Scrutinizing Strains with Science : An Objective Discussion

spurr · Nov 17, 2010

@ all,

Here are needed papers and info (#1 thru #3) for testing with TLC and using spot density measurement. Also covered in #2 is GC, HPLC, etc.

As I wrote above, TLC can be either comparative (without standards) or quantitative (with standards); but HPTLC (or OPLC) is better for quantitative planar chromatography. These papers (#2 and #3) are my main resources for the methodologies I have been working on, along with JustTLC. I plan to use Fast Blue BB salt as the stating/visualization reagent and extra preservative step.

1. JustTLC software
http://www.sweday.com/Products.aspx

^^^ software for spot density measurement for quantitative, or comparative results.

2. "Recommended methods for the identification and analysis of cannabis and cannabis products"
United Nations Office on Drugs and Crime (UNODC) 2009
http://www.unodc.org/documents/scientific/ST-NAR-40-Ebook.pdf

^^^ that paper is neat as it discuses feminzed seeds, etc., besides it's value for TLC, HPLC and GC assays, etc. This paper should be a main go-to resource.

3. (uploaded) "PHG 414 Practical course: Detection of Cannabis in samples of different sources"

^^^ that paper is a good resource, along with the one above, when one is developing testing methodologies, ex., this paper is good for info on TLC (replacing hexane might be a good idea, fwiw).

4. "Comparison of three advanced chromatographic techniques for cannabis identification"
http://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1994-01-01_2_page009.html

^^^ and older (1994) paper by the UN about TLC vs. GC vs. HPLC; the info isn't wholly accurate today with the advancement of TLC and HPTLC, but it's still a good read.

5. "TLC Testing Protocol, 2009"
http://www.democitydrug.org/uploads...rotocols/ENERGY CONTROL TLC protocol 2009.pdf

^^^ a good, basic intro into TLC

6. "Quantitative Chromatographic Analysis"
by Raymond P. W. Scott, part of the Chrom-Ed Series
http://www.chromatography-online.org/quant/contents.html

^^^ an older series that is not wholly accurate for today's TLC, HPTLC, etc., ex. using spot density measurment isn't covered. But it does have some worthwhile info.

Sam_Skunkman · Nov 18, 2010

spurr said:
Not yet, but I agree it's a no-brainier. The trick is figuring out which ones to test for, I listed a couple from the two studies I previously posted.

I didn't mean they get you high alone, I meant they affect the high from THC in a synergistic or antagonist manner. Most cultivars grown today are lacking in decent quantities of CBC, THCV, etc., esp. CBG. I'm sure this info is nothing new to you. However, being there are currently 70 identified cannabioinds (4 new ones), with too little research thus far, it's hard to say none other than THC have strong psychotropic properties (ElSohly and Slade, 2005).

First of all there are now over 90 identified Cannabinoids, I have tried maybe a dozen all alone and with pure THC, none get you high, except for THC and CBN if you call CBN a high. There are a few Cannabinoid modulators of THC, just a few. Most had zero effect on THC. Instead it is the Terpenoids I have found.

spurr said:
Yea, I've read a few papers on that, interesting stuff for sure. I wonder about the myriad of other cannabinoids found in small quantities in cannabis, too little research has been conducted on all 70 of them. I assume at least a couple are psychotropically active, besides THC.

Your assumption is wrong I would bet you.
It is the Terpenoids that with THC create all the subjective effects found in so many different varieties of Cannabis. That and CBD, CBN, THCV which are THC modulators when they are present.
Maybe we will find another terpenoid that does get you high but then why did not primitive indigenous farmers find it like they did with THC? They created pure THC varieties by eliminating anything else, all without knowing anything about THC. I suspect they would have found any other Cannabinoid that would of gotten them high, don't you?

spurr said:
What do you mean by "subjective effects"? Anything non-quantitative, or none comparative (such as TLC spot density measurement without standards) is subjective, ex., smoking a bud. AFAIK, other secondary metabolites than cannabinoids and some terpenoids affect the high, ex., some flavonoids.

I mean what a person feels when they smoke.
Can you name a single flavonoid that modulates THC's high? I don't know any.

spurr said:
I'm in the US...for now.

Where? PM me.

spurr said:
I agree, but for most people (at least in the US) using a GC is not possible, only a few states have labs that will tests cannabis (usually using GC or HPLC). Thus using comparative TLC via. spot density measurement (i.e. densitometry) with "JustTLC", is a good runner up. Also, TLC can be used in a truly quantitative manner by using densitometry and standards, but it's more cumbersome than using other planar chromatography quantitative methods I list below (Raharjo and Verpoorte, 2004).

There are other, rather simple, and inexpensive planar chromatography methods that are quantitative and provide accuracy on par with HPLC and even GC, ex. using High-Performance Thin-Layer Chromatography (HPTLC), or Optimum-Performance Laminar Chromatography (previously known as OverPressured-Layer Chromatography; OPLC). Using either HPTLC or OPLC, are superior, and more trivial for quantitative assays vs. classical TLC.

