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List of Cannabis Studies
Pharmacokinetics and cannabinoid action using oral cannabis extract
Article Date: 25 Aug 2005
A simple dosing regimen for cannabinoid therapies is complicated by individual variance in rates of breakdown in the liver and intestinal absorption. This trial measured specific plasma concentrations of orally administered cannabis extract in order to determine whether any correlation to clinical effects could be identified.
A total of 16 healthy female volunteers were given either 20mg tetrahydrocannabinol (THC) calibrated cannabis extract or an active placebo (diazepam) over a standardised breakfast. The drugs were administered in a double-blind, placebo controlled, crossover design and plasma levels of THC, cannabidiol (CBD) and another 2 active metabolites were measured before the trial began and at 2, 4 and 8 hours after administration.
In addition, at 60 minute intervals both the patient and a blinded observer recorded the prevalence of typical clinical side-effects like nausea, feeling high, sedation, vertigo and dry mouth, using a visual analogue scale (VAS). Finally, heart rate, body temperature, oxygen saturation and blood pressure were measured every 30 minutes.
Peak plasma concentrations of THC were achieved within the first 2 hours for 75% of patients, and between 2 and 4 hours in the remaining 25%. The maximum levels of side effects were seen at 2.6 hours for patients taking active cannabinoids, which differed significantly from baseline and placebo. Nevertheless, there was absolutely no recorded correlation between the relative circulating plasma concentrations of THC and either the occurrence of side-effects or their intensity. None of the other measured parameters showed any significant changes or differences between control and active groups.
The trial emphasises the enormous variation in bioavailability of orally administered cannabinoids even in strictly controlled and standardised study conditions. The investigators re-state the requirement to titrate every patient receiving cannabis extract for therapeutic pain management on an individual basis to determine the most appropriate dosage protocol.
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The chemistry and biological activity of cannabis
United Nations Office on Drugs and Crime
Author: Stig AGURELL, J. Lars G. NILSSON
Pages: 35 to 37
Creation Date: 1972/01/01
The chemistry and biological activity of cannabis
Review of the Symposium, held in Stockholm in 1971Stig AGURELL Central Military Pharmacy, Karolinska Hospital, 104 01 Stockholm 60
J. Lars G. NILSSON Faculty of Pharmacy, University of Uppsala, Box 6804, 113 86 Stockholm
The last two years has been a period when a considerable amount of basic knowledge concerning cannabis has been collected in laboratories all around the world. Most of these new, but largely unpublished, data were presented and discussed during a Symposium held in Stockholm (26-28 October 1971), under the sponsorship of Apotekarsocieteten, the Swedish Academy of Pharmaceutical Sciences. There were 40 papers covering the chemistry, biological activity and pharmacokinetics of the cannabinoids read before 100 participants from 16 countries.
The President of the Symposium was Dr. Leo Hollister, Veterans Administration Hospital, Palo Alto, California. In his opening lecture he gave the human pharmacological background to the subsequent presentations.
The clinical effects of cannabis, he said, are dose related, ranging from mild and brief states of intoxication to long-lasting hallucinogenic experiences. The initial clinical state is one of euphoria and stimulation, but later the subjects report a drowsy, sleepy state. Dr. Hollister also pointed out that the question of tolerance and sensitization to the effect of cannabis in man is not settled, although this has been studied both in his laboratory and by other groups.
Cannabis research at the Division of Narcotic Drugs, United Nations, Geneva, was presented by Dr. Olav J. Braenden. The most important function of the UN Laboratory is to coordinate, on the international level, cannabis research to avoid unnecessary duplication of effort. Reference cannabis samples for comparative studies are also provided.
On the national level, a similar but more detailed program is carried out by the National Institute of Mental Health, Bethesda, Maryland.
This was presented to the Symposium by Dr. Monique C. Braude. NIMH has at present 57 grants and 16 contracts dealing exclusively with cannabis research.
They cover a wide variety of pre-clinical and clinical studies, ranging from chemical, analytical and pre-clinical pharmacological studies, to clinical studies of physiologic, behavioural and social effects of the cannabinoids. Of particular interest are two studies now going on in Greece and Jamaica, which deal with the long-term effects of cannabis smoking.
The emphasis has been to elucidate the effects of the compounds responsible for the psychoactive effects of cannabis in man, to quantify the constituents or their metabolites in biological fluids, to determine their toxicity and side effects and to correlate these data with the social effects of the drug.
Chemistry of the cannabinoids
The three main cannabinoids present in cannabis preparations are tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Altogether, approximately 30 different cannabinoids have today been identified in cannabis.
The synthesis of many of these compounds and their metabolites have been carried out by Dr. Raphael Mechoulam, Hebrew University, Jerusalem. Among other compounds, he presented a synthesis of the recently discovered active metabolite 6-β-hydroxy-Δ 1-THC.
The presence of a number of active metabolites, Dr. Mechoulam said, makes the understanding of the pharmacological effects of cannabis in man difficult. The different effects that various people encounter may depend on the ratio between THC and its metabolites at the receptor sites. The presence of the metabolites in body fluids may also be the basis for the identification of cannabis users.
The structure-activity relationships for the tetrahydro-cannabinoids has also been studied by Dr. Mechoulam and Dr. Edery of the Tel-Aviv University Medical School. The benzopyran structure seems to be necessary for activity, since an open analogue is inactive. Other essential structural fragments of THC is the phenolic hydroxyl group and the alkyl side chain.
The position of the double band in the terpenoid ring is also important. It is known that Δ1 and Δ 1( 6) are equally potent while Δ3-THC is less active, and Δ5-THC is inactive. Dr. Mechoulam also reported that (+)-Δ1-THC and (+)-Δ 1( 6) THC are inactive, while the corresponding naturally occurring ( - )-forms have activity.
In several papers, the synthesis of the different cannabinoid metabolites were reported. Dr. J. L. G. Nilsson, Stockholm, and Dr. T. Petrzilka of the Zürich Technical High School, both described new synthetic routes to 7-hydroxy-Δ 1( 6)-THC. Dr. R. K. Razdan of the Sheehan Institute for Research at Cambridge, Mass., reported on some water-soluble derivatives of THC that could be used in pharmacological experiments. These compounds where the phenolic hydroxyl group was esterified with hydrophilic acid residues chain-formed micelles and were rapidly hydrolyzed in vivo to THC.
Three papers reported on the morphology and the constituents of Cannabis sativa. Using scanning electron microscopy, Dr. Fairbairn of London University could show some very detailed pictures of the plant leaves. He also reported that the vegetative leaves as well as the glandular hairs, contain cannabinoids. This may be of importance to workers on biosynthesis as well as to the legislators.
The natural occurrence of several cannabinoids with shorter side chains (propyl or methyl instead of n-amyl) was studied by Dr. Merkus, of the R.C. Hospital at Sittard in the Netherlands, and by Dr. Vree of the University of Nijmegen, in the Netherlands. Dr. Vree showed that by recording the mass spectra of the cannabinoids at different but low electron voltages and plotting certain peak heights against the electron voltages, curves characteristic for each type (CBD, THC, CBN) of cannabinoid were obtained. This technique was then used to identify minor constituents in the plant.
Biological activity of cannabis
The second day of the Symposium was devoted to the biological activity of the cannabinoids. Dr. Sidney Cohen, UCLA, Los Angeles, California talked first about what he called the "changing concepts of cannabis pharmacology". A number of widely held ideas about the pharmacology of cannabis must be abandoned in the light of recent findings. These include its classification as a sedative, the absence of tolerance and withdrawal effects, the notion of reversed tolerance, its ability to dilate the pupils, to produce hypoglycemia and the development of conjunctival infections.
Prolonged heavy use of cannabis may lead to an "amotivational syndrome" where the users lose interest in themselves and in the surrounding world. We still need to know more about this effect and also about the carcinogenic and teratogenic potentials of cannabis. The widely held idea that cannabis leads to the use of harder narcotics like heroin was also discussed by Cohen. He pointed out that this was a serious risk for the heavy users, while "social users" who took cannabis only occasionally normally did not start using heroin.
The behavioural effects of cannabis had been studied by several groups. Dr. E.A. Carlini from Sao Paulo Medical School, Brazil, could demonstrate that rats under stress, such as cold, starvation or morphine abstinence showed a pronounced aggressive behaviour when given cannabis extracts. Other groups reported results indicating that cannabis has a pronounced influence on the behaviour of animals and man.
A general review of the pharmacology of Cannabis was presented by Dr. Paton of Oxford University. The pharmacology is largely determined by the lipophilicity of the cannabinoids, he said. This physical property brings the cannabinoids into analogy with the anaesthetics and in this way a considerable number of its actions can be interpreted: certain neurophysiological and behavioural effects; respiratory depression; hypothermia and analgesia.
The role of the cannabinoids on the brain amines was discussed by several speakers. The over-all picture obtained from the experiments described is still not clear, although THC seems to affect both levels and turnover of the mono-amines in the brain.
Pharmacokinetics
An investigation of the stimulation of the metabolic transformation of THC in pigs was reported by Dr. J. Schou of Copenhagen University. The results indicated a metabolic adaptation of the pigs. After prolonged administration of THC, the disappearance rate of THC increases and also the appearance rate for metabolites in plasma was enhanced. Similar results were reported for humans by Dr. L. Lemberger of Lilly Laboratory for Clinical Research, Indianapolis, Indiana. He also showed that when the activity of cannabis is maximal, the amount of THC present in the plasma is very low, indicating the presence of active metabolites.
The metabolic conversions of the various cannabinoids were reported by several groups. Dr. S. Agurell, Stockholm, reported that the Δ 1( 6)-THC metabolite 7-hydroxy-Δ 1( 6-THC is pharmacologically very active and further converted in the rabbit to a carboxylic acid. This acid had also been isolated by Dr. S. Burstein of the Worchester Foundation for Experimental Biology Mass., who could show that it had a carboxyl group in the 7-position and also an additional hydroxyl group in the side chain.
The metabolism of cannabidiol was studied by Dr. I. M. Nilsson of the Faculty of Pharmacy, Stockholm. She presented three new monohydroxylated metabolites of CBD with a hydroxyl group at position 7, 10, and in the benzylic position of the side chain, respectively.
Dr. Gill of Oxford University, had also studied the metabolism of THC. He could show that CBD increased the amount of 7-hydroxy-THC in the plasma, and thus acted as a synergist. It is thus apparent that cannabis contains both the active THC and a synergist CBD.
Recently, radioactively labelled THC with high specific activity has become available. This material has been used by three groups to study its distribution using autoradiography.
Dr. McIsaac, of the Texas Research Institute of Mental Sciences at Houston, Texas, reported on the behavioural correlates of brain distribution of THC using rhesus monkeys. Dr. J. E. Indanpaan-Heikkila of the National Board of Health, Helsinki, Finland, had investigated the placental transfer and the variation in distribution of THC in mice after different routes of administration. Finally, Dr. Ryrfeldt AB Astra, Sodertalje, Sweden, had studied the whole body distribution in mice after intravenous administration.
Radioactive THC had also been used to study its binding to the plasma proteins. Dr. H. Klausner of the Vanderbilt School of Medicine, Nashville, Tennessee, and Dr. M. Widman of the Stockholm Faculty of Pharmacy both reported that THC is extensively bound to lipoproteins in plasma. Dr. Widman could also demonstrate that the metabolite 7-hydroxy-THC under the same conditions largely is bound to serum albumin.
In summary, it can be stated that the Symposium revealed that a large amount of knowledge has been accumulated on the chemistry of Cannabis and the synthesis of cannabinoid compounds and their metabolites. There remains some uncertainty as to how much THC is absorbed by smoking, to give the biological " high ". In metabolism studies it has been shown that the three compounds, however, are further converted to still others, and so far unknown metabolites, before elimination. The metabolic pattern is thus, very complex and requires much further work particularly since some metabolites are very active compounds. Quite a few other pharmacokinetic parameters are known. A number of limited clinical studies in humans, using orally administered THC or THC absorbed by smoking, have been carried out.
Thus, THC and cannabis of known potency, can now be used with confidence in large scale clinical trials. During the next few years the present clinical knowledge will be supplemented with a large amount of clinical data which undoubtedly will shed some new light on two basic questions " How dangerous is cannabis "? and " What should we do about it? ".
The abstracts of the papers presented at this Symposium will be published in Acta Pharmaceutica Suecica, No. 6, 1971. These abstracts describe most of the results presented at the Symposium.
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Differential effects of medical marijuana based on strain and route of administration
Valerie Leveroni Corral
Wo/Men's Alliance for Medical Marijuana
309 Cedar Street #39
Santa Cruz, CA 95060A collective of patients and caregivers, creating community, building hope, dissolving barriers, providing support and free medical marijuana since 1993
www.wamm.org
Abstract
Cannabis displays substantial effectiveness to affect a variety of medical symptoms. Seventy-seven patients took part in a study in California to assess the efficacy of organically grown Cannabis sativa and indica strains in treatment of various medical conditions via smoking or ingestion.
HIV/AIDS was the most frequent condition reported, at 51%. Standardized rating forms provided 1892 records that were statistically analyzed. Results demonstrated that in the case of nausea and spasm, changes in symptom expression are definitely affected by method of cannabis administration. However, while Cannabis indica strains increased energy and appetite, it is useful to note that in treating nausea in HIV/AIDS and orthopedic diagnosis groups, Cannabis sativa and C. indica strains proved equivalent.
Keywords: cannabis, medical marijuana, Cannabis sativa, Cannabis indica, AIDS, HIV
Introduction
Marijuana, whether Cannabis sativa and Cannabis indica, produces its medical and other effects by virtue of the concentration and balance of various active ingredients, especially the cannabinoids, which are unique to marijuana, but including also a wide range of terpenoids and flavonoids (McPartland and Mediavilla 2001; McPartland and Pruitt 1999). Terpenoids are cannabis constituents that provide the characteristic strong odor of marijuana and hashish. Flavonoids are any of the flavone derivatives.
The concentration and relative proportions of these ingredients depend on the plant's genetic structure and applied hybridization techniques, and as such, allow for a substantially varied outcome.
Little is known about how differences in constituent profiles translate into differences in therapeutic effectiveness. A range of differentiable effects has been ascribed to THC (tetrahydrocannabinol is the primary psychoactive component of marijuana) and CBD (cannabidiol, a compound related to THC) when administered in purified form (Iversen 2000). Studies are lacking on the differential clinical effects produced when varying "menus" of constituents are taken together.
Another factor bearing on the effects and the effectiveness of marijuana is the route of administration. Orally administered marijuana is absorbed more slowly than when delivered systemically (e.g., smoking, vaporizers). Moreover, the liver metabolizes orally ingested marijuana. This produces a potent and long-acting cannabinoid (11-hydroxy-THC), which induces varied reactions in medical marijuana patients and is not often well tolerated (Grotenhermen 2001). However, once more, there is little information available concerning the differential clinical effects of oral vs. smoked forms of marijuana.
A major obstacle to obtaining data concerning differential clinical effects produced by varying strains of cannabis and by different routes of administration is, of course, the common illegality of medical marijuana use.
Almost equally troublesome, however, is the widespread view that medical knowledge can be gained only through randomized controlled trials. It is becoming increasingly accepted that valid causal inferences can be, and frequently are drawn quite regularly in medicine without such studies. As such, observational studies are quite capable of generating useful information, provided due care is taken to keep careful track of the process. In this case, careful and consistent documentation would be required concerning: 1) which forms of marijuana are being taken, by what route, and: 2) what outcome is experienced by patients.
The passage of Proposition 215 in California in 1996 legalized medical marijuana under state law, thus clearing some legal obstacles to research. Prior to the passage of Proposition 215, two or more cannabis buyers’ clubs and our collective comprised of patients and caregivers were in operation. Several provider associations have been operating since that time despite harassment of some by law enforcement agencies.
Valerie Leveroni Corral founded the Wo/Men’s Alliance for Medical Marijuana, WAMM in 1993. WAMM is a collective of patients and caregivers attempting to create community, build hope, dissolve barriers, and provide support and medical marijuana at no cost to patient members who possess a signed and verified recommendation from a physician licensed to practice medicine in California. A genetically monitored, organic, communal garden is tended by WAMM client/ participants under the direction of Mike Corral and Valerie A. Leveroni Corral.
A primary function in this community based educational system is the creation of a database of information regarding the treatment of different symptoms with distinct cannabis varieties. This is achieved through daily effectiveness surveys and statistical analysis.
Our present collection of data also includes measures of effectiveness of cannabis on other autoimmune illnesses, such as systemic lupus erythematosis, as well the many other disorders, including muscular dystrophy, epilepsy, quadriplegia, paraplegia, Parkinson's disease, glaucoma, arthritis, fibromyalgia, depression and migraine. However, AIDS and HIV-related conditions are the most frequently represented among our clientele.
WAMM initiated a study in 1993 designed to address the question of differential clinical effects between Cannabis sativa and C. indica strains and hybrids, and also examining effects of inhaled and ingested routes of administration. This study is ongoing and now includes "blind" trials where the varieties used are not apparent to the participating patient. A statistician determined all analyses. [Tables 17 & 18]
Materials and Methods
The determination of the variety of cannabis was based on the country of origin of the seeds strains and physical characteristics of each plant variety. We assure the genetic purity through carefully controlled breeding techniques, substantiated by twenty-five years of experience in cultivation, propagation and breeding of cannabis. Personal interaction took place with patient use of cannabis in more than one hundred different terminal cases.
An assessment instrument form is provided weekly to participating patients [see Tables 17 & 18]. The patient places a label from a weekly supply on the seven day form, denoting the variety and form of cannabis (inhaled or ingested), the number of "puffs" if inhaled medicine is used and the amount or weight employed. All participants were instructed in a specific method for inhaling.
Patients were requested to use and denote dosages correlated to the relief of specific symptomatology. Participants observed and rated symptoms before and after cannabis use to record their severity. This is done upon rising from sleep in all cases except "insomnia" and prior to using any cannabis. Assessments were made weekly, at minimum, or as much as seven times per week, in order to assess effectiveness and of different strains upon different target symptoms.
Findings were derived from data gathered during the time period of June of 1993 into early 1997. Statistical analysis consisted of frequency analysis, paired T-tests of "before" and "after" scores on each measured symptom or condition, and a series of one way ANOVAs on route of administration (either inhaled or ingested, cannabis strain, and diagnosis).
Because the therapeutic effects of cannabis are sometimes ascribed to its mood-altering effects, we also performed a correlation analysis of the change in mood score with other outcome variables.
Inhalation methods of cannabis consisted mostly of smoking, with some use of vaporization, although patient reports of effectiveness appear substantially lessened when this technique was employed. This could certainly depend on the quality of the vaporizer design.
Ingested forms of cannabis consisted of baked goods and "mother's milk" (a soymilk-based liquid), and a whole cannabis tincture made with pure grain alcohol with leaf or a combined blend of leaf and flowers. Strains of marijuana were C. sativa and C. indica and their hybrids. The morphological distinction between these strains was determined by experienced cannabis cultivators associated with WAMM, based on characteristic features of the two sub-species, varieties or strains.
These sub-species varied from week to week and included the following pure strains and hybrid strains:
C. sativa, C. indica, as well as hybrids of both, being the identified female C. sativa x male C. indica, as well as the identified female C. indica x male C. sativa.
We secured a method of analysis of the chemical content of test materials, although we believe that the findings may be subject to error. Results from a drug detection laboratory indicated that C. sativa measured: THC 23.7%, CBD <0.1% and CBN <0.1%. Results indicated that C. indica strains measured THC 19.6%, CBD <0.2% and CBN <0.5%. Cannabis potency testing results by ElSohly Labs of the same sample of C. sativa after storage for eight months yielded a value of THC 17.6 %.
Results
Seventy-seven patients completed a total of 1892 forms (range 1 - 256, median 8) during the three-year study period. Of these, 43 were male (56 percent), 22 were female (29 percent) and 12 were not coded as to gender. The distribution of primary diagnosis is presented in Table 1. [Table 1]
Thirty-nine patients (51 percent) had HIV/AIDS; 14 (18 percent) had neurological diseases, and 7 (9 percent) had a principle diagnosis of cancer.
To avoid biasing results due to a large proportion of questionnaires being completed by relatively few patients, we standardized the analysis by reviewing a maximum of eight records per patient, the median number completed by study subjects.
These records were randomly chosen. Accordingly, our analysis contained 432 records. Of these, 261 (61 percent) referred to C. sativa experiences; 65 (15 percent) were C. indica, while 105 (24 percent) were coded "other". Certain types of marijuana were donated or undeclared, we labeled these as "other" and included them in our findings. Ingested forms were also recorded [Table 4]. Some entries were coded with missing information, entered as slang or incorrectly named, these were excluded.
Paired t-tests of before and after health status revealed that the following symptoms were relieved to a statistically significant extent by marijuana (without regard to strain or route of administration): pain, energy, mood, nausea, appetite, and awareness.
The remaining symptoms were not reliably relieved to this extent. Table 5 and Table 6 show the scores on each variable. The magnitude of improvement was unrelated to clinical diagnosis, as determined in ANOVA [Table 10], with one exception: the degree of relief of nausea was greater in the HIV/AIDS group (4.54 units) than in the orthopedic group (1.58 units) to a (marginally) statistically significant extent (p = 0.04).
We next performed ANOVA on the strain of marijuana ingested: C. sativa and C. indica. The mean change scores, "before" scores minus "after" scores for patients with each condition. For the most part, some observed changes were unrelated to strain of marijuana. However, two symptoms - energy and appetite - were improved to a statistically greater extent by C. indica than by either C. sativa or "other."
C. indica produced a mean improvement in energy of 3.76 units (vs. 1.53 for C. sativa and 2.22 for "other") and a mean improvement in appetite by 5.22 units (vs. 3.41 for C. sativa and 4.32 for C. indica). These differences were significant at the 0.012 and 0.005 levels, respectively [Table 8].
ANOVA was then conducted using route of administration as the independent variable [Table 6 & Table 7]. For the most part, ingested and inhaled marijuana had similar magnitudes of effects. Only one symptom - spasm - showed preferential improvement using smoked over ingested marijuana (p = .036). [Table 6]. Patients reporting "other" routes of administration, such as ingestion, had substantially less relief of nausea than patients inhaling or ingesting marijuana [Table 7].
It is reported that THC may reduce spasms associated with both neurological and non-neurological disorders (Hollister, 1986; British Medical Association Report, 1997).
It is interesting to note that the non-psychoactive cannabinoid cannabidiol has been shown to exhibit anticonvulsant properties in certain animal studies (Iversen 2000)(The Science of Marijuana, L.L. Iversen, Ph D.) In the case of some patients it has been noted to reduce or prevent the onset of both spasms and seizures when used alone or as an adjunct medicine. It appears that there are receptor sites for cannabinoids that have beneficial effects on seizure activity.
Finally, analysis of the Pearson correlation coefficients between changes in mood scores and changes in other symptom scores revealed only a single statistically significant correlation, between mood and energy level (p = 0.035). Mood was not correlated with any other outcomes, including pain relief (p = 0.817) [Table 11].
Discussion
We analyzed 432 records of therapeutic cannabis exposures, including information on strain (C. sativa, C. indica, or other), and route of administration (inhaled, ingested or other). The outcome variables consisted of scores to a series of questions on symptoms, completed by the patient both before and after administering cannabis medicines.
Results indicate that cannabis was uniformly effective in relieving symptoms across a wide range of diagnostic categories. No differences were observed in the extent to which symptoms were relieved based on diagnosis, except that patients with HIV/AIDS experienced more relief of nausea than patients with primary orthopedic diagnoses [Table 13].
On several occasions, terminally ill patients remarked upon a recurrent phenomenon, described as a “shift in consciousness" or "perception" allowing them to approach their impending death more "openly" or in a more "relaxed" manner. This is of particular interest, as each patient also reported a reduction in anxiety often associated with the dying process. Future studies will further examine measures anxiety in the cannabis patient population.
C. indica appeared to be superior to C. sativa and "other" in improving energy and appetite [Table 9]; otherwise, no differences in strain effects were observed. Route of administration had little effect on outcome in our series. Two symptoms, spasm [Table 6] and nausea [Table 7] showed preferential improvement of smoking as compared ingestion. In no condition was the ingested route superior to smoking upon symptom management.
Changes in mood were not correlated with changes in other outcomes except for a modest correlation with energy [Table 11]. The finding that mood did not correlate with other outcomes casts doubt on the theory that therapeutic cannabis effects are related primarily to improvement in mood.
Conversely, this may have something to do with the notion suggested by some patients that mood is not necessarily correlated to the concept of "feeling better." In our findings, it appeared that mood was often independent of symptom expression. This result is interesting because it appears from written testimony by patients in their surveys that they believe changes in awareness or consciousness do affect overall healing. We plan to further examine the validity of these phenomena in future studies.
These findings support that few differences were noted by patients between C. sativa and C. indica strains and between ingestion vs. inhaled routes of administration. This is likely due to modest observed differences in cannabinoid content in the supplied strains. We hope that a reliable and accessible means of analysis will become available in the near future.
This study is limited by the lack of blinding. For this reason, in 1998 a revised protocol was instituted in which patients receive a one-week supply of therapeutic cannabis at a time without knowledge of particular variety provided. Patients continue completing forms on a weekly basis. This method of blinding is expected to provide a more rigorous test of any distinctions between C. sativa and C. indica strains. Results may have implications for subsequent crossbreeding of strains to maximize therapeutic effects.
This study is only a small first step in the attempt to develop improved cannabis medicines to affected patients. The most significant current limitation to this type of research is the absence of a convenient legal mechanism in the USA for analyzing cannabis samples for biochemical constituent content. Until this limitation is overcome, progress in this area will be slow at best.
On the other hand, we should not underestimate the value of clinical observation in judging strains of cannabis and their differential clinical effects irrespective of chemical content. Thus, while the work we report here does not definitely address issues of chemical variability, we believe that our findings provide at the very least a good working hypotheses for use in future studies.
References
Grotenhermen, Franjo. 2001. Practical hints. In Cannabis and Cannabinoids: Pharmacology, toxicology and therapeutic potential, edited by F. Grotenhermen and E. Russo. Binghamton, NY: Haworth Press.
Iversen, Leslie L. 2000. The science of marijuana. Oxford ; New York: Oxford University Press.
McPartland, J. M., and P. L. Pruitt. 1999. Side effects of pharmaceuticals not elicited by comparable herbal medicines: the case of tetrahydrocannabinol and marijuana. Altern Ther Health Med 5 (4):57-62.
McPartland, John M., and Vito Mediavilla. 2001. Non-cannabinoids in cannabis. In Cannabis and cannabinoids, edited by F. Grotenhermen and E. B. Russo. Binghamton, NY: Haworth Press.
Received: September 21, 2000
Accepted in Final Form: April 29, 2001.
Purpose of the Project
- To determine if there are physical, mood and perception changes resulting from use of the test article.
- To determine if the method of delivery affects measures of effectiveness.
- To determine if different types of cannabis affect diagnoses and measures of effectiveness.
- To assess the correlation between changes in mood and other measures of effectiveness.
Summary of Population
N = 77
43 males (56%)
22 females (29%)
12 missing gender distinction (15%)
Table 1
Description of Population by Primary Diagnosis
Table 2
Description of Patient Population by Secondary Diagnosis
Description of Patient Population by Secondary Diagnosis
Table 3
Questionnaire Structure Measures of Effectiveness
Questionnaire Structure Measures of Effectiveness
|
Variable
Pain Energy Mood Nausea Appetite Muscle Spasms Seizures Ocular Insomnia Awareness Neuropathy |
None
1 1 1 1 1 1 1 1 1 1 1 |
Most
10 10 10 10 10 10 10 10 10 10 10 |
Desired Effect
Decrease Increase Increase Decrease Increase Decrease Decrease Decrease Decrease Increase Decrease |
Questionnaire Logistics
Statistical Methods
432 questionnaires analyzed
Frequency analysis, Paired t-tests, Paired t-test correlations, One Way ANOVA, Post-Hoc (Bonferroni), Pearson Correlation and Multivariate tests performed
One Way ANOVA conducted on variables using the following 3 groups
Group 1 – test article “ingested”
Muffins Mothers milk
Group 2 – test article “inhaled”
African Queen Purple Indica
Group 3 –“Other”
One Way ANOVA performed on the following test article groups:
Sativa (261 – 61%)
Other (105 – 24%)
Indica ( 65 – 15%)
Multivariate Tests performed for type of Cannabis, diagnosis, and change in variable
Pillai’s Trace
Wilks’ Lambda, and
Tests of Between-Subjects Effects
One Way ANOVA, Bonferroni, Post-Hoc tests performed for definition of diagnosis and treatment effectiveness
All tests performed using SPSS (Statistical Program for Social Scientists) Version 9.0
Question One
Are there physical, mood and perception changes resulting from use of the test article?
Table 5
Paired Samples t test
Comparing means before and after
95% confidence interval (2-tailed)
- 1892 Questionnaires Completed over 3 years
- Range of 1 to 256 questionnaires
- Average of 8 questionnaires/patient
- Analysis completed based on the average number of questionnaires completed (to normalize data for analysis)
Statistical Methods
432 questionnaires analyzed
Frequency analysis, Paired t-tests, Paired t-test correlations, One Way ANOVA, Post-Hoc (Bonferroni), Pearson Correlation and Multivariate tests performed
One Way ANOVA conducted on variables using the following 3 groups
Group 1 – test article “ingested”
Muffins Mothers milk
Group 2 – test article “inhaled”
African Queen Purple Indica
Group 3 –“Other”
One Way ANOVA performed on the following test article groups:
Sativa (261 – 61%)
Other (105 – 24%)
Indica ( 65 – 15%)
Multivariate Tests performed for type of Cannabis, diagnosis, and change in variable
Pillai’s Trace
Wilks’ Lambda, and
Tests of Between-Subjects Effects
One Way ANOVA, Bonferroni, Post-Hoc tests performed for definition of diagnosis and treatment effectiveness
All tests performed using SPSS (Statistical Program for Social Scientists) Version 9.0
Question One
Are there physical, mood and perception changes resulting from use of the test article?
Table 5
Paired Samples t test
Comparing means before and after
95% confidence interval (2-tailed)
|
Pain Energy Mood Nausea Appetite Spasm Seizure Ocular Insomnia Awareness |
Other
-3.49 2.22 2.94 -4.67 4.32 -4.33 -0.67 -3.27 -4.53 1.75 |
Sativa
-3.99 1.53 2.89 4.129 3.41 -3.53 -2.12 -2.34 -3.82 0.96 |
Indica
-2.93 1.53 3.76 -4.01 5.22 -2.23 0.50 -3.00 -3.18 1.24 |
P
0.078 0.012* 0.327 0.470 0.005* 0.071 0.316 0.646 0.221 0.173 |
One Way Anova 95% CI
*Significant
**Small sample size unable to correlate
Table 11
Interpretation of ANOVA Method for Primary Diagnostic Group
The Orthopedic and Neurologic group are different than the “Other” primary diagnostic group.
There is greater improvement in Mood (p = 0.008) for the Orthopedic group vs. “Other”
There is greater improvement in Mood (p = 0.001) for the Neurologic group vs. “Other”
Average Orthopedic 4.36
Average Neurologic 4.04
Average HIV/AIDS 2.87
Average “Other” 1.33
Average Cancer 2.64
There is no difference between the AID/HIV and Cancer groups
Table 12
Interpretation of ANOVA Method for Primary Diagnostic Group
The Orthopedic group is different than the “Other” primary diagnostic group.
There is greater improvement in Energy (p = 0.43) for the Orthopedic group than “Other”
Average Orthopedic 3.54
Average Neurologic 1.33
Average HIV/AIDS 2.31
Average “Other” 1.07
Average Cancer 1.23
There is no difference between the Neurologic, AID/HIV, and Cancer groups
Table 13
Interpretation of ANOVA Method for Primary Diagnostic Group
The HIV/AIDS group is different than the Orthopedic primary diagnostic group
There is greater improvement in Nausea (p =0.04) in the HIV/AIDS group than Orthopedic primary diagnostic group
Average Orthopedic -1.58
Average Neurologic -4.21
Average HIV/AIDS -4.54
Average “Other” -3.97
Average Cancer -4.18
There is no difference between the Neurologic, Other, and Cancer groups
Table 14
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Appetite (0.010) for all diagnostic groups
There is no difference in mean change for the Appetite variable for specific primary diagnostic groups
Average Orthopedic 4.57
Average Neurologic 3.50
Average HIV/AIDS 4.44
Average “Other” 3.08
Average Cancer 3.00
Table 15
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Insomnia (p = 0.000) for all diagnostic groups
There is no difference in mean change for the Insomnia variable for specific primary diagnostic groups
Average Orthopedic -4.68
Average Neurologic -4.66
Average HIV/AIDS -3.49
Average “Other” -2.93
Average Cancer -5.08
Table 16
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Awareness (p = 0.000) for all diagnostic groups
There is no difference in mean change for Awareness specific to primary diagnostic groups
Average Orthopedic 2.21
Average Neurologic 1.07
Average HIV/AIDS 1.15
Average “Other” 0.65
Average Cancer 2.25
Correlation Analysis Question Four
Is change in mood correlated to change in energy?
p = .035*
Is change in mood correlated to change in pain?
p = .817
Is change in mood correlated to change in nausea?
p = .434
Is change in mood correlated to change in insomnia?
P = .647
Is change in mood correlated to change in awareness?
P = .073
*Significant
Conclusions
There were observed changes in pain, energy, nausea, appetite, and awareness variables from the use of the test article.
Cannabis / Marijuana ( Δ 9 -Tetrahydrocannabinol, THC)Cannabis / Marijuana ( Δ 9 -Tetrahydrocannabinol, THC)
Marijuana is a green or gray mixture of dried shredded flowers and leaves of the hemp plant Cannabis sativa. Hashish consists of resinous secretions of the cannabis plant. Dronabinol (synthetic THC) is a light yellow resinous oil.
Synonyms: Cannabis, marijuana, pot, reefer, buds, grass, weed, dope, ganja, herb, boom, gangster, Mary Jane, sinsemilla, shit, joint, hash, hash oil, blow, blunt, green, kilobricks, Thai sticks; Marinol®
Source: Cannabis contains chemicals called cannabinoids, including cannabinol, cannabidiol, cannabinolidic acids, cannabigerol, cannabichromene, and several isomers of tetrahydrocannabinol (THC). One of these isomers, Δ 9-THC, is believed to be responsible for most of the characteristic psychoactive effects of cannabis.
Marijuana refers to the leaves and flowering tops of the cannabis plant; the buds are often preferred because of their higher THC content. Hashish consists of the THC-rich resinous secretions of the plant, which are collected, dried, compressed and smoked.
Hashish oil is produced by extracting the cannabinoids from plant material with a solvent. In the U. S., marijuana, hashish and hashish oil are Schedule I controlled substances. Dronabinol (Marinol®) is a Schedule III controlled substance and is available in strengths of 2.5, 5 or 10 mg in round, soft gelatin capsules.
Drug Class: Cannabis/ Marijuana: spectrum of behavioral effects is unique, preventing classification of the drug as a stimulant, sedative, tranquilizer, or hallucinogen. Dronabinol: appetite stimulant, antiemetic.
Medical and Recreational Uses: Medicinal: Indicated for the treatment of anorexia associated with weight loss in patients with AIDS, and to treat mild to moderate nausea and vomiting associated with cancer chemotherapy. Recreational: Marijuana is used for its mood altering effects, euphoria, and relaxation. Marijuana is the most commonly used illicit drug throughout the world.
Potency, Purity and Dose: THC is the major psychoactive constituent of cannabis. Potency is dependent on THC concentration and is usually expressed as %THC per dry weight of material. Average THC concentration in marijuana is 1-5%, hashish 5-15%, and hashish oil ³ 20%.
The form of marijuana known as sinsemilla is derived from the unpollinated female cannabis plant and is preferred for its high THC content (up to 17% THC). Recreational doses are highly variable and users often titer their own dose. A single intake of smoke from a pipe or joint is called a hit (approximately 1/20th of a gram).
The lower the potency or THC content the more hits are needed to achieve the desired effects; 1-3 hits of high potency sinsemilla is typically enough to produce the desired effects. In terms of its psychoactive effect, a drop or two of hash oil on a cigarette is equal to a single “joint” of marijuana. Medicinally, the initial starting dose of Marinol® is 2.5 mg, twice daily.
Route of Administration: Marijuana is usually smoked as a cigarette (‘joint’) or in a pipe or bong. Hollowed out cigars packed with marijuana are also common and are called `. Joints and blunts are often laced with adulterants including PCP or crack cocaine. Joints can also be dipped in liquid PCP or in codeine cough syrup. Marijuana is also orally ingested.
Pharmacodynamics: THC binds to cannabinoid receptors and interferes with important endogenous cannabinoid neurotransmitter systems. Receptor distribution correlates with brain areas involved in physiological, psychomotor and cognitive effects. Correspondingly, THC produces alterations in motor behavior, perception, cognition, memory, learning, endocrine function, food intake, and regulation of body temperature.
Pharmacokinetics: Absorption is slower following the oral route of administration with lower, more delayed peak THC levels. Bioavailability is reduced following oral ingestion due to extensive first pass metabolism. Smoking marijuana results in rapid absorption with peak THC plasma concentrations occurring prior to the end of smoking. Concentrations vary depending on the potency of marijuana and the manner in which the drug is smoked, however, peak plasma concentrations of 100-200 ng/mL are routinely encountered.
Plasma THC concentrations generally fall below 5 ng/mL less than 3 hours after smoking. THC is highly lipid soluble, and plasma and urinary elimination half-lives are best estimated at 3-4 days, where the rate-limiting step is the slow redistribution to plasma of THC sequestered in the tissues. Shorter half-lives are generally reported due to limited collection intervals and less sensitive analytical methods.
Plasma THC concentrations in occasional users rapidly fall below limits of quantitation within 8 to 12 h. THC is rapidly and extensively metabolized with very little THC being excreted unchanged from the body. THC is primarily metabolized to 11-hydroxy-THC which has equipotent psychoactivity.
The 11-hydroxy-THC is then rapidly metabolized to the 11-nor-9-carboxy-THC (THC-COOH) which is not psychoactive. A majority of THC is excreted via the feces (~65%) with approximately 30% of the THC being eliminated in the urine as conjugated glucuronic acids and free THC hydroxylated metabolites.
Molecular Interactions / Receptor Chemistry: THC is metabolized via cytochrome P450 2C9, 2C11, and 3A isoenzymes. Potential inhibitors of these isoenzymes could decrease the rate of THC elimination if administered concurrently, while potential inducers could increase the rate of elimination.
Blood to Plasma Concentration Ratio: 0.55
Interpretation of Blood Concentrations: It is difficult to establish a relationship between a person's THC blood or plasma concentration and performance impairing effects. Concentrations of parent drug and metabolite are very dependent on pattern of use as well as dose. THC concentrations typically peak during the act of smoking, while peak 11-OH THC concentrations occur approximately 9-23 minutes after the start of smoking.
Concentrations of both analytes decline rapidly and are often < 5 ng/mL at 3 hours. Significant THC concentrations (7 to 18 ng/mL) are noted following even a single puff or hit of a marijuana cigarette. Peak plasma THC concentrations ranged from 46-188 ng/mL in 6 subjects after they smoked 8.8 mg THC over 10 minutes. Chronic users can have mean plasma levels of THC-COOH of 45 ng/mL, 12 hours after use; corresponding THC levels are, however, less than 1 ng/mL. Following oral administration, THC concentrations peak at 1-3 hours and are lower than after smoking. Dronabinol and THC-COOH are present in equal concentrations in plasma and concentrations peak at approximately 2-4 hours after dosing.
It is inadvisable to try and predict effects based on blood THC concentrations alone, and currently impossible to predict specific effects based on THC-COOH concentrations. It is possible for a person to be affected by marijuana use with concentrations of THC in their blood below the limit of detection of the method. Mathematical models have been developed to estimate the time of marijuana exposure within a 95% confidence interval. Knowing the elapsed time from marijuana exposure can then be used to predict impairment in concurrent cognitive and psychomotor effects based on data in the published literature.
Interpretation of Urine Test Results: Detection of total THC metabolites in urine, primarily THC-COOH-glucuronide, only indicates prior THC exposure. Detection time is well past the window of intoxication and impairment. Published excretion data from controlled clinical studies may provide a reference for evaluating urine cannabinoid concentrations; however, these data are generally reflective of occasional marijuana use rather than heavy, chronic marijuana exposure.
It can take as long as 4 hours for THC-COOH to appear in the urine at concentrations sufficient to trigger an immunoassay (at 50ng/mL) following smoking. Positive test results generally indicate use within 1-3 days; however, the detection window could be significantly longer following heavy, chronic, use.
Following single doses of Marinol®, low levels of dronabinol metabolites have been detected for more than 5 weeks in urine. Low concentrations of THC have also been measured in over-the-counter hemp oil products – consumption of these products may produce positive urine cannabinoid test results.
Effects: Pharmacological effects of marijuana vary with dose, route of administration, experience of user, vulnerability to psychoactive effects, and setting of use.
Psychological: At recreational doses, effects include relaxation, euphoria, relaxed inhibitions, sense of well-being, disorientation, altered time and space perception, lack of concentration, impaired learning and memory, alterations in thought formation and expression, drowsiness, sedation, mood changes such as panic reactions and paranoia, and a more vivid sense of taste, sight, smell, and hearing.
Stronger doses intensify reactions and may cause fluctuating emotions, flights of fragmentary thoughts with disturbed associations, a dulling of attention despite an illusion of heightened insight, image distortion, and psychosis.
