MMJ DOCTOR THAT TESTED 15,000 POT SAMPLES SPEAKS
Lab Tested Marijuana
Dr. Paul Hornby and the Three Way Split
“My driving force behind all of this is really discovery.” - Dr. Hornby
Content by: Dr. Paul Hornby and Jason Green (QA officer Rtree Producers Ltd.)
Commentary by: Wade Reeves, Analytical Chemist and Medical writer.
Commentary on Dr. Paul Hornby, Notable Cannabis Researcher
Dr. Paul Hornby is a leading cannabis research scientist from Vancouver, Canada. Paul holds a PhD in Human Pathology.
Dr. Hornby's career began traditionally enough, conducting cancer research in the early days, then moved onto a ten year tenure as researcher / manager of herbal testing labs. About twenty years ago, Paul found his calling in the fascinating and promising world of Marijuana research, discovery and therapy.
In his career as a full-time Cannabis researcher, Paul has gained elaborate hands on experience testing cannabinoids, pesticides, terpenes and other components, has personal dealings in Canadian Marijuana Law at the Federal, Provincial and Municipal levels. Paul has been active in clinical settings where he assisted several Canadian Cannabis Clubs and their patients. He also produced unique MMJ products and formulations, cultivated and developed an assortment of strains, among other activities. What a life!
"Having worked closely with a number of local dispensaries in Vancouver, British Columbia, Canada over many years, we have come to observe repeatedly the therapeutic effects of different strains of cannabis and related this to cannabinoid profiles." - Dr. Paul
The Three Way Split, Defined
Dr. Hornby has found that clinically, MMJ strains generally fall into three categories, a " three-way split":
- 1 - Strains very high in THC but low in CBD
- 2 - Strains high in CBD but low in THC
- 3 - Strains with a 50/50 split between THC and CBD
Case in Point
Hayley’s Comet is an example of a "pioneering" strain that dramatically reduced epileptic seizures in a young female epileptic patient. Dr. Paul wrote a patent application for this 50/50 (THC/CBD) strain.
After trying more than twenty different anti-epileptic medications and still having up to 40 seizures per day, young Hayley finally found relief from cannabis provided fortuitously by a Vancouver cafe.
"Since 2008 Hayley has used medical cannabis to reduce her seizure activity dramatically and allows her to live a functioning lifestyle. After trying other CBD cultivars with limited success,
Mother Cheryl is concerned that the cannabis provided by Licensed Producers will be inadequate to treat her daughter's condition.
Hayley found one in particular was successful in treating her seizures. Hayley’s Comet is a patented cultivar with a 1:1 THC:CBD ratio that was found, tested and continues to be researched by Dr. Paul Hornby in Vancouver with the intention of making it available to all patients. Licensed producers now offer cultivars with a similar cannabinoid profile, but Cheryl and Hayley, like many other sick patients, simply cannot afford to purchase their medicine at current prices."
- Read more: Hayley's Comet, 1:1 ratio helps epilepsy
Our intent was to confirm the work of earlier molecular geneticists, who postulated a three way split in the ratio of THC to CBD. We say, “it is written” and unless the plant is genetically modified, it will never change. All three chemotypes have enormous medical value in the treatment of a multitude of illnesses. Mixing and matching of extracts of the three can yield a host of valuable medicines.
Over the past 15 years, our laboratory has observed more than 15,000 cannabinoid profiles by High Pressure Liquid. Chromatography (HPLC) and, in time, strict patterns in the THC/CBD ratios presented. In terms of these two cannabinoids, the ratio only goes three different ways; high CBD, low THC, as in hemp (Type III); roughly equal THC/CBD, as in strains like Cannatonic Tsunami or Hayley’s Comet (Type II), or high THC/low CBD, as in commercially available (recreational or street) cannabis (Type I); Molecular geneticists have noted this phenomenon in the past but it has not been documented using chromatography.
In the late 1990’s our laboratory began analyzing cannabinoid profiles in hemp samples as part of the government’s regulatory exercise, to ensure that the THC content was less than 0.3%. Over the years we became more involved with analysis of medical grade cannabis, such as that being distributed by local dispensaries in British Columbia, Canada. At roughly 1,000 samples per year we now have more than 15,000 chromatographic data files of literally hundreds of strains of cannabis that show a very distinct pattern in relation to the ratio of THC to CBD.
As medical cannabis becomes increasingly realized as a valid medicine and more research indicates the value of THC and CBD as important medical actives of the plant, their relevance to efficacy becomes more apparent. The analgesic, antiemetic, anxiolytic, appetite stimulant and psychoactive properties of THC are well characterized, as is the anxiolytic, anti-inflammatory, antioxidant and non-psychoactive properties of CBD.