I think using HPTLC, for the 'average Joe' grower, for quantitative measurement is the best bet, esp. because they can do so at home. And one can buy DEA-exempt cannabinoid standards now, thus there is little stopping a motivated and intelligent grower from carrying out truly quantitative (and accurate) assays in the US and abroad. Considering GC and HPLC are not available for most people, and that HPTLC is cheaper, and easier, then GC and HPLC, I think HPTLC is the way to go. Also using HPTLC one can quantify THC-COOH, unlike when using GC. That is why I think using HPTLC with DEA-exempt standards is the best option for most 'average Joes'; and TLC using spot density measurement comparison (via. "JustTLC") being a runner up for most people.

For me it is not about most people it is about the most accurate.

spurr said:
Using a standards and spot density measurement allows for quantitative assay of samples using classical TLC. Because one can buy DEA-exempt cannabinoid standards in the US, quantitative TLC is possible for nearly anyone to carry out for a few hundred dollars to setup the lab. That said, using classical TLC is not the best choice IMO, as I mentioned above, using HPTLC would be a better choice. I have a pretty good papers on quantitative planar chromatography (ex. HPTLC, OPLC and TLC). That said, GC is definitely the better method for people with lots of money, expertize and resources.

I am very interested in HPTLC and OPLC, vs. classical TLC, HPLC and GC. HPTLC offers benefits over GC and HPLC, such as "lower running costs [and setup costs], more rapid analysis time and the ability to analyse multiple samples simultaneously..." (Fischedick, Glas, Hazekamp, Verpoorte, 2009).

Did you use a staining/visualization reagent for TLC? Such as fast blue BB salt? Or did you rely upon UV? Did you use color (staining) reagent and scan spot density for measurement, or use spectrophotometry (UV) and/or Rf values?

Yes I used a staining reagent, and spot density. I used it for field work.

spurr said:
A great read for newbies on TLC and GC, and cannabis assays in general, is the "Recommended methods for the identification and analysis of cannabis and cannabis products", United Nations Office on Drugs and Crime (UNODC) 2009 (link). That is probably the best source for a person to develop standardized TLC and GC assay methods.

Testing standardization is one worry I have about various tests from labs, not all labs follow the same methodology, or even use the same standards or methods (i.e. GC vs. HPLC, etc.). Thus I worry about the ability to normalize and compare results from various labs...I think we need much more standardization than we have today.

Good luck, the companies like Sigma sell standards that say 96% pure but when I tested them at several labs that work with Cannabis for years, they were 85%.

spurr said:
One reason I dislike GC is you can't quantify THC-A (THC-COOH), only total THC due do the decarb of THC-A from heat of GC (Raharjo and Verpoorte, 2004). This isn't a problem with some planar chromatography if following certain methods such as HPTLC or OPLC; HPTLC might be the best option.

What do you need to test THCA for? It is all converted when made active by smoking or cooking. Maybe for products to be cooked?
I don't understand why so many people want to test for THCA?

spurr said:
I am interested not only in GC, classical TLC and HPLC, but other planar chromatography methods, esp. HTPLC and OPLC. I have a papers on OPLC, Automated Multiple Development (AMD) and HPTLC of cannabis, among other papers on GC, HPLC, et al.

spurr said:
Nice setup!

It works and keeps the work load managable.

spurr said:
Various refs., and cites from above:

Mahmoud A. ElSohly, Desmond Slade. 2005. Chemical constituents of marijuana: The complex mixture of natural cannabinoid. Life Sciences, 78, (2005), 539 – 548

Justin T. Fischedick, Ronald Glas, Arno Hazekamp, and Rob Verpoorte. 2009. A Qualitative and Quantitative HPTLC
Densitometry Method for the Analysis of Cannabinoids in Cannabis sativa L. Phytochem. Anal. 2009, 20, 421–426

Tri J. Raharjo and Robert Verpoorte. 2004. Methods for the Analysis of Cannabinoids in Biological Materials: a Review. Phytochem. Anal. 15, 79–94 (2004)

B. Szabady, E. Hidv6gi, Sz. Nyiredy. 2002. Determination of Neutral Cannabinoids in Hemp Samples by Overpressured-Layer Chromatography. Chromatographia Supplement Vol. 56, 2002

Richard Johnsson, Gustav Träff, Martin Sundén and Ulf Ellervik. 2007. Evaluation of quantitative thin layer chromatography using staining reagents. Journal of Chromatography A, Volume 1164, Issues 1-2, 14 September 2007, Pages 298-305

^^^ that last paper is looks at efficacy of using JustTLC for (stained) spot density measurement with TLC for quantification of samples with a flat bed scanner (instead of doing so spectrophotometrically), using a reagent such as Fast Blue BB salt. That is the method I suggest most people use with classical TLC, in a comparative manner if one does not have standards, and is what I have been working on for the 'average Joe'.