Physiological: The most frequent effects include increased heart rate, reddening of the eyes, dry mouth and throat, increased appetite, and vasodilatation.
Side Effect Profile: Fatigue, paranoia, possible psychosis, memory problems, depersonalization, mood alterations, urinary retention, constipation, decreased motor coordination, lethargy, slurred speech, and dizziness. Impaired health including lung damage, behavioral changes, and reproductive, cardiovascular and immunological effects have been associated with regular marijuana use.
Regular and chronic marijuana smokers may have many of the same respiratory problems that tobacco smokers have (daily cough and phlegm, symptoms of chronic bronchitis), as the amount of tar inhaled and the level of carbon monoxide absorbed by marijuana smokers is 3 to 5 times greater than among tobacco smokers. Smoking marijuana while shooting up cocaine has the potential to cause severe increases in heart rate and blood pressure.
Duration of Effects: Effects from smoking cannabis products are felt within minutes and reach their peak in 10-30 minutes. Typical marijuana smokers experience a high that lasts approximately 2 hours. Most behavioral and physiological effects return to baseline levels within 3-5 hours after drug use, although some investigators have demonstrated residual effects in specific behaviors up to 24 hours, such as complex divided attention tasks. Psychomotor impairment can persist after the perceived high has dissipated.
In long term users, even after periods of abstinence, selective attention (ability to filter out irrelevant information) has been shown to be adversely affected with increasing duration of use, and speed of information processing has been shown to be impaired with increasing frequency of use. Dronabinol has an onset of 30-60 minutes, peak effects occur at 2-4 hours, and it can stimulate the appetite for up to 24 hours.
Tolerance, Dependence and Withdrawal Effect: Tolerance may develop to some pharmacological effects of dronabinol. Tolerance to many of the effects of marijuana may develop rapidly after only a few doses, but also disappears rapidly.
Marijuana is addicting as it causes compulsive drug craving, seeking, and use, even in the face of negative health and social consequences. Additionally, animal studies suggests marijuana causes physical dependence. A withdrawal syndrome is commonly seen in chronic marijuana users following abrupt discontinuation. Symptoms include restlessness, irritability, mild agitation, hyperactivity, insomnia, nausea, cramping, decreased appetite, sweating, and increased dreaming.
Drug Interactions: Cocaine and amphetamines may lead to increased hypertension, tachycardia and possible cardiotoxicity. Benzodiazepines, barbiturates, ethanol, opioids, antihistamines, muscle relaxants and other CNS depressants increase drowsiness and CNS depression. When taken concurrently with alcohol, marijuana is more likely to be a traffic safety risk factor than when consumed alone.
Performance Effects: The short term effects of marijuana use include problems with memory and learning, distorted perception, difficultly in thinking and problem-solving, and loss of coordination. Heavy users may have increased difficulty sustaining attention, shifting attention to meet the demands of changes in the environment, and in registering, processing and using information. In general, laboratory performance studies indicate that sensory functions are not highly impaired, but perceptual functions are significantly affected.
The ability to concentrate and maintain attention are decreased during marijuana use, and impairment of hand-eye coordination is dose-related over a wide range of dosages.Impairment in retention time and tracking, subjective sleepiness, distortion of time and distance, vigilance, and loss of coordination in divided attention tasks have been reported.
Note however, that subjects can often “pull themselves together” to concentrate on simple tasks for brief periods of time. Significant performance impairments are usually observed for at least 1-2 hours following marijuana use, and residual effects have been reported up to 24 hours.
Effects on Driving: The drug manufacturer suggests that patients receiving treatment with Marinol® should be specifically warned not to drive until it is established that they are able to tolerate the drug and perform such tasks safely.
Epidemiology data from road traffic arrests and fatalities indicate that after alcohol, marijuana is the most frequently detected psychoactive substance among driving populations. Marijuana has been shown to impair performance on driving simulator tasks and on open and closed driving courses for up to approximately 3 hours.
Decreased car handling performance, increased reaction times, impaired time and distance estimation, inability to maintain headway, lateral travel, subjective sleepiness, motor incoordination, and impaired sustained vigilance have all been reported.
Some drivers may actually be able to improve performance for brief periods by overcompensating for self-perceived impairment. The greater the demands placed on the driver, however, the more critical the likely impairment.
Marijuana may particularly impair monotonous and prolonged driving. Decision times to evaluate situations and determine appropriate responses increase. Mixing alcohol and marijuana may dramatically produce effects greater than either drug on its own.
DEC Category: Cannabis
DEC Profile: Horizontal gaze nystagmus not present; vertical gaze nystagmus not present; lack of convergence present; pupil size normal to dilated; reaction to light normal to slow; pulse rate elevated; blood pressure elevated; body temperature normal to elevated. Other characteristic indicators may include odor of marijuana in car or on subject’s breath, marijuana debris in mouth, green coating of tongue, bloodshot eyes, body and eyelid tremors, relaxed inhibitions, incomplete thought process, and poor performance on field sobriety tests.
Panel’s Assessment of Driving Risks: Low doses of THC moderately impair cognitive and psychomotor tasks associated with driving, while severe driving impairment is observed with high doses, chronic use and in combination with low doses of alcohol The more difficult and unpredictable the task, the more likely marijuana will impair performance.
References and Recommended Reading:
Aceto MD, Scates SM, Lowe JA, Martin BR. Cannabinoid precipitated withdrawal by the selective cannabinoid receptor antagonist, SR 141716A. Eur J Pharmacol 1995;282(1-3): R1-2.
Adams IB, Martin BR. Cannabis: pharmacology and toxicology in animals and humans. Addiction 1996;91(11):1585-614.
Barnett G, Chiang CW, Perez-Reyes M, Owens SM. Kinetic study of smoking marijuana. J Pharmacokinet Biopharm 1982;10(5):495-506.
Baselt RC. Drug effects on psychomotor performance. Biomedical Publications, Foster City, CA; pp 403-415;2001.
Hansteen RW, Miller RD, Lonero L, Reid LD, Jones B. Effects of cannabis and alcohol on automobile driving and psychomotor tracking. Ann NY Acad Sci 1976;282:240-56.
Heishman SJ. Effects of abused drugs on human performance: Laboratory assessment. In: Drug Abuse
Handbook. Karch SB, ed. New York, NY: CRC Press, 1998, p219.
Huestis MA. Cannabis (Marijuana) - Effects on Human Performance and Behavior. Forens Sci Rev 2002;14(1/2):15-60.
Huestis MA, Sampson AH, Holicky BJ, Henningfield JE, Cone EJ. Characterization of the absorption phase of marijuana smoking. Clin Pharmacol Ther 1992;52(1):31-41.
Huestis MA, Henningfield JE, Cone EJ. Blood cannabinoids: I. Absorption of THC and formation of 11-OH-THC and THC-COOH during and after marijuana smoking. J Anal Toxicol 1992;16(5):276-82.
Huestis MA, Henningfield JE, Cone EJ. Blood cannabinoids II: Models for the prediction of time of marijuana exposure from plasma concentrations of ∆-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-∆-9-tetrahydrocannabinol (THC-COOH). J Anal Toxicol 1992;16(5):283-90.
Hunt CA, Jones RT. Tolerance and disposition of tetrahydrocannabinol in man. J Pharmacol Exp Ther 1980;215(1):35-44.
Klonoff H. Marijuana and driving in real-life situations. Science 1974;186(4161);317-24.
Leirer VO, Yesavage JA, Morrow DG. Marijuana carry-over effects on aircraft pilot performance. Aviat Space Environ Med 1991;62(3):221-7.
Mason AP, McBay AJ. Cannabis: pharmacology and interpretation of effects. J Forensic Sci 1985;30(3):615-31.
Physicians’ Desk Reference, Medical Economics Company, Montvale, NJ, 2002.
Plasse TF, Gorter RW, Krasnow SH, Lane M, Shepard KV, Wadleigh RG. Recent clinical experience with Dronabinol. Pharmacol Biochem Behav 1991;40(3):695-700.
Pope HG Jr, Yurgelun-Todd D. The residual cognitive effects of heavy marijuana use in college students. JAMA 1996;275(7):521-7.
Ramaekers JG, Robbe HW, O’Hanlon JF. Marijuana, alcohol and actual driving performance. Hum Psychopharmacol 2000;15(7):551-8.
Robbe HW, O'Hanlon JF. Marijuana and actual driving performance. US Department of Transportation/National Highway Traffic Safety Administration November: 1-133 (1993). DOT HS 808 078.
Smiley A, Moskowitz HM, Ziedman K. Effects of drugs on driving: Driving simulator tests of secobarbital, diazepam, marijuana, and alcohol. In Clinical and Behavioral Pharmacology Research Report. J.M. Walsh, Ed. U.S. Department of Health and Human Services, Rockville, 1985, pp 1-21.
Solowij N, Michie PT, Fox AM. Differential impairment of selective attention due to frequency and duration of cannabis use. Biol Psychiatry 1995;37(10):731-9.
Thornicroft G. Cannabis and psychosis. Is there epidemiological evidence for an association? Br J Psychiatry 1990;157:25-33.
Varma VK, Malhotra AK, Dang R, Das K, Nehra R. Cannabis and cognitive functions: a prospective study. Drug Alcohol Depend 1988;21(2):147-52.
WHO Division of Mental Health and Prevention of Substance Abuse: Cannabis: a health perspective and research agenda. World Health Organization 1997.
A chemotaxonomic analysis of cannabinoid variation in Cannabis
(American Journal of Botany. 2004;91:966-975.)
© 2004 Botanical Society of America, Inc
Karl W. Hillig and Paul G. Mahlberg
Department of Biology, Indiana University, Bloomington, Indiana 47405 USA
Received for publication June 19, 2003. Accepted for publication February 12, 2004.
ABSTRACT
Cannabinoids are important chemotaxonomic markers unique to Cannabis. Previous studies show that a plant's dry-weight ratio of 9-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.
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The inheritance of chemical phenotype in Cannabis sativa
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Copyright 2003 by the Genetics Society of America
The Inheritance of Chemical Phenotype in Cannabis sativa L.
Etienne P. M. de Meijer,*,1 Manuela Bagatta,† Andrea Carboni,† Paola Crucitti,†
V. M. Cristiana Moliterni,† Paolo Ranalli† and Giuseppe Mandolino†,2
*HortaPharm B.V., 1075 VS, Amsterdam, The Netherlands and †Istituto Sperimentale per le Colture Industriali, 40128 Bologna, Italy
Manuscript received April 23, 2002
Accepted for publication October 16, 2002
ABSTRACT
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure -9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F1’s were analyzed by gas chromatography for cannabinoid composition and constantly found to have amixed CBD-THC chemotype.
Ten individual F1 plants were self-fertilized, and 10 inbred F2 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 F1 to the F2’s derived from it. Dowload Complete Pdf for easier reading
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Medical Cannabis Potency Testing Project
From the Bulletin of the Multidisciplinary Association for Psychedelic Studies
MAPS - Volume 9 Number 3 Autumn 1999 - pp. 20-22Dale Gieringer, Ph.D.
California NORML, 2215-R Market St. Suite 278
San Francisco CA 94114 tel: (415) 563-5858
E-mail: canorml@igc.apc.org
Given the rapidly growing use of medical cannabis for a wide variety of indications and the manifold different underground sources currently supplying patients, there is a natural interest in investigating the potency, purity, and chemical content of the available supplies of medical cannabis. While the availability of medical cannabis has increased in the wake of the passage of California's Proposition 215 and other state medical marijuana initiatives, scientific research on its content remains frustrated by the continued federal ban on medical cannabis research.
In an effort to cast light in this obscure area, a research project was undertaken by a group of us, including researchers, growers, and medical cannabis buyers' clubs, with support from California NORML and MAPS, to analyze samples of medical cannabis from various patients' cooperatives and providers around the country. This effort proved to be a lesson in the difficulties and uncertainties of cannabis research in a society where freedom of pharmacological research has been stifled by an effectively totalitarian drug bureaucracy.
From the outset, our project was frustrated by a lack of access to qualified research labs with expertise in analysis of cannabis. The leading research lab in the country declined to do business with us for fear of compromising government contracts, while the other likely candidates were all foreign and thus not legally accessible to us because of DEA regulations. In the end, we were fortunate to obtain the services of a laboratory that had the requisite DEA license and equipment (a gas chromatograph mass spectrometer, or GCMS), but no prior experience in cannabis analysisin fact, its primary business was drug urinalysis! The analysis of our samples was accordingly a learning process for both the lab and ourselves.
Our original aim had been to obtain a broad-spectrum quantitative analysis of as many of the 60-plus naturally occurring cannabinoids as possible, in the hope of detecting differences that might produce differing therapeutic effects among the samples. To our disappointment, however, our lab could obtain laboratory standards only for the three most common cannabinoids, delta-9-THC, cannabidiol (CBD), and cannabinol (CBN).
A total of 47 different samples of medical cannabis were submitted by over a half dozen different providers and patients' cooperatives ranging from California to the East Coast. Included were 42 samples of sinsemilla bud, three samples of hashish or resin; one liquid sample of a milk-based cannabis drink ("Mother's Milk"), and one capsule of an oral whole leaf preparation.
Upon analysis by GCMS, the potency of the 42 sinsemilla samples was determined to range from 10.2% to 31.6% THC, with a mean of 19.4%. These results were surprisingly high, given that the average potency of marijuana in the U.S. has been typically estimated at around 3% to 4% by NIDA, with higher grade sinsemilla ranging towards 10% - 15%. The highest potency recorded came from a sample of hashish, which registered 68.6%. Yet even a sample of Mexican commercial grade registered a surprisingly high 11%, twice what we had expected. All of this cast a troubling shadow of doubt on our test results, although it appeared likely that we were dealing with highly potent varieties.
In contrast, the CBD levels observed were surprisingly low. Only four of the sinsemilla samples had more than 0.3% CBD, and 35 of them had only trace amounts (<0.1%). However, one sample had an astoundingly high CBD content of 28.0% (plus 11.6% THC).
Another registered 5.6% CBD and 13.4% THC. Aside from these two anomalies, the CBD results were frankly disappointing, as we had hoped to discover significant variations in the content of the samples, with accompanying variations in medical activity.
Because CBD is suspected to have peculiar efficacy for control of muscle spasms and for damping anxiety and "panic reactions" caused by THC, we had hypothesized that certain patients would tend to prefer high-CBD varieties. In fact, however, it appears that few patients are ever exposed to high-CBD cannabis. Unfortunately, we were unable to procure additional specimens of the high-CBD varieties for further testing.
As for CBN, the majority of samples showed only trace amounts. The highest level detected was 1.4%, and only one other sample tested above 1%. CBN is a breakdown product of THC, so high CBN levels are expected in old, degraded samples.
This was confirmed by the fact that one of the samples above 1% CBN was known to be a year old. The prevalence of low CBN in the samples was evidence that most available medical cannabis tends to be fresh and well-preserved. Otherwise, these results were of limited interest, as there are few if any known medical effects of CBN.
Another disappointing surprise was the failure to detect more than trace levels of THC or CBD in the liquid "Mother's Milk" sample. Upon further investigation, the lab determined that this was because it is impossible to extract cannabinoids from fat-based liquids using standard methanol extraction techniques. Consulting with other researchers, we found that there is no known method for isolating THC from fat-based liquids.
Later, we located a lab that claimed to have developed a secret, proprietary method for extracting cannabinoids from fat. With considerable difficulty, we arranged to have the lab test the Mother's Milk. To our disappointment, however, once again only trace amounts of THC and CBD were detected.
Just to make sure, one of us swallowed a sample of the Mother's Milk (which by now had spent several months in the freezer) and found it to be delightfully potent. Evidently, the lab's technique had failed. It appears that further advances in testing technology will be needed in order to properly analyze fat-based oral cannabis products such as Mother's milk, bhang, ghee, and possibly baked goods such as brownies.
Table: THC and CBD Test Results
(Round 1 vs. Rounds 2 and 3)
*Significant
**Small sample size unable to correlate
Table 11
Interpretation of ANOVA Method for Primary Diagnostic Group
The Orthopedic and Neurologic group are different than the “Other” primary diagnostic group.
There is greater improvement in Mood (p = 0.008) for the Orthopedic group vs. “Other”
There is greater improvement in Mood (p = 0.001) for the Neurologic group vs. “Other”
Average Orthopedic 4.36
Average Neurologic 4.04
Average HIV/AIDS 2.87
Average “Other” 1.33
Average Cancer 2.64
There is no difference between the AID/HIV and Cancer groups
Table 12
Interpretation of ANOVA Method for Primary Diagnostic Group
The Orthopedic group is different than the “Other” primary diagnostic group.
There is greater improvement in Energy (p = 0.43) for the Orthopedic group than “Other”
Average Orthopedic 3.54
Average Neurologic 1.33
Average HIV/AIDS 2.31
Average “Other” 1.07
Average Cancer 1.23
There is no difference between the Neurologic, AID/HIV, and Cancer groups
Table 13
Interpretation of ANOVA Method for Primary Diagnostic Group
The HIV/AIDS group is different than the Orthopedic primary diagnostic group
There is greater improvement in Nausea (p =0.04) in the HIV/AIDS group than Orthopedic primary diagnostic group
Average Orthopedic -1.58
Average Neurologic -4.21
Average HIV/AIDS -4.54
Average “Other” -3.97
Average Cancer -4.18
There is no difference between the Neurologic, Other, and Cancer groups
Table 14
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Appetite (0.010) for all diagnostic groups
There is no difference in mean change for the Appetite variable for specific primary diagnostic groups
Average Orthopedic 4.57
Average Neurologic 3.50
Average HIV/AIDS 4.44
Average “Other” 3.08
Average Cancer 3.00
Table 15
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Insomnia (p = 0.000) for all diagnostic groups
There is no difference in mean change for the Insomnia variable for specific primary diagnostic groups
Average Orthopedic -4.68
Average Neurologic -4.66
Average HIV/AIDS -3.49
Average “Other” -2.93
Average Cancer -5.08
Table 16
Interpretation of ANOVA Method for Primary Diagnostic Group
There is improvement in Awareness (p = 0.000) for all diagnostic groups
There is no difference in mean change for Awareness specific to primary diagnostic groups
Average Orthopedic 2.21
Average Neurologic 1.07
Average HIV/AIDS 1.15
Average “Other” 0.65
Average Cancer 2.25
Correlation Analysis Question Four
Is change in mood correlated to change in energy?
p = .035*
Is change in mood correlated to change in pain?
p = .817
Is change in mood correlated to change in nausea?
p = .434
Is change in mood correlated to change in insomnia?
P = .647
Is change in mood correlated to change in awareness?
P = .073
*Significant
Conclusions
There were observed changes in pain, energy, nausea, appetite, and awareness variables from the use of the test article.
Cannabis / Marijuana ( Δ 9 -Tetrahydrocannabinol, THC)Cannabis / Marijuana ( Δ 9 -Tetrahydrocannabinol, THC)
Marijuana is a green or gray mixture of dried shredded flowers and leaves of the hemp plant Cannabis sativa. Hashish consists of resinous secretions of the cannabis plant. Dronabinol (synthetic THC) is a light yellow resinous oil.
Synonyms: Cannabis, marijuana, pot, reefer, buds, grass, weed, dope, ganja, herb, boom, gangster, Mary Jane, sinsemilla, shit, joint, hash, hash oil, blow, blunt, green, kilobricks, Thai sticks; Marinol®
Source: Cannabis contains chemicals called cannabinoids, including cannabinol, cannabidiol, cannabinolidic acids, cannabigerol, cannabichromene, and several isomers of tetrahydrocannabinol (THC). One of these isomers, Δ 9-THC, is believed to be responsible for most of the characteristic psychoactive effects of cannabis.
Marijuana refers to the leaves and flowering tops of the cannabis plant; the buds are often preferred because of their higher THC content. Hashish consists of the THC-rich resinous secretions of the plant, which are collected, dried, compressed and smoked.
Hashish oil is produced by extracting the cannabinoids from plant material with a solvent. In the U. S., marijuana, hashish and hashish oil are Schedule I controlled substances. Dronabinol (Marinol®) is a Schedule III controlled substance and is available in strengths of 2.5, 5 or 10 mg in round, soft gelatin capsules.
Drug Class: Cannabis/ Marijuana: spectrum of behavioral effects is unique, preventing classification of the drug as a stimulant, sedative, tranquilizer, or hallucinogen. Dronabinol: appetite stimulant, antiemetic.
Medical and Recreational Uses: Medicinal: Indicated for the treatment of anorexia associated with weight loss in patients with AIDS, and to treat mild to moderate nausea and vomiting associated with cancer chemotherapy. Recreational: Marijuana is used for its mood altering effects, euphoria, and relaxation. Marijuana is the most commonly used illicit drug throughout the world.
Potency, Purity and Dose: THC is the major psychoactive constituent of cannabis. Potency is dependent on THC concentration and is usually expressed as %THC per dry weight of material. Average THC concentration in marijuana is 1-5%, hashish 5-15%, and hashish oil ³ 20%.
The form of marijuana known as sinsemilla is derived from the unpollinated female cannabis plant and is preferred for its high THC content (up to 17% THC). Recreational doses are highly variable and users often titer their own dose. A single intake of smoke from a pipe or joint is called a hit (approximately 1/20th of a gram).
The lower the potency or THC content the more hits are needed to achieve the desired effects; 1-3 hits of high potency sinsemilla is typically enough to produce the desired effects. In terms of its psychoactive effect, a drop or two of hash oil on a cigarette is equal to a single “joint” of marijuana. Medicinally, the initial starting dose of Marinol® is 2.5 mg, twice daily.
Route of Administration: Marijuana is usually smoked as a cigarette (‘joint’) or in a pipe or bong. Hollowed out cigars packed with marijuana are also common and are called `. Joints and blunts are often laced with adulterants including PCP or crack cocaine. Joints can also be dipped in liquid PCP or in codeine cough syrup. Marijuana is also orally ingested.
Pharmacodynamics: THC binds to cannabinoid receptors and interferes with important endogenous cannabinoid neurotransmitter systems. Receptor distribution correlates with brain areas involved in physiological, psychomotor and cognitive effects. Correspondingly, THC produces alterations in motor behavior, perception, cognition, memory, learning, endocrine function, food intake, and regulation of body temperature.
Pharmacokinetics: Absorption is slower following the oral route of administration with lower, more delayed peak THC levels. Bioavailability is reduced following oral ingestion due to extensive first pass metabolism. Smoking marijuana results in rapid absorption with peak THC plasma concentrations occurring prior to the end of smoking. Concentrations vary depending on the potency of marijuana and the manner in which the drug is smoked, however, peak plasma concentrations of 100-200 ng/mL are routinely encountered.
Plasma THC concentrations generally fall below 5 ng/mL less than 3 hours after smoking. THC is highly lipid soluble, and plasma and urinary elimination half-lives are best estimated at 3-4 days, where the rate-limiting step is the slow redistribution to plasma of THC sequestered in the tissues. Shorter half-lives are generally reported due to limited collection intervals and less sensitive analytical methods.
Plasma THC concentrations in occasional users rapidly fall below limits of quantitation within 8 to 12 h. THC is rapidly and extensively metabolized with very little THC being excreted unchanged from the body. THC is primarily metabolized to 11-hydroxy-THC which has equipotent psychoactivity.
The 11-hydroxy-THC is then rapidly metabolized to the 11-nor-9-carboxy-THC (THC-COOH) which is not psychoactive. A majority of THC is excreted via the feces (~65%) with approximately 30% of the THC being eliminated in the urine as conjugated glucuronic acids and free THC hydroxylated metabolites.
Molecular Interactions / Receptor Chemistry: THC is metabolized via cytochrome P450 2C9, 2C11, and 3A isoenzymes. Potential inhibitors of these isoenzymes could decrease the rate of THC elimination if administered concurrently, while potential inducers could increase the rate of elimination.
Blood to Plasma Concentration Ratio: 0.55
Interpretation of Blood Concentrations: It is difficult to establish a relationship between a person's THC blood or plasma concentration and performance impairing effects. Concentrations of parent drug and metabolite are very dependent on pattern of use as well as dose. THC concentrations typically peak during the act of smoking, while peak 11-OH THC concentrations occur approximately 9-23 minutes after the start of smoking.
Concentrations of both analytes decline rapidly and are often < 5 ng/mL at 3 hours. Significant THC concentrations (7 to 18 ng/mL) are noted following even a single puff or hit of a marijuana cigarette. Peak plasma THC concentrations ranged from 46-188 ng/mL in 6 subjects after they smoked 8.8 mg THC over 10 minutes. Chronic users can have mean plasma levels of THC-COOH of 45 ng/mL, 12 hours after use; corresponding THC levels are, however, less than 1 ng/mL. Following oral administration, THC concentrations peak at 1-3 hours and are lower than after smoking. Dronabinol and THC-COOH are present in equal concentrations in plasma and concentrations peak at approximately 2-4 hours after dosing.
It is inadvisable to try and predict effects based on blood THC concentrations alone, and currently impossible to predict specific effects based on THC-COOH concentrations. It is possible for a person to be affected by marijuana use with concentrations of THC in their blood below the limit of detection of the method. Mathematical models have been developed to estimate the time of marijuana exposure within a 95% confidence interval. Knowing the elapsed time from marijuana exposure can then be used to predict impairment in concurrent cognitive and psychomotor effects based on data in the published literature.
Interpretation of Urine Test Results: Detection of total THC metabolites in urine, primarily THC-COOH-glucuronide, only indicates prior THC exposure. Detection time is well past the window of intoxication and impairment. Published excretion data from controlled clinical studies may provide a reference for evaluating urine cannabinoid concentrations; however, these data are generally reflective of occasional marijuana use rather than heavy, chronic marijuana exposure.
It can take as long as 4 hours for THC-COOH to appear in the urine at concentrations sufficient to trigger an immunoassay (at 50ng/mL) following smoking. Positive test results generally indicate use within 1-3 days; however, the detection window could be significantly longer following heavy, chronic, use.
Following single doses of Marinol®, low levels of dronabinol metabolites have been detected for more than 5 weeks in urine. Low concentrations of THC have also been measured in over-the-counter hemp oil products – consumption of these products may produce positive urine cannabinoid test results.
Effects: Pharmacological effects of marijuana vary with dose, route of administration, experience of user, vulnerability to psychoactive effects, and setting of use.
Psychological: At recreational doses, effects include relaxation, euphoria, relaxed inhibitions, sense of well-being, disorientation, altered time and space perception, lack of concentration, impaired learning and memory, alterations in thought formation and expression, drowsiness, sedation, mood changes such as panic reactions and paranoia, and a more vivid sense of taste, sight, smell, and hearing.
Stronger doses intensify reactions and may cause fluctuating emotions, flights of fragmentary thoughts with disturbed associations, a dulling of attention despite an illusion of heightened insight, image distortion, and psychosis.
Physiological: The most frequent effects include increased heart rate, reddening of the eyes, dry mouth and throat, increased appetite, and vasodilatation.
Side Effect Profile: Fatigue, paranoia, possible psychosis, memory problems, depersonalization, mood alterations, urinary retention, constipation, decreased motor coordination, lethargy, slurred speech, and dizziness. Impaired health including lung damage, behavioral changes, and reproductive, cardiovascular and immunological effects have been associated with regular marijuana use.
Regular and chronic marijuana smokers may have many of the same respiratory problems that tobacco smokers have (daily cough and phlegm, symptoms of chronic bronchitis), as the amount of tar inhaled and the level of carbon monoxide absorbed by marijuana smokers is 3 to 5 times greater than among tobacco smokers. Smoking marijuana while shooting up cocaine has the potential to cause severe increases in heart rate and blood pressure.
Duration of Effects: Effects from smoking cannabis products are felt within minutes and reach their peak in 10-30 minutes. Typical marijuana smokers experience a high that lasts approximately 2 hours. Most behavioral and physiological effects return to baseline levels within 3-5 hours after drug use, although some investigators have demonstrated residual effects in specific behaviors up to 24 hours, such as complex divided attention tasks. Psychomotor impairment can persist after the perceived high has dissipated.
In long term users, even after periods of abstinence, selective attention (ability to filter out irrelevant information) has been shown to be adversely affected with increasing duration of use, and speed of information processing has been shown to be impaired with increasing frequency of use. Dronabinol has an onset of 30-60 minutes, peak effects occur at 2-4 hours, and it can stimulate the appetite for up to 24 hours.
Tolerance, Dependence and Withdrawal Effect: Tolerance may develop to some pharmacological effects of dronabinol. Tolerance to many of the effects of marijuana may develop rapidly after only a few doses, but also disappears rapidly.
Marijuana is addicting as it causes compulsive drug craving, seeking, and use, even in the face of negative health and social consequences. Additionally, animal studies suggests marijuana causes physical dependence. A withdrawal syndrome is commonly seen in chronic marijuana users following abrupt discontinuation. Symptoms include restlessness, irritability, mild agitation, hyperactivity, insomnia, nausea, cramping, decreased appetite, sweating, and increased dreaming.
Drug Interactions: Cocaine and amphetamines may lead to increased hypertension, tachycardia and possible cardiotoxicity. Benzodiazepines, barbiturates, ethanol, opioids, antihistamines, muscle relaxants and other CNS depressants increase drowsiness and CNS depression. When taken concurrently with alcohol, marijuana is more likely to be a traffic safety risk factor than when consumed alone.
Performance Effects: The short term effects of marijuana use include problems with memory and learning, distorted perception, difficultly in thinking and problem-solving, and loss of coordination. Heavy users may have increased difficulty sustaining attention, shifting attention to meet the demands of changes in the environment, and in registering, processing and using information. In general, laboratory performance studies indicate that sensory functions are not highly impaired, but perceptual functions are significantly affected.
The ability to concentrate and maintain attention are decreased during marijuana use, and impairment of hand-eye coordination is dose-related over a wide range of dosages.Impairment in retention time and tracking, subjective sleepiness, distortion of time and distance, vigilance, and loss of coordination in divided attention tasks have been reported.
Note however, that subjects can often “pull themselves together” to concentrate on simple tasks for brief periods of time. Significant performance impairments are usually observed for at least 1-2 hours following marijuana use, and residual effects have been reported up to 24 hours.
Effects on Driving: The drug manufacturer suggests that patients receiving treatment with Marinol® should be specifically warned not to drive until it is established that they are able to tolerate the drug and perform such tasks safely.
Epidemiology data from road traffic arrests and fatalities indicate that after alcohol, marijuana is the most frequently detected psychoactive substance among driving populations. Marijuana has been shown to impair performance on driving simulator tasks and on open and closed driving courses for up to approximately 3 hours.
Decreased car handling performance, increased reaction times, impaired time and distance estimation, inability to maintain headway, lateral travel, subjective sleepiness, motor incoordination, and impaired sustained vigilance have all been reported.
Some drivers may actually be able to improve performance for brief periods by overcompensating for self-perceived impairment. The greater the demands placed on the driver, however, the more critical the likely impairment.
Marijuana may particularly impair monotonous and prolonged driving. Decision times to evaluate situations and determine appropriate responses increase. Mixing alcohol and marijuana may dramatically produce effects greater than either drug on its own.
DEC Category: Cannabis
DEC Profile: Horizontal gaze nystagmus not present; vertical gaze nystagmus not present; lack of convergence present; pupil size normal to dilated; reaction to light normal to slow; pulse rate elevated; blood pressure elevated; body temperature normal to elevated. Other characteristic indicators may include odor of marijuana in car or on subject’s breath, marijuana debris in mouth, green coating of tongue, bloodshot eyes, body and eyelid tremors, relaxed inhibitions, incomplete thought process, and poor performance on field sobriety tests.
Panel’s Assessment of Driving Risks: Low doses of THC moderately impair cognitive and psychomotor tasks associated with driving, while severe driving impairment is observed with high doses, chronic use and in combination with low doses of alcohol The more difficult and unpredictable the task, the more likely marijuana will impair performance.
References and Recommended Reading:
Aceto MD, Scates SM, Lowe JA, Martin BR. Cannabinoid precipitated withdrawal by the selective cannabinoid receptor antagonist, SR 141716A. Eur J Pharmacol 1995;282(1-3): R1-2.
Adams IB, Martin BR. Cannabis: pharmacology and toxicology in animals and humans. Addiction 1996;91(11):1585-614.
Barnett G, Chiang CW, Perez-Reyes M, Owens SM. Kinetic study of smoking marijuana. J Pharmacokinet Biopharm 1982;10(5):495-506.
Baselt RC. Drug effects on psychomotor performance. Biomedical Publications, Foster City, CA; pp 403-415;2001.
Hansteen RW, Miller RD, Lonero L, Reid LD, Jones B. Effects of cannabis and alcohol on automobile driving and psychomotor tracking. Ann NY Acad Sci 1976;282:240-56.
Heishman SJ. Effects of abused drugs on human performance: Laboratory assessment. In: Drug Abuse
Handbook. Karch SB, ed. New York, NY: CRC Press, 1998, p219.
Huestis MA. Cannabis (Marijuana) - Effects on Human Performance and Behavior. Forens Sci Rev 2002;14(1/2):15-60.
Huestis MA, Sampson AH, Holicky BJ, Henningfield JE, Cone EJ. Characterization of the absorption phase of marijuana smoking. Clin Pharmacol Ther 1992;52(1):31-41.
Huestis MA, Henningfield JE, Cone EJ. Blood cannabinoids: I. Absorption of THC and formation of 11-OH-THC and THC-COOH during and after marijuana smoking. J Anal Toxicol 1992;16(5):276-82.
Huestis MA, Henningfield JE, Cone EJ. Blood cannabinoids II: Models for the prediction of time of marijuana exposure from plasma concentrations of ∆-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-∆-9-tetrahydrocannabinol (THC-COOH). J Anal Toxicol 1992;16(5):283-90.
Hunt CA, Jones RT. Tolerance and disposition of tetrahydrocannabinol in man. J Pharmacol Exp Ther 1980;215(1):35-44.
Klonoff H. Marijuana and driving in real-life situations. Science 1974;186(4161);317-24.
Leirer VO, Yesavage JA, Morrow DG. Marijuana carry-over effects on aircraft pilot performance. Aviat Space Environ Med 1991;62(3):221-7.
Mason AP, McBay AJ. Cannabis: pharmacology and interpretation of effects. J Forensic Sci 1985;30(3):615-31.
Physicians’ Desk Reference, Medical Economics Company, Montvale, NJ, 2002.
Plasse TF, Gorter RW, Krasnow SH, Lane M, Shepard KV, Wadleigh RG. Recent clinical experience with Dronabinol. Pharmacol Biochem Behav 1991;40(3):695-700.
Pope HG Jr, Yurgelun-Todd D. The residual cognitive effects of heavy marijuana use in college students. JAMA 1996;275(7):521-7.
Ramaekers JG, Robbe HW, O’Hanlon JF. Marijuana, alcohol and actual driving performance. Hum Psychopharmacol 2000;15(7):551-8.
Robbe HW, O'Hanlon JF. Marijuana and actual driving performance. US Department of Transportation/National Highway Traffic Safety Administration November: 1-133 (1993). DOT HS 808 078.
Smiley A, Moskowitz HM, Ziedman K. Effects of drugs on driving: Driving simulator tests of secobarbital, diazepam, marijuana, and alcohol. In Clinical and Behavioral Pharmacology Research Report. J.M. Walsh, Ed. U.S. Department of Health and Human Services, Rockville, 1985, pp 1-21.
Solowij N, Michie PT, Fox AM. Differential impairment of selective attention due to frequency and duration of cannabis use. Biol Psychiatry 1995;37(10):731-9.
Thornicroft G. Cannabis and psychosis. Is there epidemiological evidence for an association? Br J Psychiatry 1990;157:25-33.
Varma VK, Malhotra AK, Dang R, Das K, Nehra R. Cannabis and cognitive functions: a prospective study. Drug Alcohol Depend 1988;21(2):147-52.
WHO Division of Mental Health and Prevention of Substance Abuse: Cannabis: a health perspective and research agenda. World Health Organization 1997.
A chemotaxonomic analysis of cannabinoid variation in Cannabis
(American Journal of Botany. 2004;91:966-975.)
© 2004 Botanical Society of America, Inc
Karl W. Hillig and Paul G. Mahlberg
Department of Biology, Indiana University, Bloomington, Indiana 47405 USA
Received for publication June 19, 2003. Accepted for publication February 12, 2004.
ABSTRACT
Cannabinoids are important chemotaxonomic markers unique to Cannabis. Previous studies show that a plant's dry-weight ratio of 9-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.
iNTRODUCTION... read more
The inheritance of chemical phenotype in Cannabis sativa
LDowload Pdf for easier reading
Copyright 2003 by the Genetics Society of America
The Inheritance of Chemical Phenotype in Cannabis sativa L.
Etienne P. M. de Meijer,*,1 Manuela Bagatta,† Andrea Carboni,† Paola Crucitti,†
V. M. Cristiana Moliterni,† Paolo Ranalli† and Giuseppe Mandolino†,2
*HortaPharm B.V., 1075 VS, Amsterdam, The Netherlands and †Istituto Sperimentale per le Colture Industriali, 40128 Bologna, Italy
Manuscript received April 23, 2002
Accepted for publication October 16, 2002
ABSTRACT
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure -9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F1’s were analyzed by gas chromatography for cannabinoid composition and constantly found to have amixed CBD-THC chemotype.
Ten individual F1 plants were self-fertilized, and 10 inbred F2 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 F1 to the F2’s derived from it. Dowload Complete Pdf for easier reading
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Medical Cannabis Potency Testing Project
From the Bulletin of the Multidisciplinary Association for Psychedelic Studies
MAPS - Volume 9 Number 3 Autumn 1999 - pp. 20-22Dale Gieringer, Ph.D.
California NORML, 2215-R Market St. Suite 278
San Francisco CA 94114 tel: (415) 563-5858
E-mail: canorml@igc.apc.org
Given the rapidly growing use of medical cannabis for a wide variety of indications and the manifold different underground sources currently supplying patients, there is a natural interest in investigating the potency, purity, and chemical content of the available supplies of medical cannabis. While the availability of medical cannabis has increased in the wake of the passage of California's Proposition 215 and other state medical marijuana initiatives, scientific research on its content remains frustrated by the continued federal ban on medical cannabis research.
In an effort to cast light in this obscure area, a research project was undertaken by a group of us, including researchers, growers, and medical cannabis buyers' clubs, with support from California NORML and MAPS, to analyze samples of medical cannabis from various patients' cooperatives and providers around the country. This effort proved to be a lesson in the difficulties and uncertainties of cannabis research in a society where freedom of pharmacological research has been stifled by an effectively totalitarian drug bureaucracy.
From the outset, our project was frustrated by a lack of access to qualified research labs with expertise in analysis of cannabis. The leading research lab in the country declined to do business with us for fear of compromising government contracts, while the other likely candidates were all foreign and thus not legally accessible to us because of DEA regulations. In the end, we were fortunate to obtain the services of a laboratory that had the requisite DEA license and equipment (a gas chromatograph mass spectrometer, or GCMS), but no prior experience in cannabis analysisin fact, its primary business was drug urinalysis! The analysis of our samples was accordingly a learning process for both the lab and ourselves.
Our original aim had been to obtain a broad-spectrum quantitative analysis of as many of the 60-plus naturally occurring cannabinoids as possible, in the hope of detecting differences that might produce differing therapeutic effects among the samples. To our disappointment, however, our lab could obtain laboratory standards only for the three most common cannabinoids, delta-9-THC, cannabidiol (CBD), and cannabinol (CBN).
A total of 47 different samples of medical cannabis were submitted by over a half dozen different providers and patients' cooperatives ranging from California to the East Coast. Included were 42 samples of sinsemilla bud, three samples of hashish or resin; one liquid sample of a milk-based cannabis drink ("Mother's Milk"), and one capsule of an oral whole leaf preparation.
Upon analysis by GCMS, the potency of the 42 sinsemilla samples was determined to range from 10.2% to 31.6% THC, with a mean of 19.4%. These results were surprisingly high, given that the average potency of marijuana in the U.S. has been typically estimated at around 3% to 4% by NIDA, with higher grade sinsemilla ranging towards 10% - 15%. The highest potency recorded came from a sample of hashish, which registered 68.6%. Yet even a sample of Mexican commercial grade registered a surprisingly high 11%, twice what we had expected. All of this cast a troubling shadow of doubt on our test results, although it appeared likely that we were dealing with highly potent varieties.
In contrast, the CBD levels observed were surprisingly low. Only four of the sinsemilla samples had more than 0.3% CBD, and 35 of them had only trace amounts (<0.1%). However, one sample had an astoundingly high CBD content of 28.0% (plus 11.6% THC).
Another registered 5.6% CBD and 13.4% THC. Aside from these two anomalies, the CBD results were frankly disappointing, as we had hoped to discover significant variations in the content of the samples, with accompanying variations in medical activity.
Because CBD is suspected to have peculiar efficacy for control of muscle spasms and for damping anxiety and "panic reactions" caused by THC, we had hypothesized that certain patients would tend to prefer high-CBD varieties. In fact, however, it appears that few patients are ever exposed to high-CBD cannabis. Unfortunately, we were unable to procure additional specimens of the high-CBD varieties for further testing.
As for CBN, the majority of samples showed only trace amounts. The highest level detected was 1.4%, and only one other sample tested above 1%. CBN is a breakdown product of THC, so high CBN levels are expected in old, degraded samples.
