective for acute treatment, and TCAs for long?term treatment. As the prevalence of CHS increases, future prospective trials are greatly needed to evaluate and further define optimal pharmacologic treatment of patients with CHS.

Pharmacological and Therapeutic Secrets of Plant and Brain (Endo)Cannabinoids
Lumır Ondrej Hanus
Medicinal Research Reviews 29(2):213-71 Oct 2008
DOI 10.1002/med.20135
Research on the chemistry and pharmacology of cannabinoids and endocannabinoids has reached enormous proportions, with approximately 15,000 articles on Cannabis sativa L. and cannabinoids and over 2,000 articles on endocannabinoids. The present review deals with the history of the Cannabis sativa L. plant, its uses, constituent compounds and their biogeneses, and similarity to compounds from Radula spp. In addition, details of the pharmacology of natural cannabinoids, as well as synthetic agonists and antagonists are presented. Finally, details regarding the pioneering isolation of the endocannabinoid anandamide, as well as the pharmacology and potential therapeutic uses of endocannabinoid congeners are presented.

Pharmacological exploitation of the endocannabinoid system: new perspectives for the treatment of depression and anxiety disorders?
Viviane M. Saito, Carsten T. Wotjak, Fabrício A. Moreira
Revista Brasileira de Psiquiatria (Braz J Psychiatry. )• vol 32 • Suppl I • may2010 English & Spanish
DOI: 10.1590/S1516-44462010000500004
https://pdfs.semanticscholar.org/e73...1261dfe7e8f8f9 4b8d96.pdf
Objective: The present review provides a brief introduction into the endocannabinoid system and discusses main strategies of pharmacological interventions.
Method: We have reviewed the literature relating to the endocannabinoid system and its pharmacology; both original and review articles written in English were considered.
Discussion: Cannabinoids are a group of compounds present in Cannabis sativa (hemp), such as D9-tetrahydrocannabinol, and their synthetic analogues. Research on their pharmacological profile led to the discovery of the endocannabinoid system in the mammalian brain. This system comprises at least two G-protein coupled receptors, CB1 and CB2 their endogenous ligands (endocannabinoids; e.g. the fatty acid derivatives anandamide and 2-arachydonoyl glycerol), and the enzymes responsible for endocannabinoid synthesis and catabolism. Endocannabinoids represent a class of neuromessengers, which are synthesized on demand and released from post-synaptic neurons to restrain the release of classical neurotransmitters from pre-synaptic terminals. This retrograde signalling modulates a variety of brain functions, including anxiety, fear and mood, whereby activation of CB1 receptors was shown to exert anxiolytic- and antidepressant-like effects in preclinical studies.
Conclusion: Animal experiments suggest that drugs promoting endocannabinoid action may represent a novel strategy for the treatment of depression and anxiety disorders.

Pharmacological foundations of Cannabis chemovars. No strain, no gain
Lewis MA, Russo EB, Smith KM (2018)
Planta Med 84: 225–233
doi: 10.1055/s-0043-122240
An advanced Mendelian Cannabis breeding program has been developed utilizing chemical markers to maximize the yield of phytocannabinoids and terpenoids with the aim to improve therapeutic efficacy and safety. Cannabis is often divided into several categories based on cannabinoid content. Type I, ?9- tetrahydrocannabinol-predominant, is the prevalent offering in both medical and recreational marketplaces. In recent years, the therapeutic benefits of cannabidiol have been better recognized, leading to the promotion of additional chemovars: Type II, Cannabis that contains both ?9-tetrahydrocannabinol and cannabidiol, and cannabidiol-predominant Type III Cannabis. While high-?9-tetrahydrocannabinol and high-myrcene chemovars dominate markets, these may not be optimal for patients who require distinct chemical profiles to achieve symptomatic relief. Type II Cannabis chemovars that display cannabidiol- and terpenoid-rich profiles have the potential to improve both efficacy and minimize adverse events associated with ?9-tetrahydrocannabinol exposure. Cannabis samples were analyzed for cannabinoid and terpenoid content, and analytical results are presented via PhytoFacts, a patent-pending method of graphically displaying phytocannabinoid and terpenoid content, as well as scent, taste, and subjective therapeutic effect data. Examples from the breeding program are highlighted and include Type I, II, and III Cannabis chemovars, those highly potent in terpenoids in general, or single components, for example, limonene, pinene, terpinolene, and linalool. Additionally, it is demonstrated how Type I–III chemovars have been developed with conserved terpenoid proportions. Specific chemovars may produce enhanced analgesia, anti-inflammatory, anticonvulsant, antidepressant, and anti-anxiety effects, while simultaneously reducing sequelae of ?9-tetrahydrocannabinol such as panic

Pharmacological Interaction between Cannabidiol and A9-Tetrahydrocannabinol
I. G. Karniol, A.E. Carlini,
Psychopharmacologia (Ber].) 33, Issue 1, 53--70 (1973)
FIND DOI or LINK
The pharmacological interaction between cannabidiol (CBD) and
(--)Ag-trans-tetrahydrocannabinol (AS-TI-IC) has been studied in rabbits, mice and rats by administering mixtures containing varying amounts of both substances. CBD blocked the following effects of Ag-THC: catatonia in mice, corneal areflexia in rabbits, the increased defecation and decreased ambulation after chronic treatment and exposures of rats in an open field arena, and the aggressiveness of rats previously stressed by REM sleep deprivation. On the other hand, CBD potentiated the AS-TttC-induced analgesia in mice and the Ag-THC-impairing effect on climbing rope performance of rats. These interactions are tentatively explained by postulating that CBD directly antagonizes the excitatory effects and/or indirectly potentiates the depressant
effects of Ag-THC.

Pharmacology and potential therapeutic uses of cannabis
R. A. HIRST, D. G. LAMBERT AND W. G. NOTCUTT
British Journal of Anaesthesia 1998; 81: 77–84
https://www.researchgate.net/publica...es_of_cannabis
Throughout history, cannabis has been used as a natural therapeutic herb. Indeed the discovery of the remains of a young girl in Jerusalem who died in childbirth sometime around the third century AD, revealed residues of cannabis in the pelvic region.97 This strongly suggests that cannabis was used in medical practice in early human civilizations. Cannabis was first used as a medicine in Britain in the mid-nineteenth century by O’Shaugnessy, an army surgeon. While in India O’Shaugnessy witnessed at first hand the use of cannabis for a wide range of medical conditions such as rabies, epilepsy and muscle spasms, and for pain relief. On his return to Britain, O’Shaugnessy advocated its u se and cannabis was widely accepted as a medicine for about 70 years.61 63 In this article we shall review the pharmacology of cannabinoids and their therapeutic potential. In particular, we shall discuss their use as antinociceptive/ analgesic agents. The reader is referred to several excellent review articles and books on this subject.4 8 22 37 45 50 63 69 95 The major psychoactive constituent of Cannabis sativa was first isolated in 1964.32 The compound was identified as 9 -tetrahydrocannabinol ( 9 -THC, fig. 1), and when administered to humans produced a spectru m of effects, inclu ding increased pu lse rate, decreased blood pressure, muscle weakening, increased appetite, euphoria followed by drowsiness, depersonalization, altered time sense and decreased memory recollection; hearing became less discriminative and visual signals were sharper but distorted.45 The effects of 9 -THC at the biochemical level were virtually unknown until recently. Pharmacological studies suggested that 9 -THC and other active cannabinoids might act at specific receptor sites,38 68 but the high lipophilicity of these compounds made their use in binding assays very difficult. Some researchers were initially of the opinion that the effects observed with 9 -THC were attributable to perturbation of membrane structures.43 However, these proposals were refuted and early attempts to identify specific cannabinoid receptor binding sites in brain membranes showed encouraging results.

Physiological Rules of Endocannabinoid Action During Fetal and Neonatal Brain Development
Tibor Harkany and Valentina Cinquina
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.009
The endocannabinoid system is chiefly recognized as a homeostatic regulator of synaptic neurotransmission, primarily through the modulation of presynaptic CB1 cannabinoid neurons. Accordingly, the use of plant-derived cannabinoids received significant attention recently given the broad spectrum of physiological and pathobiological processes the endocannabinoid system is involved in. Nevertheless, a parallel line of research from a number of developmental biology groups has uncovered fundamental, evolutionarily conserved, and molecularly unique processes that endocannabinoids drive during development of the central nervous system. This lecture transcript is a concise summary of nearly 20 years of research on endocannabinoid-gated mechanisms of neurogenic specification events, which particularly define the numbers, placement, and connectivity of cortical neurons. A summary of both CB1 and alternative cannabinoid receptor contributions to neural differentiation is also discussed. Besides, insights are given into how phytocannabinoids can bypass physiologically timed and pivoted endocannabinoid action to inflict developmental errors that can significantly compromise the adaptive and computational ability of neurocircuits. By discussing specific subcellular targets of phytocannabinoid action and inferring errant glia versus neuron fate decisions and communication, a cellular basis is outlined for lifelong psychiatric phenotypes in offspring that associate with maternal cannabis seeking during pregnancy
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Phytocannabinoids: Useful Drugs for the Treatment of Obesity? Special Focus on Cannabidiol.
Bielawiec, P., Harasim-Symbor, E., & Chabowski, A.
Frontiers in Endocrinology, 11. (2020).
doi:10.3389/fendo.2020.00114
Currently, an increasing number of diseases related to insulin resistance and obesity is an alarming problem worldwide. It is well-known that the above states can lead to the development of type 2 diabetes, hypertension, and cardiovascular diseases. An excessive amount of triacylglycerols (TAGs) in a diet also evokes adipocyte hyperplasia and subsequent accumulation of lipids in peripheral organs (liver, cardiac muscle). Therefore, new therapeutic methods are constantly sought for the prevention, treatment and alleviation of symptoms of the above mentioned diseases. Currently, much attention is paid to Cannabis derivatives—phytocannabinoids, which interact with the endocannabinoid system (ECS) constituents. 19 -tetrahydrocannabinol (19 -THC) and cannabidiol (CBD) are the most abundant compounds of Cannabis plants and their therapeutic application has been suggested. CBD is considered as a potential therapeutic agent due to its anti-inflammatory, anti-oxidant, anti-tumor, neuroprotective, and potential anti-obesity properties. Therefore, in this review, we especially highlight pharmacological properties of CBD as well as its impact on obesity in different tissues

Placebo Effects of Edible Cannabis: Reported Intoxication Effects at a 30-Minute Delay
Mallory J. E. Loflin, Ph.D., Mitch Earleywine, Ph.D., Stacey Farmer, M.A., Melissa Slavin M.A., Rachel Luba, B.S. and Marcel Bonn-Miller, Ph.D.
DOI: 10.1080/02791072.2017.1354409
Previous research has demonstrated the ability of non-active smoked cannabis cigarettes to induce subjective effects of intoxication (i.e., placebo effect). No studies have been conduced to test whether edible forms of cannabis, which are associated with a significant delay in onset of effect, are able to induce a placebo effect. In the present study, 20 participants were told that they would receive an edible cannabis lollipop containing a high dose of tetrahydrocannabinol (THC), but were instead given a placebo control. Measures of intoxication and mood were taken at baseline, 30 minutes, and 60 minutes post-ingestion of the placebo lollipop. Results of four repeated-measures ANOVAs found significant and quadratic changes across time in cannabis (ARCI m-scale) intoxication (F(2,18) = 4.90, p = .01, ?2 = .22) and negative mood (F(2,18) = 3.99, p = .05, ?2 = .19). Changes in positive mood and the overall measure of general intoxication (ARCI) failed to reach significance. The present study provides preliminary evidence that a placebo effect can be induced with inert edible agents when participants are told that they are receiving active THC. This is the first known study to demonstrate an edible cannabis intoxication placebo effect.
Introduction: Inflammatory bowel diseases (IBDs) include Crohn’s disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in
detail with in vitro and ex vivo colon models.
Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.
Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the antiinflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner.
Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.
Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

Positive Effect of Cannabis sativa L. Herb Extracts on Skin Cells and Assessment of Cannabinoid-Based Hydrogels Properties
Martyna Zagórska-Dziok, Tomasz Bujak, Aleksandra Ziemlewska, and Zofia Nizio?-?ukaszewska
Molecules 2021 26(4), 802
DOI: 10.3390/molecules26040802
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The skin is an organ that is constantly exposed to many external factors that can affect its structure and function. Due to the presence of different cannabinoid receptors on many types of skin cells, cannabinoids can interact directly with them. Therefore, as part of this work, the impact of two types of Cannabis sativa L. herb extracts on keratinocytes and fibroblasts was assessed. The content of biologically active compounds such as phenols, flavonoids, chlorophylls and cannabinoids was evaluated. The antioxidant capacity of prepared extracts using the DPPH radical, H2DCFDA probe and measurement of superoxide dismutase activity was also assessed. The cytotoxicity of hemp extracts was determined using the Alamar Blue, Neutral Red and LDH assays. The ability of the extracts to inhibit the activity of matrix metalloproteinases, collagenase and elastase, was assessed. Preparations of model hydrogels were also prepared and their effect on transepidermal water loss and skin hydration was measured. The obtained results indicate that hemp extracts can be a valuable source of biologically active substances that reduce oxidative stress, inhibit skin aging processes and positively affect the viability of skin cells. The analysis also showed that hydrogels based on cannabis extracts have a positive effect on skin hydration.

Possible role of endocannabinoids in olfactory and taste dysfunctions in Alzheimer's and Parkinson's patients and volumetric changes in the brain

Emine Petekkaya, Berna Kuş, Serdar Doğan, Hanifi Bayaroğulları, Turay Mutlu, İsmet Murat Melek, Abdullah Arpacı
J Clin Neurosci
2022 Apr 7;100:52-58.
doi:10.1016/j.jocn.2022.03.047
https://pubmed.ncbi.nlm.nih.gov/35398594/
The purpose of this study is to determine the volumes of primary brain regions associated with smell and taste in Alzheimer's and Parkinson's patients and healthy controls using MR imaging and examine volumetric changes in comparison to smell/taste questionnaire and test results and endocannabinoid (EC) levels. The study included 15 AD patients with mild cognitive dysfunction scored as 18 ≤ MMSE ≤ 23, 15 PD patients with scores of 18 < MoCA < 26 and 18 ≤ MMSE ≤ 23, and 15 healthy controls. A taste and smell questionnaire was given to the participants, and their taste and smell statuses were examined using the Sniffin' Sticks smell identification test and Burghart Taste Strips. EC levels were analyzed in the blood serum samples of the participants using the ELISA method. The volumes of the left olfactory bulb (p = 0.001), left amygdala (p = 0.004), left hippocampus (p = 0.008), and bilateral insula (left p = 0.000, right p = 0.000) were significantly smaller in the Alzheimer's patients than the healthy controls. The volumes of the left olfactory bulb (p = 0.001) and left hippocampus (p = 0.009) were significantly smaller in the Parkinson's patients than the healthy controls. A significant correlation was determined between volume reduction in the left Rolandic operculum cortical region and taste dysfunction. EC levels were significantly higher in both AD (p = 0.000) and PD (p = 0.006) in comparison to the controls. Our results showed that volumetric changes occur in the brain regions associated with smell and taste in Alzheimer's and Parkinson's patients. It was observed that ECs played a role in these volumetric changes and the olfactory and taste dysfunctions of the patients.
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Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death Domenico E Pellegrini-Giampietro 1, Guido Mannaion FEBS. 2009 Jan;276(1):2-12.
doi: 10.1111/j.1742-4658.2008.06765.x
https://febs.onlinelibrary.wiley.com.. .8.2008.06765.x An emerging body of evidence supports a key role for the endocannabinoid system in numerous physiological and pathological mechanisms of the central nervous system. In the recent past, many experimental studies have examined the putative protective or toxic effects of drugs interacting with cannabinoid receptors or have measured the brain levels of endocannabinoids in in vitro and in vivo models of cerebral ischemia. The results of these studies have been rather conflicting in supporting either a beneficial or a detrimental role for the endocannabinoid system in post-ischemic neuronal death, in that cannabinoid receptor agonists and antagonists have both been demonstrated to produce either protective or toxic responses in ischemia, depending on a number of factors. Among these, the dose of the
administered drug and the specific endocannabinoid that accumulates in each particular model appear to be of particular importance. Other mechanisms that have been put forward to explain these discrepant results are the effects of cannabinoid receptor activation on the modulation of excitatory and inhibitory transmission, the vasodilatory and hypothermic effects of cannabinoids, and their activation of cytoprotective signaling pathways. Alternative mechanisms that appear to be independent from cannabinoid receptor activation have also been suggested. Endocannabinoids probably participate in the mechanisms that are triggered by the initial ischemic stimulus and lead to delayed neuronal death. However, further information is needed before pharmacological modulation of the endocannabinoid system may prove useful for therapeutic intervention in stroke and related ischemic syndrome

Potency Assessment of CBD Oils by Their Effects on Cell Signaling Pathways.
Urasaki, Beaumont, Workman, Talbot, Hill, & Le.
Nutrients, 12(2), 357.*(2020).*
doi:10.3390/nu12020357*
This study used nanofluidic protein posttranslational modification (PTM) profiling to measure the effects of six cannabidiol (CBD) oils and isolated CBD on the signaling pathways of a cultured SH-SY5Y neuronal cell line. Chemical composition analysis revealed that all CBD oils met the label claims and legal regulatory limit regarding the CBD and tetrahydrocannabinol (THC) contents, respectively. Isolated CBD was cytotoxic, with an effective concentration (EC50) of 40 µM. In contrast, the CBD oils had no effect on cell viability at CBD concentrations exceeding 1.2 mM. Interestingly, only an unadulterated CBD oil had strong and statistically significant suppressive effects on the pI3K/Akt/mTOR signaling pathway with an EC50 value of 143 µM and a slow-acting timescale requiring hours. Systematic profiling of twenty-six proteins, which served as biomarkers for nine signaling pathways, revealed that the unadulterated CBD oil downregulated seven signaling pathways but had no measurable effect on the other two signaling pathways. The remaining CBD oils, which were adulterated, and isolated CBD had weak, variable, or undetectable effects on neuronal signaling pathways. Our data clearly showed that adulteration diminished the biological activities of CBD oils. In addition, nanofluidic protein PTM profiling provided a robust means for potency assessment of CBD oils

Potential Adverse Drug Events and Drug–Drug Interactions with Medical and Consumer Cannabidiol (CBD) Use.
Brown, J. D., & Winterstein, A. G.
Journal of Clinical Medicine, 8(7), 989. (2019).
doi:10.3390/jcm8070989
Cannabidiol (CBD) is ubiquitous in state-based medical cannabis programs and consumer products for complementary health or recreational use. CBD has intrinsic pharmacologic effects and associated adverse drug events (ADEs) along with the potential for pharmacokinetic and pharmacodynamic drug–drug interactions (DDIs). Given CBD use among patients with complex conditions and treatment regimens, as well as its expanded consumer use, awareness of potential safety issues with CBD is needed. Prescribing information for federally approved products containing CBD were reviewed. Data on ADEs and DDIs were extracted and summarized. Nearly one-half of CBD users experienced ADEs, which displayed a general dose-response relationship. Common ADEs include transaminase elevations, sedation, sleep disturbances, infection, and anemia. Given CBD effects on common biological targets implicated in drug metabolism (e.g., CYP3A4/2C19) and excretion (e.g., P-glycoprotein), the potential for DDIs with commonly used medication is high. General clinical recommendations of reducing substrate doses, monitoring for ADEs, and finding alternative therapy should be considered, especially in medically complex patients. CBD is implicated as both a victim and perpetrator of DDIs and has its own ADE profile. These effects should be considered in the risk-benefit assessment of CBD therapy and patients and consumers made aware of potential safety issues with CBD use.

Potential cannabidiol (CBD) repurposing as antibacterial and promising therapy of CBD plus polymyxin B (PB) against PB‑resistant gram‑negative bacilli
NatháliaAbichabki, LuísaV. Zacharias, Natália C. Moreira, Fernando Bellissimo‑Rodrigues, Fernanda L. Moreira, Jhohann R. L. Benzi, Tânia M. C. Ogasawara, Joseane C. Ferreira, Camila M. Ribeiro, Fernando R. Pavan, Leonardo R. L. Pereira, GuilhermeT. P. Brancini, Gilberto Ú. L. Braga, AntonioW. Zuardi, Jaime E. C. Hallak, JoséA. S. Crippa, Vera L. Lanchote, Rafael Cantón, Ana Lúcia C. Darini & Leonardo N.Andrade
Nature Scientific reports (2022)
doi: 10.1038/s41598-022-10393-8
https://www.nature.com/articles/s41598-022-10393-8.pdf
This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD+polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD+PB also showed additive and/or synergistic efect against LOS-expressing GND. Time-kill assays results showed that the combination CBD+PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD+PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efcacy of the combination CBD+PB against multidrug-resistant and extensively drugresistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.

Potential Clinical Benefits of CBD-Rich Cannabis Extracts Over Purified CBD in Treatment-Resistant Epilepsy: Observational Data Meta-analysis.
Pamplona, F. A., da Silva, L. R., & Coan, A. C.
Frontiers in Neurology, 9.(2018).
doi:10.3389/fneur.2018.00759
This meta-analysis paper describes the analysis of observational clinical studies on the treatment of refractory epilepsy with cannabidiol (CBD)-based products. Beyond attempting to establish the safety and efficacy of such products, we also investigated if there is enough evidence to assume any difference in efficacy between CBD-rich extracts compared to purified CBD products. The systematic search took place in February/2017 and updated in December/2017 using the keywords “epilepsy” or “Dravet” or “Lennox-Gastaut” or “CDKL5” combined with "Cannabis", "cannabinoid", "cannabidiol" or “CBD” resulting in 199 papers. The qualitative assessment resulted in 11 valid references, with an average impact factor of 8.1 (ranging from 1.4 to 47.8). The categorical data of a total of 670 patients were analyzed by Fischer test. The average daily dose ranged between 1 and 50 mg/kg, with treatment length from 3 to 12 months (mean 6.2 months). Two thirds of patients reported improvement in the frequency of convulsive crisis (399/622, 64%). There were more reports of improvement from patients treated with CBD-rich extracts (318/447, 71%) than patients treated with purified CBD (81/223, 36%), with statistical significance

Potential Exposures Associated with Indoor Marijuana Growing Operations
John W. Martyny , Kate A. Serrano , Joshua W. Schaeffer & Mike V. Van Dyke
Journal of Occupational and Environmental Hygiene, 10:11, 622-639, (2013)
DOI: 10.1080/15459624.2013.831986
We entered a total of 30 indoor marijuana grow operations (IMGO) with law enforcement investigators in order to determine potential exposures to first responders. Samples for airborne fungal spores, volatile organic compounds, carbon dioxide, carbon monoxide, and delta-9-tetrahydrocannabinol (THC) were obtained as well as the identification of chemicals utilized in the IMGO. The chemicals utilized within the IMGOs were primarily pesticides and fertilizers with none showing high toxicity. Although several of the IMGOs had CO2 enrichment processes involving combustion, CO levels were not
elevated. THC levels were identified on surfaces within the IMGOs and on the hands of the investigators. Surface levels ranged from <0.1 ?g /100 cm2 to 2000 ?g /100 cm2 with a geometric mean of 0.37 ?g /100 cm2. THC levels on the hands of officers ranged from <0.10 ?g /wipe to 2900 ?g /wipe with a geometric mean of 15 ?g /wipe. These levels were not considered to be elevated to the point of causing a toxic exposure to responders. A total of 407 fungal spore samples were taken using both slit impactor plates and 400-
hole impactors. Both methods identified elevated fungal spore levels, especially during the removal of plants from some of the IMGOs. After plant removal, spore counts increased to levels above 50,000 spores/m3 with one sample over 500,000 spores/m3. In addition, we found that therewas a shift in species between indoor and outdoor samples with Cladosporium sp. the predominant outdoor species and Penicillium sp. the predominant indoor species. We concluded that the potential increase in fungal spore concentrations associated with the investigation and especially removal of the marijuana plants could potentially expose responders to levels of exposure consistent with those associated with mold remediation processes and that respiratory protection is advisable.

Potential occupational and respiratory hazards in a Minnesota cannabis cultivation and processing facility.
Couch, J. R., Grimes, G. R., Wiegand, D. M., Green, B. J., Glassford, E. K., Zwack, L. M., … Beezhold, D. H.
American Journal of Industrial Medicine. Am J Ind Med. 2019;1-9
doi:10.1002/ajim.23025
Background: Cannabis has been legalized in some form for much of the United States. The National Institute for Occupational Safety and Health (NIOSH) received a health hazard evaluation request from a Minnesota cannabis facility and their union to undertake an evaluation. Methods: NIOSH representatives visited the facility in August 2016 and April 2017. Surface wipe samples were collected for analysis of delta?9 tetrahydrocannabinol (?9?THC), delta?9 tetrahydrocannabinol acid (?9?THCA), cannabidiol, and cannabinol. Environmental air samples were collected for volatile organic compounds (VOCs), endotoxins (limulus amebocyte lysate assay), and fungal diversity (NIOSH two?stage BC251 bioaerosol sampler with internal transcribed spacer region sequencing analysis). Results: Surface wipe samples identified ?9?THC throughout the facility. Diacetyl and 2,3?pentanedione were measured in initial VOC screening and subsequent sampling during tasks where heat transference was greatest, though levels were well below the NIOSH recommended exposure limits. Endotoxin concentrations were highest during processing activities, while internal transcribed spacer region sequencing revealed that the Basidiomycota genus, Wallemia, had the highest relative abundance. Conclusions: To the authors’ knowledge, this is the first published report of potential diacetyl and 2,3?pentanedione exposure in the cannabis industry, most notably during cannabis decarboxylation. Endotoxin exposure was elevated during grinding, indicating that this is a potentially high?risk task. The findings indicate that potential health hazards of significance are present during cannabis processing, and employers should be aware of potential exposures to VOCs, endotoxin, and fungi. Further research into the degree of respiratory and dermal hazards and resulting health effects in this industry is recommended.

Potentiating effect of b-caryophyllene on anticancer activity of a-humulene, isocaryophyllene and paclitaxel
Jean Legault and André Pichette
Journal of Pharmacy and Pharmacology JPP 2007, 59: 1643–1647
DOI 10.1211/jpp.59.12.0005
b-caryophyllene is a sesquiterpene widely distributed in essential oils of various plants. Several biological activities are attributed to b-caryophyllene, such as anti-inflammatory, antibiotic, antioxidant, anticarcinogenic and local anaesthetic activities. In this work, the potentiating effect of bcaryophyllene on the anticancer activity of a-humulene, isocaryophyllene and paclitaxel against MCF-7, DLD-1 and L-929 human tumour cell lines was evaluated. A non-cytotoxic concentration of bcaryophyllene significantly increased the anticancer activity of a-humulene and isocaryophyllene on MCF-7 cells: a-humulene or isocaryophyllene alone (32 mgmL?1) inhibited cell growth by about 50% and 69%, respectively, compared with 75% and 90% when combined with 10 mg mL?1 b-caryophyllene. Moreover, b-caryophyllene potentiated the anticancer activity of paclitaxel on MCF-7, DLD-1 and L-929 cell lines. The highest potentiating effect was obtained in DLD-1 cells treated with paclitaxel combined with 10 mgmL?1 b-caryophyllene, which increased the paclitaxel activity about 10- fold. The intracellular accumulation of paclitaxel-oregon green was evaluated in combination with concentrations of b-caryophyllene ranging from 2.5 to 40 mgmL?1. b-Caryophyllene (10 mgmL?1) significantly increased the intracellular accumulation of paclitaxel-oregon green (about 64% over controls). Moreover, b-caryophyllene induced intracellular accumulation of calcein but not verapamil, an inhibitor of P-glycoprotein and multidrug resistance related protein transporters, suggesting that b-caryophyllene promotes drug accumulation by a different mechanism of action. These results suggest that b-caryophyllene facilitates the passage of paclitaxel through the membrane and thus potentiates its anticancer activity.

Practical use of pharmaceutically purified oral cannabidiol in Dravet syndrome and Lennox-Gastaut syndrome.
Lattanzi, S., Zaccara, G., Russo, E., La Neve, A., Lodi, M. A. M., & Striano, P.
Expert Review of Neurotherapeutics.(2020).
doi:10.1080/14737175.2021.1834383
Introduction: Pharmaceutically purified oral cannabidiol (CBD) has been recently approved by the US Food and Drug Administration and European Medicines Agency as treatment of seizures associated with Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS), which are severe and difficult-to-treat developmental and epileptic encephalopathies with onset in early childhood.
Areas covered: This review will critically review the pharmacokinetic properties of CBD, the interactions with antiseizure and non-antiseizure medications, and the main tolerability and safety issues to provide guidance for its use in everyday practice.
Expert opinion: CBD is metabolized in the liver and can influence the activity of enzymes involved in drug metabolism. The best characterized drug-drug interaction is between CBD and clobazam. The most common adverse events include somnolence, gastrointestinal discomfort and increase in serum transaminases. High-grade purified CBD oral solution represents an effective therapeutic option in patients with DS and LGS. The findings cannot be extrapolated to other cannabis-based products, synthetic cannabinoids for medicinal use and non-medicinal cannabis and CBD derivatives.

Prescribing medicinal cannabis [Correction]
DOI: 10.18773/austprescr.2020.073

Preclinical assessment of the abuse potential of purified botanical cannabidiol: self-administration, drug discrimination, and physical dependence

Royston A Gray, David J Heal, David R. Maguire, Lisa R. Gerak, Martin A. Javors, Sharon Smith and Charles P. France
Journal of Pharmacology and Experimental Therapeutics April 30, 2022, JPET-AR-2021-000988;
DOI: https://doi.org/10.1124/jpet.121.000988
https://jpet.aspetjournals.org/content/jpet/early/2022/04/29/jpet.121.000988.full.pdf
Cannabidiol (CBD) is a constituent of the cannabis plant with a diverse array of pharmacological activities as well as potential therapeutic uses. An oral formulation of CBD (Epidiolex® in the US; Epidyolex® in Europe) is approved for treating seizures associated with rare and severe forms of epilepsy. These studies, which supported the approval of the medication, investigated abuse-related effects of CBD in rats and nonhuman primates (NHPs) using drug self-administration, drug discrimination, and physical dependence procedures and characterized its pharmacokinetics. In NHPs (n=5) that self-administered midazolam (0.01 or 0.032 mg/kg/infusion), CBD (0.1-3.2 mg/kg/infusion) failed to maintain responding above vehicle levels. CBD maintained very modest levels of self-administration in rats (n=7-8) that self-administered heroin (0.015 mg/kg/infusion) and did not increase drug-lever responding, up to a dose of 150 mg/kg (p.o.), in rats (n=6) trained to discriminate 0.5 mg/kg (i.p.) midazolam. In juvenile (5-6 weeks old) and adult (10-11 weeks old) male and female rats, discontinuation of chronic treatment (twice daily for 20 days) with an oral formulation of CBD (20 or 100 mg/kg, p.o.) did not reliably produce signs of withdrawal. Pharmacokinetic studies confirmed that the dosing regimens used in these studies resulted in therapeutically relevant plasma levels. Taken together, the lack of reliable self-administration, the failure to increase drug-lever responding in rats trained to discriminate midazolam, and the absence of withdrawal signs upon discontinuation of chronic treatment indicate that CBD has very low abuse potential and is unlikely to produce physical dependence.

Preclinical investigation of β-caryophyllene as a therapeutic agent in an experimental murine model of Dravet syndrome
Cristina Alonso, Valentina Satta, Paula Díez-Guti´errez, Javier Fern´andez-Ruiz, Onintza Sagredo
Neuropharmacology 205 (2022) 108914
DOI: 10.1016/j.neuropharm.2021.108914
Dravet Syndrome (DS) is caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel
Nav1.1, which results in febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved for the management of seizures in DS patients, but it appears to be also active against associated comorbidities. In this new study, we have investigated β-caryophyllene (BCP), a cannabinoid with terpene structure that appears to also have a broad-spectrum profile, as a useful therapy against both seizuring activity and progression of associated comorbidities. This has been studied in heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the Central Nervous System (Syn-Cre/Scn1aWT/A1783V), using two experimental approaches. In the first approach, an acute treatment with BCP was effective against seizuring activity induced by pentylenetetrazole (PTZ) in wildtype (Scn1aWT/WT) and also in Syn-Cre/Scn1aWT/A1783V mice, with these last animals having a greater susceptibility to PTZ. Such benefits were paralleled by a BCP-induced reduction in PTZ-induced reactive astrogliosis (labelled with GFAP) and microgliosis (labelled with Iba-1) in the prefrontal cortex and the hippocampal dentate gyrus, which were visible in both wildtype (Scn1aWT/WT) and Syn-Cre/Scn1aWT/ A1783V mice. In the second approach, both genotypes were treated repeatedly with BCP to investigate its effects on several DS comorbidities. Thus, BCP corrected important behavioural abnormalities of Syn-Cre/Scn1aWT/ A1783V mice (e.g. delayed appearance of hindlimb grasp reflex, induction of clasping response, motor hyperactivity, altered social interaction and memory impairment), attenuated weight loss, and slightly delayed premature mortality. Again, these benefits were paralleled by a BCP-induced reduction in reactive astrogliosis and microgliosis in the prefrontal cortex and the hippocampal dentate gyrus typical of Syn-Cre/Scn1aWT/A1783V mice. In conclusion, BCP was active in Syn-Cre/Scn1aWT/A1783V mice against seizuring activity (acute treatment) and against several comorbidities (repeated treatment), in both cases in association with its capability to reduce glial reactivity in areas related to these behavioural abnormalities. This situates BCP in a promising position for further preclinical evaluation towards a close translation to DS patients.
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Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice.
Busquets-Garcia, A., Soria-Gómez, E., Redon, B., Mackenbach, Y., Vallée, M., Chaouloff, F., … Marsicano, G.
Molecular Psychiatry, 22(11), 1594–1603. (2017).
doi:10.1038/mp.2017.4
Cannabis-induced acute psychotic-like states (CIAPS) represent a growing health issue, but their underlying neurobiological mechanisms are poorly understood. The use of antipsychotics and benzodiazepines against CIAPS is limited by side effects and/or by their ability to tackle only certain aspects of psychosis. Thus, safer wide-spectrum treatments are currently needed. Although the blockade of cannabinoid type-1 receptor (CB1) had been suggested as a therapeutical means against CIAPS, the use of orthosteric CB1 receptor full antagonists is strongly limited by undesired side effects and low efficacy. The neurosteroid pregnenolone has been recently shown to act as a potent endogenous allosteric signal-specific inhibitor of CB1 receptors. Thus, we tested in mice the potential therapeutic use of pregnenolone against acute psychotic-like effects of ?9 -tetrahydrocannabinol (THC), the main psychoactive component of cannabis. We found that pregnenolone blocks a wide spectrum of THC-induced endophenotypes typically associated with psychotic-like states, including impairments in cognitive functions, somatosensory gating and social interaction. In order to capture THC-induced positive psychotic-like symptoms (e.g. perceptual delusions), we adapted a behavioral paradigm based on associations between different sensory modalities and selective devaluation, allowing the measurement of mental sensory representations in mice. Acting at hippocampal CB1 receptors, THC impaired the correct processing of mental sensory representations (reality testing) in an antipsychotic- and pregnenolone-sensitive manner. Overall, this work reveals that signal-specific inhibitors mimicking pregnenolone effects can be considered as promising new therapeutic tools to treat CIAPS.

Pregnenolone Can Protect the Brain from Cannabis Intoxication.
Vallée, M., Vitiello, S., Bellocchio, L., Hébert-Chatelain, E., Monlezun, S., Martin-Garcia, E., … Piazza, P. V.
Science, 343(6166), 94–98.(2014).
doi:10.1126/science.1243985
Pregnenolone is considered the inactive precursor of all steroid hormones, and its potential functional effects have been largely uninvestigated. The administration of the main active principle of Cannabis sativa (marijuana), ?9 -tetrahydrocannabinol (THC), substantially increases the synthesis of pregnenolone in the brain via activation of the type-1 cannabinoid (CB1) receptor. Pregnenolone then, acting as a signaling-specific inhibitor of the CB1 receptor, reduces several effects of THC. This negative feedback mediated by pregnenolone reveals a previously unknown paracrine/autocrine loop protecting the brain from CB1 receptor overactivation that could open an unforeseen approach for the treatment of cannabis intoxication and addiction.

Pregnenolone limits effects of cannabis.
Welberg, L.
Nature Reviews Neuroscience, 15(2), 66–67. (2014).
doi:10.1038/nrn3681
Pregnenolone is a precursor of neuroactive steroids that is thought to have no physiological activity itself. However, a new study shows that pregnenolone is strongly upregulated by ?9 -tetrahydrocannabinol (THC) — the main active component of cannabis — and limits several addictive and other effects of THC.

Preliminary Investigation of the Safety of Escalating Cannabinoid Doses in Healthy Dogs
Dana Vaughn, Justyna Kulpa and Lina Paulionis
Front. Vet. Sci. 7:51
doi: 10.3389/fvets.2020.00051
Objective: To determine the safety and tolerability of escalating doses of three cannabis oil formulations, containing predominantly CBD, THC, or CBD and THC (1.5:1) vs. placebo in dogs.
Design: Randomized, placebo-controlled, blinded, parallel study.
Animals: Twenty healthy Beagle dogs (10 males, 10 females).
Methods: Dogs were randomly assigned to one of five treatment groups (n = 4 dogs per group balanced by sex): CBD-predominant oil, THC-predominant oil, CBD/THC-predominant oil (1.5:1), sunflower oil placebo, medium-chain triglyceride oil placebo. Up to 10 escalating doses of the oils were planned for administration via oral gavage, with at least 3 days separating doses. Clinical observations, physical examinations, complete blood counts, clinical chemistry, and plasma cannabinoids were used to assess safety, tolerability, and the occurrence of adverse events (AEs). AEs were rated as mild, moderate, or severe/medically significant.
Results: Dose escalation of the CBD-predominant oil formulation was shown to be as safe as placebo and safer than dose escalation of oils containing THC (CBD/THC oil or THC oil). The placebo oils were delivered up to 10 escalating volumes, the CBD oil up to the tenth dose (640.5 mg; ?62 mg/kg), the THC oil up to the tenth dose (597.6 mg; ?49 mg/kg), and the CBD/THC oil up to the fifth dose (140.8/96.6 mg CBD/THC; ?12 mg/kg CBD + 8 mg/kg THC). AEs were reported in all dogs across the five groups and the majority (94.9%) were mild. Moderate AEs (4.4% of all AEs) and severe/medically significant AEs (0.8% of all AEs) manifested as constitutional (lethargy, hypothermia) or neurological (ataxia) symptoms and mainly occurred across the two groups receiving oils containing THC (CBD/THC oil or THC oil). Conclusions and clinical significance: Overall, dogs tolerated dose escalation of the CBD oil well, experiencing only mild AEs. The favorable safety profile of 10 escalating doses of a CBD oil containing 18.3–640.5 mg CBD per dose (?2–62 mg/kg) provides comparative evidence that, at our investigated doses, a CBD-predominant oil formulation was safer and more tolerated in dogs than oil formulations containing higher concentrations of THC

Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons
Adán de Salas-Quirogaa, Javier Díaz-Alonsoa, Daniel García-Rincóna, Floortje Remmersd, David Vegaa, María Gómez-Cañasa, Beat Lutzd, Manuel Guzmána, and Ismael Galve-Roperh
Proceedings of the National Academy of Sciences, 112(44), 13693–13698.
Doi: 10.1073/pnas.1514962112
The CB1 cannabinoid receptor, the main target of ?9-tetrahydrocannabinol
(THC), the most prominent psychoactive compound of marijuana, plays a crucial regulatory role in brain development as evidenced by the neurodevelopmental consequences of its manipulation in animal models. Likewise, recreational cannabis use during pregnancy affects brain structure and function of the progeny. However, the precise neurobiological substrates underlying the consequences of prenatal THC exposure remain unknown. As CB1 signaling is known to modulate long-range corticofugal connectivity, we analyzed the impact of THC exposure on cortical projection neuron development. THC administration to pregnant mice in a restricted time window interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. The identity of embryonic THC neuronal targets was determined by a Cre-mediated,
lineage-specific, CB1 expression-rescue strategy in a CB1-null background.
Early and selective CB1 reexpression in dorsal telencephalic glutamatergic neurons but not forebrain GABAergic neurons rescued the deficits in corticospinal motor neuron development of CB1-null mice and restored susceptibility to THC-induced motor alterations. In addition, THC administration induced an increase in seizure susceptibility that was mediated by its interference with CB1-dependent regulation of both glutamatergic and GABAergic neuron development. These findings demonstrate that prenatal exposure to THC has long-lasting deleterious consequences in the adult offspring solely mediated by its ability to disrupt the neurodevelopmental role of CB1 signaling

Prenatal Nicotine or Cannabis Exposure and Offspring Neurobehavioral Outcomes
Marcela C Smid, Torri D Metz, Gwen A McMillin, Lisa Mele, Brian M Casey, Uma M Reddy, Ronald J Wapner, John M Thorp, George R Saade, Alan T N Tita, Emily S Miller, Dwight J Rouse, Baha Sibai, Maged M Costantine, Brian M Mercer, Steve N Caritis
Obstet Gynecol. 2021 Dec 2.
doi: 10.1097/AOG.0000000000004632.
Objective: To study the association between nicotine or cannabis metabolite presence in maternal urine and child neurodevelopmental outcomes.
Methods: We conducted a secondary analysis of two parallel multicenter randomized controlled trials of treatment for hypothyroxinemia or subclinical hypothyroidism among pregnant individuals enrolled at 8-20 weeks of gestation. All maternal-child dyads with a maternal urine sample at enrollment and child neurodevelopmental testing were included (N=1,197). Exposure was urine samples positive for nicotine (cotinine) or cannabis 11-nor-9-carboxy-delta-9-tetrahydrocannabinol [THC-COOH]) or both metabolites. Primary outcome was child IQ at 60 months. Secondary outcomes included cognitive, motor and language, attention, behavioral and social competency, and differential skills assessments at 12, 24, 36, and 48 months. Quantile regression analysis was performed with confounder adjustment.
Results: Of 1,197 pregnant individuals, 99 (8.3%) had positive cotinine samples and 47 (3.9%) had positive THC-COOH samples; 33 (2.8%) were positive for both. Groups differed in self-reported race and ethnicity, education, marital status, insurance, and thyroid status. Median IQ was similar between cotinine-exposed and -unexposed children (90 vs 95, adjusted difference in medians -2.47, 95% CI -6.22 to 1.29) and THC-COOH-exposed and -unexposed children (89 vs 95, adjusted difference in medians -1.35, 95% CI -7.76 to 5.05). In secondary outcome analysis, children with THC-COOH exposure compared with those unexposed had higher attention scores at 48 months of age (57 vs 49, adjusted difference in medians 6.0, 95% CI 1.11-10.89).
Conclusions: Neither prenatal nicotine nor cannabis exposure was associated with a difference in IQ. Cannabis exposure was associated with worse attention scores in early childhood. Longitudinal studies assessing associations between child neurodevelopmental outcomes and prenatal nicotine and cannabis exposure with a focus on timing and quantity of exposure are needed.

Preparation and Distribution of Cannabis and Cannabis-Derived Dosage Formulations for Investigational and Therapeutic Use in the United States.
Thomas, B. F., & Pollard, G. T.
Frontiers in Pharmacology, 7. (2016)
doi:10.3389/fphar.2016.00285
Cannabis is classified as a schedule I controlled substance by the US Drug Enforcement Agency, meaning that it has no medicinal value. Production is legally restricted to a single supplier at the University of Mississippi, and distribution to researchers is tightly controlled. However, a majority of the population is estimated to believe that cannabis has legitimate medical or recreational value, numerous states have legalized or decriminalized possession to some degree, and the federal government does not strictly enforce its law and is considering rescheduling. The explosive increase in open sale and use of herbal cannabis and its products has occurred with widely variable and in many cases grossly inadequate quality control at all levels—growing, processing, storage, distribution, and use. This paper discusses elements of the analytical and regulatory system that need to be put in place to ensure standardization for the researcher and to reduce the hazards of contamination, overdosing, and underdosing for the end-user.

Prescribing medicinal cannabis
Jonathon C Arnold, Tamara Nation, Iain S McGregor
Aust Prescr VOLUME 43 : NUMBER 5 : OCTOBER 2020
DOI: 10.18773/ austprescr.2020.052
The Australian Federal Government legalised access to medicinal cannabis in 2016. More than 100 different cannabis products are now available to prescribe. Most are oral preparations (oils) or capsules containing delta-9-tetrahydrocannabinol or cannabidiol. Driedflower products are also available. As most products are unregistered drugs, prescribing requires approval under the Therapeutic Goods Administration Special Access Scheme-B or Authorised Prescriber Scheme. Special Access Scheme Category B applications can be made online, with approval usually being given within 24–48 hours. However, supply chain problems may delay dispensing by the pharmacy. By the end of 2019, over 28,000 prescribing approvals had been issued to patients, involving more than 1400 doctors, mostly GPs. More than 70,000 approvals are projected by the end of 2020. Most prescriptions are for chronic non-cancer pain, anxiety, cancer-related symptoms, epilepsy and other neurological disorders. However, the evidence supporting some indications is limited. Many doctors are cautious about prescribing cannabis. While serious adverse events are rare, there are legitimate concerns around driving, cognitive impairment and drug dependence with products containing delta-9-tetrahydrocannabinol. Cannabidiol-only products pose fewer risks.

Presenting the outputs of the IASP Presidential Task Force on Cannabis and Cannabinoid Analgesia
Soliman N, Haroutounian S, Hohmann AG, Krane E, Liao J, Macleod M, Segelcke D, Sena C, Thomas J, Vollert J, Wever K, Alaverdyan H, Barakat A, Barthlow T, Harris Bozer AL, Davidson A, Diaz-delCastillo M, Dolgorukova A, Ferdousi MI, Healy C, Hong S, Hopkins M, James A, Leake HB, Malewicz NM, Mansfield M, Mardon AK, Mattimoe D, McLoone DP, Noes-Holt G, Pogatzki-Zahn EM, Power E, Pradier B, Romanos-Sirakis E, Segelcke A, Vinagre R, Yanes JA, Zhang J, Zhang XY, Finn DP and Rice ASC
Pain March 16, 2021
DOI: 10.1097/j.pain.0000000000002210
https://journals.lww.com/pain/Citati...ial.98087.aspx
This special section of PAIN® consists of a series of linked articles that represent a rigorous and extensive appraisal of the basic and clinical science evidence pertaining to the potential analgesic effects and safety of cannabis and cannabinoids. It represents the collated outputs, gathered over a two and a half year effort, of the IASP Presidential Task Force on Cannabis and Cannabinoid Analgesia (IASP Presidential Task Force on Cannabis and Cannabinoid Analgesia membership, list available at: https://www.iasp-pain.org/About/Cont...temNumber57917. We were tasked with conducting a robust and comprehensive interrogation of the preclinical and clinical evidence for analgesic benefit and also for harm. Emphasis was placed on mitigating against the impact of bias by systemically identifying and appraising the available evidence and using the highest quality evidence available. The Task Force then used this knowledge to inform IASP’s position statement that is published concurrently with this edition of PAIN.7 The key message conveyed by the position statement is that although there are substantial preclinical data supporting the hypothesis of cannabinoid analgesia, current uncertainties in the clinical evidence base led the Task Force to conclude that we could not support the general use of cannabis nor cannabinoids and related medicines for analgesic use at this point in time.

Prevalence and self reported reasons of cannabis use for medical purposes in USA and Canada
Janni Leung, · Gary Chan · Daniel Stjepanović · Jack Yiu Chak Chung, · Wayne Hall · David Hammond Psychopharmacology 2022 DOI: 10.1007/s00213-021-06047-8
https://link.springer.com/content/pd...21-06047-8.pdf
Rationale There has been increasing attention on cannabis use for medical purposes, but there is currently a lack of data on its epidemiology.
Objectives To examine the epidemiology of self-reported cannabis use for medical purposes by (1) estimating its prevalence, (2) comparing gender and age differences, and (3) investigating what reasons they were used to manage.
Methods Participants included 27,169 respondents (aged 16–65) who completed Wave 1 of The International Cannabis Policy Study (ICPS) conducted across Canada and the USA in 2018 via online surveys. Cannabis policy conditions were “US legal–recreational” (legal for both recreational and medical uses), “US legal–medical only”, “US illegal”, and “Canada–
medical only”.
Results The overall prevalence of self-reported ever cannabis use for medical purposes was 27%, with similar rates by sex and the highest prevalence in young adults. Prevalence was higher in US legal–recreational states (34%) than US illegal states (23%), US legal–medical only states (25%), and Canada (25%). The most common physical health reasons include use to manage pain (53%), sleep (46%), headaches/migraines (35%), appetite (22%), and nausea/vomiting (21%). For mental health reasons, the most common were for anxiety (52%), depression (40%), and PTSD/trauma (17%). There were 11% who reported using cannabis for managing other drug or alcohol use and 4% for psychosis.
Conclusions A substantial proportion of the North American population self-reported cannabis use for medical purposes for a variety of medical reasons, including those living in jurisdictions without legal markets. Further research is needed to understand the safety and efficacy of these forms of medical cannabis use.

Prevalence of Cannabis Withdrawal Symptoms Among People With Regular or Dependent Use of Cannabinoids.
Bahji, A., Stephenson, C., Tyo, R., Hawken, E. R., & Seitz, D. P.
JAMA Network Open, 3(4), (2020).
doi:10.1001/jamanetworkopen.2020.2370
IMPORTANCE Cannabis withdrawal syndrome (CWS)—a diagnostic indicator of cannabis use disorder—commonly occurs on cessation of heavy and prolonged cannabis use. To date, the prevalence of CWS syndrome has not been well described, nor have the factors potentially associated with CWS. OBJECTIVES To estimate the prevalence of CWS among individuals with regular or dependent use of cannabinoids and identify factors associated with CWS. DATA SOURCES A search of literature from database inception to June 19, 2019, was performed using MEDLINE, Embase, PsycINFO, Web of Science, the Cumulative Index to Nursing and Allied Health Literature, ProQuest, Allied and Complementary Medicine, and Psychiatry online, supplemented by manual searches of reference lists of included articles. STUDY SELECTION Articles were included if they (1) were published in English, (2) reported on individuals with regular use of cannabinoids or cannabis use disorder as a primary study group, (3) reported on the prevalence of CWS or CWS symptoms using a validated instrument, (4) reported the prevalence of CWS, and (5) used an observational study design (eg, cohort or cross-sectional). DATA EXTRACTION AND SYNTHESIS All abstracts, full-text articles, and other sources were reviewed, with data extracted in duplicate. Cannabis withdrawal syndrome prevalence was estimated using a random-effects meta-analysis model, alongside stratification and meta-regression to characterize heterogeneity. MAIN OUTCOMES AND MEASURES Cannabis withdrawal syndrome prevalence was reported as a percentage with 95% CIs. RESULTS Of 3848 unique abstracts, 86 were selected for full-text review, and 47 studies, representing 23 518 participants, met all inclusion criteria. Of 23 518 participants included in the analysis, 16 839 were white (72%) and 14 387 were men (69%); median (SD) age was 29.9 (9.0) years. The overall pooled prevalence of CWS was 47% (6469 of 23 518) (95% CI, 41%-52%), with significant heterogeneity between estimates (I 2 = 99.2%). When stratified by source, the prevalence of CWS was 17% (95% CI, 13%-21%) in population-based samples, 54% in outpatient samples (95% CI, 48%-59%), and 87% in inpatient samples (95% CI, 79%-94%), which were significantly different (P < .001). Concurrent cannabis (? = 0.005, P < .001), tobacco (? = 0.002, P = .02), and other substance use disorders (? = 0.003, P = .05) were associated with a higher CWS prevalence, as was daily cannabis use (? = 0.004, P < .001).

Priapism and cannabism: A case report and review of the literature
N Chouaib , S Jidane , I Gueye , M Lekhlit , T Nebhani , A Belkouch , S Zidouh , L Belyamani
Encephale. 2020 Dec 17;S0013-7006(20)30261-X.
doi: 10.1016/j.encep.2020.09.003
Priapism is classically defined as a partial or complete erection, abnormally prolonged in the absence of desire or sexual stimulation. Several classes of drugs as well as certain psychoactive substances (e.g. alcohol, cocaine, cannabis, etc.) are accused in the occurrence of priapism. In this article, we report a case of priapism in a 37-year-old adult admitted to the emergency department after cannabis use. The patient received emergency aspiration of blood by puncturing the corpora cavernosa, followed by intra-cavernous injection of ephedrine. The evolution has been good. This case relates a rare side effect resulting from the consumption of cannabis, which requires a rapid assessment and management to avoid complications.

Primary Macrophage Chemotaxis Induced by Cannabinoid Receptor 2 Agonists Occurs Independently of the CB2 Receptor
Lewis Taylor, Ivy Christou, Theodore S. Kapellos, Alice Buchan, Maximillian H. Brodermann, Matteo Gianella-Borradori, Angela Russell, Asif J. Iqbal & David R. Greaves
Nature Scientific Reports | 5:10682
DOI: 10.1038/srep10682
Activation of CB2 has been demonstrated to induce directed immune cell migration. However, the ability of CB2 to act as a chemoattractant receptor in macrophages remains largely unexplored.Using a real-time chemotaxis assay and a panel of chemically diverse and widely used CB2 agonists, we set out to examine whether CB2 modulates primary murine macrophage chemotaxis. We report that of 12 agonists tested, only JWH133, HU308, L-759,656 and L-759,633 acted as macrophage chemoattractants. Surprisingly, neither pharmacological inhibition nor genetic ablation of CB2 had any effect on CB2 agonist-induced macrophage chemotaxis. As chemotaxis was pertussis toxin sensitive in both WT and CB2-/- macrophages, we concluded that a non-CB1/CB2, Gi/o-coupled GPCR must be responsible for CB2 agonist-induced macrophage migration. The obvious candidate receptors GPR18 and GPR55 could not mediate JWH133 or HU308-induced cytoskeletal rearrangement or JWH133-induced ? -arrestin recruitment in cells transfected with either receptor, demonstrating that neither are the unidentified GPCR. Taken together our results conclusively demonstrate that CB2 is not a chemoattractant receptor for murine macrophages. Furthermore we show for the first time that JWH133, HU308, L-759,656 and L-759,633 have off-target effects of functional consequence in primary cells and we believe that our findings have wide ranging implications for the entire cannabinoid field.

Progress toward pharmacotherapies for cannabis use disorder: an evidence-based review
Jan Copeland, Izabella Pokorski
Substance Abuse and Rehabilitation 2016:7 41–53
DOI:10.2147/SAR.S89857
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862355/pdf/sar-7-041.pdf
Cannabis is the most widely used and variably regulated drug in the world, with increasing trends of use being reported in the US, Australia, Asia, and Africa. Evidence has shown a decrease in the age of commencement of cannabis use in some developed countries and a prolongation of risk of initiation to cannabis use beyond adolescence among more recent users. Cannabis use is associated with numerous health risks and long-term morbidity, as well as risk of developing cannabis-use disorders. Cannabis users infrequently seek professional treatment, and normally do so after a decade of use. Cannabis-use disorders are currently treated using a selection of psychosocial interventions. Severity of withdrawal is a factor that increases the risk of relapse, and is the target of pharmacotherapy studies. Currently, there is no approved pharmacotherapy for cannabis-use disorders. A number of approaches have been examined, and trials are continuing to find a safe and effective medication with little abuse liability

Promoting cannabis products to pharmaceutical drugs.
Koltai, H., Poulin, P., & Namdar, D.
European Journal of Pharmaceutical Sciences,132,118–120.(2019).
doi:10.1016/j.ejps.2019.02.027
Cannabis sativa is widely used for medical purposes. However, to date, aroma, popular strain name or the content of two phytocannabinoids—?9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) are mostly considered for therapeutic activity. This is despite the hundreds of compounds in this plant and their potential synergistic interactions in mixtures. New, specific and effective cannabis-based drugs must be developed to achieve adequate medical standards for the use of cannabis. To do this, the comprehensive molecular profile of cannabis-based drugs must be defined, and mixtures of compounds should be tested for superior therapeutic activity due to synergistic effects compared to individually isolated cannabis compounds. The biological pathways targeted by these new drugs should also be characterized more accurately. For drug development and design, absorption, distribution, metabolism and elimination versus toxicity (ADME/Tox) must be characterized, and therapeutic doses identified. Promoting the quality and therapeutic activity of herbal or synthetic cannabis products to pharma grade is a pressing need worldwide.