Biologically Active Cannabinoids from High-Potency Cannabis sativa.
act Safwat A. Ahmed†, Samir A. Ross*†‡, Desmond Slade†, Mohamed M. Radwan†, Fazila Zulfiqar† and Mahmoud A. ElSohly*†§
National Center for Natural Products Research, Department of Pharmacognosy, and Department of Pharmaceutics, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677
J. Nat. Prod., 2008, 71 (4), pp 536–542
DOI: 10.1021/np070454a
Publication Date (Web): February 28, 2008
Copyright © 2008 The American Chemical Society and American Society of Pharmacognosy

Eleven new cannabinoid esters, together with three known cannabinoid acids and Δ9-tetrahydrocannabinol (Δ9-THC), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be β-fenchyl Δ9-tetrahydrocannabinolate (1), epi-bornyl Δ9-tetrahydrocannabinolate (2), α-terpenyl Δ9-tetrahydrocannabinolate (3), 4-terpenyl Δ9-tetrahydrocannabinolate (4), α-cadinyl Δ9-tetrahydrocannabinolate (5), γ-eudesmyl Δ9-tetrahydrocannabinolate (6), γ-eudesmyl cannabigerolate (7), 4-terpenyl cannabinolate (8), bornyl Δ9-tetrahydrocannabinolate (9), α-fenchyl Δ9-tetrahydrocannabinolate (10), α-cadinyl cannabigerolate (11), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA), and cannabigerolic acid (CBGA).

Do you have Arno's posters, Acidic Cannabinoids Spectroscopical Data Chart & Neutral Cannabinoids Spectroscopical Data Chart? They are pretty nice, have you seen them?

-SamS

spurr · Nov 18, 2010

Hey sam,

Sam_Skunkman said:
First of all there are now over 90 identified Cannabinoids,

Do you mind providing a reference for that claim? Thanks.

I have tried maybe a dozen all alone and with pure THC, none get you high, except for THC and CBN if you call CBN a high. There are a few Cannabinoid modulators of THC, just a few. Most had zero effect on THC. Instead it is the Terpenoids I have found.

Your assumption is wrong I would bet you.

I agree with you to the extent you have tested, but, too little research has been conducted so far on the other cannabinoids and flavonids, etc., to make an absolute claim.

My assumption might be wrong, I agree, but I could be correct too; there is too little research as of yet to say either way definitively.

It is the Terpenoids that with THC create all the subjective effects found in so many different varieties of Cannabis. That and CBD, CBN, THCV which are THC modulators when they are present.

As proven thus far, but the jury is far from out on the topic, there simply isn't enough data yet.

Maybe we will find another terpenoid that does get you high but then why did not primitive indigenous farmers find it like they did with THC? They created pure THC varieties by eliminating anything else, all without knowing anything about THC. I suspect they would have found any other Cannabinoid that would of gotten them high, don't you?

Maybe, maybe not. And maybe they didn't have cultivars with enough quantity of specific terpenoids or cannabinoids, etc., to experience a high and to selectively breed for them.

Also, quantity of secondary metabolites (ex. cannabinoids) is phenotypic expression (~50/50 genetics and environment), thus their quantity is greatly effected by growing environment and method. But, chemotype is a genotypic expression (near 100% genetics), thus the growing method and environment has little impact upon the ratios. So, if the indigenous growers had a growing environment (incl. soil and atmosphere issues) that was not conducive to high quantity of cananbinoids (other than THC, etc.) they wouldn't have be able to breed for them...

Can you name a single flavonoid that modulates THC's high? I don't know any.

I think I might be able to, IIRC, but maybe not, give me a couple of days.

Where? PM me.

OK, will do.

For me it is not about most people it is about the most accurate.

I agree 101%. However, the vast majority of people aren't able to buy and use GC, thus I am trying to help them with methods they can use. For myself, I prefer GC too.

Yes I used a staining reagent, and spot density. I used it for field work.

Cool. What reagent did you use? And how did you quantify spot density?

Good luck, the companies like Sigma sell standards that say 96% pure but when I tested them at several labs that work with Cannabis for years, they were 85%.

Yea, Sigma can be tools at times. I have three different sources for pure cananbinoids, one source you and I share I think (from Europe). IIRC, the US the source for DEA-exempt cannabinoid standards has had their standards tested by an outside lab as proof of purity of >98%.

Using HPLC or even classical TLC to make one's own standards is another, more difficult route, esp. using TLC (which is pretty hard to do correctly, thus HPLC is better).

What do you need to test THCA for? It is all converted when made active by smoking or cooking. Maybe for products to be cooked? I don't understand why so many people want to test for THCA?

For more data, that's all. I agree it's not needed due to decarb when we smoke, or heat (cook) or use alkaline water bath. That said, I like having as much data as possible.

Biologically Active Cannabinoids from High-Potency Cannabis sativa.
act Safwat A. Ahmed†, Samir A. Ross*†‡, Desmond Slade†, Mohamed M. Radwan†, Fazila Zulfiqar† and Mahmoud A. ElSohly*†§
National Center for Natural Products Research, Department of Pharmacognosy, and Department of Pharmaceutics, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677
J. Nat. Prod., 2008, 71 (4), pp 536–542
DOI: 10.1021/np070454a
Publication Date (Web): February 28, 2008
Copyright © 2008 The American Chemical Society and American Society of Pharmacognosy

Eleven new cannabinoid esters, together with three known cannabinoid acids and Δ9-tetrahydrocannabinol (Δ9-THC), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be β-fenchyl Δ9-tetrahydrocannabinolate (1), epi-bornyl Δ9-tetrahydrocannabinolate (2), α-terpenyl Δ9-tetrahydrocannabinolate (3), 4-terpenyl Δ9-tetrahydrocannabinolate (4), α-cadinyl Δ9-tetrahydrocannabinolate (5), γ-eudesmyl Δ9-tetrahydrocannabinolate (6), γ-eudesmyl cannabigerolate (7), 4-terpenyl cannabinolate (8), bornyl Δ9-tetrahydrocannabinolate (9), α-fenchyl Δ9-tetrahydrocannabinolate (10), α-cadinyl cannabigerolate (11), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA), and cannabigerolic acid (CBGA).