This was confirmed by the fact that one of the samples above 1% CBN was known to be a year old. The prevalence of low CBN in the samples was evidence that most available medical cannabis tends to be fresh and well-preserved. Otherwise, these results were of limited interest, as there are few if any known medical effects of CBN.
Another disappointing surprise was the failure to detect more than trace levels of THC or CBD in the liquid "Mother's Milk" sample. Upon further investigation, the lab determined that this was because it is impossible to extract cannabinoids from fat-based liquids using standard methanol extraction techniques. Consulting with other researchers, we found that there is no known method for isolating THC from fat-based liquids.
Later, we located a lab that claimed to have developed a secret, proprietary method for extracting cannabinoids from fat. With considerable difficulty, we arranged to have the lab test the Mother's Milk. To our disappointment, however, once again only trace amounts of THC and CBD were detected.
Just to make sure, one of us swallowed a sample of the Mother's Milk (which by now had spent several months in the freezer) and found it to be delightfully potent. Evidently, the lab's technique had failed. It appears that further advances in testing technology will be needed in order to properly analyze fat-based oral cannabis products such as Mother's milk, bhang, ghee, and possibly baked goods such as brownies.
Table: THC and CBD Test Results
(Round 1 vs. Rounds 2 and 3)
1st Round 2nd Round 3rd Round
(New Lab)
(New Lab)
|
Name of Sample |
1st Round
|
2nd Round
|
3rd Round (New Lab)
|
|
High CBD
Sinsemilla BB 006 Sinsemilla BB 008 Sinsemilla MR 001 Sinsemilla BB 009 Sinsemilla SCJ Sinsemilla BB 007 Sinsemilla Tri 501 Sinsemilla BB 010 Sinsemilla BB 004 Sinsemilla AQ Hasish Mother's Milk NIDA Leaf Low Grade Leaf |
11.6%
25.2 27.4 18.0 10.2 14.2 21.1 27.2 18.0 18.6 23.7 68.6 <1 |
28.0%
<1 <1 <1 1.3 <1 <1 <1 0.3 <1 <1 0.1 <1 |
4.0%
18.2 35.1 11.7 7.6 14.1 3.9 2.1 |
16.2%
<1 <1 <1 2.8 <1 <1 <1 |
2.8%
14.9 21.0 12.8 20.0 8.7 13 17.6 44.0 <1 |
8.8%
<1 0.07 <1 <1 <1 <1 <1 <1 |
The extraordinarily high THC potency in the sinsemilla samples raised troubling doubts about the reliability of the test results. The lab director expressed concern about the sample preparation, saying that he had noted a tendency for the oils to separate from the rest of the liquid during extraction. We therefore decided to re-submit some of the samples for a second round of testing. We selected six samples, including the one with anomalously high CBD. As a check, we added two new samples with presumably low potency: a sample of low-grade leaf, and some of the government's own marijuana, grown for NIDA, whose potency is known to be in the 2.9 - 3.9 % range.
In the second round of testing, the average THC potency for the seven samples declined slightly to 15.1% from 17.8% in the first round. For the six low-CBD samples, second-round potencies varied between 65% and 128% of their first-round values (see table). The high CBD sample registered a precipitous decline of 60 - 65% in both THC and CBD, bolstering suspicions of some kind of irregularity in the sample. NIDA's marijuana came in at 3.9%, at the high end of its expected range, and the low-grade shake came in at 2%. One sinsemilla sample registered a record 35% on re-testing.
The second round of testing failed to dispel our uncertainty about the results. Overall, the trend of the data seemed to confirm our suspicions that the first round results had been systematically too high. However, the wide variation in individual test results between the two rounds undermined confidence in any firm conclusions. While it seemed reasonable to infer that we were dealing with some genuinely potent cannabis, the high-range results for NIDA's pot suggested that the second round might still be too high.
After some months of head-scratching, we stumbled upon the opportunity to re-check our test results via a circuitous route to a second lab. This lab, recognized for its expertise in cannabis potency testing, was the same one that tested the Mother's Milk. In addition to the Mother's Milk, we submitted seven sinsemilla samples, the high-CBD sample, and the high-potency hashish. The potencies were uniformly lower in the third round than the first, by proportions ranging from 25 - 50%. All of this clearly implied that our first round test results had been systematically on the high side. Still, the average potency of the seven sinsemilla samples was an impressive 15.4%, four or five times greater than NIDA's marijuana.
From this, we can safely conclude that the marijuana currently being provided by underground cannabis clubs is far superior in quality to that currently provided by NIDA to the eight legal medical marijuana patients. Due to its higher THC content, patients need consume only a fraction of the harmful, non-medically-active tars and gases in cannabis smoke in order to achieve the same effective dose.
This is of course especially significant in light of the recent Institute of Medicine report, which singled out smoking as the major adverse health hazard of medical marijuana. Aside from THC, we could find no significant presence of the other tested cannabinoids, CBN and CBD, except in one or two anomalous samples.
There is thus little evidence that patients are currently making use of differing varieties of cannabis to treat different medical conditions, although it is possible that other, untested cannabinoids remain lurking in the background. Finally, our experience shows that laboratory measurements of cannabinoid content can vary widely from test to test and lab to lab, and are entirely undependable in the case of fat-based cannabis liquids.
LAST PARAGRAPH REVISED 12/2/02
Later, we located a lab that claimed to have developed a proprietary method for extracting cannabinoids from fat. With considerable difficulty, we arranged to have the lab test the Mother's Milk. At first, we were disappointed when it reported what seemed to be negligible traces of THC, only 50 parts per million (0.0050%) by weight. On further consideration, however, we realized that this was a reasonable concentration for a comestible product, since the weight of a one cup serving of milk (about 250 grams) was much greater than that of one cigarette (<1 gram). This works out to 12.5 milligrams of THC per cup, equivalent to two and a half standard 5 mg Marinol capsules.
Compounds found in Cannabis Sativa(-)-[delta 1]-3,4-trans-tetrahydrocannabinol (most active cannabinoid)
(-)-[delta 6]-3,4-trans-tetrahydrocannabinol
tetrahydrocannabitriol (aka cannabitriol)
cannabidiolic acid
cannabidiol
cannabinol (forms after plant dies)
THC acids A and B (inactive unless smoked)
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Minor constituents:
cannabigerol
cannabigerolic acid
cannabichromene
cannabichromenic acid
cannabicyclol (aka cannabipinol)
cannabicyclolic acid
cannabicitran
cannabielsoic acids A and B
cannabinolic acid (neutral cannabinoid)
cannabichromanon
cannabifuran
dehydrocannabifuran
2-oxo-[delta 3]-tetrahydrocannabinol
cannabigerol monomethyl ether
cannabidiol monomethyl ether
cannabinol methyl ether
propylcannabidiol (aka cannabidivarol & cannabidivarin)
propylcannabinol (aka cannabivarol & cannabivarin)
propyl-[delta 1]-THC (aka [delta 1]-tetrahydrocannabivarol & tetrahydrocannabivarin)
propylcannabigerol
propylcannabicyclol
propylcannabichromene
methylcannabidiol (aka cannabidiorcol)
methylcannabinol (aka cannabiorcol)
methyl-[delta 1]-THC (aka [delta 1]-tetrahydrocannabiorcol)
[delta 1]-tetrahydrocannabivarolic acid
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Nitrogen-containing compounds:
choline
trigonelline
muscarine
piperidine
N-(p-hydroxy-B-phenylethyl)-p-hydroxy-trans-cinnamide
neurine
L-proline
L-isoleucine betaine
hordenine
cannabisativine (alkaloid found in the roots)
[compiled from "The Botany and Chemistry of Hallucinogens" by Schultes & Hofmann]
Hemp Seed Oil : The Wonder Oil For the New Millennium
In the second round of testing, the average THC potency for the seven samples declined slightly to 15.1% from 17.8% in the first round. For the six low-CBD samples, second-round potencies varied between 65% and 128% of their first-round values (see table). The high CBD sample registered a precipitous decline of 60 - 65% in both THC and CBD, bolstering suspicions of some kind of irregularity in the sample. NIDA's marijuana came in at 3.9%, at the high end of its expected range, and the low-grade shake came in at 2%. One sinsemilla sample registered a record 35% on re-testing.
The second round of testing failed to dispel our uncertainty about the results. Overall, the trend of the data seemed to confirm our suspicions that the first round results had been systematically too high. However, the wide variation in individual test results between the two rounds undermined confidence in any firm conclusions. While it seemed reasonable to infer that we were dealing with some genuinely potent cannabis, the high-range results for NIDA's pot suggested that the second round might still be too high.
After some months of head-scratching, we stumbled upon the opportunity to re-check our test results via a circuitous route to a second lab. This lab, recognized for its expertise in cannabis potency testing, was the same one that tested the Mother's Milk. In addition to the Mother's Milk, we submitted seven sinsemilla samples, the high-CBD sample, and the high-potency hashish. The potencies were uniformly lower in the third round than the first, by proportions ranging from 25 - 50%. All of this clearly implied that our first round test results had been systematically on the high side. Still, the average potency of the seven sinsemilla samples was an impressive 15.4%, four or five times greater than NIDA's marijuana.
From this, we can safely conclude that the marijuana currently being provided by underground cannabis clubs is far superior in quality to that currently provided by NIDA to the eight legal medical marijuana patients. Due to its higher THC content, patients need consume only a fraction of the harmful, non-medically-active tars and gases in cannabis smoke in order to achieve the same effective dose.
This is of course especially significant in light of the recent Institute of Medicine report, which singled out smoking as the major adverse health hazard of medical marijuana. Aside from THC, we could find no significant presence of the other tested cannabinoids, CBN and CBD, except in one or two anomalous samples.
There is thus little evidence that patients are currently making use of differing varieties of cannabis to treat different medical conditions, although it is possible that other, untested cannabinoids remain lurking in the background. Finally, our experience shows that laboratory measurements of cannabinoid content can vary widely from test to test and lab to lab, and are entirely undependable in the case of fat-based cannabis liquids.
LAST PARAGRAPH REVISED 12/2/02
Later, we located a lab that claimed to have developed a proprietary method for extracting cannabinoids from fat. With considerable difficulty, we arranged to have the lab test the Mother's Milk. At first, we were disappointed when it reported what seemed to be negligible traces of THC, only 50 parts per million (0.0050%) by weight. On further consideration, however, we realized that this was a reasonable concentration for a comestible product, since the weight of a one cup serving of milk (about 250 grams) was much greater than that of one cigarette (<1 gram). This works out to 12.5 milligrams of THC per cup, equivalent to two and a half standard 5 mg Marinol capsules.
Compounds found in Cannabis Sativa(-)-[delta 1]-3,4-trans-tetrahydrocannabinol (most active cannabinoid)
(-)-[delta 6]-3,4-trans-tetrahydrocannabinol
tetrahydrocannabitriol (aka cannabitriol)
cannabidiolic acid
cannabidiol
cannabinol (forms after plant dies)
THC acids A and B (inactive unless smoked)
--------------------------------------------------------------------------------
Minor constituents:
cannabigerol
cannabigerolic acid
cannabichromene
cannabichromenic acid
cannabicyclol (aka cannabipinol)
cannabicyclolic acid
cannabicitran
cannabielsoic acids A and B
cannabinolic acid (neutral cannabinoid)
cannabichromanon
cannabifuran
dehydrocannabifuran
2-oxo-[delta 3]-tetrahydrocannabinol
cannabigerol monomethyl ether
cannabidiol monomethyl ether
cannabinol methyl ether
propylcannabidiol (aka cannabidivarol & cannabidivarin)
propylcannabinol (aka cannabivarol & cannabivarin)
propyl-[delta 1]-THC (aka [delta 1]-tetrahydrocannabivarol & tetrahydrocannabivarin)
propylcannabigerol
propylcannabicyclol
propylcannabichromene
methylcannabidiol (aka cannabidiorcol)
methylcannabinol (aka cannabiorcol)
methyl-[delta 1]-THC (aka [delta 1]-tetrahydrocannabiorcol)
[delta 1]-tetrahydrocannabivarolic acid
--------------------------------------------------------------------------------
Nitrogen-containing compounds:
choline
trigonelline
muscarine
piperidine
N-(p-hydroxy-B-phenylethyl)-p-hydroxy-trans-cinnamide
neurine
L-proline
L-isoleucine betaine
hordenine
cannabisativine (alkaloid found in the roots)
[compiled from "The Botany and Chemistry of Hallucinogens" by Schultes & Hofmann]
Hemp Seed Oil : The Wonder Oil For the New Millennium
By Kristin Speiser, Michael Pobeda and Laurent Sousselier, Happi, June 1999, ppg. 106-109
Summary
This perfectly balanced oil has an impressive list of proven benefits to the consumer. The product’s ideal balance as a cosmetic oil and as a fashionable ingredient meets the demands of the millennium’s market.
What’s old is new again. Hemp seed oil has been used for centuries for its medicinal and nutritional properties. Now the cosmetics industry is rediscovering this wonder oil. Not only is hemp seed oil new on the cosmetics scene, but it is a trendy product. Today’s emphasis on environmentally-sound products calls for a multipurpose ingredient such as hemp seed oil. It is a perfectly balanced oil with an impressive list of proven benefits to the consumer. The product’s ideal balance as a cosmetic oil and as a fashionable ingredient meets the demands of the millennium’s market.
Across the globe, hemp products are renowned for their versatility. This popular material is used in clothing, accessories, home furnishings and even automobiles. Hemp is no longer confused as a “cannabis” product but is relished for its own reputation.
Four thousand years ago, China’s Emperor Sheng Nung used hemp for rheumatism and constipation treatments. Buddha supposedly ate one hemp seed per day while fasting. Romans used hemp fibers in their ropes and sails. Gutenberg’s Bible, the American Constitution and the Declaration of Independence were all printed on hemp paper.
France’s Nîmes weavers used hemp in manufacturing the first denim (De Nîmes). Since hemp made up the very first jeans, contemporary fashion has turned to hemp fiber. Hemp is not a trend that any industry can afford to miss. Armani, Calvin Klein and Ralph Lauren all use hemp in their fashion lines. “I believe that hemp is going to be the fiber of choice for the millennium,” said Calvin Klein.
Summary
This perfectly balanced oil has an impressive list of proven benefits to the consumer. The product’s ideal balance as a cosmetic oil and as a fashionable ingredient meets the demands of the millennium’s market.
What’s old is new again. Hemp seed oil has been used for centuries for its medicinal and nutritional properties. Now the cosmetics industry is rediscovering this wonder oil. Not only is hemp seed oil new on the cosmetics scene, but it is a trendy product. Today’s emphasis on environmentally-sound products calls for a multipurpose ingredient such as hemp seed oil. It is a perfectly balanced oil with an impressive list of proven benefits to the consumer. The product’s ideal balance as a cosmetic oil and as a fashionable ingredient meets the demands of the millennium’s market.
Across the globe, hemp products are renowned for their versatility. This popular material is used in clothing, accessories, home furnishings and even automobiles. Hemp is no longer confused as a “cannabis” product but is relished for its own reputation.
Four thousand years ago, China’s Emperor Sheng Nung used hemp for rheumatism and constipation treatments. Buddha supposedly ate one hemp seed per day while fasting. Romans used hemp fibers in their ropes and sails. Gutenberg’s Bible, the American Constitution and the Declaration of Independence were all printed on hemp paper.
France’s Nîmes weavers used hemp in manufacturing the first denim (De Nîmes). Since hemp made up the very first jeans, contemporary fashion has turned to hemp fiber. Hemp is not a trend that any industry can afford to miss. Armani, Calvin Klein and Ralph Lauren all use hemp in their fashion lines. “I believe that hemp is going to be the fiber of choice for the millennium,” said Calvin Klein.
This “choice” plant is actually a tall weed that grows worldwide. It has many applications, aside from its excellent use in hemp seed oil form. The plant itself grows rapidly (four times faster than trees). Hemp has been highlighted lately for its environmental soundness.
A renewable biomass, hemp is grown without fertilizer or pesticides. In fact, the plant is a fertilizer itself. Therefore, without involving costly and potentially environmentally-damaging chemicals, hemp is a hardy, cost-efficient botanical that grows without damaging either the wallet or the environment.
A renewable biomass, hemp is grown without fertilizer or pesticides. In fact, the plant is a fertilizer itself. Therefore, without involving costly and potentially environmentally-damaging chemicals, hemp is a hardy, cost-efficient botanical that grows without damaging either the wallet or the environment.
It's no wonder that hemp is so widely used these days. Not only is the fiber used in paper, textiles and other products, but its hardiness makes it ideal for the building industry. Hemp is also edible and may even be found in modern food products; the nutritious oil helps reduce LDL cholesterol content.
Clearly, hemp has many beneficial uses but its full potential is realized in the form of hemp seed oil. The oil is edible, pleasing to the touch and perfectly balanced. cosmetic industry leaders recognize the desirability of high essential fatty acid contents. Hemp seed oil contains one of the highest levels of essential fatty acids: 76%.
Essential Fatty Acids and the Skin
EFAs (essential fatty acids) are very important in cell membranes. The more saturated the fatty acid, the less fluid the membrane. PUFA (poly-unsaturated fatty acids) are incorporated in the 2 position of the phospholipids constituting cell membrane. Afluid membrane is crucial for proper cell function. EFAs and their importance to the skin have been the subject of many studies.
Horrobin (J. Am. Acad. Dermatol. 1989 20 1045-1053) and later Wright (Br. J. Dermatol. 1991 125 503-515) have reviewed Essential Fatty Acid Deficiency (EFAD) consequences on the skin. They found that EFAD can lead to:
Nutgeren, et. al. (Biochim. Biophis. Acta. 1985 834 429-436) proved that EFAs are absolutely necessary for maintaining the proper skin condition of water barrier in the skin. Direct topical application on linoleic acid (LA) to the skin restores the barrier in animals with EFAD. It as been shown that radiolabeled LA is incorporated mostly in an acyl ceramide (ceramide 1) in which LA was esterified to the end position of a very long chain unsaturated omega fatty acid. In EFAD, LA is replaced by oleic acid in the ceramide, which is unable to form a normal water barrier.
PUFA supplementation influences the rate of biosynthesis of EFA derivatives as it seems to depend on the size of the precursors pool. Supplementing gamma linoleic acid (GLA) results in an increase of the less inflammatory PGE2. Similarly long chain omega-3 acids supplementation induces a marked reduction in LA and arachidonic acid (AA) in membrane lipids and also result in local generation of the less inflammatory PGE3.
Also, dihomo gamma linoleic acid (DGLA) is converted in the skin to PGE1, which is known to raise the levels of cAMP which in turn inhibits PLA2 (what’s PLA2) and so exerts anti-inflammatory effects by keeping AA locked into the phospholipidic membrane. Thus access of free AA to cyclo-oxygenase is denied and pro-inflammatory PG2 level is reduced. This implies the necessity of a well balanced mix of PUFA in the diet and in topical application.
The Right Prostaglandins are Extremely Important
Larregue (Prostaglandines et thromboxanes Masson 1997) reviewed the importance of prostaglandin (PG) in skin. PGs are not stored but are synthesized on request after being stimulated. PG2 are synthesized from AA present in cell membranes.
PG2 is a powerful vasodilator and contributes to the characteristic edema related to inflammation. It must be noted that PG1 and PG3 are less pro-inflammatory. PGs are also immune modulators: PGE2 is a powerful inhibitor of cytotoxic T cells activity. In situ PG production happens simultaneously with UV erythema. Therefore omega-3 PUFA, by helping prevent PG2, has a photo-protective effect on skin.
Clearly, hemp has many beneficial uses but its full potential is realized in the form of hemp seed oil. The oil is edible, pleasing to the touch and perfectly balanced. cosmetic industry leaders recognize the desirability of high essential fatty acid contents. Hemp seed oil contains one of the highest levels of essential fatty acids: 76%.
Essential Fatty Acids and the Skin
EFAs (essential fatty acids) are very important in cell membranes. The more saturated the fatty acid, the less fluid the membrane. PUFA (poly-unsaturated fatty acids) are incorporated in the 2 position of the phospholipids constituting cell membrane. Afluid membrane is crucial for proper cell function. EFAs and their importance to the skin have been the subject of many studies.
Horrobin (J. Am. Acad. Dermatol. 1989 20 1045-1053) and later Wright (Br. J. Dermatol. 1991 125 503-515) have reviewed Essential Fatty Acid Deficiency (EFAD) consequences on the skin. They found that EFAD can lead to:
- Scaly epidermis;
- Hypertrophy of the sebaceous glands and hyperkeratosis of sebaceous ducts;
- Weakened cutaneous capillaries;
- Increased transepidermal water loss (TEWL) and
- Thin, discolored hair, or hair loss
Nutgeren, et. al. (Biochim. Biophis. Acta. 1985 834 429-436) proved that EFAs are absolutely necessary for maintaining the proper skin condition of water barrier in the skin. Direct topical application on linoleic acid (LA) to the skin restores the barrier in animals with EFAD. It as been shown that radiolabeled LA is incorporated mostly in an acyl ceramide (ceramide 1) in which LA was esterified to the end position of a very long chain unsaturated omega fatty acid. In EFAD, LA is replaced by oleic acid in the ceramide, which is unable to form a normal water barrier.
PUFA supplementation influences the rate of biosynthesis of EFA derivatives as it seems to depend on the size of the precursors pool. Supplementing gamma linoleic acid (GLA) results in an increase of the less inflammatory PGE2. Similarly long chain omega-3 acids supplementation induces a marked reduction in LA and arachidonic acid (AA) in membrane lipids and also result in local generation of the less inflammatory PGE3.
Also, dihomo gamma linoleic acid (DGLA) is converted in the skin to PGE1, which is known to raise the levels of cAMP which in turn inhibits PLA2 (what’s PLA2) and so exerts anti-inflammatory effects by keeping AA locked into the phospholipidic membrane. Thus access of free AA to cyclo-oxygenase is denied and pro-inflammatory PG2 level is reduced. This implies the necessity of a well balanced mix of PUFA in the diet and in topical application.
The Right Prostaglandins are Extremely Important
Larregue (Prostaglandines et thromboxanes Masson 1997) reviewed the importance of prostaglandin (PG) in skin. PGs are not stored but are synthesized on request after being stimulated. PG2 are synthesized from AA present in cell membranes.
PG2 is a powerful vasodilator and contributes to the characteristic edema related to inflammation. It must be noted that PG1 and PG3 are less pro-inflammatory. PGs are also immune modulators: PGE2 is a powerful inhibitor of cytotoxic T cells activity. In situ PG production happens simultaneously with UV erythema. Therefore omega-3 PUFA, by helping prevent PG2, has a photo-protective effect on skin.
Marshall, et. al. (Progr Lipid Res 1981 20 7312-734) demonstrate that nutritional balance between omega-3 and omega-6 EFA affects prostaglandin synthesis in the immune system improving certain skin inflammatory pathologies. This is due to the competitive inhibition of cyclo-oxygenase which does not release as much pro-inflammatory AA derived PG2, favoring the less active PG3. High LNA levels in the diet led to a decreased capacity for cyclo-oxygenase produced PGE syntheses in the thymus and spleen due to the preference of desaturase and elongase enzymes for the omega-3 EFA. This causes a larger decrease in AA than may be expected on the basis of dietary LA/LNA ratio.
Finally, Ziboh (Arch. Dermatol. 1989 125 241-245) has studied the accumulation in psoriasis lesions of leukotriene B4, the major pro-inflammatory metabolite of AA. He proved that GLA and EPA present in fish oil are potent inhibitors of leukotriene B4 generation. They seem to work by competitive inhibition of 5 lipoxygenase.
PUFA Metabolism in the Skin
The enzymes involved in PUFA metabolism are crucial. Unfortunately, the key enzyme, Æ6 desaturase enzymes and cannot convert LA to GLA nor DGLA to AA, but it can convert GLA to DGLA. The epidermis is therefore dependent on the continual formation of GLA and AA by the liver and on the transport to the skin by the blood.
Kassis et. al. (Arch. Dermatol. Res. 1983 275 9-13) proved that a person’s capacity to convert LA to GLA decreases with age, as do the levels of PGE1. Æ6 desaturase is inhibited by many exogenous factors such as diet, stress and aging. Therefore, a GLA deficit leads to: a lack of PG1, an off-balance PG1/PG2 ratio and various cutaneous problems related to aging, such as skin dryness, itching, erythema and skin thinning. A well-balanced oil has to be supplemented to counter this consequence of aging by circumventing the key Æ6 desaturase stage.
Finally, Ziboh (Arch. Dermatol. 1989 125 241-245) has studied the accumulation in psoriasis lesions of leukotriene B4, the major pro-inflammatory metabolite of AA. He proved that GLA and EPA present in fish oil are potent inhibitors of leukotriene B4 generation. They seem to work by competitive inhibition of 5 lipoxygenase.
PUFA Metabolism in the Skin
The enzymes involved in PUFA metabolism are crucial. Unfortunately, the key enzyme, Æ6 desaturase enzymes and cannot convert LA to GLA nor DGLA to AA, but it can convert GLA to DGLA. The epidermis is therefore dependent on the continual formation of GLA and AA by the liver and on the transport to the skin by the blood.
Kassis et. al. (Arch. Dermatol. Res. 1983 275 9-13) proved that a person’s capacity to convert LA to GLA decreases with age, as do the levels of PGE1. Æ6 desaturase is inhibited by many exogenous factors such as diet, stress and aging. Therefore, a GLA deficit leads to: a lack of PG1, an off-balance PG1/PG2 ratio and various cutaneous problems related to aging, such as skin dryness, itching, erythema and skin thinning. A well-balanced oil has to be supplemented to counter this consequence of aging by circumventing the key Æ6 desaturase stage.
Topical application studies proved that PUFA or preferably PUFA-rich vegetable oils (released by the skin esterase) are beneficial to the skin. Prottey et. al. (J. Invest. Dermatol. 1975 64 228-234) demonstrated that, after cutaneous application of sunflower seed oil, which is rich in LA, to the right forearm of EFAD volunteers for two weeks, the level of LA in their epidermal lipids was markedly increased, the rate of TEWL was significantly lowered and the scaly lesions had disappeared. No such changes were seen in the volunteers’ left forearms after cutaneous application of olive oil (containing nearly no LA (Chart 2).
Proksch et. al. (Br. J. Dermatol. 1993 128 473-482) demonstrated that disrupting the barrier function by topical acetone treatment results in an increase of free fatty acids, sphingolipids and cholesterol in the living layer of the epidermis, leading to barrier repair. DNA synthesis is also stimulated the same way as by occlusion. This is a possible second mechanism by which the epidermis repairs its barrier function of omega-6 PUFA limits DNA synthesis and helps restore the barrier function.
Proksch et. al. (Br. J. Dermatol. 1993 128 473-482) demonstrated that disrupting the barrier function by topical acetone treatment results in an increase of free fatty acids, sphingolipids and cholesterol in the living layer of the epidermis, leading to barrier repair. DNA synthesis is also stimulated the same way as by occlusion. This is a possible second mechanism by which the epidermis repairs its barrier function of omega-6 PUFA limits DNA synthesis and helps restore the barrier function.
Coupland (Active Ingredient Conference Paris 1997 195-201) described how damaged or inflamed skin can be treated with oils containing GLA and SDA due to a reduction in inflammatory metabolites: PG. Photo-damaged skin may also benefit from these natural oils by inhibiting the secretion of TNF∝. Morganti et. al. (J. Appl. Cosm. 1985 3 211-222) showed that EFA application improves skin’s hydration capacity and protects aged skin against environmental insults. A cream containing 3% EFA prevents much better skin atrophy induced by a cortisone like compound which accelerates the skin’s aging process (Chart 3).
All these data point out the great benefits of topical PUFA supplementation with the right balance of PUFA for helping:
All these data point out the great benefits of topical PUFA supplementation with the right balance of PUFA for helping:
- correct the consequences of dry skin (more by structural change than by occlusivity);
- contribute to skin aging prevention and
- provide relief for skin inflammatory condition.
Hemp seed oil’s unique composition makes it the optimal active ingredient choice. It possess one of the highest PUFA contents but also has a perfect balance, providing the four essential fatty acids beneficial to the skin: LA, GLA, LNA, and SDA. Table II provides a comparison among the fatty acid profiles between many popular cosmetic oils.
No other oil provides the necessary EFAs with the right balance. Although any PUFA-containing oil is good, an oil such as hemp seed oil (with the right biological ratio between omega-3/omega-6) provides all the benefits. Hemp seeds oil’s fatty acid profile as well as some of the other valuable constitutes is illustrated in the pie chart above (Chart 4).
Hemp seed oil is pressed from a safe vegetable , hemp, which is a fiber-type weed of the Cannabis sativa species. The plant has dark green leaves and grows worldwide. Cannabis sativa can be separated into two categories:
So even if hemp seed oil is described by its INCI name (Cannabis sativa seed oil) it contains only traces of THC (less than 10 ppm for selected oils) and is perfectly safe for nutritional and cosmetic use.
Dr. U. Erasmus’ book: Fats that Heal, Fats that Kill, (1993, Alive Books Canada), praises hemp seed oil for its nutritional benefits. Hemp seed oil helps:
Hemp Seed Oil in Cosmetics
In addition to its outstanding composition, hemp seed oil’s unique texture imparts excellent skin feel. It is non-greasy, has high fluidity and lubricity and is absorbed quickly and efficiently in the skin. In fact, hemp seed oil is considered the “driest” vegetable oil.
Hemp seed oil's unique texture and activity on the skin (including the scalp) targets it toward many beneficial uses in cosmetic products. It is recommended in skin care formulas (up to 10%) that protect or provide anti-aging benefits, as well as dry-, mature- and sensitive skin products. It can be used at 3% in hand, foot or body creams. It can be used at 10% levels in after-sun products as well as lipsticks, lip balms and nail treatments. Hemp seed oil can also be used (3%) in cosmetic powders, liquid makeup and glossy hair conditioners that strengthen or prevent splitting and thinning. It is recommended for use (up to 10% for atopic eczema, acne and psoriasis treatment) and may be used at full strength for aromatherapy purposes and in body and massage oils.
Hemp seed oil is an excellent active ingredient in all of the above cosmetic applications. Structuring for maximum profit in the millennium means including the versatile, cost-efficient and trend-setting ingredients that today’s market demands. Hemp seed oil is the right choice. Not only is it fashionable, but it is the natural solution to the industry’s need for a rich oil that tests boundaries. Hemp seed oil is defined by unique properties that indulge the consumer in countless benefits. When used as an active ingredient, hemp seed oil follows a trend that you can bank on.
About the Authors
Kristin Speiser is a freelance public relations specialist from New York, NY. Michael Pobeda is a general manager of Teco Finance Export, a French manufacturer specialized in unique oils and butters. Laurent Sousselier is director of EX.A International - France and may be reached at + 33.1.4287.9698 or by e-mail at exacosm@club-internet.fr.
Composition of the essential oils and extracts of two populations of Cannabis sativa L. ssp. spontanea from Austria
Journal of Essential Oil Research: JEOR, May/Jun 2003Novak, Johannes, Franz, Chlodwig Abstract
The essential oil and the solvent extract of two populations of Cannabis sativa L. ssp. spontanea growing wild in Austria were analyzed comparatively. In the essential oil, myrcene (31% and 27%, respectively), (E)-beta-ocilnene (13% and 3%, respectively) and beta-caryophyllene (11 % and 16%, respectively) were found, while in the solvent extract the non-hallucinogeneous cannabidiol (77% and 59%, respectively) dominated. The hallucinogeneous delta-9-tetrahydrocannabinol (THC) was also found in the solvent extract at a level of less than 1%.
Key Word Index
Cannabissativa ssp. spontanea, Cannabaceae, essential oil composition, myrcene, (E)-beta-ocii-nene, beta-caryophyllene, cannabinoids
The Plant
In Cannabis sativa L. ssp. spontanea (formerly Cannabis ruderalis) (Cannabaceae) the perianth of the female flowers is in contrast to C. sativa ssp. sativa still present; the fruit is brownish and has a peduncle-like ringbulge. It is a ruderal, but a rare plant in the east of Austria (1).
Source
Two populations of C. sativa L. ssp. spontanea ("Albrechtsfeld" and "Schoschtolacke") from the region of lake Neusiedl, Burgenland, eastern Austria were sampled in June, 1998, at the beginning of seed ripening. At each population upper parts of approximately 10 plants were sampled. Voucher specimens were deposited in the Herbarium of the Institute for Applied Botany, University of Veterinary Medicine, Vienna.
Plant Part
For distillation and extraction, only fresh material was used, since drying results in a high loss (30-40%) of the essential oil (2). Twenty g of fresh plant material (upper plant parts) were distilled in a modified Clevenger apparatus for 3 h. The solvent extracts were prepared by adding CH^sub 2^Cl^sub 2^ to 1 g fresh material of hemp (upper plant parts); extraction was performed in an ultrasonic bath for 15 min.
The essential oil (5 (mu)L) was diluted with CH^sub 2^Cl^sub 2^ (495 (mu)L) prior to analyses. GC/MS-analyses were performed on a HP 6890 coupled with a HP 5972 MSD and fitted with a HP 30 m x 0.25 mm capillary column coated with HP-5MS (0.25 (mu)m film thickness). The analytical conditions were: carrier gas helium, injector temperature 250 deg C, split ratio 50:1, temperature programme 50 deg -140 deg C at 5 deg C/min and 140-170 deg C at 2 deg C/min. Components were identified by comparing their retention indices (RI) and mass spectra (3-5).
Previous Work
The essential oil of C. sativa has been the subject of previous studies (2, 6-15 and references cited therein).
Present Work
Mono- and sesquiterpenes: The oil of C. sativa L. ssp. spontanea contains as main compounds alpha-pinene (9% and 6%, respectively), myrcene (32% and 28%, respectively), beta-- caryophyllene (11% and 16%, respectively) and beta-caryophyllene oxide (7% and 8%, respectively) (Table I). However, the main differences between the two populations could be found in the high content of (E)-beta-ocimene with a very high content of 12.6% from "Albrechtsfeld" and a low content of 3% from "Schoschtolacke." Compared to "Schoschtolacke," the content of alpha-humulene was approximately the half at "Albrechtsfeld" (3.2%).
The oil compositions reported here differ very much from Ross et al. (2), Hendriks et al. (8) and Nigam et al. (13), where (E)-beta-ocimene was only found in traces or not at all. Hendriks et al. (8) and Nigam et al. (13) found alpha-pinene, beta-- pinene and myrcene at alevel of less than 1%, beta-caryophyllene instead reached 37% and 45%, respectively. In contrast, Ross et al. (2) noticed beta-caryophyllene to be present at only 1.3%. Myrcene (67%) and limonene (16%) were much higher than reported elsewhere (2). The Austrian populations of this report are within the range of (12) where different cultivars (especially European fiber cultivars) were analyzed.
Composition of cannabinoids: Regarding the cannabinoids in the oil, relatively high percentages of the non-- hallucinogeneous cannabidiol (CBD) (9.8% "Albrechtsfeld" and 10.9% "Schoschtolacke," respectively) could be found. The hallucinogenic delta-9-tetrahydrocannabinol (THC) was only present at "Schoschtolacke," and here only at low amounts (0.7%).
CBD in the oil was still very high, but it's content was strictly dependant on the distillation conditions. The presence of cannabinoids in oils at higher amounts (11,17 and this report) as well as the almost absence of cannabinoids (12 and 16) are also dependant on distillation conditions and the state of the plant material being distilled.
In the solvent extract, the content of CBD was extremely high (76.6% and 58.8%, respectively), while THC was always (even in the extract) below 1%. These can be regarded as being populations with a low content of THC, while the amount of CBD (especially in the extracts) was very high. So the ratio of CBD/THC, which is used for characterizing and distinguishing "fiber" from "drug" genotypes (18), is very much in favor of the fiber types.
Alkanes: Hendriks et al. (19) found nonacosane as main compound in the alkane-fraction obtained by extraction (55%) and at 11% in the oil. Nonacosane was also detected in the extracts of our study at 9% ("Albrechtsfeld") and 18% ("Schoschtolacke"), while it was absent in the oil (Table I).
*Address for correspondence
References
1. W. Adler, K. Oswald, and R. Fischer, Exkursionsflora von Osterreich. p365, Eugen Ulmer, Stuttgart, (1994).
2. S. A. Ross and M. A. ElSohly, The volatile oil composition of fresh and air-dried buds of Cannabis saliva, J. Nat. Prod., 59, 49-51 (1996).
3. R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, Illinois (1995).
4. F. W. McLafferty, Wiley Registry of Mass Spectral Data. John Wiley & Sons, Inc. New York (1989).
5. T. Mills III. and J. C. Roberson, Instrumental Data for Drug Analysis. Elsevier, Amsterdam (1987).
6. G. Fournier and M. R. Paris, Variabffite de la composition chimique de I'huile essentielle de Chanvre (Cannabis saliva Linnaeus). Rivista Ital. EPPOS, 60, 504-510 (1978).
7. H. Hendriks and A. P. Bruins, A tentative identification of components in the essential oil of Cannabis saliva L. by a combination of gas chromatography negative ion chemical ionization mass spectrometry and retention indices. Biomed. Mass Spectrom., 10, 377-381 (1983).
8. H. Hendriks, Th. M. Malingre, S. Batterman, and R. Bos, Mono- and sesqui-terpene hydrocarbons of the essential oil of Cannabis saliva, Phytochemistry, 14, 814-815 (1975).
9. L. Hanua, The presentstate of knowledge in the chemistry of substances of Cannabis saliva L. III. Terpenoid substances, Acta Universitatis Palackianae Olomucensis, 73, 233-239 (1975).
10. L Lemberkovics, P. Veszki, G. Verzar-Petri and A. Trka, Study on sesquiterpenes of the essential oil in the inflorescence and leaves of Cannabis saliva L. var. Mexico. Sci. Pharm., 49, 401-408 (1981).
11. Th. Malingre, H. Hendriks, S. Batterman, R. Bos and J. Visser, The essential oil of Cannabis sativa, Planta med. 28, 56-61 (1975).
12. V. Mediavilla, and S. Steinemann, Essential oil of Cannabis saliva L. strains, J, Internet. Hemp Assoc., 4, 82-84 (1997).
13. MC. Nigam, K. L. Handa, I. C. Nigam, K. L. Levi, Essential oils and their constituents. XXIX. The essential oil of marihuana: composition of genuine Indian Cannabis saliva L., Can. J. Chem., 43, 3372-3376 (1965).
14. M. Paris, L'essence de Cannabis: parfum mysterieux, Rivista Ital. EPPOS, 57, 83-86 (1975).
15. E. Stahl and R. Kunde, Neue Inhaltsstoffe aus dem atherischen 01 von Cannabis saliva, Tetrahedron Lett., 30, 2841-2844 (1973).
16. J. Novak, K. Zitterl-Egiseer, S.G. Deans and Ch. Franz, Essential oils of
different cultivars of Cannabis saliva L. and their antimicrobial activity, Flav. Fragr. J., 16, 259-262 (2001).
17. Th. Malingre, H. Hendriks, S. Batterman and R. Bos, The presence of cannabinoid components in the essential oil of Cannabis saliva L., Pharm. Weekbl., 108, 549-552 (1973),
18. I. Bocsa, and M. Kraus, Der Hanlanbau. Botanik, Sorten, Anbau and Emte. C.F.Miller, Heidelberg (1997).
19. H. Hendriks, Th. Malingre, S. Batterman and R. Bos, Alkanes of the essential oil of Cannabis saliva, Phytochemistry, 16, 719-721 (1977).
Johannes Novak* and Chlodwig Franz
Institute for Applied Botany, University of Veterinary Medicine, Veterinarplatz 1, A-1210 Wien, Austria
Copyright Allured Publishing Corporation May/Jun 2003
Provided by ProQuest Information and Learning Company. All rights Reserved
Non-cannabinoid constituents from a high potency Cannabis sativa variety
Radwan MM, Elsohly MA, Slade D, Ahmed SA, Wilson L, El-Alfy AT, Khan IA, Ross SA
Phytochemistry 2008 Oct; 69(14):2627-33.
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 , 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 beta-sitosteryl-3-O-beta-d-glucopyranoside-2'-O-palmitate (6). In addition, five known compounds, alpha-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and beta-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.
Cannabinoid Ester Constituents from High-Potency Cannabis sativaAhmed SA, Ross SA, Slade D, Radwan MM, Zulfiqar F, Elsohly MA
J Nat Prod 2008 Feb 28.
Eleven new cannabinoid esters, together with three known cannabinoid acids and Delta (9)-tetrahydrocannabinol ( Delta (9)-THC ), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be beta-fenchyl Delta (9)-tetrahydrocannabinolate ( 1), epi-bornyl Delta (9)-tetrahydrocannabinolate ( 2), alpha-terpenyl Delta (9)-tetrahydrocannabinolate ( 3), 4-terpenyl Delta (9)-tetrahydrocannabinolate ( 4), alpha-cadinyl Delta (9)-tetrahydrocannabinolate ( 5), gamma-eudesmyl Delta (9)-tetrahydrocannabinolate ( 6), gamma-eudesmyl cannabigerolate ( 7), 4-terpenyl cannabinolate ( 8), bornyl Delta (9)-tetrahydrocannabinolate ( 9), alpha-fenchyl Delta (9)-tetrahydrocannabinolate ( 10), alpha-cadinyl cannabigerolate ( 11), Delta (9)-tetrahydrocannabinol ( Delta (9)-THC ), Delta (9)-tetrahydrocannabinolic acid A ( Delta (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 microg/mL. CB-1 receptor assay indicated that the esters, as well as the parent acids, are not active.