As we learn more about the medical benefits of these compounds their relevance to humans and domestic animals becomes more apparent and their market value increases. Thus, it becomes essential to know the expected THC/CBD ratio of a given phenotype before planting a seed or propagating clones. Here, we are simply presenting a pattern that evolved over many years and thousands of HPLC profiles of various types and strains of cannabis.
High pressure liquid chromatography, employing a Hewlett-Packard (Agilent) 1090 Series II binary pumping system with a 79883a diode array detector, with primary absorbance at 219 nm, was used in the early years and was upgraded to a new Agilent 1220 for the past four years. Mobile phase at 0.75 ml/min was isocratic with 14% aqueous (1:25:974 phosphoric acid: methanol: distilled water) and 86% organic phase (methanol).
The column was a Zorbax C18 reverse phase 4.6mm x 25 cm. Samples were prepared by the method modified from Health Canada for the preparation of hemp samples for HPLC analysis. Calibration curves were run for a number of commercial standards (Sigma) and averages made. 0.100 gram of dry cannabis was suspended in 10 ml Methanol and placed in a sonicator bath for 3 minutes, then passed through a 0.45micron nylon syringe filter into a 1 ml HPLC sample vial. Under these conditions, suitable chromatography is achieved for the most abundant cannabinoids present.
Results and Discussion
Only through breeding experiments can the amounts of either THC or CBD be increased, by locating genotypes that tend to express the THCa synthase or CBDa synthase more than others and selecting these plants for more of the compound of interest.
Knowledge of the ratio of THC to CBD does not provide the amount (concentration) of either compound, although the ratios do not vary, one to the other, they will increase or decrease in amounts (simultaneously) depending on environment (growing conditions). Therefore, in order to obtain a plant containing a high cannabidiol count, one would breed plants of the Type III variety, selecting plants that are the largest producers of CBDa and propagating these varieties.
Conversely, growing plants with high THC values would involve crossing plants of the Type I variety, selecting the greatest producers of THCA. And finally the same would be done for what we are calling 50:50 (Type II) strains with roughly equal the THC and CBD, selectively breeding the highest producers in terms of percent THCA and CBDA. The numbering system (Type I-III) is based on the work of Small and Cronquist (2), who describe three chemical phenotypes based on the THCA and CBDA content ratios.
When we describe THC and CBD, we often refer to their acids that are the non-decarboxylated forms of the compounds. For example total THC potential of a given cannabis plant is the sum of the THC plus THC-A, when decarboxylated, the same holding true for CBD.
A similar finding demonstrating the three way split was presented by de Meijers et al. (1)
in 2003 who showed three different chemotypes (only) in breeding experiments followed by Gas Chromatographic analysis of cannabinoid profiles in the F1 and F2 generations. In all cases, they found only three different (Type I-III) chemotypes arising from the various crosses. Our data not directed at any particular endpoint in its collection verifies these genetic studies by the sheer number of data files, verifying time and time again that only three different profiles in terms of CBD and THC ratios exist. It is written.
As de Meijer et al. also pointed out that the putative enzyme synthases employed in the production of THC and CBD are coded from one locus (B) and two alleles B (D) and B (T) that are codominant. It is suggested that such co-dominance is due to the codification by the two alleles for different isoforms of the same synthase enzyme, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The cumulative HPLC data collected over 15 years, assessing numerous varieties of cannabis from British Columbia.
Canada, plus others from California, USA, Spain, UK and China consistently lead to three chemotypes (Type I-III) as proposed by Congrist et al. (2). For example, from roughly 15,000 chromatographic profiles, we never recorded a THC:CBD ratio of 80:20 or 60:40 but only ratios of greater than 75:1 for Type I (high THC, low CBD); an approximate 45:55 ratio for Type II (roughly equal THC:CBD) and a greater than 1:20 for Type III (low THC, high CBD).
A recent personal communication with Dr. Lumir Hanus (Hebrew University in Jerusalem) suggested the existence of a 4th profile, with low THC and low CBD (both less than o.5%), that may not be relevant to human medicine, other than being useful as a placebo in controlled experiments.
The relevance of these profiles to the efficacy of the herbal medicine is becoming evident from recent observational, pilot-clinical and placebo controlled clinical trials where natural product cannabis has been used as an experimental drug or intervention. Our laboratory took part in a study of persons attempting to withdraw from methadone (4), who were members of a local dispensary (Eden, Vancouver, Canada) and who used an oral preparation of Type I cannabis as a standardized oral preparation.
Anecdotal, yet accumulating data is being gathered on a young lady, who's grand mal epileptic seizures have been significantly reduced (from daily to monthly) by ingesting an oral preparation of a Type II variety (roughly equal THC:CBD) and similar findings have been reported for a pre-adolescent female using a Type III (hemp) strain. The importance of CBD in the control of epileptic seizures, convulsions, Dystonia and drug withdrawal is becoming increasingly popularized in the scientific and journalistic media.