Cool, thanks for the cite, I will download it today.

Do you have Arno's posters, Acidic Cannabinoids Spectroscopical Data Chart & Neutral Cannabinoids Spectroscopical Data Chart? They are pretty nice, have you seen them?

No, I don't, and I haven't seen them. I will try to look them up and get a hold of them. Are they digitized? Thanks for the heads up!

Sam_Skunkman · Nov 18, 2010

spurr said:
Hey sam,

Do you mind providing a reference for that claim? Thanks.

Dr Mahmoud A. ElSohly recently told me more then 90, I have seen publications that list 85, but I need to remember which one. Not sure if all 90 are published yet.

spurr said:
I agree with you to the extent you have tested, but, too little research has been conducted so far on the other cannabinoids and flavonids, etc., to make an absolute claim.

My assumption might be wrong, I agree, but I could be correct too; there is too little research as of yet to say either way definitively.

As proven thus far, but the jury is far from out on the topic, there simply isn't enough data yet.

As the researcher that has done more research on the subject then all others combined, I still stick to what I say.

spurr said:
Maybe, maybe not. And maybe they didn't have cultivars with enough quantity of specific terpenoids or cannabinoids, etc., to experience a high and to selectively breed for them.

Also, quantity of secondary metabolites (ex. cannabinoids) is phenotypic expression (~50/50 genetics and environment), thus their quantity is greatly effected by growing environment and method. But, chemotype is a genotypic expression (near 100% genetics), thus the growing method and environment has little impact upon the ratios. So, if the indigenous growers had a growing environment (incl. soil and atmosphere issues) that was not conducive to high quantity of cananbinoids (other than THC, etc.) they wouldn't have be able to breed for them...

I suspect if the environment is not good for Cannabis then it will not be easy to breed for any Cannabinoid. But what about all the areas that are good for Cannabis production? Why did it not happen at any of them? THC selection is the hand of man, plain and simple, wild Cannabis is never high in THC, unless recently escaped from cultivation by man. Same as any other Cannabinoid that man would of selected for, if you think that Cannabis was high in THC before the hand of man you are mistaken. Without the hand of man to maintain through selection Cannabis THC Cannabinoid levels fall quickly.

spurr said:
I think I might be able to, IIRC, but maybe not, give me a couple of days.

Let me know and I will try it with 100% pure THC. But to be honest I have zero faith it will work.

spurr said:
Cool, thanks for the cite, I will download it today.

No, I don't, and I haven't seen them. I will try to look them up and get a hold of them. Are they digitized? Thanks for the heads up!

Yes they are a power point poster.
-SamS

buddah · Dec 5, 2010

interesting thread :thank you:

....good info

spurr · Dec 5, 2010

@ all,

I thought I would upload the PDFs I posted on page 1 to this thread, and a couple of others that are on topic, and of interest.

Esp. of interest is the mini academic argument between M.ElSohly, et al., and E.Russo, et al. The paper by M.ElSohly, et al., ("Cannabis versus THC: response to Russo and McPartland") is an answer to the criticisms of the S.Wachtel and ElSohly, et al., paper ("A comparison of the subjective effects of tetrahydrocannabinol and marijuana in humans") by the paper from E.Russo, et al., ("Cannabis is more than simply tetrahydrocannabino").

FWIW, I plan to start a thread in this sub-forum about various secondary metabolites of cannabis, their effects, etc.

Papers I uploaded:

"Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?"
John M. McPartland and Ethan B. Russo
Journal of Cannabis Therapeutics (2001), Vol.1, No.3/4, pp. 103-132

"Comparison of the subjective effects of ∆9-tetrahydrocannabinol and marijuana in humans"
S. R. Wachtel, M. A. ElSohly, S. A. Ross and J. Ambre H. de Wit
Psychopharmacology (2002) 161:331–339

"Cannabis is more than simply ∆9-tetrahydrocannabinol"
Ethan B. Russo and John M. McPartland
Psychopharmacology (2003) 165:431–432

"Cannabis versus THC: response to Russo and McPartland"
Mahmoud A. ElSohly, Stephen R. Wachtel and Harriet Wit
Psychopharmacology (2003) 165:433–434

"Secondary metabolism in cannabis"
Isvett Joseﬁna Flores-Sanchez and Robert Verpoorte
Phytochem Rev (2008) 7:615–639

:tiphat:

spurr · Dec 5, 2010

Sam_Skunkman said:
Dr Mahmoud A. ElSohly recently told me more then 90, I have seen publications that list 85, but I need to remember which one. Not sure if all 90 are published yet.