Phytochemical and genetic analyses of ancient cannabis from Central Asia
J Exp Bot. 2008 November; 59(15): 4171–4182.
doi: 10.1093/jxb/ern260.PMCID: PMC2639026
Copyright © 2008 The Author(s).
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 Li
Visiting Professor, Institute of Botany, Chinese Academy of Sciences, erusso@gwpharm.com
GW Pharmaceuticals, Porton Down Science Park, Salisbury, Wiltshire SP4 OJQ, UK
Faculty Affiliate, Department of Pharmaceutical Sciences, University of Montana, Missoula, MT, USA
Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Bureau of Cultural Relics of Turpan Prefecture, Turpan 838000, Xinjiang, China
CRA-Centro di Recerca per le Colture Industriali, via di Corticella 133, 40128, Bologna, Italy
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
Department of Botany, University of Calcutta, Kolkata 700019, India
Xinjiang Institute of Archaeology, 4-5 South Beijing Road, Ürümqi, Xinjiang 830011, China
Institute of Palaeontology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
Department of Paleobiology, Smithsonian Institutions, Washington, DC 20560-0121, USA
College of Biological Science and Biotechnology, Beijing Forestry University, Beijing 100083, China
Beijing Museum of Natural History, Beijing 100050, China
To whom correspondence should be addressed: E-mail: lics@ibcas.ac.cn; Email: erusso@gwpharm.com
Received August 7, 2008; Revised September 24, 2008; Accepted September 25, 2008.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Abstract
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.....read more
Plant cannabinoids: a neglected pharmacological treasure trove
Br J Pharmacol. 2005 December; 146(7): 913–915.
Published online 2005 October 3. doi: 10.1038/sj.bjp.0706415.
PMCID: PMC1751232
Copyright 2005, Nature Publishing Group
Department of Medicinal Chemistry and Natural Products, Faculty of Medicine, Hebrew University, Ein Kerem Campus, Jerusalem 91120, Israel
*Author for correspondence: Email: mechou@cc.huji.ac.il
Received August 24, 2005; Revised August 31, 2005; Accepted September 1, 2005.
Abstract
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, Δ9-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.
Keywords: Anandamide, CB1 receptor antagonist, CB2 receptor antagonist, mouse vas deferens, Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabivarin, -(+)-(R)- WIN55212
Cannabis sativa L. produces more than 60 terpeno-phenols that have not been detected in any other plant. One of these constituents, Δ9-tetrahydrocannabinol (THC) (Gaoni & Mechoulam, 1964) has been the object of thousands of publications, as it is by far the major psychoactive principle in marijuana and hashish.
Cannabidiol (CBD), a nonpsychoactive component, has also been widely investigated due to its anti-inflammatory, antischizophrenic and antiepileptic properties (Pertwee, 2005). Surprisingly, the other plant cannabinoids have been mostly neglected.
Cannabinoid acids, which are precursors of the neutral cannabinoids, such as THC and CBD, were shown to be antibiotic and were actually used for some time in veterinary medicine in Czechoslovakia about 50 years ago. Most of the other plant cannabinoids were assayed for possible psychoactivity. When none was found, interest in them waned (Figure 1).
Figure 1
Structures of some cannabinoids mentioned above.
The discovery of the endocannabinoid system and the plethora of activities of...read more
Cannabis confusions
Evaluation of herbal cannabis characteristics by medical users: a randomized trial Copyright © 2006 Ware et al; licensee BioMed Central Ltd.
Abstract
Background
Cannabis, in herbal form, is widely used as self-medication by patients with diseases such as HIV/AIDS and multiple sclerosis suffering from symptoms including pain, muscle spasticity, stress and insomnia.
Valid clinical studies of herbal cannabis require a product which is acceptable to patients in order to maximize adherence to study protocols.
Methods
We conducted a randomized controlled crossover trial of 4 different herbal cannabis preparations among 8 experienced and authorized cannabis users with chronic pain. Preparations were varied with respect to grind size, THC content and humidity. Subjects received each preparation on a separate day and prepared the drug in their usual way in a dedicated and licensed clinical facility.
They were asked to evaluate the products based on appearance (smell, colour, humidity, grind size, ease of preparation and overall appearance) and smoking characteristics (burn rate, hotness, harshness and taste). Five-point Likert scores were assigned to each characteristic. Scores were compared between preparations using ANOVA.
Results
Seven subjects completed the study, and the product with highest THC content (12%), highest humidity (14%) and largest grind size (10 mm) was rated highest overall. Significant differences were noted between preparations on overall appearance and colour (p = 0.003).
Discussion
While the small size of the study precludes broad conclusions, the study shows that medical cannabis users can appreciate differences in herbal product. A more acceptable cannabis product may increase recruitment and retention in clinical studies of medical cannabis.
Background
It is now well-recognized that Cannabis sativa (marijuana, weed, pot) is being used by patients with chronic debilitating diseases such as HIV/AIDS...read more
CHEMOTAXONOMY OF CANNABIS 1 CROSSBREEDING BETWEEN CANNABIS SATIVA AND C. RUDERALIS, WITH ANALYSIS OF CANNABINOID CONTENT JOHN A. BEUTLER AND ARA H. DER MARDEROSIAN
As published in:
Economic Botany, 32(4), 1978, pp. 387-394
by the New York Botanical Garden, Bronx, NY 10458
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) con-
tent 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.
The taxonomy of Cannabis has assumed importance with the spread of marijuana as a drug of abuse, because most state and federal laws are written in terms of only one species, Cannabis sativa L. The supposed existence of more than one species in the genus has caused considerable legal difficulty. At least one article (Fullerton & Kurzman, 1974) has been written providing an arsenal of information that has proved successful in achieving acquitals in marijuana possession cases. Generally the Prosecution is forced to prove which species has been confiscated to determine if the law has actually been violated.
Much work has been done from a morphological point of view on Cannabis taxonomy. The history of the original botanical literature has been reviewed in detail (Emboden, 1974; Schultes et al., 1974).
A large variety of freshly grown specimens have been examined, both for morphology (Small & Cronquist, 1976) and chemistry (Small et al., 1975; Fetterman et al., 1971). Three species have been delineated by Schultes et al. (1974), namely C. sativa L., C. indica Lam. and C. ruderalis Janisch. They have published a key for the differentiation of these three species based on height, branching, seed coat marbling, and seed
attachment and its abscission layer.
Other investigators have held to the traditional monotypic concept (Small & Cronquist, 1976), holding that the wide variations in such characters and others are simply due to the inherent plasticity of the species.
Most phytochemical studies have revealed no major differences in the content and quality of cannabinoids (other than the ratio-of cannabidiol to tetrahydrocannabinol) that could serve to differentiate species. Other chemical markers of taxonomic significance have not been found.
Both chemical and-morphological studies have been essentially static studies of a genetically dynamic organism, with little experimental attention given to.the genetics on which the taxonomic characters are based.
In an attempt-to clarify the taxonomic situation, and to elucidate the genetics of cannabinoid production, we have carried out preliminary cross-breeding experiments between Cannabis sativa L. and Cannabis ruderalis Janisch. under greenhouse conditions with the plants in reproductive isolation from other strains.
Such a cross has been noted in the wild (R. E. Schultes, pers. comm., 1977) but was not included in the work of Small (1972) who intercrossed 38 strains of Cannabis, and found all strains to be interfertile.
Recent advances in the understanding of cannabinoid biosynthesis have made possible more meaningful experiments with the plant. Shoyama et al. (1974) have elegantly elucidated the biosynthetic pathway to the cannabinoid acids from rnevalonate, acetate, and malonate. These are well recognized as the true biosynthetic products of the plant (Fig. 1).
Many of the minor cannabinoids isolated have come to be seen as degradation products of the natural cannabinoids on storage, exposure to light, curing, and other processing. Small & Cronquist's work (1976) and the work of Fetterman et al. in Mississippi (1971) have strengthened the view that THC-acid production is more dependent on the genome of the plant than on environmental factors.
Our breeding experiments were designed to take a closer look at the breeding behavior of the plant with respect to cannabinoid production, measured by gas chromatographic techniques, and backed up by mass spectral identification of the gas chromatographic peaks as cannabinoids.
No other oil provides the necessary EFAs with the right balance. Although any PUFA-containing oil is good, an oil such as hemp seed oil (with the right biological ratio between omega-3/omega-6) provides all the benefits. Hemp seeds oil’s fatty acid profile as well as some of the other valuable constitutes is illustrated in the pie chart above (Chart 4).
Hemp seed oil is pressed from a safe vegetable , hemp, which is a fiber-type weed of the Cannabis sativa species. The plant has dark green leaves and grows worldwide. Cannabis sativa can be separated into two categories:
- Hemp (drug type): the leaves are rich in THC (Δ9 tetrahydrocannabinol) do not contain its precursor CBD (cannabidiol), and is used for its psychotropic properties;
- Hemp (fiber type): contains very low levels of THC and does contain CBD.
So even if hemp seed oil is described by its INCI name (Cannabis sativa seed oil) it contains only traces of THC (less than 10 ppm for selected oils) and is perfectly safe for nutritional and cosmetic use.
Dr. U. Erasmus’ book: Fats that Heal, Fats that Kill, (1993, Alive Books Canada), praises hemp seed oil for its nutritional benefits. Hemp seed oil helps:
- Reduce LDL cholesterol and lower blood pressure for cardiovascular disease prevention;
- Alleviate painful rheumatoid arthritis after a 12-week treatment;
- Relieve the symptoms of PNS and menopause with one teaspoon a day for three months and
- Improve health by sustaining the immune system.
Hemp Seed Oil in Cosmetics
In addition to its outstanding composition, hemp seed oil’s unique texture imparts excellent skin feel. It is non-greasy, has high fluidity and lubricity and is absorbed quickly and efficiently in the skin. In fact, hemp seed oil is considered the “driest” vegetable oil.
Hemp seed oil's unique texture and activity on the skin (including the scalp) targets it toward many beneficial uses in cosmetic products. It is recommended in skin care formulas (up to 10%) that protect or provide anti-aging benefits, as well as dry-, mature- and sensitive skin products. It can be used at 3% in hand, foot or body creams. It can be used at 10% levels in after-sun products as well as lipsticks, lip balms and nail treatments. Hemp seed oil can also be used (3%) in cosmetic powders, liquid makeup and glossy hair conditioners that strengthen or prevent splitting and thinning. It is recommended for use (up to 10% for atopic eczema, acne and psoriasis treatment) and may be used at full strength for aromatherapy purposes and in body and massage oils.
Hemp seed oil is an excellent active ingredient in all of the above cosmetic applications. Structuring for maximum profit in the millennium means including the versatile, cost-efficient and trend-setting ingredients that today’s market demands. Hemp seed oil is the right choice. Not only is it fashionable, but it is the natural solution to the industry’s need for a rich oil that tests boundaries. Hemp seed oil is defined by unique properties that indulge the consumer in countless benefits. When used as an active ingredient, hemp seed oil follows a trend that you can bank on.
About the Authors
Kristin Speiser is a freelance public relations specialist from New York, NY. Michael Pobeda is a general manager of Teco Finance Export, a French manufacturer specialized in unique oils and butters. Laurent Sousselier is director of EX.A International - France and may be reached at + 33.1.4287.9698 or by e-mail at exacosm@club-internet.fr.
Composition of the essential oils and extracts of two populations of Cannabis sativa L. ssp. spontanea from Austria
Journal of Essential Oil Research: JEOR, May/Jun 2003Novak, Johannes, Franz, Chlodwig Abstract
The essential oil and the solvent extract of two populations of Cannabis sativa L. ssp. spontanea growing wild in Austria were analyzed comparatively. In the essential oil, myrcene (31% and 27%, respectively), (E)-beta-ocilnene (13% and 3%, respectively) and beta-caryophyllene (11 % and 16%, respectively) were found, while in the solvent extract the non-hallucinogeneous cannabidiol (77% and 59%, respectively) dominated. The hallucinogeneous delta-9-tetrahydrocannabinol (THC) was also found in the solvent extract at a level of less than 1%.
Key Word Index
Cannabissativa ssp. spontanea, Cannabaceae, essential oil composition, myrcene, (E)-beta-ocii-nene, beta-caryophyllene, cannabinoids
The Plant
In Cannabis sativa L. ssp. spontanea (formerly Cannabis ruderalis) (Cannabaceae) the perianth of the female flowers is in contrast to C. sativa ssp. sativa still present; the fruit is brownish and has a peduncle-like ringbulge. It is a ruderal, but a rare plant in the east of Austria (1).
Source
Two populations of C. sativa L. ssp. spontanea ("Albrechtsfeld" and "Schoschtolacke") from the region of lake Neusiedl, Burgenland, eastern Austria were sampled in June, 1998, at the beginning of seed ripening. At each population upper parts of approximately 10 plants were sampled. Voucher specimens were deposited in the Herbarium of the Institute for Applied Botany, University of Veterinary Medicine, Vienna.
Plant Part
For distillation and extraction, only fresh material was used, since drying results in a high loss (30-40%) of the essential oil (2). Twenty g of fresh plant material (upper plant parts) were distilled in a modified Clevenger apparatus for 3 h. The solvent extracts were prepared by adding CH^sub 2^Cl^sub 2^ to 1 g fresh material of hemp (upper plant parts); extraction was performed in an ultrasonic bath for 15 min.
The essential oil (5 (mu)L) was diluted with CH^sub 2^Cl^sub 2^ (495 (mu)L) prior to analyses. GC/MS-analyses were performed on a HP 6890 coupled with a HP 5972 MSD and fitted with a HP 30 m x 0.25 mm capillary column coated with HP-5MS (0.25 (mu)m film thickness). The analytical conditions were: carrier gas helium, injector temperature 250 deg C, split ratio 50:1, temperature programme 50 deg -140 deg C at 5 deg C/min and 140-170 deg C at 2 deg C/min. Components were identified by comparing their retention indices (RI) and mass spectra (3-5).
Previous Work
The essential oil of C. sativa has been the subject of previous studies (2, 6-15 and references cited therein).
Present Work
Mono- and sesquiterpenes: The oil of C. sativa L. ssp. spontanea contains as main compounds alpha-pinene (9% and 6%, respectively), myrcene (32% and 28%, respectively), beta-- caryophyllene (11% and 16%, respectively) and beta-caryophyllene oxide (7% and 8%, respectively) (Table I). However, the main differences between the two populations could be found in the high content of (E)-beta-ocimene with a very high content of 12.6% from "Albrechtsfeld" and a low content of 3% from "Schoschtolacke." Compared to "Schoschtolacke," the content of alpha-humulene was approximately the half at "Albrechtsfeld" (3.2%).
The oil compositions reported here differ very much from Ross et al. (2), Hendriks et al. (8) and Nigam et al. (13), where (E)-beta-ocimene was only found in traces or not at all. Hendriks et al. (8) and Nigam et al. (13) found alpha-pinene, beta-- pinene and myrcene at alevel of less than 1%, beta-caryophyllene instead reached 37% and 45%, respectively. In contrast, Ross et al. (2) noticed beta-caryophyllene to be present at only 1.3%. Myrcene (67%) and limonene (16%) were much higher than reported elsewhere (2). The Austrian populations of this report are within the range of (12) where different cultivars (especially European fiber cultivars) were analyzed.
Composition of cannabinoids: Regarding the cannabinoids in the oil, relatively high percentages of the non-- hallucinogeneous cannabidiol (CBD) (9.8% "Albrechtsfeld" and 10.9% "Schoschtolacke," respectively) could be found. The hallucinogenic delta-9-tetrahydrocannabinol (THC) was only present at "Schoschtolacke," and here only at low amounts (0.7%).
CBD in the oil was still very high, but it's content was strictly dependant on the distillation conditions. The presence of cannabinoids in oils at higher amounts (11,17 and this report) as well as the almost absence of cannabinoids (12 and 16) are also dependant on distillation conditions and the state of the plant material being distilled.
In the solvent extract, the content of CBD was extremely high (76.6% and 58.8%, respectively), while THC was always (even in the extract) below 1%. These can be regarded as being populations with a low content of THC, while the amount of CBD (especially in the extracts) was very high. So the ratio of CBD/THC, which is used for characterizing and distinguishing "fiber" from "drug" genotypes (18), is very much in favor of the fiber types.
Alkanes: Hendriks et al. (19) found nonacosane as main compound in the alkane-fraction obtained by extraction (55%) and at 11% in the oil. Nonacosane was also detected in the extracts of our study at 9% ("Albrechtsfeld") and 18% ("Schoschtolacke"), while it was absent in the oil (Table I).
*Address for correspondence
References
1. W. Adler, K. Oswald, and R. Fischer, Exkursionsflora von Osterreich. p365, Eugen Ulmer, Stuttgart, (1994).
2. S. A. Ross and M. A. ElSohly, The volatile oil composition of fresh and air-dried buds of Cannabis saliva, J. Nat. Prod., 59, 49-51 (1996).
3. R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, Illinois (1995).
4. F. W. McLafferty, Wiley Registry of Mass Spectral Data. John Wiley & Sons, Inc. New York (1989).
5. T. Mills III. and J. C. Roberson, Instrumental Data for Drug Analysis. Elsevier, Amsterdam (1987).
6. G. Fournier and M. R. Paris, Variabffite de la composition chimique de I'huile essentielle de Chanvre (Cannabis saliva Linnaeus). Rivista Ital. EPPOS, 60, 504-510 (1978).
7. H. Hendriks and A. P. Bruins, A tentative identification of components in the essential oil of Cannabis saliva L. by a combination of gas chromatography negative ion chemical ionization mass spectrometry and retention indices. Biomed. Mass Spectrom., 10, 377-381 (1983).
8. H. Hendriks, Th. M. Malingre, S. Batterman, and R. Bos, Mono- and sesqui-terpene hydrocarbons of the essential oil of Cannabis saliva, Phytochemistry, 14, 814-815 (1975).
9. L. Hanua, The presentstate of knowledge in the chemistry of substances of Cannabis saliva L. III. Terpenoid substances, Acta Universitatis Palackianae Olomucensis, 73, 233-239 (1975).
10. L Lemberkovics, P. Veszki, G. Verzar-Petri and A. Trka, Study on sesquiterpenes of the essential oil in the inflorescence and leaves of Cannabis saliva L. var. Mexico. Sci. Pharm., 49, 401-408 (1981).
11. Th. Malingre, H. Hendriks, S. Batterman, R. Bos and J. Visser, The essential oil of Cannabis sativa, Planta med. 28, 56-61 (1975).
12. V. Mediavilla, and S. Steinemann, Essential oil of Cannabis saliva L. strains, J, Internet. Hemp Assoc., 4, 82-84 (1997).
13. MC. Nigam, K. L. Handa, I. C. Nigam, K. L. Levi, Essential oils and their constituents. XXIX. The essential oil of marihuana: composition of genuine Indian Cannabis saliva L., Can. J. Chem., 43, 3372-3376 (1965).
14. M. Paris, L'essence de Cannabis: parfum mysterieux, Rivista Ital. EPPOS, 57, 83-86 (1975).
15. E. Stahl and R. Kunde, Neue Inhaltsstoffe aus dem atherischen 01 von Cannabis saliva, Tetrahedron Lett., 30, 2841-2844 (1973).
16. J. Novak, K. Zitterl-Egiseer, S.G. Deans and Ch. Franz, Essential oils of
different cultivars of Cannabis saliva L. and their antimicrobial activity, Flav. Fragr. J., 16, 259-262 (2001).
17. Th. Malingre, H. Hendriks, S. Batterman and R. Bos, The presence of cannabinoid components in the essential oil of Cannabis saliva L., Pharm. Weekbl., 108, 549-552 (1973),
18. I. Bocsa, and M. Kraus, Der Hanlanbau. Botanik, Sorten, Anbau and Emte. C.F.Miller, Heidelberg (1997).
19. H. Hendriks, Th. Malingre, S. Batterman and R. Bos, Alkanes of the essential oil of Cannabis saliva, Phytochemistry, 16, 719-721 (1977).
Johannes Novak* and Chlodwig Franz
Institute for Applied Botany, University of Veterinary Medicine, Veterinarplatz 1, A-1210 Wien, Austria
Copyright Allured Publishing Corporation May/Jun 2003
Provided by ProQuest Information and Learning Company. All rights Reserved
Non-cannabinoid constituents from a high potency Cannabis sativa variety
Radwan MM, Elsohly MA, Slade D, Ahmed SA, Wilson L, El-Alfy AT, Khan IA, Ross SA
Phytochemistry 2008 Oct; 69(14):2627-33.
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 , 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 beta-sitosteryl-3-O-beta-d-glucopyranoside-2'-O-palmitate (6). In addition, five known compounds, alpha-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and beta-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.
Cannabinoid Ester Constituents from High-Potency Cannabis sativaAhmed SA, Ross SA, Slade D, Radwan MM, Zulfiqar F, Elsohly MA
J Nat Prod 2008 Feb 28.
Eleven new cannabinoid esters, together with three known cannabinoid acids and Delta (9)-tetrahydrocannabinol ( Delta (9)-THC ), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be beta-fenchyl Delta (9)-tetrahydrocannabinolate ( 1), epi-bornyl Delta (9)-tetrahydrocannabinolate ( 2), alpha-terpenyl Delta (9)-tetrahydrocannabinolate ( 3), 4-terpenyl Delta (9)-tetrahydrocannabinolate ( 4), alpha-cadinyl Delta (9)-tetrahydrocannabinolate ( 5), gamma-eudesmyl Delta (9)-tetrahydrocannabinolate ( 6), gamma-eudesmyl cannabigerolate ( 7), 4-terpenyl cannabinolate ( 8), bornyl Delta (9)-tetrahydrocannabinolate ( 9), alpha-fenchyl Delta (9)-tetrahydrocannabinolate ( 10), alpha-cadinyl cannabigerolate ( 11), Delta (9)-tetrahydrocannabinol ( Delta (9)-THC ), Delta (9)-tetrahydrocannabinolic acid A ( Delta (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 microg/mL. CB-1 receptor assay indicated that the esters, as well as the parent acids, are not active.
Phytochemical and genetic analyses of ancient cannabis from Central Asia
J Exp Bot. 2008 November; 59(15): 4171–4182.
doi: 10.1093/jxb/ern260.PMCID: PMC2639026
Copyright © 2008 The Author(s).
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 Li
Visiting Professor, Institute of Botany, Chinese Academy of Sciences, erusso@gwpharm.com
GW Pharmaceuticals, Porton Down Science Park, Salisbury, Wiltshire SP4 OJQ, UK
Faculty Affiliate, Department of Pharmaceutical Sciences, University of Montana, Missoula, MT, USA
Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Bureau of Cultural Relics of Turpan Prefecture, Turpan 838000, Xinjiang, China
CRA-Centro di Recerca per le Colture Industriali, via di Corticella 133, 40128, Bologna, Italy
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
Department of Botany, University of Calcutta, Kolkata 700019, India
Xinjiang Institute of Archaeology, 4-5 South Beijing Road, Ürümqi, Xinjiang 830011, China
Institute of Palaeontology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
Department of Paleobiology, Smithsonian Institutions, Washington, DC 20560-0121, USA
College of Biological Science and Biotechnology, Beijing Forestry University, Beijing 100083, China
Beijing Museum of Natural History, Beijing 100050, China
To whom correspondence should be addressed: E-mail: lics@ibcas.ac.cn; Email: erusso@gwpharm.com
Received August 7, 2008; Revised September 24, 2008; Accepted September 25, 2008.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Abstract
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.....read more
Plant cannabinoids: a neglected pharmacological treasure trove
Br J Pharmacol. 2005 December; 146(7): 913–915.
Published online 2005 October 3. doi: 10.1038/sj.bjp.0706415.
PMCID: PMC1751232
Copyright 2005, Nature Publishing Group
Department of Medicinal Chemistry and Natural Products, Faculty of Medicine, Hebrew University, Ein Kerem Campus, Jerusalem 91120, Israel
*Author for correspondence: Email: mechou@cc.huji.ac.il
Received August 24, 2005; Revised August 31, 2005; Accepted September 1, 2005.
Abstract
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, Δ9-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.
Keywords: Anandamide, CB1 receptor antagonist, CB2 receptor antagonist, mouse vas deferens, Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabivarin, -(+)-(R)- WIN55212
Cannabis sativa L. produces more than 60 terpeno-phenols that have not been detected in any other plant. One of these constituents, Δ9-tetrahydrocannabinol (THC) (Gaoni & Mechoulam, 1964) has been the object of thousands of publications, as it is by far the major psychoactive principle in marijuana and hashish.
Cannabidiol (CBD), a nonpsychoactive component, has also been widely investigated due to its anti-inflammatory, antischizophrenic and antiepileptic properties (Pertwee, 2005). Surprisingly, the other plant cannabinoids have been mostly neglected.
Cannabinoid acids, which are precursors of the neutral cannabinoids, such as THC and CBD, were shown to be antibiotic and were actually used for some time in veterinary medicine in Czechoslovakia about 50 years ago. Most of the other plant cannabinoids were assayed for possible psychoactivity. When none was found, interest in them waned (Figure 1).
Figure 1
Structures of some cannabinoids mentioned above.
The discovery of the endocannabinoid system and the plethora of activities of...read more
Cannabis confusions
Evaluation of herbal cannabis characteristics by medical users: a randomized trial Copyright © 2006 Ware et al; licensee BioMed Central Ltd.
Abstract
Background
Cannabis, in herbal form, is widely used as self-medication by patients with diseases such as HIV/AIDS and multiple sclerosis suffering from symptoms including pain, muscle spasticity, stress and insomnia.
Valid clinical studies of herbal cannabis require a product which is acceptable to patients in order to maximize adherence to study protocols.
Methods
We conducted a randomized controlled crossover trial of 4 different herbal cannabis preparations among 8 experienced and authorized cannabis users with chronic pain. Preparations were varied with respect to grind size, THC content and humidity. Subjects received each preparation on a separate day and prepared the drug in their usual way in a dedicated and licensed clinical facility.
They were asked to evaluate the products based on appearance (smell, colour, humidity, grind size, ease of preparation and overall appearance) and smoking characteristics (burn rate, hotness, harshness and taste). Five-point Likert scores were assigned to each characteristic. Scores were compared between preparations using ANOVA.
Results
Seven subjects completed the study, and the product with highest THC content (12%), highest humidity (14%) and largest grind size (10 mm) was rated highest overall. Significant differences were noted between preparations on overall appearance and colour (p = 0.003).
Discussion
While the small size of the study precludes broad conclusions, the study shows that medical cannabis users can appreciate differences in herbal product. A more acceptable cannabis product may increase recruitment and retention in clinical studies of medical cannabis.
Background
It is now well-recognized that Cannabis sativa (marijuana, weed, pot) is being used by patients with chronic debilitating diseases such as HIV/AIDS...read more
CHEMOTAXONOMY OF CANNABIS 1 CROSSBREEDING BETWEEN CANNABIS SATIVA AND C. RUDERALIS, WITH ANALYSIS OF CANNABINOID CONTENT JOHN A. BEUTLER AND ARA H. DER MARDEROSIAN
As published in:
Economic Botany, 32(4), 1978, pp. 387-394
by the New York Botanical Garden, Bronx, NY 10458
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) con-
tent 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.
The taxonomy of Cannabis has assumed importance with the spread of marijuana as a drug of abuse, because most state and federal laws are written in terms of only one species, Cannabis sativa L. The supposed existence of more than one species in the genus has caused considerable legal difficulty. At least one article (Fullerton & Kurzman, 1974) has been written providing an arsenal of information that has proved successful in achieving acquitals in marijuana possession cases. Generally the Prosecution is forced to prove which species has been confiscated to determine if the law has actually been violated.
Much work has been done from a morphological point of view on Cannabis taxonomy. The history of the original botanical literature has been reviewed in detail (Emboden, 1974; Schultes et al., 1974).
A large variety of freshly grown specimens have been examined, both for morphology (Small & Cronquist, 1976) and chemistry (Small et al., 1975; Fetterman et al., 1971). Three species have been delineated by Schultes et al. (1974), namely C. sativa L., C. indica Lam. and C. ruderalis Janisch. They have published a key for the differentiation of these three species based on height, branching, seed coat marbling, and seed
attachment and its abscission layer.
Other investigators have held to the traditional monotypic concept (Small & Cronquist, 1976), holding that the wide variations in such characters and others are simply due to the inherent plasticity of the species.
Most phytochemical studies have revealed no major differences in the content and quality of cannabinoids (other than the ratio-of cannabidiol to tetrahydrocannabinol) that could serve to differentiate species. Other chemical markers of taxonomic significance have not been found.
Both chemical and-morphological studies have been essentially static studies of a genetically dynamic organism, with little experimental attention given to.the genetics on which the taxonomic characters are based.
In an attempt-to clarify the taxonomic situation, and to elucidate the genetics of cannabinoid production, we have carried out preliminary cross-breeding experiments between Cannabis sativa L. and Cannabis ruderalis Janisch. under greenhouse conditions with the plants in reproductive isolation from other strains.
Such a cross has been noted in the wild (R. E. Schultes, pers. comm., 1977) but was not included in the work of Small (1972) who intercrossed 38 strains of Cannabis, and found all strains to be interfertile.
Recent advances in the understanding of cannabinoid biosynthesis have made possible more meaningful experiments with the plant. Shoyama et al. (1974) have elegantly elucidated the biosynthetic pathway to the cannabinoid acids from rnevalonate, acetate, and malonate. These are well recognized as the true biosynthetic products of the plant (Fig. 1).
Many of the minor cannabinoids isolated have come to be seen as degradation products of the natural cannabinoids on storage, exposure to light, curing, and other processing. Small & Cronquist's work (1976) and the work of Fetterman et al. in Mississippi (1971) have strengthened the view that THC-acid production is more dependent on the genome of the plant than on environmental factors.
Our breeding experiments were designed to take a closer look at the breeding behavior of the plant with respect to cannabinoid production, measured by gas chromatographic techniques, and backed up by mass spectral identification of the gas chromatographic peaks as cannabinoids.
MATERIALS AND METHODS
Seed samples. -JBC-2 was obtained from the Central Siberian Botanical Garden, Novosibirsk, USSR, and labelled Cannabis ruderalis. This is possibly identical to the C. ruderalis in Small & Beckstead (1973, p. 164, Table 5). It produced small (less than 2 ft) quick-flowering plants with low THC content (less than 0.2%). The seeds were rnarbled and dropped off at maturity, and the "fleshy caruncle-like growth at the base" was evident, though not obviously so.
JBC-3 was an alledgedly Mexican strain of C. sativa which reached 6-7 ft in height when given adequate root space, did not flower until it had reached this height, and contained a relatively high amount of THC (between 1.0 and 2.0%). Seeds were plain and indehiseent at maturity. Despite the lack of an authentic
origin for this strain, it conforms to all published criteria for C. sativa.
Growing conditions. -Seeds were sown in rows in flats containing two parts of topsoil to one part peat moss. The greenhouse was kept between 70F and 80F. When several inches tall, the seedlings were transplanted to large plastic tubs 16 inches in diameter and eight inches deep, a size that was found to minimally restrict growth. Smaller pots were not used because this produced smaller plants of C. sativa, disguising useful differences in growth and flowering behavior. Plants were watered twice daily by an automatic capillary watering system, and positioned to receive full sun in the greenhouse.
The time of sowing was not strictly controlled with regard to day-length, however the quickness of flowering was constant within the plants of each strain used in the cross. The Mexican strain (JBC-3) has shown erratic flowering behavior when grown out of step with the normal seasonal variations in day-length. The Cannabis ruderalis (JBC-2) flowered promptly when sown at any point from November to May. Convenience dictated that plants of the two strains not be started at the same time, due to the difference in time required for flower production.
Plants were harvested when mature; i.e., for males, after the pollen had been substantially shed. and for females, after sufficient ripe.seed had been collected for further breeding work.
All samples analyzed were freeze-dried and stored in the dark in scaled plastic bags at room temperature. Sampling protocols varied, especially for the Mexican strain, but all parts of the same plant contained identical cannabinoid profiles if they contained any at all.
Roots and woody stems did not contain cannabinoids, and leaves contained less than the flowering tops. All C. ruderalis plants were analyzed as the whole freeze-dried plant. Where several very small individuals were concerned, several of the same sex and age were combined into the same sample.
Gas chromatography. -The procedure used was based on that of Lerner (1969). One g of plant material was shaken with 40 ml of chloroform at room temperature for one hour in a stoppered Erlenmeyer flask. The solution was filtered, the plant material rinsed with a few ml of chloroform, and the combined solution evaporated under reduced pressure.
This residue was taken up first in 2.0 ml of chloroform, and then 2.0 ml of the internal standard solution (5.0 mg/ml androstene-3,17-dione in methanol) was added. This solution was stored in screw-capped vials with teflon inserts in the caps at freezer temperatures. Caps without
teflon inserts were leached by both methanol and chloroform. Dioetyl phthalate from the cap leachates was found to have a relative retention time of 0.45, which without mass spectral analysis could be confused with delta 8-THC.
Sample solutions were injected on a 3% OV- 17 on 100/120 Gas Chrom Q column (Supelco, Inc.) 6 ft long, 4 mm i.d., installed in an HCI Scientific gas chromatograph, or Varian model 3700 gas chromatograph, both with flame ionization detection. Injector temperature was 255C, column temperature isothermal at 220C, carrier gas flow (He) 120 ml/min. Typically one rnicroliter of sample solution was injected.
Peak areas were determined by multiplying height times half-height width. This was compared to the area of the internal standard peak and corrected for the amount of plant material taken, along with the response factor of each compound.
It should be noted that this procedure does not differentiate between cannabidiol (CBD) and cannabichromene (CBC) (RRT- 0.36) (Turner et al., 1975). To ascertain the identity of this peak, gas chromatography at I80C on a 3 % OV-101 column was performed. The results of this separation were used to get the ratio of CBD to CBC in the combined peak on OV-17.
RESULTS AND DISCUSSION
Chemistry. -The results of glc analysis are presented in Figure 2 as total percent cannabinoids on a dry-weight basis versus delta 9-THC as percent of total cannabinoids (CBD, CBC, delta 9-THC). This method of expressing cannabinoid content gives a comparison of overall production of cannabinoids, as well as an indication of how much of this cannabinoid production is delta 9-THC.
The percentage of CBC was found to be relatively constant within the experiment, from 3 to 9 % of total cannabinoids in the progeny.
Parent strains also had CBC as a non-variable cannabinoid at about the same concentration. Thus CBD and THC are the important variables in the cross. The total percent cannabinoids varies rather widely due to variation in sampling technique, but the pattern of cannabinoid production can be delimited into two groups for the cross.
Seed samples. -JBC-2 was obtained from the Central Siberian Botanical Garden, Novosibirsk, USSR, and labelled Cannabis ruderalis. This is possibly identical to the C. ruderalis in Small & Beckstead (1973, p. 164, Table 5). It produced small (less than 2 ft) quick-flowering plants with low THC content (less than 0.2%). The seeds were rnarbled and dropped off at maturity, and the "fleshy caruncle-like growth at the base" was evident, though not obviously so.
JBC-3 was an alledgedly Mexican strain of C. sativa which reached 6-7 ft in height when given adequate root space, did not flower until it had reached this height, and contained a relatively high amount of THC (between 1.0 and 2.0%). Seeds were plain and indehiseent at maturity. Despite the lack of an authentic
origin for this strain, it conforms to all published criteria for C. sativa.
Growing conditions. -Seeds were sown in rows in flats containing two parts of topsoil to one part peat moss. The greenhouse was kept between 70F and 80F. When several inches tall, the seedlings were transplanted to large plastic tubs 16 inches in diameter and eight inches deep, a size that was found to minimally restrict growth. Smaller pots were not used because this produced smaller plants of C. sativa, disguising useful differences in growth and flowering behavior. Plants were watered twice daily by an automatic capillary watering system, and positioned to receive full sun in the greenhouse.
The time of sowing was not strictly controlled with regard to day-length, however the quickness of flowering was constant within the plants of each strain used in the cross. The Mexican strain (JBC-3) has shown erratic flowering behavior when grown out of step with the normal seasonal variations in day-length. The Cannabis ruderalis (JBC-2) flowered promptly when sown at any point from November to May. Convenience dictated that plants of the two strains not be started at the same time, due to the difference in time required for flower production.
Plants were harvested when mature; i.e., for males, after the pollen had been substantially shed. and for females, after sufficient ripe.seed had been collected for further breeding work.
All samples analyzed were freeze-dried and stored in the dark in scaled plastic bags at room temperature. Sampling protocols varied, especially for the Mexican strain, but all parts of the same plant contained identical cannabinoid profiles if they contained any at all.
Roots and woody stems did not contain cannabinoids, and leaves contained less than the flowering tops. All C. ruderalis plants were analyzed as the whole freeze-dried plant. Where several very small individuals were concerned, several of the same sex and age were combined into the same sample.
Gas chromatography. -The procedure used was based on that of Lerner (1969). One g of plant material was shaken with 40 ml of chloroform at room temperature for one hour in a stoppered Erlenmeyer flask. The solution was filtered, the plant material rinsed with a few ml of chloroform, and the combined solution evaporated under reduced pressure.
This residue was taken up first in 2.0 ml of chloroform, and then 2.0 ml of the internal standard solution (5.0 mg/ml androstene-3,17-dione in methanol) was added. This solution was stored in screw-capped vials with teflon inserts in the caps at freezer temperatures. Caps without
teflon inserts were leached by both methanol and chloroform. Dioetyl phthalate from the cap leachates was found to have a relative retention time of 0.45, which without mass spectral analysis could be confused with delta 8-THC.
Sample solutions were injected on a 3% OV- 17 on 100/120 Gas Chrom Q column (Supelco, Inc.) 6 ft long, 4 mm i.d., installed in an HCI Scientific gas chromatograph, or Varian model 3700 gas chromatograph, both with flame ionization detection. Injector temperature was 255C, column temperature isothermal at 220C, carrier gas flow (He) 120 ml/min. Typically one rnicroliter of sample solution was injected.
Peak areas were determined by multiplying height times half-height width. This was compared to the area of the internal standard peak and corrected for the amount of plant material taken, along with the response factor of each compound.
It should be noted that this procedure does not differentiate between cannabidiol (CBD) and cannabichromene (CBC) (RRT- 0.36) (Turner et al., 1975). To ascertain the identity of this peak, gas chromatography at I80C on a 3 % OV-101 column was performed. The results of this separation were used to get the ratio of CBD to CBC in the combined peak on OV-17.
RESULTS AND DISCUSSION
Chemistry. -The results of glc analysis are presented in Figure 2 as total percent cannabinoids on a dry-weight basis versus delta 9-THC as percent of total cannabinoids (CBD, CBC, delta 9-THC). This method of expressing cannabinoid content gives a comparison of overall production of cannabinoids, as well as an indication of how much of this cannabinoid production is delta 9-THC.
The percentage of CBC was found to be relatively constant within the experiment, from 3 to 9 % of total cannabinoids in the progeny.
Parent strains also had CBC as a non-variable cannabinoid at about the same concentration. Thus CBD and THC are the important variables in the cross. The total percent cannabinoids varies rather widely due to variation in sampling technique, but the pattern of cannabinoid production can be delimited into two groups for the cross.
The parent strain JBC-3 (C. sativa) is seen th contain at least 70% THC of total cannabinoids. The sex appears to make no difference in cannabinoid total or
pattern in this strain.
The other parent, JBC-2 (C. ruderalis), contains less than 4O% THC of total cannabinoids, with most plants falling below the 20% level. The total percent cannabinoids was lower for C. ruderalis (mean = 0.45%) than for C. sativa (mean = 1.61%), though one "sport" of JBC-2, which was kept for several weeks under high humidity conditions (an inverted paper chromatography tank), showed 1.54% total cannabinoids on a whole plant sample. The sport's cannabinoid pattern was similar to others of its strain, however. The variation in sampling methods makes statistical comparison of the means impossible.
The offspring of the cross were from the female C. sativa. The female C. ruderalis plants were weak, and though they set seed, the number was small and of poor viability. The seedlings either did not germinate or did not survive much beyond germination. The fact that the reverse cross produced vigorous offspring, and that cannabinoid production does not seem to be linked to sex [in some strains it is linked, according to Small et al. (1975)], means that failure to generate offspring was due to generally weak plants and not to a lack of interfertility.
The progeny of the cross fell into two groups (Fig. 3), separated by a large gap in THC percentage. The total cannabinoid percent of dry weight ranged between 0.36% and 1.30%, with a mean of 0.72% for each group taken separately. All but one value was less than 1.0%. This high value was for the top of a plant selected for a voucher herbarium specimen, not for a whole plant. This underlines the importance of sampling procedure, which will be discussed in detail below.
pattern in this strain.
The other parent, JBC-2 (C. ruderalis), contains less than 4O% THC of total cannabinoids, with most plants falling below the 20% level. The total percent cannabinoids was lower for C. ruderalis (mean = 0.45%) than for C. sativa (mean = 1.61%), though one "sport" of JBC-2, which was kept for several weeks under high humidity conditions (an inverted paper chromatography tank), showed 1.54% total cannabinoids on a whole plant sample. The sport's cannabinoid pattern was similar to others of its strain, however. The variation in sampling methods makes statistical comparison of the means impossible.
The offspring of the cross were from the female C. sativa. The female C. ruderalis plants were weak, and though they set seed, the number was small and of poor viability. The seedlings either did not germinate or did not survive much beyond germination. The fact that the reverse cross produced vigorous offspring, and that cannabinoid production does not seem to be linked to sex [in some strains it is linked, according to Small et al. (1975)], means that failure to generate offspring was due to generally weak plants and not to a lack of interfertility.