Having worked closely with a number of local dispensaries in Vancouver, British Columbia, Canada over many years, we have come to observe repeatedly the therapeutic effects of different strains of cannabis and related this to cannabinoid profiles.
Type I strains primarily of the Indica subspecies were used by most persons managing chronic pain. Whereas Type II strain (Hayley’s Comet), proved effective in significantly reducing grand mal epileptic seizures as did Type III, hemp strains, apparently a result of the high CBD to THC ratio. These high cannabidiol strains also appear to be effective in treating inflammatory pain (arthritic), prostate hyperplasia, and breast cancer (3).
Persons with chronic pain Indica subspecies (sedatory) were preferred to the more stimulatory Sativa and would often use the latter as a daytime medicine to focus on daily activities, without the sedative effects of Indicas. Sativas are often preferred for mood, as they tend to counter the effects of ADHD like symptoms, allow mental focus and energy, particularly with standardized oral preparations. Interestingly, similar effects are observed with high CBD only or Type III strains when administered orally.
The difference between Indica and Sativa cannot be closely related to the THC:CBD ratio and we conclude it to be a result of different terpene profiles. Terpenes are strongly inherited and little influenced by environmental factors and, therefore, have been widely used as biochemical markers in chemosystematic studies to characterize plant species, provenances, clones, and hybrids.
Results showed a large variation between different strains in the relative contents for several monoterpenes (α-pinene, camphene, β-pinene, sabinene, Δ-3-carene, α-phellandrene, β-myrcene, α-terpinene, limonene, 1.8-cineole, γ-terpinene, cis-β-ocimene, trans-β-ocimene, α-terpinolene) and one sesquiterpene, β-caryophyllene), many of which have demonstrated medicinal as well as psychoactive properties.
If the thousands of accumulated HPLC cannabinoid profiles could be overlaid as are a few examples in Figure 1. Only three patterns would emerge (Figure 2). As a result of a number of breeding experiments between the different Types of cannabis strains we can only conclude that all are the same species (in terms of the ability to produce viable offspring) and they
are extremely diverse genetically.
And that difference in strain effects is more a result of different terpene/aromatic profiles than the ratio of THC to CBD. Regardless, we’ve never had a cannabis strain that wouldn’t mate with another, whether Indica, Sativa or Ruderalis or what we call industrial hemp, medicinal or recreational marijuana. They all interbreed, producing viable offspring…our definition of a species.
Our intent of this short dissertation was to confirm the work of earlier molecular geneticists, who postulated a three way split in the ratio of THC to CBD. We say, “it is written” and unless the plant is genetically modified, will never change. All three chemotypes have enormous medical value in the treatment of a multitude of illnesses from chronic pain to opiate withdrawal and mixing and matching of extracts of the three can yield a host of valuable medicines.
List of Abbreviations
- CBD-A Cannabidiolic acid
- CBD Cannabidiol
- CBN-A Cannabinolic Acid
- CBN Cannabinol
- THC-A Tetrahydrocannabinolic Acid
- THC Delta-9 tetrahydrocannabinol
- THF Tetrahydrofuran; HPLC High Performance Liquid Chromatography
1.) Genetics. 2003 Jan;163(1):335-46.
The inheritance of chemical phenotype in Cannabis sativa L.
de Meijer EP1, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P, Mandolino G.
2.) Genetics. 2003 January; 163(1): 335–346.
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
3.) Mol Cancer Ther. 2010 Jan;9(1):180-9. doi: 10.1158/1535-7163.MCT-09-0407. Epub 2010 Jan 6. Cannabidiol enhances the inhibitory effects of delta9-tetrahydrocannabinol on human glioblastoma cell proliferation and survival.
Marcu JP1, Christian RT, Lau D, Zielinski AJ, Horowitz MP, Lee J, Pakdel A, Allison J, Limbad C, Moore DH, Yount GL, Desprez PY, McAllister SD.
4.) Reduction in Methadone Consumption and Withdrawal Symptoms with Ingestion of Standardized Oral Cannabis. An Observational/Feasibility Study. Proceedings of the 2013 IACM conference Köln, Germany.
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© 2016 Dr. Paul Hornby and Jason Green - ALL RIGHTS RESERVED - (Content inclusive, from Abstract to References)
*** Scientific commentary and additional text was provided by Mr. Wade Reeves (former Operating Director of Cavendish Analytical Laboratories). Cavendish Labs conducted the testing of controlled substances including Marijuana, amphetamines and steroids in Vancouver, BC, Canada.
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