OK, thanks. I need to contact him this week anyway to see if he, or others, have studied ideal DLI (Daily Light Integral) for cannabis. When I do I will ask about his findings about number of identified cannabinoids. I have his two papers about PPFD (Photosynthetic Photon Flux Density) where the workers found ~1,500 PPFD is ideal for peak rate of photosynthesis, etc., but I haven't found any works from him about DLI. And it's DLI that matter more than PPFD under HID.

I also want to ask him if he has studied rates of Co2 higher than 750 ppm, and the effects upon rate of photosynthesis, stomatal conductance (higher Co2 reduces stomatal conductance), etc. I have one paper on cannabis that shows Co2 saturation is ~1,000 ppm and that agrees with Co2 saturation levels from studies on the model organism (Arabidopsis thaliana) and other C3 plants.

I also have two papers from other researchers showing ~1,500 PPFD is ideal for peak rate of photosynthesis, etc. And one paper showing ~>120,000 Lux provides highest net rate of photosynthesis and rate of photosynthesis for cannabis; but, as we know, lux, lumens and foot candles are not light measurements for plants so that paper is not of much value IMO.

Sam_Skunkman said:
spurr said:

Sam_Skunkman said:

Maybe we will find another terpenoid that does get you high but then why did not primitive indigenous farmers find it like they did with THC? They created pure THC varieties by eliminating anything else, all without knowing anything about THC. I suspect they would have found any other Cannabinoid that would of gotten them high, don't you?

Click to expand...

Maybe, maybe not. And maybe they didn't have cultivars with enough quantity of specific terpenoids or cannabinoids, etc., to experience a high and to selectively breed for them.

Also, quantity of secondary metabolites (ex. cannabinoids) is phenotypic expression (~50/50 genetics and environment), thus their quantity is greatly effected by growing environment and method. But, chemotype is a genotypic expression (near 100% genetics), thus the growing method and environment has little impact upon the ratios. So, if the indigenous growers had a growing environment (incl. soil and atmosphere issues) that was not conducive to high quantity of cananbinoids (other than THC, etc.) they wouldn't have be able to breed for them...

Click to expand...

I suspect if the environment is not good for Cannabis then it will not be easy to breed for any Cannabinoid. But what about all the areas that are good for Cannabis production? Why did it not happen at any of them? THC selection is the hand of man, plain and simple, wild Cannabis is never high in THC, unless recently escaped from cultivation by man. Same as any other Cannabinoid that man would of selected for, if you think that Cannabis was high in THC before the hand of man you are mistaken. Without the hand of man to maintain through selection Cannabis THC Cannabinoid levels fall quickly.

If an environment is good for growing cannabis it isn't necessarily good for inducing high quantities of specific terpenoids, cannabinoids, flavonoids, etc. As we know, carotenoids are under the sub-class of terpenoids called tetraterpenes*, and some carotenoids are "accessory pigments" (e.g. red, orange, or yellow pigments) that are photoreactive and help a plant carry out photoreactions (such as photosynthesis). So, light quality (wavelengths) and quantity (PPFD) have a strong effect upon some secondary metabolites of cananbis, and two separate areas that are good for growing cannabis can have different quality and quantities of light. I am not claiming light is the only factor, I am just using it as an example of an environmental factor that effects production of secondary metabolites in cannabis.

For example, lowland areas were good for growing cananbis, as well as highland areas were good for growing cannabis (ex. high elevations), but the highland areas had higher levels of UV-b and PPFD (and DLI). And UV-b has been proven to increase THC, flavonoids and possibly terpenoids too (depending on other factors); as has higher PPFD vs. lower PPFD. Flavonoids are UV-b screening agents, thus they increase in quantity under higher irradiance (as daily net quantity) of UV-b. My point is we do not know enough to make definitive claims...only hypotheses. But I do agree that there is little chance some terpenoids are psychoactive like THC.

No, I do not think originally all drug bio-type wild cannabis ecotypes (races) were high in THC as a rule. But there were some wild strains (cultivars) within those ecotypes that were high in THC, without a doubt IMO (ex. mutants). IMO it was some of those mutant wild strains, I assume, that were used by early humans for selective breeding of varieties of cannabis when they noticed a difference in the effects vs. 'normal' strains.

* Refs:

"Plant terpenoids: applications and future potentials"
Sam Zwenger and Chhandak Basu
Biotechnology and Molecular Biology Reviews Vol. 3 (1), pp. 001-007, February 2008
(uploaded to this thread)

"Chemistry Of Terpenoids And Carotenoids"
G. Singh
Discovery Publishing House (2008) ISBN 8183562795, 9788183562799
(google books) http://books.google.com/books?id=Gr...ook_result&ct=result&resnum=1&ved=0CCMQ6AEwAA

This book was written for B.Sc, B.Sc. (Hons.) and M.Sc., students. And what I find funny about that book is on the front cover is a picture of a nice cannabis bud, but when searching the book for the terms "cannabis", "marijuana" and "hemp" I found nothing. I am going to buy that book this week...