The progeny of the cross fell into two groups (Fig. 3), separated by a large gap in THC percentage. The total cannabinoid percent of dry weight ranged between 0.36% and 1.30%, with a mean of 0.72% for each group taken separately. All but one value was less than 1.0%. This high value was for the top of a plant selected for a voucher herbarium specimen, not for a whole plant. This underlines the importance of sampling procedure, which will be discussed in detail below.
One-third of the progeny (n = 5, 3 females, 2 males) had THC percentages over 60% of total cannabinoids. The other two-thirds of the progeny (n = 10, 6
females, 3 males, one died before flowering) had THC percentages between 30 and 40% of total cannabinoids.
This rather conspicuous chemical dimorphism has no easy genetic explanation. It does not seem to be a case of one enzyme being responsible for the cyclization
of cannabidiolic acid to THCA. And if it were that simple, no such "cannabidiolic acid cyclase" has ever been isolated from Cannabis of any species.
Morphology. -ln habit, the progeny resembled both parents to. some extent. They did not show the dimorphism seen in cannabinoid content. Plants were between one and two ft tall, grew vigorously on germination, and flowered after putting out several pairs of true leaves. They continued their growth after initial flowering. The plants were self-crossed to produce an F2 generation of seed, which was saved for further plantings.
Some of the F1 seeds were slower to germinate than their C. sativa parent. Four seeds remained in the flat for four months before germination. These plants
were analyzed when mature, and their cannabinoid profiles fell within either of the two groups of progeny. Delay in germination is characteristic of plants growing outside of cultivation, either in a wild or weedy state. This trait was presumably transmitted from the C. ruderalis parent, although it was not observed in the seeds sown.
A voucher specimen of one of the female progeny has been deposited in the Library of Economic Botany of the Harvard Botanical Museum, as specimen no. 35896.
Such genetic inhomogeneity has not been reported before in strains of Cannabis. The nearest case is that of South African Cannabis reported by Boucher et al. (1977), in which they found the presence of two chemotypes varying in amounts of THCA and the C3 THCA homolog tetrahydrocannabivarolic acid.
Small et al.'s breeding experiments (1975) reported only that progeny obtained in crosses were "intermediate" in cannabinoid content. The aim of chemotaxonomy is to demonstrate that the variation in chemistry between two populations has a genetic basis dependent on the presence or absence of enzymes involved with the biosynthesis of the compounds in question.
Such information cannot be gotten from studies where populations are pooled or samples are not uniform. In order to do more meaningful work on individual plant cannabinoid content, it is necessary to find sampling methods which are not subject to wide variations, and which are precisely definable and reproducible.
Sampling. -We propose that the unit of sampling for female Cannabis plants be the mature bract, at the point where the seed is no longer green and is easily separable from the bract. For the male plant, perhaps the pollen can be used. It has been found in this and other studies (Paris et al., 1975) to contain the same proportions of cannabinoids as the rest of the plant. The male flower is less reproducible for sampling, since it is continuously shedding pollen at maturity, thereby changing the relative proportions of pollen and flower tissue.
This involves working with rather small amounts, since a mature bract when fresh weighs only about 2 mg. Pollen is most easily collected in milligram quantities. But the technique makes possible reproducible multiple samplings from each plant.
As an example of the technique, single fresh bracts were put into a 4 ml screw-capped vial with 0. 1 ml of chloroform overnight at room temperaature. In the morning 0.1 ml more of chloroform was added, along with 20 microliters of a methanolic solution of the internal standard, androstene-dione at a concentration of 1 mg/ml. The sample solution was injected into the glc as with the whole plant assay.
Results for this type of assay done with single bracts from the progeny of our cross indicate that the method gives comparable results to the whole plant assay for the percentage of cannabinoids. The percent total of cannabinoids is higher than for the whole plant, as would be expected.
Assuming that the bracts are relatively uniform within the same plant, how does this sampling method compare with other methods, such as the whole plant, or the manicured plant?
The whole plant analysis involves parts (seeds, roots, and woody stems) which have little if any cannabinoid content. The relative proportion of these parts to leaf and bract varies widely with cultural conditions; e.g., if the plant is grown under dry conditions, the root will become a very short, stocky structure, with few branchings; but if it is grown with adequate moisture it will be longer, thinner, and branched with many fine rootlets that are hard to collect completely. The amount of stem and vegetative growth varies also with plant nutrition.
These factors induce error in the measurement of the plant's actual genetic capacity to produce cannabinoids, while having nothing to do with cannabinoid
production. Cannabinoids and their precursors are formed entirely in the leafy portions of the plant (Crombie & Crombie, 1975).
The bract, assuming the flower is pollinated, is the most stable part of the plant both for morphology and for sampling.
The number of flowers produced on a plant will influence the relative levels between the bract sampling method and the whole plant assay. We have observed within the progeny of the cross a variation in the number of flowers produced per plant. This is reasonable because Cannabis has been bred for higher seed and flower production.
The Mexican parent produces seed quite heavily, while the presumably wild C. ruderalis produces relatively few seeds, concentrating its energies into the production of fewer, more viable seeds. This difference in characters shows its genetic base by its mixed transmission into the offspring of the
cross.
An aim of taxonomy is to find independent, definable characters, based where possible on a single gene system. The two traits of cannabinoid production and
number of flowers per plant are mixed using a whole or manicured plant assay, but not in a bract assay, because the proportion of dry weight due to high cannabinoid bracts is greater for many-seeded varieties of Cannabis than it is for those producing relatively few seeds.
The cellular locus of cannabinoid biosynthesis is not known at present. It has been found that it does not occur exclusively in the glandular hairs, that at least the number of glandular hairs on the bract surface does not correlate positively with cannabinoid content (Turner et al., 1977). Thus, it is likely that the site of cannabinoid biosynthesis is spread throughout the cells of the epidermis of the bracts and leaves, as well as in the cells of the glandular hairs.
If the site of cannabinoid biosynthesis were the glandular hairs, the most reproducible assay for cannabinoid production would involve the isolation of the hairs. This has been done for a member of the Lamiaceac (Croteau, 1977).
Number of glandular hairs would then be a second character not controlled by cannabinoid biosynthetic ability. Since the hairs are not the specific locus of biosynthesis, the most specific assay should choose a definable unit of epidermis, and this unit of epidermis is the bract. The leaves vary in size and would be hard to define precisely for sampling. The bract is less variable. if the age on sampling (maturity of the seed) is specified.
This technique is useful for herbarium specimens, with the advantage of disturbing valuable voucher specimens less than other methods.
We would suggest that this method be adopted in further studies of Cannabis breeding and cannabinoid biosynthesis. We intend to use it in the extension of the present experiment using botanically validated stocks of Cannabis sativa, C. indica, and C. ruderalis under more tightly controlled breeding conditions and using standard plant breeding formats, with more detailed attention to individual crosses. The F2 generation of the present cross will be followed to see if its cannabinoid content is different from the F1.
Other compounds (terpenes, flavonoids) will be examined for their utility as biochemical "markers."
SUMMARY
The results of this experiment strengthen the evidence that the cannabinoid content of Cannabis species is genetically controlled, and would suggest a search for the enzyme or enzyme system responsible for "cannabidiolic -acid cyclase" activity. Feeding experiments with radiolabelled CBDA and tissue homogenate fractions could result in the isolation of such an entity. This might in turn lead to the elucidation of a specific enzyme inhibitor which would block high potency strains from producing THC.
The experiments also indicate that C. sativa and C. ruderalis are interfertile under greenhouse conditions, and that the cannabinoid profiles, while differing in the amount of cannabinoid predominating, show no novel cannabinoids. This is one piece of evidence supporting the view that Cannabis is a monotypic genus. More studies are indicated to explore this further, particularly to note if other specific biochemically significant chemotaxonomic markers (e.g., essential oils, flavonoids) can be located which might lend validity to the polytypic concept.
ACKNOWLEDGMENTS
The authors are indebted to H. Fales and D.Phillipson for gc-mass spec work performed in their laboratories, to R. E. Schultes for stimulating criticism of the project, and to the Central Siberian Botanical Garden for the Cannabis ruderalis seeds.
LITERATURE CITED
1. Boucher, F., M. Paris & L. Cosson. 1977. Mise an evidence de deux types chimiques chez le Cannabis sativa originaire d'Afrique du Sud. Phylochemistry 16: 1445-1448.
2. Crombie, L. & W. M. L. Crombie. 1975. Cannabinoid formation in Cannabis sativa grafted inter-racially, and with two Hunulus species. Phytochemistry 14: 409-412.
3. Croteau, R. 1977. Site of monoterpene biosynthesis in Marjorana hortensis leaves. Pl. Phys. 59:519-520.
4. Emboden, W. A. 1974. -Cannaabis--a polytypic genus. Econ. Bot. 28: 304-310.
5. Fetterman, P. S., E. S. Keith, C. W. Wailer, 0. Guerrtro, N. J. Doorenbos & M. W. Quimby. 1971. Mississippi-grown Cannabis sativa L.: Preliminary observation on chemical definition of phenotype and variations in tetrahydrocannabinol content versus age, sex, and plant part. J. Pharrn. Sci. 60: 1246-1249, and other publications of the Mississippi group, e.g., Turner et al., 1975.
6. Fullerton, D. 5. & M. G. Kurzman. 1974. The identification and misidentification of marijuana. Contemporary Drug Problems. A Law Quarterly 3: 291-344.
7. Lerner, P. 1969. The precise determination of tetrahydrocannabinol in marihuana and hashish. Bull. on Narcotics 21: 39-42.
8. Paris, M., F. Boucher & L. Cosson. 1975. The constituents of Cannabis saliva pollen. Econ. Bot. 29: 245-253.
9. Schultes, R. E., W. M. Klein, T. Plowman & T. E. Lockwood. 1974. Cannabis: An example of taxonomic neglect. Bot. Mus. Leafl. 23: 337~367.
10. Shoyama, Y., M. Yagi & 1. Nishioka. 1974. Biosynthesis of cannabinoid acids. Phytochemistry 14: 2189-2192.
11. Small, E. 1972. lnterfertility and chromosomal uniformity in Cannabis. Canad. J. Bot. 50: 1947-1949.
12. - & H. D. Beekstead. 1973. Common cannabinoid phenotypes in 350 stocks of Cannabis. Lloydia 36: 144-165.
13. -, - & A. Chan. 1975. The evolution of cannabinoid phenotypes in Cannabis. Econ. Bot. 29: 219-232.
14. Small, E. & A. Cronquist. 1976. A practical and natural taxonomy for Cannabis. Taxon 25: 405 -435.
15. Turner, C. E., K. W. Hadley, J. H. Holley, S. Billets & M. L. Mole. 1975. Constituents of Cannabis sativa L. VIII. Possible biological application of a new method to separate cannabidiol and cannabichromene. 3. Pharm. Sci. 64: 810-814.
16. Turner, J. C., J. K. Hemphill & P. G. Mahiberg. 1977. Gland distribution and cannabinoid content in clones of Cannabis sativa L. Amer. J. Bot. 64: 687-693.
17. Vree, T. B. 1977. Mass spectrometry of cannabinoids. J. Pharm. Sci. 66: 1444-1450.
The Philadelphia College of Pharfnacy and Science, Department of Biological Sciences, Philadelphia, PA 19104.
Submitted in partial fulfilment of the degree of Master of Science, November 28, 1977.
Received for publication December 14, 1977, accepted for publication January 18, 1978
Interrelationships of glandular trichomes and cannabinoid content II.
Developing vegetative leaves of Cannabis sativa L. (Cannabaceae) Turner, J., J. Hemphill and P. Mahlberg. Interrelationships of glandular trichomes and cannabinoid content II. Developing vegetative leaves of Cannabis sativa L. (Cannabaceae). Bulletin on narcotics, 33:3:63-71, 1981.
BY
J. C. TURNER, J. K. HF-MPHILL and P. G. MAHLBERG
Department of Biology, Indiana University, Bloomington, Indiana, USA
*This research was supported by research grants to P. G. Mahlberg from the Department of Agriculture of the United States of America and the National Institute on Drug Abuse.
ABSTRACT
Gland number and cannabinoid content were quantified during ontogeny
of vegetative leaves from three clones of Cannabis. initiation of capitate-
sessile and bulbous glands was found to occur uniformly during leaf deve-
lopinent. Cannabinoids were synthesized throughout leaf development as
well, but at a decreasing rate. A positive correlation was found for total
capitate-sessile glands per leaf as compared with total cannabinoid content
of the leaf. The data also indicated that other leaf tissues in addition to the
glands may contain cannabinoids.
Introduction
Of the three types of glandular trichomes present on Cannabis sativa, only two (bulbous and capitate-sessile) are present on vegetative leaves, while capitate-stalked glands are found in association with the inflorescence (Harnmond and Mahlberg, 1973, 1977; Turner, Hemphill and Mahlberg, 1977, 1978). Both types of capitate glands have been implicated as major reservoirs of cannabinoids (Fujita, et aL, 1967; Fairbairn, 1972; DePasquale, 1974; Malingre et al., 1975; Andre and Vercruysse, 1976; Turner et aL, 1977, 1978). The vegetative leaf, therefore, represents an experimental system in which a specific capitate gland type can be studied with relation to the cannabinoids present in the leaf.
Previous work in our laboratory has revealed that the epidermal glandular trichomes present on Cannabis appear to he a complex and dynamic system in relation to both gland ontogeny and cannabinoid content (Hammond and Mahiberg, 1973, 1977, 1978; Turner et aL, 1977, 1978). In the first part of the current study (Turner, Hemphill and Mahlberg, 1981), a positive correlation was found between the total number of capitate glands and total cannabinoids in pistillate bracts. Also, the data suggested that the glands on a bract could contain the total cannabinoid content detected in the bract (Turner et al., 1981).
The purpose of this investigation is to examine the pattern of gland distribution as well as the cannabinoid profile of vegetative leaves throughout leaf ontogeny. The results should indicate to what degree the interrelationships of glands and cannabinoids found for vegetative leaves reflect those found for pistillate bracts.
Materials and methods
Clones
Plants of three Cannabis strains were selected and cloned (Turndr et al., 1977). These clones were derived from a high delta9-tetrahydrocannabinol (delta9-THC) strain (152), a low delta9-THC, high cannabidiol (CBD) strain (79), and a high CBD strain (87). Clones were grown under ambient greenhouse conditions.
Plant parts sampled
Selected leaves from each clone were analysed for their gland number and cannabinoid content. Centre leaflets of compound leaves of increasing length were collected from vegetative plants. Lengths included 2.5 crn leaflets (very young) to 12.5 cm leaflets (mature) at 2.5 cm intervals. Leaflet samples were collected in mid-July and early September 1979. Data presented in the figures are from the September collection.
Gas-liquid chromatography (GLC) and scanning electron microscopy
Leaves to be analysed were collected at a given time of the day (3 p.m.) and processed as described previously (Turner et al., 1977). Analyses by GLC were performed on a Hewlett-Packard 5710A chromatograph equipped with a 3380A H-P integrator. Samples prepared for scanning electron microscopy (SEM) were examined with an ETEC Autoscan.
Gland quantification
Gland number per unit area on leaves was determined by counting glands directly on the SEM screen (Turner et al., 1977). On leaflets, counts were made at the midpoint of the leaf blade, from the midrib to the margin. Multiple counts (16 fields, totalling l mm2) of both the adaxial and abaxial vein as well as non-vein areas were made, and the results were averaged to provide a mean for the sample.
In the current experiments, the abaxial non-vein areas on young leaves (2.5 - 7.5 cm) were too densely covered with non-glandular trichomes to obtain an accurate gland count. Thus, for comparative reasons, all data values for the leaflet samples were averaged without including counts from the abaxial areas.
The data for each leaf length sample were calculated as glands per mm2 and also as total glands per leaflet. For the estimated cannabinoid content of the individual glands, the glands counted on the abaxial non-vein areas of older leaves (10- 12.5 cm) were included in the calculations.
Results
Gland quantification on leaflets
At intervals of leaf ontogeny, the number of each gland type per rnm2 was determined. Data collected in both July and September were essentially identical. No capitate-stalked glands were observed on any leaf samples. Capitate-sessile glands were not only present at each stage of leaf development for these clones, but also their density remained relatively constant throughout leaf ontogeny (figures I-III).
Clone 152 averaged 2 capitate-sessile glands per mm2 (figure I), while clones 79 and 87 averaged 5 capitate-sessile glands per mm2 (figures II, III). Bulbous glands also were found to have relatively stable gland densities at all stages of leaf development for these clones (figures I-III). However, the highest gland densities of bulbous glands were found on clones 152 and 79, averaging 33 and 30 bulbous glands per mrn2 respectively (figures I,II) Clone 87 was found to have a lower density of bulbous glands with 21 per mm2 (figure III).
females, 3 males, one died before flowering) had THC percentages between 30 and 40% of total cannabinoids.
This rather conspicuous chemical dimorphism has no easy genetic explanation. It does not seem to be a case of one enzyme being responsible for the cyclization
of cannabidiolic acid to THCA. And if it were that simple, no such "cannabidiolic acid cyclase" has ever been isolated from Cannabis of any species.
Morphology. -ln habit, the progeny resembled both parents to. some extent. They did not show the dimorphism seen in cannabinoid content. Plants were between one and two ft tall, grew vigorously on germination, and flowered after putting out several pairs of true leaves. They continued their growth after initial flowering. The plants were self-crossed to produce an F2 generation of seed, which was saved for further plantings.
Some of the F1 seeds were slower to germinate than their C. sativa parent. Four seeds remained in the flat for four months before germination. These plants
were analyzed when mature, and their cannabinoid profiles fell within either of the two groups of progeny. Delay in germination is characteristic of plants growing outside of cultivation, either in a wild or weedy state. This trait was presumably transmitted from the C. ruderalis parent, although it was not observed in the seeds sown.
A voucher specimen of one of the female progeny has been deposited in the Library of Economic Botany of the Harvard Botanical Museum, as specimen no. 35896.
Such genetic inhomogeneity has not been reported before in strains of Cannabis. The nearest case is that of South African Cannabis reported by Boucher et al. (1977), in which they found the presence of two chemotypes varying in amounts of THCA and the C3 THCA homolog tetrahydrocannabivarolic acid.
Small et al.'s breeding experiments (1975) reported only that progeny obtained in crosses were "intermediate" in cannabinoid content. The aim of chemotaxonomy is to demonstrate that the variation in chemistry between two populations has a genetic basis dependent on the presence or absence of enzymes involved with the biosynthesis of the compounds in question.
Such information cannot be gotten from studies where populations are pooled or samples are not uniform. In order to do more meaningful work on individual plant cannabinoid content, it is necessary to find sampling methods which are not subject to wide variations, and which are precisely definable and reproducible.
Sampling. -We propose that the unit of sampling for female Cannabis plants be the mature bract, at the point where the seed is no longer green and is easily separable from the bract. For the male plant, perhaps the pollen can be used. It has been found in this and other studies (Paris et al., 1975) to contain the same proportions of cannabinoids as the rest of the plant. The male flower is less reproducible for sampling, since it is continuously shedding pollen at maturity, thereby changing the relative proportions of pollen and flower tissue.
This involves working with rather small amounts, since a mature bract when fresh weighs only about 2 mg. Pollen is most easily collected in milligram quantities. But the technique makes possible reproducible multiple samplings from each plant.
As an example of the technique, single fresh bracts were put into a 4 ml screw-capped vial with 0. 1 ml of chloroform overnight at room temperaature. In the morning 0.1 ml more of chloroform was added, along with 20 microliters of a methanolic solution of the internal standard, androstene-dione at a concentration of 1 mg/ml. The sample solution was injected into the glc as with the whole plant assay.
Results for this type of assay done with single bracts from the progeny of our cross indicate that the method gives comparable results to the whole plant assay for the percentage of cannabinoids. The percent total of cannabinoids is higher than for the whole plant, as would be expected.
Assuming that the bracts are relatively uniform within the same plant, how does this sampling method compare with other methods, such as the whole plant, or the manicured plant?
The whole plant analysis involves parts (seeds, roots, and woody stems) which have little if any cannabinoid content. The relative proportion of these parts to leaf and bract varies widely with cultural conditions; e.g., if the plant is grown under dry conditions, the root will become a very short, stocky structure, with few branchings; but if it is grown with adequate moisture it will be longer, thinner, and branched with many fine rootlets that are hard to collect completely. The amount of stem and vegetative growth varies also with plant nutrition.
These factors induce error in the measurement of the plant's actual genetic capacity to produce cannabinoids, while having nothing to do with cannabinoid
production. Cannabinoids and their precursors are formed entirely in the leafy portions of the plant (Crombie & Crombie, 1975).
The bract, assuming the flower is pollinated, is the most stable part of the plant both for morphology and for sampling.
The number of flowers produced on a plant will influence the relative levels between the bract sampling method and the whole plant assay. We have observed within the progeny of the cross a variation in the number of flowers produced per plant. This is reasonable because Cannabis has been bred for higher seed and flower production.
The Mexican parent produces seed quite heavily, while the presumably wild C. ruderalis produces relatively few seeds, concentrating its energies into the production of fewer, more viable seeds. This difference in characters shows its genetic base by its mixed transmission into the offspring of the
cross.
An aim of taxonomy is to find independent, definable characters, based where possible on a single gene system. The two traits of cannabinoid production and
number of flowers per plant are mixed using a whole or manicured plant assay, but not in a bract assay, because the proportion of dry weight due to high cannabinoid bracts is greater for many-seeded varieties of Cannabis than it is for those producing relatively few seeds.
The cellular locus of cannabinoid biosynthesis is not known at present. It has been found that it does not occur exclusively in the glandular hairs, that at least the number of glandular hairs on the bract surface does not correlate positively with cannabinoid content (Turner et al., 1977). Thus, it is likely that the site of cannabinoid biosynthesis is spread throughout the cells of the epidermis of the bracts and leaves, as well as in the cells of the glandular hairs.
If the site of cannabinoid biosynthesis were the glandular hairs, the most reproducible assay for cannabinoid production would involve the isolation of the hairs. This has been done for a member of the Lamiaceac (Croteau, 1977).
Number of glandular hairs would then be a second character not controlled by cannabinoid biosynthetic ability. Since the hairs are not the specific locus of biosynthesis, the most specific assay should choose a definable unit of epidermis, and this unit of epidermis is the bract. The leaves vary in size and would be hard to define precisely for sampling. The bract is less variable. if the age on sampling (maturity of the seed) is specified.
This technique is useful for herbarium specimens, with the advantage of disturbing valuable voucher specimens less than other methods.
We would suggest that this method be adopted in further studies of Cannabis breeding and cannabinoid biosynthesis. We intend to use it in the extension of the present experiment using botanically validated stocks of Cannabis sativa, C. indica, and C. ruderalis under more tightly controlled breeding conditions and using standard plant breeding formats, with more detailed attention to individual crosses. The F2 generation of the present cross will be followed to see if its cannabinoid content is different from the F1.
Other compounds (terpenes, flavonoids) will be examined for their utility as biochemical "markers."
SUMMARY
The results of this experiment strengthen the evidence that the cannabinoid content of Cannabis species is genetically controlled, and would suggest a search for the enzyme or enzyme system responsible for "cannabidiolic -acid cyclase" activity. Feeding experiments with radiolabelled CBDA and tissue homogenate fractions could result in the isolation of such an entity. This might in turn lead to the elucidation of a specific enzyme inhibitor which would block high potency strains from producing THC.
The experiments also indicate that C. sativa and C. ruderalis are interfertile under greenhouse conditions, and that the cannabinoid profiles, while differing in the amount of cannabinoid predominating, show no novel cannabinoids. This is one piece of evidence supporting the view that Cannabis is a monotypic genus. More studies are indicated to explore this further, particularly to note if other specific biochemically significant chemotaxonomic markers (e.g., essential oils, flavonoids) can be located which might lend validity to the polytypic concept.
ACKNOWLEDGMENTS
The authors are indebted to H. Fales and D.Phillipson for gc-mass spec work performed in their laboratories, to R. E. Schultes for stimulating criticism of the project, and to the Central Siberian Botanical Garden for the Cannabis ruderalis seeds.
LITERATURE CITED
1. Boucher, F., M. Paris & L. Cosson. 1977. Mise an evidence de deux types chimiques chez le Cannabis sativa originaire d'Afrique du Sud. Phylochemistry 16: 1445-1448.
2. Crombie, L. & W. M. L. Crombie. 1975. Cannabinoid formation in Cannabis sativa grafted inter-racially, and with two Hunulus species. Phytochemistry 14: 409-412.
3. Croteau, R. 1977. Site of monoterpene biosynthesis in Marjorana hortensis leaves. Pl. Phys. 59:519-520.
4. Emboden, W. A. 1974. -Cannaabis--a polytypic genus. Econ. Bot. 28: 304-310.
5. Fetterman, P. S., E. S. Keith, C. W. Wailer, 0. Guerrtro, N. J. Doorenbos & M. W. Quimby. 1971. Mississippi-grown Cannabis sativa L.: Preliminary observation on chemical definition of phenotype and variations in tetrahydrocannabinol content versus age, sex, and plant part. J. Pharrn. Sci. 60: 1246-1249, and other publications of the Mississippi group, e.g., Turner et al., 1975.
6. Fullerton, D. 5. & M. G. Kurzman. 1974. The identification and misidentification of marijuana. Contemporary Drug Problems. A Law Quarterly 3: 291-344.
7. Lerner, P. 1969. The precise determination of tetrahydrocannabinol in marihuana and hashish. Bull. on Narcotics 21: 39-42.
8. Paris, M., F. Boucher & L. Cosson. 1975. The constituents of Cannabis saliva pollen. Econ. Bot. 29: 245-253.
9. Schultes, R. E., W. M. Klein, T. Plowman & T. E. Lockwood. 1974. Cannabis: An example of taxonomic neglect. Bot. Mus. Leafl. 23: 337~367.
10. Shoyama, Y., M. Yagi & 1. Nishioka. 1974. Biosynthesis of cannabinoid acids. Phytochemistry 14: 2189-2192.
11. Small, E. 1972. lnterfertility and chromosomal uniformity in Cannabis. Canad. J. Bot. 50: 1947-1949.
12. - & H. D. Beekstead. 1973. Common cannabinoid phenotypes in 350 stocks of Cannabis. Lloydia 36: 144-165.
13. -, - & A. Chan. 1975. The evolution of cannabinoid phenotypes in Cannabis. Econ. Bot. 29: 219-232.
14. Small, E. & A. Cronquist. 1976. A practical and natural taxonomy for Cannabis. Taxon 25: 405 -435.
15. Turner, C. E., K. W. Hadley, J. H. Holley, S. Billets & M. L. Mole. 1975. Constituents of Cannabis sativa L. VIII. Possible biological application of a new method to separate cannabidiol and cannabichromene. 3. Pharm. Sci. 64: 810-814.
16. Turner, J. C., J. K. Hemphill & P. G. Mahiberg. 1977. Gland distribution and cannabinoid content in clones of Cannabis sativa L. Amer. J. Bot. 64: 687-693.
17. Vree, T. B. 1977. Mass spectrometry of cannabinoids. J. Pharm. Sci. 66: 1444-1450.
The Philadelphia College of Pharfnacy and Science, Department of Biological Sciences, Philadelphia, PA 19104.
Submitted in partial fulfilment of the degree of Master of Science, November 28, 1977.
Received for publication December 14, 1977, accepted for publication January 18, 1978
Interrelationships of glandular trichomes and cannabinoid content II.
Developing vegetative leaves of Cannabis sativa L. (Cannabaceae) Turner, J., J. Hemphill and P. Mahlberg. Interrelationships of glandular trichomes and cannabinoid content II. Developing vegetative leaves of Cannabis sativa L. (Cannabaceae). Bulletin on narcotics, 33:3:63-71, 1981.
BY
J. C. TURNER, J. K. HF-MPHILL and P. G. MAHLBERG
Department of Biology, Indiana University, Bloomington, Indiana, USA
*This research was supported by research grants to P. G. Mahlberg from the Department of Agriculture of the United States of America and the National Institute on Drug Abuse.
ABSTRACT
Gland number and cannabinoid content were quantified during ontogeny
of vegetative leaves from three clones of Cannabis. initiation of capitate-
sessile and bulbous glands was found to occur uniformly during leaf deve-
lopinent. Cannabinoids were synthesized throughout leaf development as
well, but at a decreasing rate. A positive correlation was found for total
capitate-sessile glands per leaf as compared with total cannabinoid content
of the leaf. The data also indicated that other leaf tissues in addition to the
glands may contain cannabinoids.
Introduction
Of the three types of glandular trichomes present on Cannabis sativa, only two (bulbous and capitate-sessile) are present on vegetative leaves, while capitate-stalked glands are found in association with the inflorescence (Harnmond and Mahlberg, 1973, 1977; Turner, Hemphill and Mahlberg, 1977, 1978). Both types of capitate glands have been implicated as major reservoirs of cannabinoids (Fujita, et aL, 1967; Fairbairn, 1972; DePasquale, 1974; Malingre et al., 1975; Andre and Vercruysse, 1976; Turner et aL, 1977, 1978). The vegetative leaf, therefore, represents an experimental system in which a specific capitate gland type can be studied with relation to the cannabinoids present in the leaf.
Previous work in our laboratory has revealed that the epidermal glandular trichomes present on Cannabis appear to he a complex and dynamic system in relation to both gland ontogeny and cannabinoid content (Hammond and Mahiberg, 1973, 1977, 1978; Turner et aL, 1977, 1978). In the first part of the current study (Turner, Hemphill and Mahlberg, 1981), a positive correlation was found between the total number of capitate glands and total cannabinoids in pistillate bracts. Also, the data suggested that the glands on a bract could contain the total cannabinoid content detected in the bract (Turner et al., 1981).
The purpose of this investigation is to examine the pattern of gland distribution as well as the cannabinoid profile of vegetative leaves throughout leaf ontogeny. The results should indicate to what degree the interrelationships of glands and cannabinoids found for vegetative leaves reflect those found for pistillate bracts.
Materials and methods
Clones
Plants of three Cannabis strains were selected and cloned (Turndr et al., 1977). These clones were derived from a high delta9-tetrahydrocannabinol (delta9-THC) strain (152), a low delta9-THC, high cannabidiol (CBD) strain (79), and a high CBD strain (87). Clones were grown under ambient greenhouse conditions.
Plant parts sampled
Selected leaves from each clone were analysed for their gland number and cannabinoid content. Centre leaflets of compound leaves of increasing length were collected from vegetative plants. Lengths included 2.5 crn leaflets (very young) to 12.5 cm leaflets (mature) at 2.5 cm intervals. Leaflet samples were collected in mid-July and early September 1979. Data presented in the figures are from the September collection.
Gas-liquid chromatography (GLC) and scanning electron microscopy
Leaves to be analysed were collected at a given time of the day (3 p.m.) and processed as described previously (Turner et al., 1977). Analyses by GLC were performed on a Hewlett-Packard 5710A chromatograph equipped with a 3380A H-P integrator. Samples prepared for scanning electron microscopy (SEM) were examined with an ETEC Autoscan.
Gland quantification
Gland number per unit area on leaves was determined by counting glands directly on the SEM screen (Turner et al., 1977). On leaflets, counts were made at the midpoint of the leaf blade, from the midrib to the margin. Multiple counts (16 fields, totalling l mm2) of both the adaxial and abaxial vein as well as non-vein areas were made, and the results were averaged to provide a mean for the sample.
In the current experiments, the abaxial non-vein areas on young leaves (2.5 - 7.5 cm) were too densely covered with non-glandular trichomes to obtain an accurate gland count. Thus, for comparative reasons, all data values for the leaflet samples were averaged without including counts from the abaxial areas.
The data for each leaf length sample were calculated as glands per mm2 and also as total glands per leaflet. For the estimated cannabinoid content of the individual glands, the glands counted on the abaxial non-vein areas of older leaves (10- 12.5 cm) were included in the calculations.
Results
Gland quantification on leaflets
At intervals of leaf ontogeny, the number of each gland type per rnm2 was determined. Data collected in both July and September were essentially identical. No capitate-stalked glands were observed on any leaf samples. Capitate-sessile glands were not only present at each stage of leaf development for these clones, but also their density remained relatively constant throughout leaf ontogeny (figures I-III).
Clone 152 averaged 2 capitate-sessile glands per mm2 (figure I), while clones 79 and 87 averaged 5 capitate-sessile glands per mm2 (figures II, III). Bulbous glands also were found to have relatively stable gland densities at all stages of leaf development for these clones (figures I-III). However, the highest gland densities of bulbous glands were found on clones 152 and 79, averaging 33 and 30 bulbous glands per mrn2 respectively (figures I,II) Clone 87 was found to have a lower density of bulbous glands with 21 per mm2 (figure III).
The total number of capitate-sessite glands per leaflet also was determined for these clones during leaf development (figures IV-VI). On each clone, an increase in total gland number was seen as the leaf developed, although the clones differed in the specific numbers of capitate-sessile glands found (figures IV-VI).
Clone 152 had fewer glands at each development stage than clones 79. The total number of capitate-sessite glands per leaflet also was determined for these clones during leaf development (figures IV-VI).
On each clone, an increase in total gland number was seen as the leaf developed, although the clones differed in the specific numbers of capitate-sessile glands found (figures IV-VI). Clone 152 had fewer glands at each development stage than clones 79 and 87 (figure VIl). Clones 79 and 87 had similar numbers of glands except on mature leaflets (12.5 cm) where clone 87 had a higher number of capitate-sessile glands (figure VII).
Clone 152 had fewer glands at each development stage than clones 79. The total number of capitate-sessite glands per leaflet also was determined for these clones during leaf development (figures IV-VI).
On each clone, an increase in total gland number was seen as the leaf developed, although the clones differed in the specific numbers of capitate-sessile glands found (figures IV-VI). Clone 152 had fewer glands at each development stage than clones 79 and 87 (figure VIl). Clones 79 and 87 had similar numbers of glands except on mature leaflets (12.5 cm) where clone 87 had a higher number of capitate-sessile glands (figure VII).
Cannabinoid concentration, on a dry weight basis, decreased in each of the clones throughout leaflet ontogeny (figures VIII -X). Although both clones 79 and 87 characteristically have high levels of CBD, clone 79 had higher levels of CBD at most stages of leaflet development than clone 87 (figures IX, X), Clone 152, a delta9-THC strain, had levels of cannabinoids (figure VIII) comparable to cannabinoid levels found for clone 87 (figure X). The clones each contained concentrations of their characteristic cannabinoid at levels similar to the total cannabinoid concentration detected (figures VIII-X).
Expressed on a per leaflet basis, total cannabinoids increased during leaf ontogeny in each of the clones (figures IV-VI). The youngest as well as the most mature leaflets of the clones contained similar quantities of cannabinoids (figure XI). However, at intermediate stages, clones 152 and 87 had lower quantities of cannabinoids than clone 79 (figure XI). The increase in total cannabinoids per leaflet, in general, paralleled the increase in total capitate-sessile glands per leaflet throughout leaf ontogeny (figures IV - VI).
The density of non-glandular trichomes decreased slightly and at a similar rate on each clone as the leaflets enlarged (figure XII). Clones 79 and 87 were found to have comparable densities of trichomes during leaf ontogeny. However, clone 152 had approximately 25 per cent more trichornes per unit area throughout leaflet development than clone 79 or 87 (figure XIl).
Discussion
Vegetative leaves of Cannabis provide an opportunity to study one capitate gland type (capitate-sessile) with relation to cannabinoid content without the presence of capitate-stalked glands. The pattern of gland distribution during leaf ontogeny differed considerably from that found during development of the pistillate bract (Turner et al, 1981). Both gland types present on the leaf, capitate-sessile and bulbous, were found to maintain a relatively constant density throughout leaf development on each of the clones.
In comparison, both capitate-sessile and bulbous glands on bracts decreased in density during bract ontogeny. To what extent, if any, the appearance and increase in density of capitate-stalked glands during bract development influenced the decrease of capitate-sessile glands is unknown. However, on leaves, where no capitate-stalked glands were present, capitate-sessile glands did not decrease in density during leaf ontogeny. The considerations involved in determining patterns of gland density during leaf and bract development are unknown.
Although the density of the glands on each clone remained constant throughout leaf development, the specific numbers of each gland type varied from clone to clone. Clones 79 and 87, which are characteristically high in CBD, were found to have higher densities of capitate-sessile glands than clone 152 which contained high levels of delta9-THC.
Whether gland density in leaf ontogeny is influenced by the characteristic cannabinoid present in a strain is unknown. However, the presence of either delta9-THC or CBD as the characteristic cannabinoid did not appear to he a major factor in determining the cannabinoid concentration found in a clone.
For example, clone 152, a delta9-THC-containing strain, and clone 87, a CBD-containing strain, were both found to have lower concentrations of cannabinoids than clone 79, which also is a CBD-containing strain. Although cannabinoid concentrations differed quantitatively among the clones, a trend of decreasing concentration during leaf development was found in each of the clones. In contrast, the trends for cannabinoid concentrations in developing bracts were different for each of the clones (Turner et al., 1981).
Comparing cannabinoid concentrations with gland densities in these clones, it was evident that for vegetative leaves as well as pistillate bracts, there were no positive correlations between any individual gland type and any characteristic cannabinoid.
Non-glandular trichomes, reported to lack cannabinoids (Malingre, et al., 1975), were found to decrease in density at a uniform rate during leaf ontogeny. This rate of decrease was slower than the rate of increase in leaf area indicating non-glandular trichomes were continually initiated during leaf development. However, fewer non-glandular trichomes were formed at each developmental stage than glandular trichomes.
A comparison of total capitate-sessile glands per leaflet with total cannabinoids per leaflet on each of the clones indicated a positive correlation as both parameters were found to increase throughout leaf ontogeny. A similar positive correlation for these parameters was also found to exist for the pistillate bracts (Turner et al., 1981). In addition, as found for the bracts, the increase in both of these parameters indicates that initiation of glandular trichonies and synthesis of cannabinoids occurs throughout leaf ontogeny.
Although these clones were similar and showed an increase in total capitate-sessile glands per leaflet and total cannabinoids per leaflet as the leaf developed, gland number and cannabinoid levels usually differed from clone to clone.
Total gland number per leaflet showed the greatest variability among the clones at the most mature stage of leaf development. However, while clones 79 and 87 (both high in CBD) were relatively similar in gland number until the most mature stage, clone 152 (high in A)-THC) had fewer glands at almost every developmental stage.
Whether this is due to genetic or environmental factors remains to be determined. Total amounts of cannabinoids per leaflet were generally comparable among the clones throughout leaf development, although clone 79 had higher levels at intermediate leaf stages.
As demonstrated for capitate glands on bracts (Turner et al., 1981), total cannabinoids detected per leaflet can be divided by the total number of capitate-
sessile glands present per leaflet to provide an estimated amount of cannabinoids per single gland. This estimation was made for all developmental stages for each clone.
When this was done for vegetative leaves, the estimated cannabinoid content per individual gland (45 ng for clone 152, 47 ng for clone 79, and 88 ng for clone 87) was higher than the cannabinoid content found for previously analysed individual glands (Turner et al., 1978). This finding differs considerably from what was found for the bract (Turner et al., 1981).
On pistillate bracts, it was estimated that the gland population could contain essentially all of the cannabinoids detected in a bract. However, for vegetative leaves, it appears that the glands contain only part of the cannabinolds detected in the leaf, and, therefore, other leaf tissue may be involved in cannabinoid synthesis or accumulation.
In conclusion, this study has revealed several aspects of the interrelationship of glands and cannabinoids on Cannabis. On vegetative leaves, as on pistillate bracts, glands and cannabinoids are produced throughout organ development, although at different rates for the leaf as compared to the bract. Non-glandular trichomes are also initiated continuously during organ ontogeny.
A positive correlation, as was found for the bracts, exists between total numbers of glands and total cannabinoids per leaflet. However, although glands on the bract appear able to contain almost all of the cannabinoid content of the bract, the glands on the leaf apparently contain only part of the cannabinoids detected in the leaf. It seems likely that cannabinoids are present in other tissues of the leaf. Further experiments are in progress to determine the specific site of cannabinoid synthesis in the plant.
Bibliography
Andre, C. and A. Vercruysse. Histochemical study of the stalked glandular hairs of the female Cannabis plants, using fast blue salt. Planta medica, 29:361-366, 1976.
DePasquale, A. Ultrastructure of the Cannabis sativa glands. Planta' medica, 25:238-248, 1974.
Malingre, T., et al. The essential oil of Cannabis sativa. Planta medica, 28:56-61, 1975.
Fairbairn, J. The trichomes and glands of Cannabis sativa L. Bulletin on narcotics, 24:4:29-33, 1972.
Fujita, M. and others. Studies on Cannabis. II. Examination of the narcotic and its related components in hemps, crude drugs and plant organs by gas-liquid chromatography and thin-layer chromatography. Annual report of the Tokyo College of Pharmacy, 17:238-242, 1967.
Hammond, C. and P. Mahlberg. Morphology of glandular hairs of Cannabis sativa from scanning electron rnicroscopy. American journal of botany, 60:524-528, 1973.
Hammond, C. and P. Mahlberg. Morphogenesis of capitate glandular hairs of Cannabis sativa (Cannabaceae). American journal of botany, 64:1023-1031, 1977.
Hammond, C. and P. Mahlberg. Ultrastructural development of capitate glandular hairs of Cannabis sativa L. (Cannabaccae). American journal of botany, 65:140-151, 1978.