Prosocial Effects of Nonpsychotropic Cannabis sativa in Mice
Andrea Mastinu, Roberta Ascrizzi, Giovanni Ribaudo, Sara Anna Bonini, Marika Premoli, Francesca Aria, Giuseppina Maccarinelli, Alessandra Gianoncelli, Guido Flamini, Luisa Pistelli, and Maurizio Memo
Cannabis Cannabinoid Res (2021)
DOI: 10.1089/can.2021.0017
https://www.liebertpub.com/doi/epub/.../can.2021.0017
Introduction: Cannabis sativa L. (C. sativa) is used since ancient times to produce fabrics, baskets, and cords. Later, different ethnic groups used to burn the leaves and flowers of psychotropic cultivars with high Δ9-tetrahydrocannabinol (D9-THC) levels, during the religious or propitiatory rites to alter the state of consciousness. To date, it is not known whether also nonpsychotropic cultivars of C. sativa were used during these rites, and whether these varieties could have an effect on human behavior. This study aimed to evaluate the behavioral effects of an extract of nonpsychotropic C. sativa (NP-CS) in mice.
Materials and Methods: An extract of a nonpsychotropic cultivar of C. sativa dissolved in medium-chain triglyceride oil was used and the different phytochemical components were evaluated. The relative composition in terms of phytocannabinoid content was assessed by reverse phase high-performance liquid chromatography coupled to UV detection (RP-HPLC-UV), and the volatile components were analyzed by gas chromatography-mass spectrometry (GC-MS). In addition, the behavioral effect of NP-CS was assessed on a wild-type mouse model. The animals were treated for 14 days (oral gavage) and motility, anxiety, and social effects were assessed.
Results: RP-HPLC-UV analysis demonstrated that D9-THC was present in lower concentration with respect to other cannabinoids, like cannabidiol. Furthermore, the GC-MS analysis revealed the presence of several terpenoids. Concerning in vivo studies, chronic treatment with NP-CS did not alter body weight, motility, and anxiety and increased social interaction.
Conclusions: This study highlighted the prosocial effects of NP-CS.
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Protective Effects of Cannabidivarin and Cannabigerol on Cells of the Blood–Brain Barrier Under Ischemic Conditions
Nicole L Stone 1, Timothy J England 1 2, Saoirse E O'Sullivan
Cannabis Cannabinoid Res. 2021 Mar 17.
Doi: 10.1089/can.2020.0159.
Background and Objectives: Preclinical studies have shown cannabidiol is protective in models of ischemic stroke. Based on results from our recent systematic review, we investigated the effects of two promising neuroprotective phytocannabinoids, cannabigerol (CBG) and cannabidivarin (CBDV), on cells of the blood-brain barrier (BBB), namely human brain microvascular endothelial cells (HBMECs), pericytes, and astrocytes.
Experimental Approach: Cultures were subjected to oxygen-glucose deprivation (OGD) protocol to model ischemic stroke and cell culture medium was assessed for cytokines and adhesion molecules post-OGD. Astrocyte cell lysates were also analyzed for DNA damage markers. Antagonist studies were conducted where appropriate to study receptor mechanisms.
Results: In astrocytes CBG and CBDV attenuated levels of interleukin-6 (IL-6) and lactate dehydrogenase (LDH), whereas CBDV (10 nM-10 μM) also decreased vascular endothelial growth factor (VEGF) secretion. CBDV (300 nM-10 μM) attenuated levels of monocyte chemoattractant protein (MCP)-1 in HBMECs. In astrocytes, CBG decreased levels of DNA damage proteins, including p53, whereas CBDV increased levels of DNA damage markers. Antagonists for CB1, CB2, PPAR-γ, PPAR-α, 5-HT1A, and TRPV1 had no effect on CBG (3 μM) or CBDV (1 μM)-mediated decreases in LDH in astrocytes. GPR55 and GPR18 were partially implicated in the effects of CBDV, but no molecular target was identified for CBG.
Conclusions: We show that CBG and CBDV were protective against OG mediated injury in three different cells that constitute the BBB, modulating different hallmarks of ischemic stroke pathophysiology. These data enhance our understanding of the protective effects of CBG and CBDV and warrant further investigation into these compounds in ischemic stroke. Future studies should identify other possible

Psychiatric Disorders and Cannabinoid Receptors
Neal Joshi , Emmanuel S Onaivi
Adv Exp Med Biol. 2021;1264:131-153.
doi: 10.1007/978-3-030-57369-0_9
With the increasing global use of medical and adult recreational use of cannabis and cannabinoids, this chapter provides overview of evidence from animal and human studies on psychiatric disorders and cannabinoid receptors. We review and present evaluation of the relationship between changes in the ECS and psychiatric disorders. Evidence suggests the existence of a relationship between changes in components of the ECS, and some of the symptoms present in psychiatric disorders. Both CB1Rs and CB2Rs are components of the endocannabinoid system with different cellular and tissue localization patterns that are differentially expressed in the CNS and PNS and are emerging targets for the treatment of number psychiatric disorders. As cannabis preparations are widely used for recreation globally, it is predictable that cannabis use disorders (CUDs) will increase and there is currently no available treatment for CUDs. Although major advances have been reported from cannabinoid and ECS research, there are gaps in scientific knowledge on long-term consequences of cannabis use. Adolescent and cannabis use during pregnancy presents further challenges, and more research will uncover the signaling pathways that couple the gut microbiota with the host ECS. Development of cannabis and cannabinoid nanomedicine for nanotherapy will certainly overcome some of the shortcomings and challenges in medicinal and recreational use of cannabis and cannabinoids. Thus, nanotechnology will allow targeted delivery of cannabinoid formulations with the potential to elevate their use to scientifically validated nanotherapeutic applications as the field of cannabis nanoscience matures

Psychopharmacology of the endocannabinoids: far beyond anandamide
F A Pamplona , R N Takahashi
J Psychopharmacol 2012 Jan;26(1):7-22.
DOI: 10.1177/0269881111405357
The study of endocannabinoid pharmacology has proceeded from the discovery of Δ9-tetrahydrocannabinol, the main psychoactive compound in Cannabis sativa, to the identification of an endogenous endocannabinoid system that is essential for physiological modulation of neuronal functions. We have not yet achieved a complete understanding of the various roles of the endocannabinoids, but this is one of the fastest-growing fields in psychopharmacology. This review starts with a brief historical description of the discovery of the endocannabinoids and then focuses on recent pharmacological advances and recently discovered endocannabinoid mechanisms of action (e.g. functional selectivity, allosterism, and receptor trafficking). Finally, we will discuss the contention that the existence of evidence-based therapeutic applications for cannabinoids and the wide range of physiological functions affected by endocannabinoids suggests that the careful study of the endocannabinoid system may lead to the development of novel therapeutic drugs with higher societal acceptability and lower side effects profiles.

Pulmonary Aspergillosis, Inhalation of Contaminated Marijuana Smoke, Chronic Granulomatous Disease.
CHUSID, M. J.
Annals of Internal Medicine, 82(5), 682.(1975).
Doi:10.7326/0003-4819-82-5-682
INCREASED SUSCEPTIBILITY to infection is a hallmark of chronic granulomatous disease of childhood (1), an inherited disorder in which phagocytic cells are unable to kill certain types of ingested organisms (2). We present a case of Aspergillus fumigatus pneumonitis in a 17-year-old boy with chronic granulomatous disease, whose infection may have been acquired through inhalation of smoke from marijuana contaminated with fungi. A 17-year-old boy was admitted to the National Institutes of Health with fever and exertional dyspnea. Two weeks before admission, he noted the onset of malaise 12 hours after smoking several pipefulls of marijuana that had been buried in the earth for "aging." One week before admission, while continuing heavy marijuana use, he developed a nonproductive cough and night sweats. On admission, physical examination was unremarkable except for a respiratory rate of 32/min. No rales, rhonchi, or wheezes were heard during the chest examination. Temperature was 38.3 °C [101 °F]. Leukocyte count was 8500/mm3 with 65% segmented neutrophils, 8% bands, and 5% eosinophils. Erythrocyte sedimentation rate was 50 mm/min.

Pulmonary effects of inhaled cannabis smoke.
Tashkin, D. P., & Roth, M. D.
The American Journal of Drug and Alcohol Abuse, 1–14. (2019).
doi:10.1080/00952990.2019.1627366
Background: The smoke generated from cannabis delivers biologically active cannabinoids and a number of combustion-derived toxins, both of which raise questions regarding the impact of cannabis smoking on lung function, airway inflammation and smoking-related lung disease.
Objectives: Review the potential effects of cannabis smoking on respiratory symptoms, lung function, histologic/molecular alterations in the bronchial mucosa, smoking-related changes in alveolar macrophage function and the potential clinical impact of cannabis smoking on chronic obstructive pulmonary disease, lung cancer and pulmonary infections.
Methods: Focused literature review.
Results: The carcinogens and respiratory toxins in cannabis and tobacco smoke are similar but the smoking topography for cannabis results in higher per-puff exposures to inhaled tar and gases. The frequency of chronic cough, sputum and wheeze and the presence of airway mucosal inflammation, goblet cell and vascular hyperplasia, metaplasia and cellular disorganization are similar between cannabis smokers and tobacco smokers. Cannabis smoke has modest airway bronchodilator properties but of unclear clinical significance. While clear evidence exists for progression to obstructive lung disease and emphysema in chronic tobacco smokers, the effects from habitual cannabis use are less clear. Evidence suggests that alveolar macrophages from cannabis smokers have deficits in cytokine production and antimicrobial activity not present in cells from tobacco smokers.
Conclusions: Solid conclusions regarding the respiratory consequences of regular cannabis smoking are difficult to make due to a relative paucity of literature, confounding by concurrent tobacco smoking and reports of conflicting outcomes. Additional well-controlled clinical studies on the pulmonary consequences of habitual cannabis use are needed.

Pure ?9-tetrahydrocannabivarin and a Cannabis sativa extract with high content in ?9-tetrahydrocannabivarin inhibit nitrite production in murine peritoneal macrophages
Barbara Romanok, Ester Pagano, Pierangelo Orlando, Raffaele Capasso, Maria Grazia, Cascio, Roger Pertwee, Vincenzo Di Marzok, Angelo A. Izzo, Francesca Borrelli
DOI: 10.1016/j.phrs.2016.07.045
Historical and scientific evidence suggests that Cannabis use has immunomodulatory and antiinflammatory effects. We have here investigated the effect of the non-psychotropic phytocannabinoid
9-tetrahydrocannabivarin (THCV) and of a Cannabis sativa extract with high (64.8%) content in THCV (THCV-BDS) on nitric oxide (NO) production, and on cannabinoid and transient receptor potential (TRP) channel expression in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. THCV-BDS and THCV exhibited similar affinity in radioligand binding assays for CB1 and CB2 receptors, and inhibited, via CB2 but not CB1 cannabinoid receptors, nitrite production evoked by LPS in peritoneal macrophages. THCV down-regulated the over-expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin 1 (IL-1) proteins induced by LPS. Furthermore, THCV counteracted LPS-induced up-regulation of CB1 receptors, without affecting the changes in CB2, TRPV2 or TRPV4 mRNA expression caused by LPS. Other TRP channels, namely, TRPA1, TRPV1, TRPV3 and TRPM8 were poorly expressed or undetectable in both unstimulated and LPS-challenged macrophages. It is concluded that THCV ? via CB2 receptor activation ? inhibits nitrite production in macrophages. The effect of this phytocannabinoid was associated with a down-regulation of CB1, but not CB2 or TRP channel mRNA expression.

Quality of Life, Mental Health, Personality and Patterns of Use in Self-Medicated Cannabis Users with Chronic Diseases: A 12-Month Longitudinal Study.
Bouso, J. C., Jiménez-Garrido, D., Ona, G., Wo?nica, D., dos Santos, R. G., Hallak, J. E. C., … Farré, M.
Phytotherapy Research.(2020).
doi:10.1002/ptr.6639
The number of patients using cannabis for therapeutic purposes is growing worldwide. While research regarding the treatment of certain diseases/disorders with cannabis and cannabinoids is also expanding, only a few longitudinal studies have assessed the mid-term impacts of medical cannabis use on psychological variables and quality of life (QoL). The aim of the study was to assess the psychological safety and QoL of patients with chronic diseases who self-medicate with cannabis over time. We recruited patients with various chronic diseases who use cannabis and collected data regarding patterns of cannabis use as well as mental health, personality and QoL. Participants were followed-up at baseline, 4, 8 and 12 months. Hair analysis was conducted to confirm the presence of cannabinoids. Personality assessment showed a consistent decrease in self-transcendence and self-directedness scores. Neither cognitive nor psychopathological deterioration was found. There were also no variations in QoL. Mid-term use of medical cannabis seems to show adequate tolerability regarding cognitive and psychopathological abilities, and it may help patients with chronic diseases to maintain an acceptable QoL.

Quality Standards in State Programs Permitting Cannabis for Medical Uses
Schuyler A. Pruyn, Qiang Wang, Charles G. Wu, and Cassandra L. Taylor
Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0164
Currently in the United States, there exists a patchwork of state-level laws and regulations surrounding cannabis use. Although cannabis (excluding hemp under the Agricultural Improvement Act of 2018, Public Law 115-334) is illegal at the federal level and is not FDA (U.S. Food and Drug Administration) approved for any indication, many states allow patients with qualifying conditions to register for their medical cannabis program(MCP). To better understand the quality of cannabis found in these programs, we collected laws, regulations, and guidance documents available on public state-run websites and compared them with current good manufacturing practices (CGMPs) applicable to finished drug products. CGMPs for human drugs contain minimum requirements for the methods, facilities, and controls used in manufacturing, processing, and packaging of a drug product to assure it is safe for use. Such a comparison will aid the development of consistent quality standards that could, in turn, improve the quality of a wide range of cannabis medical products in development that may be sold in the United States. States may likewise choose to have the cannabis and cannabis-derived products that fall within their MCP to follow quality-focused guidelines, such as those listed in CGMPs, to ensure the quality of these products and promote public health.
This study further solidifies the need for standardized testing protocols and methodologies to keep consumers safe.
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Randomized, dose-ranging safety trial of cannabidiol in Dravet syndrome.
Devinsky, O., Patel, A. D., Thiele, E. A., Wong, M. H., Appleton, R., … Harden, C. L.
Neurology, 90(14), e1204–e1211. (2018).
doi:10.1212/wnl.0000000000005254
Objective To evaluate the safety and preliminary pharmacokinetics of a pharmaceutical formulation of purified cannabidiol (CBD) in children with Dravet syndrome.
Methods Patients aged 4–10 years were randomized 4:1 to CBD (5, 10, or 20 mg/kg/d) or placebo taken twice daily. The double-blind trial comprised 4-week baseline, 3-week treatment (including titration), 10-day taper, and 4-week follow-up periods. Completers could continue in an openlabel extension. Multiple pharmacokinetic blood samples were taken on the first day of dosing
and at end of treatment for measurement of CBD, its metabolites 6-OH-CBD, 7-OH-CBD, and 7-COOH-CBD, and antiepileptic drugs (AEDs; clobazam and metabolite N-desmethylclobazam [N-CLB], valproate, levetiracetam, topiramate, and stiripentol). Safety assessments were clinical laboratory tests, physical examinations, vital signs, ECGs, adverse events (AEs), seizurerequency, and suicidality.
Results Thirty-four patients were randomized (10, 8, and 9 to the 5, 10, and 20 mg/kg/d CBD groups, and 7 to placebo); 32 (94%) completed treatment. Exposure to CBD and its metabolites was dose-proportional (AUC0–t). CBD did not affect concomitant AED levels, apart from an increase in N-CLB (except in patients taking stiripentol). The most common AEs on CBD were pyrexia, somnolence, decreased appetite, sedation, vomiting, ataxia, and abnormal behavior. Six patients taking CBD and valproate developed elevated transaminases; none met criteria for drug-induced liver injury and all recovered. No other clinically relevant safety signals were observed.
Conclusions Exposure to CBD and its metabolites increased proportionally with dose. An interaction with N-CLB was observed, likely related to CBD inhibition of cytochrome P450 subtype 2C19. CBD resulted in more AEs than placebo but was generally well-tolerated

Rare genetic variants in the endocannabinoid system genes CNR1 and DAGLA are associated with neurological phenotypes in humans.
Douglas R. Smith, Christine M. Stanley, Theodore Foss, Richard G. Boles, Kevin McKernan
PLoS One. 2017 Nov 16;12(11):e0187926.
DOI: 10.1371/journal.pone.0187926
Rare genetic variants in the core endocannabinoid system genes CNR1, CNR2, DAGLA, MGLL and FAAH were identified in molecular testing data from up to 6.032 patients with a broad spectrum of neurological disorders. The variants were evaluated for association with phenotypes similar to those observed in the orthologous gene knockouts in mice. Heterozygous rare coding variants in CNR1, which encodes the type 1cannabinoid receptor (CB1), were found to be significantly associated with pain sensitivity (especially migraine), sleep and memory disorders - alone or in combination with anxiety - compared to a set of controls without such CNR1 variants. Similarly, heterozygous rare variants in DAGLA, which encodes diacylglycerol lipase alpha, were found to be significantly associated with seizures and developmental disorders, including abnormalities of brain morphology, compared to controls. Rare variants in MGLL, FAAH and CNR2 were not associated with any neurological phenotypes in the patients tested. Diacylglycerol lipase alpha synthesizes the endocannabinoid 2-AG in the brain, which interacts with CB1 receptors. The phenotypes associated with rare CNR1 variants are reminiscent of those implicated in the theory of clinical endocannabinoid deficiency syndrome. The severe phenotypes associated with rare DAGLA variants underscore the critical role of rapid 2-AG synthesis and the endocannabinoid system in regulating neurological function and development. Mapping of the variants to the 3D structure of the type 1 cannabinoid receptor, or primary structure of diacylglycerol lipase alpha, reveals clustering of variants in certain structural regions and is consistent with impacts to function..

Rates of Prenatal Cannabis Use Among Pregnant Women Before and During the COVID-19 Pandemic
JAMA November 2, 2021 Volume 326, Number 17
https://jamanetwork.com/journals/jam...rticle/2784637
Methods | The sample comprised all KPNC pregnant women screened for prenatal cannabis use via a universal urine toxicology test from January 1, 2019, through December 31, 2020, during standard prenatal care (at ≈8weeks’ gestation). The institutional review board of KPNC approved this study and
waived the need for informed consent. We computed monthly rates of prenatal cannabis use standardized to the year 2020 age and race and ethnicity distribution. We fit interrupted time-series (ITS) models to monthly rate data using negative binomial regression, adjusted for age (<25, 25 to <35, ≥35 years) and self-reported race and ethnicity (Asian/Pacific Islander, Black, Hispanic, non-Hispanic White, or other or unknown), which were included because of the known age and race and ethnicity differences in the prevalence of prenatal cannabis use. The pre pandemic period was defined as urine toxicology tests conducted from January 2019 to March 2020 and the pandemic period from April through December 2020 (see Laboratory Methods in the Supplement).
The rate ratio and corresponding 95% CIs are reported herein.
We conducted the analyses using SAS version 9.4 (SAS Institute Inc). A 2-sided P < .05 was considered statistically significant.
Results | Of 100 005 pregnancies (95 412 women), 26% were Asian or Pacific Islander; 7%, Black; 28%, Hispanic; 34%, non- Hispanic White; and 5%, other, unknown, or multiracial. The Patients were a mean age of 31 years (median, 31 years). There were negligible differences in age or race and ethnicity in the 2 periods. During the pandemic, patients completed toxicology
testing slightly earlier in their pregnancies (before pandemic mean, 8.51 weeks’ gestation; during pandemic mean, 8.04 weeks’ gestation). Before the pandemic, the standardized rate of prenatal cannabis use was 6.75% of pregnancies (95% CI, 6.55%-6.95%); that rate increased to 8.14% of pregnancies (95% CI, 7.85%- 8.43%) during the pandemic (Figure). In the ITS analysis, we found that prenatal cannabis use increased by 25% (95% CI,
12%-40%; Table) during the pandemic over prenatal cannabis use during the 15 months before the pandemic. The ITS analysis confirmed that these rates before and during the pandemic were stable, with no statistically significant month-to month trends (Table).
Discussion | Rates of biochemically verified prenatal cannabis use increased significantly during the COVID-19 pandemic among pregnant women in Northern California. Results are consistent with the rise in cannabis sales seen in California during the same period.5 When the toll of the COVID-19 pandemic begins to fade and restrictions are lifted, it is unknown whether pandemic-related increases in rates of cannabis use during pregnancy will reverse or remain elevated. Continued monitoring of trends is critical as the pandemic continues to evolve. This study is limited to pregnant women universally screened in the KPNC system for prenatal cannabis use via
urine toxicology testing early in pregnancy (≈8 weeks’ gestation) as part of standard prenatal care, and data do not reflect continued use throughout pregnancy. In some cases, positive toxicology test results may detect prenatal cannabis use that occurred prior to pregnancy recognition. Additional studies
that capture pandemic-related changes infrequency of and reasons for cannabis use during pregnancy and among non pregnant women are also needed. Prenatal cannabis use is associated with health risks, including low infant birth weight and potential effects on offspring neurodevelopment.6 Clinicians should educate pregnant women about the harms of prenatal cannabis use, support women to quit, and provide resources for stress reduction.

Real life Experience of Medical Cannabis Treatment in Autism: Analysis of Safety and Efficacy.
Bar-Lev Schleider, L., Mechoulam, R., Saban, N., Meiri, G., & Novack, V.
Scientific Reports, 9(1).(2019).
doi:10.1038/s41598-018-37570-y
There has been a dramatic increase in the number of children diagnosed with autism spectrum disorders (ASD) worldwide. Recently anecdotal evidence of possible therapeutic efects of cannabis products has emerged. The aim of this study is to characterize the epidemiology of ASD patients receiving medical cannabis treatment and to describe its safety and efcacy. We analysed the data prospectively collected as part of the treatment program of 188 ASD patients treated with medical cannabis between 2015 and 2017. The treatment in majority of the patients was based on cannabis oil containing 30% CBD and 1.5% THC. Symptoms inventory, patient global assessment and side efects at 6 months were primary outcomes of interest and were assessed by structured questionnaires. After six months of treatment 82.4% of patients (155) were in active treatment and 60.0% (93) have been assessed; 28 patients (30.1%) reported a signifcant improvement, 50 (53.7%) moderate, 6 (6.4%) slight and 8 (8.6%) had no change in their condition. Twenty-three patients (25.2%) experienced at least one side efect; the most common was restlessness (6.6%). Cannabis in ASD patients appears to be well tolerated, safe and efective option to relieve symptoms associated with ASD.

Recent cannabis use and nightly sleep duration in adults: a population analysis of the NHANES from 2005 to 2018
Calvin Diep, Chenchen Tian, Kathak Vachhani, Christine Won, Duminda N Wijeysundera, Hance Clarke, Mandeep Singh, Karim S Ladha
Regional Anesthesia & Pain Medicine 2021
doi: 10.1136/rapm-2021-103294
DOI: 10.1136/rapm-2021-103161
https://rapm.bmj.com/content/early/2...pm-2021-103161
Background While popularly consumed for its perceived benefits as a sleeping aid, the impact of cannabis on sleep-wake regulation in clinical studies is inconclusive. The purpose of this study was to determine the relationship between cannabis use and nightly sleep duration in a nationally representative dataset.
Methods A cross-sectional analysis of adults was undertaken using the National Health and Nutrition Examination Survey data from 2005 to 2018. Respondents were dichotomized as recent users or non-users if they had used or not used cannabis in the past 30 days, respectively. The primary outcome was nightly sleep duration, categorized as short (<6 hours), optimal (6–9 hours), and long (>9 hours). Multinomial logistic regression was used to adjust for sociodemographic and health-related covariates, and survey sample weights were used in modeling.
Results From a sample representing approximately 146 million adults in the USA, 14.5% reported recent cannabis use. In an adjusted analysis, recent users were more likely than non-users to report both short sleep (OR 1.34, 95% CI 1.12 to 1.59, p<0.001) and long sleep (OR 1.56, 95% CI 1.25 to 1.96, p<0.001). Heavy users (≥20 of the past 30 days) were even more likely to be at the extremes of nightly sleep duration.
Discussion Recent cannabis use was associated with the extremes of nightly sleep duration in a nationally representative sample of adults, with suggestions of a dose–response relationship. Our findings highlight the need to further characterize the sleep health of regular cannabis users in the population.
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Reduction of Benzodiazepine Use in Patients Prescribed Medical Cannabis
Chad Purcell, Andrew Davis, Nico Moolman, and S. Mark Taylor
Cannabis and Cannabinoid Research Volume 4, Number 3, 2019
DOI: 10.1089/can.2018.0020
Background: Benzodiazepines are a class of medication with sedative properties, commonly used for anxiety and other neurological conditions. These medications are associated with several well-known adverse effects. This observational study aims to investigate the reduction of benzodiazepine use in patients using prescribed medical cannabis.
Methods: A retrospective analysis was performed on a cohort of 146 medical cannabis patients (average age 47 years, 61% female, 54% reporting prior use of cannabis) who reported benzodiazepine use at initiation of cannabis therapy. These data are a part of a database gathered by a medical cannabis clinic (Canabo Medical). Descriptive statistics were used to quantify associations of the proportion of benzodiazepine use with time on medical cannabis therapy.
Results: After completing an average 2-month prescription course of medical cannabis, 30.1% of patients had discontinued benzodiazepines. At a follow-up after two prescriptions, 65 total patients (44.5%) had discontinued benzodiazepines. At the final follow-up period after three medical cannabis prescription courses, 66 total patients (45.2%) had discontinued benzodiazepine use, showing a stable cessation rate over an average of 6 months. Conclusion: Within a cohort of 146 patients initiated on medical cannabis therapy, 45.2% patients successfully discontinued their pre-existing benzodiazepine therapy. This observation merits further investigation into the risks and benefits of the therapeutic use of medical cannabis and its role relating to benzodiazepine use

Regulation of Brain Reward by the Endocannabinoid System: A Critical Review of Behavioral Studies in Animals
Styliani (Stella) Vlachou, George Panagi
Current Pharmaceutical Design 20(13) June 2013
DOI: 10.2174/13816128113199990433
The endocannabinoid system has been implicated in the regulation of a variety of physiological processes, including a crucial involvement in brain reward systems and the regulation of motivational processes. Behavioral studies have shown that cannabinoid reward may involve the same brain circuits and similar brain mechanisms with other drugs of abuse, such as nicotine, cocaine, alcohol and heroin, as well as natural rewards, such as food, water and sucrose, although the conditions under which cannabinoids exert their rewarding effects may be more limited. The purpose of the present review is to briefly describe and evaluate the behavioral and pharmacological research concerning the major components of the endocannabinoid system and reward processes. Special emphasis is placed on data received from four procedures used to test the effects of the endocannabinoid system on brain reward in animals; namely, the intracranial self-stimulation paradigm, the self-administration procedure, the conditioned place preference procedure and the drug-discrimination procedure. The effects of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor agonists, antagonists and endocannabinoid modulators in these procedures are examined. Further, the involvement of CB1 and CB2 receptors, as well the fatty acid amid hydrolase (FAAH) enzyme in reward processes is investigated through presentation of respective genetic ablation studies in mice. We suggest that the endocannabinoid system plays a major role in modulating motivation and reward processes. Further research will provide us with a better understanding of these processes and, thus, could lead to the development of potential therapeutic compounds for the treatment of reward-related disorders.

Regulation of Cannabinoid CB1 Receptors in the Central Nervous System by Chronic Cannabinoids
Laura J. Sim-Selley
Critical Reviews™ in Neurobiology Volume 15, 2003 Issue 2 30 pages
DOI: 10.1615/CritRevNeurobiol.v15.i2.10
Marijuana produces a number of characteristic behaviors in humans and animals, including memory impairment, antinociception, and locomotor and psychoactive effects. However, tolerance and dependence to cannabinoids develops after chronic use, as demonstrated both clinically and in animal models. The potential therapeutic benefits of certain cannabinoid-mediated effects, as well as the use of marijuana for its psychoactive properties, has raised interest in understanding the cellular adaptations produced by chronic administration of this class of drugs. The primary active constituent of marijuana, delta9-tetrahydrohydrocannabinol (THC), binds to specific G-protein-coupled receptors. The central nervous system (CNS) effects of THC are mediated by CB1 receptors, which couple primarily to inhibitory G-proteins. High levels of CB1 receptors are found in the basal ganglia, hippocampus, cortex, and cerebellum, consistent with the profile of behavioral effects. Studies over the past decade have determined that CB1 receptors undergo downregulation and desensitization following chronic administration of THC or synthetic cannabinoid agonists. In general, these adaptations are regionally widespread and of considerable magnitude, and are thought to contribute to tolerance to cannabinoid- mediated behavioral effects. Adaptation at the effector level has been more difficult to characterize, although it appears that alterations in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity may be particularly important in cannabinoid dependence. A striking characteristic of CB1 receptor adaptation is the region dependence of the magnitude and rate of development of downregulation and desensitization. These regional differences may provide interesting insights into the mechanisms of CB1 receptors receptor signaling in different brain regions. Moreover, region-specific adaptations in CB1 receptors following chronic cannabinoid administration may produce differential adaptations at the in vivo level.

Relative pharmacological potency in mice of optical isomers of Δ1-tetrahydrocannabinol.
Jones, G., Pertwee, R. G., Gill, E. W., Paton, W. D. M., Nilsson, I. M., Widman, M., & Agurell, S.
Biochemical Pharmacology, 23(2), 439–446.(1974).
doi:10.1016/0006-2952(74)90435-3
The tritium-labelled unnatural enantiomorph of A’-tetrahydrocannabinol (A’-THC) was synthesized. The ‘H-( +)-A’-THC had a specific activity of I.3 Ci,‘mmole and an optical purity of ca. 97”,,. The equipotent molar ratio for (f ) and ( -)-At-tetrahydrocannabinols was determined in mice by an established behavioural bioassay. The (+)-A’-THC was found to be significantly less potent than the laevorotatory Isomer, the mean potency ratio being 13 (95 per cent confidence limits: 7 and 24). Brain levels of (+)-A’-THC and its metabolites were measured in mice 20 min after intravenous injection of 3H-( +)-At-THC (2 mg kg) and were compared with the corresponding levels of (-I-A’-THC and its metabolites. With the exception of the concentrations of one metabolite. no statistically significant differences were observed between the mean levels of enantiomorphs of the cannabinoidsin the brain. In the case of the single metabolite (which was tentatively assigned the structure of 7-hydroxy-A’-THC) the brain level of the dextrorotatoryisomer was l,8-times higher than that of the laevorotatory isomer, a difference which was statistically significant. On incubation in vitro vvith an enriched mouse liver homogenate. (+ )-A’-THC was partially metabolized to more polar compounds; the principal metabolite was shown to be (+)-7-hydroxy-A’-THC. It was concluded that the differences in the psychopharmacological potencies in uiuo of the optical isomers of A’-THC are determined within the central nervous system and are not due to gross differences in metabolism or body distribution.

Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy
Brenda E. Porter, Catherine Jacobson
2013, Epilepsy & Behavior, 29(3) 574-577
DOI: 10.1016/j.yebeh.2013.08.037
Severe childhood epilepsies are characterized by frequent seizures, neurodevelopmental delays, and impaired
quality of life. In these treatment-resistant epilepsies, families often seek alternative treatments. This survey explored the use of cannabidiol-enriched cannabis in children with treatment-resistant epilepsy. The survey was presented to parents belonging to a Facebook group dedicated to sharing information about the use of cannabidiol-enriched cannabis to treat their child's seizures. Nineteen responses met the following inclusion criteria for the study: a diagnosis of epilepsy and current use of cannabidiol-enriched cannabis. Thirteen children had Dravet syndrome, four had Doose syndrome, and one each had Lennox–Gastaut syndrome and idiopathic epilepsy. The average number of antiepileptic drugs (AEDs) tried before using cannabidiol-enriched cannabis was 12. Sixteen (84%) of the 19 parents reported a reduction in their child's seizure frequency while taking cannabidiol-enriched cannabis. Of these, two (11%) reported complete seizure freedom, eight (42%) reported a greater than 80% reduction in seizure frequency, and six (32%) reported a 25–60% seizure reduction. Other beneficial effects included increased alertness, better mood, and improved sleep. Side effects included drowsiness and fatigue. Our survey shows that parents are using cannabidiol-enriched cannabis as a treatment for their children with treatment-resistant epilepsy. Because of the increasing number of states that allow access to medical cannabis, its use will likely be a growing concern for the epilepsy community. Safety and tolerability data for cannabidiol-enriched cannabis use among children are not available. Objective measurements of a standardized preparation of pure cannabidiol are needed to determine whether it is safe, well tolerated, and efficacious at controlling seizures in this pediatric population with difficult-to-treat seizures.

Resolution of Cannabinoid Hyperemesis Syndrome with Benzodiazepines: A Case Series.
Kheifets M, Karniel E, Landa D, Vons SA, Meridor K, Charach G
Isr Med Assoc J. 2019 Jun;21(6):404-407.
https://www.ima.org.il/FilesUpload/I...363/181650.pdf
Background: Cannabinoid hyperemesis syndrome (CHS) is under-recognized by clinicians. It is characterized by nausea, severe abdominal pain, and cyclical vomiting in the context of chronic cannabis use. Oral benzodiazepine is a proposed treatment for CHS. It decreases activation of Cannabinoid Type 1 Receptor (CB1) in the frontal cortex, has a sedative and hypnotic effect and reduces the anticipation of nausea and vomiting. These effects on the central nervous system (CNS) might explain its beneficial antiemetic effect for this syndrome.
Objectives: To increase the index of suspicion for CHS, a unique syndrome that requires a unique treatment with benzodiazepines and not antiemetics.
Methods: We describe a series of four patients with documented cannabis use, who were admitted to an internal medicine department of Meir Medical Center due to symptoms consistent with abdominal pain, nausea, and vomiting. They were initially treated with conventional antiemetics and proton pump inhibitors without response. Intensive investigations were conducted to exclude common and sometimes urgent gastrointestinal or CNS syndromes.
Results: After excluding urgent gastrointestinal and CNS origins for the vomiting, we suspected CHS. All four patients experienced similar symptoms and failure of conventional treatment with antiemetics and proton pump inhibitors. They experienced relief after administration of benzodiazepines.
Conclusions: A high index of suspicion for CHS allows for rapid, appropriate treatment with benzodiazepines, which in turn may lead to cessation of the debilitating symptoms caused by this syndrome.
BACKGROUND:
Cannabinoid hyperemesis syndrome (CHS) is under-recognized by clinicians. It is characterized by nausea, severe abdominal pain, and cyclical vomiting in the context of chronic cannabis use. Oral benzodiazepine is a proposed treatment for CHS. It decreases activation of Cannabinoid Type 1 Receptor (CB1) in the frontal cortex, has a sedative and hypnotic effect and reduces the anticipation of nausea and vomiting. These effects on the central nervous system (CNS) might explain its beneficial antiemetic effect for this syndrome.
OBJECTIVES:
To increase the index of suspicion for CHS, a unique syndrome that requires a unique treatment with benzodiazepines and not antiemetics.
METHODS:
We describe a series of four patients with documented cannabis use, who were admitted to an internal medicine department of Meir Medical Center due to symptoms consistent with abdominal pain, nausea, and vomiting. They were initially treated with conventional antiemetics and proton pump inhibitors without response. Intensive investigations were conducted to exclude common and sometimes urgent gastrointestinal or CNS syndromes.
RESULTS:
After excluding urgent gastrointestinal and CNS origins for the vomiting, we suspected CHS. All four patients experienced similar symptoms and failure of conventional treatment with antiemetics and proton pump inhibitors. They experienced relief after administration of benzodiazepines.
CONCLUSIONS:
A high index of suspicion for CHS allows for rapid, appropriate treatment with benzodiazepines, which in turn may lead to cessation of the debilitating symptoms caused by this syndrome.

Rimonabant: Just an Antiobesity Drug? Current Evidence on Its Pleiotropic Effects
Maurizio Bifulco, Claudia Grimaldi, Patrizia Gazzerro, Simona Pisanti, and Antonietta Santoro Molecular Pharmacology 71(6):1445-56
DOI: 10.1124/mol.106.033118
The advent of the highly selective cannabinoid receptor (CB1) antagonist, rimonabant (SR141716; Acomplia) can revolutionize the ability of the clinicians to manage obesity. Large-scale clinical trials have demonstrated that rimonabant therapy can reduce obesity. Although, the precise mechanisms of action of rimonabant have to be further dissected, it is emerging, from both preclinical and clinical research, that not only is rimonabant an antiobesity drug, but also its pleiotropic functions affect a broad range of diseases, from obesity-related comorbidities to drug dependence and cancer. Here we review recent data from the literature and discuss the full pharmacological potential of this drug.

Not Cannabis specific
Rising Host Resistance against Coronavirus
Gershom Zajicek M.D,
https://www.growkudos.com/projects/r...st-coronavirus
https://www.youtube.com/watch?v=0Otn...PrKGvs&index=1

Risk-thresholds for the association between frequency of cannabis use and the development of psychosis: a systematic review and meta-analysis
https://www.cambridge.org/core/journals/psychological-medicine/article/riskthresholds-for-the-association-between-frequency-of-cannabis-use-and-the-development-of-psychosis-a-systematic-review-and-metaanalysis/4EB34100E58C5C9E1370CAFE83C7F7 05
Tessa Robinson, Muhammad Usman Ali, Bethany Easterbrook, Wayne Hall, Didier Jutras-Aswad, and Benedikt Fischer
Published online by Cambridge University Press: 24 March 2022
Background
Epidemiological studies show a dose–response association between cannabis use and the risk of psychosis. This review aimed to determine whether there are identifiable risk-thresholds between the frequency of cannabis use and psychosis development.
Methods
Systematic search of Embase, MEDLINE, PsycINFO, CINAHL, and Web of Science for relevant studies (1 January 2010–26 April 2021). Case–control or cohort studies that investigated the relationship between cannabis use and the risk of psychosis development that reported effect estimates [odds ratios (OR), hazard ratios (HR), risk ratios (RR)] or the raw data to calculate them, with information on the frequency of cannabis consumption were included. Effect estimates were extracted from individual studies and converted to RR. Two-stage dose–response multivariable meta-analytic models were utilized and sensitivity analyses conducted. The Newcastle Ottawa Scale was used to assess the risk of bias of included studies.
Results
Ten original (three cohorts, seven case–control) studies were included, including 7390 participants with an age range of 12–65 years. Random-effect model meta-analyses showed a significant log-linear dose–response association between cannabis use frequency and psychosis development. A restricted cubic-splines model provided the best fit for the data, with the risk of psychosis significantly increasing for weekly or more frequent cannabis use [RR = 1.01, 95% confidence interval (CI) 0.93–1.11 yearly; RR = 1.10, 95% CI 0.97–1.25 monthly; RR = 1.35, 95% CI 1.19–1.52 weekly; RR = 1.76, 95% CI 1.47–2.12 daily]
Conclusion
Individuals using cannabis frequently are at increased risk of psychosis, with no significant risk associated with less frequent use. Public health prevention messages should convey these risk-thresholds, which should be refined through further work.

Risks of harm with cannabinoids, cannabis, and cannabis-based medicine for pain management relevant to patients receiving pain treatment: protocol for an overview of systematic reviews
Ian Gilrona, Fiona M. Blythd, Louisa Degenhardte, Marta Di Fortif, Christopher Ecclestoni, Simon Haroutounianj, Andrew Moorek, Andrew S.C. Ricel, Mark Wallacem
PAIN Reports, 4(3), e742. (2019)
doi:10.1097/pr9.0000000000000742
Introduction: With the increasing availability of cannabis and cannabinoids and their potential utility for pain treatment, there is a growing need to evaluate the risk-benefit considerations of cannabinoids for the management of pain. As part of the IASP Cannabis and Cannabinoids Task Force, this protocol describes a planned overview of systematic reviews summarizing the risks of harm with cannabinoids that are relevant to patients receiving pain treatment.
Methods: This overview will involve literature searches of several databases and a defined search strategy that will target systematic reviews or meta-analyses of cannabinoids where harms are the primary focus. Data extraction will include various features of the cannabinoid(s) and the harm(s) being studied as well as other methodological features of each included systematic review. Methodological quality of each included review will be assessed using AMSTAR-2 as well as compliance with the PRISMA harms checklist. Prospero registration pending.
Discussion: The broad overview of reviews defined by this protocol is expected to synthesize available good quality evidence of harms that will help inform risk-benefit considerations about the use of cannabinoids for pain management.

Role of acylethanolamides in the gastrointestinal tract with special reference to food intake and energy balance.
Francesca Borrelli, Angelo A. Izzo
Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 33–49
Doi: 10.1016/j.beem.2008.10.003
https://www.researchgate.net/publica...l_reference_to _food_intake_and_energy_balanc e
Acylethanolamides (AEs) are a group of lipids occurring in both plants and animals. The best-studied AEs are the endocannabinoid anandamide (AEA), the anti-inflammatory compound palmitoylethanolamide (PEA), and the potent anorexigenic molecule oleoylethanolamide (OEA). AEs are biosynthesized in the gastrointestinal tract, and their levels may change in response to noxious stimuli, food deprivation or diet-induced obesity. The biological actions of AEs within the gut are not limited to the modulation of food intake and energy balance. For example, AEs exert potential beneficial effects in the regulation of intestinal motility, secretion, inflammation and cellular proliferation. Molecular targets of AEs, which have been identified in the gastrointestinal tract, include cannabinoid CB1 and CB2 receptors
(activated by AEA), transient receptor potential vanilloid type 1 (TRPV1, activated by AEA and OEA), the nuclear receptor peroxisome proliferators-activated receptor-a (PPAR-a, activated by OEA and, to a less extent, by PEA), and the orphan G-coupled receptors GPR119 (activated by OEA) and GPR55 (activated by PEA and, with lower potency, by AEA and OEA). Modulation of AE levels in the gut may provide new pharmacological strategies not only for the treatment of feeding disorders but also for the prevention or cure of widespread intestinal diseases such as inflammatory bowel disease and colon cancer.

Role of Cannabidiol and Tetrahydrocannabivarin on Paclitaxel-induced neuropathic pain in rodents
Anil Kumar Kalvala, Arvind Bagde, Peggy Arthur, Sunil Kumar Surapaneni, Nimma Ramesh, Aakash Nathani, Mandip Singh
International Immunopharmacology Volume 107, June 2022
DOI: 10.1016/j.intimp.2022.108693

THCV, when combined with CBD, provides excellent neuroprotection against paclitaxel-induced peripheral neuropathy in mice.

THCV and CBD improve mitochondrial health and neuron function by modulating CB1 and 5HT1A receptors, respectively.

After blocking CB1 and 5HT1A receptors in PTX-injured DRG primary cultures, THCV and CBD have no effect on neurite outgrowths, mitochondrial membrane potentials, or mitochondrial superoxide production.
The purpose of this study was to evaluate if phytocannabinoids, synthetic cannabidiol (CBD), and tetrahydrocannabivarin (THCV), and their combination, could protect mice from Paclitaxel-induced peripheral neuropathy (PIPN). Six groups of C57BL/6J mice (n = 6) were used in this study. The mice were given paclitaxel (PTX) (8 mg/kg/day, i.p.) on days 1, 3, 5, and 7 to induce neuropathy. Mice were evaluated for behavioral parameters, and dorsal root ganglions (DRG) were collected from the animals and subjected to RNA sequencing and westernblot analysis at the end of the study. On cultured DRGs derived from adult male rats, immunocytochemistry and mitochondrial functional assays were also performed. When compared to individual treatments, the combination of CBD and THCV improved thermal and mechanical neurobehavioral symptoms in mice by twofold. Targets for CBD and THCV therapy were identified by KEGG (RNA sequencing). PTX reduced the expression of p-AMPK, SIRT1, NRF2, HO1, SOD2, and catalase while increasing the expression of PI3K, p-AKT, p-P38 MAP kinase, BAX, TGF-β, NLRP3 inflammasome, and caspase 3 in DRG homogenates of mice. Combination therapy outperformed monotherapy in reversing these protein expressions. The addition of CBD and THCV to DRG primary cultures reduced mitochondrial superoxides while increasing mitochondrial membrane potentials. WAY100135 and rimonabant altered the neuroprotective effects of CBD and THCV respectively by blocking 5-HT1A and CB1 receptors in mice and DRG primary cultures. The entourage effect of CBD and THCV against PIPN appears to protect neurons in mice via 5HT1A and CB1 receptors respectively.
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Role of Cannabidiol in response to 3-nitro proponic acid-induced neurotoxicity in BV2 microglial cells
Tanya Keyur Desai Thesis
https://www.academia.edu/40414002/MAIN_DISSERTATION?auto=downloa d&email_work_card=download-paper
CONTENTS:
1) Introduction 2) Materials and methods 3) Results
4) Discussion
5) Conclusion
A number of data concerning the neuro-protective actions of cannabinoids against mitochondrial toxins, substances that disrupt the mitochondrial respiratory chain, have emerged in the last few years. Cannabinoids can be defined as naturally occurring compounds present in Cannabis sativaplant; the main ones being delta-tetrahydrocannabinoid ( D9-THC) and cannabidiol. Cannabidiol, in particular, is a phytocannabinoid which belongs to the Cannabis species. This compound is found to exhibit variety of properties such as anti- convulsant, anti-inflammatory, anti-neoplastic, anti-proliferative which will be later explained. This paper explains the neuroprotective and anti-convulsant actions and effects of cannabidiol against 3-Nitropropionic acid which acts as a mitochondrial toxin in this case; an irreversible inhibitor of the enzyme, succinate dehydrogenase. Hence, 3-Nitropropionic acid is utilized as a mitochondrial toxin in this case. It is administered in excess in the BV2 microglial cells, thus leading to drug-induced neurotoxicity. Cannabidiol are then introduced and the role of cannabidiol is then studied in response to drug- induced neurotoxicity. Cannabidiol can be utilized as an effective measure to prevent and cure epilepsy, considering the positive results it has shown against a number of drugs and toxins. Thus, it can be contemplated as a potent remedy for future purposes.

Role of Cannabis and Its Derivatives in Gastrointestinal and Hepatic Disease.
Gotfried, J., Naftali, T., & Schey, R.
Gastroenterology. (2020).
doi:10.1053/j.gastro.2020.03.087
Medical and recreational cannabis use has increased dramatically over the last decade, resulting from mainstream cultural acceptance
and legalization in several countries worldwide. Cannabis and its derivatives affect many gastrointestinal processes, via the
endocannabinoid system (ECS). The ECS influences gastrointestinal homeostasis through anti-inflammatory, anti-nociceptive, and
anti-secretory effects. Some gastrointestinal disorders might therefore be treated with cannabinoids. Despite numerous studies in cell
lines and animals, few human studies have evaluated the therapeutic effects of cannabinoids. Cannabis’ schedule 1 drug status has
limited its availability in research; cannabis has been only recently legalized, in some states, for medicinal and/or recreational use.
Cannabinoids can alleviate chemotherapy-induced nausea and emesis and chronic pain. Studies have demonstrated the important roles
of the ECS in metabolism, obesity, and non-alcoholic fatty liver disease and the anti-inflammatory effects of cannabis have been
investigated in patients with inflammatory bowel diseases. Despite its potential benefits, undesired or even detrimental effects of
cannabis can limit its use. Side effects such as cannabinoid hyperemesis syndrome affect some users. We review the ECS and the
effects of cannabis and its derivatives on gastrointestinal and hepatic function in health and disease.

Role of Caryophyllane Sesquiterpenes in the Entourage Effect of Felina 32 Hemp Inflorescence Phytocomplex in Triple Negative MDA-MB-468 Breast Cancer Cells
Silvia Di Giacomo, Alessia Mariano, Marco Gullì, Caterina Fraschetti, Annabella Vitalone, Antonello Filippi, Luisa Mannina, Anna Scotto d’Abusco, and Antonella Di Sotto
Molecules. 2021 Nov; 26(21): 6688. 2021 Nov 5.
doi: 10.3390/molecules26216688
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8587411/
Cannabis sativa L. crops have been traditionally exploited as sources of fibers, nutrients, and bioactive phytochemicals of medical interest. In the present study, two terpene-rich organic extracts, namely FOJ and FOS, obtained from Felina 32 hemp inflorescences collected in June and September, respectively, have been studied for their in vitro anticancer properties. Particularly, their cytotoxicity was evaluated in different cancer cell lines, and the possible entourage effect between nonintoxicating phytocannabinoids (cannabidiol and cannabichromene) and caryophyllane sesquiterpenes (β-caryophyllene, β-caryophyllene oxide and α-humulene), as identified at GC/MS analysis, was characterized. Modulation of cannabinoid CB1 and CB2 receptors was studied as a mechanistic hypothesis. Results highlighted marked cytotoxic effects of FOJ, FOS, and pure compounds in triple negative breast cancer MDA-MB-468 cells, likely mediated by a CB2 receptor activation. Cannabidiol was the main cytotoxic constituent, although low levels of caryophyllane sesquiterpenes and cannabichromene induced potentiating effects; the presence in the extracts of unknown antagonistic compounds has been highlighted too. These results suggest an interest in Felina 32 hemp inflorescences as a source of bioactive phytocomplexes with anticancer properties and strengthen the importance of considering the possible involvement of minor terpenes, such as caryophyllane sesquiterpenes, in the entourage effect of hemp-based extracts.

Role of chronic cannabis use: Cyclic vomiting syndrome vs cannabinoid hyperemesis syndrome.
Venkatesan, T., Levinthal, D. J., Li, B. U. K., Tarbell, S. E., Adams, K. A., Issenman, R. M., … Hasler, W. L.
Neurogastroenterology & Motility, 31, e13606. (2019).
doi:10.1111/nmo.13606
Cannabis is commonly used in cyclic vomiting syndrome (CVS) due to its antiemetic and anxiolytic properties. Paradoxically, chronic cannabis use in the context of cyclic vomiting has led to the recognition of a putative new disorder called cannabinoid hy? peremesis syndrome (CHS). Since its first description in 2004, numerous case series and case reports have emerged describing this phenomenon. Although not pathog? nomonic, a patient behavior called “compulsive hot water bathing” has been associ? ated with CHS. There is considerable controversy about how CHS is defined. Most of the data remain heterogenous with limited follow?up, making it difficult to ascertain whether chronic cannabis use is causal, merely a clinical association with CVS, or unmasks or triggers symptoms in patients inherently predisposed to develop CVS. This article will discuss the role of cannabis in the regulation of nausea and vomiting, specifically focusing on both CVS and CHS, in order to address controversies in this context. To this objective, we have collated and analyzed published case series and case reports on CHS in order to determine the number of reported cases that meet current Rome IV criteria for CHS. We have also identified limitations in the existing diagnostic framework and propose revised criteria to diagnose CHS. Future research in this area should improve our understanding of the role of cannabis use in cyclic vomiting and help us better understand and manage this disorder.

Role of endocannabnoid system in mental diseases
JORGE MANZANARES*, LEYRE URIGÜEN, GABRIEL RUBIO and TOMÁS PALOMO
Neurotoxicity Research, 2004, VOL. 6(3). pp. 001-012
https://www.researchgate.net/publica...endocannabinoi d_system_in_mental_diseases
In the last decade, a large number of studies using Δ9-tetrahydrocannabinol (THC), the main active principle derivative of the marijuana plant, or cannabinoid synthetic derivatives have substantially contributed to advance the understanding of the pharmacology and neurobiological mechanisms produced by cannabinoid receptor activation. Cannabis has been historically used to relieve some of the symptoms associated with central nervous system disorders. Nowadays, there are anecdotal evidences for the use of cannabis in many patients suffering from multiple sclerosis or chronic pain. Following the historical reports on the use of cannabis for medicinal purposes, recent research has highlighted the potential of cannabinoids to treat a wide variety of clinical disorders. Some of these disorders that are being investigated are pain, motor dysfunctions, or psychiatric illness. On the other hand, cannabis abuse has been related to several psychiatric disorders such as dependence, anxiety, depression, cognitive impairment, and psychosis. Considering that cannabis or cannabinoid pharmaceutical preparations may no longer be exclusively recreational drugs but may also present potential therapeutic uses, it has become of great interest to analyze the neurobiological and behavioral consequences of their administration. This review attempts to link current un

[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Role of Endocannabinoids in Neuron-Glial Crosstalk
Livio Luongo, Enza Palazzo, Vito de Novellis and Sabatino Maione
The
Open Pain Journal, 2010, 3, 29-36
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]DOI: 10.2174/1876386301003010029[/FONT]
Evidence shows bidirectional crosstalk between neurons and glia, suggesting that glia play an active role in synaptic plasticity leading to chronic pain. Importantly, gliosis has been implicated in the development and maintenance [/FONT]of hyperalgesia or allodynia following chronic inflammation or nerve injury. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG), or the lipoamino acid N-arachydonoyldopamine (NADA), are fatty acid derivative neurotransmitters, named endocannabinoids (eCBs). These perform several biological actions, via the activation of cannabinoid type 1 and 2 (CB1/CB2) receptors belonging to the G-protein-coupled receptor family. The eCBs are produced on demand by neurons or glial cells and it has been suggested that they might be involved in the crosstalk between astrocytes, microglia, oligodendrocytes and neurons. In chronic pain, the modified glial or neural activity also seems to be associated with changes in eCB levels in pain processing areas either in the spinal cord or the brain. The activation of the eCB system in microglia or astrocytes could be crucial in modulating axonal growth and synaptogenesis at the base of neural phenotypic changes. Furthermore, changes in eCBs levels have been suggested to affect the destiny of cells: death or survival may depend on a specific pain condition. Thus, although eCBs are emerging as neurotransmitters responsible for the regulation of glia-neuron crosstalk in chronic pain, the precise mechanisms leading to eCB production, the origin and the timecourse of eCB release, the eCB release switch from one cell type to the other and their movement or catabolism across the glial or neural cell membrane nevertheless still remain unknown. These issues together with alternative eCB targets will
be addressed in the current review.

Role of the Endogenous Cannabinoid System in Nicotine Addiction: Novel Insights
Islam Hany Gamaleddin, Jose M. Trigo, Aliou B. Gurye, Alexander Zvonok, Alexandros Makriyannis, Steven R. Goldberg and Bernard Le Foll
DOI: 10.3389/fpsyt.2015.00041
https://www.ncbi.nlm.nih.gov/pmc/art...t-06-00041.pdf
Several lines of evidence have shown that the endogenous cannabinoids are implicated in several neuropsychiatric diseases. Notably, preclinical and human clinical studies have shown a pivotal role of the cannabinoid system in nicotine addiction. The CB1 receptor inverse agonist/antagonist rimonabant (also known as SR141716) was effective to decrease nicotine-taking and nicotine-seeking in rodents, as well as the elevation of dopamine induced by nicotine in brain reward area. Rimonabant has been shown to improve the ability of smokers to quit smoking in randomized clinical trials. However, rimonabant was removed from the market due to increased risk of psychiatric side-effects observed in humans. Recently, other components of the endogenous cannabinoid system have been explored. Here, we present the recent advances on the understanding of the role of the different components of the cannabinoid system on nicotine’s effects. Those recent findings suggest possible alternative ways of modulating the cannabinoid system that could have implication for nicotine dependence treatment. . .

Role of marijuana components on the regenerative ability of stem cells
Henry Miller, Nicholas De Leo, Jeremy Badach, Andrew Lin, John Williamson, Steven Bonawitz, Olga Ostrovsky
Cell Biochemistry and Function. (2020)
doi:10.1002/cbf.3609
Stem cell therapy promotes tissue regeneration and wound healing. Efforts have been made to prime stem cells to enhance their regenerative abilities. Certain marijuana components, namely the non-psychoactive cannabidiol (CBD) and psychoactive tetrahydrocannabinol (THC), are defined as immunomodulators.9 We test whether two sources of stem cells, primed with CBD or THC, would demonstrate improved regenerative abilities. Human adipose-derived stem cells (ASCs) and bone marrowderived stem cells (BMDSCs), not obtained from the same individual, were treated with low (300 nM) or high (3 ?M) concentration CBD. Porcine ASCs and BMDSCs were isolated from a single pig, and treated with either low or high concentrations of CBD or THC. Transwell migration and MTT proliferation assays were performed on the human ASCs and BMDSCs. Also, transwell migration assay was performed on the porcine ASCs and BMDSCs. Finally, a wound healing scratch assay in porcine primary fibroblasts (PFs) was performed, co-cultured with the cannabinoid-treated ASCs. CBD priming at low concentration induces migration by 180% (P < .01) in porcine ASCs, and by only 93% (P < .02) in porcine BMDSCs. In porcine stem cells, THC priming at low concentration induces migration by 91.6% (P < .01) in ASCs but by only 44.3% (P < .03) in BMDSCs. Compared to PFs co-cultured with untreated ASCs, PFs co-cultured with low CBD-primed ASCs had 75% faster wound closure at 18 hours (P < .01). CBD and THC priming of ASCs and BMDSCs, particularly at lower doses, enhances a number of regenerative parameters, suggesting that these major marijuana components may improve stem cell-based therapies. Significance of the study: Our study demonstrates that cannabinoids can enhance the regenerative capacity of two major sources of stem cells, adipose- and bone marrow-derived, from human and porcine donors. Stem cell isolation and expansion is invasive, costly and time consuming. Stem cells with improved regenerative properties may be effective in the treatment of acute or chronic wounds. This is the first study to compare the priming potential of two sources of stem cells from the same animal, with the same genetic and epigenetic profile, as well as the first to prime with THC.

Role of the Cannabinoid System in Pain Control and Therapeutic Implications for the Management of Acute and Chronic Pain Episodes.
Manzanares, J., Julian, M., & Carrascosa, A.
Current Neuropharmacology, 4(3), 239–257. (2006).
doi:10.2174/157015906778019527
Cannabis extracts and synthetic cannabinoids are still widely considered illegal substances. Preclinical and clinical studies have suggested that they may result useful to treat diverse diseases, including those related with acute or chronic pain. The discovery of cannabinoid receptors, their endogenous ligands, and the machinery for the synthesis, transport, and degradation of these retrograde messengers, has equipped us with neurochemical tools for novel drug design. Agonist-activated cannabinoid receptors, modulate nociceptive thresholds, inhibit release of pro-inflammatory molecules, and display synergistic effects with other systems that influence analgesia, especially the endogenous opioid system. Cannabinoid receptor agonists have shown therapeutic value against inflammatory and neuropathic pains,
conditions that are often refractory to therapy. Although the psychoactive effects of these substances have limited clinical progress to study cannabinoid actions in pain mechanisms, preclinical research is progressing rapidly. For example, CB1-mediated suppression of mast cell activation responses, CB2-mediated indirect stimulation of opioid receptors located in primary afferent pathways, and the discovery of inhibitors for either the transporters or the enzymes degrading endocannabinoids, are recent findings that suggest new therapeutic approaches to avoid central nervous system side effects. In this review, we will examine promising indications of cannabinoid receptor agonists to alleviate acute and chronic pain episodes. Recently, Cannabis sativa extracts, containing known doses of tetrahydrocannabinol and cannabidiol, have granted approval in Canada for the relief of neuropathic pain in multiple sclerosis. Further double-blind placebo-controlled clinical trials are needed to evaluate the potential therapeutic effectiveness of various cannabinoid agonists-based medications for controlling different types of pain.

Role of the Endocannabinoid System in Depression: from Preclinical to Clinical Evidence
Vincenzo Micale, Katarina Tabiova, Jana Kucerova and Filippo Drago
in book: Cannabinoid Modulation of Emotion, Memory, and Motivation DOI: 10.1007/978-1-4939-2294-9_5 https://www.researchgate.net/publica...f_the_Endocann abinoid_System_in_Depression_f rom_Preclinical_to_Clinical_Ev idence?enrichId=rgreq-dd7385c81288a56dfb6d7380b68182 49-XXX&enrichSource=Y292ZXJQYWdlO zI3OTgzOTczNztBUzoyNDg0OTIwNjk yMjQ0NDhAMTQzNjI1NjUzNDgzNA%3D %3D&el=1_x_3&_esc=publicationC overPdf
The endogenous cannabinoid system (ECS) works as pro-homeostatic and pleiotropic signaling system activated in a time- and tissue-specific way during physiological conditions, which include cognitive, emotional and motivational processes. It is composed of two G protein-coupled receptors (the cannabinoid receptors types 1 and 2 [CB1 and CB2] for marijuana’s psychoactive ingredient Δ9-tetrahydrocannabinol [Δ9-THC]), their endogenous small lipid ligands (anandamide [AEA] and 2-arachidonoylglycerol [2-AG], also known as endocannabinoids), and the proteins for endocannabinoid biosynthesis and deactivation. Data from preclinical and clinical studies have reported that a hypofunction of the endocannabinoid signaling could induce a depressive-like phenotype; consequently, enhancement of endocannabinoid signaling could be a novel therapeutic avenue for the treatment of depression. To this aim there have been proposed cannabinoid receptor agonists or synthetic molecules that inhibit endocannabinoid degradation. The latter ones do not induce the psychotropic side effects by direct CB1 receptor activation, but rather elicit antidepressant-like effects by enhancing the monoaminergic neurotransmission, promoting hippocampal neurogenesis and normalizing the hyperactivity of hypothalamic-pituitary-adrenal axis, similarly as the standard antidepressants. The dysfunction of elements belonging to the ECS and the possible therapeutic use of endocannabinoid deactivation inhibitors and phytocannabinoids in depression is discussed in this chapter .

S-Adenosyl-L-Methionine (SAMe), Cannabidiol (CBD), and Kratom in Psychiatric Disorders: Clinical and Mechanistic Considerations.
Taylor Levine, M., Gao, J., Satyanarayanan, S. K., Berman, S., Rogers, J. T., & Mischoulon, D.
Brain, Behavior, and Immunity. (2019).
DOI:10.1016/j.bbi.2019.07.013
Given the limitations of prescription antidepressants, many individuals have turned to natural remedies for the management of their mood disorders. We review three selected natural remedies that may be of potential use as treatments for depressive disorders and other psychiatric or neurological conditions. The best studied and best supported of these three remedies is S-adenosyl-L-methionine (SAMe), a methyldonor with a wide range of physiological functions in the human organism. With the increasing legalization of cannabis-related products, cannabidiol (CBD) has gained popularity for various potential indications and has even obtained approval in the United States and Canada for certain neurological conditions. Kratom, while potentially useful for certain individuals
with psychiatric disorders, is perhaps the most controversial of the three remedies, in view of its greater potential for abuse and dependence. For each remedy, we will review indications, doses and delivery systems, potential anti-inflammatory and immunomodulatory action, adverse effects, and will provide recommendations for clinicians who may be considering prescribing these remedies in their practice.