Microbeman · Dec 5, 2010

if you think that Cannabis was high in THC before the hand of man you are mistaken. Without the hand of man to maintain through selection Cannabis THC Cannabinoid levels fall quickly.

I am skeptical regarding this statement. I'd be interested in reading some objective literature which substantiates this. It is similar in my opinion, to the blanket statements that the quality of domesticated races of corn are superior to the original North American 'wild' corn.

Would the same hypothesis apply to the papavar somniferum which is used in opium production?

bendoslendo · Dec 11, 2010

It's hard to find a testable neolithic cannabis sample, I'm quite sure

Probably equally difficult to find a landrace untouched by man ever in it's evolutionary history.

I guess the argument lies in the cost/benefit of producing copious amounts of THC in that species' environment. If high levels of THC (or it's precursors) provide a greater benefit to an individual's fitness than low levels of THC, genes that increase THC production will become more frequent in the population. Whether the physically protective, antibiotic, antifungal properties of cannabinoids benefit the plant enough to allow a selective pressure to evolve high THC is I guess, up for debate. However, I think there can be little doubt that today's high THC cultivars owe a large degree of their THC content to man even before breeding, by means of accidental dispersal of preferred individuals seeds.

Note the last two words of this chapter title. A very good read.
http://books.google.com/books?id=AznCzOxvrtwC&lpg=PA71&ots=zo6hvLrOzu&lr&pg=PA71#v=onepage&q&f=false

Aren't domesticated races of corn far superior in production of grain than wild types? I am confused by what you mean by superior microbeman.

scurred, I'm totally psyched about TLC for qualitative and semi-quantitative analysis. I'm going to be following your posts on this very carefully and reading all the studies/guides you put out here. Please keep it coming.

I've done a few TLC labs in organic chem, it's a pretty straightforward and simple process that I'm pretty confident I could perform if I can obtain all the materials required.

Totally psyched.

Cannabologist · Dec 17, 2010

Probably equally difficult to find a landrace untouched by man ever in it's evolutionary history.
[/quote] - If I remember correctly in my reading, Schultz said that there is no such thing as an “untouched” landrace; all Cannabis that exists today has ancestral origins in cultivated populations.

- Cannabis has been one of the earliest cultivated crops in existence, and one would be hard pressed to find a truly “wild” population of Cannabis that is not simply an escaped cultivar.

- Hence, there is no population of Cannabis in the wild in existence today that has only had natural selection pressures acting upon that population since Cannabis first evolved.

- Besides, an escaped cultivar will acclimatize to any particular environment over a number of generations (this is an ecotype). This ecotype is synonymous with landrace.

- As Sam has stated, this does not mean (high) THC production will be improved, or in any way maintained, via natural selection.

- Cannabis has been improved by artificial selection (ie. the hand of man). This is what has given us all the variety seen in the various cultivars, and high THC content, etc.

- Is it reasonable to think that natural selection could produce stable lines of Cannabis churning out consistent rates of THC higher than 10%? 15%? 20%? If memory (and sourcing) serves the highest recorded dry weight sample was 29% THC, recorded in Canada.

- Realistically, nature could perhaps create a freak mutant with high (10-15+%) THC content that could perhaps pass on it’s genes to further generations (will it - no). This would be an extreme rarity, whereas artificial selection garners stable cultivars that consistently produce 10, 15+% THC.

Cannabologist · Dec 17, 2010

Apologies for the double post, 2 separate topics

Sam, Spurr, a line of reasoning if I may interject concerning THC, accessory cannabinoids, and terpenes;
- My experience has garnered “rare” varieties of Cannabis that appear to possess no appreciable effect that typical users would describe as “tolerance”, or as Sam would describe “ceiling”.

- This lack of “tolerance” or “ceiling” remains even after a considerable amount of time using that strain as compared to most other strains.

- Smoking other certain strains with the “special” strain will result in the typical “tolerance” or “ceiling” effect.

-Thus, terpenes and/or accessory cannabinoids (like CBD) play a role in causing a “ceiling” effect.

- My thought, that chemicals creating this “tolerance”/”ceiling” are in fact common to most Cannabis populations/strains/cultivars.

-Hence, most users experience “tolerance” when smoking any particular kind over time because they are imbibing this common "tolerance" creating chemical.

Question;
Could it be true that “ceiling”/”tolerance” is the result of terpene(s) and/or accessory cannabinoid(s), not just saturation of receptors by THC?

-If so, what are the terpene(s)/cannabinoid(s) that give such an effect?
-Could it be possible then, that Cannabis that is “most desired” or “most potent” (no tolerance, no ceiling), in fact lacks a terpene common to most varieties (and one wishes to artificially select out this trait)?

-I feel given some of the concerns you have had Sam about corporate agro-industry and its potential ability to misuse such information, that you may opt not to answer or answer privately.

spurr · Dec 17, 2010

Cannabologist said:
- Is it reasonable to think that natural selection could produce stable lines of Cannabis churning out consistent rates of THC higher than 10%? 15%? 20%?

I think it's reasonable to speculate that wild ecotype populations could have had > 10% THC as a rule. But we are just speculating so it's not worth much.

Cannabologist said:
If memory (and sourcing) serves the highest recorded dry weight sample was 29% THC, recorded in Canada.