Ledbetter, M. and A. Krikorian. Trichornes of Cannabis sativa as viewed with scanning electron microscopy. Phytomorphology, 25:166-176, 1975.
Turner, J., J. Hemphill and P. Mahlberg. Gland distribution and cannabinoid content in clones of Cannabis sativa L. American journal of botany, 64:687-693, 1977.
Turner, J., J. Hemphill and P. Mahlberg. Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. (Cannabaccae). American joumal of botany, 65:1103-1106, 1978.
Turner, J., J. Hemphill and P. Mahlberg Interrelationships of glandular trichomes and cannabinoid content. I: Developing pistillate bracts of Cannabis sativa L. (Cannabaceac). Bulletin on narcotics, 33:2:59-69, 1981.
Constituents of Cannabis sativa L., XX: the cannabinoid content of Mexican variants grown in Mexico and in Mississippi, United States of AmericaTurner, C.E., H.N. Elsohly, and G.S. Lewis. "Constituents of Cannabis sativa L., XX: the cannabinoidcontent of Mexican variants grown in Mexico and in
Mississippi, United States of America" Bulletin on Narcotics, vol. 34, No.1 pp 45-59 1982
C. E. TURNER, H. N. ELSOHLY and G. S. LEWIS
,Research Institute of Pharmaceutical Sciences. School of Pharmacy, University of
Mississippi, University, Mississippi, United Stages of America
1. LOPEZ-SANTIBANEZ
office of Procuraduria General de la Republica, Mexico City, D. F., Mexico
I.CARRANZA
,Research and Education Department, Instituto Mexicano del Seguro Social, Mexico City, Mexico
ABSTRACT
Cannabis growing in 12 different localities in Mexico was analysed quantitatively for
10 cannabinoids by gas-liquid chromatography. Cannabinoid profiles of 12 variants
from Mexico were compared with profiles derived when 7 out of these 12 variants
were grown in Mississippi, United States of America. The effects of temperature and
rainfall on the delta9-THC content of these variants are presented.
Introduction
The crude marijuana produced from Mexican Cannabis is well known for its potent drug quality. Previous studies by Turner and others on a Mexican variant (ME-A) and Indian variants showed that the major cannabinoids cannabichromene (CBC), cannabidiol (CBD), delta9-tetrahydrocannabinol (delta9-THC) and cannabinot (CBN), vary in a cyclic pattern and are a function of time of day, sex, age of plants at sampling, and parts of plants sampled.
Cannabis has been divided into three distinct types - the drug, fibre and an intermediate type. The last is mostly used to produce hashish. The fibre type is used to produce fibres, and the drug type is used for its psychoactive properties.
The psychoactive agent in Cannabis is generally referred to as (-)-trans-,delta9-tetrahydrocannabinol (A9-THC). As the supplier of research marijuana for the National Institute on Drug Abuse (NIDA), we have the responsibility to correlate analytical data, when possible, with biological data.
Previous to recent work in these laboratories demonstrating that a Cannabis plant could be a fibre type one day and another type on a different day, marijuana samples from a particular geographical location were assunned to be identical and delta9-THC was thought to be the only cannabinoid of importance in understanding the pharmacology of the drug. The delta9-THC content of any marijuana sample cannot explain all the biological properties experimentally observed.
Therefore, the study of the fluctuation of cannabinoids in different types becomes important in correlating the pharmacology of marijuana with the analytical data.
The research findings discussed above prompted a complete evaluation of the cannabinoid profile and chemical classification of Cannabis grown in Mexico and subsequently grown in Mississippi, United States of America.
The drug type Cannabis, grown in Mississippi and used to produce NIDA's standard research marijuana, originated from a seed stock obtained near Acapulco in the state of Guerrere, Mexico. Since Acapulco is on the west coast of Mexico, and since considerable illicit Mexican marijuana originates on the east coast near Veracruz, it was necessary to investigate Cannabis growing in diverse locations within Mexico.
Material and methods
Plant material
Twelve Mexican-grown Cannabis samples were collected near 12 cities in 10 different states in 1973, plus one Cannabis seed sample from the state of Oaxaca (see table 1).
The dry plant material, mostly flowering tops, was manicured, and leaves were separated from stems and seeds by passing it through a 14-mesh sieve. During 1974 and 1976, seeds of seven Mexican variants of known geographic origin were grown in the medicinal plant garden at the Research Institute of Pharmaceutical Sciences, University of Mississippi, under the same soil and fertilization conditions.
Leaves were randomly collected from bottom to top, from six different plants of the same variant at the same time and day of each week. The age of the plants was recorded from the dates of planting. Samples from vegetative, staminate and pistillate plants of each variant were collected, air dried and manicured.
For this investigation, 7 seed stocks were selected from 12 Mexican Cannabis samples originally collected in 1973 from the following 10 states: Aquascalientes, Campeche, Chihuahua, Durango, Guerrero, Jalisw, Nayarit, Puebla, Sinaloa and Veracruz.
These locations provided a relatively good cross-section of different geographical locations within Mexico. A weekly cannabinoid analysis was performed on the randomly collected leaves in order to determine the cannabinoid growth profiles for these variants grown in Mississippi. Data from these experiments were compared with data from the original analysis of the sample obtained in Mexico.
Vegetative leaves of Cannabis provide an opportunity to study one capitate gland type (capitate-sessile) with relation to cannabinoid content without the presence of capitate-stalked glands. The pattern of gland distribution during leaf ontogeny differed considerably from that found during development of the pistillate bract (Turner et al, 1981). Both gland types present on the leaf, capitate-sessile and bulbous, were found to maintain a relatively constant density throughout leaf development on each of the clones.
In comparison, both capitate-sessile and bulbous glands on bracts decreased in density during bract ontogeny. To what extent, if any, the appearance and increase in density of capitate-stalked glands during bract development influenced the decrease of capitate-sessile glands is unknown. However, on leaves, where no capitate-stalked glands were present, capitate-sessile glands did not decrease in density during leaf ontogeny. The considerations involved in determining patterns of gland density during leaf and bract development are unknown.
Although the density of the glands on each clone remained constant throughout leaf development, the specific numbers of each gland type varied from clone to clone. Clones 79 and 87, which are characteristically high in CBD, were found to have higher densities of capitate-sessile glands than clone 152 which contained high levels of delta9-THC.
Whether gland density in leaf ontogeny is influenced by the characteristic cannabinoid present in a strain is unknown. However, the presence of either delta9-THC or CBD as the characteristic cannabinoid did not appear to he a major factor in determining the cannabinoid concentration found in a clone.
For example, clone 152, a delta9-THC-containing strain, and clone 87, a CBD-containing strain, were both found to have lower concentrations of cannabinoids than clone 79, which also is a CBD-containing strain. Although cannabinoid concentrations differed quantitatively among the clones, a trend of decreasing concentration during leaf development was found in each of the clones. In contrast, the trends for cannabinoid concentrations in developing bracts were different for each of the clones (Turner et al., 1981).
Comparing cannabinoid concentrations with gland densities in these clones, it was evident that for vegetative leaves as well as pistillate bracts, there were no positive correlations between any individual gland type and any characteristic cannabinoid.
Non-glandular trichomes, reported to lack cannabinoids (Malingre, et al., 1975), were found to decrease in density at a uniform rate during leaf ontogeny. This rate of decrease was slower than the rate of increase in leaf area indicating non-glandular trichomes were continually initiated during leaf development. However, fewer non-glandular trichomes were formed at each developmental stage than glandular trichomes.
A comparison of total capitate-sessile glands per leaflet with total cannabinoids per leaflet on each of the clones indicated a positive correlation as both parameters were found to increase throughout leaf ontogeny. A similar positive correlation for these parameters was also found to exist for the pistillate bracts (Turner et al., 1981). In addition, as found for the bracts, the increase in both of these parameters indicates that initiation of glandular trichonies and synthesis of cannabinoids occurs throughout leaf ontogeny.
Although these clones were similar and showed an increase in total capitate-sessile glands per leaflet and total cannabinoids per leaflet as the leaf developed, gland number and cannabinoid levels usually differed from clone to clone.
Total gland number per leaflet showed the greatest variability among the clones at the most mature stage of leaf development. However, while clones 79 and 87 (both high in CBD) were relatively similar in gland number until the most mature stage, clone 152 (high in A)-THC) had fewer glands at almost every developmental stage.
Whether this is due to genetic or environmental factors remains to be determined. Total amounts of cannabinoids per leaflet were generally comparable among the clones throughout leaf development, although clone 79 had higher levels at intermediate leaf stages.
As demonstrated for capitate glands on bracts (Turner et al., 1981), total cannabinoids detected per leaflet can be divided by the total number of capitate-
sessile glands present per leaflet to provide an estimated amount of cannabinoids per single gland. This estimation was made for all developmental stages for each clone.
When this was done for vegetative leaves, the estimated cannabinoid content per individual gland (45 ng for clone 152, 47 ng for clone 79, and 88 ng for clone 87) was higher than the cannabinoid content found for previously analysed individual glands (Turner et al., 1978). This finding differs considerably from what was found for the bract (Turner et al., 1981).
On pistillate bracts, it was estimated that the gland population could contain essentially all of the cannabinoids detected in a bract. However, for vegetative leaves, it appears that the glands contain only part of the cannabinolds detected in the leaf, and, therefore, other leaf tissue may be involved in cannabinoid synthesis or accumulation.
In conclusion, this study has revealed several aspects of the interrelationship of glands and cannabinoids on Cannabis. On vegetative leaves, as on pistillate bracts, glands and cannabinoids are produced throughout organ development, although at different rates for the leaf as compared to the bract. Non-glandular trichomes are also initiated continuously during organ ontogeny.
A positive correlation, as was found for the bracts, exists between total numbers of glands and total cannabinoids per leaflet. However, although glands on the bract appear able to contain almost all of the cannabinoid content of the bract, the glands on the leaf apparently contain only part of the cannabinoids detected in the leaf. It seems likely that cannabinoids are present in other tissues of the leaf. Further experiments are in progress to determine the specific site of cannabinoid synthesis in the plant.
Bibliography
Andre, C. and A. Vercruysse. Histochemical study of the stalked glandular hairs of the female Cannabis plants, using fast blue salt. Planta medica, 29:361-366, 1976.
DePasquale, A. Ultrastructure of the Cannabis sativa glands. Planta' medica, 25:238-248, 1974.
Malingre, T., et al. The essential oil of Cannabis sativa. Planta medica, 28:56-61, 1975.
Fairbairn, J. The trichomes and glands of Cannabis sativa L. Bulletin on narcotics, 24:4:29-33, 1972.
Fujita, M. and others. Studies on Cannabis. II. Examination of the narcotic and its related components in hemps, crude drugs and plant organs by gas-liquid chromatography and thin-layer chromatography. Annual report of the Tokyo College of Pharmacy, 17:238-242, 1967.
Hammond, C. and P. Mahlberg. Morphology of glandular hairs of Cannabis sativa from scanning electron rnicroscopy. American journal of botany, 60:524-528, 1973.
Hammond, C. and P. Mahlberg. Morphogenesis of capitate glandular hairs of Cannabis sativa (Cannabaceae). American journal of botany, 64:1023-1031, 1977.
Hammond, C. and P. Mahlberg. Ultrastructural development of capitate glandular hairs of Cannabis sativa L. (Cannabaccae). American journal of botany, 65:140-151, 1978.
Ledbetter, M. and A. Krikorian. Trichornes of Cannabis sativa as viewed with scanning electron microscopy. Phytomorphology, 25:166-176, 1975.
Turner, J., J. Hemphill and P. Mahlberg. Gland distribution and cannabinoid content in clones of Cannabis sativa L. American journal of botany, 64:687-693, 1977.
Turner, J., J. Hemphill and P. Mahlberg. Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. (Cannabaccae). American joumal of botany, 65:1103-1106, 1978.
Turner, J., J. Hemphill and P. Mahlberg Interrelationships of glandular trichomes and cannabinoid content. I: Developing pistillate bracts of Cannabis sativa L. (Cannabaceac). Bulletin on narcotics, 33:2:59-69, 1981.
Constituents of Cannabis sativa L., XX: the cannabinoid content of Mexican variants grown in Mexico and in Mississippi, United States of AmericaTurner, C.E., H.N. Elsohly, and G.S. Lewis. "Constituents of Cannabis sativa L., XX: the cannabinoidcontent of Mexican variants grown in Mexico and in
Mississippi, United States of America" Bulletin on Narcotics, vol. 34, No.1 pp 45-59 1982
C. E. TURNER, H. N. ELSOHLY and G. S. LEWIS
,Research Institute of Pharmaceutical Sciences. School of Pharmacy, University of
Mississippi, University, Mississippi, United Stages of America
1. LOPEZ-SANTIBANEZ
office of Procuraduria General de la Republica, Mexico City, D. F., Mexico
I.CARRANZA
,Research and Education Department, Instituto Mexicano del Seguro Social, Mexico City, Mexico
ABSTRACT
Cannabis growing in 12 different localities in Mexico was analysed quantitatively for
10 cannabinoids by gas-liquid chromatography. Cannabinoid profiles of 12 variants
from Mexico were compared with profiles derived when 7 out of these 12 variants
were grown in Mississippi, United States of America. The effects of temperature and
rainfall on the delta9-THC content of these variants are presented.
Introduction
The crude marijuana produced from Mexican Cannabis is well known for its potent drug quality. Previous studies by Turner and others on a Mexican variant (ME-A) and Indian variants showed that the major cannabinoids cannabichromene (CBC), cannabidiol (CBD), delta9-tetrahydrocannabinol (delta9-THC) and cannabinot (CBN), vary in a cyclic pattern and are a function of time of day, sex, age of plants at sampling, and parts of plants sampled.
Cannabis has been divided into three distinct types - the drug, fibre and an intermediate type. The last is mostly used to produce hashish. The fibre type is used to produce fibres, and the drug type is used for its psychoactive properties.
The psychoactive agent in Cannabis is generally referred to as (-)-trans-,delta9-tetrahydrocannabinol (A9-THC). As the supplier of research marijuana for the National Institute on Drug Abuse (NIDA), we have the responsibility to correlate analytical data, when possible, with biological data.
Previous to recent work in these laboratories demonstrating that a Cannabis plant could be a fibre type one day and another type on a different day, marijuana samples from a particular geographical location were assunned to be identical and delta9-THC was thought to be the only cannabinoid of importance in understanding the pharmacology of the drug. The delta9-THC content of any marijuana sample cannot explain all the biological properties experimentally observed.
Therefore, the study of the fluctuation of cannabinoids in different types becomes important in correlating the pharmacology of marijuana with the analytical data.
The research findings discussed above prompted a complete evaluation of the cannabinoid profile and chemical classification of Cannabis grown in Mexico and subsequently grown in Mississippi, United States of America.
The drug type Cannabis, grown in Mississippi and used to produce NIDA's standard research marijuana, originated from a seed stock obtained near Acapulco in the state of Guerrere, Mexico. Since Acapulco is on the west coast of Mexico, and since considerable illicit Mexican marijuana originates on the east coast near Veracruz, it was necessary to investigate Cannabis growing in diverse locations within Mexico.
Material and methods
Plant material
Twelve Mexican-grown Cannabis samples were collected near 12 cities in 10 different states in 1973, plus one Cannabis seed sample from the state of Oaxaca (see table 1).
The dry plant material, mostly flowering tops, was manicured, and leaves were separated from stems and seeds by passing it through a 14-mesh sieve. During 1974 and 1976, seeds of seven Mexican variants of known geographic origin were grown in the medicinal plant garden at the Research Institute of Pharmaceutical Sciences, University of Mississippi, under the same soil and fertilization conditions.
Leaves were randomly collected from bottom to top, from six different plants of the same variant at the same time and day of each week. The age of the plants was recorded from the dates of planting. Samples from vegetative, staminate and pistillate plants of each variant were collected, air dried and manicured.
For this investigation, 7 seed stocks were selected from 12 Mexican Cannabis samples originally collected in 1973 from the following 10 states: Aquascalientes, Campeche, Chihuahua, Durango, Guerrero, Jalisw, Nayarit, Puebla, Sinaloa and Veracruz.
These locations provided a relatively good cross-section of different geographical locations within Mexico. A weekly cannabinoid analysis was performed on the randomly collected leaves in order to determine the cannabinoid growth profiles for these variants grown in Mississippi. Data from these experiments were compared with data from the original analysis of the sample obtained in Mexico.
Cannabinoid analysis
Manicured plant material (1 g) of each variant was extracted with chloroform. The residue was dissolved in 1 ml of internal standard solution (androst-4-ene-3, 17-dione 1 per cent in ethanol). The resulting solution was quantitatively analysed by gas-liquid chromatography, using a Beckman GC-65 equipped with a 2 per cent OV- 1 7 column. A 6 per cent OV-1 column was also used to quantitate cannabicyclol (CBL), (-)-trans-delta9-tetrahydrocannabivarin (delta9-THCV), CBC, and CBD [2].
Results
As a result of cannabinoid analysis of different Mexican Cannabis variants (see table 2) collected in different locations in Mexico, delta9-THC was found to be the most abundant cannabinoid. The average content of delta9-THC was 1.69 per cent, with the highest 2.97 per cent, and the lowest 0. 14 per cent. Cannabinol (CBN) and (-)-trans- delta8-tetrahydrocannabinol (delta8-THC) were not present in fresh samples of Cannabis.
However, during storage and the analytical process, delta9-THC acid decarboxylates to give delta9-THC, which then went through hydroxy intermediates to give CBN by air oxidation or delta8-THC by isomerization [51. When we combined the delta9-THC, delta9-THCV, delta8-THC, and CBN content, the average percentage of the psychologically active cannabinoids in samples of Mexican Cannabis grown in Mexico was 2.02, with the highest 3.27 and the lowest 0.71.
All but one of the Mexican-grown Cannabis variants contained only trace, or undetectable, amounts of CBD. The exception, ME-H, was collected from Poza Rica, Veracruz, and had 0.54 per cent of CBD (see table 2). Plants of this variant grown in Mississippi also showed significant amounts of CBD.
In 1974, only vegetative plants showed maximum CBD levels at 12 weeks of age (0.38 per cent) and 16 weeks of age (0.37 per cent), while staminate and pistillate plants contained only trace amounts of CBD (see table 3). Other Mexican variants planted in 1974 and 1976 in Mississippi usually contained less than 0.05 per cent of CBD (see tables 3 and 4). In 1976, vegetative plants contained 0.90 per cent of CBD at 13 weeks of age and 0.38 per cent at 16 weeks of age (see table 4). For staminate plants, maximum CBD amounts were found at 19 weeks of age (1.19 per cent) and 24 weeks of age (0.91 per cent).
For pistillate plants, maximum CBD percentages were found at 19 weeks of age (0.67 per cent) and 21 weeks of age (0.63 per cent) (see table 4).
Manicured plant material (1 g) of each variant was extracted with chloroform. The residue was dissolved in 1 ml of internal standard solution (androst-4-ene-3, 17-dione 1 per cent in ethanol). The resulting solution was quantitatively analysed by gas-liquid chromatography, using a Beckman GC-65 equipped with a 2 per cent OV- 1 7 column. A 6 per cent OV-1 column was also used to quantitate cannabicyclol (CBL), (-)-trans-delta9-tetrahydrocannabivarin (delta9-THCV), CBC, and CBD [2].
Results
As a result of cannabinoid analysis of different Mexican Cannabis variants (see table 2) collected in different locations in Mexico, delta9-THC was found to be the most abundant cannabinoid. The average content of delta9-THC was 1.69 per cent, with the highest 2.97 per cent, and the lowest 0. 14 per cent. Cannabinol (CBN) and (-)-trans- delta8-tetrahydrocannabinol (delta8-THC) were not present in fresh samples of Cannabis.
However, during storage and the analytical process, delta9-THC acid decarboxylates to give delta9-THC, which then went through hydroxy intermediates to give CBN by air oxidation or delta8-THC by isomerization [51. When we combined the delta9-THC, delta9-THCV, delta8-THC, and CBN content, the average percentage of the psychologically active cannabinoids in samples of Mexican Cannabis grown in Mexico was 2.02, with the highest 3.27 and the lowest 0.71.
All but one of the Mexican-grown Cannabis variants contained only trace, or undetectable, amounts of CBD. The exception, ME-H, was collected from Poza Rica, Veracruz, and had 0.54 per cent of CBD (see table 2). Plants of this variant grown in Mississippi also showed significant amounts of CBD.
In 1974, only vegetative plants showed maximum CBD levels at 12 weeks of age (0.38 per cent) and 16 weeks of age (0.37 per cent), while staminate and pistillate plants contained only trace amounts of CBD (see table 3). Other Mexican variants planted in 1974 and 1976 in Mississippi usually contained less than 0.05 per cent of CBD (see tables 3 and 4). In 1976, vegetative plants contained 0.90 per cent of CBD at 13 weeks of age and 0.38 per cent at 16 weeks of age (see table 4). For staminate plants, maximum CBD amounts were found at 19 weeks of age (1.19 per cent) and 24 weeks of age (0.91 per cent).
For pistillate plants, maximum CBD percentages were found at 19 weeks of age (0.67 per cent) and 21 weeks of age (0.63 per cent) (see table 4).
The fluctuation of delta9-THC levels in ME-N planted in 1976, was similar to that of ME-A planted in 1971 [1). Both ME-N and ME-A seeds came from Acapulco, Guerrero.
Vegetative plants showed maximum levels of delta9-THC at week 15 and minimum levels at weeks 17 and 18. Staminate plants contained maximum amounts of delta9-THC at week 20 and minimum at week 22. The pistillate plants showed maximum amounts at week 19 and minimum at week 23.The highest delta9-THC level was in the vegetative plant prior to any sexual differentiation at week 15. However, in 1974, ME-N vegetative plants showed a maximum amount of delta9-THC at week 18. Staminate and pistillate plants had maximum levels of delta9-THC at weeks 21 and 18 respectively, and then dropped sharply in succeeding weeks. In this case, the highest content of delta9-THC was in the staminate plants at week 21. This pattern of two cyclic peaks is consistent with the data obtained in 1971 with ME-A seed stock.
In 1974, vegetative plants of ME-H, ME-K, ME-L, ME-N and ME-0, at 13 weeks of age had higher delta9-THC content than at weeks 12 and 14.
They showed minimum delta9-THC content at week 15. For the most part, 1974 staminate and pistillate plants grown in Mississippi produced a low delta9-THC concentration. None of these plants had a delta9-THC content higher than the original samples of material grown in Mexico in 1973 (see table 2).
However, these same variants grown in 1976 showed a higher delta9-THC content in at least one growth week than the original analysis. In general, variants grown in 1976 showed two periods of maximum delta9-THC levels between weeks 13 and 15, and weeks 19 and 20. The minimum amounts were between weeks 17 and 18, and weeks 22 and 23 respectively.
In all variants, the average delta9-THC content was higher in 1976 than in 1974. Also, a greater fluctuation of delta9-THC levels was observed in 1976 than in 1974.
More importantly, the major cannabinoid growth profiles for the same variant were found to differ significantly between 1974 and 1976. Since biosynthesis and rnetabolism of delta9-THC in individual plants varies hourly no simple explanation for the differences in delta9-THC growth profiles is possible. Small amounts of cannabigerol monomethyl ether (CBGM) also existed in each Mexican variant. Such levels were also found to exist in a previous study of Indian variants].
Small and Beckstead [61 classified Cannabis containing a small amount of CBGM, especially variants originating in north-east Asia, as phenotype IV.
In 1976, all Mississippi-grown Mexican variants (see table 4) showed increased CBC content when compared to the 1974 data. In most cases, pistillate plants showed less CBC content than staminate plants of the same variant.
From this study on Mexican Cannabis grown both in Mexico and in Mississippi, United States of America, we conclude that Mexican Cannabis is almost exclusively of the drug type (see tables 3 and 4), except in three cases. These exceptions were samples from staminate plants derived from ME-H at weeks 19, 23 and 24 (see table 4). This is the only variant in which CBD was detected. Although it is generally believed that the genetic character determines cannabinoid profile [7], these data indicate that environmental factors can influence the cannabinoid profile.
The results of the effect of temperature and rainfall on the delta9-THC content of the different Mexican variants, ME-H through ME-0, are indicated in table 5.
Thus it can be seen that, with a fairly high temperature and a reasonable amount of rain, an increase in delta9-THC can be anticipated. In 1974, the temperature showed a cooler trend, starting from week 15 from the date of planting, when compared with that of the growing season of 1976.
Also, the total amount of rainfall in 1976 (19.57 in.) was 28 percent less than in 1974 (27.22 in.). This might explain why the delta9-THC content of Cannabis grown in 1974 was much lower than that grown in 1976, although all plants were produced from the same seed stock and in the same garden using identical agricultural procedures.
From this study it is concluded that all these variants, with the exception of ME-H, had similar growth profiles.
Accordingly, Mexican Cannabis could possibly include two variants. Using the Wailer method, one variant is always a drug type no matter when samples are collected. Using the Small and Beckstead method, this variant belongs to different phenotypes. The second variant can be a drug or a fibre type, depending on when the collection was made, according to the Wailer method.
This variant generally contains appreciable amounts of CBD, whereas the other variants contain appreciable ainounts of CBC and negligible amounts of CBD.
Thus, seed code ME-H is of variant two and all others are of variant one. If we use both the Wailer and the Small and Beekstead methods and apply these methods to all seed codes, we find that all seed codes fall predominantly into phenotype 1, or phenotypes 1 and IV, depending on the age of the sample when analysed; whereas all fit in the drug type, according to Wailer.
The exception of both of these is the variant which contains sufficient quantities of CBD (ME-H) which fits in phenotypes I, II or IV and in the drug or fibre type in the Wailer system. Tlese data strongly indicate that a single analysis gives a classification for this sample only and not for the variant.
Vegetative plants showed maximum levels of delta9-THC at week 15 and minimum levels at weeks 17 and 18. Staminate plants contained maximum amounts of delta9-THC at week 20 and minimum at week 22. The pistillate plants showed maximum amounts at week 19 and minimum at week 23.The highest delta9-THC level was in the vegetative plant prior to any sexual differentiation at week 15. However, in 1974, ME-N vegetative plants showed a maximum amount of delta9-THC at week 18. Staminate and pistillate plants had maximum levels of delta9-THC at weeks 21 and 18 respectively, and then dropped sharply in succeeding weeks. In this case, the highest content of delta9-THC was in the staminate plants at week 21. This pattern of two cyclic peaks is consistent with the data obtained in 1971 with ME-A seed stock.
In 1974, vegetative plants of ME-H, ME-K, ME-L, ME-N and ME-0, at 13 weeks of age had higher delta9-THC content than at weeks 12 and 14.
They showed minimum delta9-THC content at week 15. For the most part, 1974 staminate and pistillate plants grown in Mississippi produced a low delta9-THC concentration. None of these plants had a delta9-THC content higher than the original samples of material grown in Mexico in 1973 (see table 2).
However, these same variants grown in 1976 showed a higher delta9-THC content in at least one growth week than the original analysis. In general, variants grown in 1976 showed two periods of maximum delta9-THC levels between weeks 13 and 15, and weeks 19 and 20. The minimum amounts were between weeks 17 and 18, and weeks 22 and 23 respectively.
In all variants, the average delta9-THC content was higher in 1976 than in 1974. Also, a greater fluctuation of delta9-THC levels was observed in 1976 than in 1974.
More importantly, the major cannabinoid growth profiles for the same variant were found to differ significantly between 1974 and 1976. Since biosynthesis and rnetabolism of delta9-THC in individual plants varies hourly no simple explanation for the differences in delta9-THC growth profiles is possible. Small amounts of cannabigerol monomethyl ether (CBGM) also existed in each Mexican variant. Such levels were also found to exist in a previous study of Indian variants].
Small and Beckstead [61 classified Cannabis containing a small amount of CBGM, especially variants originating in north-east Asia, as phenotype IV.
In 1976, all Mississippi-grown Mexican variants (see table 4) showed increased CBC content when compared to the 1974 data. In most cases, pistillate plants showed less CBC content than staminate plants of the same variant.
From this study on Mexican Cannabis grown both in Mexico and in Mississippi, United States of America, we conclude that Mexican Cannabis is almost exclusively of the drug type (see tables 3 and 4), except in three cases. These exceptions were samples from staminate plants derived from ME-H at weeks 19, 23 and 24 (see table 4). This is the only variant in which CBD was detected. Although it is generally believed that the genetic character determines cannabinoid profile [7], these data indicate that environmental factors can influence the cannabinoid profile.
The results of the effect of temperature and rainfall on the delta9-THC content of the different Mexican variants, ME-H through ME-0, are indicated in table 5.
Thus it can be seen that, with a fairly high temperature and a reasonable amount of rain, an increase in delta9-THC can be anticipated. In 1974, the temperature showed a cooler trend, starting from week 15 from the date of planting, when compared with that of the growing season of 1976.
Also, the total amount of rainfall in 1976 (19.57 in.) was 28 percent less than in 1974 (27.22 in.). This might explain why the delta9-THC content of Cannabis grown in 1974 was much lower than that grown in 1976, although all plants were produced from the same seed stock and in the same garden using identical agricultural procedures.
From this study it is concluded that all these variants, with the exception of ME-H, had similar growth profiles.
Accordingly, Mexican Cannabis could possibly include two variants. Using the Wailer method, one variant is always a drug type no matter when samples are collected. Using the Small and Beckstead method, this variant belongs to different phenotypes. The second variant can be a drug or a fibre type, depending on when the collection was made, according to the Wailer method.
This variant generally contains appreciable amounts of CBD, whereas the other variants contain appreciable ainounts of CBC and negligible amounts of CBD.
Thus, seed code ME-H is of variant two and all others are of variant one. If we use both the Wailer and the Small and Beekstead methods and apply these methods to all seed codes, we find that all seed codes fall predominantly into phenotype 1, or phenotypes 1 and IV, depending on the age of the sample when analysed; whereas all fit in the drug type, according to Wailer.
The exception of both of these is the variant which contains sufficient quantities of CBD (ME-H) which fits in phenotypes I, II or IV and in the drug or fibre type in the Wailer system. Tlese data strongly indicate that a single analysis gives a classification for this sample only and not for the variant.
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Acknowledgements
This work has been supported by the National Institute on Drug Abuse, contract numbers HSM-42-70-109 and 271-78-3527, and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi.
References
1 . C. E. Turner and others, "Constituents of Cannabis sativa L. X. Cannabinoid profile of a Mexican variant and its possible correlation to pharmacological activity", Acia Pharmareurica Jugoslavica, vol. 25,1975, p. 7.
2. C. E. Turner and others, "Constituents of Cannabis sativa XV. Botanical and chemical profile of Indian variants", Planta Medica, vol. 37,1979, p. 217.
3. C. E. Turner and others, "Constituents of Cannabis sativa XIV. Intrinsic problems in classifying Cannabis based on a single cannabinoid analysis", Journal of Natural Products, vol. 42, 1979, p. 217.
4. N. J. Doorenbos and others, "Cultivation, extraction, and analysis of Cannabis saliva L.". New York Academy of Sciences Annals. vol. 191, 197 1, pp. 3-14.
5. C. E. Turner and M. A. Elsobly, "Constituents of Cannabis sativa L. XXI. A possible decomposition pathway of delta9-tetrahydrocannabinol to cannabinol". Journal of Heterocyclic Chemistry, vol. 16,1979, p. 1667.
6. E. Small and H. D. Beekstead. "Common cannabinoid phenotypes in 350 stocks of Cannabis". Lloydia, vol. 36,1973, p. 144.
7. 5. Guerrero Dayalos and others, "Analysis of a population of Cannabis sativa L. originating from Mexico and cultivated in France", Experientia, vol. 33, No. 12 (1977), pp. 1562-1563
Some features of Cannabis plants grown in the United Kingdom from seeds of known origin
Pitts JE, Neal JD, Gough TA
Pharm Pharmacol 1992 Dec; 44(12):947-51
The cannabinoid content of UK-grown plants (up to the 6th generation) from Moroccan, Sri Lankan and Zambian seedstock was determined by TLC, GLC and HPLC. All plants from the 5th and 6th series resembled their parents, and UK-grown plants were always much greener than those grown overseas. Cannabinoid content remained broadly typical of the source countries.
However, tetrahydrocannabinolic acid (THCA) consistently predominated over tetrahydrocannabinol (THC) to a far greater extent than in the original plants; the THCA/THC ratio was 17 in UK-grown plants compared with 2.0 in the plants from the original areas.
Two types of plant emerged from the Moroccan seedstock, one tending to increased cannabidiol (CBD), the other tending to zero levels of this component. The first generation Sri Lankan plants revealed one type of plant with an increased CBD/THC ratio (1.7 compared with 0.11) but this returned to the original value in the succeeding generations. Other Sri Lankan plants had low or undetectable levels of CBD. Moroccan and Sri Lankan CBD-rich plants did not contain cannabichromene, although this cannabinoid was found in THC-rich plants.
Zambian plants did not appear to show such a pattern. Zambian seedstock plants had total tetrahydrocannabivarin (diol and acid) levels greater than THC but the ratio was progressively reversed in succeeding generations. The study concludes that the ratios of particular cannabinoids is greatly influenced by the environment.
Characterisation of cannabis plants phenotypes from illegal cultivations in Crete
Boll Chim Farm. 2000 May-Jun;139(3):140-5.
Tsatsakis AM, Tutudaki M, Stiakakis I, Dimopoulou M, Tzatzarakis M, Michalodimitrakis M.
Laboratory of Toxicology, Medical School, University of Crete, Heraklion, Greece.
AbstractIn the present study samples of cannabis plants presented to us by the Drug Enforcement Units were characterised, based on the analysis of active substances. The fresh samples were dried in a dark room were they were kept until analysis. The samples included leaves, flowers roots and trunks.
The analysis was performed by High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) using standard solutions of cannabidiol, D-9 tetrahydrocannabinol, D-8 tetrahydrocannabinol and cannabinol. Chemical analysis of the flowers revealed that 80% of the plants were classified as resinous phenotype while the remaining 20% were found to be of the textile phenotype (low concentration of active cannabinoids).
The concentration of D-9 tetrahydrocannabinol in the flowers and leaves ranged from 0.014 to 21.06 mg/g, of cannabinol from 0.0002 to 0.350 mg/g and of cannabidiol from 0.03 to 29.6 mg/g. Roots and trunks contained very small quantities of active substances and should not be used for phenotype identification. No delta-8 THC was detected in any sample. Leaves gave less resinous phenotypes than flowers. The use of either mathematical formula, A or B produced the same phenotype character for each separate part of the plant.
NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa
Phytochem Anal. 2004 Nov-Dec;15(6):345-54.
Choi YH, Hazekamp A, Peltenburg-Looman AM, Frédérich M, Erkelens C, Lefeber AW, Verpoorte R.
Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.
AbstractThe complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-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.
Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa LPhytochem Anal.
2005 Jan-Feb;16(1):45-8.
Ross SA, ElSohly MA, Sultana GN, Mehmedic Z, Hossain CF, Chandra S.
National Center for Natural Products Research, University of Mississippi, University, Mississippi 38677-1848, USA. sross@olemiss.edu
AbstractChemical 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 delta9-tetrahydrocannabiorcol, cannabidivarin, cannabicitran, delta9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, delta9-tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol, 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-O-ethylcannabitriol.
Biochemical differences in Cannabis sativa L. depending on sexual phenotypeJ. Appl. Genet. 43(4), 2002, pp. 451-462
Biochemical differences in Cannabis sativa L.
depending on sexual phenotype
Elena TRU1, Elvira GILLE2, Ecaterina TOTH2, Marilena MANIU3
1Department of Genetics, Institute of Biological Research, Iai, Romania
2Department of Genetics, Centre of Research Stejarulh, Piatra Neamt, Romania
3Department of Genetics, Faculty of Biology, University Al. I. Cuzah, Iai, Romania
Abstract. Hemp (Cannabis sativa L.) is a species considered as having one of the most
complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in
leaves of the two sexual phenotypes of hemp were studied. Significant differences
in isoperoxidase and isoesterase patterns were found between male and female plants,
both in the number and stain intensity of bands. For both esterase and peroxidase,
the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious
between the two sexes concerning catalase and peroxidase activities, as well as
the level of soluble protein. The quantitative analysis of flavones, polyholozides
and polyphenols emphasized differences depending not only on sex, but also on tested
organ.
Key words: electrofocusing, hemp, isoenzymatic pattern, secondary metabolites, sexual
phenotype.
Introduction
The chemical composition of hemp (Cannabis sativa L.) is very complex, including
about a hundred of compounds isolated from hemp organs: flavonoids, fatty
acids, phenolic spiroindans, dihydrostilbenes, nitrate substances (amines, ammonium
salts, spermidine-derived alkaloids), etc. The hemp flavour is due to volatile
terpenic compounds of essential oils, monoterpenes representing 47.9-92.1%
and sesquiterpenes 52-48.6% of total terpenes. Compounds like friedeline,
epifriedelinol, -sitosterol, carvone and dihydrocarvone were isolated from roots
(SETHI et al. 1978). Seeds contain oils (PETRI 1988), while among plant organs
Received: February 12, 2002. Accepted: July 2, 2002.
Correspondence: E. TRU, Department of Genetics, Biological Research Institute Bd. Carol I 20A,
6600 Iai, Romania, e-mail: elentru@email.ro
flowers are richer in oils than leaves (LEMBERKOVICS et al. 1979). The fatty acid
composition of fruits is of great interest, because of their use for nutritive
and pharmaceutical purposes. If the complete fruit and seed are similar in this aspect,
some differences are in the outer layer (MOLLEKEN, THEIMER 1997). Although
we have not found any systematic study on flavonoid synthesis in
the genus Cannabis there are a few papers regarding these compounds in hemp
(BATE-SMITH 1962, PARIS et al. 1975, 1976, PARIS, PARIS 1976, SEGELMAN
et al. 1978), but the findings are contradictory, having a limited systematic value,
because of the use of different analytic methods or of different plant organs or
of various provenances.
The hemp-specific substances, cannabinoids, include more than 70 substances,
such as 9-THC (tetrahydrocannabinol), CBD (cannabidiol) and CBN
(cannabinol), which are the criteria distinguishing between the hemp chemotypes
(especially 9-THC and CBD and THC/CBD ratio).
Although the hemp is a dioecious species, as a consequence of intensive
improvement, a lot of sexual phenotypes are cultivated, the most frequent being
the monoecious forms, classified in more categories, on a five-point scale, depending
on female flowers/male flowers ratio. Cannabis sativa L. has a very complex
genetic constitution and heredity, which explains the dioicism, amplitude
of phenotypical variability, polymorphism and the great biological plasticity
of this species.WESTERGAARD (1958) considered the sex inheritance in hemp as
being one of the most complicated mechanisms among all dioecious plants.
For hemp we could not find any consistent study on differentiation between sexual
phenotypes, regarding morphological, physiological or biochemical traits, in spite
of some disparate data. It is known that, by specific reactions, it becomes possible
to make the distinction between male and female individuals of Populus, as well
as between the . and + Mucor hyphae (SINNOTT 1960). Certain differences also
exist between staminate and pistillate plants of Lychnis dioica (STANFIELD 1944,
cited in SINNOTT 1960). For genera Cannabis and Spinacia, variable levels of cellular
extract pH are cited, depending on sex (CHEUVART 1954). In other plant species,
the analyses evidenced different values of oxidase activities in female and
male individuals (AITCHINSON 1953, cited in SINNOTT 1960).
For these reasons, the principal objective of this study was to identify the existence
of some biochemical differences (enzymes, secondary metabolites)
in hemp, depending on sexual phenotype.
Material and methods
The studied material was collected from female and male plants of hemp (Cannabis
sativa L. subsp. sativa var. sativa), randomly chosen from a population grown
in the experimental field of the Botanical Garden of University �gAl. I. Cuza�h Iai.
The seeds belonging to a fiber hemp cultivar were provided by the Agricultural
452 E. Tru et al.
Research Centre of Secuieni at Neam. To estimate in vivo catalase and peroxidase
activities, as well as soluble proteins, leaves of female and male hemp plants were
used. These determinations were carried out individually, in leaves collected from
20 females and 15 males of the same age (20 weeks old). Because of the lack
of simultaneity in maturity and flowering, specific for this species, the plants were
in different developmental stages. The females were in the early fruit formation
phase and the male plants were in full bloom.
To obtain the crude extract for the determination of the enzymatic activities
and the amount of soluble protein, known quantities of well ground plant material
(fine powder) in 0.01 M sodium phosphate (pH 7) were homogenized. The homogenate
was maintained at 4oC for 4 hours, and then it was centrifuged at
22 0000 rpm for 10 minutes. The supernatant was used as extract.
Catalase activity was determined by the iodometric method (ARTENIE,
TANASE 1981). The principle of this method is based on potassium iodide oxidation
by undecomposed hydrogen peroxide, after an incubation interval with
catalase, followed by titration of delivered iodine with sodium thiosulfate, in
the presence of starch solution as an indicator. The mixture: 0.01 M phosphate
buffer pH 7, enzymatic extract and 3% hydrogen peroxide was incubated for
5 minutes. The reaction was blocked with 10% sulfuric acid. Then 10% potassium
iodide and 1% ammonium molybdate were added. Titration was made with 0.1 sodium
thiosulfate. The catalase activity was calculated knowing that one catalase
unit is equivalent to the amount of enzyme which decomposes 1 ƒÊmol (0.034 mg)
H2O2 during 1 minute. The results are expressed in mg H2O2/g fresh matter.