Safety and Efficacy of Lenabasum, a Cannabinoid Receptor Type 2 Agonist, in Dermatomyositis Patients with Refractory Skin Disease A Randomized Clinical Trial

Victoria P Werth, Emily Hejazi, Sandra M Pena, Jessica Haber, Majid Zeidi, Nithin Reddy, Joyce Okawa, Rui Feng, Muhammad M Bashir, Kirubel Gebre, Arvin S Jadoo, Josef Symon S Concha, Nancy Dgetluck, Scott Constantine, Barbara White
J Invest Dermatol 2022 Apr 28;S0022-202X(22)
DOI: 10.1016/j.jid.2022.03.029
https://pubmed.ncbi.nlm.nih.gov/35490744/
Background: Treatment options are limited for skin disease in dermatomyositis (DM). Lenabasum is a cannabinoid receptor type 2 agonist that triggers resolution of inflammation.
Objective: Evaluate the safety and efficacy of lenabasum in patients with refractory cutaneous DM.
Design: This study was a single-center, double-blind, randomized, placebo-controlled Phase 2 study conducted from July 2015 to August 2017.
Population: Subjects ≥ 18 years of age with at least moderately active DM skin activity by Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) activity ≥ 14 and failure or intolerance to hydroxychloroquine.
Intervention: Participants received lenabasum 20 mg daily for 28 days, then 20 mg BID for 56 days, or placebo.
Main outcomes and measures: The primary outcome was change in CDASI activity. Safety and other secondary efficacy assessments were performed to Day 113.
Results: 22 subjects were randomized to lenabasum (n=11) or placebo (n=11). No serious or severe adverse events (AEs) were related to lenabasum, and no participants discontinued the study. The adjusted least squares mean for CDASI activity decreased more for lenabasum, and the difference was significant at Day 113 (least squares mean [SE] difference -6.5 [3.1], p = 0.038). Numerically greater improvements were seen in multiple secondary efficacy outcomes and biomarkers with lenabasum.
Conclusion: Lenabasum treatment was well tolerated and was associated with greater improvement in CDASI activity and multiple efficacy outcomes.
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Safety and Efficacy of Medical Cannabis in Autism Spectrum Disorder Compared with Commonly Used Medications
Richard Holdman, Daniel Vigil, Kelsey Robinson, Puja Shah, and Alexandra Elyse Contreras
DOI: 10.1089/can.2020.0154
https://www.liebertpub.com/doi/10.1089/can.2020.0154
https://www.researchgate.net/publica..._and_Efficacy_ of_Cannabis_in_Autism_Spectrum _Disorder
Objective: The objective of this study was to evaluate the safety and efficacy of medications commonly used in autism spectrum disorder (ASD) and compare this to what current research has shown regarding medical cannabis use in this population.
Methods: Searches were performed to collect information surrounding currently used medications and their safety and efficacy profiles, biologic plausibility of cannabis use for symptoms of ASD, and studies detailing cannabis' safety and efficacy profile for use in the ASD population. Results were used to compare medications to cannabis as a proposed treatment.
Results: The heterogeneity of ASD produces great difficulties in finding appropriate treatment, leading to many medication changes or treatment trials throughout a patient's life. Commonly prescribed medications display varying levels of efficacy, safety, and tolerability between patients and symptoms targeted. Some of the most common side effects cited are also considered the most troubling symptoms associated with ASD; aggression, anxiety, irritability, and a negative effect on cognition, leading many patients to discontinue use as the side effects outweigh benefits. Recent case reports and retrospective studies have displayed the potential efficacy, safety, and tolerability of cannabidiol (CBD)-rich medical cannabis use for treating both core symptoms of ASD and many comorbid symptoms such as irritability and sleep problems. Studies have also identified circulating endocannabinoids as a possible biomarker for ASD, providing another possible method of diagnosis.
Conclusions: Currently, there are no approved medications for the core symptoms of ASD and only two medications Food and Drug Administration approved for associated irritability. Prescribed medications for symptoms associated with ASD display varying levels of efficacy, safety, and tolerability among the heterogeneous ASD population. At the time of this study there are no published placebo-controlled trials of medical cannabis for ASD and the observational studies have limitations. CBD-rich medical cannabis seems to be an effective, tolerable, and relatively safe option for many symptoms associated with ASD, however, the long-term safety is unknown at this time.
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Safety and Efficacy of Medicinal Cannabis in the Treatment of Fibromyalgia: A Systematic Review.
Kurlyandchik, I., Tiralongo, E., & Schloss, J.
The Journal of Alternative and Complementary Medicine. (2020). doi:10.1089/acm.2020.0331
Background: Fibromyalgia (FMS) is a complex condition that is characterized by various pain syndromes and fatigue, among other symptoms experienced. Current medical treatment of FMS involves both pharmacological and nonpharmacological approaches, but often with ineffective outcomes. Medicinal cannabis has the potential to be a therapeutic option for patients with FMS due to the positive research in chronic pain management. In addition, it has been found to have fewer adverse effects compared with currently available pain medications. This literature review aims at answering whether medicinal cannabis is reported to be safe and effective for the treatment of pain and symptomology experienced by people with FMS. Methods: A systematic review was conducted on human trials utilizing cannabis in FMS. MEDLINE, Embase, CINAHL, AMED, Scopus, and Cochrane CENTRAL were used for databases search, and mesh terms were used for cannabis and FMS. The search was limited to studies conducted from 2000 to 2020. Results: From the 181 citations identified, 10 studies were included after title, abstract, and full text screening occurred. A total of 1136 of patients (intervention n = 945, control n = 108, crossover n = 83) participated in the 10 studies ranging from 9 to 383 patients (mean = 114, median = 36). Of these studies, there were three randomized controlled trials, six observational studies, and one study that compared the management of chronic pain patients with FMS patients. Cannabis was found to be safe and well tolerated in FMS. The main adverse events identified included feeling ‘‘high,’’ dizziness/vertigo, dry mouth, cough, red eyes, and drowsiness with no serious adverse events reported. Conclusions: This literature review identified that medical cannabis may be beneficial for some people with FMS. Further studies are required to confirm its efficacy, what type of cannabis is the most effective form to use, and what assessment tools need to be utilized to understand how to quantify clinical outcomes.

Safety and pharmacokinetics of oral delta-9-tetrahydrocannabinol in healthy older subjects: A randomized controlled trial.
Ahmed, A. I. A., van den Elsen, G. A. H., Colbers, A., van der Marck, M. A., Burger, D. M., Feuth, T. B., … Kramers, C.
European Neuropsychopharmacology, 24(9), 1475–1482.(2014).
doi:10.1016/j.euroneuro.2014.06.00
There is a great concern about the safety of THC-based drugs in older people (Z65 years), as most of THC-trials did not include such group. In this phase 1, randomized, double-blind, double-dummy, placebo-controlled, cross-over trial, we evaluated the safety and pharmacokinetics of three oral doses of Namisols, a novel THC in tablet form, in older subjects. Twelve healthy older subjects (6 male; mean age 7275 years) randomly received a single oral dose of 3 mg, 5 mg, or 6.5 mg of THC or matching placebo, in a crossover manner, on each intervention day. The data for 11 subjects were included in the analysis. The data of 1 subject were excluded due to non-compliance to study medication. THC was safe and well tolerated. The most frequently reported adverse events (AEs) were drowsiness (27%) and dry mouth (11%). Subjects reported more AEs with THC 6.5 mg than with 3 mg (p=0.048), 5 mg (p=0.034) and placebo (p=0.013). There was a wide inter-individual variability in plasma concentrations of THC. Subjects for whom the Cmax fell within the sampling period (over 2 h), Cmax was 1.42– 4.57 ng/mL and Tmax was 67–92 min. The AUC0–2 h (n=11) was 1.67–3.51 ng/mL. Overall, the pharmacodynamic effects of THC were smaller than effects previously reported in young adults.

Safety and tolerability of escalating cannabinoid doses in healthy cats
Justyna E K, Lina J Paulionis, Graham ML Eglit, Dana M Vaughn
Journal of Feline Medicine and Surgery (March 2021)
DOI: 10.1177/1098612X211004215
Objectives: The aim of this study was to determine the safety and tolerability of escalating doses of orally delivered cannabis oils predominant in cannabidiol (CBD), tetrahydrocannabinol (THC), or both CBD and THC in healthy cats.
Methods:In this placebo-controlled, blinded study, 20 healthy adult cats were randomized to one of five treatment groups (n = 4 per group): two placebo groups (sunflower oil [SF] or medium-chain triglyceride oil [MCT]), or three plant-derived cannabinoid oil groups (CBD in MCT, THC in MCT or CBD/THC [1.5:1] in SF). Up to 11 escalating doses of each formulation were delivered orally via syringe to fasted subjects, with at least 3 days separating doses. Safety and tolerability were determined from clinical observations, complete blood counts (CBCs) and clinical chemistry. Plasma cannabinoids (CBD, THC) and metabolites (7-COOH-CBD, 11-OH-THC) were assessed.
Results:Titration to maximum doses of 30.5 mg/kg CBD (CBD oil), 41.5 mg/kg THC (THC oil) or 13.0:8.4 mg/kg CBD:THC (CBD/THC oil) was safely achieved in all subjects. All observed adverse events (AEs) were mild, transient and resolved without medical intervention. Gastrointestinal AEs were more common with formulations containing MCT. Constitutional (lethargy, hypothermia), neurologic (ataxia) and ocular (protrusion membrana nictitans) AEs were more common with oils containing THC (CBD/THC and THC oils). There were no clinically significant changes in CBC or clinical chemistry across treatment groups. Higher plasma levels of the cannabinoids and their metabolites following administration of the CBD/THC combination product are suggestive of a pharmacokinetic interaction.
Conclusions and relevance:This is the first feline study to explore the safety and tolerability of CBD and THC, alone and in combination, in a controlled research setting. These findings will inform veterinarians of the safety profile of cannabinoids, particularly when considering the potential therapeutic use of CBD in cats or recognizing clinical signs associated with accidental exposure to THC-containing products.
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Safety Assessment of a Hemp Extract using Genotoxicity and Oral Repeat-Dose Toxicity Studies in Sprague-Dawley Rats.
Dziwenka, M., Coppock, R., Alexander, M., Palumbo, E., Ramirez, C., & Lermer, S.
Toxicology Reports, 7, 376–385.(2020).
doi:10.1016/j.toxrep.2020.02.014
Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p < 0.05) in adrenal-to-body weight ratios was observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not correlate with any of the pathology findings. The NOAEL for the test article is 800 mg/kg bw/day for female and 400 mg/kg bw/day for male Sprague Dawley rats.

Safety, Pharmacokinetics and Pharmacodynamics of Spectrum Yellow Oil in Healthy Participants
Erica N. Peters, Irina Mosesova, Laura MacNair, Ryan Vandrey, M. Hunter Land, Mark A. Ware, Cynthia Turcotte and Marcel O. Bonn-Miller
Journal of Analytical Toxicology, 2022, 46, 393–407
DOI: 10.1093/jat/bkab026
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9021973/pdf/bkab026.pdf
Due to a lack of published pharmacokinetic (PK) and/or pharmacodynamic (PD) data, decision-making surrounding appropriate dosing of cannabis used for medical purposes is limited. This multiple-dose study evaluated the safety, tolerability, PK and PD of Spectrum Yellow oil [20 mg/mL cannabidiol (CBD)/<1 mg/mL ∆9 -tetrahydrocannabinol (THC)]. Participants (n = 43) were randomized to one of five groups: 120 mg CBD and 5.4 mg THC daily, 240 mg CBD and 10.8 mg THC daily, 360 mg CBD and 16.2 mg THC daily, 480 mg CBD and 21.6 mg THC daily or placebo. Study medication was administered every 12 h for 7 consecutive days. Treatment-emergent adverse events (TEAEs); plasma and urine concentrations of THC, CBD and metabolites; and self-reported subjective effects were collected. Nearly all TEAEs (44/45) were of mild or moderate severity; none was serious. The highest incidence of TEAEs (67%) was in the two higher-dose treatment groups. The highest number of TEAEs (17/45) occurred on the first treatment day. Steady-state plasma CBD concentrations were reached by Day 7. On Day 7, CBD exposure showed dose proportionality (AUC0–t slope = 1.03 [0.70, 1.36], Cmax slope = 0.92 [0.53, 1.31]). Most plasma THC concentrations were below the limit of quantification. Across Days 1 and 7, there were no consistent differences in subjective effects between placebo and active study medication. A prudent approach to improve tolerability with Spectrum Yellow oil might involve initial doses no higher than 240 mg total CBD and 10.8 mg total THC daily in divided doses, with titration upward over time as needed based on tolerability

Safety, Tolerability, and Pharmacokinetics of FAAH Inhibitor BIA 10-2474: A Double-Blind, Randomized, Placebo-Controlled
Study in Healthy Volunteers

José-Francisco Rocha, Ana Santos, Helena Gama, Paul Moser, Amílcar Falcão, Peter Pressman, A. Wallace Hayes and Patricio Soares-da-Silva CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 111 NUMBER 2 | February 2022 doi:10.1002/cpt.2290 https://ascpt.onlinelibrary.wiley.co.. ..1002/cpt.2290
This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of BIA 10-2474, a fatty acid amide hydrolase (FAAH) inhibitor, after first administration to healthy male and female participants. Participants (n = 116) were recruited into this phase I, double-blind, randomized, placebo-controlled, single ascending dose and multiple ascending dose (10-day) study. The primary outcome was the safety and tolerability of BIA 10-2474. Secondary outcomes were pharmacokinetics of BIA 10-2474
and pharmacodynamics, considering plasma concentrations of anandamide and three other fatty acid amides (FAAs) and leukocyte FAAH activity. Single oral doses of 0.25–100 mg and repeated oral doses of 2.5–50 mg were evaluated. BIA 10-2474 was well tolerated up to 100 mg as a single dose and up to 20 mg once daily for 10 days. In the cohort receiving repeated administrations of 50 mg, there were central nervous system adverse events in five of six participants, one with fatal outcome, which led to early termination of the study. BIA 10-2474 showed a linear relationship between dose and area under plasma concentration-time
curve (AUC) across the entire dose range and reached steady state within 5–6 days of administration, with an accumulation ratio, based on AUC0–24h, of <2 on Day 10. BIA 10-2474 was rapidly absorbed with a mean terminal elimination half-life of 8–10 hours (Day 10). BIA 10-2474 caused reversible, dose-related increases in plasma FAAs. In conclusion, we propose that these data, as well as the additional data generated since the clinical trial was stopped, do not provide a complete mechanistic explanation for the tragic fatality.

Scandalous decisions: explaining shifts in UK medicinal cannabis policy
Mark Monaghan, Emma Wincup, Ian Hamilton
Addiction 06 January 2021
doi: 10.1111/add.15350.
Background and aims
Opening up access to scheduled drugs such as cannabis in the United Kingdom rarely happens, yet on 1 November 2018 the United Kingdom changed the law to allow cannabis?derived products to be prescribed for medicinal purposes, albeit in tightly controlled circumstances. This followed substantial media interest in the cases of two children with epilepsy. This article focuses upon the role of scandal in bringing about legislative change.
Methods
We used political science and social policy theories (punctuated equilibrium theory and scandal theory) to guide a qualitative content analysis of media articles published in 2018 in UK national newspapers. We focused in particular on the 6?month period prior to the policy change.
Results
The concentrated attention by the media given to the suffering of children with epilepsy appears to have prompted the rapid change in policy by the UK government. A variety of strategies were used to develop a highly emotive response to garner support for reform. Media stories emphasized the injustice of two extremely sick children being unable to access the medicine they apparently needed to enable them to have a ‘normal’ childhood. Three groups of ‘claim?makers’ were identified as important in influencing public opinion: families, high?profile individuals and campaigning groups.
Conclusions
The case of medicinal cannabis in the United Kingdom suggests that policy reform can occur when a scandal is successfully manufactured. We must be cautious, however, about over?emphasizing the role of scandal as a driver of policy change in this context: only a limited set of circumstances will permit a prescription for cannabis?based medicine to be issued in the United Kingdom.

Schizotypy but not Cannabis Use Modestly Predicts Psychotogenic Experiences: A Cross-Sectional Study Using the Oxford-Liverpool Inventory of Feelings and Experiences (O-LIFE)
Nicola D. Airey, Richard Hammersley, and Marie Reid
Hindawi Journal of Addiction Volume 2020, Article ID 5961275, 7 pages
DOI: 10.1155/2020/5961275
Objective. Cannabis use predicts psychosis in longitudinal studies, but it is difficult to infer causation. Some precursor variables predict both, including childhood trauma and adversity. Additionally, some of the desired effects of cannabis use resemble the symptoms of psychosis. It would be preferable to assess psychotomimetic or “unusual” experiences that include psychotic symptoms but without assuming pathology. Finally, it is possible that similar people are prone to psychosis and drawn to cannabis use, perhaps, because they are sensitive or attracted to unusual experiences. Schizotypy provides a trait measure of proneness to unusual experiences. +e study aimed to examine cross-sectionally relationships between cannabis use, schizotypy, and unusual experiences whilst controlling for current trauma symptoms. Method. A volunteer online sample (n ? 129, 64% women, predominantly students) who had used cannabis at least once was recruited. People who reported active effects of past trauma were excluded with a brief primary care posttraumatic stress disorder screen. Participants completed the Oxford-Liverpool Inventory of Feelings and Experience, the Cognitive Failures Questionnaire, and measures of substance use and sociodemographics. Results. +e majority of respondents recounted unusual experiences after cannabis use, and many of these might have been considered symptoms of psychosis if they had received medical attention. In regression analysis, the only predictor of the unusual experiences scale of O-LIFE was schizotypy (measured by the remaining subscales; 4% of variance). +ere were no correlations between cannabis use frequency and schizotypy or unusual experiences. Conclusions. +ese findings suggest that, after controlling for schizotypy and excluding people who are actively experiencing the effects of past trauma, frequency of cannabis use does not predict unusual experiences. However, individuals with schizotypal personality traits may have more unusual experiences when using cannabis.

Scientific Prospects for Cannabis-Microbiome Research to Ensure Quality and Safety of Products
Vladimir Vujanovic, Darren R. Korber, Silva Vujanovic, Josko Vujanovic and Suha Jabaji Microorganisms 2020, 8, 290; doi:10.3390/microorganisms8020290
https://pdfs.semanticscholar.org/763...879.1582090724
Cannabis legalization has occurred in several countries worldwide. Along with steadily growing research in Cannabis healthcare science, there is an increasing interest for scientific-based knowledge in plant microbiology and food science, with work connecting the plant microbiome and plant health to product quality across the value chain of cannabis. This review paper provides an overview of the state of knowledge and challenges in Cannabis science, and thereby identifies critical risk management and safety issues in order to capitalize on innovations while ensuring product quality control. It highlights scientific gap areas to steer future research, with an emphasis on plant-microbiome sciences committed to using cutting-edge technologies for more efficient Cannabis production and high-quality products intended for recreational, pharmaceutical, and medicinal use

Scoping Review and Meta-Analysis Suggests that Cannabis Use May Reduce Cancer Risk in the United States
Thomas M. Clark
Cannabis and Cannabinoid Research Volume X, Number X, 2020
DOI: 10.1089/can.2019.0095
https://www.liebertpub.com/doi/pdfpl.../can.2019.0095
Introduction: Cannabis smoke contains carcinogens similar to tobacco, in addition to compounds with antitumor activity. Cannabis use reduces the risk of obesity and cannabinoids inhibit chronic inflammation, known causes of cancer. The net effect of Cannabis use on cancer risk is not known.
Objective: To examine the association between Cannabis use and cancer risk in the United States.
Methods: Identify and analyze published data on cancer risk in Cannabis users.
Results: A total of 55 data points, consisting of risk ratios of cancer in Cannabis users and nonusers, were identified from 34 studies. Of these, 5 did not contain data essential for inclusion in the meta-analysis. The remaining data showed a nonsignificant trend to an association with reduced risk (relative risk [RR] = 0.90, p > 0.06, N = 50) although heterogeneity is high (I2 = 72.4%). Removal of data with high risk of selection bias (defined as those from North Africa and those that failed to adjust for tobacco) and data with high risk of performance bias (defined as those with fewer than 20 cases or controls among Cannabis users) resulted in an RR < 1.0 (RR = 0.86, p < 0.017, N = 24) and large effect size (Hedges g = 0.66), but did not decrease heterogeneity (I2 = 74.9). Of all cancer sites, only testicular cancer showed an RR value > 1, although this was not significant and had a negligible effect size (RR = 1.12, p = 0.3, Hedges g = 0.02). Following removal of testicular cancers the remaining data showed a decrease in risk (RR = 0.87, p < 0.025, N = 41). Cancers of the head and neck showed a negative association with cancer risk (RR = 0.83, p < 0.05), with a large effect size (Hedges g = 0.55), but high heterogeneity (I2 = 79.2%). RR did not reach statistical significance in the remaining cancer site categories (lung, testicular, obesity-associated, other). The data are consistent with a negative association between Cannabis use and nontesticular cancer, but there is low confidence in this result due to high heterogeneity and a paucity of data for many cancer types.

Secondary Metabolites Profiled in Cannabis Inflorescences, Leaves, Stem Barks, and Roots for Medicinal Purposes.
Jin, D., Dai, K., Xie, Z., & Chen, J.
Scientific Reports, 10(1).(2020).
doi:10.1038/s41598-020-60172-6
Cannabis research has historically focused on the most prevalent cannabinoids. However, extracts with a broad spectrum of secondary metabolites may have increased efcacy and decreased adverse efects compared to cannabinoids in isolation. Cannabis’s complexity contributes to the length and breadth of its historical usage, including the individual application of the leaves, stem barks, and roots, for which modern research has not fully developed its therapeutic potential. This study is the frst attempt to profle secondary metabolites groups in individual plant parts comprehensively. We profled 14 cannabinoids, 47 terpenoids (29 monoterpenoids, 15 sesquiterpenoids, and 3 triterpenoids), 3 sterols, and 7 favonoids in cannabis fowers, leaves, stem barks, and roots in three chemovars available. Cannabis inforescence was characterized by cannabinoids (15.77–20.37%), terpenoids (1.28–2.14%), and favonoids (0.07–0.14%); the leaf by cannabinoids (1.10–2.10%), terpenoids (0.13–0.28%), and favonoids (0.34–0.44%); stem barks by sterols (0.07–0.08%) and triterpenoids (0.05–0.15%); roots by sterols (0.06–0.09%) and triterpenoids (0.13–0.24%). This comprehensive profle of bioactive compounds can form a baseline of reference values useful for research and clinical studies to understand the “entourage efect” of cannabis as a whole, and also to rediscover therapeutic potential for each part of cannabis from their traditional use by applying modern scientifc methodologies.

Self-Assembly System Based on Cyclodextrin for Targeted Delivery of Cannabidiol
Panyong Zhu, Pin Lv, Yazhou Zhang, Rongqiang Liao, Jing Liu, Rong Guo, Xuan Chen, Xiali Liao, Chuanzhu Gao, Kun Zhang, Ming Yang and Bo Yang
Front. Chem. November 2021 | Volume 9
doi: 10.3389/fchem.2021.754832
https://www.frontiersin.org/articles...21.754832/full
Cannabidiol (CBD) is one specifi c kind of the cannabinoid in Cannabis sativa L with a wide range of pharmacological activities. However, the poor water solubility and specifi city of CBD limits its application in pharmaceutical fi eld. For solving these problems, in this work, we successfully prepared a targeted carrier by grafting biotin (BIO) onto ethylenediamine-β -Cyclodextrin (EN-CD) in a single step to generate a functionalized supramolecule, named BIO-CD. Subsequently, an amantadine-conjugated cannabinoids (AD-CBD) was prepared and self-assembled with the BIO-CD. A series of methods were used to characterize the inclusion behavior and physicochemical properties of AD-CBD and BIO-CD. The results showed that AD-CBD entered the cavity of BIO-CD and formed a 1:1 host-guest inclusion complex. MTT assay and confocal laser scanning microscopy (CLSM) revealed that the targeting effect and anticancer activity of AD-CBD/BIO-CD inclusion complex against three human cancer cell lines were higher than BIO-CD, ADCBD and free CBD. Moreover, the inclusion complex could release drugs under weakly acidic conditions. These results demonstrated that AD-CBD/BIO-CD inclusion complex possess excellent targeted and anticancer activity, which is hopeful to be applied in clinic as a new therapeutic approach.

Self-initiated use of topical cannabidiol oil for epidermolysis bullosa.
Malcolm P. Chelliah | Zachary Zinn | Phoung Khuu ,Joyce M. C. Teng
Pediatric Dermatology. 2018;1–4.
DOI: 10.1111/pde.13545
Epidermolysis bullosa is a rare blistering skin disorder that is challenging to
manage because skin fragility and repeated wound healing cause itching, pain,
limited mobility, and recurrent infections. Cannabidiol, an active cannabinoid found in cannabis, is postulated to have antiinflammatory and analgesic effects. We report 3 cases of self-initiated topical cannabidiol use in patients with epidermolysis bullosa in an observational study. One patient was weaned completely off oral opioid analgesics. All 3 reported faster wound healing, less blistering, and amelioration of pain with cannabidiol use. Although these results demonstrate promise, further randomized, double-blind clinical trials are necessary to provide scientific evidence of our observed benefits of cannabidiol for the treatment of epidermolysis bullosa.

Self-reported cannabis use and biomarkers of inflammation among adults in the United States.
Okafor, C. N., Li, M., & Paltzer, J.
Brain, Behavior, & Immunity - Health, 7, 100109. (2020).
doi:10.1016/j.bbih.2020.100109
Background: Cannabis is among the most frequently used substance in United States (U.S.). Studies evaluating the association between cannabis use and inflammation in humans have been few and have not explored potential sex-dependent effects. Objective: To examine the relationship between self-reported cannabis use and high-sensitivity C-reactive protein (hsCRP), Interleukin 6 (IL-6) and fibrinogen. Methods: We used Wave 1 of the Population Assessment of Tobacco and Health (PATH) – a nationally representative sample of adults in the U.S. Weighted linear regression models were used to determine associations of self-reported cannabis use with natural log-transformed hs-CRP, IL-6 and fibrinogen, adjusting for sociodemographic and psychosocial factors. Results: Self-reported cannabis use, particularly cannabis use within the past 30 days, was associated with lower levels of each biomarker of systemic inflammation, although findings were imprecise. Specifically, in multivariable models, the associations between respondents who self-reported cannabis use in the past 30 days compared to never use was imprecise for hs-CRP (? ¼ 0.15, 95% confidence interval (CI): 0.32, 0.00), IL-6 (? ¼ 0.02, 95% CI: 0.10, 0.05) and fibrinogen (? ¼ 0.01, 95% CI: 0.04, 0.02). We did not find that these associations differed significantly by sex. Discussions: Data from this nationally representative study suggest potential anti-inflammatory effects of recent cannabis use. Additional studies that biologically measure the THC and CBD concentrations of the cannabis used and employ prospective and or experimental study designs investigate cannabis and inflammation associations are needed.

Semen endocannabinoids are correlated to sperm quality in a cohort of 200 young Swiss men.
Zufferey, F., Donzé, N., Rahban, R., Senn, A., Stettler, E., Rudaz, S., … Rossier, M. F.
Andrology. (2020).
doi:10.1111/andr.12785
A role for endocannabinoids in the male and female reproductive systems has been highlighted during the recent decades. Some of these compounds bind the cannabinoid CB1 receptor, which is abundantly expressed in the central nervous system but also present in the reproductive system, while others act as “entourage compounds” modulators.
Objectives
The present study aimed at evaluating the relationship between sperm quality andendocannabinoid profiles in a cohort of 200 young Swiss men and whether the presence of specific xenobiotics could influence these profiles.
Design & Methods
Semen analysis was performed according to WHO guidelines. Endocannabinoid profiles in blood and semen, as well as bisphenol A and S in urine, were determined by LC-MSMS methods. The presence of selected drugs was tested in urine by immunological screening, and urinary
tetrahydrocannabinol (THC) metabolites were quantified by GC-MS.
Results
Anandamide concentrations in seminal fluid and oleoylethanolamide (OEA) concentrations in blood serum appeared inversely correlated with sperm motility, while semen palmytoylethanolamide (PEA) was positively linked to sperm concentration. Moreover, OEA and PEA in seminal fluid were associated with better sperm morphology. Interestingly, the concentrations of the same endocannabinoids measured in both blood and semen were not
correlated, and the presence of THC metabolites in some individuals was linked to lower concentrations of endocannabinoids.
Conclusions
In the context of the general decline of the sperm count observed within the male population, endocannabinoids in semen constitute a class of promising biochemical markers that open new perspectives as a complement for the usual evaluation of semen quality or for the toxicological screening of individuals’ exposure to putative endocrine disruptors.

Serum cannabidiol, tetrahydrocannabinol (THC), and their native acid derivatives after transdermal application of a low‐THC Cannabis sativa extract in beagles.
Hannon, M. B., Deabold, K. A., Talsma, B. N., Lyubimov, A., Iqbal, A., Zakharov, A., … Wakshlag, J. J.
Journal of Veterinary Pharmacology and Therapeutics. (2020).
doi:10.1111/jvp.12896
Cannabinoids hold promise for treating health problems related to inflammation and chronic pain in dogs, in particular cannabidiol (CBD), and its native acid derivative cannabidiolic acid (CBDA). Information regarding systemic delivery of cannabinoids through transdermal routes is sparse. The purpose of this study was to determine pharmacokinetics of transdermal administration of a low-THC Cannabis sativa extract in healthy dogs. Six purpose-bred research beagles were treated with a transdermal CBD-CBDA-rich extract, and serum concentrations of CBD, CBDA, tetrahydrocannabinol (THC), and its acid derivative tetrahydrocannabinolic acid (THCA) were examined prior to and at the end of weeks 1 and 2. A 4 mg/kg dose of total cannabinoids twice daily resulted in appx 10 ng/ml of CBD, 21–32 ng/ml of CBDA, trace amounts of THCA, and unquantifiable amounts of THC in serum at the end of weeks 1 and 2 of treatment. Results showed that CBDA and THCA were absorbed better systemically than CBD or THC.

Serum Endocannabinoid Content is Altered in Females with Depressive Disorders: A Preliminary Report.
Hill, M., Miller, G., Ho, W.-S., Gorzalka, B., & Hillard, C.
Pharmacopsychiatry, 41(2), 48–53. (2008).
doi:10.1055/s-2007-993211
Background: Preclinical research has suggested that the endocannabinoid system may be involved in the etiology and / or treatment of depression; however, there are no published studies examining circulating endocannabinoid content in patients with clinical depression.
Methods: This study examined the endocannabinoids (anandamide; AEA) and 2-arachidonylglycerol (2-AG) in serum from ambulatory, medication-free female patients diagnosed with minor or major depression, and in controls matched for demographic characteristics.
Results: Serum 2-AG content was significantly decreased in patients diagnosed with major depression, and this decrease was correlated significantly and negatively with duration of the depressive episode, such that 2-AG content was progressively lower the longer the depressive episode. While AEA was not associated with major depression per se , a strong negative correlation was found between serum AEA content and Hamilton ratings for cognitive and somatic anxiety, suggesting that AEA content may relate to the anxiety dimension of affective disorders. In subjects with minor depression, serum AEA
was signifi cantly elevated, with 2-AG content demonstrating a similar, but statistically insignificant trend.
Discussion: These are the fi rst clinical data to indicate that the endocannabinoid system may be disturbed in affective disease, and suggest that future research is required to determine the relevance of these changes with respect to disease manifestation and pharmacotherapy.

Sex differences in the acute effects of intravenous (IV) delta-9 tetrahydrocannabinol (THC)
Psychopharmacology April 2022 DOI: 10.1007/s00213-022-06135-3
Anahita Bassir Nia, Maria J. Orejarena, Leigh Flynn, Christina Luddy, Deepak Cyril D’Souza, Patrick D. Skosnik, Brian Pittman, Mohini Ranganathan
https://www.researchgate.net/public...travenous_IV_delta-9_tetrahydrocannabinol_THC
Background Cannabis is the most common illicit drug used in the USA and its use has been rising over the past decade, while the historical gap in rates of use between men and women has been decreasing. Sex differences in the effects of cannabinoids have been reported in animal models, but human studies are sparse and inconsistent. We investigated the sex differences in the acute subjective, psychotomimetic, cognitive, and physiological effects of intravenous (IV) delta-9 tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis. Methods Healthy male and female individuals, with limited exposure to cannabis, participated in a double blind, placebo-controlled study of intravenous (IV) placebo or THC at two doses (0.015 mg/kg and 0.03 mg/kg). Visual analog scale (VAS) was used to measure subjective effects, Psychotomimetic States Inventory (PSI) and the Clinician-Administered Dissociative Symptoms Scale (CADSS) were used to assess the psychotomimetic effects and perceptual alterations, respectively, and Rey Auditory Verbal Learning Task (RAVLT) was used to evaluate cognitive effects. Outcome variables were represented as the peak change from baseline for each variable, except RAVLT which was used only once per the test day after the subjective effects. Results A total of 42 individuals participated in this study. There were no significant differences between male and female participants in background characteristics. There was a significant main effect of sex on the VAS scores for THC-induced “High” (F1,38 = 4.27, p < 0.05) and a significant dose × sex interaction (F2,77 = 3.38, p < 0.05) with female participants having greater “High” scores than male participants at the lower THC dose (0.015 mg/kg). No other sex differences were observed in acute subjective, psychotomimetic, cognitive, or physiological effects of THC. Conclusion There were significant sex differences in subjective effects of feeling “High” at a lower dose of THC. However, there were no other sex-related differences in the subjective, physiological, or cognitive effects of THC.
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Sex Differences in the Association Between Cannabis Use and Diabetes Mellitus among U.S. Adults: The National Health and Nutritional Examination Survey, 2013–2018 Ayobami S. Ogunsola, Samuel Smith, Olatunji A. Eniola, Udeh C. Mercy, and Ibraheem M. Karaye Cannabis and Cannabinoid Research 2022 DOI: 10.1089/can.2021.0134 Background: Diabetesmellitus is an important public health problem in the United States, accounting for 87,647 deaths in 2019. This study aimed to assess the association between cannabis use and diabetes mellitus by sex among U.S. adults.
Methods: Data were abstracted from the National Health and Nutrition Examination Survey (NHANES) from 2013 through 2018. Cannabis use was estimated using exposure status and frequency of use. Diabetes mellitus was assessed based on physician diagnosis or laboratory results, per the American Diabetes Association guidelines. A multivariable survey logistic regression model was fitted to estimate adjusted odds ratios (aOR) and confidence intervals (95% CIs).
Results: A total of 15,062 participants were included in this study. The majority were female (n = 7845; 51.1%), > 40 years of age (n = 8564; 56.3%), non-Hispanic white (n = 4873; 61.5%), with at least a college-level education (n = 8239; 62.5%). Female participants who used cannabis heavily were less likely to be diagnosed with diabetes mellitus than female noncannabis users (aOR = 0.49; 95% CI: 0.30–0.81; aOR = 0.51; 95% CI: 0.31–0.84). However, no significant association was found for female adults who engaged in light use of cannabis (aOR = 0.98; 95% CI: 0.55–1.75; aOR = 1.01; 95% CI: 0.57–1.79). Among male adults, cannabis use, irrespective of the degree of exposure, was not significantly associated with diabetes mellitus (heavy users: aOR = 0.89; 95% CI = 0.56–1.41; light users: aOR = 0.53; 95% CI = 0.22–1.29).
Conclusions: Heavy cannabis use is inversely associated with diabetes mellitus in females but not males. Further studies are needed to explore the sex-based heterogeneity—and individual and contextual factors responsible—in the association between cannabis use and diabetes mellitus Find Pdf

Shifting the Paradigm on Cannabis Safety.
Bannigan, P., Evans, J. C., & Allen, C.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2020.0003
The global movement toward legalization of cannabis is resulting in an ever-increasing public perception that cannabis is safe. Cannabis is not the first drug to be available for nonmedical use, nor is it the first to have such an unfounded safety profile. The safety of long-term exposure to phytocannabinoids is misunderstood by, and under reported to, the general public. There is evidence to suggest that long-term use of recreational cannabis may be associated with an increased risk of undesirable side effects. This evidence warrants both appropriate caution from the general public and investment in further research by government and industry sectors that are profiting from the sale of these potent psychoactive agents. There is no doubt that these compounds have medical potential. However, in addition to the medical potential, we must also remain aware of the adverse health effects that are becoming synonymous with recreational cannabis use. This perspective highlights the privileged role that cannabis has as a perceived ‘‘safe drug’’ in society and summarizes some concerning side effects that are becoming associated with regular nonprescribed cannabis use

Short and Long-Term Effects of Cannabis on Symptoms of Post-Traumatic Stress Disorder.
LaFrance, E. M., Glodosky, N. C., Bonn-Miller, M., & Cuttler, C.
Journal of Affective Disorders.(2020).
doi:10.1016/j.jad.2020.05.132
Background: Many individuals use cannabis to manage symptoms of post-traumatic stress disorder (PTSD), and evidence indicates that the endocannabinoid system represents a viable target for treating these symptoms.
Method: Data from 404 medical cannabis users who selfidentified
as having PTSD were obtained from Strainprint®, a medical cannabis app that patients use to track changes in symptoms as a function of different strains and doses of cannabis across time. This sample collectively used the app 11,797 times over 31 months to track PTSD-related symptoms (intrusive thoughts, flashbacks, irritability, and/or anxiety) immediately before and after inhaling cannabis. Latent change score models were used to examine changes in symptom severity and predictors of these changes (gender, dose, cannabis constituents, time). Multilevel models were used to explore long-term consequences of repeatedly using cannabis to manage these symptoms.
Results: All symptoms were reduced by more than 50% immediately after cannabis use. Time predicted larger decreases in intrusions and irritability, with later cannabis use sessions predicting greater symptom relief than earlier sessions. Higher doses of cannabis predicted larger reductions in intrusions and anxiety, and dose used to treat anxiety increased over time. Baseline severity of all symptoms remained constant across time.
Limitations: The sample was self-selected, self-identified as having PTSD, and there was no placebo control group. Conclusions: Cannabis provides temporary relief from PTSD-related symptoms. However, it may not be an effective long-term remedy as baseline symptoms were maintained over time and dose used for anxiety increased over time, which is indicative of development of tolerance.

Short-term effects of cannabis consumption on cognitive performance in medical cannabis Patients
Phillip Olla, Nicholas Rykulski, Jessica L. Hurtubise, Stephen Bartol, Rachel Foote, Laura Cutler, Kaitlyn Abeare, Nora McVinnie, Alana G. Sabelli, Maurissa Hastings & Laszlo A. Erdodi
Applied Neuropsychology: Adult, (2019)
DOI: 10.1080/23279095.2019.1681424
This observational study examined the acute cognitive effects of cannabis. We hypothesized that cognitive performance would be negatively affected by acute cannabis intoxication. Twenty-two medical cannabis patients from Southwestern Ontario completed the study. The majority (n.13) were male. Mean age was 36.0 years, and mean level of education was 13.7 years. Participants were administered the same brief neurocognitive battery three times during a six-hour period: at baseline (“Baseline”), once after they consumed a 20% THC cannabis product (“THC”), and once again several hours later (“Recovery”). The average self-reported level of cannabis intoxication prior to the second assessment (i.e., during THC) was 5.1 out of 10. Contrary to expectations, performance on neuropsychological tests remained stable or even improved during the acute intoxication stage (THC; d: .49_.65, medium effect), and continued to increase during Recovery (d: .45_.77, medium-large effect). Interestingly, the failure rate on performance validity indicators increased during THC. Contrary to our hypothesis, there was no psychometric evidence for a decline in cognitive ability following THC intoxication. There are several possible explanations for this finding but, in the absence of a control group, no definitive conclusion can be reached at this time.

Should donors who have used marijuana be considered candidates for living kidney donation?
Ruckle, D., Keheila, M., West, B., Baron, P., Villicana, R., Mattison, B., … Baldwin, D. D.
Clinical Kidney Journal. CKJ (2018). 1–6
doi:10.1093/ckj/sfy107
Background. The use of marijuana in the USA has been steadily increasing over the last 10 years. This study is the first to investigate the effect of marijuana use by live kidney donors upon outcomes in both donors and recipients. Methods. Living kidney donor transplants performed between January 2000 and May 2016 in a single academic institution were retrospectively reviewed. Donor and recipient groups were each divided into two groups by donor marijuana usage. Outcomes in donor and recipient groups were compared using t-test, Chi-square and mixed linear analysis (P < 0.05 considered significant). Results. This was 294 living renal donor medical records were reviewed including 31 marijuana-using donors (MUD) and 263 non-MUDs (NMUD). It was 230 living kidney recipient records were reviewed including 27 marijuana kidney recipients (MKRs) and 203 non-MKRs (NMKR). There was no difference in donor or recipient perioperative characteristics or postoperative outcomes based upon donor marijuana use (P > 0.05 for all comparisons). There was no difference in renal function between NMUD and MUD groups and no long-term difference in kidney allograft function between NMKR and MKR groups. Conclusions. Considering individuals with a history of marijuana use for living kidney donation could increase the donor pool and yield acceptable outcomes

Should Oncologists Recommend Cannabis?
Donald I. Abrams
Current Treatment Options in Oncology 20(7) July 2019
DOI: 10.1007/s11864-019-0659-9
Cannabis is a useful botanical with a wide range of therapeutic potential. Global prohibition over the past century has impeded the ability to study the plant as medicine. However, delta-9-tetrahydrocannabinol (THC) has been developed as a stand-alone pharmaceutical initially approved for the treatment of chemotherapy-related nausea and vomiting in 1986. The indication was expanded in 1992 to include treatment of anorexia in patients with the AIDS wasting syndrome. Hence, if the dominant cannabinoid is available as a schedule III prescription medication, it would seem logical that the parent botanical would likely have similar therapeutic benefits. The system of cannabinoid receptors and endogenous cannabinoids (endocannabinoids) has likely developed to help us modulate our response to noxious stimuli. Phytocannabinoids also complex with these receptors, and the analgesic effects of cannabis are perhaps the best supported by clinical evidence. Cannabis and its constituents have also been reported to be useful in assisting with sleep, mood, and anxiety. Despite significant in vitro and animal model evidence supporting the anti-cancer activity of individual cannabinoids—particularly THC and cannabidiol (CBD)—clinical evidence is absent. A single intervention that can assist with nausea, appetite, pain, mood, and sleep is certainly a valuable addition to the palliative care armamentarium. Although many healthcare providers advise against the inhalation of a botanical as a twenty-first century drug-delivery system, evidence for serious harmful effects of cannabis inhalation is scant and a variety of other methods of ingestion are currently available from dispensaries in locales where patients have access to medicinal cannabis. Oncologists and palliative care providers should recommend this botanical remedy to their patients to gain first-hand evidence of its therapeutic potential despite the paucity of results from randomized placebo-controlled clinical trials to appreciate that it is both safe and effective and really does not require a package insert.

Sleep and Neurochemical Modulation by Cannabidiolic Acid Methyl Ester in Rats
Eric Murillo-Rodr´?guez, Gloria Arankowsky-Sandoval, Roger G. Pertwee, Linda Parker, Raphael Mechoulam
Brain Research Bulletin Volume 155, February 2020, Pages 166-173
doi: 10.1016/j.brainresbull.2019.12.006
Cannabidiolic acid methyl ester (HU-580) is a more stable compound than cannabidiolic acid (CBDA) which has been shown to be effective in reducing nausea, anxiety, depression behaviors in animal models. Here we extend the investigation of this compound to determine its effect on the sleep-wake cycle in male Wistar rats. HU-580 dose-dependently (0.1, 1.0 or 100 ?g/Kg, i.p.) prolonged wakefulness (W) and decreased slow wave sleep (SWS) duration whereas rapid eye movement sleep (REMS) showed no statistical change. In addition, the brain microdialysis probes either placed at nucleus accumbens (NAc) or into the basal forebrain in freely moving animals were used to evaluate the effects of HU-580 treatment on neurotransmitters related to the sleep-wake cycle modulation. HU-580 enhanced extracellular levels of dopamine, serotonin collected from NAc while adenosine and acetylcholine were increased in basal forebrain. In summary, HU-580 seems to possess wake-promoting pharmacological properties and enhances the levels of wake-related neurochemicals. This is the first report of effects of HU-580 on sleep modulation expanding the very limited existent data on the neurobiological effects of HU-580 on rats.

Small bowel intussusception in marijuana users
Daniel Kakish1,2 , Marwan Alaoudi1,2, *, Brian Welch1,2 , David Fan1,2 , Melissa Meghpara1,2 , Nageswara Mandava1,2 and Narendra Kumthekar2
Journal of Surgical Case Reports, 2020;9, 1–4
doi: 10.1093/jscr/rjaa335
Intussusception occurs when one portion of bowel ‘telescopes’ into another due to a lead point created by a range of benign or pathologic process. Intussusception mostly occurs in children. Although adult intussusception (AI) is rare, accounting for <5% of intestinal obstructions, it is more concerning in adults as malignancy accounts for nearly 65% of lead points in AI. Patients present with severe abdominal pain concerning for an acute abdomen along with a degree of bowel obstruction. We have experienced a total of 11 patients within recent years presenting with symptoms of an acute abdomen due to AI. None of these patients were found to have a pathologic process creating a lead point. However, we found that all of them were marijuana users. In this report, we compare their management, hospital course and review of the literature discussing proposed mechanisms that suggest an association between cannabis and intussusception.

Smoked cannabis for spasticity in multiple sclerosis: a randomized, placebo-controlled trial
Jody Corey-Bloom, Tanya Wolfson, Anthony Gamst, Shelia Jin,
Thomas D. Marcotte, Heather Bentley, Ben Gouaux
CMAJ, July 10, 2012, 184(10)
DOI:10.1503/cmaj.110837
Background: Spasticity is a common and poorly controlled symptom of multiple sclerosis. Our objective was to determine the short-term effect of smoked cannabis on this symptom.
Methods: We conducted a placebo-controlled, crossover trial involving adult patients with multiple sclerosis and spasticity. We recruited participants from a regional clinic or by referral from specialists. We randomly assigned
participants to either the intervention (smoked cannabis, once daily for three days) or control (identical placebo cigarettes, once daily for three days). Each participant was assessed daily before and after treatment. After a washout interval of 11 days, participants crossed over to the op posite group. Our primary outcome was change in spasticity as measured by patient score on the modified Ashworth scale. Our secondary outcomes included patients’ perception of pain (as measured using a visual analogue scale), a timed
walk and changes in cognitive function (as measured by patient performance on the Paced Auditory Serial Addition Test), in addition to ratings of fatigue.
Results: Thirty-seven participants were randomized at the start of the study, 30 of whom completed the trial. Treatment with smoked cannabis resulted in a reduction in patient scores on the modified Ashworth scale by an average of 2.74 points more than placebo (p <0.0001). In addition, treatment reduced pain scores on a visual analogue scale by an average of 5.28 points more than placebo (p = 0.008). Scores for the timed walk did not differ significantly
between treatment and placebo (p = 0.2). Scores on the Paced Auditory Serial Addition Test decreased by 8.67 points more with treatment than with placebo (p = 0.003). No serious adverse events occurred during the trial.
Interpretation: Smoked cannabis was superior to placebo in symptom and pain reduction in participants with treatment-resistant spasticity. Future studies should examine whether different doses can result in similar beneficial
effects with less cognitive impact.

Smoked Medicinal Cannabis for Neuropathic Pain in HIV: A Randomized, Crossover Clinical Trial
Ronald J Ellis, Will Toperoff, Florin Vaida, Geoffrey van den Brande, James Gonzales, Ben Gouaux, Heather Bentley and J Hampton Atkinson
Neuropsychopharmacology (2009) 34, 672–680
doi:10.1038/npp.2008.120
Despite management with opioids and other pain modifying therapies, neuropathic pain continues to reduce the quality of life and daily functioning in HIV-infected individuals. Cannabinoid receptors in the central and peripheral nervous systems have been shown to modulate pain perception. We conducted a clinical trial to assess the impact of smoked cannabis on neuropathic pain in HIV. This was a phase II, double-blind, placebo-controlled, crossover trial of analgesia with smoked cannabis in HIV associated distal sensory predominant polyneuropathy (DSPN). Eligible subjects had neuropathic pain refractory to at least two previous analgesic classes; they continued on their prestudy analgesic regimens throughout the trial. Regulatory considerations dictated that subjects smoke under direct observation in a hospital setting. Treatments were placebo and active cannabis ranging in potency between 1 and 8% D-9-tetrahydrocannabinol, four times daily for 5 consecutive days during each of 2 treatment weeks, separated by a 2-week washout. The primary outcome was change in pain intensity as measured by the Descriptor Differential Scale (DDS) from a pretreatment baseline to the end of each treatment week. Secondary measures included assessments of mood and daily functioning. Of 127 volunteers screened, 34 eligible subjects enrolled and 28 completed both cannabis and placebo treatments. Among the completers, pain relief was greater with cannabis than placebo (median difference in DDS pain intensity change, 3.3 points, effect size.0.60; p.0.016). The proportions of subjects achieving at least 30% pain relief with cannabis versus placebo were 0.46 (95%CI 0.28, 0.65) and 0.18 (0.03, 0.32). Mood and daily functioning improved to a similar extent during both treatment periods. Although most side effects were mild and self-limited, two subjects experienced treatment-limiting toxicities. Smoked cannabis was generally

Smokeless consumption of medical cannabis pharmacokinetics, safety and feasibility of the CannaHALER© a phase 1a study
Offir Ben-Ishay , Ortal Bar-On and Yoram Kluger
Journal of Cannabis Research (2020) 2:15
doi 10.1186/s42238-020-00022-4
Background: Substantial advancements were achieved in the management of postoperative pain, however the need for further improvement remains. This study explores the pharmacokinetics and safety of the CannaHaler, a metered dose inhaler for plant material made by Kite-Systems situated in Tel-Aviv, Israel.
Methods: The study was conducted on 12 healthy adult volunteers divided into four arms (each arm/group holds 3 volunteers) with the evaporated plant material being Alaska strain provided by “Tikun Olam”. This strain is a hybrid of 70% Sativa and 30% Indika strains, consisting of 20–22% THC and 0% CBD. Each arm received a single dose and groups were divided in an ascending dose fashion: Group I-IV receiving 10, 15, 20, 25 mg of THC respectively. The volunteers inhaled a single dose of THC using the CannaHaler, device. Blood samples for ?9 – Tetrahydrocannabinol (THC) and 9-THCCOOH were taken at base line and up to 30 min after dosing. Adverse events were monitored following the inhalation. Pharmacokinetics profile was obtained for each patient in all arms.
Results: Ascending doses of THC produced a linear increase in the maximum concentration 10, 15, 20 and 25 mg of THC. (35.43 ± 5.97, 51.47 ± 13.79, 72.37 ± 15.93, 88.63 ± 14.75 respectively) with the same linear increase in the dimension of the AUC (441.59 ± 88.49, 624 ± 123.56, 698.35 ± 174.98, 971.36 ± 310.4 respectively) both with no change in the time needed to reach such concentration. No adverse events were recorded in all of study subjects. The CannaHaler achieved high Cmax (35.43–88.63 ng/mL) values and low coefficient of variations (16.64–26.79%) in comparison to both smoking and oral preparations, thus reaching the potential of a pharmaceutical grade device for inhaled substance.
Conclusions: The current study showed that the use of Kite-Systems CannaHaler as a smokeless medical cannabis inhalation device is feasible and efficient. The low coefficient of variation together with the high Cmax values, suggest the potential use of the CannaHaler device as a pharmaceutical cannabis dosing administrator

Not Cannabis specific but if no sense of smell Cannabis effects are reduced
Sniffing out pharmacology: Interactions of drugs with human olfaction
Jorn Lotsch, Gerd Geisslinger and Thomas Hummel
Trends in Pharmacological Sciences, April 2012, Vol. 33, No. 4
DOI: 10.1016/j.tips.2012.01.004
Advances in the understanding of the sense of smell have increased awareness of the role of olfaction in human life. Odors are perceived via specific G proteincoupled receptors (GPCRs) with cAMP as the second messenger. Drugs that interact with this signaling cascade, such as opioids, cannabinoids and sildenafil, are known to reduce olfactory function. Drugs that are active
in the central nervous system (CNS) may also hinder the complex processing of olfactory information to distinguish, via pattern recognition, thousands of odors from the signals of only _400 distinct olfactory receptors. Many other interactions with drug targets expressed at the olfactory bulb are also likely to occur. However, olfactory drug effects have rarely been explored in controlled
studies. In the current activities of drug development and re-purposing, olfaction could become highly important because it can impact significantly upon the enjoyment of food. With an established molecular basis and using available tools, the assessment of olfaction in drug development and approval is advised.

So near yet so far: why won’t the UK prescribe medical cannabis?
Nutt, D., Bazire, S., Phillips, L. D., & Schlag, A. K.
BMJ Open, 10(9), e038687. (2020).
doi:10.1136/bmjopen-2020-038687
Although cannabis-based products for medicinal use are now legal in the UK, it is still challenging for patients to gain access, and only very few National Health Service prescriptions have been written to date. This paper attempts to make sense of why the UK lags behind so many other countries which also have legalised medical cannabis. From consulting with parents and patients, prescribers, pharmacists and decision-makers it seems that there are a series of distinct barriers to prescribing that need to be overcome in order to improve patient access to medical cannabis in the UK. These include concerns about the perceived lack of scientific evidence. To alleviate these concerns, we highlight the importance of patient-centred approaches including patient-reported outcomes, pharmacoepidemiology and n=1 trials, which can contribute to the development of the evidence base for medical cannabis. We hope that this paper will help policymakers and prescribers understand the challenges to prescribing and so help them develop approaches to overcome the current situation which is detrimental to patients

Some Historical Aspects of Marijuana.
Winek, C. L.
Clinical Toxicology, 10(2), 243–253.(1977).
doi:10.3109/1556365770898796
Marijuana, by whatever species of Cannabis it is called, has been known to science and medicine for almost 5000 years. It is reported to have been contained in the Chinese Emperor Nuna' s Herbal dated 2737 B.C. It was not given the name Cannabis sativa L. until Linnaeus named it in 1753 A.D. In searching some of the older Materia Medica texts and journals, one finds reference to the medicinal uses of the plant and its products. In the 1868 edition (3rd) of Materia Medica for the Use of Students (Published by Lindsay & Blakiston, Philadelphia) by John B. Biddle, M.D. of the Jefferson Medical College of Philadelphia the following is given on pp. 66-67:

Spinal astroglial cannabinoid receptors control pathological tremor
Nature Neuroscience.
DOI: 10.1038/s41593-021-00818-4
https://www.azolifesciences.com/news...ing-aging.aspx
Medical cannabis is a highly debated topic, but there is still a lot more to learn about this compound. A research team from the Department of Neuroscience at the Faculty of Health and Medical Sciences has now made a recent finding that may prove crucial to upcoming studies and treatments using medical cannabis.
Cannabinoids are compounds present in cannabis and they can also be found in the central nervous system. The researchers used a mouse model to prove that a particular synthetic cannabinoid, called cannabinoid WIN55,212¬2, decreases essential tremors by stimulating the support cells of the brain and spinal cord, called astrocytes. Prior studies into medicinal cannabis have targeted the nerve cells or the supposed neurons.

Spinal cannabinoid CB1 or CB2 receptors activation attenuates mechanical allodynia in streptozotocin-induced diabetic rats
Maryna Rodrigues Gonçalves, Milena Santana da Conceição, Carlos Henrique Alves Jesus, Aléxia Thamara Gasparin, Evelize Stacoviaki Rosa, Joice Maria da Cunha
Behav Pharmacol. 2020 Aug 13.
doi: 10.1097/FBP.0000000000000580.
Diabetes is a chronic disease associated with a high number of complications such as peripheral neuropathy, which causes sensorial disturbances and may lead to the development of diabetic neuropathic pain (DNP). The current treatment for DNP is just palliative and the drugs may cause severe adverse effects, leading to discontinuation of treatment. Thus, new therapeutic targets need to be urgently investigated. Studies have shown that cannabinoids have promising effects in the treatment of several pathological conditions, including chronic pain. Thus, we aimed to investigate the acute effect of the intrathecal injection of CB1 or CB2 cannabinoid receptor agonists N-(2-chloroethyl)-5Z, 8Z, 11Z, 14Z-eicosatetraenamide (ACEA) or JWH 133, respectively (10, 30 or 100 ?g/rat) on the mechanical allodynia associated with experimental diabetes induced by streptozotocin (60 mg/kg; intraperitoneal) in rats. Cannabinoid receptor antagonists CB1 AM251 or CB2 AM630 (1 mg/kg) were given before treatment with respective agonists to confirm the involvement of cannabinoid CB1 or CB2 receptors. Rats with diabetes exhibited a significant reduction on the paw mechanical threshold 2 weeks after diabetes induction, having the maximum effect observed 4 weeks after the streptozotocin injection. This mechanical allodynia was significantly improved by intrathecal treatment with ACEA or JWH 133 (only at the higher dose of 100 ?g). Pre-treatment with AM251 or AM630 significantly reverted the anti-allodynic effect of the ACEA or JWH 133, respectively. Considering the clinical challenge that the treatment of DPN represents, this study showed for the first time, that the intrathecal cannabinoid receptors agonists may represent an alternative for the treatment of DNP.

Standardized Cannabis Smoke Extract Induces Inflammation in Human Lung Fibroblasts

Front. Pharmacol., 28 March 2022 https://doi.org/10.3389/fphar.2022.852029
https://www.frontiersin.org/articles/10.3389/fphar.2022.852029/full?xnpe_tifc=4.V8OIV7h.VD4dYXb.nJxypZhfEWVjQsVuU_O.VjOIolhkUstIYDhIbA4FP_hNl8bdhNtIHpbDPNxfb7bDHdb9p_4fe.bubXxFzZxFVpbdnD&utm_source=eNews&utm_campaign=TCS Newsletter Week 16 2022&utm_medium=email

Studies and Case Reports IAMC
https://www.cannabis-med.org/english/studies.pdf
Almost 700 medical Cannabis studies by indication.