IIRC that was a claim by Dr. Hornsby, and I have spoken to a few people who know him and his protocols, that question the validity of his testing due to his sourcing of standards and his protocols. He is famous for testing samples that have very high THC, I think he is suspect...

Cannabologist said:
- Realistically, nature could perhaps create a freak mutant with high (10-15+%) THC content that could perhaps pass on it’s genes to further generations (will it - no). This would be an extreme rarity, whereas artificial selection garners stable cultivars that consistently produce 10, 15+% THC.

The main factor would be "what purpose does THC serve the cannabis plant in nature?". For example, terpenoids often are used by some plants to attract insects for various reasons, e.g., pollination. Thus, if THC was used by the plant for some important purpose then it could indeed have been high in wild strains (i.e., within wild ecotype populations) before THC was human-bred to be high in non-wild cultivars (i.e., within varietal populations).

The fact THC is increased under sufficient UV-b irradiation (e.g., greater conversion of CBG into THC vs. CBG into CBD) could have made high altitude ecotypes higher in THC as rule, not as an exception to a rule.

spurr · Dec 18, 2010

Cannabologist said:
Apologies for the double post, 2 separate topics

Sam, Spurr, a line of reasoning if I may interject concerning THC, accessory cannabinoids, and terpenes;
- My experience has garnered “rare” varieties of Cannabis that appear to possess no appreciable effect that typical users would describe as “tolerance”, or as Sam would describe “ceiling”.

- This lack of “tolerance” or “ceiling” remains even after a considerable amount of time using that strain as compared to most other strains.

- Smoking other certain strains with the “special” strain will result in the typical “tolerance” or “ceiling” effect.

-Thus, terpenes and/or accessory cannabinoids (like CBD) play a role in causing a “ceiling” effect.

Yup, I agree. And not only terpenoids (IMO terpenes are too large of a class for this type of discussion, terpene and terpenoid are not synonymous), but also flavonids could also be a factor. FWIW, CBD does hinder the psychoactivity of THC at CB1 receptors*, even though CBD binds poorly to CB1 receptors; that is why one hypothesis is that "non-CB1" receptors are the primary receptors for CBD.

Why did you use the term "accessory" cannabinoids? CBD, CBC, THCV, CBDV, etc., are all equal cannabinoids to THC. Do you mean accessory in terms of what we, as humans, see as the primary cannabinoid of interest (i.e. THC)?

* Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?
by John M. McPartland and Ethan B. Russo

"...and (3) CBD is a potent inhibitor of cytochrome P450 3A11 metabolism, thus it blocks the hydroxylation of THC to its 11-hydroxy metabolite (Bornheim et al. 1995). The 11-hydroxy metabolite is four times more psychoactive than unmetabolized THC (Browne and Weissman 1981), and four times more immunosuppressive (Klein et al. 1987)."

FWIW, I am still holding out the very outside possibility that there could be psychoactive terpenoids from cannabis. Currently there are no known terpenoids from cannabis that are psychoactive, but I think psychoactive terpenoids could exist in cannabis, mostly because there are psychoactive terpenoids in other plants. Ex., it's a specific terpenoid from Saliva that provides the psychoactive/psychotropic effects from Saliva (i.e., "Salvinorin A", aka "Divinorin A").

I have been planning to look into the potentiating effects certain terpenoids can have upon the psychoactive effects of THC; but at this time I think terpenoids do not make THC get us higher, I think they do however seem to make the high 'better'.

Here is my poor analogy of tepenoids to THC: Kind of like adding spices to meat: the spices (i.e. terpenoids) do not make the meat (i.e. THC) have more protein (i.e. psychoactive effects of THC), but the spices do make the meat taste better (i.e. potentiates some affects from THC) and thus we enjoy eating (i.e. smoking) the meat more than without spices.

Cannabologist said:
- My thought, that chemicals creating this “tolerance”/”ceiling” are in fact common to most Cannabis populations/strains/cultivars.

-Hence, most users experience “tolerance” when smoking any particular kind over time because they are imbibing this common "tolerance" creating chemical.

If that were true it would seem smoking (close to) pure THC (i.e. > 98%) would have no ceiling. Sam can comment here because he has smoked pure THC. And IIRC, he has written he didn't like the high as much as smoking whole cannabis (or whole cannabis extracts). He can probably comment on wither or not there is a ceiling with pure THC.

Cannabologist said:
Question;
Could it be true that “ceiling”/”tolerance” is the result of terpene(s) and/or accessory cannabinoid(s), not just saturation of receptors by THC?

Possibly, I will look through some studies I have and see if I find anything. I didn't get a chance to email Dr. ElSohly last week (re: DLI, PPFD, Co2), but I will this coming week and I will ask him about your question regarding possible saturation of CB1/CB2 receptors by THC in terms of a limit to 'highness'.

Sam can probably provide some good insights to your question.

Cannabologist said:
-If so, what are the terpene(s)/cannabinoid(s) that give such an effect?