Peroxidase activity was quantified by the photometric method, based on
benzidine oxidation under the peroxidase activity, in the presence of H2O2/time
unit. The mixture, composed of 1% benzidine in glacial acetic acid, 3% hydrogen
peroxide, and the enzymatic extract, was incubated for 3 minutes. 30% NaOH was
used to stop the reaction. Absolute ethanol was added. The values of extinctions
were determined with a SPEKOL 20 spectrophotometer, at = 470 nm. After
the estimation of the ratio: sample extinction/control extinction, the peroxidase
activity was expressed in mg H2O2 /g fresh matter.
The specific activities for catalase and peroxidase were estimated by reporting
the quantity of substratum consumed by enzyme to the concentration of soluble
protein in 1 g of tissue. They were expressed in mg H2O2 /mg protein.
The obtaining of enzymatic extracts used in electrofocusing involved very fine
grinding of the plant material with a Potter homogenizer. The homogenate (1 : 3,
w/v, in 0.1 M Tris/HCl buffer pH 7.2) was centrifuged at 17 000 rpm, with a refrigerated
JANETZKI K-24 centrifuge. The supernatant was used to identify
the izoenzymatic patterns. To assess the isoenzymatic pattern for esterase
and peroxidase, we used electrofocusing on polyacrylamidic gel containing urea,
H2O, acrylamide, ampholine, and ammonium persulfate, according to the Wrigley
method (TOTH 1992). Ampholine pH 3.5-10 (LKB) was used to establish the pH
gradient. The pH was controlled with a digital RADELKIS 20 pH meter. The sep-
Biochemical differences in Cannabis sativa L. 453
aration was achieved in the glass test tubes 0.5 ~ 7.5 cm of the electrophoretic apparatus,
in a disk of SHANDON type. The peroxidase visualization was
performed with o-dianisidine (MCDONALD, SMITH 1972), at pH 4.8, established
with 0.2 M sodium acetate buffer. The esterase visualization was conducted according
to SCANDALIOS (1969). In the solution for incubation (0.15Mphosphate
buffer, pH 7), 1% -naphthylacetate and Fast blue RR stain (2 mg/ml) were introduced.
The isoenzymatic fractions separated by electrofocusing were drawn and represented
as zymograms. The stain intensity is indicated by hatching.
The phytochemical analyses were conducted on biological material dried at
40oC, powdered and then subjected to extraction with different solvents. Determination
of polyholozidic content was realized in aqueous extract, the results being
expressed as 'absent', +, ++ or +++, depending on reaction intensity. Methanolic
extracts were processed to permit the quantification of flavones by colorimetric
method (on account of complexation with AlCl3) and of catechin-like
polyphenolic derivatives by the colorimetric method, in the presence
of 4-dimethyl-amino-antipyrine and ammonium persulfate, at pH = 8-9 (GRIGORESCU,
STANESCU 1982).
For the determination of soluble protein the Lowry method (LOWRY et al.
1951) was used. The enzymatic extract was treated with Folin-Ciocalteu solution.
The extinctions were registered with a SPEKOL 20 spectrophotometer, at
= 500 nm. The amounts of soluble proteins are expressed in mg/g fresh matter.
These values are required to estimate the specific activities of the two
hydroperoxidases.
The statistical analysis of the obtained data was performed using the method
described by RAICU et al. (1973). The arithmetical mean (x), the standard deviation
(SD), the standard error of the mean (SE), the coefficient of variation (CV)
and the standard error of the mean, expressed in % (SE %), were calculated.
Results and discussion
Quantitative analysis of catalase and peroxidase activities
As shown in Table 1, some differences are obvious between the two groups
of plants of different sex. First, the male individuals have a greater catalase activity
in relation to fresh biomass unit, as compared to females. The difference between
mean values for males and females was 2.27 mg H2O2/g fresh matter.
The peroxidase activity registered superior values in female plants, but the difference
between mean values of female and male plants was smaller (0.9 mg H2O2/g
fresh matter). The specific activities of the two enzymes were also different. Thus,
the mean and the standard error of the mean of catalase were 4.88 } 0.10 mg
H2O2/mg protein, for the group of female plants, and 6.02 } 0.10 mgH2O2/mg pro-
454 E. Tru et al.
Table 1. Average values of the catalase and peroxidase activities and the amount of soluble proteins in male and female hemp plants
Plant sex n
Catalase Peroxidase Soluble proteins
mg H2O2/g fresh matter mg H mg/g fresh matter 2O2/mg protein mg H2O2/g fresh matter mg H2O2/mg protein
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x �} SE SD CV
%
SE
%
Female 20
27.62
}
0.38
1.71 6.21 1.39
4.88
}
0.10
0.45 9.22 2.04
10.53
}
0.38
1.69 16.04 3.60
1.86
}
0.02
0.10 5.37 1.07
5.659
}
0.16
0.72 12.72 2.83
Male 15
29.89
}
0.61
2.39 7.99 2.04
6.02
}
0.10
0.39 6.47 1.66
9.63
}
0.40
1.55 16.09 4.15
1.94
}
0.05
0.21 10.82 2.57
4.961
}
0.07
0.30 6.04 1.41
n = number of studied plants; x = mean; SE = standard error of the mean; SD = standard deviation; CV = coefficient of variation; SE % = standard error of the mean, expressed in %
tein, for the male individuals. The specific peroxidase activities registered values
of 1.86 } 0.02 mg H2O2/mg protein in pistillate plants and 1.94 } 0.05 mg
H2O2/mg protein in staminate plants. For both catalase and peroxidase, the specific
activity was greater in males, but the increase is more important for catalase
(1.14 mg H2O2/mg protein), while for peroxidase the increase is only 0.08 mg
H2O2/mg protein.
The amount of soluble protein was greater in pistillate plants (5.659
} 0.16 mg/g fresh matter). In staminate plants, these values were 4.961
} 0.07 mg/g fresh matter, namely with 0.698 mg protein/g fresh matter smaller
than those noted for female sexual phenotypes (Table 1).
The standard deviation gives indications on the spread of the observations
around the mean. For the three analysed quantitative characters, the greatest concentration
of the observations around the mean was registered for proteins. In this
case, SD had the smallest values (0.30 for males, and 0.73 for females). The greatest
deviations were noted for catalase: SD = 2.39 in males, and SD = 1.71 in females.
The values of CV showed that the most variable character seems to be
the peroxidase activity (CV = 16.09% for males, and CV = 16.04% for females).
Besides that, the SE% had the highest values for this biochemical trait (4.15 for
males and 3.60 for female plants) (Table 1).
The commentary on the obtained results must start from the fact that
peroxidase activity is related to the developmental processes. Thus, in
organogenesis, the role of peroxidase is frequently explained by the double function
of this enzyme, involved both in oxidizing of some substrata and in auxine catabolism
(LEGRAND, BOUAZZA 1991).The latter function enables the modulation
of morphogenesis by peroxidase, as a result of intervention on endogenous hormonal
balance. In hemp, as well as in other monoecious or dioecious plants,
the gibberellins, auxins, ethylene and cytokinins have an important contribution to
sex expression (MOHAN RAM, SETT 1982a, b, DURAND, DURAND 1984,
CHAILAKHYAN 1985). These hormones generally intervene in the derepression of
reglator genes, which enable the synthesis of specific proteins that control flower
organogenesis. Because of its intervention in the regulation of IAA
(indole-3-acetic acid) level, peroxidase has an indirect role in the sex-determining
mechanism in hemp, more exactly in stamenogenesis and carpellogenesis. Hemp
is one of the species in which a high level of IAA induces the female sex
phenotypisation. The idea of a strong peroxidase activity associated with an increased
auxine catabolism is generally accepted. Concerning catalase, the specific
reaction catalysed by this enzyme is the direct degradation of the toxic H2O2
(the final product of biological oxidization), with release of water and oxygen, that
is taken over to oxygenate the tissues. It seems, however, that the peroxidative activity
of catalases (the reason for which the two enzymes are known as
ghydroperoxidasesh) prevails in tissue. In the case of a smallerH2O2 concentration
and of greater quantities of other substrata, catalase can use a hydrogen donor
other than H2O2.
456 E. Tru et al.
The isoesterase and isoperoxidase patterns
The isoenyzmatic patterns were done for single individuals. The differences between
individuals of the same sex were not significant. Therefore, the schematic
zymograms of izoesterases and isoperoxidases, for one male and one female, are
compared in Figure 1. The esterases are hydrolases that catalyse the hydrolytic
splitting of molecules of substrata at the level of esteric bonds, with formation of
one alcohol molecule and one acid molecule. For both esterase (A) and peroxidase
(B), the isoenzymatic spectrum is richer for staminate plants. Thus, in female
plants, eight multiple isoesterase forms appear, six of them being placed in the domain
of pH = 5.5-6.0 and the other two in the interval of pH = 6.5-7.0. The most
active isoesterase band is placed in the weak acid domain, having pI (isoelectric
point) at pH = 6.0. The eleven isoesterase forms of staminate plants are situated in
the interval of pH = 4.5-7.2, the most active esterase forms having pI situated in
the range of pH = 4.5-5.5, namely more acid that in the case of female plants.
A specific aspect is the presence of three well outlined isoesterase bands at pH
= 6.1-6.5 in male plants . bands that have no correspondence in the isoesterase
pattern of pistillate plants.
Differences also exist for the isoperoxidase pattern. Thus, the female plant has,
as in the case of esterase, fewer bands, two isoforms being in the range of
pH = 4.5-5.5, one at pH = 7.4 and one at pH = 8.9 (extremely basic). The ten fractions
of male genotypes were distributed in the following manner: seven at
pH = 4.7-6.1 (acid), one isoform at pH = 7.4-7.6 (weakly basic) and two bands at
the extremely basic value (pH = 8.9). The presence of four isoperoxidase fractions
at pH = 5.8-6.1 confers a strong physiological advantage of male genotypes over
the female genotypes. The isoesterase and isoperoxidase patterns reveal some differences
at the metabolic level, related to a specific multigenic determinism.
Peroxidase is distributed both in the cytosol and cell wall, as different genetic
isoenzymatic forms (OKEY et al. 1997). Cell wall is regarded as the site of primary
plant peroxidase activity (FRY 1986). Generally it is agreed that acid peroxidases
(especially those found in the cell wall) intervene in lignin biosynthesis, while
the basic ones (with cytoplasmic and vacuolar distribution) are involved in IAA
catabolism, through a decarboxylation step (LIMAM et al. 1998). It is obvious
(Figure 1) that, although there are fewer isoenzymatic bands in the female genotype,
the bands associated with cell walls (active in the acid range) prevail in both
analysed genotypes . a fact in accordance with data suggesting that cell walls
are a principal site of plant peroxidases. Concerning acid peroxidases it is not clear
if they are the products of different genes or if they are modified post-translational
products of a small number of genes (ROS BARCELO et al. 1987). In hemp, like in
other plant species, peroxidase has several isoforms, each with a well-defined
role. The isoperoxidase pattern is complex, just this complexity being the element
amplifying the difficulty to decipher all specific functions of this enzyme
(CLEMENTE 1998, YUN et al. 1998). The genetic determinism of these isoforms is
multigenic. If there are still unexplained details for hemp, for Brassica napus
Biochemical differences in Cannabis sativa L. 457
the existence of at least four distinct genes has been established (HAMED et al.
1998). The isoenzymes of Petunia are under the control of three genes, while in
wheat isoperoxidases are controlled by different genomes and the environmental
conditions do not modify the isoenzymatic pattern (HAMED et al. 1998).
Quantitative analysis of polyphenols, flavones and crude polyholozides
The data regarding the amount of some secondary metabolites, depending on sex
and organ of the plant, were obtained from the individuals whose zymograms are
presented in Figure 1. For every quantitative essay, two replications were made.
Table 2 presents the average values of these phytochemical determinations, depending
on sexual phenotype and on analysed organ, in dry and fresh matter.
The comparative analysis of these results exhibits important differences. Thus,
leaves from female plants have an increased level of polyphenols (1.560 mg%, expressed
in catechin) and flavones (1.084 mg%, expressed in rutosid), while in
male leaves polyphenols are not present, and the quantity of flavones represents
2/3 of the value for female leaves (0.680 mg%). Regarding the stem, no
polyphenols were identified in the terminal (top) part of male plants. In female
plants, however, their level was high (0.940 mg%, expressed in catechin). For flavones,
the situation is inversed: in pistillate plants these compounds are not found,
458 E. Tru et al.
Figure 1. Zymograms of isoesterases (A) and isoperoxidases (B), in female (1) and male (2)
of individuals Cannabis sativa L.
and in staminate plants their level was 0.460 mg%. In middle parts of stems,
the results were negative for all three categories of tested compounds in female
plants, whereas in male plants, polyphenols are lacking. The flavones of
the rutosid type have a value of 0.525 mg%, and the crude polyholozides have
a good representation (+++). Generally, higher levels of polyholozides were present
in male plants (for example, ++ for terminal part of stem, +++ for leaves, +++
for middle part of stem, and for female plants, respectively: +, ++, absent).
The quantitative analyses conducted in fresh matter were negative for
polyphenols for all tested organs, both for male and female plants, but the flavones
and crude polyholozides were present, the latter in high levels (+++) in all samples.
Thus, between sexual phenotypes as well as between the organs of hemp
plants, visible differences exist. It is also important that in the fresh and dry matter
of the male plants, polyphenols were absent . an aspect possibly related to the fact
that in hemp species, in which a high IAA level favours the phenotypisation of female
sex, the capacity to degrade IAA is counterbalanced by auxine protectors
(phenols).
As in the case of other secondary metabolites, the hemp callus was unable to
synthetize polyphenols and flavones (TRUTA, unpublished), which is in accordance
with results of other studies (BRAEMER et al. 1985, BRAUT-BOUCHER et al.
1981, GILLE 1996).
Biochemical differences in Cannabis sativa L. 459
Table 2. Average values registered for polyphenols, flavones and crude polyholozides,
depending on sexual phenotype and on analysed organ in hemp
Plant sex Organ
Polyphenols
mg % catechin
Flavones
mg % rutosid
Polyholozides
Dry matter
Male terminal part of stem 0 0.460 ++
Female terminal part of stem 0.940 0 +
Male leaves 0 0.680 +++
Female leaves 1.560 1.084 ++
Male middle part of stem 0 0.525 +++
Female middle part of stem 0 0 absent
Male inflorescence 0 1.006 absent
Fresh matter
Male leaves 0 0.340 +++
Female leaves 0 0.525 +++
Male inflorescence 0 0.444 +++
Conclusions
In this study, the isoenzymatic pattern of esterase and peroxidase is richer in hemp
male plants, as compared to female plants. For both analysed sexes,
the isoperoxidase bands localized in acid domain are prevalent. The relative
and specific activity of catalase have more reduced values in female plants.
The specific peroxidasic activity is greater in male plants. The average level
of soluble protein was higher in female plants. Significant differences are registered,
depending not only on sex, but also on tested organ in the same plant, in respect
of level of polyphenols, flavones and polyholozides. In male plant,
polyphenols were absent.
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Download PDF here
The Inheritance of Chemical Phenotype in Cannabis sativa L
Genetics, Vol. 163, 335-346, January 2003, Copyright © 2003
Etienne P. M. de Meijer, Manuela Bagatta, Andrea Carboni, Paola Crucitti, V. M. Cristiana Moliterni, Paolo Ranall, and Giuseppe Mandolino
aHortaPharm B.V., 1075 VS, Amsterdam, The Netherlands
Istituto Sperimentale per le Colture Industriali, 40128 Bologna, Italy
Corresponding author: Giuseppe Mandolino, Via di Corticella 133, 40128 Bologna, Italy., g.mandolino@isci.it (E-mail)
Communicating editor: C. S. GASSER
ABSTRACT
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure -9-tetrahydrocannabinol (THC) chemotypes.
All the plants belonging to the F1's were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F1 plants were self-fertilized, and 10 inbred F2 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 F1 to the F2's derived from it. A model involving one locus, B, with two alleles, BD and BT, is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype BD/BT at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either BD/BD or BT/BT). 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 F2 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.
CHEMOTYPICAL diversity in Cannabis: The class of secondary products unique to the dioecious species Cannabis sativa L. (hemp) is the terpenophenolic substances known as cannabinoids, which accumulate mainly in the glandular trichomes of the plant (MECHOULAM 1970...read more
Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?John M. McPartland Ethan B. Russo
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, £G9-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.
[Article copies available for a fee from The Haworth Document Delivery
Service: 1-800-342-9678. E-mail address: <getinfo@haworthpressinc.com>
Website: <http://www.HaworthPress.com> 2001 by The Haworth Press, Inc.
All rights reserved.]
John M. McPartland, DO, MS, is affiliated with GW Pharmaceuticals, Ltd., Porton
Down Science Park, Salisbury, Wiltshire, SP4 0JQ, UK.
Ethan B. Russo, MD, is affiliated with Montana Neurobehavioral Specialists, 900
North Orange Street, Missoula, MT 59802 USA.
Address correspondence to: John M. McPartland, DO, Faculty of Health&Environmental
Science, UNITEC, Private Bag 92025, Auckland, New Zealand (E-mail: jmcpartland
@unitec.ac.nz).
The authors thank David Pate and Vincenzo Di Marzo for pre-submission reviews.
[Haworth co-indexing entry note]: ¡§Cannabis and Cannabis Extracts: Greater Than the Sum of Their
Parts?¡¨ McPartland, John M., and Ethan B. Russo. Co-published simultaneously in Journal of Cannabis Therapeutics
(The Haworth Integrative Healing Press, an imprint of The Haworth Press, Inc.) Vol. 1, No. 3/4,
2001, pp. 103-132; and: Cannabis Therapeutics in HIV/AIDS (ed: Ethan Russo) The Haworth Integrative
Healing Press, an imprint of The Haworth Press, Inc., 2001, pp. 103-132. Single or multiple copies of this article are available for a fee from The Haworth Document Delivery Service [1-800-342-9678, 9:00 a.m. - 5:00
p.m. (EST). E-mail address: getinfo@haworthpressinc.com].
2001 by The Haworth Press, Inc. All rights reserved. 103
INTRODUCTION
Cannabis is an herb; it contains hundreds of pharmaceutical compounds
(Turner et al. 1980). Herbalists contend that polypharmaceutical herbs provide
two advantages over single-ingredient synthetic drugs:
(1) therapeutic effects
of the primary active ingredients in herbs may be synergized by other compounds,
and
(2)side effects of the primary active ingredients may be mitigated
by other compounds.
Thus, cannabis has been characterized as a ¡§synergistic
shotgun,¡¨ in contrast to Marinol. (£G9-tetrahydrocannabinol, THC), a synthetic,
single-ingredient ¡§silver bullet¡¨ (McPartland and Pruitt 1999).
Mechoulam et al. (1972) suggested that other compounds present in herbal
cannabis might influence THC activity. Carlini et al. (1974) determined that
cannabis extracts produced effects ¡§two or four times greater than that expected
from their THC content.¡¨ Similarly, Fairbairn and Pickens (1981) detected
the presence of unidentified ¡§powerful synergists¡¨ in cannabis extracts
causing 330% greater activity in mice than THC alone.
Other compounds in herbal cannabis may ameliorate the side effects of
THC. Whole cannabis causes fewer psychological side effects than synthetic
THC, seen as symptoms of dysphoria, depersonalization, anxiety, panic reactions,
and paranoia (Grinspoon and Bakalar 1997). This difference in side effect
profiles may also be due, in part, to differences in administration: THC
taken by mouth undergoes ¡§first pass metabolism¡¨ in the small intestine and
liver, to 11-hydroxy THC; the metabolite is more psychoactive than THC itself
(Browne and Weissman 1981). Inhaled THC undergoes little first-pass metabolism,
so less 11-hydroxy THC is formed. Thus, ¡§smoking cannabis is a satisfactory
expedient in combating fatigue, headache and exhaustion, whereas the
oral ingestion of cannabis results chiefly in a narcotic effect which may cause
serious alarm¡¨ (Walton 1938, p. 49).
Respiratory side effects from inhaling cannabis smoke may be ameliorated by
both cannabinoid and non-cannabinoid components in cannabis. For instance,
throat irritation may be diminished by anti-inflammatory agents, mutagens in
the smoke may be mitigated by antimutagens, and bacterial contaminants in
cannabis may be annulled by antibiotic compounds (McPartland and Pruitt
1997). The pharmaceutically active compounds in cannabis that enhance beneficial
THC activity and reduce side effects are relatively unknown. The pur-
104 CANNABIS THERAPEUTICS IN HIV/AID.... read full PDF
Effects of Gibberellic Acid on Primary Terpenoids and Delta-Tetrahydrocannabinol in Cannabis sativa at Flowering StageMansouri H, Asrar Z, Mehrabani M
J Integr Plant Biol 2009 Jun; 51(6):553-61.
Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway.
However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA(3)) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways.
Our results showed GA(3) caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Delta(9)-tetrahydrocannabinol (THC) contents and an increase in alpha-tocopherol content. The treated plants with GA(3) showed an increase in HMGR activity.
This increase in HMGR activity was followed by accumulation of stigmasterol and beta-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA(3) can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.
A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa LFischedick JT, Glas R, Hazekamp A, Verpoorte R
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 Delta(9)-tetrahydrocannabinol (Delta(9)-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: Delta(9)-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 r(2) = 0.9968.
CONCLUSION: Results have shown that the HPTLC method is reproducible and accurate for the quantification of Delta(9)-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.
Comparative Proteomics of Cannabis sativa Plant Tissues J Biomol Tech. 2004 June; 15(2): 97–106.
Tri J. Raharjo,ab Ivy Widjaja,c Sittiruk Roytrakul,b and Robert Verpoorteb
aDepartment of Chemistry, Gadjah Mada University, Yogyakarta, Indonesia;
bDivision of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands
cCurrent address: Genome Institute of Singapore, 1 Science Park Road, #05–01, The Capricorn, Singapore Science Park II, Singapore 117528.
Robert Verpoorte, Division of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands (fax: 31-71-5274511; e-mail: verpoort@ lacdr.leidenuniv.nl).
This article has been cited by other articles in PMC.
Abstract
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.
Keywords: Cannabis sativa, comparative, mass fingerprint, proteomic
The biosynthesis of cannabinoids, a class of terpenephenolic compounds found in Cannabis sativa, is not yet fully known. Cannabinoids are found in all tissues of the C. sativa plant, but the amount in which they are present differs considerably among the tissues.
Cannabinoids are most abundant in flowers, especially in the glands. This raises the question of whether biosynthesis of cannabinoids occurs in all tissues but in different quantities, or only in one tissue and is then transported to the others. In both scenarios it is assumed that the expression level of the genes involved in the cannabinoid biosynthesis is different among the tissues. In any case, the differential expression of cannabinoid biosynthesis may be used to further clarify this pathway by comparing, on the level of proteins or mRNAs, the tissues with varying amounts of cannabinoids with the tissues that do not produce cannabinoids.
Gene expression can be studied by measuring mRNA levels using methods such as microarrays, serial analysis of gene expression, and real-time polymerase chain reaction. However, studies in yeast revealed the absence of a strong correlation between the abundance of the protein and the corresponding mRNA. Alternative methods of study involve the use of enzyme assays or proteome analysis to identify expressed proteins.
The enzymes known to be involved in cannabis biosynthesis are olivetolic acid prenylase, tetrahydrocannabinolic acid synthase (THCA synthase), cannabidiolic acid synthase (CBDA synthase), and cannabichromenic acid synthase (CBCA synthase). Though assays are available for several of the enzymatic steps of the cannabinoid biosynthesis, it would be an immense task to purify each of these enzymes for sequencing. Moreover, not all of the steps are known. Thus, proteome analysis (proteomics) seems to be preferable to enzyme assaying in obtaining sequence information from all proteins connected with cannabinoid biosynthesis.
The use of proteomics in the study of secondary metabolite biosynthesis has been reviewed by Jacobs et al.
Proteomics is a new tool used to identify and characterize all proteins expressed in an organism or cell. Single proteins can be separated using column chromatography or two-dimensional (2D) electrophoresis prior to mass spectrometric (MS) analysis. Advanced MS allows ionization of macromolecules such as proteins and peptides. Proteins can be identified by matching peptide mass fingerprinting with database sequences or by sequencing whole-length proteins with tandem MS.
Peptide fingerprints can be obtained by ionization of the peptides that result from enzymatic digestion, usually by trypsin. Accurate peptide masses of peptide fingerprints can be used for searching matching proteins in the databases resulting in molecular weight search (Mowse) score. The peptides themselves can be fragmented using tandem MS resulting in the amino acid sequences.
Thousands of proteins occur in the cell, and to choose and separate the protein responsible for a particular function is not an easy task. Using 2D electrophoresis proteins are separated based on pI and molecular weight (MW), which results in a proteome pattern of the cells or tissues under a certain condition. Proteins involved in the production of metabolites can be studied by comparing producing with nonproducing conditions of the cells or tissues:
Proteins that are present in the producing conditions but not in the nonproducing conditions might be involved in the production of the compounds of interest. This comparison can be performed more easily with cell cultures, as they tend to have a less complex matrix than plant tissues. Unfortunately, cannabinoids are not produced by cell cultures. Another option is to compare high-producing tissues, such as flowers, with low-producing tissues, such as leaves. The pI and MW of THCA synthase, CBDA synthase, and CBCA synthase are available (Table 11).). Therefore, these proteins might be identified from the tissues of flowers and glands using 2D electrophoresis and confirmed by MS analysis.
This work has been supported by the National Institute on Drug Abuse, contract numbers HSM-42-70-109 and 271-78-3527, and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi.
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3. C. E. Turner and others, "Constituents of Cannabis sativa XIV. Intrinsic problems in classifying Cannabis based on a single cannabinoid analysis", Journal of Natural Products, vol. 42, 1979, p. 217.
4. N. J. Doorenbos and others, "Cultivation, extraction, and analysis of Cannabis saliva L.". New York Academy of Sciences Annals. vol. 191, 197 1, pp. 3-14.
5. C. E. Turner and M. A. Elsobly, "Constituents of Cannabis sativa L. XXI. A possible decomposition pathway of delta9-tetrahydrocannabinol to cannabinol". Journal of Heterocyclic Chemistry, vol. 16,1979, p. 1667.
6. E. Small and H. D. Beekstead. "Common cannabinoid phenotypes in 350 stocks of Cannabis". Lloydia, vol. 36,1973, p. 144.
7. 5. Guerrero Dayalos and others, "Analysis of a population of Cannabis sativa L. originating from Mexico and cultivated in France", Experientia, vol. 33, No. 12 (1977), pp. 1562-1563
Some features of Cannabis plants grown in the United Kingdom from seeds of known origin
Pitts JE, Neal JD, Gough TA
Pharm Pharmacol 1992 Dec; 44(12):947-51
The cannabinoid content of UK-grown plants (up to the 6th generation) from Moroccan, Sri Lankan and Zambian seedstock was determined by TLC, GLC and HPLC. All plants from the 5th and 6th series resembled their parents, and UK-grown plants were always much greener than those grown overseas. Cannabinoid content remained broadly typical of the source countries.
However, tetrahydrocannabinolic acid (THCA) consistently predominated over tetrahydrocannabinol (THC) to a far greater extent than in the original plants; the THCA/THC ratio was 17 in UK-grown plants compared with 2.0 in the plants from the original areas.
Two types of plant emerged from the Moroccan seedstock, one tending to increased cannabidiol (CBD), the other tending to zero levels of this component. The first generation Sri Lankan plants revealed one type of plant with an increased CBD/THC ratio (1.7 compared with 0.11) but this returned to the original value in the succeeding generations. Other Sri Lankan plants had low or undetectable levels of CBD. Moroccan and Sri Lankan CBD-rich plants did not contain cannabichromene, although this cannabinoid was found in THC-rich plants.
Zambian plants did not appear to show such a pattern. Zambian seedstock plants had total tetrahydrocannabivarin (diol and acid) levels greater than THC but the ratio was progressively reversed in succeeding generations. The study concludes that the ratios of particular cannabinoids is greatly influenced by the environment.
Characterisation of cannabis plants phenotypes from illegal cultivations in Crete
Boll Chim Farm. 2000 May-Jun;139(3):140-5.
Tsatsakis AM, Tutudaki M, Stiakakis I, Dimopoulou M, Tzatzarakis M, Michalodimitrakis M.
Laboratory of Toxicology, Medical School, University of Crete, Heraklion, Greece.
AbstractIn the present study samples of cannabis plants presented to us by the Drug Enforcement Units were characterised, based on the analysis of active substances. The fresh samples were dried in a dark room were they were kept until analysis. The samples included leaves, flowers roots and trunks.
The analysis was performed by High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) using standard solutions of cannabidiol, D-9 tetrahydrocannabinol, D-8 tetrahydrocannabinol and cannabinol. Chemical analysis of the flowers revealed that 80% of the plants were classified as resinous phenotype while the remaining 20% were found to be of the textile phenotype (low concentration of active cannabinoids).
The concentration of D-9 tetrahydrocannabinol in the flowers and leaves ranged from 0.014 to 21.06 mg/g, of cannabinol from 0.0002 to 0.350 mg/g and of cannabidiol from 0.03 to 29.6 mg/g. Roots and trunks contained very small quantities of active substances and should not be used for phenotype identification. No delta-8 THC was detected in any sample. Leaves gave less resinous phenotypes than flowers. The use of either mathematical formula, A or B produced the same phenotype character for each separate part of the plant.
NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa
Phytochem Anal. 2004 Nov-Dec;15(6):345-54.
Choi YH, Hazekamp A, Peltenburg-Looman AM, Frédérich M, Erkelens C, Lefeber AW, Verpoorte R.
Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.
AbstractThe complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-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.
Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa LPhytochem Anal.
2005 Jan-Feb;16(1):45-8.
Ross SA, ElSohly MA, Sultana GN, Mehmedic Z, Hossain CF, Chandra S.
National Center for Natural Products Research, University of Mississippi, University, Mississippi 38677-1848, USA. sross@olemiss.edu
AbstractChemical 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 delta9-tetrahydrocannabiorcol, cannabidivarin, cannabicitran, delta9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, delta9-tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol, 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-O-ethylcannabitriol.
Biochemical differences in Cannabis sativa L. depending on sexual phenotypeJ. Appl. Genet. 43(4), 2002, pp. 451-462
Biochemical differences in Cannabis sativa L.
depending on sexual phenotype
Elena TRU1, Elvira GILLE2, Ecaterina TOTH2, Marilena MANIU3
1Department of Genetics, Institute of Biological Research, Iai, Romania
2Department of Genetics, Centre of Research Stejarulh, Piatra Neamt, Romania
3Department of Genetics, Faculty of Biology, University Al. I. Cuzah, Iai, Romania
Abstract. Hemp (Cannabis sativa L.) is a species considered as having one of the most
complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in
leaves of the two sexual phenotypes of hemp were studied. Significant differences
in isoperoxidase and isoesterase patterns were found between male and female plants,
both in the number and stain intensity of bands. For both esterase and peroxidase,
the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious
between the two sexes concerning catalase and peroxidase activities, as well as
the level of soluble protein. The quantitative analysis of flavones, polyholozides
and polyphenols emphasized differences depending not only on sex, but also on tested
organ.
Key words: electrofocusing, hemp, isoenzymatic pattern, secondary metabolites, sexual
phenotype.
Introduction
The chemical composition of hemp (Cannabis sativa L.) is very complex, including
about a hundred of compounds isolated from hemp organs: flavonoids, fatty
acids, phenolic spiroindans, dihydrostilbenes, nitrate substances (amines, ammonium
salts, spermidine-derived alkaloids), etc. The hemp flavour is due to volatile
terpenic compounds of essential oils, monoterpenes representing 47.9-92.1%
and sesquiterpenes 52-48.6% of total terpenes. Compounds like friedeline,
epifriedelinol, -sitosterol, carvone and dihydrocarvone were isolated from roots
(SETHI et al. 1978). Seeds contain oils (PETRI 1988), while among plant organs
Received: February 12, 2002. Accepted: July 2, 2002.
Correspondence: E. TRU, Department of Genetics, Biological Research Institute Bd. Carol I 20A,
6600 Iai, Romania, e-mail: elentru@email.ro
flowers are richer in oils than leaves (LEMBERKOVICS et al. 1979). The fatty acid
composition of fruits is of great interest, because of their use for nutritive
and pharmaceutical purposes. If the complete fruit and seed are similar in this aspect,
some differences are in the outer layer (MOLLEKEN, THEIMER 1997). Although
we have not found any systematic study on flavonoid synthesis in
the genus Cannabis there are a few papers regarding these compounds in hemp
(BATE-SMITH 1962, PARIS et al. 1975, 1976, PARIS, PARIS 1976, SEGELMAN
et al. 1978), but the findings are contradictory, having a limited systematic value,
because of the use of different analytic methods or of different plant organs or
of various provenances.
The hemp-specific substances, cannabinoids, include more than 70 substances,
such as 9-THC (tetrahydrocannabinol), CBD (cannabidiol) and CBN
(cannabinol), which are the criteria distinguishing between the hemp chemotypes
(especially 9-THC and CBD and THC/CBD ratio).
Although the hemp is a dioecious species, as a consequence of intensive
improvement, a lot of sexual phenotypes are cultivated, the most frequent being
the monoecious forms, classified in more categories, on a five-point scale, depending
on female flowers/male flowers ratio. Cannabis sativa L. has a very complex
genetic constitution and heredity, which explains the dioicism, amplitude
of phenotypical variability, polymorphism and the great biological plasticity
of this species.WESTERGAARD (1958) considered the sex inheritance in hemp as
being one of the most complicated mechanisms among all dioecious plants.
For hemp we could not find any consistent study on differentiation between sexual
phenotypes, regarding morphological, physiological or biochemical traits, in spite
of some disparate data. It is known that, by specific reactions, it becomes possible
to make the distinction between male and female individuals of Populus, as well
as between the . and + Mucor hyphae (SINNOTT 1960). Certain differences also
exist between staminate and pistillate plants of Lychnis dioica (STANFIELD 1944,
cited in SINNOTT 1960). For genera Cannabis and Spinacia, variable levels of cellular
extract pH are cited, depending on sex (CHEUVART 1954). In other plant species,
the analyses evidenced different values of oxidase activities in female and
male individuals (AITCHINSON 1953, cited in SINNOTT 1960).
For these reasons, the principal objective of this study was to identify the existence
of some biochemical differences (enzymes, secondary metabolites)
in hemp, depending on sexual phenotype.
Material and methods
The studied material was collected from female and male plants of hemp (Cannabis
sativa L. subsp. sativa var. sativa), randomly chosen from a population grown
in the experimental field of the Botanical Garden of University �gAl. I. Cuza�h Iai.
The seeds belonging to a fiber hemp cultivar were provided by the Agricultural
452 E. Tru et al.
Research Centre of Secuieni at Neam. To estimate in vivo catalase and peroxidase
activities, as well as soluble proteins, leaves of female and male hemp plants were
used. These determinations were carried out individually, in leaves collected from
20 females and 15 males of the same age (20 weeks old). Because of the lack
of simultaneity in maturity and flowering, specific for this species, the plants were
in different developmental stages. The females were in the early fruit formation
phase and the male plants were in full bloom.
To obtain the crude extract for the determination of the enzymatic activities
and the amount of soluble protein, known quantities of well ground plant material
(fine powder) in 0.01 M sodium phosphate (pH 7) were homogenized. The homogenate
was maintained at 4oC for 4 hours, and then it was centrifuged at
22 0000 rpm for 10 minutes. The supernatant was used as extract.
Catalase activity was determined by the iodometric method (ARTENIE,
TANASE 1981). The principle of this method is based on potassium iodide oxidation
by undecomposed hydrogen peroxide, after an incubation interval with
catalase, followed by titration of delivered iodine with sodium thiosulfate, in
the presence of starch solution as an indicator. The mixture: 0.01 M phosphate
buffer pH 7, enzymatic extract and 3% hydrogen peroxide was incubated for
5 minutes. The reaction was blocked with 10% sulfuric acid. Then 10% potassium
iodide and 1% ammonium molybdate were added. Titration was made with 0.1 sodium
thiosulfate. The catalase activity was calculated knowing that one catalase
unit is equivalent to the amount of enzyme which decomposes 1 ƒÊmol (0.034 mg)
H2O2 during 1 minute. The results are expressed in mg H2O2/g fresh matter.
Peroxidase activity was quantified by the photometric method, based on
benzidine oxidation under the peroxidase activity, in the presence of H2O2/time
unit. The mixture, composed of 1% benzidine in glacial acetic acid, 3% hydrogen
peroxide, and the enzymatic extract, was incubated for 3 minutes. 30% NaOH was
used to stop the reaction. Absolute ethanol was added. The values of extinctions
were determined with a SPEKOL 20 spectrophotometer, at = 470 nm. After
the estimation of the ratio: sample extinction/control extinction, the peroxidase
activity was expressed in mg H2O2 /g fresh matter.
The specific activities for catalase and peroxidase were estimated by reporting
the quantity of substratum consumed by enzyme to the concentration of soluble
protein in 1 g of tissue. They were expressed in mg H2O2 /mg protein.
The obtaining of enzymatic extracts used in electrofocusing involved very fine
grinding of the plant material with a Potter homogenizer. The homogenate (1 : 3,
w/v, in 0.1 M Tris/HCl buffer pH 7.2) was centrifuged at 17 000 rpm, with a refrigerated
JANETZKI K-24 centrifuge. The supernatant was used to identify
the izoenzymatic patterns. To assess the isoenzymatic pattern for esterase
and peroxidase, we used electrofocusing on polyacrylamidic gel containing urea,
H2O, acrylamide, ampholine, and ammonium persulfate, according to the Wrigley
method (TOTH 1992). Ampholine pH 3.5-10 (LKB) was used to establish the pH
gradient. The pH was controlled with a digital RADELKIS 20 pH meter. The sep-
Biochemical differences in Cannabis sativa L. 453
aration was achieved in the glass test tubes 0.5 ~ 7.5 cm of the electrophoretic apparatus,
in a disk of SHANDON type. The peroxidase visualization was
performed with o-dianisidine (MCDONALD, SMITH 1972), at pH 4.8, established
with 0.2 M sodium acetate buffer. The esterase visualization was conducted according
to SCANDALIOS (1969). In the solution for incubation (0.15Mphosphate
buffer, pH 7), 1% -naphthylacetate and Fast blue RR stain (2 mg/ml) were introduced.
The isoenzymatic fractions separated by electrofocusing were drawn and represented
as zymograms. The stain intensity is indicated by hatching.
The phytochemical analyses were conducted on biological material dried at
40oC, powdered and then subjected to extraction with different solvents. Determination
of polyholozidic content was realized in aqueous extract, the results being
expressed as 'absent', +, ++ or +++, depending on reaction intensity. Methanolic
extracts were processed to permit the quantification of flavones by colorimetric
method (on account of complexation with AlCl3) and of catechin-like
polyphenolic derivatives by the colorimetric method, in the presence
of 4-dimethyl-amino-antipyrine and ammonium persulfate, at pH = 8-9 (GRIGORESCU,
STANESCU 1982).
For the determination of soluble protein the Lowry method (LOWRY et al.
1951) was used. The enzymatic extract was treated with Folin-Ciocalteu solution.
The extinctions were registered with a SPEKOL 20 spectrophotometer, at
= 500 nm. The amounts of soluble proteins are expressed in mg/g fresh matter.
These values are required to estimate the specific activities of the two
hydroperoxidases.
The statistical analysis of the obtained data was performed using the method
described by RAICU et al. (1973). The arithmetical mean (x), the standard deviation
(SD), the standard error of the mean (SE), the coefficient of variation (CV)
and the standard error of the mean, expressed in % (SE %), were calculated.
Results and discussion
Quantitative analysis of catalase and peroxidase activities
As shown in Table 1, some differences are obvious between the two groups
of plants of different sex. First, the male individuals have a greater catalase activity
in relation to fresh biomass unit, as compared to females. The difference between
mean values for males and females was 2.27 mg H2O2/g fresh matter.
The peroxidase activity registered superior values in female plants, but the difference
between mean values of female and male plants was smaller (0.9 mg H2O2/g
fresh matter). The specific activities of the two enzymes were also different. Thus,
the mean and the standard error of the mean of catalase were 4.88 } 0.10 mg
H2O2/mg protein, for the group of female plants, and 6.02 } 0.10 mgH2O2/mg pro-
454 E. Tru et al.
Table 1. Average values of the catalase and peroxidase activities and the amount of soluble proteins in male and female hemp plants
Plant sex n
Catalase Peroxidase Soluble proteins
mg H2O2/g fresh matter mg H mg/g fresh matter 2O2/mg protein mg H2O2/g fresh matter mg H2O2/mg protein
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x } SE SD CV
%
SE
%
x �} SE SD CV
%
SE
%
Female 20
27.62
}
0.38
1.71 6.21 1.39
4.88
}
0.10
0.45 9.22 2.04
10.53
}
0.38
1.69 16.04 3.60
1.86
}
0.02
0.10 5.37 1.07
5.659
}
0.16
0.72 12.72 2.83
Male 15
29.89
}
0.61
2.39 7.99 2.04
6.02
}
0.10
0.39 6.47 1.66
9.63
}
0.40
1.55 16.09 4.15
1.94
}
0.05
0.21 10.82 2.57
4.961
}
0.07
0.30 6.04 1.41
n = number of studied plants; x = mean; SE = standard error of the mean; SD = standard deviation; CV = coefficient of variation; SE % = standard error of the mean, expressed in %
tein, for the male individuals. The specific peroxidase activities registered values
of 1.86 } 0.02 mg H2O2/mg protein in pistillate plants and 1.94 } 0.05 mg
H2O2/mg protein in staminate plants. For both catalase and peroxidase, the specific
activity was greater in males, but the increase is more important for catalase
(1.14 mg H2O2/mg protein), while for peroxidase the increase is only 0.08 mg
H2O2/mg protein.