Sudden unexpected death under acute influence of cannabis.
Hartung, B., Kauferstein, S., Ritz-Timme, S., & Daldrup, T.
Forensic Science International, 237, (2014).
doi:10.1016/j.forsciint.2014.02.001
The acute toxicity of cannabinoids is said to be low and there is little public awareness of the potentially hazardous cardiovascular effects of cannabis, e.g. marked increase in heart rate or supine blood pressure. We describe the cases of two young, putative healthy men who died unexpectedly under the acute influence of cannabinoids. To our knowledge, these are the first cases of suspected fatal cannabis intoxications where full postmortem investigations, including autopsy, toxicological, histological, immunohistochemical and genetical examinations, were carried out. The results of these examinations are presented. After exclusion of other causes of death we assume that the young men experienced fatal cardiovascular complications evoked by smoking cannabis

Survey of Patients Employing Cannabigerol-Predominant Cannabis Preparations:
Perceived Medical Effects, Adverse Events, and Withdrawal Symptoms
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0058
https://www.liebertpub.com/doi/abs/1...ournalCode=can
Introduction: Cannabigerol (CBG), and its precursor before decarboxylation, cannabigerolic acid is sometimes labeled the ‘‘mother of all cannabinoids.’’ The purpose of the present study was to investigate reasons for use and self-reported therapeutic effects in CBG-predominant cannabis users. Usage patterns and adverse effects, including withdrawal symptoms were also explored.
Methods: Cannabidiol-predominant cannabis users were recruited online to complete an online survey assessing CBG use patterns, conditions treated with CBG-predominant cannabis (containing > 50% CBG), perceived efficacy, associated adverse events, and withdrawal symptoms. One hundred twenty-seven eligible participants (U.S. residents ages 21 + who reported using CBG-predominant cannabis in the past 6 months) completed the survey.
Results: Most of the samples (n = 65; 51.2%) reported use of CBG-predominant products solely for medical purposes
(n = 46; 36.2% reported use for medical and recreational purposes; n = 8; 6.3% reported recreational use only, and n = 8 were missing). The most common conditions the complete sample reported using CBG to treat were anxiety (51.2%), chronic pain (40.9%), depression (33.1%), and insomnia/disturbed sleep (30.7%). Efficacy was highly rated, with the majority reporting their conditions were ‘‘very much improved’’ or ‘‘much improved’’ by CBG. Furthermore, 73.9% claimed superiority of CBG-predominant cannabis over conventional medicines for chronic pain, 80% for depression, 73% for insomnia, and 78.3% for anxiety. Forty-four percent
of CBG-predominant cannabis users reported no adverse events, with 16.5% noting dry mouth, 15% sleepiness, 11.8% increased appetite, and 8.7% dry eyes. Around 84.3% reported no withdrawal symptoms, with sleep difficulties
representing the most frequently endorsed withdrawal symptom (endorsed by two respondents).
Conclusions: This is the first patient survey of CBG-predominant cannabis use to date, and the first to document self-reported efficacy of CBG-predominant products, particularly for anxiety, chronic pain, depression, and insomnia. Most respondents reported greater efficacy of CBG-predominant cannabis over conventional pharmacotherapy, with a benign adverse event profile and negligible withdrawal symptoms. This study establishes that
humans are employing CBG and suggests that CBG-predominant cannabis-based medicines should be studied in randomized controlled trials.
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Sweet Leaf Relief - Guide to Using Cannabis as a Medicine
https://canna-base.com/
This FREE guide (and website) was started when the founders got weary of the “one size fits all” approach offered by their doctors who just handed out pills to combat their pain.
Tired, stressed and unable to continue with the only option their doctors gave them they found their own path to reduced prescription medication, and the guide is the sum total of the journey taken to escape the opiate maze. Their findings were pieced together from research, learning and some experimentation. Leading to significantly improved quality of life and general well being.
The information contained within Canna-Base has most definitely been learned the hard way. The main team that operates the site, suffer from long-term chronic conditions that limit their lifestyles; they literally feel your pain.
A FREE downloadable guide to help you manage chronic pain the natural way. Research your own path to a reduced pain existence using cannabis.

Systematic Review of Cannabis Use and Risk of Occupational Injury
Wade R. Biasutti, Kurt S. H. Leffers & Russell C. Callaghan
SUBSTANCE USE & MISUSE (2020)
DOI: 10.1080/10826084.2020.1759643
Aim: A range of nations, including countries of the European Union, Australia, and the Americas have recently implemented or proposed reforms to how they control cannabis use, thereby departing from traditional approaches of criminal prohibition that have dominated throughout most of the twentieth century. Given these policy developments and the widespread global use of cannabis, it is critically important to understand the possible risks associated with cannabis use in relation to major societal harms. Methods: This systematic review investigates the potential link between cannabis use and occupational injury. Consequently, it appraises all available current literature from five databases, following Cochrane and PRISMA guidelines.
Results: Seven of the 16 reviewed studies show evidence supporting a positive association between cannabis use and occupational injury. One study shows evidence supporting a negative association and the remaining eight studies show no evidence of a significant relation. None of the studies assessed cannabis-related impairment. Only three of the reviewed studies show clear evidence that cannabis use preceded the occupational-injury event. Conclusion: The current body of evidence does not provide sufficient evidence to support the position that cannabis users are at increased risk of occupational injury. Further, the study quality assessment suggests significant biases in the extant literature are present due to potential confounding variables, selection of participants, and measurement of exposures and outcomes. Future high-quality evidence will be needed to elucidate the relation between cannabis use and occupational injury.

Symptoms and Extraintestinal Manifestations in Active Cannabis Users with Inflammatory Bowel Disease
DOI: 10.1089/can.2020.0155
https://www.liebertpub.com/doi/pdf/1...?download=true
Cannabis and Cannabinoid Research 2021
Introduction: Cannabis use is common in the setting of inflammatory bowel disease (IBD). Patients frequently use cannabis to treat IBD-associated symptoms, and there is evidence that cannabis and its derivatives are helpful for this purpose. However, it is unclear how the symptom profiles of active IBD cannabis users and nonusers compare and how these symptoms may relate to their underlying disease state and/or complications.
Materials and Methods: We performed a retrospective cohort study using a consented IBD natural history registry from a single tertiary care referral center between January 1, 2015 and August 31, 2020. We asked patients about current cannabis use and frequency. We also abstracted demographic and clinical characteristic information, including endoscopic severity, and totals and subscores of surveys assessing IBD characteristics, presence of anxiety/depression, and IBD-associated symptoms. We compared clinical and demographic factors of cannabis users and nonusers and developed a logistic regression model to evaluate for independent associations with cannabis use.
Results: Three hundred eighty-three IBD patients met the inclusion criteria (206 females, 177 males; 258 Crohn's disease [CD], 118 ulcerative colitis, and 7 indeterminate colitis). Thirty patients (7.8%) were active cannabis users, consuming it for an average of 2.7 times per week. Cannabis users were more likely to report abdominal pain (83.3% vs. 61.7%), gas (66.7% vs. 45.6%), tenesmus (70.0% vs. 47.6%), and arthralgias (53.3% vs. 20.3%) compared to those that did not use cannabis (p<0.05 for each). Incidence of moderate-severe endoscopic inflammation was similar between cannabis users and nonusers, while CD-associated complications were more common in nonusers (39.1% vs. 69.7%, p<0.05). The only factor that demonstrated a significant association with cannabis use on multivariable analysis was arthralgia (p<0.01).
Discussion: Active IBD cannabis users were more likely to report a variety of symptoms, including abdominal pain, gas, tenesmus, and arthralgias. However, they did not demonstrate more frequent active disease or IBD-associated complications, suggesting that other nonluminal factors influence their symptoms and/or decision to use cannabis. These findings demonstrate the importance of evaluating for extraintestinal contributors to symptom burden in IBD cannabis users, as well as the ongoing need to develop safer and more effective methods for recognizing and managing abdominal pain and other symptoms in this setting.
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Targeting cannabinoid receptors in brain tumors
Guillermo Velasco, Arkaitz Carracedo, Cristina Blázquez, Mar Lorente, Tania Aguado, Cristina Sánchez, Ismael Galve-Roperh, and Manuel Guzmán In book Cannabinoids and the brain 2008 Attila Köfalvi Editor
DOI: 10.1007/978-0-387-74349-3_17 https://www.researchgate.net/publication/227141784_Targeting_Cannabinoi d_Receptors_in_Brain_Tumors Cannabinoids, the active components of Cannabis sativa L., act in the body by mimicking endogenous substances – the endocannabinoids – that activate specific cell surface receptors. Cannabinoids exert various palliative effects in cancer patients. In addition, cannabinoids inhibit the growth of different types of tumor cells, including glioma cells, in laboratory animals. They do so by modulating key cell signaling pathways, mostly the endoplasmic reticulum stress response, thereby inducing antitumoral actions such as the apoptotic death of tumor cells and the inhibition of tumor angiogenesis. Of interest, cannabinoids seem to be selective antitumoral compounds as they kill glioma cells but not their nontransformed astroglial counterparts. On the basis of these preclinical findings, a pilot clinical study of D 9- tetrahydrocannabinol (D 9 -THC) in patients with recurrent glioblastoma multiforme has been recently run. The fair safety profile of D 9 -THC, together with its possible growth-inhibiting action on tumor cells, may set the basis for future trials aimed at evaluating the potential antitumoral activity of cannabinoids.

Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease.
McKallip, R. J.
Blood, 100(2), 627–634.(2002).
doi:10.1182/blood-2002-01-0098
In the current study, we examined whether ligation of CB2 receptors would lead to induction of apoptosis in tumors of immune origin and whether CB2 agonist could be used to treat such cancers. Exposure of murine tumors EL-4, LSA, and P815 to delta-9-tetrahydrocannabinol (THC) in vitro led to a significant reduction in cell viability and an increase in apoptosis. Exposure of EL-4 tumor cells to the synthetic cannabinoid HU-210 and the endogenous cannabinoid anandamide led to significant induction of apoptosis, whereas exposure to WIN55212 was not effective. Treatment of EL-4 tumorbearing mice with THC in vivo led to a significant reduction in tumor load, increase in tumor-cell apoptosis, and increase in survival of tumor-bearing mice. Examination of a number of human leukemia and lymphoma cell lines, including Jurkat, Molt-4, and Sup-T1, revealed that they expressed CB2 receptors but not CB1. These human tumor cells were also susceptible to apoptosis induced by THC, HU-210, anandamide, and the CB2-selective agonist JWH-015. This effect was mediated at least in part through the CB2 receptors because pretreatment with the CB2 antagonist SR144528 partially reversed the THC-induced apoptosis. Culture of primary acute lymphoblastic leukemia cells with THC in vitro reduced cell viability and induced apoptosis. Together, the current data demonstrate that CB2 cannabinoid receptors expressed on malignancies of the immune system may serve as potential targets for the induction of apoptosis. Also, because CB2 agonists lack psychotropic effects, they may serve as novel anticancer agents to selectively target and kill tumors of immune origin.

Targeting the endocannabinoid system: future therapeutic strategies Oier Aizpurua-Olaizola, Izaskun Elezgarai, Irantzu Rico-Barrio, Iratxe Zarandona, Nestor Etxebarria and Aresatz Usobiaga DOI: 10.1016/j.drudis.2016.08.005 Drug Discovery Today Volume 00, Number 00 August 2016 https://www.academia.edu/28280146/Targeting_the_endocannabinoid_ system_future_therapeutic_stra tegies The endocannabinoid system (ECS) is involved in many physiological regulation pathways in the human body, which makes this system the target of many drugs and therapies. In this review, we highlight the latest studies regarding the role of the ECS and the drugs that target it, with a particular focus on the basis for the discovery of new cannabinoid-based drugs. In addition, we propose some key steps, such as the creation of a cannabinoid-receptor interaction matrix (CRIM) and the use of metabolomics, toward the development of improved and more specific drugs for each relevant disease.

Techniques and technologies for the bioanalysis of Sativex(®), metabolites and related compounds
Anna Molnar, Shanlin Fu
Bioanalysis 8(8) March 2016
DOI:10.4155/bio-2015-0021
Sativex(®) is an oromucosal spray indicated for the treatment of moderate-to-severe spasticity in multiple sclerosis and is also an effective analgesic for advanced cancer patients. Sativex contains ?(9)-tetrahydrocannabinol (THC) and cannabidiol in an approximately 1:1 ratio. The increasing prevalence of medicinal cannabis products highlights the importance of reliable bioanalysis and re-evaluation of the interpretation of positive test results for THC, as legal implications may arise in workplace, roadside and sports drug testing situations. This article summarizes published research on the bioanalysis of THC and cannabidiol, with particular focus on Sativex. Common screening and confirmatory testing of blood, urine, oral fluid and hair samples are outlined. Correlations between matrices and current analytical pitfalls are also addressed.

Terpenes and derivatives as a new perspective for pain treatment: a patent review
Adriana G Guimaraes, Mairim R Serafini & Lucindo J Quintans-Junior
Expert Opin. Ther. Patents (2013) 24(3)
DOI: 10.1517/13543776.2014.870154
Introduction: Terpenes are natural compounds found in several organisms belonging to the animal and plant kingdoms. They constitute the largest class of natural products with > 55,000 known compounds structurally diversified. Several studies have attributed to this big family of compounds a range of pharmacological properties, such as anticancer, antimicrobial, antifungal, antiviral, antihyperglycemic, analgesic, anti-inflammatory and antiparasitic. Areas covered: In this review, the authors summarize therapeutic patent
applications concerning the employment of terpenes for pain relief, focusing on the perspective for these compounds to become candidates for new drugs
intended to control painful syndromes.
Expert opinion: Over years of tremendous academic and industrial investment in the characterization of the analgesic action of terpenes, there was the development of a successful product that has been well-accepted clinically. Furthermore, there is still hope that new therapeutic options for the control of painful syndromes will be developed from terpenes, which have been shown to be great candidates for this purpose because of the range of pharmacological mechanisms in important target sites.

Terpenoids, ‘minor’ cannabinoids contribute to ‘entourage effect’ of cannabis-based medicines
Fred Gardner
O’Shaughnessy’s The Journal of Cannabis in Clinical Practice autumn 2011
https://www.beyondthc.com/wp-content...rageEffect.pdf
The chemical structure of tetrahyrdocannabinol (THC) was determined in 1964 by Raphael Mechoulam and Yechiel Gaoni. For more than three decades thereafter, the blatant psychoactivity of THC induced scientists to define it as the active ingredient in the plant. Experienced marijuana smokers who tried the drug Marinol (pure, synthetic THC) when it became prescribable in the mid-1980s reported that the effects were dissimilar. But it wasn’t until the late 1990s that the research establishment acknowledged that another compound, cannabidiol
(CBD), also exerted effects when present in significant amounts.
In 1999 a British start-up, GW Pharmaceuticals, began clinical trials of a whole-plant extract containing roughly equal amounts of THC and CBD. Multiple Sclerosis patients found the combination extract —dubbed “Sativex”— more effective in reducing pain and spasticity than a high-THC extract devoid of CBD, and less psychoactive.

Tetrahydrocannabinolic acid A (THCA-A) reduces adiposity and prevents metabolic disease caused by diet-induced obesity.
Palomares, B., Ruiz-Pino, F., Garrido-Rodriguez, M., Eugenia Prados, M., Sánchez-Garrido, M. A., Velasco, I., … Muñoz, E.
Biochemical Pharmacology, (2019).
doi:10.1016/j.bcp.2019.113693
Medicinal cannabis has remarkable therapeutic potential, but its clinical use is limited by the psychotropic activity of ?9 -tetrahydrocannabinol (?9 -THC). However, the biological profile of the carboxylated, non-narcotic native precursor of ?9 -THC, the ?9 - THC acid A (?9 -THCA-A), remains largely unexplored. Here we present evidence that ?9 -THCA-A is a partial and selective PPAR? modulator, endowed with lower adipogenic activity than the full PPAR? agonist rosiglitazone (RGZ) and enhanced osteoblastogenic effects in hMSC. Docking and in vitro functional assays indicated that ?9 -THCA-A binds to and activates PPAR? by acting at both the canonical and the alternative sites of the ligand-binding domain. Transcriptomic signatures in iWAT from mice treated with ?9 -THCA-A confirmed its mode of action through PPAR?. Administration of ?9 -THCA-A in a mouse model of HFD-induced obesity significantly reduced fat mass and body weight gain, markedly ameliorating glucose intolerance and insulin resistance, and largely preventing liver steatosis, adipogenesis and macrophage infiltration in fat tissues. Additionally, immunohistochemistry, transcriptomic, and plasma biomarker analyses showed that treatment with ?9 -THCA-A caused browning of iWAT and displayed potent anti-inflammatory actions in HFD mice. Our data validate the potential of ?9 -THCA-A as a low adipogenic PPAR? agonist, capable of substantially improving the symptoms of obesity-associated metabolic syndrome and inflammation.

THC and CBD afect metabolic syndrome parameters including microbiome in mice fed high fat-cholesterol diet
Jonathan Gorelick , Tal Assa‑Glazer , Gil Zandani , Anna Altberg , Noa Sela , Abraham Nyska and Zecharia Madar
Journal of Cannabis Research (2022) 4:27
https://jcannabisresearch.biomedcentral.com/track/pdf/10.1186/s42238-022-00137-w.pdf
Background: Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome, which often includes obesity, diabetes, and dyslipidemia. Several studies in mice and humans have implicated the involvement of the gut microbiome in NAFLD. While cannabis and its phytocannabinoids may potentially be benefcial for treating metabolic disorders such as NAFLD, their efects on liver diseases and gut microbiota profle have yet to be addressed. In this study, we evaluated the therapeutic efects of the two major cannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on NAFLD progression. Methods: NAFLD was induced by feeding mice a high fat-cholesterol diet (HFCD) for 6 weeks. During this period, the individual cannabinoids, THC or CBD, were added to the experimental diets at a concentration of 2.5 or 2.39 mg/kg. Profle of lipids, liver enzymes, glucose tolerance, and gene expression related to carbohydrate lipids metabolism and liver infammation was analyzed. The efect of THC or CBD on microbiota composition in the gut was evaluated. Results: While not alleviating hepatic steatosis, THC or CBD treatment infuenced a number of parameters in the HFCD mouse model. CBD increased food intake, improved glucose tolerance, reduced some of the infammatory response including TNFa and iNOS, and partially mitigated the microbiome dysbiosis observed in the HFCD fed mice. THC produced a much weaker response, only slightly reducing infammatory-related gene expression and microbi‑ ome dysbiosis. Conclusions: The results of this study indicate the potential therapeutic efects of individual phytocannabinoids are diferent from the efects of the cannabis plant possessing a mixture of compounds. While CBD may help ameliorate symptoms of NAFLD, THC alone may not be as efective. This disparity can putatively be explained based on changes in the gut microbiota. .

THC and endocannabinoids differentially regulate neuronal activity in the prefrontal cortex and hippocampus in the subchronic PCP model of schizophrenia.
Aguilar DD, Giuffrida A, Lodge DJ.
J Psychopharmacol. 2016 Feb;30(2):169-81.
DOI:10.1177/0269881115612239
Cannabis use has been associated with an increased risk to develop schizophrenia as well as symptom exacerbation in patients. In contrast, clinical studies have revealed an inverse relationship between the cerebrospinal fluid levels of the endocannabinoid anandamide and symptom severity, suggesting a therapeutic potential for endocannabinoid-enhancing drugs. Indeed, preclinical studies have shown that these drugs can reverse distinct behavioral deficits in a rodent model of schizophrenia. The mechanisms underlying the differences between exogenous and endogenous cannabinoid administration are currently unknown. Using the phencyclidine (PCP) rat model of schizophrenia, we compared the effects on neuronal activity of systematic administration of delta-9-tetrahydrocannabinol (THC) with the fatty acid amide hydrolase inhibitor URB597. Specifically, we found that the inhibitory response in the prefrontal cortex to THC administration was absent in PCP-treated rats. In contrast, an augmented response to endocannabinoid upregulation was observed in the prefrontal cortex of PCP-treated rats. Interestingly, differential effects were also observed at the neuronal population level, as endocannabinoid upregulation induced opposite effects on coordinated activity when compared with THC. Such information is important for understanding why marijuana and synthetic cannabinoid use may be contraindicated in schizophrenia patients while endocannabinoid enhancement may provide a novel therapeutic approach.

THC shows activity against cultured Plasmodium falciparum Ana Carolina C. de Sousa, Jill M. Combrinck, Keletso Maepa, Timothy J. Egan Bioorg. Med. Chem. Lett. 54 (2021) 128442
DOI: 10.1016/j.bmcl.2021.128442 https://www.researchgate.net/publica...ium_falciparum
The FDA approved drug Dronabinol was identified in a previous study applying virtual screening using the haemozoin crystal as a target against malaria parasites. The active ingredient of dronabinol is synthetic tetrahydrocannabinol (THC), which is one of the major cannabinoids from Cannabis sativa. Traditional use of cannabis for malaria fever was reported in the world’s oldest pharmacopoeia, dating to around 5000 years ago. In this research we report that THC inhibits β-haematin (synthetic haemozoin) and malaria parasite growth. Due the psychoactivity of THC, CBD, the other major naturally occurring cannabinoid that lacks the off-target psychoactive effects of THC, was also tested and inhibited β-haematin but showed only a mild antimalarial activity. To evaluate whether THC inhibit haemozoin formation, we performed a cellular haem fractionation assay that indicated that is not the likely mechanism of action. For the first time, the cannabinoid chemical structure is raised as a new chemical class to be further studied for malaria treatment, aiming to overcome the undesirable psychoactive effects of THC and optimize the antimalarial effects.

The anxiolytic effect of cannabidiol depends on the nature of the trauma when patients with post-traumatic stress disorder recall their trigger event
Lívia Maria Bolsoni, José Alexandre S. Crippa,Jaime Eduardo Cecílio HallakFrancisco Silveira Guimarães,Antonio Waldo Zuardi
Braz J Psychiatry (2022)
DOI: 10.1590/1516-4446-2021-2317
https://www.scielo.br/j/rbp/a/y4LLjM...at=pdf&lang=en
Objectives:
We assessed whether administering cannabidiol (CBD) before recalling the traumatic event that triggered their disorder attenuates anxiety in patients with post-traumatic stress disorder (PTSD). As an exploratory pilot analysis, we also investigated whether this effect depends on the nature of the event (sexual vs. nonsexual trauma).
Methods:
Thirty-three patients of both sexes with PTSD were recruited and randomized 1:1 into two groups. One group received oral CBD (300 mg), and the other received a placebo before listening to a digital audio playback of their previously recorded report of the trigger event. Subjective and physiological measurements were taken before and after recall. We analyzed the data in two subsamples: trigger events involving sexual and nonsexual trauma.
Results:
In the nonsexual trauma group, the differences between measurements before and after recall were significantly smaller with CBD than placebo; this held true for anxiety and cognitive impairment. However, in the sexual trauma group, the differences were non-significant for both measurements.
Conclusion:
A single dose of CBD (300mg) attenuated the increased anxiety and cognitive impairment induced by recalling a traumatic event in patients with PTSD when the event involved nonsexual trauma.

The association between cannabis use and depression: a review of the evidence
Louisa Degenhardt, Wayne Hall, Michael Lynskey, Carolyn Coffey and George Patton
Chapter 4 in book Marijuana and Madness Psychiatry and Neurobiology 2004
Doi: 10.1017/CBO9780511543630.006
The association between cannabis and depression has not received as much attention as the links between cannabis use and psychosis. One of the reasons may be that the depressed are much less likely to come to the attention of treatment services than are those who are psychotic. Furthermore, some symptoms of cannabis dependence may mimic those of depression and so comorbid depression may go undiagnosed. Rising rates of cannabis use (Donnelly and Hall, 1994; Hall et al., 1999; Degenhardt et al., 2000; Johns, 2001), depression (Andrews et al., 1998; Cicchetti and Toth, 1998) and suicide among young adults (Diekstra et al., 1995; Lynskey et al., 2000) have increased public concern about the role of substance abuse, including cannabis, in non-psychotic mental disorders. There has also been increasing advocacy for interventions to prevent and treat problematic cannabis use and depressed mood among young people. Given these parallel rises, recent speculation that the two may be linked is understandable. Given the high prevalence of both cannabis use and depression there remains a question why any comorbid relationship has received little clinical attention? It may reflect a lack of association between the two. However, until recently, there was disagreement as to whether cannabis dependence (or problematic cannabis use) existed, with few treatments available. A lack of clinical attention may therefore have simply reflected a lack of services that might have detected an association. Lastly, due to its illegal status, cannabis use may remain unreported by clients presenting with depression. .

The association between cannabis use and depression: a systematic review and meta-analysis of longitudinal studies
S. Lev-Ran, M. Roerecke, B. Le Foll, T. P. George, K. McKenzie and J. Rehm
Psychological Medicine, 2013
doi:10.1017/S0033291713001438
Background. Longitudinal studies reporting the association between cannabis use and developing depression provide mixed results. The objective of this study was to establish the extent to which different patterns of use of cannabis are associated with the development of depression using meta-analysis of longitudinal studies.
Method. Peer-reviewed publications reporting the risk of developing depression in cannabis users were located using searches of EMBASE, Medline, PsychINFO and ISI Web of Science. Only longitudinal studies that controlled for depression at baseline were included. Data on several study characteristics, including measures of cannabis use, measures of depression and control variables, were extracted. Odds ratios (ORs) were extracted by age and length of follow-up.
Results. After screening for 4764 articles, 57 articles were selected for full-text review, of which 14 were included in the quantitative analysis (total number of subjects=76058). The OR for cannabis users developing depression compared with controls was 1.17 [95% confidence interval (CI) 1.05–1.30]. The OR for heavy cannabis users developing depression was 1.62 (95% CI 1.21–2.16), compared with non-users or light users. Meta-regression revealed no significant differences in effect based on age of subjects and marginal difference in effect based on length of follow-up in the individual studies.
There was large heterogeneity in the number and type of control variables in the different studies.
Conclusions. Cannabis use, and particularly heavy cannabis use, may be associated with an increased risk for developing depressive disorders. There is need for further longitudinal exploration of the association between cannabis use and developing depression, particularly taking into account cumulative exposure to cannabis and potentially significant confounding factors.

The association between cannabis use and testicular function in men: a systematic review and meta-analysis
Federico Belladellia, Francesco Del Giudicec, Alex Kasmanc, Tina Kold Jensene, Niels Jørgensenf, Andrea Saloniaa, Michael L Eisenberg
Andrology Nov 2020
doi: 10.1111/ANDR.12953
Objective: To evaluate the association between cannabis use and testicular function (as assessed through semen quality and serum hormone levels) in different populations.
Evidence Review: Systematic review and meta-analysis of population-based retrospective cohort studies. PRISMA guidelines were used for abstracting data and assessing data quality and validity. Data were pooled using a fixed-effect or random-effects model depending on the heterogeneity of studies included. Pooled risk ratio (RR) of having any sperm abnormality and Testosterone, FSH, and LH standardized mean differences among male cannabis users and non-users, and metaregression analysis according to age, and year of publication.
Results: Nine studies were evaluted which included 4014 men with semen data and 4787 with hormonal data. Overall among 1158 cannabis users, 44.9% had impaired semen parameters. Compared to 24.5% of the 2856 nonusers. The relative risk among cannabis users for any abnormal semen parameter was 1.159 (95%CI: 0.840; 1.599, p=0.369). The standardized mean difference between users and non-users testosterone levels was -0.139 (95%CI: -0.413; 0.134, p= 0.318). For FSH, the standardized mean difference estimate was -0.142 (95%CI: -0.243; -0.0425, p=0.005), while for LH the standardized mean difference estimate was -0.318 (95%CI: -0.810- 0.175; p= 0.206). Conclusions: The current evidence does not suggest clinically significant associations between cannabis use and testicular function. However, we cannot exclude an effect of cannabis due to the limited and heterogeneous studies. Additional, well-designed studies will be needed to define the association between cannabis use and the male reproductive system.

The antimicrobial potential of cannabidiol
Mark A. T. Blaskovich, Angela M. Kavanagh, Alysha G. Elliott, Bing Zhang, Soumya Ramu, Maite Amado, Gabrielle J. Lowe, Alexandra O. Hinton, Do Minh Thu Pham, Johannes Zuegg, Neil Beare, Diana Quach, Marc D. Sharp, Joe Pogliano, Ashleigh P. Rogers, Dena Lyras, Lendl Tan, Nicholas P. West, David W. Crawford, Marnie L. Peterson, Matthew Callahan & Michael Thurn
COMMUNICATIONS BIOLOGY (2021)
DOI: 10.1038/s42003-020-01530-y
Antimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol’s primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the ‘urgent threat’ pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics


The anxiolytic effect of cannabidiol depends on the nature of the trauma when patients with post-traumatic stress disorder recall their trigger event
Lívia Maria Bolsoni, José Alexandre S. Crippa,Jaime Eduardo Cecílio HallakFrancisco Silveira Guimarães,Antonio Waldo Zuardi
Braz J Psychiatry (2022)
DOI: 10.1590/1516-4446-2021-2317
https://www.scielo.br/j/rbp/a/y4LLjM...at=pdf&lang=en
Objectives:
We assessed whether administering cannabidiol (CBD) before recalling the traumatic event that triggered their disorder attenuates anxiety in patients with post-traumatic stress disorder (PTSD). As an exploratory pilot analysis, we also investigated whether this effect depends on the nature of the event (sexual vs. nonsexual trauma).
Methods:
Thirty-three patients of both sexes with PTSD were recruited and randomized 1:1 into two groups. One group received oral CBD (300 mg), and the other received a placebo before listening to a digital audio playback of their previously recorded report of the trigger event. Subjective and physiological measurements were taken before and after recall. We analyzed the data in two subsamples: trigger events involving sexual and nonsexual trauma.
Results:
In the nonsexual trauma group, the differences between measurements before and after recall were significantly smaller with CBD than placebo; this held true for anxiety and cognitive impairment. However, in the sexual trauma group, the differences were non-significant for both measurements.
Conclusion:
A single dose of CBD (300mg) attenuated the increased anxiety and cognitive impairment induced by recalling a traumatic event in patients with PTSD when the event involved nonsexual trauma.

The association between cannabis use and depression: a review of the evidence
Louisa Degenhardt, Wayne Hall, Michael Lynskey, Carolyn Coffey and George Patton
chapter 4 in Book Marijuana and madness
Doi: 10.1017/CBO9780511543630.006
The association between cannabis and depression has received less attention than the links between cannabis use and psychosis. Some have suggested that cannabis use may be a contributory cause of suicidal behaviors. A number of studies have found association between cannabis use and suicide, but the quality of control for confounding variables has varied widely. There is increasing evidence that regular cannabis use and depression occur together more often than we might expect by chance. The association between cannabis use and depression may arise because the same factors that predispose people to use cannabis also increase their risk of depression. Cross-sectional and longitudinal studies have provided mixed evidence on the association between cannabis use and depression. There is a need for longitudinal and twin studies that assess the relationship between cannabis use, depression and confounding factors.

The Atypical Cannabinoid Abn-CBD Reduces Inflammation and Protects Liver, Pancreas, and Adipose Tissue in a Mouse Model of Prediabetes and Non-alcoholic Fatty Liver Disease
Romero-Zerbo SY, García-Fernández M, Espinosa-Jiménez V, Pozo-Morales M, Escamilla-Sánchez A, Sánchez-Salido L, Lara E, Cobo-Vuilleumier N, Rafacho A, Olveira G, Rojo-Martínez G, Gauthier BR, González-Mariscal, and Bermúdez-Silva FJ
Front. Endocrinol. 11:103. (2020)
doi: 10.3389/fendo.2020.00103
Background and Aims: The synthetic atypical cannabinoid Abn-CBD, a cannabidiol (CBD) derivative, has been recently shown to modulate the immune system in different organs, but its impact in obesity-related meta-inflammation remains unstudied. We investigated the effects of Abn-CBD on metabolic and inflammatory parameters utilizing a diet-induced obese (DIO) mouse model of prediabetes and non-alcoholic fatty liver disease (NAFLD). Materials and Methods: Ten-week-old C57Bl/6J mice were fed a high-fat diet for 15 weeks, following a 2-week treatment of daily intraperitoneal injections with Abn-CBD or vehicle. At week 15 mice were obese, prediabetic and developed NAFLD. Body weight and glucose homeostasis were monitored. Mice were euthanized and blood, liver, adipose tissue and pancreas were collected and processed for metabolic and inflammatory analysis.
Results: Body weight and triglycerides profiles in blood and liver were comparable between vehicle- and Abn-CBD-treated DIO mice. However, treatment with Abn-CBD reduced hyperinsulinemia and markers of systemic low-grade inflammation in plasma and fat, also promoting white adipose tissue browning. Pancreatic islets from AbnCBD-treated mice showed lower apoptosis, inflammation and oxidative stress than vehicle-treated DIO mice, and beta cell proliferation was induced. Furthermore, AbnCBD lowered hepatic fibrosis, inflammation and macrophage infiltration in the liver when compared to vehicle-treated DIO mice. Importantly, the balance between hepatocyte proliferation and apoptosis was improved in Abn-CBD-treated compared to vehicletreated DIO mice.
Conclusions: These results suggest that Abn-CBD exerts beneficial immunomodulatory actions in the liver, pancreas and adipose tissue of DIO prediabetic mice with NAFLD, thus protecting tissues. Therefore, Abn-CBD and related compounds could represent novel pharmacological strategies for managing obesity-related metabolic disorders.

The Big Pharma Take over of Medical Cannabis
Katie Jones
https://www.visualcapitalist.com/the...ical-cannabis/
Medical Cannabis is on the verge of radical transformation. As evidence of its many benefits mount, so too does the interest of the global pharmaceutical industry, know as Big Pharma. For the most part, Big Pharma has watched by the sidelines, deterred by regulatory concerns. Now, the sleeping giant’s takeover is intensifying as patent, partnerships, and sponsored clinical trials come to fruition.

The biology that underpins the therapeutic potential of cannabis-based medicines for the control of spasticity in multiple sclerosis
David Baker, Gareth Pryce, Samuel J. Jackson, Chris Bolton, Gavin Giovannoni
Multiple Sclerosis and Related Disorders 1 (2012) 64–75
http://files7.design-editor.com/92/9...0ED319F5EE.pdf
Cannabis-based medicines have recently been approved for the treatment of pain and spasticity in multiple sclerosis (MS). This supports the original perceptions of people with MS, who were using illegal street cannabis for symptom control and pre-clinical testing in animal models of MS. This activity is supported both by the biology of the disease and the biology of the cannabis plant and the endocannabinoid system. MS results from disease that impairs neurotransmission and this is controlled by cannabinoid receptors and endogenous cannabinoid ligands. This can limit spasticity and may also influence the processes that drive the accumulation of progressive disability.

The Bivalent Rewarding and Aversive properties of ?9-tetrahydrocannabinol are Mediated Through Dissociable Opioid Receptor Substrates and Neuronal Modulation Mechanisms in Distinct Striatal Sub-Regions
Christopher Norris, Hanna J. Szkudlarek, Brian Pereira, Walter Rushlow &
Steven R. Laviolette
Nature Scientific Reports volume 9, Article number: 9760 (2019)
DOI: 10.1038/s41598-019-46215-7
The primary psychoactive compound in cannabis, ?9 tetrahydrocannabinol (THC), is capable of producing bivalent rewarding and aversive affective states through interactions with the mesolimbic
system. However, the precise mechanisms underlying the dissociable effects of THC are not currently understood. In the present study, we identify anatomically dissociable effects of THC within the rat nucleus accumbens (NAc), using an integrative combination of behavioral pharmacology and in vivo neuronal electrophysiology. We report that the rewarding vs. aversive stimulus properties of THC are both anatomically and pharmacologically dissociable within distinct anterior vs. posterior subregions of the NAc. While the rewarding effects of THC were dependent upon local ?-opioid receptor signaling, the aversive effects of THC were processed via a ?-opioid receptor substrate. Behaviorally, THC in the posterior NASh induced deficits in social reward and cognition whereas THC in the anterior NAc, potentiated opioid-related reward salience. In vivo neuronal recordings demonstrated that THC decreased medium spiny neuron (MSN) activity in the anterior NAc and increased the power of gamma (?) oscillations. In contrast, THC increased MSN activity states in the posterior NASh and decreased ?-oscillation power. These findings reveal critical new insights into the bi-directional neuronal and pharmacological mechanisms controlling the dissociable effects of THC in mesolimbic-mediated affective processing

The bright side of psychoactive substances: cannabinoid-based drugs in motor diseases
R. Coccurello & T. Bisogno Expert Review of Clinical Pharmacology, DOI: 10.1080/17512433.2016.1209111
http://eprints.bice.rm.cnr.it/15457/...%26Bisogno.pdf
https://sci-hub.se/10.1080/17512433.2016.1209111
Introduction: Psychoactive substances are associated with the idea of drugs with high addictive liability, affecting mental states, cognition, emotion and motor behavior. However these substances can modify synaptic transmission and help to disclose some mechanisms underlying alterations in brain processing and pathophysiology of motor disease. Hence, the ‘bright side’ of e cannabinoid-based drugs must be thoroughly examined to be identified within the latter framework.
Areas covered: We will analyze the preclinical and clinical evidence of cannabinoid-based drugs, discussing their therapeutic value in basal ganglia motor disorders such as Parkinson’s disease and Huntington disease.
Expert commentary: Despite the knowledge acquired in the last years, the therapeutic potential of cannabinoid-based drugs should be further tested by novel routes of investigation. This should be focused on the role of cannabinoid signaling system in mitochondrial function as well as on the physical and functional interaction with other key receptorial targets belonging to this network.
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The cannabinoid ?9-tetrahydrocannabivarin (THCV) ameliorates insulin sensitivity in two mouse models of obesity.
Wargent, E. T., Zaibi, M. S., Silvestri, C., Hislop, D. C., Stocker, C. J., Stott, C. G., … Cawthorne, M. A.
Nutrition & Diabetes, 3(5), (2013).
doi:10.1038/nutd.2013.9
BACKGROUND: Cannabinoid type-1 (CB1) receptor inverse agonists improve type 2 diabetes and dyslipidaemia but were discontinued due to adverse psychiatric effects. D9 -Tetrahydrocannabivarin (THCV) is a neutral CB1 antagonist producing hypophagia and body weight reduction in lean mice. We investigated its effects in dietary-induced (DIO) and genetically (ob/ob) obese mice. METHODS: We performed two dose-ranging studies in DIO mice; study 1: 0.3, 1, 2.5, 5 and 12.5 mg kg 1 , oral twice daily for 30 days and study 2: 0.1, 0.5, 2.5 and 12.5 mg kg 1 , oral, once daily for 45 days. One pilot (study 3: 0.3 and 3 mg kg 1 , oral, once daily) and one full dose-ranging (study 4: 0.1, 0.5, 2.5 and 12.5 mg kg 1 , oral, once daily) studies in ob/ob mice for 30 days. The CB1 inverse agonist, AM251, oral, 10 mg kg 1 once daily or 5 mg kg 1 twice daily was used as the positive control. Cumulative food and water intake, body weight gain, energy expenditure, glucose and insulin levels (fasting or during oral glucose tolerance tests), plasma high-density lipoprotein and total cholesterol, and liver triglycerides were measured. HL-5 hepatocytes or C2C12 myotubes made insulin-resistant with chronic insulin or palmitic acid were treated with 0, 1, 3 and 10 mM THCV or AM251. RESULTS: THCV did not significantly affect food intake or body weight gain in any of the studies, but produced an early and transient increase in energy expenditure. It dose-dependently reduced glucose intolerance in ob/ob mice and improved glucose tolerance and increased insulin sensitivity in DIO mice, without consistently affecting plasma lipids. THCV also restored insulin signalling in insulin-resistant hepatocytes and myotubes. CONCLUSIONS: THCV is a new potential treatment against obesity-associated glucose intolerance with pharmacology different from that of CB1 inverse agonists/antagonists.

The cannabinoid receptor-1 gene interacts with stressful life events to increase the risk for problematic alcohol use
Lisa Bornscheuer, Andreas Lundin, Yvonne Forsell, Catharina Lavebratt, Philippe A Melas Nature 2022
https://www.nature.com/articles/s41598-022-08980-w.pdf
Problematic alcohol use is a major contributor to the global burden of death and disabilities, and it represents a public health concern that has grown substantially following the COVID-19 pandemic. The available treatment options remain limited and to develop better pharmacotherapies for alcohol misuse we need to identify suitable biological targets. Previous research has implicated the brain's endocannabinoid system (ECS) in psychiatric and stress-related outcomes, including substance use and habituation to repeated stress. Moreover, genetic variants in the cannabinoid-1 receptor gene (CNR1; CB1R) have been associated with personality traits, which are in turn predictors of substance use disorders. To date, however, no human genome-wide association study has provided evidence for an involvement of the ECS in substance use outcomes. One reason for this ECS-related "missing heritability" may be unexamined gene-environment interactions. To explore this possibility, we conducted cross-sectional analyses using DNA samples and stress-exposure data from a longitudinal Swedish population-based study (N = 2,915). Specifically, we genotyped rs2023239, a functional C/T single nucleotide polymorphism in CNR1, previously reported to be associated with CNR1 binding in the brain, subjective reward following alcohol intake, and alcohol cue-elicited brain activation. Our two outcomes of interest were (i) problematic alcohol use based on the Alcohol Use Disorders Identification Test (AUDIT), and (ii) personality trait scores based on the Five Factor Model. We found no baseline association between rs2023239 and problematic alcohol use or personality traits. However, there was a clear trend for interaction between rs2023239's risk allele (C) and stressful life events (SLEs) in both childhood and adulthood, which predicted problematic alcohol use. Although not significant, there was also some indication that the risk allele interacted with child SLEs to increase scores on neuroticism. Our study supports the notion that the ECS can affect alcohol intake behaviors by interacting with life adversities and is-to the best of our knowledge-the first to focus on the interaction between CNR1 and stressors in both childhood and adulthood in humans. Further studies are warranted to confirm these findings.

The cannabinoid system and visual processing: A review on experimental findings and clinical presumptions
Thomas Schwitzer, Raymund Schwan, Karine Angioi-Duprez, Isabelle Ingster-Moati, Laurence Lalanne, Anne Giersch, Vincent Laprevote
European Neuropsychopharmacology Volume 25, Issue 1, January 2015, Pages 100-112
DOI: 10.1016/j.euroneuro.2014.11.002
Cannabis is one of the most prevalent drugs used worldwide. Regular cannabis use is associated with impairments in highly integrative cognitive functions such as memory, attention and executive functions. To date, the cerebral mechanisms of these deficits are still poorly understood. Studying the processing of visual information may offer an innovative and relevant approach to evaluate the cerebral impact of exogenous cannabinoids on the human brain. Furthermore, this knowledge is required to understand the impact of cannabis intake in everyday life, and especially in car drivers. Here we review the role of the endocannabinoids in the functioning of the visual system and the potential involvement of cannabis use in visual dysfunctions. This review describes the presence of the endocannabinoids in the critical stages of visual information processing, and their role in the modulation of visual neurotransmission and visual synaptic plasticity, thereby enabling them to alter the transmission of the visual signal. We also review several induced visual changes, together with experimental dysfunctions reported in cannabis users. In the discussion, we consider these results in relation to the existing literature. We argue for more involvement of public health research in the study of visual function in cannabis users, especially because cannabis use is implicated in driving impairments .

The Cannabis Constituent Cannabigerol Does Not Disrupt Fear Memory Processes or Stress-Induced Anxiety in Mice
Cilla Zhou, Neda Assareh, and Jonathon C. Arnold
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0027
https://www.liebertpub.com/doi/epub/.../can.2021.0027
Introduction: Medicinal cannabis has proliferated around the world and there is increasing interest in the therapeutic potential of individual plant-derived cannabinoids (phytocannabinoids). Preclinical evidence suggests the phytocannabinoid cannabigerol (CBG) could be useful in treating brain disorders, including stress and anxiety-related disorders. In this study, we aimed to explore whether CBG disrupts various contextually conditioned fear memory processes and trauma-induced anxiety-related behavior in a mouse model of post-traumatic stress disorder (PTSD).
Materials and Methods: All mice underwent contextual fear conditioning. CBG was administered between 1 and 60 mg/kg intraperitoneally (i.p.). We 􀂦rst assessed the effects of repeated CBG exposure on long-term fear memories. We also examined whether acute CBG affected various fear memory processes, namely expression, acquisition, consolidation, and reconsolidation of conditioned fear. Finally, the effect of acute CBG administration on stress-induced anxiety in the light/dark test was assessed.
Results: Repeated CBG exposure did not affect long-term conditioned fear that was observed 24 days after the conditioning session. Moreover, acute CBG administration did not in􀂧uence the acquisition, consolidation, reconsolidation, or expression of contextually conditioned fear. Acute CBG treatment also did not affect stress-induced anxiety-related behaviors in the light/dark test.
Conclusions: CBG was ineffective in disrupting long-term fear memories, various conditioned fear memory processes, or stress-induced anxiety-related behavior in mice. These preclinical data suggest CBG may have limited scope in the treatment of PTSD and stress-related anxiety.

The CANNA-TICS Study Protocol: A Randomized Multi-Center Double-Blind Placebo Controlled Trial to Demonstrate the Efficacy and Safety of Nabiximols in the Treatment of Adults With Chronic Tic Disorders
Ewgeni Jakubovski, Anna Pisarenko, Carolin Fremer, Martina Haas, Marcus May, Carsten Schumacher, Christoph Schindler, Sebastian Häck, Lukas Aguirre Davila, Armin Koch, Alexander Brunnauer, Camelia Lucia Cimpianu, Beat Lutz, Laura Bindila and Kirsten Müller-Vahl
Front. Psychiatry 11:575826.
doi: 10.3389/fpsyt.2020.575826
Background: Gilles de la Tourette syndrome (TS) is a chronic neuropsychiatric disorder characterized by motor and vocal tics. First-line treatments for tics are antipsychotics and tic-specific behavioral therapies. However, due to a lack of trained therapists and adverse events of antipsychotic medication many patients seek alternative treatment options including cannabis. Based on the favorable results obtained from case studies on different cannabis-based medicines as well as two small randomized controlled trials using delta-9-tetrahydrocannabinol (THC), we hypothesize that the cannabis extract nabiximols can be regarded as a promising new and safe treatment strategy in TS.
Objective: To test in a double blind randomized clinical trial, whether treatment with the cannabis extract nabiximols is superior to placebo in patients with chronic tic disorders.
Patients and Methods: This is a multicenter, randomized, double-blind, placebo controlled, parallel-group, phase IIIb trial, which aims to enroll 96 adult patients with chronic tic disorders (TS or chronic motor tic disorder) across 6 centers throughout Germany. Patients will be randomized with a 2:1 ratio into a nabiximols and a placebo arm. The primary efficacy endpoint is defined as tic reduction of at least 30% (compared to baseline) according to the Total Tic Score of the Yale Global Tic Severity Scale (YGTSS-TTS) after 13 weeks of treatment. In addition, several secondary endpoints will be assessed including changes in different psychiatric comorbidities, quality of life, driving ability, and safety assessments.
Discussion: This will be the first large, controlled study investigating efficacy and safety of a cannabis-based medicine in patients with TS. Based on available data using different cannabis-based medicines, we expect not only a reduction of tics, but also an improvement of psychiatric comorbidities. If the cannabis extract nabiximols is proven to be safe and effective, it will be a valuable alternative treatment option. The results of this study will be of high health-economic relevance, because a substantial number of patients uses cannabis (illegally) as self-medication.
Conclusion: The CANNA-TICS trial will clarify whether nabiximols is efficacious and safe in the treatment of patients with chronic tic disorders.

Not Cannabis specific
The Case for Product-Specific Surveillance
Nabarun Dasgupta
AJPH March 2020, Vol 110, No. 3
doi: 10.2105/AJPH.2019.305542
https://www.ncbi.nlm.nih.gov/pmc/art...019.305542.pdf
Until the synthetic chemistry revolution of the 1800s expanded the palette, humans consumed a handful of naturally derived substances: caffeine-like stimulants, tobacco, opium, alcohol, cannabis, and ethnohallucinogens. Those who devise inertia-bound surveillance systems seem content to categorize modern renditions using broad historical categories. For example, differentiating between cannabis products modifies our understanding of safety, edibles emerging as more problematic. Public health data systems have not distinguished between heroin types (e.g., black tar, cold shake), creating a blind spot to illicitly manufactured fentanyl analogs. Blood alcohol concentrations and milligrams of morphine equivalents can homogenize productspecific risks, losing nuance worth probing

The clinical toxicology of cannabis
Leo J Schep, Robin J Slaughter, Paul Glue, Paul Gee
NZMJ 9 October 2020, Vol 133 No 1523
https://assets-global.website-files....ep%20FINAL.pdf
Cannabis is one of the most widely used recreational drugs in the world. Tetrahydrocannabinol (THC) is the psychoactive principal constituent of the cannabis plant (Cannabis sativa). It is taken either orally or by inhalation, resulting in sedation, euphoria, relaxation and loss of social inhibition. Adverse e¬ ects from higher doses can include fear, distrust and a profound state of unease, hallucinations, ataxia, stupor and seizures. Long-term use can result in respiratory and cardiovascular toxicity and has been associated with a range of psychiatric conditions. Cannabinoid hyperemesis syndrome can occur with chronic use. Driving under the influence of THC is associated with approximately double the risk of motor vehicle crashes. The intensity and duration of symptoms is proportional to the concentration of THC in the blood. Following acute use, THC only remains in the blood for several hours before it is converted into a carboxylic derivative of THC and this partitions into the fat, from where it leaches out and can be detected in urine for weeks after use. Treatment of acute intoxication mainly consists of appropriate symptom-directed supportive care. Children are more susceptible to cannabis toxicity, particularly seizures and coma, and therefore may require additional supportive care for these potential symptoms. The aim of this narrative review is to provide a brief overview of the acute and chronic e¬ffects of cannabis, its pharmacokinetics, toxicity and the medical management of intoxication.

The contribution of cannabis use to variation in the incidence of psychotic disorder across Europe (EU-GEI): a multicentre case-control study
Marta Di Forti, Diego Quattrone, Tom P Freeman, Giada Tripoli, Charlotte Gayer-Anderson, Harriet Quigley, Victoria Rodriguez, Hannah E Jongsma,
Laura Ferraro, Caterina La Cascia, Daniele La Barbera, Ilaria Tarricone, Domenico Berardi, Andrei Szöke, Celso Arango, Andrea Tortelli, Eva Velthorst, Miguel Bernardo, Cristina Marta Del-Ben, Paulo Rossi Menezes, Jean-Paul Selten, Peter B Jones, James B Kirkbride, Bart PF Rutten, Lieuwe de Haan, Pak C Sham, Jim van Os, Cathryn M Lewis, Michael Lynskey, Craig Morgan, Robin M Murray,
Lancet Psychiatry. 2019 May;6(5):427-436.
doi: 10.1016/S2215-0366(19)30048-3
Background Cannabis use is associated with increased risk of later psychotic disorder but whether it affects incidence of the disorder remains unclear. We aimed to identify patterns of cannabis use with the strongest effect on odds of
psychotic disorder across Europe and explore whether differences in such patterns contribute to variations in the incidence rates of psychotic disorder.
Methods We included patients aged 18–64 years who presented to psychiatric services in 11 sites across Europe and Brazil with first-episode psychosis and recruited controls representative of the local populations. We applied adjusted
logistic regression models to the data to estimate which patterns of cannabis use carried the highest odds for psychotic disorder. Using Europe-wide and national data on the expected concentration of ??-tetrahydrocannabinol (THC) in the different types of cannabis available across the sites, we divided the types of cannabis used by participants into two categories: low potency (THC <10%) and high potency (THC ?10%). Assuming causality, we calculated the population attributable fractions (PAFs) for the patterns of cannabis use associated with the highest odds of psychosis and the correlation between such patterns and the incidence rates for psychotic disorder across the study sites.
Findings Between May 1, 2010, and April 1, 2015, we obtained data from 901 patients with first-episode psychosis across 11 sites and 1237 population controls from those same sites. Daily cannabis use was associated with increased odds of psychotic disorder compared with never users (adjusted odds ratio [OR] 3.2, 95% CI 2.2–4.1), increasing to nearly five-times increased odds for daily use of high-potency types of cannabis (4.8, 2.5–6.3). The PAFs calculated indicated that if high-potency cannabis were no longer available, 12.2% (95% CI 3.0–16.1) of cases of first-episode psychosis could be prevented across the 11 sites, rising to 30.3% (15.2–40.0) in London and 50.3% (27.4–66.0) in Amsterdam. The adjusted incident rates for psychotic disorder were positively correlated with the prevalence in controls across the 11 sites of use of high-potency cannabis (r = 0.7; p=0.0286) and daily use (r = 0.8; p=0.0109).
Interpretation Differences in frequency of daily cannabis use and in use of high-potency cannabis contributed to the striking variation in the incidence of psychotic disorder across the 11 studied sites. Given the increasing availability of high-potency cannabis, this has important implications for public health.

The cytoprotective effects of oleoylethanolamide in insulin-secreting cells do not require activation of GPR119
Virginia M Stone, Shalinee Dhayal, David M Smith, Carol Lenaghan, Katy J Brocklehurst and Noel G Morgan
British Journal of Pharmacology (2012) 165 2758–2770
DOI:10.1111/j.1476-5381.2011.01755.x
BACKGROUND AND PURPOSE b-cells express a range of fatty acid-responsive G protein-coupled receptors, including GPR119, which regulates insulin secretion and is seen as a potential therapeutic target in type 2 diabetes. The long-chain unsaturated fatty acid derivative oleoylethanolamide (OEA) is an endogenous agonist of GPR119 and, under certain conditions, some long-chain unsaturated fatty acids can promote b-cell cytoprotection. It is not known, however, if OEA is cytoprotective in b-cells. The present study has examined this and determined whether GPR119 is involved.
METHODS Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1E, were exposed to fatty acids complexed with BSA. cAMP levels, insulin release and cell viability were measured. Protein expression was studied by Western blotting and receptor expression by RT-PCR.
KEY RESULTS GPR119 was expressed in both BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA per se is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate.
CONCLUSIONS AND IMPLICATIONS Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection

The dose-dependent effect of a stabilized cannabidiol nanoemulsion on ocular surface inflammation and intraocular pressure
Leslie Rebibo, Marina Frušić-Zlotkin, Ron Ofri, Taher Nassar,
Int J Pharm. 2022 Apr 5;617:121627
DOI: 10.1016/j.ijpharm.2022.121627
Cannabidiol (CBD) is a phytocannabinoid that has a great clinical therapeutic potential. Few studies have been published on its efficacy in ocular inflammations while its impact on intraocular pressure (IOP), a major risk factor for glaucoma, remains unclear. Moreover, due to its lability and high lipophilicity, its formulation within a prolonged stable topical ophthalmic solution or emulsion able to penetrate the highly selective corneal barrier is challenging. Therefore, various CBD nanoemulsions (NEs) were designed and evaluated for stability in accelerated conditions. Further, the optimal formulation was tested on a murine LPS-induced keratitis inflammation model. Lastly, increasing CBD concentrations were topically applied, for two weeks, on mice eyes, for IOP measurement. CBD NEs exhibited optimal physicochemical characteristics for ocular delivery. A specific antioxidant was required to obtain the stable, final, formulation. In vivo, 0.4 to 1.6% CBD w/v reduced the levels of key inflammatory cytokines, depending on the concentration applied. These concentrations decreased or did not affect the IOP. Our results showed that a well-designed CBD ocular dosage form can be stabilized for an extended shelf life. Furthermore, the significant decrease in inflammatory cytokines levels could be exploited, provided that an adequate therapeutic dosage regimen is identified in humans.
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The effect of cannabidiol (CBD) on low-frequency activity and functional connectivity in the brain of adults with and without autism spectrum disorder (ASD).
Pretzsch, C. M., Voinescu, B., Mendez, M. A., Wichers, R., Ajram, L., Ivin, G., … McAlonan, G. M.
Journal of Psychopharmacology, (2019).
doi:10.1177/0269881119858306
Background: The potential benefits of cannabis and its major non-intoxicating component cannabidiol (CBD) are attracting attention, including as a potential treatment in neurodevelopmental disorders such as autism spectrum disorder (ASD). However, the neural action of CBD, and its relevance to ASD, remains unclear. We and others have previously shown that response to drug challenge can be measured using functional magnetic resonance imaging (fMRI), but that pharmacological responsivity is atypical in ASD.
Aims: We hypothesized that there would be a (different) fMRI response to CBD in ASD.
Methods: To test this, task-free fMRI was acquired in 34 healthy men (half with ASD) following oral administration of 600 mg CBD or matched placebo (random order; double-blind administration). The ‘fractional amplitude of low-frequency fluctuations’ (fALFF) was measured across the whole brain, and, where CBD significantly altered fALFF, we tested if functional connectivity (FC) of those regions was also affected byCBD.
Results: CBD significantly increased fALFF in the cerebellar vermis and the right fusiform gyrus. However, post-hoc within-group analyses revealed that this effect was primarily driven by the ASD group, with no significant change in controls. Within the ASD group only, CBD also significantly altered vermal FC with several of its subcortical (striatal) and cortical targets, but did not affect fusiform FC with other regions in either group.
Conclusion: Our results suggest that, especially in ASD, CBD alters regional fALFF and FC in/between regions consistently implicated in ASD. Future studies should examine if this affects the complex behaviours these regions modulate.

The effect of cannabis in the treatment of Hodgkin’s lymphoma in a pregnant patient - extensive case report and literature review
Anca Huniadi, Andrea Sorian, Cristian Iuhas, Alin Bodog, Mircea Ioan Sandor
JBUON 2021; 26(1): 11-16
https://jbuon.com/archive/26-1-0011.pdf
Purpose: Hodgkin lymphoma (HL) is the fourth most frequent cancer diagnosis among pregnant females. A multidisciplinary team is mandatory to obtain the best treatment and prognosis for the mother and for the baby. Here, we present the case of a patient diagnosed with HL and its evolution during 2 pregnancies.
Case presentation: Herein we present the case of a 21-yearold female Caucasian patient, with free history, diagnosed with HL stage IIB. The patient started first line chemotherapy and radiotherapy, with incomplete remission. She refused any other treatment. Five years later, the patient became pregnant and was offered chemotherapy in the 2nd trimester of pregnancy, that she refused, and delivered by C-section at 37 weeks. In the same year, the patient became pregnant again and was proposed termination of pregnancy, that she also refused. The MRI scan revealed progression of HL and she was admitted in the hospital several times for altered general condition, respiratory infections and increased need of painkillers including opioids. At 26 weeks of pregnancy, the patient began on her own a treatment with pure cannabis. Her pain and general status got better and the tumor tissue decreased. She delivered by C-section at 34 weeks a boy that presented in the first 24 h postpartum a withdrawal syndrome and intestinal invagination, requiring care in NICU and surgery with bowel resection.
Conclusion: Therefore, we can conclude that cannabis could be part of oncological treatment. No other case like this, as far as we know, has been previously reported.