See this post for a breakdown of some common terpenoids and the effects they have: https://www.icmag.com/ic/showpost.php?p=4055858&postcount=1

Cannabologist said:
-Could it be possible then, that Cannabis that is “most desired” or “most potent” (no tolerance, no ceiling), in fact lacks a terpene common to most varieties (and one wishes to artificially select out this trait)?

I doubt it, but maybe. A ceiling I am sure is also a factor of the of the person getting high, e.g., two people smoking the same bud might not experience the same lack of ceiling.

Cannabologist said:
-I feel given some of the concerns you have had Sam about corporate agro-industry and its potential ability to misuse such information, that you may opt not to answer or answer privately.

I feel any worries about agro-industry are unfounded on this board, and limiting the free flow of information is counter productive to the goals of this sub-forum. AFAIK there is only one person here who fits the bill of being from "corporate agro-industry", and I am sure Sam isn't worried about him...

:tiphat:

spurr · Dec 18, 2010

bendoslendo said:
scurred, I'm totally psyched about TLC for qualitative and semi-quantitative analysis. I'm going to be following your posts on this very carefully and reading all the studies/guides you put out here. Please keep it coming.

I've done a few TLC labs in organic chem, it's a pretty straightforward and simple process that I'm pretty confident I could perform if I can obtain all the materials required.

Totally psyched.

Cool, glad you are into it. I will make public my protocol at a later date, and how/where to source the needed chemicals, etc. For now I can suggest the SIRCHIE THC TLC test kit (see below). I bought a few of them quite a while ago to see if they were any good. While they do not use great TLC plates, they do have all needed chemicals (incl. Fast Blue BB salt visualization reagent) and the protocol they suggest is sound (incl. dipping the plates into the reagent and not spraying the plates with the reagent).

SIRCHIE is a company for LEA, but they sell to anyone

The kit includes everything needed, incl. all glassware, chemicals, plates, capillary pipette, etc. And within the links I provided (ex. the UN guides) you can find the color code for various cannabinoids when using Fast Blue BB salt, as well as Rf values too.

If using the SIRCHIE THC TLC test kit (~$82), you can also download the free trial of JustTLC (full price is ~$400) and using a good flatbed scanner you can carry out comparitive TLC at home for under ~$100 (that is, if you already have a good flatbed scanner).

Here is the order page for the test kit, and I uploaded a PDF detailing the protocol and such of the test kit to this post.
http://store.sirchie.com/Thin-Layer-Chromatography-of-Marijuana-Kit-with-6-Tests-P1468.aspx

bendoslendo · Dec 18, 2010

Thanks scurred, great find on the kit! I've seen less inclusive kits online for over $200, of course marketed to cultivators. I've ordered one so I can get started with practicing, keeping me busy until you post your protocol.

I don't suppose we can infer the location of the solvent front in this image, can we? I'd like to compare the rf values to the ones in the UN doc.

Eugenics · Dec 18, 2010

Just want to throw this out there, scientifically speaking; as a person with experience in the botanical studies. We should really stop mislabeling and accepting "strain" as proper nomenclature, it drives me nuts since I'm a breeder with a formal education. "Cultivar" is the proper, correct, scientific use of the word.

Look into it, there is a big difference. The whole time everyone is trying to be articulate, and display legitimacy with the community, the wrong term has been perpetuated too long.

Thanks, Eugenics Genetics, breeder.

Microbeman · Dec 18, 2010

Eugenics said:
Just want to throw this out there, scientifically speaking; as a person with experience in the botanical studies. We should really stop mislabeling and accepting "strain" as proper nomenclature, it drives me nuts since I'm a breeder with a formal education. "Cultivar" is the proper, correct, scientific use of the word.

Look into it, there is a big difference. The whole time everyone is trying to be articulate, and display legitimacy with the community, the wrong term has been perpetuated too long.

Thanks, Eugenics Genetics, breeder.

or in many of these cases 'cultigen'? (until an actual name is accepted) or 'race' as has been used here and there in this thread.

spurr · Dec 18, 2010

bendoslendo said:
Thanks scurred, great find on the kit! I've seen less inclusive kits online for over $200, of course marketed to cultivators. I've ordered one so I can get started with practicing, keeping me busy until you post your protocol.

I don't suppose we can infer the location of the solvent front in this image, can we? I'd like to compare the rf values to the ones in the UN doc.

I'm curious, why do you call me "scurred"? I wouldn't try to use Rf with the image in the PDF. I for one don't like Rf values if we can use color. Glad you ordered one of the kits, they are much better than the cheese kits sold to cannabis growers.

bendoslendo · Dec 18, 2010

spurr said:
I'm curious, why do you call me "scurred"?

wow... I'm curious too. :dunno:

I just checked and there is a member "scurred", though none of his posts are prescient in my mind. Maybe I read something of his the day I started reading this thread. I must have attributed the name with this thread initially and it stuck.

Sorry about that, ha! :redface:

onegreenday · Dec 18, 2010

Phytochemical and genetic analyses of ancient cannabis
from Central Asia (the ancient shaman's stash)

http://jxb.oxfordjournals.org/content/59/15/4171.full.pdf+html

Edit: Talk about 'amber' trichomes. Those are dark, dark amber without a stalk.
Maybe this 'shaman' knew something about 'fully maturing' the flower.......

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