The amount of soluble protein was greater in pistillate plants (5.659
} 0.16 mg/g fresh matter). In staminate plants, these values were 4.961
} 0.07 mg/g fresh matter, namely with 0.698 mg protein/g fresh matter smaller
than those noted for female sexual phenotypes (Table 1).
The standard deviation gives indications on the spread of the observations
around the mean. For the three analysed quantitative characters, the greatest concentration
of the observations around the mean was registered for proteins. In this
case, SD had the smallest values (0.30 for males, and 0.73 for females). The greatest
deviations were noted for catalase: SD = 2.39 in males, and SD = 1.71 in females.
The values of CV showed that the most variable character seems to be
the peroxidase activity (CV = 16.09% for males, and CV = 16.04% for females).
Besides that, the SE% had the highest values for this biochemical trait (4.15 for
males and 3.60 for female plants) (Table 1).
The commentary on the obtained results must start from the fact that
peroxidase activity is related to the developmental processes. Thus, in
organogenesis, the role of peroxidase is frequently explained by the double function
of this enzyme, involved both in oxidizing of some substrata and in auxine catabolism
(LEGRAND, BOUAZZA 1991).The latter function enables the modulation
of morphogenesis by peroxidase, as a result of intervention on endogenous hormonal
balance. In hemp, as well as in other monoecious or dioecious plants,
the gibberellins, auxins, ethylene and cytokinins have an important contribution to
sex expression (MOHAN RAM, SETT 1982a, b, DURAND, DURAND 1984,
CHAILAKHYAN 1985). These hormones generally intervene in the derepression of
reglator genes, which enable the synthesis of specific proteins that control flower
organogenesis. Because of its intervention in the regulation of IAA
(indole-3-acetic acid) level, peroxidase has an indirect role in the sex-determining
mechanism in hemp, more exactly in stamenogenesis and carpellogenesis. Hemp
is one of the species in which a high level of IAA induces the female sex
phenotypisation. The idea of a strong peroxidase activity associated with an increased
auxine catabolism is generally accepted. Concerning catalase, the specific
reaction catalysed by this enzyme is the direct degradation of the toxic H2O2
(the final product of biological oxidization), with release of water and oxygen, that
is taken over to oxygenate the tissues. It seems, however, that the peroxidative activity
of catalases (the reason for which the two enzymes are known as
ghydroperoxidasesh) prevails in tissue. In the case of a smallerH2O2 concentration
and of greater quantities of other substrata, catalase can use a hydrogen donor
other than H2O2.
456 E. Tru et al.
The isoesterase and isoperoxidase patterns
The isoenyzmatic patterns were done for single individuals. The differences between
individuals of the same sex were not significant. Therefore, the schematic
zymograms of izoesterases and isoperoxidases, for one male and one female, are
compared in Figure 1. The esterases are hydrolases that catalyse the hydrolytic
splitting of molecules of substrata at the level of esteric bonds, with formation of
one alcohol molecule and one acid molecule. For both esterase (A) and peroxidase
(B), the isoenzymatic spectrum is richer for staminate plants. Thus, in female
plants, eight multiple isoesterase forms appear, six of them being placed in the domain
of pH = 5.5-6.0 and the other two in the interval of pH = 6.5-7.0. The most
active isoesterase band is placed in the weak acid domain, having pI (isoelectric
point) at pH = 6.0. The eleven isoesterase forms of staminate plants are situated in
the interval of pH = 4.5-7.2, the most active esterase forms having pI situated in
the range of pH = 4.5-5.5, namely more acid that in the case of female plants.
A specific aspect is the presence of three well outlined isoesterase bands at pH
= 6.1-6.5 in male plants . bands that have no correspondence in the isoesterase
pattern of pistillate plants.
Differences also exist for the isoperoxidase pattern. Thus, the female plant has,
as in the case of esterase, fewer bands, two isoforms being in the range of
pH = 4.5-5.5, one at pH = 7.4 and one at pH = 8.9 (extremely basic). The ten fractions
of male genotypes were distributed in the following manner: seven at
pH = 4.7-6.1 (acid), one isoform at pH = 7.4-7.6 (weakly basic) and two bands at
the extremely basic value (pH = 8.9). The presence of four isoperoxidase fractions
at pH = 5.8-6.1 confers a strong physiological advantage of male genotypes over
the female genotypes. The isoesterase and isoperoxidase patterns reveal some differences
at the metabolic level, related to a specific multigenic determinism.
Peroxidase is distributed both in the cytosol and cell wall, as different genetic
isoenzymatic forms (OKEY et al. 1997). Cell wall is regarded as the site of primary
plant peroxidase activity (FRY 1986). Generally it is agreed that acid peroxidases
(especially those found in the cell wall) intervene in lignin biosynthesis, while
the basic ones (with cytoplasmic and vacuolar distribution) are involved in IAA
catabolism, through a decarboxylation step (LIMAM et al. 1998). It is obvious
(Figure 1) that, although there are fewer isoenzymatic bands in the female genotype,
the bands associated with cell walls (active in the acid range) prevail in both
analysed genotypes . a fact in accordance with data suggesting that cell walls
are a principal site of plant peroxidases. Concerning acid peroxidases it is not clear
if they are the products of different genes or if they are modified post-translational
products of a small number of genes (ROS BARCELO et al. 1987). In hemp, like in
other plant species, peroxidase has several isoforms, each with a well-defined
role. The isoperoxidase pattern is complex, just this complexity being the element
amplifying the difficulty to decipher all specific functions of this enzyme
(CLEMENTE 1998, YUN et al. 1998). The genetic determinism of these isoforms is
multigenic. If there are still unexplained details for hemp, for Brassica napus
Biochemical differences in Cannabis sativa L. 457
the existence of at least four distinct genes has been established (HAMED et al.
1998). The isoenzymes of Petunia are under the control of three genes, while in
wheat isoperoxidases are controlled by different genomes and the environmental
conditions do not modify the isoenzymatic pattern (HAMED et al. 1998).
Quantitative analysis of polyphenols, flavones and crude polyholozides
The data regarding the amount of some secondary metabolites, depending on sex
and organ of the plant, were obtained from the individuals whose zymograms are
presented in Figure 1. For every quantitative essay, two replications were made.
Table 2 presents the average values of these phytochemical determinations, depending
on sexual phenotype and on analysed organ, in dry and fresh matter.
The comparative analysis of these results exhibits important differences. Thus,
leaves from female plants have an increased level of polyphenols (1.560 mg%, expressed
in catechin) and flavones (1.084 mg%, expressed in rutosid), while in
male leaves polyphenols are not present, and the quantity of flavones represents
2/3 of the value for female leaves (0.680 mg%). Regarding the stem, no
polyphenols were identified in the terminal (top) part of male plants. In female
plants, however, their level was high (0.940 mg%, expressed in catechin). For flavones,
the situation is inversed: in pistillate plants these compounds are not found,
458 E. Tru et al.
Figure 1. Zymograms of isoesterases (A) and isoperoxidases (B), in female (1) and male (2)
of individuals Cannabis sativa L.
and in staminate plants their level was 0.460 mg%. In middle parts of stems,
the results were negative for all three categories of tested compounds in female
plants, whereas in male plants, polyphenols are lacking. The flavones of
the rutosid type have a value of 0.525 mg%, and the crude polyholozides have
a good representation (+++). Generally, higher levels of polyholozides were present
in male plants (for example, ++ for terminal part of stem, +++ for leaves, +++
for middle part of stem, and for female plants, respectively: +, ++, absent).
The quantitative analyses conducted in fresh matter were negative for
polyphenols for all tested organs, both for male and female plants, but the flavones
and crude polyholozides were present, the latter in high levels (+++) in all samples.
Thus, between sexual phenotypes as well as between the organs of hemp
plants, visible differences exist. It is also important that in the fresh and dry matter
of the male plants, polyphenols were absent . an aspect possibly related to the fact
that in hemp species, in which a high IAA level favours the phenotypisation of female
sex, the capacity to degrade IAA is counterbalanced by auxine protectors
(phenols).
As in the case of other secondary metabolites, the hemp callus was unable to
synthetize polyphenols and flavones (TRUTA, unpublished), which is in accordance
with results of other studies (BRAEMER et al. 1985, BRAUT-BOUCHER et al.
1981, GILLE 1996).
Biochemical differences in Cannabis sativa L. 459
Table 2. Average values registered for polyphenols, flavones and crude polyholozides,
depending on sexual phenotype and on analysed organ in hemp
Plant sex Organ
Polyphenols
mg % catechin
Flavones
mg % rutosid
Polyholozides
Dry matter
Male terminal part of stem 0 0.460 ++
Female terminal part of stem 0.940 0 +
Male leaves 0 0.680 +++
Female leaves 1.560 1.084 ++
Male middle part of stem 0 0.525 +++
Female middle part of stem 0 0 absent
Male inflorescence 0 1.006 absent
Fresh matter
Male leaves 0 0.340 +++
Female leaves 0 0.525 +++
Male inflorescence 0 0.444 +++
Conclusions
In this study, the isoenzymatic pattern of esterase and peroxidase is richer in hemp
male plants, as compared to female plants. For both analysed sexes,
the isoperoxidase bands localized in acid domain are prevalent. The relative
and specific activity of catalase have more reduced values in female plants.
The specific peroxidasic activity is greater in male plants. The average level
of soluble protein was higher in female plants. Significant differences are registered,
depending not only on sex, but also on tested organ in the same plant, in respect
of level of polyphenols, flavones and polyholozides. In male plant,
polyphenols were absent.
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The Inheritance of Chemical Phenotype in Cannabis sativa L
Genetics, Vol. 163, 335-346, January 2003, Copyright © 2003
Etienne P. M. de Meijer, Manuela Bagatta, Andrea Carboni, Paola Crucitti, V. M. Cristiana Moliterni, Paolo Ranall, and Giuseppe Mandolino
aHortaPharm B.V., 1075 VS, Amsterdam, The Netherlands
Istituto Sperimentale per le Colture Industriali, 40128 Bologna, Italy
Corresponding author: Giuseppe Mandolino, Via di Corticella 133, 40128 Bologna, Italy., g.mandolino@isci.it (E-mail)
Communicating editor: C. S. GASSER
ABSTRACT
Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure -9-tetrahydrocannabinol (THC) chemotypes.
All the plants belonging to the F1's were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F1 plants were self-fertilized, and 10 inbred F2 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 F1 to the F2's derived from it. A model involving one locus, B, with two alleles, BD and BT, is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype BD/BT at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either BD/BD or BT/BT). 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 F2 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.
CHEMOTYPICAL diversity in Cannabis: The class of secondary products unique to the dioecious species Cannabis sativa L. (hemp) is the terpenophenolic substances known as cannabinoids, which accumulate mainly in the glandular trichomes of the plant (MECHOULAM 1970...read more
Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?John M. McPartland Ethan B. Russo
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, £G9-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.
[Article copies available for a fee from The Haworth Document Delivery
Service: 1-800-342-9678. E-mail address: <getinfo@haworthpressinc.com>
Website: <http://www.HaworthPress.com> 2001 by The Haworth Press, Inc.
All rights reserved.]
John M. McPartland, DO, MS, is affiliated with GW Pharmaceuticals, Ltd., Porton
Down Science Park, Salisbury, Wiltshire, SP4 0JQ, UK.
Ethan B. Russo, MD, is affiliated with Montana Neurobehavioral Specialists, 900
North Orange Street, Missoula, MT 59802 USA.
Address correspondence to: John M. McPartland, DO, Faculty of Health&Environmental
Science, UNITEC, Private Bag 92025, Auckland, New Zealand (E-mail: jmcpartland
@unitec.ac.nz).
The authors thank David Pate and Vincenzo Di Marzo for pre-submission reviews.
[Haworth co-indexing entry note]: ¡§Cannabis and Cannabis Extracts: Greater Than the Sum of Their
Parts?¡¨ McPartland, John M., and Ethan B. Russo. Co-published simultaneously in Journal of Cannabis Therapeutics
(The Haworth Integrative Healing Press, an imprint of The Haworth Press, Inc.) Vol. 1, No. 3/4,
2001, pp. 103-132; and: Cannabis Therapeutics in HIV/AIDS (ed: Ethan Russo) The Haworth Integrative
Healing Press, an imprint of The Haworth Press, Inc., 2001, pp. 103-132. Single or multiple copies of this article are available for a fee from The Haworth Document Delivery Service [1-800-342-9678, 9:00 a.m. - 5:00
p.m. (EST). E-mail address: getinfo@haworthpressinc.com].
2001 by The Haworth Press, Inc. All rights reserved. 103
INTRODUCTION
Cannabis is an herb; it contains hundreds of pharmaceutical compounds
(Turner et al. 1980). Herbalists contend that polypharmaceutical herbs provide
two advantages over single-ingredient synthetic drugs:
(1) therapeutic effects
of the primary active ingredients in herbs may be synergized by other compounds,
and
(2)side effects of the primary active ingredients may be mitigated
by other compounds.
Thus, cannabis has been characterized as a ¡§synergistic
shotgun,¡¨ in contrast to Marinol. (£G9-tetrahydrocannabinol, THC), a synthetic,
single-ingredient ¡§silver bullet¡¨ (McPartland and Pruitt 1999).
Mechoulam et al. (1972) suggested that other compounds present in herbal
cannabis might influence THC activity. Carlini et al. (1974) determined that
cannabis extracts produced effects ¡§two or four times greater than that expected
from their THC content.¡¨ Similarly, Fairbairn and Pickens (1981) detected
the presence of unidentified ¡§powerful synergists¡¨ in cannabis extracts
causing 330% greater activity in mice than THC alone.
Other compounds in herbal cannabis may ameliorate the side effects of
THC. Whole cannabis causes fewer psychological side effects than synthetic
THC, seen as symptoms of dysphoria, depersonalization, anxiety, panic reactions,
and paranoia (Grinspoon and Bakalar 1997). This difference in side effect
profiles may also be due, in part, to differences in administration: THC
taken by mouth undergoes ¡§first pass metabolism¡¨ in the small intestine and
liver, to 11-hydroxy THC; the metabolite is more psychoactive than THC itself
(Browne and Weissman 1981). Inhaled THC undergoes little first-pass metabolism,
so less 11-hydroxy THC is formed. Thus, ¡§smoking cannabis is a satisfactory
expedient in combating fatigue, headache and exhaustion, whereas the
oral ingestion of cannabis results chiefly in a narcotic effect which may cause
serious alarm¡¨ (Walton 1938, p. 49).
Respiratory side effects from inhaling cannabis smoke may be ameliorated by
both cannabinoid and non-cannabinoid components in cannabis. For instance,
throat irritation may be diminished by anti-inflammatory agents, mutagens in
the smoke may be mitigated by antimutagens, and bacterial contaminants in
cannabis may be annulled by antibiotic compounds (McPartland and Pruitt
1997). The pharmaceutically active compounds in cannabis that enhance beneficial
THC activity and reduce side effects are relatively unknown. The pur-
104 CANNABIS THERAPEUTICS IN HIV/AID.... read full PDF
Effects of Gibberellic Acid on Primary Terpenoids and Delta-Tetrahydrocannabinol in Cannabis sativa at Flowering StageMansouri H, Asrar Z, Mehrabani M
J Integr Plant Biol 2009 Jun; 51(6):553-61.
Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway.
However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA(3)) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways.
Our results showed GA(3) caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Delta(9)-tetrahydrocannabinol (THC) contents and an increase in alpha-tocopherol content. The treated plants with GA(3) showed an increase in HMGR activity.
This increase in HMGR activity was followed by accumulation of stigmasterol and beta-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA(3) can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.
A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa LFischedick JT, Glas R, Hazekamp A, Verpoorte R
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 Delta(9)-tetrahydrocannabinol (Delta(9)-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: Delta(9)-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 r(2) = 0.9968.
CONCLUSION: Results have shown that the HPTLC method is reproducible and accurate for the quantification of Delta(9)-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.
Comparative Proteomics of Cannabis sativa Plant Tissues J Biomol Tech. 2004 June; 15(2): 97–106.
Tri J. Raharjo,ab Ivy Widjaja,c Sittiruk Roytrakul,b and Robert Verpoorteb
aDepartment of Chemistry, Gadjah Mada University, Yogyakarta, Indonesia;
bDivision of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands
cCurrent address: Genome Institute of Singapore, 1 Science Park Road, #05–01, The Capricorn, Singapore Science Park II, Singapore 117528.
Robert Verpoorte, Division of Pharmacognosy, Institute of Biology, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands (fax: 31-71-5274511; e-mail: verpoort@ lacdr.leidenuniv.nl).
This article has been cited by other articles in PMC.
Abstract
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.
Keywords: Cannabis sativa, comparative, mass fingerprint, proteomic
The biosynthesis of cannabinoids, a class of terpenephenolic compounds found in Cannabis sativa, is not yet fully known. Cannabinoids are found in all tissues of the C. sativa plant, but the amount in which they are present differs considerably among the tissues.
Cannabinoids are most abundant in flowers, especially in the glands. This raises the question of whether biosynthesis of cannabinoids occurs in all tissues but in different quantities, or only in one tissue and is then transported to the others. In both scenarios it is assumed that the expression level of the genes involved in the cannabinoid biosynthesis is different among the tissues. In any case, the differential expression of cannabinoid biosynthesis may be used to further clarify this pathway by comparing, on the level of proteins or mRNAs, the tissues with varying amounts of cannabinoids with the tissues that do not produce cannabinoids.
Gene expression can be studied by measuring mRNA levels using methods such as microarrays, serial analysis of gene expression, and real-time polymerase chain reaction. However, studies in yeast revealed the absence of a strong correlation between the abundance of the protein and the corresponding mRNA. Alternative methods of study involve the use of enzyme assays or proteome analysis to identify expressed proteins.
The enzymes known to be involved in cannabis biosynthesis are olivetolic acid prenylase, tetrahydrocannabinolic acid synthase (THCA synthase), cannabidiolic acid synthase (CBDA synthase), and cannabichromenic acid synthase (CBCA synthase). Though assays are available for several of the enzymatic steps of the cannabinoid biosynthesis, it would be an immense task to purify each of these enzymes for sequencing. Moreover, not all of the steps are known. Thus, proteome analysis (proteomics) seems to be preferable to enzyme assaying in obtaining sequence information from all proteins connected with cannabinoid biosynthesis.
The use of proteomics in the study of secondary metabolite biosynthesis has been reviewed by Jacobs et al.
Proteomics is a new tool used to identify and characterize all proteins expressed in an organism or cell. Single proteins can be separated using column chromatography or two-dimensional (2D) electrophoresis prior to mass spectrometric (MS) analysis. Advanced MS allows ionization of macromolecules such as proteins and peptides. Proteins can be identified by matching peptide mass fingerprinting with database sequences or by sequencing whole-length proteins with tandem MS.
Peptide fingerprints can be obtained by ionization of the peptides that result from enzymatic digestion, usually by trypsin. Accurate peptide masses of peptide fingerprints can be used for searching matching proteins in the databases resulting in molecular weight search (Mowse) score. The peptides themselves can be fragmented using tandem MS resulting in the amino acid sequences.
Thousands of proteins occur in the cell, and to choose and separate the protein responsible for a particular function is not an easy task. Using 2D electrophoresis proteins are separated based on pI and molecular weight (MW), which results in a proteome pattern of the cells or tissues under a certain condition. Proteins involved in the production of metabolites can be studied by comparing producing with nonproducing conditions of the cells or tissues:
Proteins that are present in the producing conditions but not in the nonproducing conditions might be involved in the production of the compounds of interest. This comparison can be performed more easily with cell cultures, as they tend to have a less complex matrix than plant tissues. Unfortunately, cannabinoids are not produced by cell cultures. Another option is to compare high-producing tissues, such as flowers, with low-producing tissues, such as leaves. The pI and MW of THCA synthase, CBDA synthase, and CBCA synthase are available (Table 11).). Therefore, these proteins might be identified from the tissues of flowers and glands using 2D electrophoresis and confirmed by MS analysis.
Western blot analysis using antibodies against a group of protein...read more
Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa
Copyright © 2009 The Author(s).
M. David Marks, Li Tian, Jonathan P. Wenger, Stephanie N. Omburo, Wilfredo Soto-Fuentes, Ji He, David R. Gang, George D. Weiblen, and Richard A. Dixon
Department of Plant Biology, University of Minnesota, 1445 Gortner Ave, St Paul, MN 55108, USA
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721-0036, USA
To whom correspondence should be addressed: E-mail: marks004@umn.edu
Present address: Department of Plant Sciences, University of California Davis, One Shield Avenue, Davis, CA 95616, USA.
Received May 14, 2009; Revised June 3, 2009; Accepted June 5, 2009.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Abstract
RNA isolated from the glands of a Δ9-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.
Introduction
Cannabis sativa has a long history of cultivation for a variety of uses including food, fibre, medicine, and recreational drugs...read more
Cannabis: A source of useful pharma compounds neglected in India
Express Pharma Pulse
In Europe and in the New World where Cannabis was introduced very late is being cultivated on an increasing scale as a valuable crop for industrial products, while in India where it has been cultivated since time immemorial as a fibre and food crop the cultivation is dwindling, writes N C Shah in the first part of the article
Cannabis sativa commonly known as cannabis is the earliest food, fibre, medicinal, psychoactive and oil yielding cultivated plant and for centuries ranked as one of the most important agricultural crop of the orient. It is interesting to note that in Europe and in the New World where it was introduced very late is being cultivated on an increasing scale as a valuable crop for industrial products, while in India in the Himalayan states like, Himachal Pradesh, Uttaranchal, Darjeeling (WB) and Sikkim, where it has been cultivated since time immemorial as a fiber and food crop the cultivation is dwindling. Certain useful pharmaceutical compounds found in different parts of the plants are as follows.
Female inflorescence, seed, seed oil and seed cake: Chemical composition
Female inflorescence: The chemical composition hemp inflorescence of female flowers contain about 15-20 per cent of resin and a total of 483 natural chemical components, which have been isolated and identified. The cannabinoids are the most distinctive active constituent found only in the Cannabis plant and the most important one is (-)-D9-trans-tetrahydrocanabinol, commonly referred to as D9-THC. The total 483 chemical constituents can further be grouped into the following distinct classes; cannabinoids - 66; nitrogenous compounds - 27; amino acids - 18; proteins, glycoproteins and enzymes - 11; sugars and related compounds - 34; hydrocarbons - 50; simple alcohols - 7; simple aldehydes - 12; simple ketones - 13; Simple acids - 21; fatty acids - 22; simple esters and lactones - 13; steroids - 11; terpenes -120; non-cannabinoid phenols - 25; flavonoids - 21; vitamins - 1; pigments - 2; elements-9, (ElSohly 2002).
Seed composition : According to Duke (1983) the composition of Asian seeds per 100 g is: moisture - 13.6 g and protein - 27.1 g; fat - 25.6 g; carbohydrate total - 27.6 g; fiber - 20.3 g; ash - 6.1 g; calcium - 120 mg; phosphorus - 970 mg; iron - 12.0 mg; beta-carotene - 5 mg; thiamine - 0.32 mg; riboflavin - 0.17 mg; niacin - 2.1 mg and K calories 421 have been reported.
Seed oil composition: The hemp seed oil contains 25 per cent to 35 per cent of oil and it is the lowest in saturated fats 9-11 per cent of total volume of oil. The oil pressed from the seed contains, a number of saturated and unsaturated essential fatty acids such as; oleic acid, linoleic acid (LA), linolenic and isolinolenic acids (LNA & ILNA), respectively.
The composition of seed cake or defated meal: According to Duke (1983) the seed cake contains water - 10.8 per cent; fat - 10.2 per cent; protein - 30.8 per cent; N-free extract - 40.6 per cent; and ash - 7.7 per cent; (K20 - 20.3 per cent; Na20 - 0.8 per cent; CaO - 23.6 per cent; MgO - 5.7 per cent; Fe2O3 - 1.0 per cent; P2O5 - 36.5 per cent; SO3 - 0.2 per cent; SiO2 - 11.9 per cent; Cl - 0.1 per cent; and a trace of Mn2O3). A crystalline protein globulin has been isolated from defatted meal and it contains; glycocol - 3.8 per cent; alanine - 3.6 per cent; valine and leucine - 20.9 per cent; phenylalanine - 2.4 per cent; tyrosine -2.1 per cent; serine - 0.3 per cent; cystine - 0.2 per cent; proline - .1 per cent; oxyproline - 2.0 per cent; aspartic acid - 4.5 per cent; glutamic acid - 18.7 per cent; tryptophane and arginine - 14.4 per cent; lysine -1.7 per cent; and histidine - 2.4 per cent.
Unrefined seed oil composition after Leson & Pless, et al, (2002 p. 411)
Analyses Hemp Oil
Saturated fatty acids Individual saturated fatty acids percentage
Palmitic acid (16:0) 6-9 per cent
Stearic acid (18:0) 2-3.5 per cent
Arachidic acid (20:0) <1-3 per cent
Behenic acid (22:0) 0.3 per cent
Total saturated fatty acid 9-11 per cent
Unsaturated fatty acids Individual unsaturated fatty acids percentage
Oleic acid (18:1 w-9) 8.5-16 per cent
Linoleic acid (18:2 w-6) 53-60 per cent
g-Linolenic acid GLA (18:3 w-6) 1-4 per cent
a-Linolenic acid (18:3 w-3) 15-25 per cent
Stearidonic acid (18:4 w-3) 0.4-2 per cent
Eicosanoic acid (20:1) <0.5 per cent
Total unsaturated fatty acids 89-91 per cent
Chemical Analyses
Vitamin E 100-150 mg/100 g (mostly g-tocopherol)
13-20 IU/100g (as a-tocopherol equivalents)
Chlorophyll 50-20 ppm
THC content 2-20 ppm
Specific gravity 0.92 kg/1
Iodine value 155-170
Peroxide value 4-7 meq 02/kg
Free fatty acids 1.5-2.0% as Oleic acid
Phosphatides 100-400 ppm
Essential oils: Novak et al, (2001) extracted and reported the main constituents from the essential oil as alpha-pinene, myrcene, trans-beta-ocimene, alpha-terpinolene, trans-caryophyllene and alpha-humulene. The content of alpha-terpinolene divided the cultivars in two distinct groups, an Eastern European group with 8 per cent and a French group of cultivars of around 16 per cent, respectively. The antimicrobial activity of the essential oil was reported as modest. However, delta-9-tetrahydrocannabinol could not be detected in any of the essential oils and the amount of other cannabinoids was very poor.
Pharmacological, clinical and nutritional values
According to Burger (1986 p.81) the mind-affecting constituents of cannabis are the tetrahydro-cannabinols, etc., and it is reported to lower the elevated intraocular pressure during glaucoma. D9 - THC has specific anti-emetic property. Vomiting is a serious side effect of irradiation or of drug administration in cancer therapy and other antiemetic drugs usually produce incomplete and variable results. For the last 15 years patients undergoing such treatments, have claimed that marijuana smoking is quite beneficial in alleviating or preventing such emesis. The antiemetic effect of D9- THC is presently being evaluated in numerous clinics and quite possibly will become a standard treatment in the near future.
In modern medicine possible uses of cannabis are; in glaucoma, alleaviating the pains of cancer and in chemotherapy. It has been observed Lewis lung adenocarcinonoma growth has been retarded by oral administration of D9-tetrahydrocannabinol, D9-tetrahydrocannabinol and cannabinol, but not by cannabidiol. The D9-THC also inhibits the replication of Herpes simplex virus, which generally observed after chemotherapy to the cancer patients.
Pate (1995) has given the potential uses of Cannabis and its chemicals in eighteen ailments and diseases such as; Aid’s patients appetite stimulation; amelioration; nausea; chemotherapy; approved uses of THC in cancer; anxiety and psychosis; asthma; epilepsy; glaucoma; inflammation and swelling; microbial infections; movement disorder; spasticity and other neuromuscular disorders; multiple sclerosis; niemann-pick disease; opiate and alcohol addiction and pain and ulcer.
According to Bayer (2001) Cannabis helps to cope with some of the difficult symptoms and treatment associated with AIDS. In spite of a need for more rigorous scientifically controlled research, an increasing number of persons with AIDS are using cannabis because they find it controls nausea, increases appetite, promotes weight gain, decreases pain and improves mood.
D9-THC is the pharmacologically and toxicologically most relevant constituent of the hemp plant, responsible for most of the effects of natural cannabis preparations. According to Grotenhermen (2002) the following dose-dependent effects were observed in clinical studies in vivo or in vitro, respectively, as described under:
Intraocular pressure: Marijuana smoking reduced intraocular pressure in patients with glaucoma and added to the effects of conventional glaucoma medications. It has been argued that since all of the conventional medications and further support the use of marijuana as a medicinal adjunct to the treatment of the second leading cause of blindness in the United States.
Brachycardia syndromes and insomonia: Numerous other medicinal applications of the D9-tetrahydrocannabinol (THC) the major active constituent of marijuana and other marijuana derived cannabinoids have been described. In man these derivatives produce tachycardia, an effect of possible therapeutic benefit for a variety of bradycardia syndromes, since the tachycardia effect is centrally mediated. In the treatment of insomnia, THC reduces the time required to fall asleep.
As analgesic: The analgesic effects of cannabinoids have prompted their use in the treatment of headache, dysmennorhea and is associated with metastatic cancer. Some therapeutic benefits of THC has also been shown for the treatment of asthma.
Essential oil: The antibacterial activity of the essential oil of C sativa was assessed on Staphylococcus aureus, Streptococcus faecalis, Mycobacterium smegmatis, Pseudomonas flurescens and Escherichia coli. The oil was found to be active on Gram-positive bacteria and has been used against these bacteria in cases of resistance against penicillin. The antibacterial agent appears to be cannabidiolic acid, (Oliver-Bever 1986).
Synthetic preparations
According to Grotenhermen & Russo (2002 p XXVIII) there are synthetically manufactured (-)-trans-isomer of D9-THC named as dronabinol. If it is dissolved in sesame oil and filled in capsule and known as marinol, which is available in USA, Canada and some European countries. Another synthetic drug is nabilone, which is a synthetic derivative of D9-THC with a slightly modified molecular structure. It is a registered trade mark of Eli Lilly & Co. and marketed under the name Cesamet. It is available in UK and Canada and in some other European countries.
The author is ex-founder director of Herbal Research & Development Institute (Govt of UP now Uttaranchal), retired scientist (CIMAP,C SIR) and also hon.coordinator, Centre for Indigenous Knowledge of Indian Herbal Resources. (CIKIHR).
Email: ncshah@sancharnet.in
Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa
Copyright © 2009 The Author(s).
M. David Marks, Li Tian, Jonathan P. Wenger, Stephanie N. Omburo, Wilfredo Soto-Fuentes, Ji He, David R. Gang, George D. Weiblen, and Richard A. Dixon
Department of Plant Biology, University of Minnesota, 1445 Gortner Ave, St Paul, MN 55108, USA
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721-0036, USA
To whom correspondence should be addressed: E-mail: marks004@umn.edu
Present address: Department of Plant Sciences, University of California Davis, One Shield Avenue, Davis, CA 95616, USA.
Received May 14, 2009; Revised June 3, 2009; Accepted June 5, 2009.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Abstract
RNA isolated from the glands of a Δ9-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.
Introduction
Cannabis sativa has a long history of cultivation for a variety of uses including food, fibre, medicine, and recreational drugs...read more
Cannabis: A source of useful pharma compounds neglected in India
Express Pharma Pulse
In Europe and in the New World where Cannabis was introduced very late is being cultivated on an increasing scale as a valuable crop for industrial products, while in India where it has been cultivated since time immemorial as a fibre and food crop the cultivation is dwindling, writes N C Shah in the first part of the article
Cannabis sativa commonly known as cannabis is the earliest food, fibre, medicinal, psychoactive and oil yielding cultivated plant and for centuries ranked as one of the most important agricultural crop of the orient. It is interesting to note that in Europe and in the New World where it was introduced very late is being cultivated on an increasing scale as a valuable crop for industrial products, while in India in the Himalayan states like, Himachal Pradesh, Uttaranchal, Darjeeling (WB) and Sikkim, where it has been cultivated since time immemorial as a fiber and food crop the cultivation is dwindling. Certain useful pharmaceutical compounds found in different parts of the plants are as follows.
Female inflorescence, seed, seed oil and seed cake: Chemical composition
Female inflorescence: The chemical composition hemp inflorescence of female flowers contain about 15-20 per cent of resin and a total of 483 natural chemical components, which have been isolated and identified. The cannabinoids are the most distinctive active constituent found only in the Cannabis plant and the most important one is (-)-D9-trans-tetrahydrocanabinol, commonly referred to as D9-THC. The total 483 chemical constituents can further be grouped into the following distinct classes; cannabinoids - 66; nitrogenous compounds - 27; amino acids - 18; proteins, glycoproteins and enzymes - 11; sugars and related compounds - 34; hydrocarbons - 50; simple alcohols - 7; simple aldehydes - 12; simple ketones - 13; Simple acids - 21; fatty acids - 22; simple esters and lactones - 13; steroids - 11; terpenes -120; non-cannabinoid phenols - 25; flavonoids - 21; vitamins - 1; pigments - 2; elements-9, (ElSohly 2002).
Seed composition : According to Duke (1983) the composition of Asian seeds per 100 g is: moisture - 13.6 g and protein - 27.1 g; fat - 25.6 g; carbohydrate total - 27.6 g; fiber - 20.3 g; ash - 6.1 g; calcium - 120 mg; phosphorus - 970 mg; iron - 12.0 mg; beta-carotene - 5 mg; thiamine - 0.32 mg; riboflavin - 0.17 mg; niacin - 2.1 mg and K calories 421 have been reported.
Seed oil composition: The hemp seed oil contains 25 per cent to 35 per cent of oil and it is the lowest in saturated fats 9-11 per cent of total volume of oil. The oil pressed from the seed contains, a number of saturated and unsaturated essential fatty acids such as; oleic acid, linoleic acid (LA), linolenic and isolinolenic acids (LNA & ILNA), respectively.
The composition of seed cake or defated meal: According to Duke (1983) the seed cake contains water - 10.8 per cent; fat - 10.2 per cent; protein - 30.8 per cent; N-free extract - 40.6 per cent; and ash - 7.7 per cent; (K20 - 20.3 per cent; Na20 - 0.8 per cent; CaO - 23.6 per cent; MgO - 5.7 per cent; Fe2O3 - 1.0 per cent; P2O5 - 36.5 per cent; SO3 - 0.2 per cent; SiO2 - 11.9 per cent; Cl - 0.1 per cent; and a trace of Mn2O3). A crystalline protein globulin has been isolated from defatted meal and it contains; glycocol - 3.8 per cent; alanine - 3.6 per cent; valine and leucine - 20.9 per cent; phenylalanine - 2.4 per cent; tyrosine -2.1 per cent; serine - 0.3 per cent; cystine - 0.2 per cent; proline - .1 per cent; oxyproline - 2.0 per cent; aspartic acid - 4.5 per cent; glutamic acid - 18.7 per cent; tryptophane and arginine - 14.4 per cent; lysine -1.7 per cent; and histidine - 2.4 per cent.
Unrefined seed oil composition after Leson & Pless, et al, (2002 p. 411)
Analyses Hemp Oil
Saturated fatty acids Individual saturated fatty acids percentage
Palmitic acid (16:0) 6-9 per cent
Stearic acid (18:0) 2-3.5 per cent
Arachidic acid (20:0) <1-3 per cent
Behenic acid (22:0) 0.3 per cent
Total saturated fatty acid 9-11 per cent
Unsaturated fatty acids Individual unsaturated fatty acids percentage
Oleic acid (18:1 w-9) 8.5-16 per cent
Linoleic acid (18:2 w-6) 53-60 per cent
g-Linolenic acid GLA (18:3 w-6) 1-4 per cent
a-Linolenic acid (18:3 w-3) 15-25 per cent
Stearidonic acid (18:4 w-3) 0.4-2 per cent
Eicosanoic acid (20:1) <0.5 per cent
Total unsaturated fatty acids 89-91 per cent
Chemical Analyses
Vitamin E 100-150 mg/100 g (mostly g-tocopherol)
13-20 IU/100g (as a-tocopherol equivalents)
Chlorophyll 50-20 ppm
THC content 2-20 ppm
Specific gravity 0.92 kg/1
Iodine value 155-170
Peroxide value 4-7 meq 02/kg
Free fatty acids 1.5-2.0% as Oleic acid
Phosphatides 100-400 ppm
Essential oils: Novak et al, (2001) extracted and reported the main constituents from the essential oil as alpha-pinene, myrcene, trans-beta-ocimene, alpha-terpinolene, trans-caryophyllene and alpha-humulene. The content of alpha-terpinolene divided the cultivars in two distinct groups, an Eastern European group with 8 per cent and a French group of cultivars of around 16 per cent, respectively. The antimicrobial activity of the essential oil was reported as modest. However, delta-9-tetrahydrocannabinol could not be detected in any of the essential oils and the amount of other cannabinoids was very poor.
Pharmacological, clinical and nutritional values
According to Burger (1986 p.81) the mind-affecting constituents of cannabis are the tetrahydro-cannabinols, etc., and it is reported to lower the elevated intraocular pressure during glaucoma. D9 - THC has specific anti-emetic property. Vomiting is a serious side effect of irradiation or of drug administration in cancer therapy and other antiemetic drugs usually produce incomplete and variable results. For the last 15 years patients undergoing such treatments, have claimed that marijuana smoking is quite beneficial in alleviating or preventing such emesis. The antiemetic effect of D9- THC is presently being evaluated in numerous clinics and quite possibly will become a standard treatment in the near future.
In modern medicine possible uses of cannabis are; in glaucoma, alleaviating the pains of cancer and in chemotherapy. It has been observed Lewis lung adenocarcinonoma growth has been retarded by oral administration of D9-tetrahydrocannabinol, D9-tetrahydrocannabinol and cannabinol, but not by cannabidiol. The D9-THC also inhibits the replication of Herpes simplex virus, which generally observed after chemotherapy to the cancer patients.
Pate (1995) has given the potential uses of Cannabis and its chemicals in eighteen ailments and diseases such as; Aid’s patients appetite stimulation; amelioration; nausea; chemotherapy; approved uses of THC in cancer; anxiety and psychosis; asthma; epilepsy; glaucoma; inflammation and swelling; microbial infections; movement disorder; spasticity and other neuromuscular disorders; multiple sclerosis; niemann-pick disease; opiate and alcohol addiction and pain and ulcer.
According to Bayer (2001) Cannabis helps to cope with some of the difficult symptoms and treatment associated with AIDS. In spite of a need for more rigorous scientifically controlled research, an increasing number of persons with AIDS are using cannabis because they find it controls nausea, increases appetite, promotes weight gain, decreases pain and improves mood.
D9-THC is the pharmacologically and toxicologically most relevant constituent of the hemp plant, responsible for most of the effects of natural cannabis preparations. According to Grotenhermen (2002) the following dose-dependent effects were observed in clinical studies in vivo or in vitro, respectively, as described under:
Intraocular pressure: Marijuana smoking reduced intraocular pressure in patients with glaucoma and added to the effects of conventional glaucoma medications. It has been argued that since all of the conventional medications and further support the use of marijuana as a medicinal adjunct to the treatment of the second leading cause of blindness in the United States.
Brachycardia syndromes and insomonia: Numerous other medicinal applications of the D9-tetrahydrocannabinol (THC) the major active constituent of marijuana and other marijuana derived cannabinoids have been described. In man these derivatives produce tachycardia, an effect of possible therapeutic benefit for a variety of bradycardia syndromes, since the tachycardia effect is centrally mediated. In the treatment of insomnia, THC reduces the time required to fall asleep.
As analgesic: The analgesic effects of cannabinoids have prompted their use in the treatment of headache, dysmennorhea and is associated with metastatic cancer. Some therapeutic benefits of THC has also been shown for the treatment of asthma.
Essential oil: The antibacterial activity of the essential oil of C sativa was assessed on Staphylococcus aureus, Streptococcus faecalis, Mycobacterium smegmatis, Pseudomonas flurescens and Escherichia coli. The oil was found to be active on Gram-positive bacteria and has been used against these bacteria in cases of resistance against penicillin. The antibacterial agent appears to be cannabidiolic acid, (Oliver-Bever 1986).
Synthetic preparations
According to Grotenhermen & Russo (2002 p XXVIII) there are synthetically manufactured (-)-trans-isomer of D9-THC named as dronabinol. If it is dissolved in sesame oil and filled in capsule and known as marinol, which is available in USA, Canada and some European countries. Another synthetic drug is nabilone, which is a synthetic derivative of D9-THC with a slightly modified molecular structure. It is a registered trade mark of Eli Lilly & Co. and marketed under the name Cesamet. It is available in UK and Canada and in some other European countries.
The author is ex-founder director of Herbal Research & Development Institute (Govt of UP now Uttaranchal), retired scientist (CIMAP,C SIR) and also hon.coordinator, Centre for Indigenous Knowledge of Indian Herbal Resources. (CIKIHR).
Email: ncshah@sancharnet.in