The effect of five day dosing with THCV on THC-induced cognitive, psychological and physiological effects in healthy male human volunteers: a placebo-controlled, double-blind, crossover pilot trial.
Amir Englund, Zerrin Atakan, Aleksandra Kralj, Nigel Tunstall, Robin Murray and Paul Morrison
DOI: 10.1177/0269881115615104
In this small pilot study with healthy infrequent cannabis users, results indicate that the dose of 10mg oral THCV is well tolerated with no serious adverse reactions, and was subjectively not distinguishable from placebo. Furthermore, the lower dose of 1mg iv THC did not produce any significant short-term memory impairment, or psychotic or paranoid reactions. THCV significantly inhibited THC-induced impairment to delayed recall as well as THC-induced increase of heart rate. THCV on its own showed signs towards improved performance on the harder working-memory task, while also producing a slight increase in
anxiety. However, these effects were small and need to be further studied in a larger sample.
RATIONALE:
Cannabis is mostly grown under illegal and unregulated circumstances, which seems to favour a product increasingly high in its main cannabinoid ?-9-tetrahydrocannabinol (THC). ?-9-tetrahydrocannabivarin (THCV) is a relatively untestedcannabinoid which is said to be a cannabinoid receptor neutral antagonist, and may inhibit the effects of THC.
RATIONALE:
Cannabis is mostly grown under illegal and unregulated circumstances, which seems to favour a product increasingly high in its main cannabinoid ?-9-tetrahydrocannabinol (THC). ?-9-tetrahydrocannabivarin (THCV) is a relatively untestedcannabinoid which is said to be a cannabinoid receptor neutral antagonist, and may inhibit the effects of THC.
OBJECTIVES:
To explore the safety and tolerability of repeated THCV administration and its effects on symptoms normally induced by THC in a sample of healthy volunteers.
METHODS:
Ten male cannabis users (<25 use occasions) were recruited for this within-subjects, placebo-controlled, double-blind, cross-over pilot study. 10mg oral pure THCV or placebo were administered daily for five days, followed by 1mg intravenous THCon the fifth day.
RESULTS:
THCV was well tolerated and subjectively indistinguishable from placebo. THC did not significantly increase psychotic symptoms, paranoia or impair short-term memory, while still producing significant intoxicating effects. Delayed verbal recall was impaired by THC and only occurred under placebo condition (Z=-2.201, p=0.028), suggesting a protective effect of THCV. THCV also inhibited THC-induced increased heart rate (Z=-2.193, p=0.028). Nine out of ten participants reported THC underTHCV condition (compared to placebo) to be subjectively weaker or less intense (?2=6.4, p=0.011). THCV in combination with THC significantly increased memory intrusions (Z=-2.155, p=0.031).
CONCLUSION:
In this first study of THC and THCV, THCV inhibited some of the well-known effects of THC, while potentiating others. These findings need to be interpreted with caution due to a small sample size and lack of THC-induced psychotomimetic and memory-impairing effect, probably owing to the choice of dose.

The Effect of Medical Cannabis on Pain Level and Quality of Sleep among Rheumatology Clinic Outpatients
George Habib , Fadi Khazin , Suheil Artul
Pain Res Manag. 2021 Sep 6;2021:1756588.
doi: 10.1155/2021/1756588.
https://www.ncbi.nlm.nih.gov/pmc/art...21-1756588.pdf
Introduction: Medical cannabis (MC) is becoming increasingly popular for the treatment of chronic pain conditions. In this study, we evaluated the effect of MC treatment on pain level and quality of sleep of patients with different medical conditions at the rheumatology clinic.
Methods: Patients licensed for the use of MC at the rheumatology clinics at different settings were located and contacted. Their demographic and clinical parameters were documented, including type of medical cannabis consumed, way of consumption, and current monthly consumed amount. These patients were contacted by phone and asked about the effect on pain level and quality of sleep.
Results: A total of 351 patients were located, and 319 completed the questionnaire. Mean age was 46 ± 12 years, 76% were female, 82% had fibromyalgia, ∼9% had mechanical problems, ∼4% had inflammatory problems, ∼4% had neurological problems, and ∼1% had other problems. The average monthly consumed dose of MC was 31, 35, 36, and 32 g, with mean pain level reduction of 77%, 82%, 83%, and 57%, and mean sleep quality improvement of 78%, 71%, 87%, and 76% among patients with fibromyalgia, mechanical, neuropathic, and inflammatory problems, respectively. Mean THC and CBD contents were 18.38% ± 4.96 and 2.62% ± 4.87, respectively. The THC concentration, duration of MC consumption, and MC consumption dose had independent significant correlations with pain reduction while only the duration of MC consumption had an independent significant correlation with sleep quality improvement.
Conclusions: MC had a favorable effect on pain level and quality of sleep among all spectrums of problems at the rheumatology clinic.

Not directly Cannabis related
The Effect of Omega-3 Fatty Acids on ARDS: A Randomized DoubleBlind Study
Masoud Parish, Farnaz Valiyi, Hadi Hamishehkar, Sarvin Sanaie, Mohammad Asghari Jafarabadi, Samad EJ Golzari, Ata Mahmoodpoor
Adv Pharm Bull, 2014, 4(Suppl 2), 555-561
doi: 10.5681/apb.2014.082
https://www.ncbi.nlm.nih.gov/pmc/art.../apb-4-555.pdf
Purpose: The aim of this study was to evaluate the effect of an enteral nutrition diet, enriched with omega-3 fatty acids because of its anti-inflammatory effects on treatment of patients with mild to moderate ARDS. Methods: This randomized clinical trial was performed in two ICUs of Tabriz University of Medical Sciences from Jun 2011 until Sep 2013 in north west of Iran. Fifty-eight patients with mild to moderate ARDS were enroled in this clinical trial. All patients received standard treatment for ARDS based on ARDS network trial. In intervention group, patients received 6 soft-gels of omega-3/day in addition to the standard treatment.
Results: Tidal volume, PEEP, pH, PaO2 /FiO2 , SaO2, P platue and PaCO2 on the 7th and 14th days didn’t have significant difference between two groups. Indices of lung mechanics (Resistance, Compliance) had significant difference between the groups on the 14th day. Pao2 had significant difference between two groups on both 7th and 14th days. Trend of PaO2 changes during the study period in two groups were significant. We showed that adjusted mortality rate did not have significant difference between two groups. Conclusion: It seems that adding omega-3 fatty acids to enteral diet of patients with ARDS has positive results in term of ventilator free days, oxygenation, lung mechanic indices; however, we need more multi center trials with large sample size and different doses of omega-3 fatty acids for their routine usage as an adjuant for ARDS treatment.

The Effect of Piperine on Oral Absorption of Cannabidiol following Acute vs. Chronic Administration.
Izgelov, D., Domb, A. J., & Hoffman, A.
European Journal of Pharmaceutical Sciences, (2020). doi:10.1016/j.ejps.2020.105313
The piperine is an alkaloid naturally found mostly in black pepper with a myriad of pharmacological attributes. The most far reaching indication of piperine is its use as an absorption enhancer, with supportive data regarding piperine’s ability to inhibit first pass effect mechanisms. However, alongside these findings, the role of piperine as an absorption enhancer is undermined with publications stating an effect of a metabolic inducer. The aim of this commentary is to investigate the effect of repeated administration of piperine on oral absorption of cannabidiol (CBD), compared to single dose administration. The effect of piperine on absorption was determined preclinically in the freely moving rat model. Repeated administration of piperine, dissolved in a lipid-based formulation did not differ from acute piperine demonstration in its effect on CBD absorption, with a 2.5-fold increase in oral bioavailability in comparison to control group without piperine.

The effect of tetrahydrocannabinol on testosterone among men in the united states: results from the national health and nutrition examination survey.
Fantus, R. J., Kohn, T. P., & Ramasamy, R.
Fertility and Sterility, 112(3), e62–e63.(2019).
doi:10.1016/j.fertnstert.2019.07.286
https://sci-hub.tw/10.1016/j.fertnstert.2019.07.286
OBJECTIVE: Smoking tetrahydrocannabinol (THC) causes central suppression of gonadotropins resulting in testosterone deficiency. Emerging literature suggests that this effect may not occur, and that men using THC may actually have increased testosterone (T). Given this discrepancy, we sought to determine the association between different levels of THC usage and T levels using a nationally representative cohort.
DESIGN: This is a retrospective review of a cross-sectional data set, the National Health and Nutrition Examination Survey (NHANES). A survey designed by the center for disease control (CDC) to determine the health of the United States.
MATERIALS AND METHODS: All men ages 18-80 years who answered the substance use questionnaire and underwent laboratory testing for T were included. THC use was self-reported and categorized by number of times used monthly. Multivariate modeling, controlling for confounders identified on univariate analysis, was then used to determine the relationship between THC use and T levels.
RESULTS: Among the 5,146 men who met inclusion criteria, 1477 (28.7%) endorsed smoking THC at least once in their lifetime, 809 endorse smoking in the last year (15.7%), and 625 (12.1%) reported smoking the last month. Mean T level of the cohort was 430 185 ng/dL. Univariate analysis revealed that men who reported smoking THC in the last year on average had a higher T (497) compared to those who did not report using THC (414 ng/ dL, p¼0.002). Multivariate analysis controlling for age, body mass index, exercise level, alcohol use, and race demonstrated an inverse U association between THC use in the past year and T (Table), (p<0.001)
CONCLUSIONS: Analysis of a nationally representative cohort suggests that there is a dose-dependent effect of THC on T levels. While there is an increase in T in all THC users, increased amounts of THC usage appear to have a detrimental effect on serum testosterone levels. Future prospective work using specific doses of THC and studies elucidating the mechanism of the association is required to corroborate these findings.
TABLE. The effects of THC use on testosterone when controlling for age, body mass index, exercise level, alcohol use, and race.
THC use within last year Difference in T Level (ng/dL)
Never — Once a month 49.96 2-3 times a month 66.77
4-8 times a month 52.18
9-24 times a month 41.81
25-30 times a month 33.44
Controlling for age, body mass index, exercise, alcohol use, race, comorbidities, study years

The Effectiveness and Safety of Medical Cannabis for Treating Cancer Related Symptoms in Oncology Patients
Joshua Aviram, Gil M. Lewitus, Yelena Vysotski, Mahmoud Abu Amna, Anton Ouryvaev, Shiri Procaccia, Idan Cohen, Anca Leibovici, Luiza Akria, Dimitry Goncharov, Neomi Mativ, Avia Kauffman, Ayelet Shai, Gil Bar-Sela and David Meiri
Front. Pain Res., 20 May 2022 |
DOI: 10.3389/fpain.2022.861037
https://www.frontiersin.org/articles/10.3389/fpain.2022.861037/full
The use of medical cannabis (MC) to treat cancer-related symptoms is rising. However, there is a lack of long-term trials to assess the benefits and safety of MC treatment in this population. In this work, we followed up prospectively and longitudinally on the effectiveness and safety of MC treatment. Oncology patients reported on multiple symptoms before and after MC treatment initiation at one-, three-, and 6-month follow-ups. Oncologists reported on the patients' disease characteristics. Intention-to-treat models were used to assess changes in outcomes from baseline. MC treatment was initiated by 324 patients and 212, 158 and 126 reported at follow-ups. Most outcome measures improved significantly during MC treatment for most patients (p < 0.005). Specifically, at 6 months, total cancer symptoms burden declined from baseline by a median of 18%, from 122 (82–157) at baseline to 89 (45–138) at endpoint (−18.98; 95%CI= −26.95 to −11.00; p < 0.001). Reported adverse effects were common but mostly non-serious and remained stable during MC treatment. The results of this study suggest that MC treatment is generally safe for oncology patients and can potentially reduce the burden of associated symptoms with no serious MC-related adverse effects.

The Effectiveness of Common Cannabis Products for Treatment of Nausea
Stith, Sarah S. PhD[SUP]*[/SUP]; Li, Xiaoxue PhD[SUP]*[/SUP]; Orozco, Joaquin MS[SUP]†[/SUP]; Lopez, Victoria BS[SUP]‡[/SUP]; Brockelman, Franco BS[SUP]§[/SUP]; Keeling, Keenan BS[SUP]§[/SUP]; Hall, Branden[SUP]§[/SUP]; Vigil, Jacob M. PhD[SUP]†[/SUP]
Journal of Clinical Gastroenterology: April 2022 - Volume 56 - Issue 4 - p 331-338
doi: 10.1097/MCG.0000000000001534
Goals:
We measure for the first time how a wide range of cannabis products affect nausea intensity in actual time.
Background:
Even though the Cannabis plant has been used to treat nausea for millennia, few studies have measured real-time effects of common and commercially available cannabis-based products.
Study:
Using the Releaf App, 886 people completed 2220 cannabis self-administration sessions intended to treat nausea between June 6, 2016 and July 8, 2019. They recorded the characteristics of self-administered cannabis products and baseline symptom intensity levels before tracking real-time changes in the intensity of their nausea.
Results:
By 1 hour postconsumption, 96.4% of people had experienced symptom relief with an average symptom intensity reduction of −3.85 points on a 0 to 10 visual analog scale (SD=2.45, d=1.85, P<0.001). Symptom relief was statistically significant at 5 minutes and increased with time. Among product characteristics, flower and concentrates yielded the strongest, yet similar results; products labeled as Cannabis indica underperformed those labeled as Cannabis sativa or hybrid; and joints were associated with greater symptom relief than pipes or vaporizers. In sessions using flower, higher tetrahydrocannbinol and lower cannabidiol were generally associated with greater symptom relief (eg, within 5 min).
Conclusions:
The findings suggest that the vast majority of patients self-selecting into cannabis use for treatment of nausea likely experience relief within a relative short duration of time, but the level of antiemetic effect varies with the characteristics of the cannabis products consumed in vivo. Future research should focus on longer term symptom relief, including nausea-free intervals and dosing frequency; the risks of consumption of medical cannabis, especially among high-risk populations, such as pregnant women and children; and potential interactions between cannabis, conventional antiemetics, other medications, food, tobacco, alcohol, and street drugs among specific patient populations.
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The effectiveness of self-directed medical cannabis treatment for pain
Xiaoxue Li, Jacob M.Vigil, Sarah S.Stith, Franco Brockelman, Keenan Keeling, Branden Hall
Complementary Therapies in Medicine Volume 46, October 2019, Pages 123-130
doi: 10.1016/j.ctim.2019.07.022
The prior medical literature offers little guidance as to how pain relief and side effect manifestation may vary across commonly used and commercially available cannabis product types. We used the largest dataset in the United States of real-time responses to and side effect reporting from patient-directed cannabis consumption sessions for the treatment of pain under naturalistic conditions in order to identify how cannabis affects momentary pain intensity levels and which product characteristics are the best predictors of therapeutic pain relief. Between 06/06/2016 and 10/24/2018, 2987 people used the ReleafApp to record 20,513 cannabis administration measuring cannabis’ effects on momentary pain intensity levels across five pain categories: musculoskeletal, gastrointestinal, nerve, headache-related, or non-specified pain. The average pain reduction was –3.10 points on a 0–10 visual analogue scale (SD?=?2.16, d?=?1.55, p?<?.001). Whole Cannabis flower was associated with greater pain relief than were other types of products, and higher tetrahydrocannabinol (THC) levels were the strongest predictors of analgesia and side effects prevalence across the five pain categories. In contrast, cannabidiol (CBD) levels generally were not associated with pain relief except for a negative association between CBD and relief from gastrointestinal and non-specified pain. These findings suggest benefits from patient-directed, cannabis therapy as a mid-level analgesic treatment; however, effectiveness and side effect manifestation vary with the characteristics of the product used.

The effects of cannabidiol on male reproductive system: A literature review.
Carvalho, R. K., Andersen, M. L., & Mazaro?Costa, R.
Journal of Applied Toxicology. (2019).
doi:10.1002/jat.3831
Cannabidiol (CBD) is one of the most abundant phytocannabinoids present in the plant Cannabis sativa (marijuana). There have been several studies of CBD in the last few decades, mainly focused on its neuroprotective properties, particularly after the identification of the endocannabinoid system and its participation in the central nervous system. On the other hand, the peripheral effects of CBD, particularly on reproductive physiology, were also evidenced. A narrative review was conducted using the PubMed database to identify studies that analyzed the pharmacological effects of CBD on the male reproductive system of vertebrates and invertebrates. Thirty?two citations (in vivo and in vitro) were identified. Among the vertebrates, the studies were carried out with men, monkeys, rats and mice. Studies with invertebrates are centered exclusively on the sea urchin. The CBD treatment periods includemostly acute and subacute evaluations. Exposure to CBD is associated with a reduction in mammalian testis size, the number of germ and Sertoli cells in spermatogenesis, fertilization rates, and plasma concentrations of hypothalamic, pituitary and gonadal hormones. Moreover, chronic doses of CBD have impaired sexual behavior in mice. From the studies identified in this review, it is possible to conclude that CBD has negative effects on the reproductive system of males. However, knowledge is still limited, and additional research is required to elucidate fully the mechanisms of action, as well as the reversibility of CBD effects on the reproductive system

The effects of cannabis and alcohol on simulated driving: Influences of dose and experience.
Downey, L. A., King, R., Papafotiou, K., Swann, P., Ogden, E., Boorman, M., & Stough, C.
Accident Analysis & Prevention, 50, 879–886.(2013).
doi:10.1016/j.aap.2012.07.016
Background: Cannabis and alcohol are the most popular drugs amongst recreational users, and most prevalent in injured and deceased drivers. Clarification of the interactive effects of these drugs upon driving behaviour is critical for reducing drug-related road deaths.
Objectives: The current study had two objectives, to examine the effects of cannabis and alcohol on driving performance, and identify if any differences between the effects of cannabis and alcohol on driving performance exist between regular cannabis users and non-regular cannabis users.
Methods: The projectinvolved 80 participants (49 male, 31 female) who were abstinent recreational users of alcohol and marijuana. They participated in six experimental sessions that involved the consumption of cannabis cigarettes containing no THC, 1.8% THC or 3% THC together with the consumption of alcohol to obtain either 0% BAC, 0.03% BAC or 0.05% BAC. The six sessions were double-blind, counter-balanced, placebo-controlled and medically supervised. Forty participants were allocated to the cannabis with low alcohol (0.03% BAC) group, and 40 participants were allocated to the cannabis with high alcohol (0.05% BAC) group. Driving simulator performance was assessed at 20 min post-drug administration and blood samples were taken before and after driving.
Results: Driving simulator performance was more impaired in the THC and alcohol combined conditions. Consistent with past research, the level of THC detected in blood is higher when THC is consumed with alcohol, than when cannabis is consumed alone, and regular cannabis users returned higher levels of THC in plasma than non-regular users. Generally, regular cannabis users displayed more driving errors than non-regular cannabis users.

The Effects of Cannabis on Female Reproductive Health Across the Life Course
Daniel J. Corsi, Malia S.Q. Murphy, and Jocelynn Cook
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0065
https://www.liebertpub.com/doi/pdf/1.../can.2020.0065
Introduction: Cannabis is commonly used for its medicinal and therapeutic benefits and is also widely used as a recreational drug. Cannabis use has been increasing in Canada, including among Canadian women of reproductive age. Post-legalization, further increases in cannabis use are expected due to increased availability and lowered perceptions of harm. Although cannabinoids are well known for their effects on the central and peripheral nervous systems, endocannabinoid receptors have also been characterized throughout the female reproductive tract. Cannabinoids may affect many aspects of female reproductive health, including fertility, pregnancy outcomes with neonatal implications, and menopause. Purpose: To provide a comprehensive review of trends in cannabis use among women and review the impact of cannabis across the female reproductive lifespan. Methods: We searched PubMed and Cochrane Library databases using keywords and MeSH terms. Included studies reported the potential impact of cannabinoids on female fertility, pregnancy, transmission to breast milk, neonatal outcomes, and menopause. Results: The existing literature is primarily concentrated on the effect of cannabis use in pregnancy and breastfeeding, with little exploration of its impact on fertility and in later life. Studies are limited in number, with small sample sizes, and are hampered by methodological challenges related to confounding and other potential biases. Conclusions: There remain critical gaps in the literature about the potential risks of cannabis use, particularly in vulnerable populations, including pregnant women, women who are breastfeeding, and their infants. Given the rise in the prevalence of cannabis use, new, robust investigations into the consequences of cannabis exposure on female reproductive health are needed

The efficacy and safety of cannabidivarin treatment on epilepsy in girls with Rett syndrome: A phase 1 clinical trial
Ellen N Hurley, Carolyn J Ellaway, Alexandra M Johnson, Linda Truong, Rebecca Gordon, Peter Galettis, Jennifer H Martin, John A Lawson
Epilesia April 2022
doi: 10.1111/EPI.17247
https://onlinelibrary.wiley.com/doi/10.1111/epi.17247
Objective: Rett Syndrome (RTT), commonly caused by methyl-CpG-binding protein 2 (MECP2) pathogenic variants, has many co-morbidities. 50-90% of children with RTT have epilepsy which is often drug resistant. Cannabidivarin (CBDV), a non-hallucinogenic phytocannabinoid has shown benefit in MECP2 animal models. This Phase I trial assessed the safety and tolerability of CBDV in female children with RTT and drug resistant epilepsy, as well as the effect on mean monthly seizure frequency (MMSF), the electroencephalogram (EEG), and non-epilepsy comorbid symptoms.
Methods: Five female children with drug resistant epilepsy and a pathogenic MECP2 variant were enrolled. Baseline clinical and laboratory assessments, including monthly seizure frequency, were recorded. CBDV oral solution (50mg/mL) was prescribed and titrated to 10mg/kg/day. Data collected over 15 months included pharmacokinetics, seizure type and frequency, adverse events, EEG, and responses to Rett syndrome behaviour questionnaire (RSBQ) and Rett syndrome symptom severity index, and was compared to baseline data.
Results: All five children reached the maximum CBDV dose of 10mg/kg/day and had a reduction in MMSF (median 79% reduction). Three children had MMSF reduction >75%. This corresponded to an overall reduction in seizure frequency from 32 to 7.2 seizures per month. 91% of adverse events were mild or moderate and none required drug withdrawal. 62% were judged unrelated to CBDV. 31% of adverse events were identified as possibly related, of which nearly all were mild, and the remainder were later assessed as RTT symptoms. Hypersomnolence and drooling were identified as related to CBDV. No serious adverse events reported were related to CBDV. No significant change was noted in EEG or non-epilepsy related symptoms of RTT.
Significance: 10mg/kg/day of CBDV is safe and well tolerated in a paediatric Rett syndrome cohort and suggests improved seizure control in children with MECP2-related Rett syndrome.

The endocannabinoid system: a general view and latest additions
Luciano De Petrocellis, Maria Grazia Cascio & Vincenzo Di Marzo
British Journal of Pharmacology (2004) 141, 765–774
doi:10.1038/sj.bjp.0705666
https://www.ncbi.nlm.nih.gov/labs/pm...1-0705666a.pdf
After the discovery, in the early 1990s, of specific G-protein-coupled receptors for marijuana’s psychoactive principle D9 -tetrahydrocannabinol, the cannabinoid receptors, and of their endogenous agonists, the endocannabinoids, a decade of investigations has greatly enlarged our understanding of this altogether new signalling system. Yet, while the finding of the endocannabinoids resulted in a new effort to reveal the mechanisms regulating their levels in the brain and peripheral organs under physiological and pathological conditions, more endogenous substances with a similar action, and more molecular targets for the previously discovered endogenous ligands, anandamide and 2- arachidonoylglycerol, or for some of their metabolites, were being proposed. As the scenario becomes subsequently more complicated, and the experimental tasks to be accomplished correspondingly more numerous, we briefly review in this article the latest ‘additions’ to the endocannabinoid system together with earlier breakthroughs that have contributed to our present knowledge of the biochemistry and pharmacology of the endocannabinoids

The Endocannabinoid System: A New Treatment Target for Obsessive Compulsive Disorder?
Kayser, R. R., Snorrason, I., Haney, M., Lee, F. S., & Simpson, H. B.
Cannabis and Cannabinoid Research.(2019).
doi:10.1089/can.2018.0049
Introduction: Obsessive-compulsive disorder (OCD) is a disabling illness that is associated with significant functional impairment. Although evidence-based pharmacotherapies exist, currently available medications are ineffective in some patients and may cause intolerable side effects in others. There is an urgent need for new treatments.
Discussion: A growing body of basic and clinical research has showed that the endocannabinoid system (ECS) plays a role in anxiety, fear, and repetitive behaviors. At the same time, some patients with OCD who smoke cannabis anecdotally report that it relieves their symptoms and mitigates anxiety, and several case reports describe patients whose OCD symptoms improved after they were treated with cannabinoids. Taken together, these findings suggest that the ECS could be a potential target for novel medications for OCD. In this study, we review evidence from both animal and human studies that suggests that the ECS may play a role in OCD and related disorders. We also describe findings from studies in which cannabinoid drugs were shown to impact symptoms of these conditions.
Conclusions: An emerging body of evidence suggests that the ECS plays a role in OCD symptoms and may be a target for the development of novel medications. Further exploration of this topic through well-designed human trials is warranted

The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain
Austen Lowell Thompson, Shaness A Grenald, Haley Ciccone, Todd Vanderah
Journal of Pharmacology and Experimental Therapeutics February 2020
DOI: 10.1124/jpet.119.262337
https://jpet.aspetjournals.org/conte...62337.full.pdf
Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis are long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer induced bone pain (CIBP). Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activate CB1 and CB2 receptors inhibiting inflammation and pain. We demonstrate that administration of MJN110 significantly and dose-dependently alleviates spontaneous pain behavior during acute administration compared to vehicle control. In addition, the MJN110 maintains its efficacy in a chronic dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose dependent and significant decrease in cell viability of 66.1 breast adenocarcinoma cells and to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden evidenced by radiograph or histological analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid based therapies is essential and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.

The Endocannabinoid System: An Ancient Signaling Involved in the Control of Male Fertility
Silvia Fasano, Rosaria Meccariello, Gilda Cobellis, Rosanna Chianese, Giovanna Cacciola, Teresa Chioccarelli, and Riccardo Pierantonia
Trends in Comparative Endocrinology and Neurobiology: Ann. N.Y. Acad. Sci. 1163: 112–124 (2009)
https://sites.oxy.edu/clint/physio/a...noidsystem.pdf
doi: 10.1111/j.1749-6632.2009.04437.x

The effects of cannabinoids on human health have been known since the antiquities when the extract of the plant Cannabis sativa was used because of its psychoactivity. The scientific story of the cannabinoids started in the 1960s with the isolation and characterization of the active component of the plant. After the synthesis of cannabinoid analogues, the analysis of structure–effect relationships was implemented, and this had a similar effect to a positive “Pandora’s box” opening. To date, numerous roles have been ascribed to the “endocannabinoid system.” Here we describe its involvement in the control of male reproduction, taking into consideration possible evolutionary speculations. Indeed, the endocannabinoid system is a very ancient signaling system, being clearly present from the divergence of the protostomian/deuterostomian.

The endocannabinoid system: an overview
Natalia Battista, Monia Di Tommaso, Monica Bari and Mauro Maccarrone
Frontiers in Behavioral Neuroscience March 2012 | Volume 6 | Article 9 | 1
doi: 10.3389/fnbeh.2012.00009
https://www.ncbi.nlm.nih.gov/labs/pm...h-06-00009.pdf
Upon the identification of anandamide (AEA) in the porcine brain, numerous studies contributed to the current state of knowledge regarding all elements that form the “endocannabinoid system (ECS).” How this complex system of receptors, ligands, and enzymes is integrated in helping to regulate fundamental processes at level of central nervous and peripheral systems and how its regulation and dysregulation might counteract disturbances of such functions, is nowadays still under investigation. However, the most recent advances on the physiological distribution and functional role of ECS allowed the progress of various research tools aimed at the therapeutic exploitation of endocannabinoid (eCB) signaling, as well as the development of novel drugs with pharmacological advantages. Here, we shall briefly overview the metabolic and signal transduction pathways of the main eCBs representatives, AEA, and 2-arachidonoylglycerol (2-AG), and we will discuss the therapeutic potential of new ECS-oriented drugs.

The endocannabinoid system and amyloid-related disease Lourdes Ruiz-Valdepeñas, Cristina Benito, Rosa María Tolón, José Antonio Martínez Orgado, Julián Romero
Experimental Neurology 224 (2010) 66–73
doi: 10.1016/j.expneurol.2010.03.024
The endocannabinoid system may be the target of novel therapies in a wide variety of diseases. Among them, those related with amyloid accumulation will be discussed in the present review. Several components of this system (CB1 and CB2 receptors, endocannabinoids, FAAH enzyme) may participate in different aspects of amyloid pathophysiology such as, for instance, synaptic activity, cell migration, cytokine production or phagocytic activity. Consistent with recent data, putative lines of research and hypothesis will be discussed.

The endocannabinoid system and anxiety
S F Lisboa, F V Gomes, A L B Terzian, D C Aguiar, F A Moreira, L B M Resstel, F S Guimarães
Vitam Horm. 2017;103:193-279

doi: 10.1016/bs.vh.2016.09.006
The medical properties of Cannabis sativa is known for centuries. Since the discovery and characterization of the endogenous cannabinoid system, several studies have evaluated how cannabinoid compounds and, particularly, how the modulation of the endocannabinoid (eCB) system influences a wide range of functions, from metabolic to mental disorders. Cannabinoids and eCB system often exert opposite effects on several functions, such as anxiety. Although the mechanisms are not completely understood, evidence points to different factors influencing those effects. In this chapter, the recent advances in research about the relationship between eCB system and anxiety disorders in humans, as well as in animal models, will be discussed. The recent data addressing modulation of the eCBs in specific brain areas, such as the medial prefrontal cortex, amygdaloid complex, bed nucleus of stria terminalis, hippocampus, and dorsal periaqueductal gray, will be summarized. Finally, data from animal models addressing the mechanisms through which the eCB system modulates anxiety-related behavior dependent on stressful situations, such as the involvement of different receptors, distinct eCBs, modulation of neurotransmitters release, HPA axis and immune system activation, and plastic mechanisms, will also be discussed.
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The endocannabinoid system and cancer: therapeutic implication

Josée Guindon, Andrea G Hohmann
British Journal of Pharmacology (2011) 163 1447–1463
doi: 10.1111/j.1476-5381.2011.01327.x
The endocannabinoid system is implicated in a variety of physiological and pathological conditions (inflammation, immunomodulation, analgesia, cancer and others). The main active ingredient of cannabis, ?9?tetrahydrocannabinol (?9?THC), produces its effects through activation of CB1 and CB2 receptors. CB1 receptors are expressed at high levels in the central nervous system (CNS), whereas CB2 receptors are concentrated predominantly, although not exclusively, in cells of the immune system. Endocannabinoids are endogenous lipid?signalling molecules that are generated in the cell membrane from phospholipid precursors. The two best characterized endocannabinoids identified to date are anandamide (AEA) and 2?arachidonoylglycerol (2?AG). Here we review the relationship between the endocannabinoid system and anti?tumour actions (inhibition of cell proliferation and migration, induction of apoptosis, reduction of tumour growth) of the cannabinoids in different types of cancer. This review will focus on examining how activation of the endocannabinoid system impacts breast, prostate and bone cancers in both In vito and in vivo systems. The therapeutic potential of cannabinoids for cancer, as identified in clinical trials, is also discussed. Identification of safe and effective treatments to manage and improve cancer therapy is critical to improve quality of life and reduce unnecessary suffering in cancer patients. In this regard, cannabis?like compounds offer therapeutic potential for the treatment of breast, prostate and bone cancer in patients. Further basic research on anti?cancer properties of cannabinoids as well as clinical trials of cannabinoidtherapeutic efficacy in breast, prostate and bone cancer is therefore warranted.

The Endocannabinoid System and Its Relevance for Nutrition
Mauro Maccarrone, Valeria Gasperi, Maria Valeria Catani, Thi Ai Diep, Enrico Dainese, Harald S. Hansen, and Luciana Avigliano Annu. Rev. Nutr. 2010. 30:423–40 doi:10.1146/annurev.nutr.012809.104701 https://www.researchgate.net/publica..._for_Nutrition
Endocannabinoids bind to cannabinoid, vanilloid, and peroxisome
proliferator-activated receptors.The biological actions of these polyunsaturated lipids are controlled by key agents responsible for their synthesis, transport and degradation,which together form an endocannabinoid system (ECS). In the past few years, evidence has been accumulated for a role of the ECS in regulating food intake and energy balance, both centrally and peripherally. In addition, up-regulation of the ECS in the gastrointestinal tract has a potential impact on inflammatory bowel diseases. In this review, the main features of the ECS are summarized in order to put in better focus our current knowledge of the nutritional relevance of endocannabinoid signaling and of its role in obesity, cardiovascular pathologies, and gastrointestinal diseases. The central and peripheral pathways that underlie these effects are discussed, as well as the possible exploitation of ECS components as novel drug targets for therapeutic intervention in eating disorders

The endocannabinoid system and its therapeutic exploitation
Vincenzo Di Marzo , Maurizio Bifulco, Luciano De Petrocellis
Nature Reviews Drug Discovery 2004 Sep;3(9):771-84.
DOI: 10.1038.nrd1485
https://www.researchgate.net/publica...c_exploitation
The term 'endocannabinoid' - originally coined in the mid-1990s after the discovery of membrane receptors for the psychoactive principle in Cannabis, Delta9-tetrahydrocannabinol and their endogenous ligands - now indicates a whole signalling system that comprises cannabinoid receptors, endogenous ligands and enzymes for ligand biosynthesis and inactivation. This system seems to be involved in an ever-increasing number of pathological conditions. With novel products already being aimed at the pharmaceutical market little more than a decade since the discovery of cannabinoid receptors, the endocannabinoid system seems to hold even more promise for the future development of therapeutic drugs. We explore the conditions under which the potential of targeting the endocannabinoid system might be realized in the years to come.

The endocannabinoid system and migraine
Rosaria Greco, Valeria Gasperi, Mauro Maccarrone, Cristina Tassorelli
Experimental Neurology 224 (2010) 85–91
doi:10.1016/j.expneurol.2010.03.029
The recently discovered endocannabinoid system (ECS), which includes endocannabinoids and the proteins that metabolize and bind them, has been implicated in multiple regulatory functions both in health and disease. Several studies have suggested that ECS is centrally and peripherally involved in the processing of pain signals. This finding is corroborated by the evidence that endocannabinoids inhibit, through a cannabinoid type-1 receptor (CB1R)-dependent retrograde mechanism, the release of neurotransmitters controlling nociceptive inputs and that the levels of these lipids are high in those regions (such as sensory terminals, skin, dorsal root ganglia) known to be involved in transmission and modulation of pain signals. In this review we shall describe experimental and clinical data that, intriguingly, demonstrate the link between endocannabinoids and migraine, a neurovascular disorder characterized by recurrent episodic headaches and caused by abnormal processing of sensory information due to peripheral and/or central sensitization. Although the exact ECS-dependent mechanisms underlying migraine are not fully understood, the available results strongly suggest that activation of ECS could represent a promising therapeutical tool for reducing both the physiological and inflammatory components of pain that are likely involved in migraine attacks

The endocannabinoid system and nondrug rewarding behaviours
Liana Fattore, Miriam Melis, Paola Fadda, Marco Pistis, Walter Fratta
Experimental Neurology 224 (2010) 23–36
doi:10.1016/j.expneurol.2010.03.020
https://d1wqtxts1xzle7.cloudfront.ne.. .oid_system_and _nondrug_r20151201-3878-1t6az70-libre.pdf?1448957870=&response-content-disposition=inline%3B+filename %3DThe_endocannabinoid_system_ and_nondrug_r.pdf&Expires=1648 399025&Signature=MwC~WQOO1h-eazHdaPsEv6gsvGPKjheCZoRR6XUNt EGHuhTg4YpXCp5fB-K-GETHEi7lF-zg6O21-APPgHZP~gUQxEWgZzn1ABRnX50qi-m2o9EHLMdnILhYduytK~AGtLDEu5Nw 3LqgYZOlFR3aFIx5~4qQ0lnNyL4F9N s9AxLVzqZ02aIb4JLPSORMn-5SkfaC2ENfVeOV6EQywhscsKJyMeMv 6XV97eL4abiLcKdPyePNsNp~~Z0nPN lwLFkvVr0~xUJkXNYZCKRP06971w8g nbb6ZfHBD9nDIBWdwIJEJl32Zsvsbf cxb~nIwvt~LY-Q8jPLj~n~X0swPGroyQ__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA
Rewarding behaviours such as sexual activity, eating, nursing, parenting, social interactions, and play activity are conserved strongly in evolution, and they are essential for development and survival. All of these behaviours are enjoyable and represent pleasant experiences with a high reward value. Remarkably, rewarding behaviours activate the same brain circuits that mediate the positive reinforcing effects of drugs of abuse and of other forms of addiction, such as gambling and food addiction. Given the involvement of the endocannabinoid system in a variety of physiological functions of the nervous system, it is not surprising that it takes part in the complex machinery that regulates gratification and perception of pleasure. In this review, we focus first on the role of the endocannabinoid system in the modulation of neural activity and synaptic functions in brain regions that are involved in natural and nonnatural rewards (namely, the ventral tegmental area, striatum, amygdala, and prefrontal cortex). Then, we examine the role of the endocannabinoid system in modulating behaviours that directly or indirectly activate these brain reward pathways. More specifically, current knowledge of the effects of the pharmacological manipulation of the endocannabinoid system on natural (eating, sexual behaviour, parenting, and social play) and pathological (gambling) rewarding behaviours is summarised and discussed. .
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The endocannabinoid system and psychiatric disorders
Daniela Parolaro, Natalia Realini, Daniela Vigano, Tiziana Rubino
Exp Neurol. 2010 Jul. 2010 Jul;224(1):3-14.
DOI: 10.1016/j.expneurol.2010.03.018
The present review summarizes the latest information on the role and the pharmacological modulation of the endocannabinoid system in mood disorders and its potential implication in psychotic disorders such as schizophrenia. Reduced functionality might be considered a predisposing factor for major depression, so boosting endocannabinoid tone might be a useful alternative therapeutic approach for depressive disorders. The picture regarding endocannabinoids and anxiety is more complicated since either too much or too little anandamide can lead to anxiety states. However, a small rise in its level in specific brain areas might be beneficial for the response to a stressful situation and therefore to tone down anxiety. This effect might be achieved with low doses of cannabinoid indirect agonists, such as blockers of the degradative pathway (i.e. FAAH) or re-uptake inhibitors. Moreover several lines of experimental and clinical evidence point to a dysregulation of the endocannabinoid system in schizophrenia. The high anandamide levels found in schizophrenic patients, negatively correlated with psychotic symptoms, point to a protective role, whereas the role of 2-arachidonoyl glycerol is still unclear. There is a potential for pharmacological manipulation of the endocannabinoid system as a novel approach for treating schizophrenia, although experimental findings are still controversial, often with different effects depending on the drug, the dose, the species and the model used for simulating positive or negative symptoms. Besides all these limitations, SR141716A and cannabidiol show the most constant antipsychotic properties in dopamine- and glutamate-based models of schizophrenia, with profiles similar to an atypical antipsychotic drug.

The endocannabinoid system and the regulation of neural development: Potential implications in psychiatric disorders European Archives of Psychiatry and Clinical Neuroscience 259(7):371-82 August 2009 Ismael Galve-Roperh, Javier Palazuelos, Tania Aguado, Manuel Guzmán DOI: 10.1007/s00406-009-0028-y https://www.researchgate.net/publica...tric_disorders During brain development, functional neurogenesis is achieved by the concerted action of various steps that include the expansion of progenitor cells, neuronal specification, and establishment of appropriate synapses. Brain patterning and regionalization is regulated by a variety of extracellular signals and morphogens that, together with neuronal activity, orchestrate and regulate progenitor proliferation, differentiation, and neuronal maturation. In the adult brain, CB(1) cannabinoid receptors are expressed at very high levels in selective areas and are engaged by endocannabinoids, which act as retrograde messengers controlling neuronal function and preventing excessive synaptic activity. In addition, the endocannabinoid system is present at early developmental stages of nervous system formation. Recent studies have provided novel information on the role of this endogenous neuromodulatory system in the control of neuronal specification and maturation. Thus, cannabinoid receptors and locally produced endocannabinoids regulate neural progenitor proliferation and pyramidal specification of projecting neurons. CB(1) receptors also control axonal navigation, migration, and positioning of interneurons and excitatory neurons. Loss of function studies by genetic ablation or pharmacological blockade of CB(1) receptors interferes with long-range subcortical projections and, likewise, prenatal cannabinoid exposure induces different functional alterations in the adult brain. Potential implications of these new findings, such as the participation of the endocannabinoid system in the pathogenesis of neurodevelopmental disorders (e.g., schizophrenia) and the regulation of neurogenesis in brain depression, are discussed herein

The endocannabinoid system as a key mediator during liver diseases: new insights and therapeutic openings
A Mallat, F Teixeira?Clerc, V Deveaux, S Manin, S Lotersztajn
British Journal of Pharmacology (2011) 163 1432–1440
doi: 10.1111/j.1476-5381.2011.01397.x
Chronic liver diseases represent a major health problem due to cirrhosis and its complications. During the last decade, endocannabinoids and their receptors have emerged as major regulators of several pathophysiological aspects associated with chronic liver disease progression. Hence, hepatic cannabinoid receptor 2 (CB2) receptors display beneficial effects on alcoholic fatty liver, hepatic inflammation, liver injury, regeneration and fibrosis. Cannabinoid receptor 1 (CB1) receptors have been implicated in the pathogenesis of several lesions such as alcoholic and metabolic steatosis, liver fibrogenesis, or circulatory failure associated with cirrhosis. Although the development of CB1 antagonists has recently been suspended due to the high incidence of central side effects, preliminary preclinical data obtained with peripherally restricted CB1 antagonists give real hopes in the development of active CB1 molecules devoid of central adverse effects. CB2?selective molecules may also offer novel perspectives for the treatment of liver diseases, and their clinical development is clearly awaited. Whether combined treatment with a peripherally restricted CB1 antagonist and a CB2 agonist might result in an increased therapeutic potential will warrant further investigation.

The Endocannabinoid System as a Target in Cancer Diseases: Are We There Yet?
Estefanía Moreno, Milena Cavic, Ana Krivokuca, Vicent Casadó, and Enric Canela
Front Pharmacol. 2019; 10: 339
doi: 10.3389/fphar.2019.00339 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459931/pdf/fphar-10-00339.pdf The endocannabinoid system (ECS) has been placed in the anti-cancer spotlight in the last decade. The immense data load published on its dual role in both tumorigenesis and inhibition of tumor growth and metastatic spread has transformed the cannabinoid receptors CB1 (CB1R) and CB2 (CB2R), and other members of the endocannabinoid-like system, into attractive new targets for the treatment of various cancer subtypes. Although the clinical use of cannabinoids has been extensively documented in the palliative setting, clinical trials on their application as anti-cancer drugs are still ongoing. As drug repurposing is significantly faster and more economical than de novo introduction of a new drug into the clinic, there is hope that the existing pharmacokinetic and safety data on the ECS ligands will contribute to their successful translation into oncological healthcare. CB1R and CB2R are members of a large family of membrane proteins called G protein-coupled receptors (GPCR). GPCRs can form homodimers, heterodimers and higher order oligomers with other GPCRs or non-GPCRs. Currently, several CB1R and CB2R-containing heteromers have been reported and, in cancer cells, CB2R form heteromers with the G protein-coupled chemokine receptor CXCR4, the G protein-coupled receptor 55 (GPR55) and the tyrosine kinase receptor (TKR) human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2). These protein complexes possess unique pharmacological and signaling properties, and their modulation might affect the antitumoral activity of the ECS. This review will explore the potential of the endocannabinoid network in the anti-cancer setting as well as the clinical and ethical pitfalls behind it, and will develop on the value of cannabinoid receptor heteromers as potential new targets for anti-cancer therapies and as prognostic biomarkers.

The endocannabinoid system – current implications for drug development.
Fowler, C. J.
Journal of Internal Medicine. (2020).
doi:10.1111/joim.13229
In this review, the state of the art for compounds affecting the endocannabinoid (eCB) system is described with a focus on the treatment of pain. Among directly acting CB receptor ligands, clinical experience with ?9 -tetrahydracannabinol and medical cannabis in chronic noncancer pain indicates that there are differences between the benefits perceived by patients and the at best modest effect seen in meta-analyses of randomised controlled trials. The reason for this difference is not known but may involve differences in the type of patients that are recruited, the study conditions that are chosen and the degree to which biases such as reporting bias are operative. Other directly acting CB receptor ligands such as biased agonists and allosteric receptor modulators have not yet reached the clinic. Among indirectly acting compounds targeting the enzymes responsible for the synthesis and catabolism of the eCBs anandamide and 2- arachidonoylglycerol, fatty acid amide hydrolase (FAAH) inhibitors have been investigated clinically but were per se not useful for the treatment of pain, although they may be useful for the treatment of post-traumatic stress disorder and cannabis use disorder. Dual acting compounds targeting this enzyme and other targets such as cyclooxygenase-2 or transient potential vanilloid receptor 1 may be a way forward for the treatment of pain.

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome
Lyndsey L Anderson, Peter T Doohan, Nicole A Hawkins, Dilara Bahceci, Sumanta Garai, Ganesh A Thakur, Jennifer A Kearney, Jonathon C Arnold
Neuropharmacology . 2021 Nov 22;205:108897.
doi: 10.1016/j.neuropharm.2021.
https://www.sciencedirect.com/scienc...548?via%3Dihub
Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.
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The endocannabinoid system in brain reward processes

M Solinas, SR Goldberg and D Piomelli
British Journal of Pharmacology (2008) 154, 369–383;
doi:10.1038/bjp.2008.130;
https://escholarship.org/content/qt0...ash_5e1a39c7a2 7c814bfb7a8613533b0bad.pdf
Food, drugs and brain stimulation can serve as strong rewarding stimuli and are all believed to activate common brain circuits that evolved in mammals to favour fitness and survival. For decades, endogenous dopaminergic and opioid systems have been considered the most important systems in mediating brain reward processes. Recent evidence suggests that the endogenous cannabinoid (endocannabinoid) system also has an important role in signalling of rewarding events. First, CB1 receptors are found in brain areas involved in reward processes, such as the dopaminergic mesolimbic system. Second, activation of CB1 receptors by plant-derived, synthetic or endogenous CB1 receptor agonists stimulates dopaminergic neurotransmission, produces rewarding effects and increases rewarding effects of abused drugs and food. Third, pharmacological or genetic blockade of CB1 receptors prevents activation of dopaminergic neurotransmission by several addictive drugs and reduces rewarding effects of food and these drugs. Fourth, brain levels of the endocannabinoids anandamide and 2- arachidonoylglycerol are altered by activation of reward processes. However, the intrinsic activity of the endocannabinoid system does not appear to play a facilitatory role in brain stimulation reward and some evidence suggests it may even oppose it. The influence of the endocannabinoid system on brain reward processes may depend on the degree of activation of the different brain areas involved and might represent a mechanism for fine-tuning dopaminergic activity. Although involvement of the various components of the endocannabinoid system may differ depending on the type of rewarding event investigated, this system appears to play a major role in modulating reward processes. .


The endocannabinoid signaling system in cancer
Simona Pisanti, Paola Picardi, Alba D’Alessandro, Chiara Laezza, and Maurizio Bifulco,
Trends in Pharmacological Sciences, May 2013, Vol. 34, No. 5
DOI: 10.1016/j.tips.2013.03.003
Changes in lipid metabolism are intimately related to cancer. Several classes of bioactive lipids play roles in the regulation of signaling pathways involved in neoplastic transformation and tumor growth and progression. The endocannabinoid system, comprising lipid-derived endocannabinoids, their G-protein-coupled receptors (GPCRs), and the enzymes fortheir metabolism, is emerging as a promising therapeutic target in cancer. This report highlights the main signaling pathways for the antitumor effects of the endocannabinoid system in cancer and its basic role in cancer pathogenesis, and discusses the alternative view of cannabinoid receptors as tumor promoters. We focus on new players in the antitumor action ofthe endocannabinoidsystem and on emerging crosstalk among cannabinoid receptors and other membrane or nuclear receptors involved in cancer. We also discuss the enzyme MAGL, a key player in endocannabinoid metabolism that was recently recognized as a marker of tumor lipogenic phenotype.

The endocannabinoid system in gp120-mediated insults and HIV-associated dementia
Monica Bari, Cinzia Rapino, Pamela Mozetic, Mauro Maccarrone
Experimental Neurology 224 (2010) 74–84
doi:10.1016/j.expneurol.2010.03.025
Endocannabinoids (eCBs) include a group of lipid mediators that act as endogenous agonists at cannabinoid (CB1, CB2) and vanilloid (TRPV1) receptors. In the last two decades a number of eCBs-metabolizing enzymes have been discovered that, together with eCBs and congeners, target receptors and proteins responsible for their transport and intracellular trafficking form the so-called “endocannabinoid system” (ECS). Within the central nervous system ECS elements participate in neuroprotection against neuroinflammatory/neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. More recently, a role for eCBs has been documented also in human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120-mediated insults, and in HIV-associated dementia (HAD). The modulation of ECS in the latter disease conditions is the subject of this review, that will also address the molecular mechanisms underlying the neuroprotective effects of eCBs. In particular, the interactions between neurons and glia during neuroinflammation, and the alterations of ECS in these cells upon gp120 insults. and HAD will be discussed, along with the potential therapeutic exploitation of ECS-oriented drugs for the treatment of HAD and related disorders


The Endocannabinoid System in Huntington’s Disease
M.R. Pazos, O. Sagredo and J. Fernández-Ruiz
Current Pharmaceutical Design, 2008, 14, 2317-2325
DOI:10.2174/138161208785740108
The hypokinetic profile of certain cannabinoid agonists becomes these compounds as promising medicines to attenuate the hyperkinesia that characterizes the first grades of Huntington’s disease (HD) and that represents the major neurological abnormality in this disease. The fact that CB1 receptors, the receptor type involved in motor effects of cannabinoid agonists, are significantly reduced in the basal ganglia during the progression of HD represents a convincing explanation for the hyperkinesia typical of this disorder and supports the usefulness of enhancing CB1 receptor signaling in HD. However, further studies revealed that the key property that enables certain cannabinoid agonists to reduce hyperkinesia is their capability to directly activate vanilloid TRPV1 receptors. Cannabinoids may also serve to delay/arrest the progression of HD by protecting striatal projection neurons from death. Several cannabinoid agonists have been tested for this purpose in various animal models of HD, and these studies revealed that the major characteristics that enable cannabinoids to provide neuroprotection are three: (i) a reduction in inflammatory events exerted through activating CB2 receptors located in glial cells; (ii) a normalization of glutamate homeostasis, then limiting excitotoxicity, an effect that would be exerted through CB1 receptors; and (iii) an antioxidant effect exerted by cannabinoid receptor-independent mechanisms. The changes experienced by the endocannabinoid signaling system during the striatal degeneration support this neuroprotective effect, particularly the up-regulatory responses proved by CB2 receptors in glial cells recruited at lesioned sites. The present article will review the neurochemical and pharmacological bases that sustain the importance of the endocannabinoid system in the pathophysiology of HD, trying to collect the present information and the future lines for research on the therapeutic potential of this system in this disorder.

The endocannabinoid system in targeting inflammatory neurodegenerative diseases

Diego Centonze , Alessandro Finazzi-Agrò, Giorgio Bernardi, Mauro Maccarrone

TRENDS in Pharmacological Sciences Vol.28 No.4

doi: 10.1016/j.tips.2007.02.004

http://www.thehempresourcecenter.co...degenerative-diseases_centonze_et_al_2007.pdf

The classical divide between degenerative and inflammatory disorders of the CNS is vanishing as accumulating evidence shows that inflammatory processes are important in the pathophysiology of primarily degenerative disorders, and neurodegeneration complicates primarily inflammatory diseases of the brain and spinal cord. Here, we review the contribution of degenerative and inflammatory processes to CNS disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis and HIV-associated dementia. An early combination of neuroprotective and anti-inflammatory approaches to these disorders seems particularly desirable because isolated treatment of one pathological process might worsen another. We also discuss the apparently unique opportunity to modify neurodegeneration and neuroinflammation simultaneously by pharmacological manipulation of the endocannabinoid system in the CNS and in peripheral immune cells. Current knowledge of this system and its involvement in the above CNS disorders are also reviewed. .


The endocannabinoid system in the physiology and pathophysiology of the gastrointestinal tract
Federico Massa . Martin Storr . Beat Lutz
J Mol Med (2005) 83: 944–954
DOI 10.1007/s00109-005-0698-5
Numerous investigations have recently demonstrated the important roles of the endocannabinoid system in the gastrointestinal (GI) tract under physiological and pathophysiological conditions. In the GI tract, cannabinoid type 1 (CB1) receptors are present in neurons of the enteric nervous system and in sensory terminals of vagal and spinal neurons, while cannabinoid type 2 receptors are located in immune cells. Activation of CB1 receptors was shown to modulate several functions in the GI tract, including gastric secretion, gastric emptying and intestinal motility. Under pathophysiological conditions induced experimentally in rodents, the endocannabinoid system conveys protection to the GI tract (e.g. from inflammation and abnormally high gastric and enteric secretions). Such protective activities are largely in agreement with anecdotal reports from folk medicine on the use of Cannabis sativa extracts by subjects suffering from various GI disorders. Thus, the endocannabinoid system may serve as a potentially promising therapeutic target against different GI disorders, including frankly inflammatory bowel diseases (e.g. Crohn’s disease), functional bowel diseases (e.g. irritable bowel syndrome) and secretion- and motilityrelated disorders. As stimulation of this modulatory system by CB1 receptor agonists can lead to unwanted psychotropic side effects, an alternative and promising avenue for therapeutic applications resides in the treatment with CB1 receptor agonists that are unable to cross the blood–brain barrier, or with compounds that inhibit the degradation of endogenous ligands (endocannabinoids) of CB1 receptors, hence prolonging the activity of the endocannabinoid system.

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone
WM Olango, M Roche, GK Ford, B Harhen and DP Finn1
British Journal of Pharmacology (2012) 165 2549–2560
DOI:10.1111/j.1476-5381.2011.01478.x
BACKGROUND AND PURPOSE Endocannabinoids in the midbrain periaqueductal grey (PAG) modulate nociception and unconditioned stress-induced analgesia; however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats.
EXPERIMENTAL APPROACH Rats received intra-dlPAG administration of the CB1 receptor antagonist/inverse agonist rimonabant, or vehicle, before re-exposure to a context paired 24 h previously with foot shock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22 kHz ultrasonic vocalization) were assessed. In a separate cohort, levels of endocannabinoids [2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamide (anandamide; AEA)] and the related N-acylethanolamines (NAEs) [N-palmitoyl ethanolamide (PEA) and N-oleoyl ethanolamide (OEA)] were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone.
KEY RESULTS Re-exposure of rats to the context previously associated with foot shock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear without formalin-evoked nociceptive tone was associated with increased levels of 2-AG, AEA, PEA and OEA in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG.
CONCLUSIONS AND IMPLICATIONS Conditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG. These data support a role for endocannabinoids in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts.

The endocannabinoid system of the skin. A potential approach for the treatment of skin disorders.
Río, C. del, Millán, E., García, V., Appendino, G., DeMesa, J., & Muñoz, E.
Biochemical Pharmacology. (2018).
doi:10.1016/j.bcp.2018.08.022*
The skin is the largest organ of the body and has a complex and very active structure that contributes to homeostasis and provides the first line defense against injury and infection. In the past few years it has become evident that the endocannabinoid system (ECS) plays a relevant role in healthy and diseased skin. Specifically, we review how the dysregulation of ECS has been associated to dermatological disorders such as atopic dermatitis, psoriasis, scleroderma and skin cancer. Therefore, the druggability of the ECS could open new research avenues for the treatment of the pathologies mentioned. Numerous studies have reported that phytocannabinoids and their biological analogues modulate a complex network pharmacology involved in the modulation of ECS, focusing on classical cannabinoid receptors, transient receptor potential channels (TRPs), and peroxisome proliferator-activated receptors (PPARs). The combined targeting of several end-points seems critical to provide better chances of therapeutically success, in sharp contrast to the one-disease-one-target dogma that permeates current drug discovery campaigns.

The Endogenous Cannabinoid System Regulates Seizure Frequency and Duration in a Model of Temporal Lobe Epilepsy
Melisa J. Wallace, Robert E. Blair, Katherine W. Falenski, Billy R. Martin and Robert J. DeLorenzo
Journal of Pharmacology and Experimental Therapeutics October 2003, 307 (1) 129-137;
DOI: 10.1124/jpet.103.051920
Several lines of evidence suggest that cannabinoid compounds are anticonvulsant. However, the anticonvulsant potential of cannabinoids and, moreover, the role of the endogenous cannabinoid system in regulating seizure activity has not been tested in an in vivo model of epilepsy that is characterized by spontaneous, recurrent seizures. Here, using the rat pilocarpine model of epilepsy, we show that the marijuana extract ?9-tetrahydrocannabinol (10 mg/kg) as well as the cannabimimetic, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one [R(+)WIN55,212 (5 mg/kg)], completely abolished spontaneous epileptic seizures. Conversely, application of the cannabinoid CB1 receptor (CB1) antagonist, N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A), significantly increased both seizure duration and frequency. In some animals, CB1 receptor antagonism resulted in seizure durations that were protracted to a level consistent with the clinical condition status epilepticus. Furthermore, we determined that during an short-term pilocarpine-induced seizure, levels of the endogenous CB1ligand 2-arachidonylglycerol increased significantly within the hippocampal brain region. These data indicate not only anticonvulsant activity of exogenously applied cannabinoids but also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of the CB1 receptor. Furthermore, Western blot and immunohistochemical analyses revealed that CB1 receptor protein expression was significantly increased throughout the CA regions of epileptic hippocampi. By demonstrating a role for the endogenous cannabinoid system in regulating seizure activity, these studies define a role for the endogenous cannabinoid system in modulating neuroexcitation and suggest that plasticity of the CB1 receptor occurs with epilepsy.

FYI, I did THCV/THC trials 15 years ago, unpublished, but we found much the same, THCV is not for recreational users unless they like their THC tuned down. THCV delays THC onset, reduces peak experiences, and maybe lengthens the reduced effects.
-SamS

The “Entourage Effect”: Terpenes Coupled with Cannabinoids for the Treatment of Mood Disorders and Anxiety Disorders
Sari Goldstein Ferber, DvoraNamdar, Danielle Hen-Shoval, Gilad Eger, Hinanit Koltai, Gal Shoval, Liat Shbiro and Aron Weller
Current Neuropharmacology, 2020, 18, 87-96
DOI: 10.2174/1570159X17666190903103923
https://www.ncbi.nlm.nih.gov/labs/pm...f/CN-18-87.pdf
Mood disorders are the most prevalent mental conditions encountered in psychiatric practice. Numerous patients suffering from mood disorders present with treatment-resistant forms of depression, co-morbid anxiety, other psychiatric disorders and bipolar disorders. Standardized essential oils (such as that of Lavender officinalis) have been shown to exert clinical efficacy in treating anxiety disorders. As endocannabinoids are suggested to play an important role in major depression, generalized anxiety and bipolar disorders, Cannabis sativa was suggested for their treatment. The endocannabinoid system is widely distributed throughout the body including the brain, modulating many functions. It is involved in mood and related disorders, and its activity may be modified by exogenous cannabinoids. CB1 and CB2 receptors primarily serve as the binding sites for endocannabinoids as well as for phytocannabinoids, produced by cannabis inflorescences. However, ‘cannabis’ is not a single compound product but is known for its complicated molecular profile, producing a plethora of phytocannabinoids alongside a vast array of terpenes. Thus, the “entourage effect” is the suggested positive contribution derived from the addition of terpenes to cannabinoids. Here, we review the literature on the effects of cannabinoids and discuss the possibility of enhancing cannabinoid activity on psychiatric symptoms by the addition of terpenes and terpenoids. Possible underlying mechanisms for the anti-depressant and anxiolytic effects are reviewed. These natural products may be an important potential source for new medications for the treatment of mood and anxiety disorders.

The Essential Medicinal Chemistry of Cannabidiol (CBD)
Kathryn M. Nelson, Jonathan Bisson, Gurpreet Singh, James G. Graham, Shao-Nong Chen, J. Brent Friesen, Jayme L. Dahlin, Matthias Niemitz, Michael A. Walters, and Guido F. Pauli
J. Med. Chem. 2020, 63, 12137−12155
Doi: 10.1021/acs.jmedchem.0c00724
This Perspective of the published essential medicinal chemistry of cannabidiol (CBD) provides evidence that the popularization of CBD-fortified or CBD-labeled health products and CBD-associated health claims lacks a rigorous scientific foundation. CBD’s reputation as a cure-all puts it in the same class as other “natural” panaceas, where valid ethnobotanicals are reduced to single, purportedly active ingredients. Such reductionist approaches oversimplify useful, chemically complex mixtures in an attempt to rationalize the commercial utility of natural compounds and exploit the “natural” label. Literature evidence associates CBD with certain semiubiquitous, broadly screened, primarily plantbased substances of undocumented purity that interfere with bioassays and have a low likelihood of becoming therapeutic agents. Widespread health challenges and pandemic crises such as SARS-CoV-2 create circumstances under which scientists must be particularly vigilant about healing claims that lack solid foundational data. Herein, we offer a critical review of the published medicinal chemistry properties of CBD, as well as precise definitions of CBD-containing substances and products, distilled to reveal the essential factors that impact its development as a therapeutic agent.

The failings of per se limits to detect cannabis-induced driving impairment: Results from a simulated driving study
Thomas R Arkell , Tory R Spindle,, Richard C Kevin , Ryan Vandrey , Iain S McGregor
Traffic Inj . 2021 Feb
DOI: 10.1080/15389588.2020.1851685
Objective: Many jurisdictions use per se limits to define cannabis-impaired driving. Previous studies, however, suggest that THC concentrations in biological matrices do not reliably reflect cannabis dose and are poorly correlated with magnitude of driving impairment. Here, we first review a range of concerns associated with per se limits for THC. We then use data from a recent clinical trial to test the validity of a range of extant blood and oral fluid THC per se limits in predicting driving impairment during a simulated driving task.
Methods: Simulated driving performance was assessed in 14 infrequent cannabis users at two timepoints (30 min and 3.5 h) under three different conditions, namely controlled vaporization of 125 mg (i) THC-dominant (11% THC; <1% CBD), (ii) THC/CBD equivalent (11% THC; 11% CBD), and (iii) placebo (<1% THC & CBD) cannabis. Plasma and oral fluid samples were collected before each driving assessment. We examined whether per se limits of 1.4 and 7 ng/mL THC in plasma (meant to approximate 1 and 5 ng/mL whole blood) and 2 and 5 ng/mL THC in oral fluid reliably predicted impairment (defined as an increase in standard deviation of lateral position (SDLP) of >2 cm relative to placebo).
Results: For all participants, plasma and oral fluid THC concentrations were over the per selimits used 30 min after vaporizing THC-dominant or THC/CBD equivalent cannabis. However, 46% of participants failed to meet SDLP criteria for driving impairment. At 3.5 h post-vaporization, 57% of participants showed impairment, despite having low concentrations of THC in both blood (median = 1.0 ng/mL) and oral fluid (median = 1.0 ng/mL). We highlight two individual cases illustrating how (i) impairment can be minimal in the presence of a positive THC result, and (ii) impairment can be profound in the presence of a negative THC result.
Conclusions: There appears to be a poor and inconsistent relationship between magnitude of impairment and THC concentrations in biological samples, meaning that per se limits cannot reliably discriminate between impaired from unimpaired drivers. There is a pressing need to develop improved methods of detecting cannabis intoxication and impairment.

The Health Effects of Cannabis and Cannabinoids: The Current State of Evidence and Recommendations for Research
National Academies of Sciences, book
https://www.nap.edu/catalog/24625/th...-current-state
doi: 10.17226/24625.
At the time of this report’s release in January 2017, 28 states and the District of Columbia have legalized cannabis for the treatment of medical conditions. Eight of these states and the District of Columbia have also legalized cannabis for recreational use. In addition to the growing availability of legalized cannabis, there has also been a rapid expansion in the types of available cannabis products, including edibles, oils, and a variety of inhaled substances. The growing acceptance, accessibility, and use of cannabis raise important public health concerns and there is a clear need to establish what is known and what needs to be known about the health effects of cannabis use. The committee was tasked with conducting a comprehensive review of the current evidence regarding the health effects of using cannabis and cannabis-derived products. The study was conducted in a limited time frame in order to respond to a quickly moving landscape, but as described in the report’s methods section, the amount of work that this report entailed and the volume of literature reviewed clearly indicates the substantial effort involved and the importance of this issue to the committee. In the current report, the committee presents a rigorous and thoughtful summary of the landscape of cannabis and health and puts forth recommendations to help advance the research field and better inform public health decisions. I wish to express my deepest gratitude to my fellow committee members who worked so hard and with good grace to accomplish this task. As with other National Academies of Sciences, Engineering, and Medicine reports, the work of the committee would have been far more difficult, if not impossible, without the support of a dedicated, knowledgeable and also very hard working National Academies staff.

The heterogeneity and complexity of Cannabis extracts as antitumor agents
Liran Baram, Ella Peled, Paula Berman, Ben Yellin, Elazar Besser, Maya Benami, Igal Louria-Hayon, Gil M. Lewitus, and David Meiri
Oncotarget. 2019; 10:4091-4106.
doi.:10.18632/oncotarget.26983
The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant’s therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-?9-trans-tetrahydrocannabinol (?9-THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of ?9-THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract’s composition as well as on certain characteristics of the targeted cells.

The Highs and Lows of Cannabis in Cancer Treatment and Bone Marrow Transplantation
Osnat Almogi-Hazan, Iman Khuja, Sivan Ritter, and Reuven Or
Rambam Maimonides Med J January 2020 Volume 11 Issue 1
doi: 10.5041/RMMJ.10391
https://www.ncbi.nlm.nih.gov/pmc/art...11-1-e0009.pdf
In the last decade, we have observed an increased public and scientific interest in the clinical applications of medical cannabis. Currently, the application of cannabinoids in cancer patients is mainly due to their analgesic and anti-emetic effects. The direct effects of phyto-cannabinoids on cancer cells are under intensive research, and the data remain somewhat inconsistent. Although anti-proliferative properties were observed in vitro, conclusive data from animal models and clinical trials are lacking. Since immunotherapy
of malignant diseases and bone marrow transplantation are integral approaches in hemato-oncology, the immuno-modulatory characteristic of cannabinoids is a fundamental aspect for consideration. The effect of
cannabinoids on the immune system is presently under investigation, and some evidence for its immunoregulatory properties has been shown. In addition, the interaction of cannabinoids and classical cytotoxic agents is a subject for further investigation. Here we discuss the current knowledge of cannabinoid-based treatments in preclinical models and the limited data in oncological patients. Particularly, we address the possible contradiction between the direct anti-tumor and the immune-modulatory effects of cannabinoids.

The impact of medical cannabis consumption on the oral flora and saliva.
Habib G, Steinberg D, Jabbour A
PLoS ONE 16(2):e0247044. (2021)
Doi: 10.1371/journal.pone.0247044
Objective To evaluate the effect of medical cannabis consumption on oral flora and saliva.
Design A clinical prospective study, at the rheumatology clinic of the Nazareth Hospital in Nazareth, recruiting consecutively patients approved for medical cannabis, evaluating their saliva flow, pH and microbial load of Streptococcus mutans and Lactobacillus, prior to and under medical cannabis treatment.
Methods Patients recently licensed for medical cannabis treatment, were recruited just prior to starting medical cannabis consumption (week 0), 1 and 4 weeks later, patients provided 5-minute time saliva samples, which were measured for their volume and pH, and cultured on a special microbial kit, evaluating the growth of Streptococcus mutans and Lactobacillus.
Results Out of 16 patients enrolled, 14 were female and had fibromyalgia. The mean age of the patients was 52.8±12.9 years. The mean saliva flow at week 0, week 1 and week 4 were 5.38±3.36 ml/5-minutes, 6 (p = 0.769) and 5.45 (p = 0.391), respectively, and for saliva pH were 6.28, 5.94 (p = 0.51) and 5.5 (p = 0.07) respectively also. The mean Streptococcus mutans growth score at weeks 0, 1 and 4 was1.8±0.75, 1.6±0.83 (p = 0.234), and 2.4±0.84 (p = 0.058), respectively. The mean Lactobacilli growth score at weeks 0, 1 and 4 was 2.59±0.88, 3.1±0.69 (p = 0.033) and 3.3±0.67 (p = 0.025), respectively.
Conclusions The results of this study show that medical cannabis consumption has no significant effect on saliva volume or pH, but it may be associated with changes in salivary levels of oral microbes such as Streptococcus mutans and Lactobacilli.

The Impact of Marijuana Use on Liver Transplant Recipients: A 900 Patient Single Center Experience
Jacob Guorgui, Takahiro Ito, Daniela Markovic, Antony Aziz, Stephanie Younan, Alyscia Severance, Michelle Lu, Jane Lee, Joseph DiNorcia 3rd, Vatche G Agopian, Douglas G Farmer, Ronald W Busuttil, Fady M Kaldas
Clin Transplant. 2021 Jan 6;e14215.
doi: 10.1111/ctr.14215
This study examined the impact of pre and post-transplant marijuana use on liver transplant (LT) outcomes. While there were significant differences between marijuana users and non-users with respect to recipient demographics, including the indication for LT, the rate of pre-LT ICU admission, and MELD score, there were no statistical differences in post-operative outcomes, including patient/graft survival and post-LT complications in pre- or post-LT users. These findings may help guide future policies regarding marijuana use in LT candidates, although confirmation utilizing larger cohorts is warranted.

The impact of naturalistic cannabis use on self-reported opioid withdrawal.
Bergeria, C. L., Huhn, A. S., & Dunn, K. E. (2020).
Journal of Substance Abuse Treatment, 113, (2020).
doi:10.1016/j.jsat.2020.108005
Objectives: Four states have legalized medical cannabis for the purpose of treating opioid use disorder. It is unclear whether cannabinoids improve or exacerbate opioid withdrawal. A more thorough examination of cannabis and its impact on specific symptoms of opioid withdrawal is warranted.
Method: Two hundred individuals recruited through Amazon Mechanical Turk with past month opioid and cannabis use and experience of opioid withdrawal completed the survey. Participants indicated which opioid withdrawal symptoms improved or worsened with cannabis use and indicated the severity of their opioid withdrawal on days with and without cannabis.
Results: 62.5% (n = 125) of 200 participants had used cannabis to treat withdrawal. Participants most frequently indicated that cannabis improved: anxiety, tremors, and trouble sleeping. A minority of participants (6.0%, n = 12) indicated cannabis worsened opioid withdrawal, specifically symptoms of yawning, teary eyes, and runny nose. Across all symptoms, more participants indicated that symptoms improved with cannabis compared to those that indicated symptoms worsened with cannabis. Women reported greater relief from withdrawal with cannabis use than men.
Discussion: These results show that cannabis may improve opioid withdrawal symptoms and that the size of the effect is clinically meaningful. It is important to note that symptoms are exacerbated with cannabis in only a minority of individuals. Prospectively designed studies examining the impact of cannabis and cannabinoids on opioid withdrawal are warranted.

The influence of cannabinoids on generic traits of neurodegeneration
S G Fagan and V A Campbell
British Journal of Pharmacology (2014) 171 1347–136
DOI:10.1111/bph.12492
https://www.ncbi.nlm.nih.gov/pmc/art...h0171-1347.pdf
In an increasingly ageing population, the incidence of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are rising. While the aetiologies of these disorders are different, a number of common mechanisms that underlie their neurodegenerative components have been elucidated; namely neuroinflammation, excitotoxicity, mitochondrial dysfunction and reduced trophic support. Current therapies focus on treatment of the symptoms and attempt to delay the progression of these diseases but there is currently no cure. Modulation of the endogenous cannabinoid system is emerging as a potentially viable option in the treatment of neurodegeneration. Endocannabinoid signalling has been found to be altered in many neurodegenerative disorders. To this end, pharmacological manipulation of the endogenous cannabinoid system, as well as application of phytocannabinoids and synthetic cannabinoids have been investigated. Signalling from the CB1 and CB2 receptors are known to be involved in the regulation of Ca2+ homeostasis, mitochondrial function, trophic support and inflammatory status, respectively, while other receptors gated by cannabinoids such as PPARγ, are gaining interest in their anti-inflammatory properties. Through multiple lines of evidence, this evolutionarily conserved neurosignalling system has shown neuroprotective capabilities and is therefore a potential target for neurodegenerative disorders. This review details the mechanisms of neurodegeneration and highlights the beneficial effects of cannabinoid treatment.

The in vitro efect of delta-9-tetrahydrocannabinol and cannabidiol on whole blood viscosity, elasticity and membrane integrity
Tameika R. James, Andrea A. Richards, Dwight A. Lowe, Walton A. Reid, Charah T. Watson and Dagogo J. Pepple
Journal of Cannabis Research (2022) 4:15
DOI: 10.1186/s42238-022-00126-z
Background: The main biological activities of cannabis are due to the presence of several compounds known as cannabinoids. Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two of the main cannabinoids. Studies have shown that the efects of THC can be modulated by CBD. Objective: This study aims to look at the efect of diferent concentrations of THC and CBD separately and in combination, on blood viscosity, elasticity and membrane integrity. Methods: Blood samples were collected from twenty-four healthy adult non-smokers. Blood viscosity and elasticity were determined using the Vilastic Scientifc Bioprofler for diferent concentrations (0, 2.5, 25, 50 and 100ng/ml) of CBD and THC respectively, as well as in extracts with combinations of CBD and THC in 4:1 and 1:1 ratios respectively. Repeated measures analysis of variance (ANOVA) was used to determine the diference between the means of the groups. Results: Blood viscosity increased signifcantly with increasing concentrations of both THC and CBD from 25ng/ ml up to 100ng/ml ranging from 6.45±0.36mPa·s to 11.60±1.12mPa·s for THC and ranging from 5.46±0.24mPa·s to 9.91±1.10mPa·s for CBD respectively, being more pronounced in the extracts at 21.33±2.17mPa·s for the 4THC:1CBD extract and 21.76±1.88mPa·s for the 1THC:1CBD extract. There was no signifcant increase in elasticity for THC and CBD separately. However, a signifcant increase in elasticity was observed in the extracts. THC and CBD afected red cell morphology resulting in complete disintegration at the highest concentrations. Conclusions: THC and CBD increased red blood cell viscosity and elasticity separately and in combination. They also adversely afected membrane integrity.

The major endocannabinoid anandamide (AEA) induces apoptosis of human granulosa cells
L Costa , B Moreia-Pinto , E Felgueira , A Ribeiro , I Rebelo , B M Fonseca
Prostaglandins Leukot Essent Fatty Acids 2021
DOI: 10.1016/j.plefa.2021.102311
The endocannabinoid system (ECS) plays a crucial role in human reproduction. Changes in anandamide (AEA) levels affect reproductive events and has already been suggested as biomarker of reproductive potential of male and female gametes. Although cannabinoid-receptor 1 (CB1) was already identified in human granulosa cells (hGCs) the ECS was not characterized on granulosa cells line COV434 nor the effects of AEA on GCs viability and function depicted. Therefore, the aim of this study was to characterize the ECS elements and explore the effects of AEA on both COV434 and hGCs. Our results revealed that hGCs express the full enzymatic machinery responsible for AEA metabolism as well as cannabinoid receptors. In addition, AEA induced a reduction in both COV434 and hGCs viability in a concentration and time-dependent manner. Nevertheless, the effects of AEA in cell viability was independent of either CB1 or CB2 receptors. There was no ROS release in both cell models; however, AEA induced morphological changes, presenting chromatin condensation at 72 h, and variation on mitochondrial membrane potential. Moreover, AEA induced an increase in caspase -3/-7 activities in both cell models, but in hGCs there was also an increase in caspase 8 activity. This study supports the idea that ECS balance is crucial for folliculogenesis and oocyte quality as dysregulated AEA levels may compromise female fertility.
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The Management of Cancer Symptoms and Treatment Induced Side Effects With Cannabis or Cannabinoids
Michelle Sexton, Jose M. Garcia, MD, Aminah Jatoi, Carey S. Clark, Mark S. Wallace
J Natl Cancer Inst Monogr (2021) 2021(58): lgab011
DOI: 10.1093/jncimonographs/lgab011
https://academic.oup.com/jncimono/ar.../58/86/6446200
Cannabis and cannabinoids are increasingly being accessed and used by patients with advanced cancer for various symptoms and general quality of life. Specific symptoms of pain, nausea and vomiting, loss of appetite and cachexia, anxiety, sleep disturbance, and medical trauma are among those that have prompted patients with cancer to use cannabis. This conference report from the National Cancer Institute’s “Cannabis, Cannabinoid and Cancer Research Symposium” on the topic of “Cancer Symptom/Treatment Side Effect Management” is an expert perspective of cannabis intervention for cancer and cancer treatment-related symptoms. The purpose of the symposium was to identify research gaps, describe the need for high-quality randomized prospective studies of medical cannabis for palliative care in patients with cancer, and evaluate the impact of medical cannabis on cancer survivors’ quality of life. Further, education of clinicians and affiliated health-care providers in guiding cancer patients in using cannabis for cancer care would benefit patients. Together, these steps will further aid in refining the use of cannabis and cannabinoids for symptom palliation and improve safety and efficacy for patients.

The Medical Geography of Cannabinoid Botanicals in Washington State: Access, Delivery, and Distress
Sunil Kumar Aggarwal
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
https://www.academia.edu/819092/The_...y_and_Distress
Though rendered dormant by a post-1937 Cannabis sativa L. prohibition, the emerging field of cannabinoid medicine is growing in the United States as ever greater numbers of physicians become educated about the physiologic importance of the endogenous cannabinoid system and about the wide safety margins and broad clinical efficacies of cannabinoid drugs, available in both purely botanical and purely chemical varieties and useful for managing pain and other conditions in the growing chronically and critically ill patient population. Research presented here is focused on medical access and delivery of cannabinoid botanicals in Washington State and seeks to map the geography of this developing cannabinoid medical care system by taking medical geographic “snapshots” of two purposefully chosen locations: a rural clinic site in Washington State where patients currently access cannabinoid botanicals for medical use in the treatment of chronic pain syndromes with acceptable safety under medical supervision and another site where qualifying patients are delivered environmentally culled cannabinoid botanicals. At the former site, retrospective chart reviews were conducted with 139 patients with chronic pain, and at the latter site, a convenience sample of 37 qualifying patients delivered a monoclonal lot of cannabinoid botanical medicine were prospectively studied using standard and tailored survey instruments. A political ecology of disease approach was employed to rationalize and depathologize patients’ mental distress at potentially facing possession-related legal problems due to their consumption of the still-contraband biota. Results provide quantitative and qualitative insight into the frail health status in both samples of qualifying patients and give a grounded understanding of the lengths that patients and care providers go, despite multiple hurdles, to access and deliver treatment with cannabinoid botanicals that relieves patients’ diverse symptoms and improves their health-related quality-of life.

The Medicalization of Cannabis
Edited by S M Crowther, L A Reynolds and E M Tansey
The transcript of a Witness Seminar held by the Wellcome Trust Centre for the History of Medicine at UCL, London, on 24 March 2009
https://qmro.qmul.ac.uk/xmlui/bitst...icalizationofCannabis2010FINAL.pdf?sequence=2
CONTENTS Illustrations and credits v
Abbreviations vii
Witness Seminars: Meetings and publications; Acknowledgements E M Tansey, L A Reynolds and S M Crowther ix
Introduction Leslie Iversen xix
Transcript Edited by S M Crowther, L A Reynolds and E M Tansey 1
Appendix 1 Diagrams of the structures of some major plant cannabinoids and of certain structurally related synthetic cannabinoids 73
References 75
Biographical notes 89
Index 97
The medical use of cannabis has a very long history; it was used for thousands of years in Indian and other Asian medicine and was first introduced to the west in the mid-nineteenth century by a brilliant young doctor, W B O’Shaughnessy, returning to England after service in India. Cannabis was taken up enthusiastically by physicians in Europe and the US and was widely used for almost a hundred years until it fell out of favour as new and more easily standardized medicines became available and government regulations were imposed. Tincture of cannabis finally left the British Pharmacopoeia in the mid-1970s.
This Witness Seminar, however, was focused not on this early history but on the resurgence of interest in medical cannabis that has occurred in the past few decades. It brought together a group of people with diverse expertise who had witnessed at first hand the development of this field. Although the seminar did not deal at all with the recreational use of cannabis, it is impossible to consider the history of medical cannabis without considering the impact that the rapid growth of the illicit recreational use of the drug in the latter part of the twentieth century has had. The ‘cannabis wars’ have been fought between those who believe it to be harmless and medically useful, and those who see it as a danger to health and to society without any legitimate medical use. For many years the stigmatization of cannabis had a negative influence on the availability of research funding and promoted reluctance on the part of doctors and pharmaceutical companies to be involved in research on the medical uses of cannabis.

The Medicinal Use of Cannabis and Cannabinoids—An International Cross-Sectional Survey on Administration Forms.
Hazekamp, A., Ware, M. A., Muller-Vahl, K. R., Abrams, D., & Grotenhermen, F.
Journal of Psychoactive Drugs, 45(3), 199–210. (2013).
doi:10.1080/02791072.2013.805976
Cannabinoids, including tetrahydrocannabinol and cannabidiol, are the most important active constituents of the cannabis plant. Over recent years, cannabinoid-based medicines (CBMs) have become increasingly available to patients in many countries, both as pharmaceutical products and as herbal cannabis (marijuana). While there seems to be a demand for multiple cannabinoid-based therapeutic products, specifically for symptomatic amelioration in chronic diseases, therapeutic effects of different CBMs have only been directly compared in a few clinical studies. The survey presented here was performed by the International Association for Cannabinoid Medicines (IACM), and is meant to contribute to the understanding of cannabinoid-based medicine by asking patients who used cannabis or cannabinoids detailed questions about their experiences with different methods of intake. The survey was completed by 953 participants from 31 countries, making this the largest international survey on a wide variety of users of cannabinoid-based medicine performed so far. In general, herbal non-pharmaceutical CBMs received higher appreciation scores by participants than pharmaceutical products containing cannabinoids. However, the number of patients who reported experience with pharmaceutical products was low, limiting conclusions on preferences. Nevertheless, the reported data may be useful for further development of safe and effective medications based on cannabis and single cannabinoids.

The Metabolic Efficacy of a Cannabidiolic Acid (CBDA) Derivative in Treating Diet- and Genetic-Induced Obesity
Elad Ben-Cnaan , Anna Permyakova, Shahar Azar, Shira Hirsch, Saja Baraghithy, Liad Hinden and Joseph Tam
Int. J. Mol. Sci. 2022, 23, 5610.
https://doi.org/10.3390/ijms23105610
Obesity is a global medical problem; its common form is known as diet-induced obesity (DIO); however, there are several rare genetic disorders, such as Prader–Willi syndrome (PWS), that are also associated with obesity (genetic-induced obesity, GIO). The currently available therapeutics
for treating DIO and GIO are very limited, and they result in only a partial improvement. Cannabidiolic acid (CBDA), a constituent of Cannabis sativa, gradually decarboxylates to cannabidiol (CBD). Whereas the anti-obesity properties of CBD have been reasonably identified, our knowledge of the pharmacology of CBDA is more limited due to its instability. To stabilize CBDA, a new derivative, CBDA-O-methyl ester (HU-580, EPM301), was synthesized. The therapeutic potential of EPM301 in appetite reduction, weight loss, and metabolic improvements in DIO and GIO was tested in vivo. EPM301 (40 mg/kg/d, i.p.) successfully resulted in weight loss, increased ambulation, as well as improved glycemic and lipid profiles in DIO mice. Additionally, EPM301 ameliorated DIO-induced hepatic dysfunction and steatosis. Importantly, EPM301 (20 and 40 mg/kg/d, i.p.) effectively reduced body weight and hyperphagia in a high-fat diet-fed Magel2null mouse model for PWS. In addition, when given to standard-diet-fed Magel2null mice as a preventive treatment, EPM301 completely inhibited weight gain and adiposity. Lastly, EPM301 increased the oxidation of different nutrients in each strain. All together, EPM301 ameliorated obesity and its metabolic abnormalities in both DIO and GIO. These results support the idea to further promote this synthetic CBDA derivative toward clinical evaluation in humans. .

The Mystery of Low Phosphate: Marijuana is the Smoking Gun
Talha Ahmed, Safwan Muhammad, Ayesha Safdar, Amna Shaukat
Cureus 12(3): (2020)
DOI: 10.7759/cureus.7392
Hypophosphatemia is a rarely reported side-effect of cannabis use. The potential mechanisms of hypophosphatemia include enteric malabsorption/loss, excessive urinary excretion, or rapid trans-cellular shifts. Severe hypophosphatemia from daily marijuana use is a rare side-effect. A trans-cellular shift is the most likely proposed mechanism. Although it tends to self-correct fairly rapidly, close observation for the dreaded consequences related to hypophosphatemia is required. Both the users and providers must be aware of this rare association of hypophosphatemia with daily marijuana use

The National Cancer Institute and Cannabis and Cannabinoids Research
Gary L. Ellison, L. Alejandro Salicrup, Andrew N. Freedman, Yali Fu, Sharon Ross, Ronald L. Johnson, Alexis Bakos, Jeffrey D. White
J Natl Cancer Inst Monogr (2021) 2021(58): lgab014
DOI: 10.1093/jncimonographs/lgab014
https://academic.oup.com/jncimono/ar.../58/35/6446198
The landscape of both recreational and medicinal cannabis use has changed dramatically over the past decade; however, research examining the risks and benefits of cannabis and cannabinoid use has lagged significantly behind the increased media promotion and their use by the general public and cancer patients. The National Cancer Institute (NCI) has supported cannabis-related research projects and funding opportunity announcements. In addition, NCI organized a virtual symposium on December 15-18, 2020, to discuss recent research findings on the use of cannabis and cannabinoids in relationship to cancer risk, prevention, and care. Specifically, the symposium sought to highlight the state of the science regarding cannabis, including the chemical constituents of cannabis (eg, cannabinoids), and cancer research involving cannabis, including cancer epidemiology, use in cancer patients, cancer biology and prevention, and preclinical and clinical cancer symptom and treatment side effect management with cannabis and cannabinoids as therapeutics. The symposium identified promising areas of future study, current barriers to conducting the research, and strategies to overcome those barriers. The series of papers in this special edition provide a summary of the symposium sessions as well as a synopsis of opportunities and challenges related to conducting research in this area.

The Pharmacological Basis of Cannabis Therapy for Epilepsy
Doodipala Samba Reddy and Victoria M. Golub
J Pharmacol Exp Ther. 2016 Apr;357(1):45-55.
DOI: 10.1124/jpet.115.230151
Recently, cannabis has been suggested as a potential alternative therapy for refractory epilepsy, which affects 30% of epilepsy patients including children who do not respond to current medications. There is a large unmet medical need for new antiepileptics for refractory epilepsy and conditions associated with refractory seizures that would not interfere with normal function. The two chief cannabinoids are delta-9-tetrahyrdrocannabinol, the major psychoactive component of marijuana, and cannabidiol (CBD), the major non-psychoactive component of marijuana. There are claims of clinical efficacy of CBD-predominant cannabis or medical marijuana for epilepsy, mostly from limited studies, surveys or case reports. However, the mechanisms underlying the antiepileptic efficacy of cannabis remain unclear. This article highlights the pharmacological basis of cannabis therapy, with an emphasis on the endocannabinoid mechanisms underlying the emerging neurotherapeutics of CBD in epilepsy. CBD is anticonvulsant, but it has a low affinity for the cannabinoid CB1 and CB2 receptors; therefore the exact mechanism by which it affects seizures remains poorly understood. A rigorous clinical evaluation of pharmaceutical CBD products is needed to establish the safety and efficacy for the treatment of epilepsy. Identification of mechanisms underlying the anticonvulsant efficacy of CBD is additionally critical to identify other potential treatment options.

The Pharmacology and Therapeutic Potential of Cannabidiol
R.G. Pertwee
In: Di Marzo, V., Ed., Cannabinoids, Kluwer Academic/Plenum Publishers, New York, 32-83.
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This review surveys the evidence that the non-psychotropic plant cannabinoid, cannabidiol (CBD), has therapeutic potential as a neuroprotective agent and for the management of epilepsy, glaucoma, central and peripheral inflammatory disorders, anxiety, acute schizophrenia, dystonia, nausea and cancer. Current information about the pharmacological effects of CBD and about the molecular targets with which this cannabinoid and its synthetic
analogues interact is also presented. These targets include various receptor types, both established and postulated, release processes for certain neurotransmitters and cytokines, receptor signalling mechanisms, membrane transporters, and enzymes responsible for catalysing the biosynthesis and/or metabolism of prostaglandins, endogenous cannabinoids and other eicosanoids. The ability of CBD to alter membrane fluidity and stability and to interact with cytochrome P450 (CYP) enzymes is also discussed as is its ability to attenuate or enhance some effects of the main psychotropic plant cannabinoid, ?9-tetrahydrocannabinol.

The phytocannabinoid (-)-cannabidiol (CBD) operates as a complex, differential modulator of human hair growth: Anti-inflammatory submicromolar versus hair growth inhibitory micromolar effects.
Szabó, I. L., Herczeg-Lisztes, E., Béke, G., Tóth, K. F., Paus, R., Oláh, A., & Bíró, T.
Journal of Investigative Dermatology.(2019).
doi:10.1016/j.jid.2019.07.690
Most cases of excessive hair loss and unwanted hair growth (e.g., effluvium, alopecia, hirsutism, hypertrichosis) result in part from major disturbances in the cyclic transformation of hair follicles (HFs), namely in their switch from active growth and pigmented hair shaft production (anagen) to apoptosisdriven HF involution (catagen) (Oh et al., 2016; Paus and Cotsarelis, 1999). It is now clear that this switch is profoundly influenced by complex neuroendocrine pathways, which still await systematic therapeutic targeting (Paus et al., 2014). In this context, the endocannabinoid system is of special interest (Maccarrone et al., 2015; Ola´h and Bı´ro´, 2017; To´th et al., 2019). Specifically, activation of CB1 receptors by the endocannabinoid anandamide, and the plant-derived (e)-D9 -trans-tetrahydrocannabinol, promotes premature catagen entry in human scalp HFs ex vivo (Telek et al., 2007). However, 2- arachidonoylglycerol, another prototypic endocannabinoid, does not modulate HF growth ex vivo, suggesting functional heterogeneity between different cannabinoids in terms of human hair growth modulation (Telek et al., 2007).

The prevalence of cannabinoid hyperemesis syndrome among regular marijuana smokers in an urban public hospital. Joseph Habboushe, Ada Rubin, Haoming Liu and Robert S. Hoffman
Basic & Clinical Pharmacology & Toxicology, 2018, 122, 660–662
Doi: 10.1111/bcpt.12962
https://onlinelibrary.wiley.com/doi/...111/bcpt.12962
Epidemiological data, including prevalence, for cannabinoid hyperemesis syndrome (CHS) remain largely unknown. Without these data, clinicians often describe CHS as ‘rare’ or ‘very rare’ without supporting evidence. We seek to estimate the prevalence of CHS in a population of patients presenting to a socio-economically and racially diverse urban Emergency Department of a public hospital. This study consisted of a questionnaire administered to a convenience sample of patients presenting to the ED of the oldest public hospital in the United States. Trained Research Associates (RAs) administered the questionnaire to patients between the ages of 18–49 years who reported smoking marijuana at least 20 days per month. The survey included questions related to CHS symptoms (nausea and vomiting) and Likert scale rankings on eleven symptom relief methods, including ‘hot showers’. Patients were classified as experiencing a phenomenon consistent with CHS if they reported smoking marijuana at least 20 days per month and also rated ‘hot showers’ as five or more on the ten-point symptom relief method Likert scale for nausea and vomiting. Among 2127 patients approached for participation, 155 met inclusion criteria as smoking 20 or more days per month. Among those surveyed, 32.9% (95% CI, 25.5–40.3%) met our criteria for having experienced CHS. If this is extractable to the general population, approximately 2.75 million (2.13–3.38 million) Americans may suffer annually from a phenomenon similar to CHS.

The Potential of Salivary Lipid-Based Cannabis-Responsive Biomarkers to Evaluate Medical Cannabis Treatment in Children with Autism Spectrum Disorder
Michael Siani-Rose, Robert McKee, Stephany Cox, Bonni Goldstein,
Donald Abrams, Myiesha Taylor, and Itzhak Kurek Cannabis and Cannabinoid Research (march 2022)
https://doi.org/10.1089/can.2021.0224
https://www.liebertpub.com/doi/10.10...m_medium=email
Introduction: Autism spectrum disorder (ASD) is a group of heterogeneous neurodevelopmental conditions affecting social communication and social interaction. Medical cannabis (MC) treatment shows promising results as an approach to reduce behavioral difficulties, as determined mainly by subjective observations. We have recently shown the potential of cannabis-responsive biomarkers detected in saliva of children with ASD to objectively quantify the impact of successful MC treatment using a metabolomics approach. Since the pathology of ASD is associated with abnormal lipid metabolism, we used lipidomics on the same samples to (1) expand the repertoire of cannabis-responsive biomarkers and (2) provide preliminary insight into the role of MC on lipid metabolism.
Materials and Methods: Saliva samples collected from children with ASD (n=15) treated with MC (both before and at the time of maximal impact of treatment) and an age-matched group of typically developing (TD) children (n=9) were subjected to untargeted lipidomics. The study was observational. Each child from the ASD group was receiving a unique individualized MC treatment regimen using off-the-shelf products as permitted by California law under physician supervision for at least 1 year. Doses of tetrahydrocannabinol (THC) ranged from 0.05 to 50 mg and cannabidiol (CBD) from 7.5 to 200 mg per treatment. The ASD group was evaluated for signs of improvement using parental brief Likert scale surveys.
Results: Twenty-two potential lipid-based cannabis-responsive biomarkers exhibiting a shift toward the TD physiological levels in children with ASD after MC treatment were identified. Members from all five lipid subclasses known to be present in saliva were characterized. Preliminary lipid association network analysis suggests involvement of two subnetworks previously linked to (1) inflammation and/or redox regulation and (2) oxidative stress. The significant changes in sphingomyelin in this study and in N-acetyl-aspartate (NAA) previously detected in the metabolomics analysis of the same saliva samples may indicate a role of MC in neuron function.
Conclusions: Our findings suggest that lipid metabolites in saliva can potentially serve as cannabis-responsive biomarkers and objectively quantify the impact of MC treatment, and indicate a possible mechanism of action for MC. This preliminary study requires further investigation with a larger population and appropriate clinical trial monitoring. Link with paper
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The potential therapeutic effects of THC on Alzheimer's disease.
Cao C, Li Y, Liu H, Bai G, Mayl J, Lin X, Sutherland K, Nabar N, Cai J.
J Alzheimers Dis. 2014;42(3):973-84
doi: 10.3233/JAD-140093.
The purpose of this study was to investigate the potential therapeutic qualities of ?9-tetrahydrocannabinol (THC) with respect to slowing or halting the hallmark characteristics of Alzheimer's disease. N2a-variant amyloid-? protein precursor (A?PP) cells were incubated with THC and assayed for amyloid-? (A?) levels at the 6-, 24-, and 48-hour time marks. THC was also tested for synergy with caffeine, in respect to the reduction of the A? level in N2a/A?PPswe cells. THC was also tested to determine if multiple treatments were beneficial. The MTT assay was performed to test the toxicity of THC. Thioflavin T assays and western blots were performed to test the direct anti-A? aggregation significance of THC. Lastly, THC was tested to determine its effects on glycogen synthase kinase-3? (GSK-3?) and related signaling pathways. From the results, we have discovered THC to be effective at lowering A? levels in N2a/A?PPswe cells at extremely low concentrations in a dose-dependent manner. However, no additive effect was found by combining caffeine and THC together. We did discover that THC directly interacts with A? peptide, thereby inhibiting aggregation. Furthermore, THC was effective at lowering both total GSK-3? levels and phosphorylated GSK-3? in a dose-dependent manner at low concentrations. At the treatment concentrations, no toxicity was observed and the CB1 receptor was not significantly upregulated. Additionally, low doses of THC can enhance mitochondria function and does not inhibit melatonin's enhancement of mitochondria function. These sets of data strongly suggest that THC could be a potential therapeutic treatment option for Alzheimer's disease through multiple functions and pathways.

The protective mechanism of cannabidiol in cardiac injury: A systematic review of non-clinical studies.
Shayesteh MR, Haghi-Aminjan, Mousavi MJ, Momtaz S, Abdollahi M.
Curr Pharm Des. 2019 Jul 9. doi: 10.2174/2210327909666190710103103. [Epub ahead of print]
BACKGROUND: Cardiac disease is accounted as the leading cause of worldwide morbidity and mortality, mainly in association with induction of inflammation and oxidative stress. The disease is characterized by the overproduction of reactive oxygen and/or nitrogen species (ROS/RNS), and reduced antioxidant capacity. Cannabidiol (CBD) is a non-psychoactive ingredient of marijuana that reported to be safe and well tolerated in patients. Due to its pleiotropic effect, CBD has been shown to exert cytoprotective effects. This study intended to clarify the mechanisms and the potential role of CBD regarding cardiac injuries treatment.
METHODS: A systematic literature search was conducted, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, in the electronic databases including PubMed, Web of Science, Scopus, and Embase up to July 2018 using predefined search terms in the titles and abstracts by two independent investigators. Accordingly, a set of pre-specified inclusion and exclusion criteria were considered and 8 articles were ultimately included in this study.
RESULTS: Our findings obviously demonstrate that CBD has multi-functional protective assets to improve cardiac injuries; preliminary through scavenging of free radicals, and reduction of oxidative stress, apoptosis, and inflammation.
CONCLUSION: CBD can protect against cardiac injuries, mainly through its anti-oxidant, anti-inflammatory, and anti-apoptotic effects on the basis of non-clinical studies.

The Relationship between Cannabis and Tobacco Co-administration and Long-Term Patterns of Cannabis Use in Young Adults Who Use Stimulants: A Prospective Population-Based Study.
Abdullah Ahmed Alghamdi, Coral Gartner, Jake M. Najman, Gary Chan, Andrew Smirnov
Substance Use & Misuse Nov. 2021
DOI: 10.1080/10826084.2021.1975744
https://www.tandfonline.com/doi/abs/...75744?journalC ode=isum20
Background: Co-administering cannabis with tobacco (i.e. co-administration of the substances mixed together) is a common practice among cannabis users, but the consequences of this practice are not well understood. This study examines the relationship between co-administering cannabis with tobacco and the long-term frequency of cannabis use in a young adult population group with high rates of cannabis and tobacco use.
Methods: The data are from an Australian prospective population-based study of young adults who recurrently used amphetamine-type stimulants (ATS). The mean age of participants was 20.8 years at baseline, sample size ( = 277), and 47% were female. We examined the frequency and quantity of cannabis consumption over 4 ½ years. Negative binomial regression analysis was conducted to examine the frequency of cannabis use at 12-month follow-up and at 4 ½ years, with co-administering practices as the predictor.
Results: At every time interval, participants who always co-administered their cannabis with tobacco used cannabis on more days in the last month than those who only sometimes co-administered, rarely co-administered, or never co-administered these substances ( < 0.001). Sometimes co-administering cannabis with tobacco at baseline predicted more frequent cannabis use at 12-month follow-up (adjusted IRR: 2.25, 95% CI: 1.05, 4.78), independently of the baseline frequency of cannabis use. However, levels of co-administering cannabis with tobacco at 12-month follow-up (rarely, sometimes, and always) did not predict high levels of cannabis use at 4 ½ years follow-up after adjusting for cannabis use at 12-month follow-up.
Conclusions: Among people who use ATS and cannabis, frequent cannabis use may be a marker of the practice of co-administering cannabis with tobacco, and can be used to target tobacco cessation interventions in these populations.
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Not cannabis specific
The Revaluation of Plant-Derived Terpenes to Fight Antibiotic-Resistant Infections.
Cappiello, F., Loffredo, M. R., Del Plato, C., Cammarone, S., Casciaro, B., Quaglio, D., … Ghirga, F.
Antibiotics, 9(6), 325.(2020).
doi:10.3390/antibiotics9060325
The discovery of antibiotics has revolutionized the medicine and treatment of microbial infections. However, the current scenario has highlighted the difficulties in marketing new antibiotics and an exponential increase in the appearance of resistant strains. On the other hand, research in the field of drug-discovery has revaluated the potential of natural products as a unique source for new biologically active molecules and scaffolds for the medicinal chemistry. In this review, we first contextualized the worldwide problem of antibiotic resistance and the importance that natural products of plant origin acquire as a source of new lead compounds. We then focused on terpenes and their potential development as antimicrobials, highlighting those studies that showed an activity against conventional antibiotic-resistant strains

The Role of Cannabinoid Receptor Type 2 in the Bone Loss Associated With Pediatric Celiac Disease
Chiara Tortora, Francesca Punzo, y Maura Argenziano, y Alessandra Di Paola, Carlo Tolone, Caterina Strisciuglio, and Francesca Rossi AB
JPGN Volume 71, Number 5, November 2020
DOI: 10.1097/MPG.0000000000002863
Objectives: In this study, we investigated the role of the cannabinoid receptor type 2 (CB2) in the bone loss associated with celiac disease (CD) evaluating the effect of its pharmacological modulation on osteoclast activity. We previously demonstrated a significant association between the CB2 Q63R variant and CD, suggesting it as a possible disease biomarker. Moreover, CB2 stimulation is beneficial for reducing osteoclast activity in several bone pathologic conditions.
Methods: In vitro osteoclasts (OCs) were differentiated from peripheral blood mononuclear cells of healthy donors, CD children at diagnosis and after 1 year of gluten-free diet (GFD) and characterized by real-time PCR and western blot for the expression of CB2 and specific osteoclastic markers, TRAP and Cathepsin K. TRAP assay and Bone Resorption assay were performed to evaluate osteoclast activity before and after 48 h exposure to CB2 selective drugs (JWH-133 and AM630) and Vitamin D.
Results: We found in CD patients an osteoclast hyperactivation and low levels of CB2. CB2 stimulation with JWH-133 agonist is more effective than Vitamin D in reducing osteoclast activity whereas CB2 blockade with AM630 increases osteoclast activation. The anti-osteoporotic effect of JWH-133 decreases when used in co-treatment with vitamin D. GFD reduces osteoclast activity without restore CB2 expression.
Conclusions: CB2 could be a molecular marker to predict the risk of bone alterations in CD and a pharmacological target to reduce bone mass loss in patients who need a direct intervention on bone metabolism, in addition to the GFD.

The role of cannabinoids in adult neurogenesis: Cannabinoids and Neurogenesis
Br J Pharmacol. 2015;172(16):3950-3963.
doi:10.1111/bph.13186
https://www.researchgate.net/publica...d_Neurogenesis The processes underpinning post-developmental neurogenesis in the mammalian brain continue to be defined. Such processes involve the proliferation of neural stem cells (NSCs) and neural progenitor cells (NPCs), neuronal migration, differentiation and integration into a network of functional synapses within the brain. Both intrinsic (cell signalling cascades) and extrinsic (neurotrophins, neurotransmitters, cytokines, hormones) signalling molecules are intimately associated with adult neurogenesis and largely dictate the proliferative activity and differentiation capacity of neural cells. Cannabinoids are a unique class of chemical compounds incorporating plant-derived cannabinoids (the active components of Cannabis sativa), the endogenous cannabinoids and synthetic cannabinoid ligands, and these compounds are becoming increasingly recognized for their roles in neural developmental processes. Indeed, cannabinoids have clear modulatory roles in adult neurogenesis, likely through activation of both CB1 and CB2 receptors. In recent years a large body of literature has deciphered the signalling networks involved in cannabinoid-mediated regulation of neurogenesis. This timely review summarises the evidence that the cannabinoid system is intricately associated with neuronal differentiation and maturation of NPCs, and highlights intrinsic/extrinsic signalling mechanisms that are cannabinoid targets. Overall these findings identify the central role of the cannabinoid system in adult neurogenesis in the hippocampus and the lateral ventricles, and hence provide insight into the processes underlying post-developmental neurogenesis in the mammalian brain.

The role of cannabinoids in epilepsy treatment: a critical review of efficacy results from clinical trials.
Nabbout R1, Thiele EA2.
Epileptic Disord. 2020 Jan 8.
doi: 10.1684/epd.2019.1124
CBD was shown to have anti-seizure activity based on in vitro and in vivo models. However, several reports of small series or case reports of the use of cannabis extracts in epilepsy yielded contradictory results and the efficacy of cannabis use in patients with epilepsy have also been inconclusive. In 2013, the first Phase 1 trial for a purified form of CBD (Epidiolex/Epidyolex; >99% CBD), developed by GW Pharma, showed some efficacy signals and subsequently, a comprehensive program on the efficacy and tolerability of this compound for the treatment of drug-resistant epilepsies was initiated. Results of these trials led to the FDA and EMA approval respectively in 2018 and 2019 for the treatment of seizures associated with two rare epilepsies: Lennox-Gastaut syndrome (LGS) or Dravet syndrome (DS) in patients two years of age and older. Thus, CBD became the first FDA-approved purified drug substance derived from cannabis and also the first FDA-approved drug for the treatment of seizures in DS. We detail the clinical studies using purified CBD (Epidiolex/Epidyolex), including the first open interventional exploratory study and Randomized Control Ttrials for DS and LGS.

The Role of Cannabinoids in Inflammatory Modulation of Allergic Respiratory Disorders, Inflammatory Pain and Ischemic Stroke
Alessandro Pini, Guido Mannaioni, Domenico Pellegrini-Giampietro, Maria Beatrice Passani, Rosanna Mastroianni, Daniele Bani and Emanuela Masini
Current Drug Targets Volume 13 , Issue 7 , 2012 Page: [984 - 993]
DOI: 10.2174/138945012800675786
This review is intended to offer updated information on the involvement of cannabinoids in the process of inflammation, focusing on immune/allergic reactions, inflammatory pain and neuroinflammation and discussing the interactions among endocannabinoid metabolism, prostanoids and nitric oxide. Two types of cannabinoid receptors, CB1 and CB2, which belong to the G protein-coupled receptor family, have been identified and are targeted by numerous exogenous and endogenous ligands. The activation of CB2 receptors on mast cells has direct antiinflammatory effects, causing decreased release of pro-inflammatory mediators by these cells. The activation of CB1 receptors on bronchial nerve endings has bronchodilator effects by acting on the airway smooth muscle and may be beneficial in airway hyperreactivity and asthma. Moreover, pharmacologic interference with endocannabinoid metabolism has been demonstrated to result in anti-nociceptive activity, mediated by CB1 and CB2 receptors, in animal models of inflammatory pain. The presence of endocannabinoid machinery in the central nervous system, together with high levels of CB1 expression, suggests that the endocannabinoid system is an important modulator of neuroinflammation and a possible drug target. In selected conditions, the activation of CB1 receptors in cerebral blood vessels can have beneficial antiischemic effects. However, as endocannabinoids can also bind to vanilloid receptors, they may also mediate neurotoxic effects.

The Role of Cannabinoids in Neuroanatomic Alterations in Cannabis Users
Valentina Lorenzetti, Nadia Solowij, and Murat Yu?cel
Biological Psychiatry April 1, 2016
Doi: 10.1016/j.biopsych.2015.11.013
The past few decades have seen a marked change in the composition of commonly smoked cannabis. These changes primarily involve an increase of the psychoactive compound ?9-tetrahydrocannabinol (THC) and a decrease
of the potentially therapeutic compound cannabidiol (CBD). This altered composition of cannabis may be linked to persistent neuroanatomic alterations typically seen in regular cannabis users. In this review, we summarize recent
findings from human structural neuroimaging investigations. We examine whether neuroanatomic alterations are 1) consistently observed in samples of regular cannabis users, particularly in cannabinoid receptor–high areas, which
are vulnerable to the effects of high circulating levels of THC, and 2) associated either with greater levels of cannabis use (e.g., higher dosage, longer duration, and earlier age of onset) or with distinct cannabinoid compounds (i.e., THC and CBD). Across the 31 studies selected for inclusion in this review, neuroanatomic alterations emerged across regions that are high in cannabinoid receptors (i.e., hippocampus, prefrontal cortex, amygdala, cerebellum). Greater dose and earlier age of onset were associated with these alterations. Preliminary evidence shows that THC exacerbates, whereas CBD protects from, such harmful effects. Methodologic differences in the quantification of levels of cannabis use prevent accurate assessment of cannabis exposure and direct comparison of findings across studies. Consequently, the field lacks large “consortium-style” data sets that can be used to develop reliable neurobiological models of cannabis-related harm, recovery, and protection. To move the field forward, we encourage a coordinated approach and suggest the urgent development of consensus-based guidelines to accurately and comprehensively quantify cannabis use and exposure in human studies.

The Role of Cannabinoids in the Setting of Cirrhosis
Pratima Dibba, Andrew A. Li, George Cholankeril, Umair Iqbal, Chiranjeevi Gadiparthi, Muhammad Ali Khan, Donghee Kim and Aijaz Ahmed
Medicines June 2018
https://pdfs.semanticscholar.org/e2c...879.1582090724
Although the mortality rates of cirrhosis are underestimated, its socioeconomic burden has demonstrated a significant global impact. Cirrhosis is defined by the disruption of normal liver architecture after years of chronic insult by different etiologies. Treatment modalities are recommended primarily in decompensated cirrhosis and specifically tailored to the different manifestations of hepatic decompensation. Antifibrogenic therapies are within an active area of investigation. The endocannabinoid system has been shown to play a role in liver disease, and cirrhosis specifically, with intriguing possible therapeutic benefits. The endocannabinoid system comprises cannabinoid receptors 1 (CB1) and cannabinoid receptor 2 (CB2) and their ligands, endocannabinoids and exocannabinoids. CB1 activation enhances fibrogenesis, whereas CB2 activation counteracts progression to fibrosis. Conversely, deletion of CB1 is associated with an improvement of hepatic fibrosis and steatosis, and deletion of CB2 results in increased collagen deposition, steatosis, and enhanced inflammation. CB1 antagonism has also demonstrated vascular effects in patients with cirrhosis, causing an increase in arterial pressure and vascular resistance as well as a decrease in mesenteric blood flow and portal pressure, thereby preventing ascites. In mice with hepatic encephalopathy, CB1 blockade and activation of CB2 demonstrated improved neurologic score and cognitive function. Endocannabinoids, themselves also have mechanistic roles in cirrhosis. Arachidonoyl ethanolamide (AEA) exhibits antifibrogenic properties by inhibition of HSC proliferation and induction of necrotic death. AEA induces mesenteric vasodilation and hypotension via CB1 induction. 2-arachidonoyl glycerol (2-AG) is a fibrogenic mediator independent of CB receptors, but in higher doses induces apoptosis of HSCs, which may actually show antifibrotic properties. 2-AG has also demonstrated growth-inhibitory and cytotoxic effects. The exocannabinoid, THC, suppresses proliferation of hepatic myofibroblasts and stellate cells and induces apoptosis, which may reveal antifibrotic and hepatoprotective mechanisms. Thus, several components of the endocannabinoid system have therapeutic potential in cirrhosis.

The role of endocannabinoid signaling in the molecular mechanisms of neurodegeneration in Alzheimer's disease
Gaurav Bedse, Adele Romano, Angelo M Lavecchia, Tommaso Cassano, Silvana Gaetani
J Alzheimers Dis . 2015;43(4):1115-36.

doi: 10.3233/JAD-141635
https://www.researchgate.net/publication/265019450_The_Role_of_Endocann abinoid_Signaling_in_the_Molec ular_Mechanisms_of_Neurodegene ration_in_Alzheimer's_Disease
Alzheimer's disease (AD) is the most common form of progressive neurodegenerative disease characterized by cognitive impairment and mental disorders. The actual cause and cascade of events in the progression of this pathology is not fully determined. AD is multifaceted in nature and is linked to different multiple mechanisms in the brain. This aspect is related to the lack of efficacious therapies that could slow down or hinder the disease onset/progression. The ideal treatment for AD should be able to modulate the disease through multiple mechanisms rather than targeting a single dysregulated pathway. Recently, the endocannabinoid system emerged as a novel potential therapeutic target to treat AD. In fact, exogenous and endogenous cannabinoids seem to be able to modulate multiple processes in AD, although the mechanisms that are involved are not fully elucidated. This review provides an update of this area. In this review, we recapitulate the role of endocannabinoid signaling in AD and the probable mechanisms through which modulators of the endocannabinoid system provide their effects, thus highlighting how this target might provide more advantages over other therapeutic targets.

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis

Natalia Battista, Rosaria Meccariello Gilda Cobellis, Silvia Fasano Monia Di Tommaso Valentina Pirazzi, Justin C.Konje, Riccardo Pierantoni, Mauro Maccarrone

Molecular and Cellular Endocrinology Volume 355, Issue 1, 15 May 2012, Pg 1-14

DOI: 10.1016/j.mce.2012.01.014
https://www.sciencedirect.com/science/article/pii/S0303720712000445?via=ihub
Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.
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The role of structure and molecular properties of terpenoids in determining their antimicrobial activity.
Griffin, S. G., Wyllie, S. G., Markham, J. L., & Leach, D. N.
Flavour and Fragrance Journal, 14(5), 322–332.(1999).
doi:10.1002/(sici)1099-1026(199909/10)14:5<322::aid-ffj837>3.0.co;2-4
The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into ®ve groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to con®rm these groupings and to show the dierences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans.

Role of the Adipocyte in Development of Type 2 Diabetes
(2011) Book
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Robert Dinu[/FONT], [FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Maria Mota,Simona Popa[/FONT]
DOI: 10.5772/1543

Not Cannabis specific
The Role of the Endocannabinoid System in the Pathogeny of Type 2 Diabetes
Robert Dinu. Maria Mota, Simona Popa
Chapter 15 in book Role of the Adipocyte in Development of Type 2 Diabetes (2011) Robert Dinu, Maria Mota, Simona Popa
DOI: 10.5772/1543 .
Diabetes mellitus is characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both (Bennett & Knowler, 2005). The pathophysiology of type 2 diabetes mellitus (T2DM) includes three mechanisms: insulin deficiency due to insufficient pancreatic insulin production, excessive hepatic glucose release and insulinresistance in peripheral tissues and liver (Gurber, 2010). Usually, T2DM is associated with a cluster of cardiovascular and metabolic risks including obesity (especially abdominal obesity), decreased levels of high-density lipoprotein cholesterol (HDL), high levels of triglycerides, elevated blood pressure and silent
inflammation and diabetes itself is considered to be a cardiovascular disease risk equivalent (Task Force on Diabetes and Cardiovascular Diseases of the European Society of Cardiology and the European Society for the Study of Diabetes, 2007). Taking into account the fact that T2DM is associated with obesity and excess fat accumulation may be the main cause of T2DM, one can evaluate the role of the endocannabinoid system (ECS) assessing (1) its role
in the pathogeny of obesity and (2) the role of ECS on hyperglycemia; this last part can be split in (a) the implication of ECS on insulin resistance and (b) its effects on insulin release. .

The Science of Marijuana
Leslie L. Iversen
2000 by Oxford University Press, Inc.
https://www.academia.edu/9803324/Science_of_Marijuana
Marijuana (cannabis) is among the most widely used of all psy choactive drugs. Despite the fact that its possession and use is illegal in most countries, cannabis is used regularly by as many
as 20 million people in the United States and Europe, and by millions more in other parts of the world. In recent years thousands of patients with acquired immunodeficiency syndrome (AIDS), multiple sclerosis, and a variety of other disabling diseases have begun to smoke marijuana illegally in the firm belief that it makes their symptoms better, despite the relative paucity of medical evidence to substantiate such belief.
The writer Stephen Jay Gould described his use of marijuana in suppressing the nausea associated with cancer treatment:
I had surgery, followed by a month of radiation, chemotherapy, more sur gery, and a subsequent year of additional chemotherapy. I found that I could control the less severe nausea of radiation by conventional medi cines. But when I started intravenous chemotherapy (Adriamycin),abso lutely nothing in the available arsenal of antiemetics worked at all. I was miserable and came to dread the frequent treatments with an almost per verse intensity.
. . . marijuana worked like a charm. I disliked the " side effect" of mental blurring (the "main effect" for recreational users), but the sheer bliss of not experiencing nausea —and then not having to fear it for all the days inter vening between treatments —was the greatest boost I received in all my year of treatment, and surely had a most important effect upon my eventual cure. (Grinspoon and Bakalar, 1993)
In California as part of the 1996 election voters approved "Proposi tion 215," which sought to make it legal to smoke marijuana with a doctor's recommendation. During the following year "cannabis buyers clubs" were established throughout the state to provide supplies of cannabis for medicinal use. On the whole these were run by well intentioned people and were strictly regulated, patients were checked for identity, medical records, and doctor's diagnosis and only then were they allowed to purchase a small quantity of marijuana. In the 1998 state elections a further six states voted to permit access to medical Marijuana, and in early 1999, such laws began to be put into effect, despite opposition from the federal government.

The Therapeutic Aspects of the Endocannabinoid System (ECS) for Cancer and their Development: From Nature to Laboratory
Mohammed I. Khan, Anna A. Sobociska, Anna M. Czarnecka, Magdalena Król, Bruno Botta and Cezary Szczylik
Current Pharmaceutical Design, 2016, 22, 1756-1766
https://www.eurekaselect.net/public/article/72458
doi: 10.2174/1381612822666151211094901
The endocannabinoid system (ECS) is a group of neuromodulatory lipids and their receptors, which are
widely distributed in mammalian tissues. ECS regulates various cardiovascular, nervous, and immune system functions inside cells. In recent years, there has been a growing body of evidence for the use of synthetic and natural cannabinoids as potential anticancer agents. For instance, the CB1 and CB2 receptors are assumed to play an important role inside the endocannabinoid system. These receptors are abundantly expressed in the brain and fatty tissue of the human body. Despite recent developments in molecular biology, there is still a lack of knowledge about the distribution of CB1 and CB2 receptors in the human kidney and their role in kidney cancer. To address this gap, we explore and demonstrate the role of the endocannabinoid system in renal cell carcinoma (RCC). In this brief overview, we elucidate the therapeutic aspects of the endocannabinoid system for various cancers and explain how this system can be used for treating kidney cancer. Overall, this review provides new insights into cannabinoids’ mechanisms of action in both in vivo and in vitro models, and focuses on recent discoveries in the field.

The Therapeutic Effectiveness of Full Spectrum Hemp Oil Using a Chronic Neuropathic Pain Model
Jacob M. Vigil, Marena A. Montera, Nathan S. Pentkowski, Jegason P. Diviant, Joaquin Orozco, Anthony L. Ortiz, Lawrence J. Rael and Karin N. Westlund
Life 2020, 10, 69;
doi:10.3390/life10050069
Background: Few models exist that can control for placebo and expectancy effects commonly observed in clinical trials measuring ‘Cannabis’ pharmacodynamics. We used the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT-ION) model to measure the effect of “full-spectrum” whole plant extracted hemp oil on chronic neuropathic pain sensitivity in mice. Methods: Male BALBc mice were submitted to the FRICT-ION chronic neuropathic pain model with oral insertion through an incision in the buccal/cheek crease of 3 mm of chromic gut suture (4-0). The suture, wedged along the V2 trigeminal nerve branch, creates a continuous irritation that develops into secondary mechanical hypersensitivity on the snout. Von Frey filament stimuli on the mouse whisker pad was used to assess the mechanical pain threshold from 0–6 h following dosing among animals (n = 6) exposed to 5 µL of whole plant extracted hemp oil combined with a peanut butter vehicle (0.138 mg/kg), the vehicle alone (n = 3) 7 weeks post-surgery, or a naïve control condition (n = 3). Results: Mechanical allodynia was alleviated within 1 h (d = 2.50, p < 0.001) with a peak reversal effect at 4 h (d = 7.21, p < 0.001) and remained significant throughout the 6 h observation window. There was no threshold change on contralateral whisker pad after hemp oil administration, demonstrating the localization of anesthetic response to affected areas. Conclusion: Future research should focus on how whole plant extracted hemp oil affects multi-sensory and cognitive-attentional systems that process pain

The therapeutic potential of cannabis.
Baker D, Pryce G, Giovannoni G, Thompson AJ.
Lancet Neurol. 2003 May;2(5):291-8.
Research of the cannabinoid system has many similarities with that of the opioid system. In both instances, studies into drug-producing plants led to the discovery of an endogenous control system with a central role in neurobiology. Few compounds have had as much positive press from patients as those of the cannabinoid system. While these claims are investigated in disorders such as multiple sclerosis spasticity and pain, basic research is discovering interesting members of this family of compounds that have previously unknown qualities, the most notable of which is the capacity for neuroprotection. Large randomised clinical trials of the better known compounds are in progress. Even if the results of these studies are not as positive as many expect them to be, that we are only just beginning to appreciate the huge therapeutic potential of this family of compounds is clear.

The therapeutic potential of cannabis
Jarnail Singh and Janardhan Singh
ejbps, 2017, Volume 4, Issue 03, 139-146.
https://www.cannabisskunksense.co.uk/uploads/site-files/THERAPEUTIC_POTENTIAL_OF_CANNA BIS.pdf
Medical marijuana use is controversial in the American society. While the states move to legalize marijuana for medical or recreational use, research is needed to elucidate the adverse effects and potential therapeutic benefits of marijuana therapy. This review provides the current information on potential indications, pharmacology, toxicity, and contraindications for medical cannabis. Understanding of the potential uses of cannabinoids in various medical conditions will benefit the patients and healthcare providers. Legalization of marijuana use in 21 states of the US and the district of Columbia has called attention for health care providers.

The therapeutic potential of cannabinoids for integumentary wound management
Maximillian A. Weigelt | Raja Sivamani | Hadar Lev-Tov
Experimental Dermatology. 2021;30:201–211
DOI: 10.1111/exd.14241
https://onlinelibrary.wiley.com/doi/...1111/exd.14241
The increasing legalization of Cannabis for recreational and medicinal purposes in the United States has spurred renewed interest in the therapeutic potential of cannabinoids (CBs) for human disease. The skin has its own endocannabinoid system (eCS) which is a key regulator of various homeostatic processes, including those necessary for normal physiologic wound healing. Data on the use of CBs for wound healing are scarce. Compelling pre-clinical evidence supporting the therapeutic potential of CBs to improve wound healing by modulating key molecular pathways is herein reviewed. These findings merit further exploration in basic science, translational and clinical studies.

The Therapeutic Potential of Cannabis and Cannabinoids
Franjo Grotenhermen, and Kirsten Müller-Vahl,
Dtsch Arztebl Int. 2012 Jul; 109(29-30): 495–501.
doi: 10.3238/arztebl.2012.0495
Background Cannabis-based medications have been a topic of intense study since the endogenous cannabinoid system was discovered two decades ago. In 2011, for the first time, a cannabis extract was approved for clinical use in Germany.
Methods Selective literature review
Results
Cannabis-based medications exert their effects mainly through the activation of cannabinoid receptors (CB1 and CB2). More than 100 controlled clinical trials of cannabinoids or whole-plant preparations for various indications have been conducted since 1975. The findings of these trials have led to the approval of cannabis-based medicines (dronabinol, nabilone, and a cannabis extract [THC:CBD=1:1]) in several countries. In Germany, a cannabis extract was approved in 2011 for the treatment of moderate to severe refractory spasticity in multiple sclerosis. It is commonly used off label for the treatment of anorexia, nausea, and neuropathic pain. Patients can also apply for government permission to buy medicinal cannabis flowers for self-treatment under medical supervision. The most common side effects of cannabinoids are tiredness and dizziness (in more than 10% of patients), psychological effects, and dry mouth. Tolerance to these side effects nearly always develops within a short time. Withdrawal symptoms are hardly ever a problem in the therapeutic setting.
Conclusion
There is now clear evidence that cannabinoids are useful for the treatment of various medical conditions.
Knowledge about the therapeutic potential of cannabis products has been greatly improved by a large number of clinical trials in recent years (1– 5). In October 2008, the German Medical Association, the National Association of Statutory Health Insurance Physicians, and the Drug Commission of the German Medical Association issued the following statement at a hearing of the Health Committee of the German Federal Parliament (Bundestag): “The benefit of treatment with cannabinoids for a number of medical indications has been shown in controlled trials in which predominantly standardized and/or synthetic cannabinoid preparations were used. The use of such preparations may therefore be reasonable for patients in whom conventional treatment does not achieve adequate relief of symptoms such as spasticity, pain, nausea, vomiting, or loss of appetite” (6). The first cannabis-based medication was approved for use in Germany in 2011. In this article we present the current state of knowledge on the therapeutic application of cannabinoid medications.

The Transdermal Delivery of Therapeutic Cannabinoids
Haleh Mahmoudinoodezh, Srinivasa Reddy Telukutla, Sukhvir Kaur Bhangu, Ava Bachari, Francesca Cavalieri and Nitin Mantri
Pharmaceutics 2022, 14,
DOI: 10.3390/pharmaceutics14020438 https://www.researchgate.net/public...nsdermal_Delivery_of_Therapeutic_Cannabinoids
Recently, several studies have indicated an increased interest in the scientific community regarding the application of Cannabis sativa plants, and their extracts, for medicinal purposes. This plant of enormous medicinal potential has been legalised in an increasing number of countries globally.
Due to the recent changes in therapeutic and recreational legislation, cannabis and cannabinoids are now frequently permitted for use in clinical settings. However, with their highly lipophilic features and very low aqueous solubility, cannabinoids are prone to degradation, specifically in solution, as they are light-, temperature-, and auto-oxidation-sensitive. Thus, plant-derived cannabinoids have been developed for oral, nasal-inhalation, intranasal, mucosal (sublingual and buccal), transcutaneous (transdermal), local (topical), and parenteral deliveries. Among these administrations routes, topical and transdermal products usually have a higher bioavailability rate with a prolonged steady-state plasma concentration. Additionally, these administrations have the potential to eliminate the psychotropic impacts of the drug by its diffusion into a nonreactive, dead stratum corneum. This modality avoids oral administration and, thus, the first-pass metabolism, leading to constant cannabinoid plasma levels. This review article investigates the practicality of delivering therapeutic cannabinoids via skin in accordance with existing literature.

The transition of cannabis into the mainstream of Australian healthcare: framings in professional medical publications
Monique Lewis and John Flood
Journal of Cannabis Research (2021) 3:48
DOI: 10.1186/s42238-021-00105-w
https://jcannabisresearch.biomedcentra l.com/track/pdf/10.1186/s42238-021-00105-w.pdf
Background: Medicinal cannabis has been legalised for use for a range of specified medical conditions in Australia since 2016. However, the nature of the government regulations and the subsequent complexity of prescribing, as
well as doctors’ safety uncertainties and the stigma of the plant, remain contributing barriers to patient access. Media representations can offer insights into the nature of the discourse about new medical products and therapies and how ideas and understandings about social phenomena become constructed. Focusing on professional medical publications, this study sought to investigate how medicinal cannabis is being represented in professional medical publications.
Methods: Using a content analysis approach, we investigated articles about medicinal cannabis from 2000 to the end of 2019 in the Medical Journal of Australia, Australian Doctor, Medical Observer, Australian Journal of General Practice, Australian Family Physician, and Australian Medicine. Articles were coded according to article type, framings of cannabis, headline and article tone, and key sources used in the article. We also used manifest textual analysis to search for
Results: A total of 117 articles were retrieved for analysis, the majority of which were news stories for a physician audience. Across the longitudinal period, we found that most reports carried a positive tone towards medicinal cannabis. Cannabis is most frequently framed as a legitimate therapeutic option that is complex to prescribe and access, does not have a strong evidence base to support its use, and also carries safety concerns. At the same time, the outlook on cannabis research data is largely positive. Primary sources most frequently used in these reports are peerreviewed journals or government reports, voices from medical associations or foundations, as well as government and university researchers. Chronic pain or pain were the conditions most frequently mentioned in articles about cannabis, followed by epilepsy, cancer or cancer pain, and nausea and chemotherapy.
Conclusions: This analysis offers evidence that medicinal cannabis is being framed as a valid medicine advocated by the community, with potential for addressing a range of conditions despite the lack of evidence, and a medicine that is not free of risk.

The Treatment of Cannabis Use Disorder
DOI: 10.1016/B978-0-323-54856-4.00007-9
In book: The Assessment and Treatment of Addiction January 2019
Itai Danovitch
David Gorelick
Cannabis is a flowering plant containing a class of lipophilic hydrocarbons called “cannabinoids.” The stem, leaves, and flowers from cannabis have been cultivated throughout history for therapeutic as well as intoxicating purposes. There are dozens of unique cannabinoid substances in cannabis, only several of which have been well characterized.1 The primary psychoactive component of cannabis is D9 -tetrahydrocannabinol (THC). THC produces an intoxication syndrome and promotes release of dopamine in the nucleus accumbens, one of the cornerstone features of reinforcing drugs.2 Cannabadiol (CBD), which has been associated with some of the therapeutic effects ascribed to cannabis, may influence the effects of THC but does not itself produce intoxication. Cannabis can be bred to contain varying levels of THC, CBD, and other cannabinoids. For this chapter, the term “cannabis” will refer to THC or cannabis species that are bred predominantly for their intoxicating effects.

The use of cannabinoids as anticancer agents
Guillermo Velasco, Sonia Hernández-Tiedra, David Dávila, Mar Lorente
Progress in Neuro-Psychopharmacology & Biological Psychiatry 64 (2016) 259–266
DOI: 10.1016/j.pnpbp.2015.05.010
It is well-established that cannabinoids exert palliative effects on some cancer-associated symptoms. In addition evidences obtained during the last fifteen years support that these compounds can reduce tumor growth in animal models of cancer. Cannabinoids have been shown to activate an ER-stress related pathway that leads to the stimulation of autophagy-mediated cancer cell death. In addition, cannabinoids inhibit tumor angiogenesis and decrease cancer cell migration. The mechanisms of resistance to cannabinoid anticancer action as well as the possible strategies to develop cannabinoid-based combinational therapies to fight cancer have also started to be explored. In this review we will summarize these observations (that have already helped to set the bases for the development of the first clinical studies to investigate the potential clinical benefit of using cannabinoids in anticancer therapies) and will discuss the possible future avenues of research in this area.

The Use of Cannabis and Cannabinoid-based Products by Pregnant Women: A Patent Review
Diana Babini Lapa de Albuquerque Britto, Fernanda das Chagas Angelo Mendes Tenorio, Bruno Tenorio, Larissa Rolim, Valdemiro
Silva Júnior
Recent Patents on Biotechnology Volume 15 , Issue 3 , 2021
DOI : 10.2174/1872208315666210719110606
Background: Plants of the genus Cannabis have been gaining attention in medicine. The most outstanding treatments include antispasmodic, analgesic, antiemetic, neuroprotective, and anti-inflammatory actions.
Objective: To describe patents associated cannabis and cannabinoids as a treatment for pregnant women.
Method: a search was carried out on four patent bases: National Institute of Industrial Property, Latipat, Espacenet, and Patentscope. The search keys used were "Cannabis," "Cannabidiol," "Cannabinoids," "CBD," and "THC" associated with "pregnant women." Patents in any language and year were considered.
Results: a total of 1,402 patents were found in all used databases. After reading the title and abstract, patents were excluded if they did not meet the eligibility criteria and/or if they were duplicates, thus leaving 11 patents on the use of cannabis and/or cannabinoids as a treatment in pregnant women. In addition, a critical analysis was carried out. All selected patents were from the Asian continent (China), focusing on traditional Chinese medicine for treating various possible changes during pregnancy and also disease preventions in the postpartum period. It was found that the leading causes for the need of such treatments were related to spleen deficiencies, inability, and moisture stagnation. Finally, the vast majority of treatments used Cannabis fruits associated with other plants.
Conclusion: Given the above, patents related to cannabis and cannabinoids for the treatment of pregnant women in the peri- and post-natal period were analyzed and found to be associated with traditional Chinese medicine.
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The use of cannabis and perceptions of its effect on fertility among infertility patients.
Jordan, T., Ngo, B., & Jones, C. A.
Human Reproduction Open, 2020(1).
doi:10.1093/hropen/hoz041
STUDY QUESTION: What is the prevalence of cannabis use and the perceptions of its impact on fertility among infertility patients? SUMMARY ANSWER: A total of 13% of infertility patients used cannabis within the last year, and current usage is associated with patient perceptions of negative effects of cannabis on fertility and pregnancy.
WHAT IS KNOWN ALREADY: Cannabis use is increasing among the general population and pregnant women, particularly in places where cannabis use is legal despite having known and potential negative effects on fertility and pregnancy.
STUDY DESIGN, SIZE, DURATION: A cross-sectional patient survey study was performed between July 2017 and September 2017. Patients attending a university-affiliated hospital-based fertility clinic (n = 290) were invited to complete a written survey. Inclusion criteria were limited to the ability to read English. There were no exclusion criteria.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 290 patients approached, 270 (93%) agreed to participate. The questions covered demographics, cannabis usage, perceptions of the effect of cannabis on fertility and pregnancy, cessation of use due to infertility and personal history of disclosing cannabis use to healthcare providers (HCP).
MAIN RESULTS AND THE ROLE OF CHANCE: The results showed that 13% of respondents disclosed use of cannabis in the past year (past year users) and 38% had not used cannabis in the past year but had previously used cannabis (>1 year users) while 49% had never used cannabis (never users). Baseline demographics were similar for the three groups, but across four measures of fertility and pregnancy health, past-year users perceived less of a negative effect compared to >1 year users, and never users (P values of 0.02, 0.03, 0.01, <0.01 for questions on pregnancy, offspring health, male fertility and female fertility, respectively). Of past year users, 72% said they had or would disclose use to their HCP, but only 9.4% reported that their HCP had actually instructed them to discontinue use.
LIMITATIONS, REASONS FOR CAUTION: Self-reported patient surveys are subject to reporting bias and may not reflect actual use and perceptions. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that cannabis use is common among infertility patients. Given the known negative impacts of cannabis on pregnancy, the authors would have expected informed infertility patients to cease cannabis use as part of their efforts to conceive. As the prevalence of cannabis use in the last year among infertility patients is similar to that in the general Canadian population, it is unclear whether the prevalence of cannabis use in the sample population merely reflects the average usage in society or, after taking into account those who reduced their usage to improve their fertility, is a factor contributing to infertility and thus prompting fertility referral. Given concern about the potential negative impact of cannabis use on fertility, and that only 9% of past year users had been instructed by an HCP to cease cannabis use, HCPs should consider the benefits of counselling about cannabis cessation for patients who are attempting to conceive. Future research should focus on analysing the effects of cannabis use on female fertility and determining whether a reduction in use among patients with infertility can improve conception rates.
STUDY FUNDING/COMPETING INTEREST(S): Michelle Shin, Clinical Research Associate, is supported by the University of Toronto GREI Fellowship Fund, which is sponsored by unrestricted research grants from EMD Serono, Merck Canada and Ferring Pharmaceuticals. The authors have no potential conflicts of interest to disclose.

The use of cannabis for Hyperemesis Gravidarum (HG)
Gideon Koren and Rana Cohen
Journal of Cannabis Research 2020
https://jcannabisresearch.biomedcent.. .020-0017-6.pdf
DOI: 10.1186/s42238-020-0017-6
Background: Hyperemesis Gravidarum (HG) responds only partially to standard antiemetic medications. Cannabis has been known to possess antiemetic effects and there are several medicinal cannabinoids used as anti -emetics for cancer chemotherapy. Its favorable use for HG has been described in social media, but not in the medical literature.
Methods: We evaluated 4 women with HG counseled by the Motherisk Program, before and following the use of cannabis. Using the validated Pregnancy Unique Quantification of Emesis (PUQE) scoring system and employing the Student’s paired t test, we compared changes in symptoms following initiation of cannabis.
Results: There was a highly significant improvement in symptoms: PUQE score improved from 14.5+/− 1 to 7.5+/ − 0.58(p = 0.0004). Cannabis use was associated with a significant increase in the PUQE Quality of Life scale, from 2+/− 0.82 to 7+/− 0.82 (p = 0.0012).
Conclusions: The results suggest that cannabis may be effective for HG, and should be studied in appropriately powered, controlled studies, fully considering potential fetal risks.

The use of medical cannabis in pediatric palliative care: a case series
Antuan Divisic, Irene Avagnina, Valentina De Tommasi, Anna Santini, Laura Brogelli, Luca Giacomelli and Franca Benini
Italian Journal of Pediatrics (2021) 47:229
DOI: 10.1186/s13052-021-01179-1
https://www.researchgate.net/publica...e_of_medical_c annabis_in_pediatric_palliativ e_care_a_case_series
Background: Medical cannabis may be a useful tool for managing treatment-resistant epilepsy and chronic pain, which affect many patients in pediatric palliative care (PPC); however, little evidence is available in this setting.
Case presentation: We aimed to describe a clinical experience in a setting where high-level evidence may not be obtained. We report our clinical experience in a pediatric palliative care department in Italy. Caregivers reported changes in intensity and frequency of pain and epilepsy events. Six patients received a titrated plant extract of cannabis sativa for 1 year. Only mild and transient adverse events occurred: drowsiness, euphoria, restlessness and tachycardia; the resolution was either spontaneous or obtained by modifying the administration schedule. Treatment was never discontinued. No overdoses occurred. All patients experienced seizures during the pretreatment observation period, and obtained a reduction in seizure frequency, although with variable extent while receiving cannabis. In addition, a benefit on pain was observed, based on the caregiver’s evaluation, and a reduction of analgesic use.
Conclusion: Our experience suggests that a titrated plant extract preparation of medical cannabis may be useful to control treatment-resistant pain and epilepsy in PPC patients.

The wellness revolution: hemp and Cannabinoids introduction -
https://www.academia.edu/24964395/T..._Hemp_and_Cannabinoids_Introduction_281_Words

Paul Benhaim
Use of cannabis, or hemp, for medicinal purposes has gained increased attention in recent years due to a surge in anecdotal reports documenting possible benefits in treating a wide variety of diseases, as well as an increase in scientific research focusing on cannabinoids. This review will explore the current research supporting the use of non-psychoactive cannabinoids, in particular cannabidiol, in the management of a number of chronic health conditions. Research suggests that cannabinoids such as cannabidiol may exert therapeutic effects against inflammatory bowel diseases, some cancers, skin conditions, bone fractures, )+epilepsy, autism, AlzheimerÕs disease, depression, anxiety and addictions, as well as chronic pain and inflammation. Cannabidiol, in particular as a component of whole hemp extracts, has been found to exhibit a superior safety profile to many pharmaceutical drugs, including synthetic cannabinoids. However, human clinical trials and research are still scarce or lackin

Therapeutic application of cannabidiol on UVA and UVB irradiated rat skin. A proteomic study.
Atalay, S., G?gotek, A., Wro?ski, A., Domigues, P., & Skrzydlewska, E.
Journal of Pharmaceutical and BiomedicalAnalysis,192,113656. (2021).
doi:10.1016/j.jpba.2020.113656
UV phototherapy used in chronic skin diseases causes redox imbalance and pro-inflammatory reactions, especially in the case of unchanged skin cells. To prevent the harmful effects of UV radiation, cannabidiol (CBD) has been used, which has antioxidant and anti-inflammatory properties. Therefore, the aim of this study was to evaluate the effect of CBD on the metabolism of skin keratinocytes in nude rats exposed to UVA/UVB radiation using a proteomic approach. The results obtained with SDSPAGE/nanoHPLC/QexactiveOrbiTrap show that exposure of rat’s skin to UVA/UVB radiation, as well as the action of CBD, significantly modified the expression of proteins involved in inflammation, redox balance and apoptosis. UVA/UVB radiation significantly increased the expression and biological effectiveness of the nuclear factor associated with erythroid factor 2 (Nrf2) and cytoprotective proteins being products of its transcriptional activity, including superoxide dismutase (Cu,Zn-SOD) and the inflammatory response (nuclear receptor coactivator-3 and paralemmin-3), while CBD treatment counteracted and partially eliminated these changes. Moreover, cannabidiol reversed changes in the UV-induced apoptotic pathways by modifying anti-apoptotic and pro-apoptotic factors (apoptosis regulator Bcl-2 and transforming growth factor-). The results show that CBD maintains keratinocyte proteostasis and therefore could be suggested as a protective measure in the prevention of UV-induced metabolic changes in epidermal keratinocytes.

Therapeutic opportunities through modulation of the endocannabinoid system
Alexandros Makriyannis, Raphael Mechoulam, Daniele Piomelli
Neuropharmacology 48 (2005) 1068e1071
doi:10.1016/j.neuropharm.2005.03.012
The discovery of the cannabinoid receptors and endocannabinoid ligands has generated a great deal of interest in identifying opportunities for the development of novel cannabinergic therapeutic drugs. Such an effort was first undertaken three decades ago by a number of pharmaceutical industries, but was rewarded with only modest success. However, the newly acquired knowledge on the physiological roles of the endocannabinoid system has significantly enhanced these prospects

Therapeutic potential of cannabidivarin for epilepsy and autism spectrum disorder
Erica Zamberletti, Tiziana Rubino, Daniela Parolaro
Pharmacology Therapeutics 226:107878 April 2021
DOI: 10.1016/j.pharmthera.2021.107878
https://www.researchgate.net/publica...ctrum_disorder
Recent years have seen a renewed interest on the possible therapeutic exploitations of specific cannabinoids derived from the Cannabis sativa plant. Thus far, the most studied non-psychotomimetic cannabinoid is cannabidiol (CBD), which has shown promising therapeutic potential for relieving a variety of neurological diseases. However, also its propyl analogue, cannabidivarin (CBDV), has recently gained much attention as a potential therapeutic agent for the management of disabling neurological conditions. This review aims at providing a comprehensive and updated overview of the available animal and human data, which have investigated the possible therapeutic potential of CBDV for the management of epilepsy and autism spectrum disorder.
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Therapeutic Potential and Medical Uses of Marijuana
Tod Mikuriya
Journal of Psychoactive Drugs 14 (1982): 239-241
https://www.ukcia.org/research/Thera...edicalUses.php
Inscribed on the facade of the Ministry of Truth, these words summarize the federal medical and scientific policies in the field of moral pharmacology. With this most recent groupthink revision of scientific newspeak, cannabis history now starts in 1981 in response to the introduction of a bill in Congress (H.R. 4498) "to provide for the therapeutic use of marijuana in situations involving life-threatening or sense-threatening illness and to provide adequate supplies of marijuana for such use," and promises a review of the literature, which, except for one citation in 1889, one in 1947 and one in 1953, the expunged literature is now comprised mostly of research conducted in the 1970's. Thus, the perspective of the Committee is based on minimal experience in therapeutic applications, deprived of the practical experience from clinical access enjoyed by their colleagues of half a century ago when cannabis was available by prescription. Most important in therapeutic potential and medical uses of marijuana are the omissions. Left out was the fact that cannabis presentations were widely used in Western medicine from 1839 to the early 1940's. Omitted: concise and accurate descriptions of the medicinal applications of cannabis that appeared in the U.S. Pharmacopoeia and Goodman and Gilman's textbook of pharmacology, second edition (1955). Forgotten: primary scientific, structure-activity and pharmacologic studies by Professor S. Loewe (1950) in the 1940's and early 1950's. Neglected: comprehensive clinical research by the Mayor's Committee on Marihuana in 1944.

Therapeutic Potential of Cannabidiol, Cannabidiolic Acid, and Cannabidiolic Acid Methyl Ester as Treatments for Nausea and Vomiting
Erin M. Rock, Cheryl L. Limebeer, Roger G. Pertwee, Raphael Mechoulam, and Linda A. Parker
Cannabis and Cannabinoid Research Volume X, Number X, June 2021
DOI: 10.1089/can.2021.0041
https://www.liebertpub.com/doi/pdf/1.../can.2021.0041
Introduction: Nausea and vomiting are the most distressing symptoms reported by oncology patients undergoing anticancer treatment. With the currently available treatments, vomiting and especially nausea remain problematic, highlighting the need for alternative treatments. Discussion: Here we review in vitro and in vivo evidence for the effectiveness of the nonpsychoactive cannabinoid cannabidiol (CBD) in managing nausea and vomiting. In addition, we also review the evidence for CBD’s acidic precursor, cannabidiolic acid (CBDA), and a methylated version of CBDA (CBDA-ME) in these phenomena. Finally, we explore the potential role of CBD in the treatment of cannabinoid hyperemesis syndrome. Conclusions: CBD has demonstrated efficacy in reducing nausea and vomiting, with CBDA and CBDA-ME being more potent. The data suggest a need for these compounds to be evaluated in clinical trials for their ability to reduce nausea and/or vomiting.

Therapeutic Potential of Cannabidiol (CBD) for Skin Health and Disorders
Sudhir M Baswan, Allison E Klosner, Kelly Glynn, Arun Rajgopal, Kausar Malik, Sunghan Yim, Nathan Stern
Clinical, Cosmetic and Investigational Dermatology Dec 2020
DOI: 10.2147/CCID.S286411
Though there is limited research confirming the purported topical benefits of cannabinoids, it is certain that cutaneous biology is modulated by the human endocannabinoid system (ECS). Receptors from the ECS have been identified in the skin and systemic abuse of synthetic cannabinoids, and their analogs, have also been associated with the manifestation of dermatological disorders, indicating the effects of the ECS on cutaneous biology. In particular, cannabidiol (CBD), a non-psychoactive compound from the cannabis plant, has garnered significant attention in recent years for its anecdotal therapeutic potential for various pathologies, including skin and cosmetic disorders. Though a body of preclinical evidence suggests topical application of CBD may be efficacious for some skin disorders, such as eczema, psoriasis, pruritis, and inflammatory conditions, confirmed clinical efficacy and elucidation of underlying molecular mechanisms have yet to be fully identified. This article provides an update on the advances in CBD research to date and the potential areas of future exploration

Therapeutic Potential of Cannabinoids in Psychosis
F. Markus Leweke, Juliane K. Mueller, Bettina Lange, and Cathrin Rohleder
Biological Psychiatry April 1, 2016; 79:604–612 w
DOI: 10.1016/j.biopsych.2015.11.018
https://addictiondomain.com/wp-conte...-Psychosis.pdf
Over recent years, the interest in the endocannabinoid system (ECS) as a new target for the treatment of schizophrenia has evolved. The ECS represents one of the most relevant neurotransmitter systems in the brain and mainly fulfills a homeostatic role in terms of neurotransmission but also with respect to inflammatory processes. Two main approaches to the modulation of endocannabinoid functioning have been chosen so far. First, the selective blockade or inverse agonism of the type 1 cannabinoid receptor has been tested for the improvement of acute psychotic symptoms, as well as for the improvement of cognitive functions in schizophrenia. This was not effective in either case. Second, the modulation of endocannabinoid levels by use of the phytocannabinoid cannabidiol and selective fatty acid amide hydrolase inhibitors has been proposed, and the antipsychotic properties of cannabidiol are currently being investigated in humans. Unfortunately, for most of these trials that have focused on psychopathological and cognitive effects of cannabidiol, no published data are available. However, there is first evidence that cannabidiol may ameliorate psychotic symptoms with a superior side-effect profile compared with established antipsychotics. In conclusion, several clinical trials targeting the ECS in acute schizophrenia have either been completed or are underway. Although publicly available results are currently limited, preliminary data indicate that selected compounds modulating the ECS may be effective in acute schizophrenia. Nevertheless, so far, sample sizes of patients investigated are not sufficient to come to a final judgment, and no maintenance studies are available to ensure long-term efficacy and safety.

Therapeutic Potential of Cannabinoids in Schizophrenia
Jana Kucerova, Katarina Tabiova, Filippo Drago and Vincenzo Micale
Recent Patents on CNS Drug Discovery, 2014, 9, 13-25
DOI: 10.2174/1574889809666140307115532
https://www.ingentaconnect.com/conte...pplication/pdf
Increasing evidence suggests a close relationship between the endocannabinoid system and schizophrenia. The endocannabinoid system comprises of two G protein-coupled receptors (the cannabinoid receptors 1 and 2 [CB1 and CB2] for marijuana’s psychoactive principle 9 -tetrahydrocannabinol), their endogenous small lipid ligands (namely anandamide [AEA] and 2-arachidonoylglycerol [2-AG], also known as endocannabinoids), and proteins for endocannabinoid biosynthesis and degradation. It has been suggested to be a pro-homeostatic and pleiotropic signalling system activated in a time- and tissue-specific manner during pathophysiological conditions. In the brain, activation of this system impacts the release of numerous neurotransmitters in various systems and cytokines from glial cells. Hence, the endocannabinoid system is strongly involved in neuropsychiatric disorders, such as schizophrenia. Therefore, adolescence use of Cannabis may alter the endocannabinoid signalling and pose a potential environmental risk to develop psychosis. Consistently, preclinical and clinical studies have found a dysregulation in the endocannabinoid system such as changed expression of CB1 and CB2 receptors or altered levels of AEA and 2-AG . Thus, due to the partial efficacy of actual antipsychotics, compounds which modulate this system may provide a novel therapeutic target for the treatment of schizophrenia. The present article reviews current available knowledge on herbal, synthetic and endogenous cannabinoids with respect to the modulation of schizophrenic symptomatology. Furthermore, this review will be highlighting the therapeutic potential of cannabinoid-related compounds and presenting some promising patents targeting potential treatment options for schizophrenia.

Therapeutic potential of medicinal marijuana: An educational primer for health care professionals
Yara Mouhamed, Andrey Vishnyakov, Bessi Qorri, Manpreet Sambi, SM Signy Frank, Catherine Nowierski, Anmol Lamba, Umrao Bhatti, Myron R Szewczuk
Drug, Healthcare and Patient Safety Volume 10:45-66 June 2018
DOI: 10.2147/DHPS.S158592
With the proposed Canadian July 2018 legalization of marijuana through the Cannabis Act, a thorough critical analysis of the current trials on the efficacy of medicinal marijuana (MM) as a treatment option is necessary. This review is particularly important for primary care physicians whose patients may be interested in using MM as an alternative therapy. In response to increased interest in MM, Health Canada released a document in 2013 for general practitioners (GPs) as an educational tool on the efficacy of MM in treating some chronic and acute conditions. Although additional studies have filled in some of the gaps since the release of the Health Canada document, conflicting and inconclusive results continue to pose a challenge for physicians. This review aims to supplement the Health Canada document by providing physicians with a critical yet concise update on the recent advancements made regarding the efficacy of MM as a potential therapeutic option. An update to the literature of 2013 is important given the upcoming changes in legislation on the use of marijuana. Also, we briefly highlight the current recommendations provided by Canadian medical colleges on the parameters that need to be considered prior to authorizing MM use, routes of administration as well as a general overview of the endocannabinoid system as it pertains to cannabis. Lastly, we outline the appropriate medical conditions for which the authorization of MM may present as a practical alternative option in improving patient outcomes as well as individual considerations of which GPs should be mindful. The purpose of this paper is to offer physicians an educational tool that provides a necessary, evidence-based analysis of the therapeutic potential of MM and to ensure physicians are making decisions on the therapeutic use of MM in good faith.

Tic Reduction in Adult Onset Gilles De La Tourette Syndrome Using as Required Nabiximols Spray
MAXIMILIAN A. SCHWITTAY, ANDREAS STEINBRECHER, ELMAR LOBSIEN
Tremor Other Hyperkinet Mov (N Y). 2021; 11: 33.
doi: 10.5334/tohm.613
https://www.ncbi.nlm.nih.gov/pmc/art...m-11-1-613.pdf
Background: Tourette syndrome (TS) manifests with motor and vocal tics that can reach disabling intensity. Established therapies may show insufficient relief or side effects. Cannabinoids have demonstrated therapeutic potential in small studies. This report presents buccal Nabiximols “as required” in the treatment of tics in TS.
Case report: A 25-year-old man presented with stigmatizing motor and phonic tics after cessation of daily Cannabis use. After Tiaprid 300 mg per day had shown no sufficient effect a trial of Nabiximols reduced tics by >90%.
Discussion: Nabiximols could be an adjunct treatment in TS for situations were tics are severly disabling.

Time Dependent Metabolomics and Transcriptional Analysis of Cannabinoid Biosynthesis in Cannabis sativa var. Bedrobinol and Bediol Grown under Standardized Condition and with Genetic Homogeneity
Remco Muntendam, Nizar Happyana, Tjalling Erkelens, Freerk Bruining, Oliver Kayser
Volume 1, Issue 2, pp. 31-40; December 2012 International Journal of Medicinal Plants Research
DOI: 10.13140/RG.2.1.2263.5360
Cannabis sativa L. is mostly grown under non standardized conditions resulting in high fluctuations of cannabinoid contents. The exact measure of these metabolites is hampered as most of the research has been conducted on seized, extracted or non-standardized cultivated C. sativa L. material. In this
report legal standardized cultivation of two C. sativa L. chemotypes is investigated by HPLC-UV, qPCR and 1H-NMR, respectively during two cultivation phases of four (4) weeks each. Based on HPLC the cannabinoid spectrum over 4 week cultivation period is monitored under specific light conditions and in correlation with the expression profile of thca and cbda genes in both chemotypes. Cannabinoid accumulation is observed specifically after shortening of the light period. Gene transcript based chemotyping was only possible comparing the thca and cbda transcription level ratio at the middle of the cultivation period, while at the end differences in gene transcription ratio diminished. 1H-NMR results indicate a clear difference between both chemotypes and cultivation phases by applying pattern
recognition software. The differences detected by using simple and non-destructive 1H-NMR were used to build a model, which can be applied to chemotyping and cultivation phase separation using selective 1H-NMR signals.

Tolerability profile of topical cannabidiol and palmitoylethanolamide: a compilation of single-centre randomized evaluator-blinded clinical and in vitro studies in normal skin
J Maghfour , H Rietcheck, M D Szeto , C W Rundle , T E Sivesind , R P Dellavalle , P Lio , C A Dunnick , J Fernandez , H Yardley
Clin Exp Dermatol . 2021 Dec;46(8):1518-1529.
doi: 10.1111/ced.14749
Background: An increasing number of studies have investigated the adverse effect profile of oral cannabinoids; however, few studies have provided sufficient data on the tolerability of topical cannabinoids in human participants.
Aim: To assess the tolerability profile of several commercial topical formulations containing cannabidiol (CBD) and palmitoylethanolamide (PEA) on the skin of healthy human participants.
Methods: Three human clinical trials and one in vitro study were conducted. The potential for skin irritation, sensitization and phototoxicity of several products, were assessed via patch testing on healthy human skin. The products assessed included two formulations containing CBD and PEA, one containing hemp seed oil and four concentrations of CBD alone. Ocular toxicity was tested using a traditional hen's egg chorioallantoic membrane model with three CBD, PEA and hemp seed oil formulations.
Results: There was no irritation or sensitization of the products evident via patch testing on healthy participants. Additionally, mild phototoxicity of a hemp seed oil product was found at the 48-h time point compared with the negative control. The in vitro experiment demonstrated comparable effects of cannabinoid products with historically nonirritating products.
Conclusion: These specific formulations of CBD- and PEA-containing products are nonirritating and nonsensitizing in healthy adults, and further encourage similar research assessing their long-term safety and efficacy in human participants with dermatological diseases. There are some limitations to the study: (i) external validity may be limited as formulations from a single manufacturer were used for this study, while vast heterogeneity exists across unregulated, commercial CBD products on the market; and (ii) products were assessed only on normal, nondiseased human skin, and therefore extrapolation to those with dermatological diseases cannot be assumed.
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Too Many Mouldy Joints – Marijuana and Chronic Pulmonary Aspergillosis
Yousef Gargani, Paul Bishop and David W. Denning
Mediterr J Hematol Infect Dis 2011, 3; Open Journal System
DOI 10.4084/MJHID.2011.005
Chronic pulmonary aspergillosis is a progressive debilitating disease with multiple underlying pulmonary diseases described. Here we report the association of chronic pulmonary aspergillosis and long term marijuana smoking in 2 patients and review the literature related to invasive and allergic aspergillosis.

Too young for Cannabis? Choice of minimum legal age for legalized nonmedical Cannabis in Canada
Hai V. Nguyen, Stephen Bornstein, John-Michael Gamble, Maria Mathews, Lisa Bishop and Shweta Mital
BMC Public Health (2020) 20:557
DOI: 10.1186/s12889-020-08639-z
Background: Choice of minimum legal age (MLA) for cannabis use is a critical and contentious issue in legalization of non-medical cannabis. In Canada where non-medical cannabis was recently legalized in October 2018, the federal government recommended age 18, the medical community argued for 21 or even 25, while public consultations led most Canadian provinces to adopt age 19. However, no research has compared later life outcomes of first using cannabis at these different ages to assess their merits as MLAs. Methods: We used doubly robust regression techniques and data from nationally representative Canadian surveys to compare educational attainment, cigarette smoking, self-reported general and mental health associated with different ages of first cannabis use. Results: We found different MLAs for different outcomes: 21 for educational attainment, 19 for cigarette smoking and mental health and 18 for general health. Assuming equal weight for these individual outcomes, the ‘overall’ MLA for cannabis use was estimated to be 19 years. Our results were robust to various robustness checks. Conclusion: Our study indicated that there is merit in setting 19 years as MLA for non-medical cannabis.

Topical Cannabidiol (CBD) After Total Knee Arthroplasty Does Not Decrease Pain or Opioid Use: A Prospective Randomized Double-Blinded Placebo-Controlled Trial

Amer Haffar, Irfan A Khan, Mohammad S Abdelaal, Samik Banerjee, Peter F Sharkey
J Arthroplasty April 2022 doi: 10.1016/j.arth.2022.03.081

https://pubmed.ncbi.nlm.nih.gov/35390457/

https://www.arthroplastyjournal.org/article/S0883-5403(22)00383-7/fulltext
Background: Multimodal analgesia has become the standard of care for pain management following total knee arthroplasty (TKA). Cannabidiol (CBD) is increasingly utilized in the postoperative period. The purpose of this study was to analyze the analgesic benefits of topical CBD following primary TKA.
Methods: In this randomized double-blinded placebo-controlled trial, 80 patients undergoing primary unilateral TKA applied topical CBD (CBD; n=19), essential oil (EO; n=21), CBD and essential oil (CBD+EO; n=21), or placebo (PLA; n=19) thrice daily around the knee for two weeks postoperatively. This supplemented a standardized multimodal analgesic protocol. Outcomes included visual analog scale (VAS) pain and numeric rating scale (NRS) sleep scores (collected on postoperative day [POD] 0, 1, 2, 7, 14, 42), and cumulative postoperative opioid use (42 days).
Results: Demographic characteristics were similar among the four cohorts. Preoperative VAS and NRS scores were similar among groups. The CBD cohort had a higher mean VAS pain score on POD 2 compared to the EO cohort (CBD: 69.9 ± 19.3 vs. EO: 51.0 ± 18.2; p=0.013). No significant differences existed for VAS scores at other times, and no significant differences were observed for postoperative NRS sleep scores or postoperative opioid use at any time point.
Conclusions: Utilization of topical CBD in supplement to multimodal analgesia did not reduce pain or opioid consumption, or improve sleep scores following TKA. These results suggest the local effects of topical CBD are not beneficial for providing additional pain relief after TKA.
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Topical application of THC containing products is not able to cause positive cannabinoid finding in blood or urine
C. Hess, M. Kr¨amer, B. Madea
Forensic Science International 2017
DOI: 10.1016/j.forsciint.2017.01.008
A male driver was checked during a traffic stop. A blood sample was collected 35 minutes later and contained 7.3 ng/mL THC, 3.5 ng/mL 11-hydroxy-THC and 44.6 ng/mL 11-nor-9-carboxy-THC. The subject claimed to have used two commercially produced products topically that contained 1.7 ng and 102 ng THC per mg, respectively. In an experiment, three volunteers (25, 26 and 34 years) applied both types of salves over a period of 3 days every 2-4 hours. The application was extensive (50-100 cm2). Each volunteer applied the products to different parts of the body (neck, arm/leg and trunk, respectively). After the first application blood and urine samples of the participants were taken every 2-4 hours until 15 hours after the last application (overall n = 10 urine and n=10 blood samples, respectively, for each participant). All of these blood and urine samples were tested negative for THC, 11-hydroxy-THC and 11-nor-9-carboxy-THC by a GC-MS method (LoD (THC) = 0.40 ng/mL; LoD (11-hydroxy-THC) = 0.28 ng/mL; LoD (THC-COOH) = 1.6 ng/mL;. LoD (THC-COOH in urine) = 1.2 ng/mL). According to our studies and further literature research on in vitro testing of transdermal uptake of THC, the exclusive application of (these two) topically applied products did not produce cannabinoid findings in blood or urine.

Topical cannabis based medicines – A novel paradigm and treatment for non-uremic calciphylaxis leg ulcers: An open label trial.
Maida, V., Shi, R. B., Fazzari, F. G. T., & Zomparelli, L.
International Wound Journal. (2020).
doi:10.1111/iwj.13484
Non-Uremic Calciphylaxis (NUC) is a rare condition that often manifests as intractable and painful integumentary wounds, afflicting patients with a high burden of co-morbidity. The Endocannabinoid System (ECS) is a ubiquitous signalling system that is theorised to be dysregulated within wound beds and associated peri-wound tissues. Preclinical research has shown that the dominant chemical classes derived from the cannabis plant, cannabinoids, terpenes, and flavonoids, interact with the integumentary ECS to promote wound closure and analgesia. This is a prospective open label cohort study involving two elderly Caucasian females with recalcitrant NUC leg ulcers of greater than 6 months duration. Topical Cannabis-Based Medicines (TCBM) composed of cannabinoids, terpenes, and flavonoids were applied daily to both the wound bed and peri-wound tissues until complete wound closure was achieved. Wounds were photographed regularly, and the digital images were subjected to planimetric analysis to objectively quantify the degree of granulation and epithelization. Analgesic utilisation, as a surrogate/proxy for pain scores, was also tracked. The cohort had a mean M3 multimorbidity index score of 3.31. Complete wound closure was achieved in a mean of 76.3 days. Additionally, no analgesics were required after a mean of 63 days. The treatments were well tolerated with no adverse reactions. The positive results demonstrated in very challenging wounds such as NUC, among highly complex patients, suggest that TCBM may have an even broader role within integumentary and wound management. This treatment paradigm warrants being trialled in other wound types and classes, and ultimately should be subjected to randomised controlled trials.

Totality of the Evidence Suggests Prenatal Cannabis Exposure Does Not Lead to Cognitive Impairments: A Systematic and Critical Review
Ciara A. Torres, Christopher Medina-Kirchner, Kate Y. O’Malley, and Carl L. Har
Frontiers in Psychology 1 May 2020 | Volume 11 | Article 816
DOI: 10.3389/fpsyg.2020.00816/full
Background: Despite limited data demonstrating pronounced negative effects of prenatal cannabis exposure, popular opinion and public policies still reflect the belief that cannabis is fetotoxic. Methods: This article provides a critical review of results from longitudinal studies examining the impact of prenatal cannabis exposure on multiple domains of cognitive functioning in individuals aged 0 to 22 years. A literature search was conducted through PsycINFO, PubMed, and Google Scholar. Articles were included if they examined the cognitive performance of offspring exposed to cannabis in utero. Results: An examination of the total number of statistical comparisons (n = 1,001) between groups of participants that were exposed to cannabis prenatally and non-exposed controls revealed that those exposed performed differently on a minority of cognitive outcomes

Transfer of Inhaled Cannabis Into Human Breast Milk.
Baker, T., Datta, P., Rewers-Felkins, K., Thompson, H., Kallem, R. R., & Hale, T. W.
Obstetrics & Gynecology, 131(5), 783–788. (2018)
doi:10.1097/aog.0000000000002575
To evaluate the transfer of delta-9- tetrahydrocannabinol and its metabolites into human breast milk after maternal inhalation of 0.1 g cannabis containing 23.18% delta-9-tetrahydrocannabinol. METHODS: In this pilot pharmacokinetic study, breast milk samples were collected from mothers who regularly consumed cannabis, were 2–5 months postpartum, and exclusively breastfeeding their infants. Women were anonymously recruited for the study. After discontinuing cannabis for at least 24 hours, they were directed to obtain a baseline breast milk sample, then smoke a preweighed, analyzed, standardized strain of cannabis from one preselected dispensary, and collect breast milk samples at specific time points: 20 minutes and 1, 2, and 4 hours. Quantification of delta-9-tetrahydrocannabinol and its metabolites in these collected breast milk samples was performed by high-performance liquid chromatography tandem mass spectrometry. RESULTS: A total of eight women were enrolled. Most were occasional cannabis smokers and one a chronic user. Delta-9-tetrahydrocannabinol was detected at low concentrations at all the time points beyond time zero. No metabolites were detected at any time point. Delta- 9-tetrahydrocannabinol was transferred into mother’s milk such that exclusively breastfeeding infants ingested an estimated mean of 2.5% of the maternal dose (the calculated relative infant dose52.5%, range 0.4–8.7%). The estimated daily infant dose was 8 micrograms per kilogram per day. CONCLUSION: This study documents inhaled delta-9-tetrahydrocannabinol transfer into the mother’s breast milk. Low concentrations of delta-9-tetrahydrocannabinol were detected. The long-term neurobehavioral effect of exposure to delta-9-tetrahydrocannabinol on the developing brain is unclear. Mothers should be cautious using cannabis during pregnancy and breastfeeding.

Treating insomnia symptoms with medical cannabis: a randomized, crossover trial of the efficacy of a cannabinoid medicine compared with placebo
Jennifer H Walsh, Kathleen J Maddison, Tim Rankin, Kevin Murray, Nigel McArdle, Melissa J Ree, David R Hillman, Peter R Eastwood
Sleep 44(9821) (June 2021)
DOI: 10.1093/sleep/zsab149/6296857
https://www.researchgate.net/publica...d_with_Placebo
Study Objectives: This randomized, double-blind, placebo-controlled, cross-over study was conducted to evaluate the safety and efficacy of two-weeks of nightly sublingual cannabinoid extract (ZTL-101) in treating chronic insomnia (symptoms ≥three months).
Methods: Co-primary study endpoints were safety of the medication based on adverse event reporting and global insomnia symptoms (Insomnia Severity Index; ISI). Secondary endpoints included: self-reported (sleep diary), actigraphy-derived and polysomnography measurements of sleep onset latency (SOL), wake after sleep onset (WASO), total sleep time (TST), sleep efficiency (SE); and self-reported assessments of sleep quality (sSQ) and feeling rested upon waking. Adjusted mean differences between placebo and ZTL-101 were calculated.
Results: Twenty-three of 24 randomized participants (n=20 female, mean age 53±9years) completed the protocol. No serious adverse events were reported. Forty mild, non-serious, adverse events were reported (36 during ZTL-101) with all but one resolving overnight or soon after waking. Compared to placebo, ZTL-101 decreased ISI (-5.07units [95%CI: -7.28 to -2.86]; p=0.0001) and self-reported SOL (-8.45mins [95%CI: -16.33 to -0.57]; p=0.04) and increased self-reported TST (64.6mins [95%CI: 41.70 to 87.46]; p<.0001), sSQ, (0.74units [95%CI: 0.51 to 0.97]; p<0.0001) and feeling of being rested on waking (0.51units [95%CI: 0.24 to 0.78]; p=0.0007). ZTL-101 also decreased actigraphy-derived WASO (-10.2mins [95%CI: -16.2 to -4.2]; p=0.002), and increased actigraphy-derived TST (33.4mins [95%CI: 23.07 to 43.76]; p<0.001) and SE (2.9% [95%CI: 2.0 to 3.8]; p=0.005).
Conclusion: Two-weeks of nightly sublingual administration of a cannabinoid extract (ZTL-101) is well tolerated and improves insomnia symptoms and sleep quality in individuals with chronic insomnia symptoms.

Treatment of Cannabis Use Disorder: Current Science and Future Outlook.
Sherman, B. J., & McRae-Clark, A. L.
Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 36(5), 511–535. (2016).
doi:10.1002/phar.1747
Cannabis is the most commonly used illicit substance in the United States. Rates of cannabis use and cannabis use disorder (CUD) have increased in the past decade, paralleling changes in the legal and political climate favoring legalization. Almost 20 million people 12 years or older report past-month cannabis use, and 8 million report daily or near-daily use. Concurrently, the perception that cannabis use poses a significant risk of negative consequences has decreased. Contrary to this perception, heavy cannabis use is associated with cognitive impairment, increased risk for psychotic disorders and other mental health problems, lower education attainment, and unemployment. Clinical trials of various treatments for CUD have likewise increased, focusing primarily on psychotherapy treatments, specifically motivational enhancement therapy, cognitive behavioral therapy, and contingency management. Their findings suggest that a combination of these three modalities produces the best abstinence outcomes, although abstinence rates remain modest and decline after treatment. More recently, pharmacotherapy trials have been conducted as adjunctive interventions to psychosocial treatment. N-acetylcysteine and gabapentin are two of the most promising medications, although no pharmacologic treatment has emerged as clearly efficacious. In this review, we provide a detailed summary of clinical trials that evaluated psychotherapy and pharmacotherapy for treating CUD and discuss emerging areas of clinical research and cannabis-specific barriers to treatment

Treatment of Fragile X Syndrome with Cannabidiol: A Case Series Study and Brief Review of the Literature.
Tartaglia, N., Bonn-Miller, M., & Hagerman, R.
Cannabis and Cannabinoid Research, 4(1), 3–9.(2019).
doi:10.1089/can.2018.0053
Fragile X syndrome (FXS) is an X-linked dominant disorder caused by a mutation in the fragile X mental retardation 1 gene. Cannabidiol (CBD) is an exogenous phytocannabinoid with therapeutic potential for individuals with anxiety, poor sleep, and cognitive deficits, as well as populations with endocannabinoid deficiencies, such as those who suffer from FXS. The objective of this study was to provide a brief narrative review of recent literature on endocannabinoids and FXS and to present a case series describing three patients with FXS who were treated with oral CBD-enriched (CBD+) solutions. We review recent animal and human studies of endocannabinoids in FXS and present the cases of one child and two adults with FXS who were treated with various oral botanical CBD+ solutions delivering doses of 32.0 to 63.9 mg daily. Multiple experimental and clinical models of FXS combine to highlight the therapeutic potential of CBD for management of FXS. All three patients described in the case series exhibited functional benefit following the use of oral CBD+ solutions, including noticeable reductions in social avoidance and anxiety, as well as improvements in sleep, feeding, motor coordination, language skills, anxiety, and sensory processing. Two of the described patients exhibited a reemergence of a number of FXS symptoms following cessation of CBD+ treatment (e.g., anxiety), which then improved again after reintroduction of CBD+ treatment. Findings highlight the importance of exploring the therapeutic potential of CBD within the context of rigorous clinical trials.

Treatment of Tourette Syndrome with Delta-9- Tetrahydrocannabinol (D9 -THC): No Influence on Neuropsychological Performance
Kirsten R Mu¨ller-Vahl, Heidrun Prevedel, Karen Theloe, Hans Kolbe, Hinderk M Emrich, and Udo Schneider
Neuropsychopharmacology (2003) 28, 384–388
DOI: 10.1038/sj.npp.1300047
Previous studies provide evidence that marijuana (Cannabis sativa) and delta-9-tetrahydrocannabinol (D9 -THC), the major psychoactive ingredient of marijuana, respectively, are effective in the treatment of tics and behavioral problems in Tourette syndrome (TS). It, therefore, has been speculated that the central cannabinoid receptor system might be involved in TS pathology. However, in healthy marijuana users there is an ongoing debate as to whether the use of cannabis causes acute and/or long-term cognitive deficits. In this randomized double-blind placebo-controlled study, we investigated the effect of a treatment with up to 10 mg D9 -THC over a 6-week period on neuropsychological performance in 24 patients suffering from TS. During medication and immediately as well as 5–6 weeks after withdrawal of D9 -THC treatment, no detrimental effect was seen on learning curve, interference, recall and recognition of word lists, immediate visual memory span, and divided attention. Measuring immediate verbal memory span, we even found a trend towards a significant improvement during and after treatment. Results from this study corroborate previous data suggesting that in patients suffering from TS, treatment with D9 -THC causes neither acute nor long-term cognitive deficits. Larger and longer-duration controlled studies are recommended to provide more information on the adverse effect profile of THC in patients suffering from TS.

Treatment studies with cannabinoids in anorexia nervosa: a systematic review
Emilie Vangsgaard Rosager, Christian Møller, Magnus Sjögren
Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity
Doi: 10.1007/s40519-020-00891-x
Introduction Anorexia nervosa (AN) is a psychiatric disorder with a high mortality and unknown etiology, and efective treatment is lacking. For decades, cannabis has been known to cause physical efects on the human body, including increasing appetite, which may be benefcial in the treatment of AN.
Objective To systematically review the literature for evidence of an efect of cannabinoids on (1) weight gain, and (2) other outcomes, in AN.
Method A systematic review was done using three databases Embase, PubMed and Psychinfo. The review was registered in PROSPERO with ID number CRD42019141293 and was done according to PRISMA guidelines. Results There were 1288 studies identifed and after thorough review and exclusion of copies, 4 studies met the inclusion criteria. Three studies used the same AN population and utilized data from one original study, leaving only two original studies. Both of these were Randomized Controlled Trials that explored the efects of delta-9-tetrahydrocannabinol (?9-THC) or dronabinol in AN, whereof one study was properly designed and powered and showed a weight increase of an added 1 kg over 4 weeks over placebo.
Discussion and conclusion There are few studies and the level of evidence is low. The only properly designed, low bias and highly powered study found a weight increasing efect of dronabinol in AN, while the other, using ?9-THC at a high dose, found no efect and where the dose may have counteracted the weight gaining efects due to adverse events. More research on cannabinoids in anorexia nervosa is warranted, especially its efects on psychopathology.
Level of evidence Level I, systematic review.

Trends in Prevalence and Outcomes of Cannabis Use Among Chronic Obstructive Pulmonary Disease Hospitalizations: A Nationwide Population-Based Study 2005–2014
Kulothungan Gunasekaran, Dinesh C. Voruganti, Mandeep Singh Rahi, Kalaimani Elango, Sathishkumar Ramalingam, Adiba Geeti, and Jeff Kwon
Cannabis and Cannabinoid Research Volume 6, Number 4, 2021
DOI: 10.1089/can.2020.0133
https://www.liebertpub.com/doi/pdf/1.../can.2020.0133
Background: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality in the United States. Due to the ongoing legalization of cannabis, its acceptance, availability, and use in the in-patient population are on the rise. In this retrospective study, we investigated the association of cannabis use with important outcomes in COPD hospitalizations. Methods: The National Inpatient Sample (NIS) data were analyzed from 2005 to 2014. The primary outcome of interest was the trends and outcomes of cannabis use among COPD hospitalizations, including in-hospital mortality, pneumonia, sepsis, and respiratory failure. Results: We identified 6,073,862 hospitalizations, 18 years of age or older, with COPD using hospital discharge codes. Of these, 6,049,316 (99.6%) were without cannabis use, and 24,546 (0.4%) were admitted with cannabis use. The majority of COPD hospitalizations with cannabis use were aged 50–64 (60%). Cannabis use was associated with lower odds of in-hospital mortality (odds ratio [OR] 0.624 [95% confidence interval (CI) 0.407– 0.958]; p = 0.0309) and pneumonia (OR 0.882 [95% CI 0.806–0.964]; p = 0.0059) among COPD hospitalizations. Cannabis use also had lower odds of sepsis (OR 0.749 [95% CI 0.523–1.071]; p = 0.1127) and acute respiratory failure (OR 0.995 [95% CI 0.877–1.13]; p = 0.9411), but it was not statistically significant. Conclusions: Among hospitalized patients with a diagnosis of COPD, cannabis users had statistically significant lower odds of in-hospital mortality and pneumonia compared to noncannabis users. The association between cannabis use and these favorable outcomes deserves further study to understand the interaction between cannabis use and COPD.

TRPV1 and Endocannabinoids: Emerging Molecular Signals that Modulate Mammalian Vision
Daniel A. Ryskamp, Sarah Redmon, Andrew O. Jo, David Križaj
Cells 2014, 3(3), 914-938;
DOI:10.3390/cells3030914
https://www.mdpi.com/2073-4409/3/3/914/htm
Transient Receptor Potential Vanilloid 1 (TRPV1) subunits form a polymodal cation channel responsive to capsaicin, heat, acidity and endogenous metabolites of polyunsaturated fatty acids. While originally reported to serve as a pain and heat detector in the peripheral nervous system, TRPV1 has been implicated in the modulation of blood flow and osmoregulation but also neurotransmission, postsynaptic neuronal excitability and synaptic plasticity within the central nervous system. In addition to its central role in nociception, evidence is accumulating that TRPV1 contributes to stimulus transduction and/or processing in other sensory modalities, including thermosensation, mechanotransduction and vision. For example, TRPV1, in conjunction with intrinsic cannabinoid signaling, might contribute to retinal ganglion cell (RGC) axonal transport and excitability, cytokine release from microglial cells and regulation of retinal vasculature. While excessive TRPV1 activity was proposed to induce RGC excitotoxicity, physiological TRPV1 activity might serve a neuroprotective function within the complex context of retinal endocannabinoid signaling. In this review we evaluate the current evidence for localization and function of TRPV1 channels within the mammalian retina and explore the potential interaction of this intriguing nociceptor with endogenous agonists and modulators.

Two-weeks treatment with cannabidiol improves biophysical and behavioral deficits associated with experimental type-1 diabetes
Yane Costa Chavesa, Karina Genarob, Cristina Aparecida Sterna, Gisele de Oliveira Guaitaa, José Alexandre de Souza Crippad, Joice Maria da Cunhaa, Janaína Menezes Zanoveli
Neuroscience Letters 729 (2020) 135020
DOI: 10.1016/j.neulet.2020.135020
The prevalence rates of depression and anxiety are at least two times higher in diabetic patients, increasing morbidity and mortality. Cannabidiol (CBD) has been identified as a therapeutic agent viable to treat diverse psychiatric disorders. Thus, this study aimed to investigate the effect of CBD treatment (once a day for 14 days starting two weeks after diabetes induction; at doses of 0, 3, 10 or 30 mg/kg, i.p.) on depression- and anxiety-like behaviors associated with experimental diabetes induced by streptozotocin (60 mg/kg; i.p.) in rats. Levels of plasma insulin, blood glucose, and weight gain were evaluated in all experimental groups, including a positive control group treated with imipramine. The rats were tested in the modified forced swimming test (mFST) and elevated plus maze (EPM) test. Besides, the levels of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in two emotion-related brain regions, the prefrontal cortex (PFC) and hippocampus (HIP) were evaluated using high-pressure liquid chromatography. Our results showed that CBD treatment (only at the higher dose of 30 mg/ kg) reduced the exaggerated depressive- and anxiogenic-like behaviors of diabetic (DBT) rats, which may be associated with altered 5-HT, NA and/or DA levels observed in the PFC and HIP. Treatment with CBD (higher dose) also induced a significant increase in weight gain and the insulin levels (and consequently reduced glycemia) in DBT rats. The long-term CBD effects gave rise to novel therapeutic strategies to limit the physiological and neurobehavioral deficits in DBT rats. This approach provided evidence that CBD can be useful for treating psychiatry comorbidities in diabetic patients

Uncovering the hidden antibiotic potential of Cannabis
Maya A. Farha, Omar M. El-Halfawy, Robert T. Gale, Craig R. MacNair, Lindsey A.Carfrae, Xiong Zhang, Nicholas G. Jentsch, Jakob Magolan, Eric D. Brown
https://www.biorxiv.org/content/10.1...392v3.full.pdf
Public Health agencies around the globe have identified antimicrobial resistance as one of the most critical challenges of our time. The rapid and global spread of antimicrobial-resistant organisms in recent years has been unprecedented. So much so that the world health organization (WHO) published its first ever list of antibiotic-resistant "priority pathogens", made up of 12 families of bacteria that pose the greatest threat to human health. Among them, Staphylococcus aureus is the leading cause of both healthcare and community-associated infections worldwide and a major cause for morbidity and, especially with the emergence and rapid spread of methicillin-resistant S. aureus (MRSA), which is resistant to all known ?-lactam antibiotics. Worse yet, resistance to vancomycin, linezolid and daptomycin has already been reported in MRSA clinical strains, compromising the therapeutic alternatives for life-threatening MRSA infections. Further, antibiotic-resistant Gram-negative infections have increasingly become a pressing issue in the clinic. Indeed, of the bacteria highlighted by the WHO, 75% are Gram-negative organisms. Among the currently approved antibiotics in clinical use, the latest discovery of a new drug class dates back to more than 30 years ago. The rapid loss of antibiotic effectiveness and diminishing pipeline beg for the exploration of alternative therapies.

Understanding dabs: contamination concerns of cannabis concentrates and cannabinoid transfer during the act of dabbing.
Raber, J. C., Elzinga, S., & Kaplan, C.
The Journal of Toxicological Sciences, 40(6), 797–803.(2015).
doi:10.2131/jts.40.797
Cannabis concentrates are gaining rapid popularity in the California medical cannabis market. These extracts are increasingly being consumed via a new inhalation method called 'dabbing'. The act of consuming one dose is colloquially referred to as "doing a dab". This paper investigates cannabinoid transfer efficiency, chemical composition and contamination of concentrated cannabis extracts used for dabbing. The studied concentrates represent material available in the California medical cannabis market. Fifty seven (57) concentrate samples were screened for cannabinoid content and the presence of residual solvents or pesticides. Considerable residual solvent and pesticide contamination were found in these concentrates. Over 80% of the concentrate samples were contaminated in some form. THC max concentrations ranged from 23.7% to 75.9% with the exception of one outlier containing 2.7% THC and 47.7% CBD. Up to 40% of the theoretically available THC could be captured in the vapor stream of a dab during inhalation experiments. Dabbing offers immediate physiological relief to patients in need but may also be more prone to abuse by recreational users seeking a more rapid and intense physiological effect.

Understanding the Medical Chemistry of the Cannabis Plant is Critical to Guiding Real World Clinical Evidence.
Ladha, K. S., Ajrawat, P., Yang, Y., & Clarke, H.
Molecules, 25(18), 4042. (2020).
doi:10.3390/molecules25184042
While cannabis has been consumed for thousands of years, the medical-legal landscape surrounding its use has dramatically evolved over the past decades. Patients are turning to cannabis as a therapeutic option for several medical conditions. Given the surge in interest over the past decades there exists a major gap in the literature with respect to understanding the products that are currently being consumed by patients. The current perspective highlights the lack of relevance within the current literature towards understanding the medical chemistry of the products being consumed. The cannabis industry must rigorously invest into understanding what people are consuming from a chemical composition standpoint. This will inform what compounds in addition to ? 9 -tetrahydrocannabinol and cannabidiol may be producing physiologic/therapeutic effects from plant based extracts. Only through real-world evidence and a formalized, granular data collection process within which we know the chemical inputs for patients already using or beginning to use medical cannabis, we can come closer to the ability to provide targeted clinical decision making and design future appropriate randomized controlled trials

Unexpected improvement of hyperhidrosis with cannabidiol
Rubens Pitliuk, Christina Morotomi Funatsu Coelho, Tatyanny Paula Pinto da Costa Santos Fucci
einstein (São Paulo). 2022;20:
DOI: 10.31744/einstein_journal/2022RC5795
https://www.scielo.br/j/eins/a/Qn7Nf...at=pdf&lang=en
Hyperhidrosis is characterized by excessive sweating and it affects almost 5% of the population. The affected age group is wide, and it can affect from children to elderlies. There are two types of hyperhidrosis: generalized and focal. Treatment depends on the symptoms presented. In more severe cases, radiofrequency sympatholysis and bilateral thoracic sympathectomy are the options. However, recurrence is possible or the postoperative appearance of conditions called compensatory hyperhidrosis or reflex hyperhidrosis. We describe two cases of patients treated with Cannabidiol who had significant and unexpected improvement of hyperhidrosis. The first patient received Cannabidiol specific for public presentations at work, and the second patient had a diagnosis of autism spectrum disorder. The hyperhidrosis improved in both patients immediately after using Cannabidiol.

Unheated Cannabis sativa extracts and its major compound THC-acid have potential immuno-modulating properties not mediated by CB1 and CB2 receptor coupled pathways.
Verhoeckx, K. C. M., Korthout, H. A. A. J., van Meeteren-Kreikamp, A. P., Ehlert, K. A., Wang, M., van der Greef, J., … Witkamp, R. F.
International Immunopharmacology, 6(4), 656–665. (2006).
doi:10.1016/j.intimp.2005.10.002
There is a great interest in the pharmacological properties of cannabinoid like compounds that are not linked to the adverse effects of D9 -tetrahydrocannabinol (THC), e.g. psychoactive properties. The present paper describes the potential immunomodulating activity of unheated Cannabis sativa extracts and its main non-psychoactive constituent D9 -tetrahydrocanabinoid acid (THCa). By heating Cannabis extracts, THCa was shown to be converted into THC. Unheated Cannabis extract and THCa were able to inhibit the tumor necrosis factor alpha (TNF-a) levels in culture supernatants from U937 macrophages and peripheral blood macrophages after stimulation with LPS in a dose-dependent manner. This inhibition persisted over a longer period of time, whereas after prolonged exposure time THC and heated Cannabis extract tend to induce the TNF-a level. Furthermore we demonstrated that THCa and THC show distinct effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibit the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. These results suggest that THCa and THC exert their immuno-modulating effects via different metabolic pathways

Unsubstantiated health claims for COVID-19 infections are led by cannabidiol: return of snake oil medicine
Allan Tran, Natasha Y. Sheikhan, Tania Sheikhan, Dominik A. Nowak and Theodore J. Witek Jr
Journal of Cannabis Research (2021) 3:49
DOI: 10.1186/s42238-021-00109-6
https://jcannabisresearch.biomedcentra l.com/track/pdf/10.1186/s42238-021-00109-6.pdf
Background: The United States Food and Drug Administration (FDA) monitors, inspects, and enforces the promotion of products by companies that claim to mitigate, prevent, treat, diagnose, or cure COVID-19. The introduction of COVID-19-related diagnostics and therapeutics during the pandemic has highlighted the signifcance of rigorous clinical trials to ensure safety and efcacy of such interventions. The objective of this report is to provide a descriptive review of promotional violations of health products for COVID-19 infection.
Methods: Warning letters issued by the FDA’s Center for Drug Evaluation and Research were retrieved over an 18month period (March 6, 2020, to August 30, 2021) to identify promotional violations. FDA violation letters categorized as “Unapproved and Misbranded Products Related to Coronavirus Disease 2019 (COVID-19)” were reviewed. A content analysis was performed for each letter to identify categories for product type, promotional venue, violation type, and country of origin. For cannabidiol-related violations, a content analysis was repeated within its own product category.
Results: A total of 130 letters were reported. Across all letters, cannabidiol products were the most frequent subject of violation (15/130; 11.5%). Of the cannabidiol letters, all reported the promotion of unapproved products (15/15; 100%), misbranding (15/15; 100%), and/or had claims that lacked scientifc substantiation (14/15; 93.3%). All promotional violations were linked to websites (15/15; 100%), along with other mainstream venues: Facebook, Instagram, YouTube, Twitter, LinkedIn, and email. Lastly, the cannabidiol products were described to provide therapeutic beneft to COVID-19, by acting as an anti-viral (5; 33.3%), pro-infammatory (1; 6.7%), anti-infammatory (7; 46.7%), immunebooster (5; 40%), immune-suppressor (2; 13.3%), and/or other (2; 13.3%).
Conclusion: Despite the urgent need for COVID-19 treatments, promotional material by companies must comply with standard regulatory requirements, namely substantiation of claims. As the pandemic persists, the FDA must continue their eforts to monitor, inspect, and enforce violative companies. Cannabidiol-related substances led the spectrum of products with unsubstantiated claims to treat COVID-19 infection. Improving awareness among the public, healthcare providers, and stakeholders highlights the value of drug approval process, while protecting public safety

Untargeted metabolomics of prostate cancer zwitterionic and positively charged compounds in urine
Andrea Cerrato, Carmen Bedia, Anna Laura Capriotti, Aldo Laganà
Analytica Chimica Acta 1158: March 2021
DOI: 10.1016/j.aca.2021.338381
Prostate cancer, a leading cause of cancer-related deaths worldwide, principally occurs in over 50-year-old men. Nowadays there is urgency to discover biomarkers alternative to prostate-specific antigen, as it cannot discriminate patients with benign prostatic hyperplasia from clinically significant forms of prostatic cancer. In the present paper, 32 benign prostatic hyperplasia and 41 prostatic cancer urine samples were collected and analyzed. Polar and positively charged metabolites were therein investigated using an analytical platform comprising an up to 40-fold analyte enrichment step by graphitized carbon black solid-phase extraction, HILIC separation, and untargeted high-resolution mass spectrometry analysis. These classes of compounds are often neglected in common metabolomics experiments even though previous studies reported their significance in cancer biomarker discovery. The complex metabolomics big datasets, generated by the UHPLC-HRMS, were analyzed with the ROIMCR procedure, based on the selection of the MS regions of interest data and their analysis by the Multivariate Curve-Resolution Alternating Least Squares chemometrics method. This approach allowed the resolution and tentative identification of the metabolites differentially expressed by the two data sets. Among these, amino acids and carnitine derivatives were tentatively identified highlighting the importance of the proposed methodology for cancer biomarker research.
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Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol.
Citti, C., Palazzoli, F., Licata, M., Vilella, A., Leo, G., Zoli, M., … Cannazza, G.
Journal of Pharmaceutical and Biomedical Analysis, 161, 1–11.(2018).
doi:10.1016/j.jpba.2018.08.021
Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (?9 -tetrahydrocannabinol (THC), 11- hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

Update on the endocannabinoid system as an anticancer target
Anna Maria Malfitano, Elena Ciaglia, Giuseppina Gangemi, Patrizia Gazzerro, Chiara Laezza & Maurizio Bifulco
Expert Opin. Ther. Targets (2011) 15(3)
DOI: 10.1517/14728222.2011.553606
Introduction: Recent studies have shown that the endocannabinoid system (ECS) could offer an attractive antitumor target. Numerous findings suggest the involvement of this system (constituted mainly by cannabinoid receptors, endogenous compounds and the enzymes for their synthesis and degradation) in cancer cell growth in vitro and in vivo.
Areas covered: This review covers literature from the past decade which highlights the potential of targeting the ECS for cancer treatment. In particular, the levels of endocannabinoids and the expression of their receptors in several types of cancer are discussed, along with the signaling pathways involved in the endocannabinoid antitumor effects. Furthermore, the beneficial and adverse effects of old and novel compounds in clinical use are discussed. Expert opinion: One direction that should be pursued in antitumor therapy is to select compounds with reduced psychoactivity. This is known to be connected to the CB1 receptor; thus, targeting the CB2 receptor is a popular objective. CB1 receptors could be maintained as a target to design new compounds, and mixed CB1--CB2 ligands could be effective if they are able to not cross the BBB. Furthermore, targeting the ECS with agents that activate cannabinoid receptors or inhibitors of endogenous degrading systems such as fatty acid amide hydrolase inhibitors may have relevant therapeutic impact on tumor growth. Additional studies into the downstream consequences of endocannabinoid treatment are required and may illuminate other potential therapeutic targets

Update on the Role of Cannabinoid Receptors after Ischemic Stroke
Luciano S. A. Capettini, Silvia Q. Savergnini, Rafaela F. da Silva, Nikos Stergiopulos, Robson A. S. Santos, Franc¸ois Mach, and Fabrizio Montecucco
Mediators of Inflammation Volume 2012, 8 pg.
doi:10.1155/2012/824093
https://downloads.hindawi.com/journa...012/824093.pdf
Cannabinoids are considered as key mediators in the pathophysiology of inflammatory diseases, including atherosclerosis. In particular, they have been shown to reduce the ischemic injury after acute cardiovascular events, such as acute myocardial infarction and ischemic stroke. These protective and anti-inflammatory properties on peripheral tissues and circulating inflammatory have been demonstrated to involve their binding with both selective cannabinoid type 1 (CB1) and type 2 (CB2) transmembrane receptors. On the other hands, the recent discoveries of novel different classes of cannabinoids and receptors have increased the complexity of this system in atherosclerosis. Although only preliminary data have been reported on the activities of novel cannabinoid receptors, several studies have already investigated the role of CB1 and CB2 receptors in ischemic stroke. While CB1 receptor activation has been shown to directly reduce atherosclerotic plaque inflammation, controversial data have been shown on neurotransmission and neuroprotection after stroke. Given its potent anti-inflammatory activities on circulating leukocytes, the CB2 activation has been proven to produce protective effects against acute poststroke inflammation. In this paper, we will update evidence on different cannabinoid-triggered avenues to reduce inflammation and neuronal injury in acute ischemic stroke.


Urinary Elimination of 11-Nor-9-Carboxy-?9-tetrahydrocannnabinol in Cannabis Users During Continuously Monitored Abstinence
Journal of analytical toxicology 32(8):562-9 November 2008
DOI: 10.1093/jat/32.8.562
Marilyn A. Huestis, Robert S. Goodwin, William D. Darwin, C. Nora Chiang, Ming Shih, Shou-Hua Li, and Marilyn A. Huestis,
The time course of 11-nor-9-carboxy-?9-tetrahydrocannnabinol (THCCOOH) elimination in urine was characterized in 60 cannabis users during 24 h monitored abstinence on a closed research unit for up to 30 days. Six thousand, one hundred fiftyeight individual urine specimens were screened by immunoassay with values ? 50 ng/mL classified as positive. Urine specimens
were confirmed for THCCOOH by gas chromatography–mass
spectrometry following base hydrolysis and liquid–liquid or solidphase
extraction. In 60%, the maximum creatinine normalized concentration occurred in the first urine specimen; in 40%, peaks occurred as long as 2.9 days after admission. Data were divided into three groups, 0–50, 51–150, and > 150 ng/mg, based on the creatinine corrected initial THCCOOH concentration. There were statistically significant correlations between groups and number of days until first negative and last positive urine specimens; mean number of days were 0.6 and 4.3, 3.2 and 9.7, and 4.7 and 15.4 days, respectively, for the three groups. These data provide guidelines for interpreting urine cannabinoid test results and suggest appropriate detection windows for differentiating new cannabis use from residual drug excretion

Urinary Tetrahydrocannabinol After 4 Weeks of a Full-Spectrum, High-Cannabidiol Treatment in an Open-Label Clinical Trial
M. Kathryn Dahlgren, Kelly A. Sagar, Ashley M. Lambros, Rosemary T. Smith, Staci A. Gruber
JAMA Psychiatry Published online November 4, 2020
doi: 10.1001/jamapsychiatry.2020.3567
Methods This study was approved by the Partners Healthcare institutional review board, and all participants provided written informed consent. Study enrollment was conducted at McLean Hospital between June 2018 and February 2020. Participants were required to be 18 years or older, report at least moderate levels of anxiety assessed using well-validated measures,3,4 and test negative at baseline for 11-nor-9-carboxy- ?9 -tetrahydrocannabinol (THC-COOH), a major metabolite of ?9-THC. Patients did not use cannabis and could not use any other cannabis/cannabinoid–based products throughout the 4-week trial. Women were required to have a negative pregnancy test result. Exclusion criteria included serious medical illness (eg, kidney or liver disease, neurological disorder). The open-label phase was capped at 15 participants to determine dosing and tolerability. The CONSORT guidelines were followed. A protocol is available in the Supplement. The study product was formulated using a full-spectrum, high-CBD extract containing 9.97mg/mLofCBD (1.04%) and0.23 mg/mL of ?9-THC (0.02%), as confirmed by ProVerde Laboratories. Patients self-administered 1mL of the study product sublingually 3 times per day, for a targeted daily dose of approximately 30 mg of CBD and less than 1 mg of ?9-THC. The actual dosage was quantified using outgoing vs incoming bottle weights, cross-referenced with weekly drug diaries. Urine drug assays (a 12-panel test, waived by the Clinical Laboratory Improvement Amendments5 ) assessed the presence of THC-COOH,which was confirmed via gas chromatography–mass spectrometry (Quest Diagnostics). Exploratory logistic regression analyses (SPSS version 25 [IBM]; ? = .05, 2-tailed) assessed associations between THC-positive status, demographic variables, and creatinine, which is reflective of kidney function and hydration.
Results Of 15 patients enrolled (11 women [79%]; 12White individuals [86%]), 1 discontinued participation because of use of another cannabinoid product; the remaining 14 patients completed all study procedures (Figure). The study drug was well tolerated; no serious adverse events were reported, and no patients reported psychoactivity. Patients used amean (SD) of 3.48 (0.60)mL of the studyproductperday, equivalent to amean (SD) of 34.73 (6.03) mg of CBD per day and 0.80 (0.14) mg of ?9-THC per day.Results revealed that after 4weeks, 7 participants (50%) tested positive forTHC-COOH,while 7 tested negative. Gas chromatography–mass spectrometry results confirmed assay findings but indicated that the drug screen was often more sensitive than its stated lower limit of detection (50 ng/mL). Participants’ THC status was only significantly associated with creatinine levels (B, 1.92; P < .001; Table).
Discussion The results suggest that patients consistently using full-spectrum, hemp-derived products may have positive test results for THC-COOH on a urinary drug screen. Studies with larger sample sizes are needed tomore thoroughly assesswhich variables (product use, body mass index, age, sex, race, medication use, etc) contribute to positive findings in only some individuals, particularly those with higher creatinine levels. Importantly, the study product contained 0.02% of ?9-THC by weight; in the US, hemp-derived products can legally contain 0.30% or less of ?9-THC by weight, more than 10 times the amount of ?9-THC as the current study product. Despite limitations in sample size and diversity, these findings have important public health implications. It is often assumed individuals using hemp-derived products will test negative for THC. Current results indicate this may not be true, especially if assays are more sensitive than advertised, underscoring the potential for adverse consequences, including loss of employment and legal or treatment ramifications, despite the legality of hemp-derived products.

Use of Cannabidiol for the Treatment of Anxiety: A Short Synthesis of Pre-Clinical and Clinical Evidence.
Wright, M., Di Ciano, P., & Brands, B.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2019.0052
Anxiety disorders have the highest lifetime prevalence of any mental illness worldwide, leading to high societal costs and economic burden. Current pharmacotherapies for anxiety disorders are associated with adverse effects and low efficacy. Cannabidiol (CBD) is a constituent of the Cannabis plant, which has potential therapeutic properties for various indications. After the recent legalization of cannabis, CBD has drawn increased attention as a potential treatment, as the majority of existing data suggest it is safe, well tolerated, has few adverse effects, and demonstrates no potential for abuse or dependence in humans. Pre-clinical research using animal models of innate fear and anxiety-like behaviors have found anxiolytic, antistress, anticompulsive, and panicolytic-like effects of CBD. Preliminary evidence from human trials using both healthy volunteers and individuals with social anxiety disorder, suggests that CBD may have anxiolytic effects. Although these findings are promising, future research is warranted to determine the efficacy of CBD in other anxiety disorders, establish appropriate doses, and determine its long-term efficacy. The majority of pre-clinical and clinical research has been conducted using males only. Among individuals with anxiety disorders, the prevalence rates, symptomology, and treatment response differ between males and females. Thus, future research should focus on this area due to the lack of research in females and the knowledge gap on sex and gender differences in the effectiveness of CBD as a potential treatment for anxiety.

Use of cannabidiol (CBD) for the treatment of chronic pain.
Urits, I., Gress, K., Charipova, K., Habib, K., Lee, D., Lee, C., … Viswanath, O.
Best Practice & Research Clinical Anaesthesiology.(2020).
doi:10.1016/j.bpa.2020.06.004
Chronic pain can be recurrent or constant pain that lasts for longer than three months and can result in disability, suffering, and a physical disturbance. Related to the complex nature of chronic pain, treatments have a pharmacological and non-pharmacological approach. Due to the opioid epidemic, alternative therapies have been introduced, and components of the cannabis Sativa, ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have gained recent interest as a choice of treatment. The exact mechanism for cannabidiol is currently unknown, but unlike the CBD's psychoactive counterpart, THC, the side effects of CBD itself have been shown to be overall much more benign. The current pharmaceutical products for the treatment of chronic pain are known as nabiximols, and they contain a ratio of THC combined with CBD, which has been promising. This review focuses on the treatment efficacy of CBD, THC: CBD based treatments for chronic pain and adverse events with each.

Use of cannabinoid-based medicine among older residential care recipients diagnosed with dementia: study protocol for a double-blind randomised crossover trial.
Timler, A., Bulsara, C., Bulsara, M., Vickery, A., Smith, J., & Codde, J.
Trials, 21(1). (2020)
doi:10.1186/s13063-020-4085-x
Background: Dementia is a neurological condition that affects the cognitive and functional ability of the brain and is the leading cause of disability among those aged 65 years and above. More effective ways to manage dementia symptoms are needed because current treatment options (antidepressants and antipsychotics) can be ineffective and are associated with substantial side effects, including increased rate of mortality. Cannabinoid-based medicine (CBM) has shown an ability to inhibit some symptoms associated with dementia, and the adverse effects are often minimal; yet, little research has explored the use of CBM among this population. Aim: To monitor the safety of a purified dose of CBM oil (3:2 delta-9-tetrahydrocannabinol:cannabidi ol) on behaviour symptoms, quality of life and discomfort caused by pain. Methods/design: We will carry out an 18-week, randomised, double-blind crossover trial that consists of a 2-week eligibility period, two 6-week treatment cycles, and two 2-week washout periods (between both cycles and after the second treatment cycle). We aim to recruit 50 participants with dementia who are living in residential agedcare facilities. The participants will be randomised into two groups and will receive a dose of either CBM oil or placebo for the first treatment cycle and the opposite medication for the second. Data will be collected using the Neuropsychiatric Inventory Questionnaire, the Cohen-Mansfield Agitation Inventory, the Quality of Life in Alzheimer’s Disease questionnaire, and the Abbey Pain Scale on seven occasions. These will be completed by the participants, aged-care staff, and nominated next of kin or family members. The participants’ heart rate and blood pressure will be monitored weekly, and their body composition and weight will be monitored fortnightly by a research nurse, to assess individual dose response and frailty. In addition, pre- and post-surveys will be administered to aged-care staff and family members to understand their perceptions of CBM and to inform proposed focus groups consisting of the aged-care staff and next of kin. Discussion: The study design has been informed by medical professionals and key stakeholders, including those working in the residential aged-care industry to ensure patient safety, collection of non-invasive measures, and methodological rigor and study feasibility

Use of cannabinoids in cancer care: palliative care
S.K. Aggarwal
Current Oncology Vol. 23, Supp. 2, March 2016
DOI: 10.3747/co.23.2962
All too often neglected, maximal quality improvement in the setting of life-limiting illness and noxious symptomatology is a worthy medical, public health, and humanitarian goal. The World Health Organization estimates that, globally, about 60% of all people who die would benefit from palliative care before death; however, palliative care is not exclusively reserved for patients at the end of life1. Given the relative newcomer status of this field of medical care, some definition would be helpful. Palliative care is a multidisciplinary medical specialty—often involving social work and spiritual care—that is concerned with impeccable symptom management and supportive care for patients and their families facing life-limiting illness. It focuses on the amelioration of physical, emotional, psychological, and spiritual suffering2. Palliative care is not strictly reserved for practice by palliative care specialists; non–palliative care treating specialists can and should be trained to provide what is called “primary palliative care” such as basic pain management and facilitation of clinical discussions with patients and families about disease prognosis and advance care planning

Use of Cannabis for Self-Management of Chronic Pelvic Pain.
Carrubba, A. R., Ebbert, J. O., Spaulding, A. C., DeStephano, D., & DeStephano, C. C.
Journal of Women’s Health. (2020).
doi:10.1089/jwh.2020.8737
Background: Chronic pelvic pain (CPP) affects up to 15% of women in the United States. The endocannabinoid system is a potential pharmacological target for pelvic pain as cannabinoid receptors are highly expressed in the uterus and other nonreproductive tissues. We hypothesize that cannabis use is common for self-management of CPP, and our primary objective was to determine the prevalence of cannabis use in this population. Materials and Methods: A cross-sectional survey of women with pelvic and perineal pain, dyspareunia, or endometriosis was performed between March and August 2019. Subjects were recruited in an outpatient gynecology office. An anonymous, confidential, electronic survey was performed using a tablet. Statistical analysis was performed using JMP (SAS, Cary, NC). Results: A total of 240 patients were approached, with 113 responses (47.1% response rate). There were 26 patients who used cannabis (23%). The majority used at least once per week (n = 18, 72%). Most users (n = 24, 96%) reported improvement in symptoms, including pain, cramping, muscle spasms, anxiety, depression, sleep disturbances, libido, and irritability. Over one-third (35%) stated that cannabis use decreased the number of phone calls or messages sent to their provider, and 39% reported decreased number of clinical visits. Side effects, including dry mouth, sleepiness, and feeling ‘‘high,’’ were reported by 84% (n = 21).
Conclusions: Almost one-quarter of patients with CPP report regular use of cannabis as an adjunct to their prescribed therapy. Although side effects are common, most users report improvement in symptoms. Our study highlights the potential of cannabis as a therapeutic option for patients with CPP.

Use of Cannabis sativa L. for the treatment of Alzheimer's disease
Laura Jauregui
Conference: MOL2NET 2019, International Conference on Multidisciplinary Sciences, 5th edition
DOI: 10.3390/mol2net-05-06238
Alzheimer´s Disease (AD) is a neurodegenerative disorder which represents a problem for the public health system because it affects more than 50 million people in the world, currently there is no successful treatment to treat this disease. The most accepted hypothesis regarding the development of this disease is the accumulation of plaques A? in the brain, have studied different molecules of natural origin as treatment based on this hypothesis. Cannabis sativa L. is a plant that has great potential as a treatment for this disease due to its antioxidant and neuroprotective properties and it has been shown to reduce the accumulation of A? plaques

Use of Cannabis to Relieve Pain and Promote Sleep by Customers at an Adult Use Dispensary.
Marcus Bachhuber, Julia H. Arnsten & Gwen Wurm
Journal of Psychoactive Drugs. (2019).
doi:10.1080/02791072.2019.1626953
Medical cannabis patients consistently report using cannabis as a substitute for prescription medications; however, little is known about individuals accessing cannabis through adult-use markets. A survey at two retail stores was conducted in Colorado, United States. Between August 2016 and October 2016, store staff asked customers if they wanted to participate and, if so, provided an electronic survey link. All customers reporting medical certification were excluded. Of 1,000 adult-use only customer respondents, 65% reported taking cannabis to relieve pain and 74% reported taking cannabis to promote sleep. Among respondents taking cannabis for pain, 80% reported that it was very or extremely helpful, and most of those taking over-the-counter pain medications (82%) or opioid analgesics (88%) reported reducing or stopping use of those medications. Among respondents taking cannabis for sleep, 84% found it very or extremely helpful, and most of those taking over-the-counter (87%) or prescription sleep aids (83%) reported reducing or stopping use of those medications. De facto medical use of cannabis for symptom relief was common among adult-use dispensary customers and the majority reported that cannabis decreased their medication use. Adult use cannabis laws may broaden access to cannabis for the purpose of symptom relief.

Use of highly-potent cannabis concentrate products: More common in U.S. states with recreational or medical cannabis laws.
Deborah S.Hasin, Jacob Borodovsky, Dvora Shmulewitz, Claire Walsh, Cara A. Struble, Efrat Aharonovich, David S. Fink, Alan Budney
Drug and Alcohol Dependence Volume 229, Part B, 1 December 2021, 109159
DOI: 10.1016/j.drugalcdep.2021.109159
Background
Highly-potent cannabis products, e.g., concentrates, entail greater risks of cannabis-related harms than lower-potency products such as plant or flower material. However, little information is available on whether individuals in U.S. states with recreational cannabis laws (RCL) or medical cannabis laws (MCL) are more likely than individuals in U.S. states without cannabis legalization (no-CL) to use highly-potent forms of cannabis.
Methods
Cannabis-using adults in a 2017 online survey (N = 4064) provided information on state of residence and past-month cannabis use, including types of products used, categorized as low-potency (smoked or vaped plant cannabis) or high-potency (vaping or dabbing concentrates). Multivariable logistic regression models generated adjusted odds ratios (aOR) and 95% confidence intervals (CI) for use of high-potency cannabis products by state cannabis legalization status (RCL, MCL, no-CL).
Results
Compared to participants in no-CL states, participants in RCL states had greater odds of using high-potency concentrate products (aOR=2.61;CI=1.77-3.86), as did participants in MCL-only states (aOR=1.55;CI=1.21-1.97). When participants in RCL states and MCL states were directly compared, those in RCL states had greater odds of using high-potency concentrate products (aOR=1.69;CI=1.27-2.42).
Discussion
Although the sample was not nationally representative and the cross-sectional data precluded determining the direction of effect, results suggest that use of high-potency cannabis concentrates is more likely among those in RCL states. Clinicians in RCL states should screen cannabis users for harmful patterns of use. Policymakers in states that do not yet have RCL should consider these findings when drafting new cannabis laws, including the specific products permitted and how best to regulate them.
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Use of non-psychoactive residual biomass from Cannabis sativa L. for obtaining phenolic rich-extracts with antioxidant capacity

Guillermo A. Vega, Javier A. Dávila
Natural Products Research Aug 2021
DOI: 10.1080/14786419.2021.1969562
The present research aims to give an added value to the chain production of Cannabis sativa L plant, taking advantage of the non-psychoactive residual biomass (stems and leaves). Total phenolic content (TPC) and total antioxidant capacity (TAC) were analysed. A factorial design 23 was carried out using extraction time (T), Particle size (PS), and solid-solvent ratio (SS) as factors. The extractions were made with ethanol at 96% as solvent. The maximum concentration of TPC found was 1264.61 mg GA/g DW at 6:250 g/mL, 8 h, and 109.28 µm for SS, T and PS, respectively. Similarly, the maximum TAC obtained was 0.467 mM Trolox equivalent, at 8 h, 6:250 g/mL and 109.28 µm for T, SS and PS, respectively. Additionally, the presence of oil was found in some samples of cannabis extracts for which FITR was performed, obtaining the presence of C-OH bonds associated with alcohols, phenols and possible cannabinoids.
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Using cannabis for pain management after spinal cord injury: a qualitative study.
Bourke, J. A., Catherwood, V. J., Nunnerley, J. L., Martin, R. A., Levack, W. M. M., Thompson, B. L., & Acland, R. H.
Spinal Cord Series and Cases, 5(1). (2019).
doi:10.1038/s41394-019-0227-3
Study design A descriptive qualitative study.
Objectives To explore why individuals with spinal cord injury (SCI) choose to use cannabis to manage their pain and their experiences in doing so. Setting Community-dwelling adults with SCI in New Zealand.
Methods Semi-structured interviews were conducted with individuals who had a SCI, experienced pain, and self-reported use of cannabis to manage their pain. Interviews were recorded, transcribed, and subject to thematic analysis. Results Eight individuals participated in this study. We interpreted six themes that captured the participants’ perspectives regarding their choice to, and perceptions of, using cannabis to manage SCI pain. Participants were motivated to use cannabis when other pain management strategies had been ineffective and were well-informed, knowledgeable cannabis consumers. Participants reported cannabis reduced their pain quickly and enabled them to engage in activities of daily living and participate in life roles without the drowsiness of traditional prescribed pain medication. Despite the positive aspects, participants were concerned about the irregularity of supply and inconsistent dosage.
Conclusions Findings show that cannabis is used to reduce pain after SCI and enable increased community participation. Findings suggest that future studies examining the efficacy of cannabinoids in managing pain include function and participation outcome measures rather than solely focusing on measuring pain intensity. Focusing on meaningful outcomes may contribute to a greater understanding of the experiences of people with SCI.

Utilization of medicinal cannabis for pain by individuals with spinal cord injury
Michael Stillman ? Maclain Capron ? Michael Mallow ? Tracy Ransom ? Kristin Gustafson ? Alison Bell ? Daniel Graves
Spinal Cord Series and Cases volume 5, (2019) NATURE
https://www.nature.com/articles/s41394-019-0208-6.pdf
Study design A cross-sectional multi-center study using an on-line survey addressing utilization, knowledge, and perceptions of medicinal cannabis (MC) by people with spinal cord injury (SCI).
Objective To characterize differences between current (CU), past (PU), and never users (NU) of MC with SCI; to determine why people with SCI use MC; to examine reports of MCs’ efficacy and tolerability by individuals with SCI.
Setting Three academic medical centers in the United States.
Methods Comparison of demographic and attitudinal differences between CU, PU, and NU and differences in the groups’ reports of pain, health, and quality of life (QOL). Evaluation of utilization patterns and perceived efficacy of MC among CU and PU and reports of side effects of MC versus prescription medications. Data were analyzed using either Chi Square, distribution-free exact statistics, or t-tests for continuous data.
Results Among a nationwide sample (n = 353) of individuals with SCI, NU were less likely than CU and PU to believe that cannabis ought to be legalized and more likely to endorse risks of use. Current users and PU reported greater pain interference in daily life than did NU, but there were no between group differences in QOL or physical or emotional health. Current users and PU took MC to address pain (65.30%), spasms (63.30%), sleeplessness (32.70%), and anxiety (24.00%), and 63.30% reported it offered “great relief” from symptoms. Participants reported that MC is more effective and carries fewer side effects than prescription medications.
Conclusions Medicinal cannabis is an effective and well-tolerated treatment for a number of SCI-related symptoms.

Vaping Is Not Safe: A Case of Acute Eosinophilic Pneumonia following Cannabis Vapor Inhalation.
Antwi-Amoabeng, D., & Islam, R.
Case Reports in Pulmonology, 2020, 1–5.
doi:10.1155/2020/9496564
¬ere is a well-established association between inhalational exposures and acute eosinophilic pneumonia (AEP). ¬e most reported exposure is cigarette smoking. Here, we present a case of progressive shortness of breath and nonproductive cough in a college student with no significant medical history, approximately 10 days aer inhaling cannabis aerosols on two separate occasions. He was started on empiric antibiotics and bronchodilators without improvement. He was diagnosed with AEP based on peripheral eosinophilia and high-resolution CT image results. He made rapid recovery on intravenous glucocorticoids. Vaping has gained popularity among young adults mainly due to the perception that it is a safe alternative to smoking. ¬is case shows that there may be a false sense of security with vaping. Vaping poses a yet-to-be quantified public health threat, which requires further studies.


Vapor inhalation of cannabidiol (CBD) in rats.
Javadi-Paydar, M., Creehan, K. M., Kerr, T. M., & Taffe, M. A.
Pharmacology Biochemistry and Behavior, 172741. (2019).
doi:10.1016/j.pbb.2019.172741
Rationale: Cannabidiol (CBD), a compound found in many strains of the Cannabis genus, is increasingly available in e-cigarette liquids as well as other products. CBD use has been promoted for numerous purported benefits which have not been rigorously assessed in preclinical studies. Objective: To further validate an inhalation model to assess CBD effects in the rat. The primary goal was to determine plasma CBD levels after vapor inhalation and compare that with the levels observed after injection. Secondary goals were to determine if hypothermia is produced in male Sprague-Dawley rats and if CBD affects nociception measured by the warm water tail-withdrawal assay. Methods: Blood samples were collected from rats exposed for 30 minutes to vapor generated by an ecigarette device using CBD (100, 400 mg/mL in the propylene glycol vehicle). Separate experiments assessed the body temperature response to CBD in combination with nicotine (30 mg/mL) and the anti-nociceptive response to CBD. Results: Vapor inhalation of CBD produced concentration-related plasma CBD levels in male and female Wistar rats that were within the range of levels produced by 10 or 30 mg/kg, CBD, i.p.. Dose-related hypothermia was produced by CBD in male Sprague-Dawley rats, and nicotine (30 mg/mL) inhalation enhanced this effect. CBD inhalation had no effect on anti-nociception alone or in combination with ? 9 - tetrahydrocannabinol inhalation. Conclusions: The vapor-inhalation approach is a suitable pre-clinical model for the investigation of the effects of inhaled CBD. This route of administration produces hypothermia in rats, while i.p. injection does not, at comparable plasma CBD levels

Vapor Pressure, Vaping, and Corrections to Misconceptions Related to Medical Cannabis’ Active Pharmaceutical Ingredients’ Physical Properties and Compositions
Aharon M. Eyal, Dana Berneman Zeitouni, Dor Tal, Daniel Schlesinger, Elyad M. Davidson, and Noa Raz Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0173 https://www.liebertpub.com/doi/epdf/10.1089/can.2021.0173
Medical cannabis products contain dozens of active pharmaceutical ingredients (APIs) derived from the cannabis plant. However, their actual compositions and relative doses significantly change according to the production methods. Product compositions are strongly dependent on processing step conditions and on components’ evaporation during those steps. Review of the documentation presented to caregivers and to patients show erroneous data or misinterpretation of data related to the evaporation, for example, cannabinoids’ boiling points, as well as confusions between terms, such as boiling, vaporization, and evaporation. Clarifying these aspects is
essential for caregivers, for researchers, and for developers of manufacturing processes. Original and literature data were analyzed, comparing composition changes during various processing steps and correlating the extent of change to components’ vapor pressures at the corresponding temperature. Evaporation-related composition changes start at temperatures as low as those of drying and curing and become extensive during decarboxylation.
The relative rate of components’ evaporation is determined by their relative vapor pressure and monoterpenes are lost first. On vaping, terpenes are inhaled before cannabinoids do. Commercial medical cannabis products are deficient in terpenes, mainly monoterpenes, compared with the cannabis plants used to produce them. Terms, such as ‘‘whole plant’’ and ‘‘full spectrum,’’ are misleading since no product actually reflects the original cannabis plant composition. There are important implications for medical cannabis manufacturing and for the ability to make the most out of the terpene API contribution. Medical cannabis products’ composition and product delivery are controlled by the relative vapor pressure of the various APIs. Quantitative data provided in this study can be used for improvement to reach better accuracy, reproducibility, and preferred medical cannabis compositions.
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Vaporization as a Smokeless Cannabis Delivery
System: A Pilot Study
DI Abrams, HP Vizoso, SB Shade, C Jay, ME Kelly and NL Benowitz
Nature VOLUME 82 NUMBER 5 | NOVEMBER 2007
doi:10.1038/sj.clpt.6100200
Although cannabis may have potential therapeutic value, inhalation of a combustion product is an undesirable delivery system. The aim of the study was to investigate vaporization using the Volcanos device as an alternative means of delivery of inhaled Cannabis sativa . Eighteen healthy inpatient subjects enrolled to compare the delivery of cannabinoids by vaporization to marijuana smoked in a standard cigarette. One strength (1.7, 3.4, or 6.8%
tetrahydrocannabinol (THC)) and delivery system was randomly assigned for each of the 6 study days. Plasma concentrations of D -9-THC, expired carbon monoxide (CO), physiologic and neuropsychologic effects were the main
outcome measures. Peak plasma concentrations and 6-h area under the plasma concentration–time curve of THC were similar. CO levels were reduced with vaporization. No adverse events occurred. Vaporization of cannabis is a
safe and effective mode of delivery of THC. Further trials of clinical effectiveness of cannabis could utilize vaporization as a smokeless delivery system.

Vaporized cannabis extracts have reinforcing properties and support conditioned drug-seeking behavior in rats.
Freels TG, Baxter-Potter LN, Lugo JM, Glodosky NC, Wright HR, Baglot SL Petrie GN, Zhihao Y, Clowers BH, Cuttler C, Fuchs RA, Hill MN, McLaughlin RJ
J Neurosci. 2020 Jan 16. pii: 2416-19.
doi: 10.1523/JNEUROSCI.2416-19.2020.
Recent trends in cannabis legalization have increased the necessity to better understand the effects of cannabis use. Animal models involving traditional cannabinoid self-administration approaches have been notoriously difficult to establish and differences in the drug employed and its route of administration have limited the translational value of preclinical studies. To address this challenge in the field, we have developed a novel method of cannabis self-administration using response-contingent delivery of vaporized ?9-tetrahydrocannabinol-rich (CANTHC) or cannabidiol-rich (CANCBD) whole-plant cannabis extracts. Male Sprague Dawley rats were trained to nosepoke for discrete puffs of CANTHC, CANCBD, or vehicle (VEH) in daily one-hour sessions. Cannabis vapor reinforcement resulted in strong discrimination between active and inactive operanda. CANTHC maintained higher response rates under fixed ratio schedules and higher break points under progressive ratio schedules compared to CANCBD or VEH, and the number of vapor deliveries positively correlated with plasma THC concentrations. Moreover, metabolic phenotyping studies revealed alterations in locomotor activity, energy expenditure, and daily food intake that are consistent with effects in human cannabis users. Furthermore, both cannabis regimens produced ecologically relevant brain concentrations of THC and CBD and CANTHC administration decreased hippocampal CB1 receptor binding. Removal of CANTHCreinforcement (but not CANCBD) resulted in a robust extinction burst and an increase in cue-induced cannabis-seeking behavior relative to VEH. These data indicate that volitional exposure to THC-rich cannabis vapor has bona fide reinforcing properties and collectively support the utility of the vapor self-administration model for the preclinical assessment of volitional cannabis intake and cannabis-seeking behaviors.SIGNIFICANCE STATEMENTThe evolving legal landscape concerning recreational cannabis use has increased urgency to better understand its effects on the brain and behavior. Animal models are advantageous in this respect; however, current approaches typically employ forced injections of synthetic cannabinoids or isolated cannabis constituents that may not capture the complex effects of volitionalcannabis consumption. We have developed a novel model of cannabis self-administration using response-contingent delivery of vaporizedcannabis extracts containing high concentrations of ?9 tetrahydrocannabinol (THC) or cannabidiol (CBD). Our data indicate that THC-richcannabis vapor has reinforcing properties that support stable rates of responding and conditioned drug-seeking behavior. This approach will be valuable for interrogating effects of cannabis and delineating neural mechanisms that give rise to aberrant cannabis-seeking behavior.

VCE-004.3, A CANNABIDIOL AMINOQUINONE DERIVATIVE, PREVENTS BLEOMYCIN-INDUCED SKIN FIBROSIS AND INFLAMMATION TROUGH PPAR?- AND CB2 -DEPENDENT PATHWAYS.
Del Rio, C., Cantarero, I., Palomares, B., Gómez-Cañas, M., Fernández-Ruiz, J., Pavicic, C., … Muñoz, E.
British Journal of Pharmacology.(2018).*
doi:10.1111/bph.14450*
Background and Purpose: The endocannabinoid system (ECS) as well as PPAR? are relevant targets for the development of novel compounds against fibrotic diseases such as Systemic Sclerosis (SSc), also called Scleroderma. The aim of this study was to characterize VCE-004.3, a novel cannabidiol derivative, and to study it anti-inflammatory and anti-fibrotic activities. Experimental Approach: CB1, CB2 and PPAR? binding and functional activities were studied in vitro and in silico. The anti-fibrotic effects of VCE-004.3 were investigated in NIH-3T3 fibroblasts and in human dermal fibroblasts. To assess the anti-inflammatory and anti-fibrotic efficacy of VCE-004.3 in vivo we used two complementary models of bleomycin-induced fibrosis. The effect of VCE-004.3 on ERK1/2 phosphorylation induced by IgG from SSc patients and PDGF was also investigated. Key Results: VCE-004.3 binds and activates PPAR? and CB2 receptors and antagonizes CB1 receptor. VCE-004.3 binds to an alternative site at the PPAR? ligand-binding pocket (LBP). VCE-004.3 inhibits collagen gene transcription and synthesis and prevents TGF?-induced fibroblast migration and differentiation to myofibroblasts. VCE-004.3 prevents skin fibrosis, myofibroblast differentiation and ERK1/2 phosphorylation in bleomycin-induced skin fibrosis. In addition, VCE-004.3 reduces mast cell degranulation, macrophage activation, T lymphocytes infiltration, and the expression of inflammatory and profibrotic factors. Topical application of VCE-004.3 also alleviates skin fibrosis. Finally, VCE-004.3 inhibits PDGFBB- and SSc IgG-induced ERK1/2 activation in fibroblasts. Conclusion and Implications: VCE-004.3 is a novel semi-synthetic cannabidiol derivative behaving as a dual PPAR?/CB2 agonist and CB1 receptor modulator that could be considered for the development of novel therapies against different forms of Scleroderma.

VCE-004.8, A Multitarget Cannabinoquinone, Attenuates Adipogenesis and Prevents Diet-Induced Obesity.
Palomares, B., Ruiz-Pino, F., Navarrete, C., Velasco, I., Sánchez-Garrido, M. A., Jimenez-Jimenez, C., … Muñoz, E.
Scientific Reports, 8(1). (2018).
doi:10.1038/s41598-018-34259-0*
Over the past few years, the endocannabinoid system (ECs) has emerged as a crucial player for the regulation of food intake and energy metabolism, and its pharmacological manipulation represents a novel strategy for the management of metabolic diseases. The discovery that VCE-004.8, a dual PPAR? and CB2 receptor agonist, also inhibits prolyl-hydroxylases (PHDs) and activates the HIF pathway provided a rationale to investigate its efect in in vitro models of adipogenesis and in a murine model of metabolic syndrome, all processes critically regulated by these targets of VCE-004.8. In accordance with its diferent binding mode to PPAR? compared to rosiglitazone (RGZ), VCE-004.8 neither induced adipogenic diferentiation, nor afected osteoblastogenesis. Daily administration of VCE-004.8 (20mg/ kg) to HFD mice for 3-wks induced a signifcant reduction in body weight gain, total fat mass, adipocyte volume and plasma triglycerides levels. VCE-004.8 could also signifcantly ameliorate glucose tolerance, reduce leptin levels (a marker of adiposity) and increase adiponectin and incretins (GLP-1 and GIP) levels. Remarkably, VCE-004.8 increased the FGF21 mRNA expression in white and brown adipose, as well as in a BAT cell line, qualifying cannabinoaminoquinones as a class of novel therapeutic candidates for the management of obesity and its common metabolic co-morbidities

Very low doses of ?8-THC increase food consumption and alter neurotransmitter levels following weight loss.
Avraham, Y., Ben-Shushan, D., Breuer, A., Zolotarev, O., Okon, A., Fink, N., … Berry, E. M. (2004)
Pharmacology Biochemistry and Behavior, 77(4), 675–684.
doi:10.1016/j.pbb.2004.01.015
We have investigated the effect of 0.001 mg/kg D8 -tetrahydrocannabinol (THC) on food consumption, cognitive function, and neurotransmitters in mice. Sabra mice were treated with vehicle, THC, or THC+CB1 antagonist (SR141716A). The mice were fed for 2.5 h a day for 9 or 50 days. In the 9-day schedule, THC-treated mice showed a 16% increase in food intake compared with controls ( P<001) was recorded. During the course of the 9- and 50-day experimental protocol, all mice lost about 20% and 10% of their original weight, respectively, to reach approximately the same weights, which were not significantly different between the different treatment groups. In addition, THC caused an increase in activity ( P<05) Cognitive function showed a tendency to improve (P<06) in the THC-treated mice, which was reversed by the antagonist for Days 4 and 5 of the maze (P<01 and P<05, ) , respectively). Significant decreases in dopamine and serotonin (5-HT) levels were found both in the hypothalamus (P<01) and the hippocampus (P<01, P<05) ), respectively, while norepinephrine (NE) levels showed tendency to increase in both the hypothalamus and hippocampus. D8 -THC increased food intake significantly more (P<05) than did D9 -THC, while performance and activity were similar. Thus, D8 -THC (0.001 mg/kg) caused increased food consumption and tendency to improve cognitive function, without cannabimimetic side effects. Hence, a low dose of THC might be a potential therapeutic agent in the treatment of weight disorders.

Vitamin D mitigates diabetes-associated metabolic and cognitive dysfunction by modulating gut microbiota and colonic cannabinoid receptor 1
Hebatallah M. Hussein, Mohammed F. Elyamany, Laila A. Rashed , Nada A. Sallam
European Journal of Pharmaceutical Sciences (2021),
doi: 10.1016/j.ejps.2021.106105
https://reader.elsevier.com/reader/s...1?token=BAA679 B94091C131644C21B0B80A1029F72D B5A0C2AA032D80DBF3C84F913876C2 58961E36B88480EB3BA24C4CD4FD3E &originRegion=us-east-1&originCreation=20220103044 23 0
Introduction: Obesity is associated with elevated endocannabinoid tone, gut dysbiosis, and inflammation predisposing to diabetes. The endocannabinoid system mediates the effects of gut microbiota and regulates the gut barrier integrity. We examined the effects of vitamin D (VD) on colonic cannabinoid receptor 1(CB1R), tight junction proteins, gut dysbiosis, metabolic and cognitive dysfunction in a model of type 2 diabetes compared with metformin.
Methods: Rats received high-fat, high-sucrose diet (HFSD) and either VD (500 IU/kg/day; p.o.), or metformin (200 mg/kg/day; p.o.) for 8 weeks. After 6 weeks, streptozotocin (STZ) (40 mg/kg; i.p) was injected. Behavioral, cognitive, and metabolic assessments were carried out. Finally, fecal, blood, and tissue samples were collected to examine Bacteroidetes/Firmicutes ratio, colonic CB1R, zonula occludens-1 (ZO-1), occludin, and Toll-like receptor 4 (TLR4); serum lipopolysaccharides (LPS), peptidoglycan (PGN), tumor necrosis factor-alpha (TNF-ɑ), glucagon-like peptide-1 (GLP-1), lipids, and VD; hippocampal brain-derived neurotrophic factor (BDNF) and inflammatory markers.
Results: VD ameliorated HFSD/STZ-induced dysbiosis/gut barrier dysfunction as indicated by lower circulating LPS, PGN and TNF-ɑ levels, likely by downregulating colonic CB1R and upregulating ZO-1 and occludin expressions. Additionally, VD suppressed HFSD/STZ-induced hyperglycemia, hyperinsulinemia, dyslipidemia, and hippocampal neuroinflammation. These changes culminated in improved glycemic control and cognitive function. VD was more effective than metformin in decreasing serum LPS and TNF-ɑ levels; whereas metformin resulted in better glycemic control.
Conclusion: Targeting gut microbiota by VD could be a successful strategy in the treatment of diabetes and associated cognitive deficit. The crosstalk between VD axis and the endocannabinoid system needs further exploration.

Wakey, Wakey, Sleepy Head
Data from the National Health and Nutrition Examination Survey sheds light on the role of cannabis in sleep–wake regulation

Diep C, et al. Reg Anesth Pain Med, 0, 1-5 (2021). DOI: 10.1136/rapm-2021-103161 Find Pdf Not available yet


What are the differences between full spectrum CBD and broad spectrum CBD?
MedicalNewsToday
https://www.medicalnewstoday.com/art...um-cbd#summary
There are three main types of cannabidiol (CBD). CBD isolate is the pure form of CBD, while full-spectrum CBD is an extract containing other compounds of the cannabis plant, such as terpenes and other cannabinoids. The third type is broad-spectrum CBD.
People use CBD for a variety of health reasons, such as pain and anxiety. While more research could clarify their anecdotally reported benefits, some evidence suggests that CBD products can help with certain conditions.
This article aims to help demystify the terms CBD isolate and full-spectrum CBD. We explain the differences and potential benefits of both forms. Full spectruspectrum CBD are two of the three main types of CBD extract available. The main difference between the two relates to the other naturally occurring plant compounds in the extract.
Although much of the research into CBD is still in its early stages, many people are eager to try CBD products. Anecdotal evidence suggests that some people have found CBD helpful in relieving ailments such as anxiety and pain.
In fact, one 2018 study Trusted Source in people who use CBD revealed that 62% of people use it for medical symptoms, including pain, depression, and anxiety.
CBD is one of many compounds in the cannabis plant. Other than tetrahydrocannabinol (THC), it is the most abundant compound in the plant.

What Do You Know About Maryjane? A Systematic Review of the Current Data on the THC:CBD Ratio.
Zeyl, V., Sawyer, K., & Wightman, R. S.
Substance Use & Misuse, 1–5. (2020).
doi:10.1080/10826084.2020.1731547
Background: Ratios of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) impact metabolism and therapeutic effects of cannabis. Currently, no states with legalized medical or recreational cannabis consider ratios THC:CBD in regulations. Objective: Determine what THC:CBD ratios are selected for use in clinical cannabis trials and what is the rationale. Methods: This is a systematic literature review of Central, CINAHL, Embase, PsycInfo, and PubMed of the last 10 years of English language medical cannabis publications highlighting THC:CBD ratios. Included were clinical studies of products containing and listing both THC and CBD ratios, percentages, or weighted amounts. Case reports and series, abstracts, reviews, and meta-analysis were excluded. Non-human, nontherapeutic, or studies examining approved cannabis pharmaceuticals were excluded. Results: Four hundred and seventy-nine (479) unique references were found, of which 11 met inclusion criteria. THC:CBD ratios listed and/or calculated: 1:0, 22:1, 2:1, 1:1, 1:2, 1:6, 1:9, 1:20, 1:33, 1:50, and 0:1. Rationale for ratios selected was often not listed, or simply trivialized as the ratios available to patients in the area, or ratios that were pharmaceutically available throughout the study country. One study compared ratios of high and low THC:CBD, but did not specify the ratios. Conclusion: The medical and scientific communities have not drawn substantive conclusions nor thoroughly explored THC:CBD ratios for “best practice” treatment of different disease processes and their sequelae. While there is evidence that cannabis provides medical benefits, research is lacking on standardization of medical cannabis use in modern medical practices.

WHAT HAPPENS TO CBD IN THE BODY?
FRED GARDNER
O’Shaughnessy’s Online
https://beyondthc.com/what-happens-to-cbd-in-the-body/

What is the prevalence and risk of cannabis use disorders among people who use cannabis? A systematic review and meta-analysis.
Leung, J., Chan, G. C. K., Hides, L., & Hall, W. D.
Addictive Behaviors, 106479. (2020).
doi:10.1016/j.addbeh.2020.106479
Background and Aims: We aim to quantify the prevalence and risk of having a cannabis use disorder (CUD), cannabis abuse (CA) or cannabis dependence (CD) among people in the general population who have used cannabis. Method: We conducted a systematic review of epidemiological cross-sectional and longitudinal studies on the prevalence and risks of CUDs among cannabis users. We identified studies published between 2009-2019 through PubMed, the Global Burden Disease (GBD) Database, and supplementary searches up to 2020. The outcomes of interest were CUDs based on DSM or ICD criteria. Estimates were synthesized using random-effects meta-analyses, followed by meta-regression of study characteristics on effect sizes.
Results; From 1383 records identified, 21 studies were included. Meta-analyses showed that among people who used cannabis, 22% (18-26%) have CUD, 13% (8-18%) have CA, and 13% (10- 15%) have CD. Estimates from cohort studies, showed that the risk of developing CD increased to 33% (22-44%) among young people who engaged in regular (weekly or daily) use of cannabis. There was a lack of data from cohort studies to estimate the risk of CUD or CA among regular cannabis users.
Conclusions: Cannabis users need to be informed about the risks of developing CUDs and the higher risks among those who initiate early and use frequently during adolescence. Future studies are 3 needed to examine how changes in cannabis policies may affect the risks of CUDs in the population.

Where there’ s smoke, there’ s fire: cannabis allergy through passive exposure
Ine I. Decuyper, Margaretha A. Faber, Vito Sabato, Chris H. Bridts,
Margo M. Hagendorens, Hans-Peter Rihs, Luc S. De Clerck, and Didier G.
DOI: https://doi.org/10.1016/j.jaip.2016.10.019
The effects of indirect exposure to cannabis (respiratory/cutaneous) on allergic sensitization are unknown. We report on 2 cases in whom cannabis sensitization and allergy with subsequent cross-reactive food allergies seem
to result from exposure to cannabis smoke and/or indirect cutaneous transmission.

White Paper – Technology for Medical Cannabis
10 critical succesfactors for achieving the best results!
CODEMA
https://codema.nl/products/medicinal-cannabis/
https://codema.nl/wp-content/uploads...nnabis-ENG.pdf
Codema has been supplying technology to medicinal cannabis growers for more than 15 years. In the last number of years we are involved in increasing number of medicinal and legalized Cannabis projects in both Canada and the US. We believe in our contribution to creating a healthier and more sustainable world for future generations. That is why we develop horticultural solutions for the cultivation of medicinal cannabis. A crop that ensures that people who benefit from it can lead a better life.

Wound healing activity of terpinolene and α-phellandrene by attenuating inflammation and oxidative stress in vitro.
De Christo Scherer, M. M., Marques, F. M., Figueira, M. M., Peisino, M. C. O., Schmitt, E. F. P., Kondratyuk, T. P., … Fronza, M.
Journal of Tissue Viability.(2019).
doi:10.1016/j.jtv.2019.02.003
This study was undertaken to investigate the in vitro wound healing effects and the anti-inflammatory and antioxidant activities of terpinolene and α-phellandrene. The in vitro stimulatory effects on the proliferation and migration of fibroblasts were assessed using the scratch assay. The anti-inflammatory activity was evaluated using cell-based assays by investigating their influence on nitric oxide (NO), superoxide anion (O2 •- ), tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) production and using the TNF-α-induced nuclear factor kappa (NF-κB) assay. Antioxidant activity was determined by the ABTS cation radical scavenging capacity, ferric reducing/antioxidant potential (FRAP), and NO free radical scavenging assays. Terpinolene and α-phellandrene significantly increased the migration and proliferation of fibroblasts and suppressed the pro-inflammatory cytokines IL-6 and TNF-α in a dose-dependent manner. Terpinolene and α-phellandrene at a concentration of 100 μM significantly inhibited NO production (41.3 and 63.8%, respectively) in a macrophage cell-culture-based assay, and resulted in reductions in O2 •- production of 82.1 ± 3.5% and 70.6 ± 4.3%, respectively. Moreover, these monoterpenes were verified to suppress NF-κB activity. In summary, terpinolene and α-phellandrene may contribute to broadening clinical options in the treatment of wounds by attenuating inflammation and oxidative stress in vitro.
 
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