Experimental
Results and discussion
Acknowledgements
Author: A. OHLSSON , C.I. ABOU-CHAAR , S. AGURELL , I.M. NILSSON , K. OLOFSSON , F. SANDBERG
Pages: 29 to 32
Creation Date: 1971/01/01
There occur in nature a large number of types of both cultivated and wild hemp differing morphologically as well as chemically. It is now suggested (R. E. Schultes in ref. [ 1] ) that there is only one species of Cannabis, C. sativa L. and that the different types of Cannabis may be regarded as "chemotypes" i.e. chemical varieties.
As pointed out in a recent paper by Valle et al. ( [ 2] ), there is a general belief ( [ 3] , [ 4] ) that only the female plants of C. sativa produce the psychotomimetically active compounds of Cannabis preparations (hashish, marihuana). Valle et al. ( [ 2] ) investigated the resin, derived from male and female Cannabis, using pharmacological tests and concluded that the resin from male and female plants exhibited similar pharmacological activity.
Variations in the chemical constituents of Cannabis preparations from different varieties and countries have been investigated in detail by a large number of authors ( [ 4] , [ 5] - [ 8] and others). In particular, there has been an interest in trying to establish by chemical analysis of the cannabinoids, from what country a Cannabis sample originates (e.g. [ 3] , [ 9] , [ 10] ). Although excellent methods are available for the qualitative and quantitative analysis of Cannabis samples ( [ 4] , [ 5] , [ 9] , [ 10] , [ 16] and others) by TLC and GLC, it would seem that there has been found no general correlation between the geographical origin of a Cannabis sample and its constituents.
In the present study, we have analysed qualitatively and quantitatively the chemical constituents of fresh male and female plants of Cannabis sativa by TLC and GLC as well as by mass spectrometry. We also include some data and aspects on the possibility to correlate the chemical constituents and geographical origin of a Cannabis sample.
* Part V of Metabolism of cannabis. Part IV: Acta Chem. Scand. 23, 2207 (1969).
1 Department of Pharmacognosy, Faculty of Pharmacy, Box 6804, 113 86 Stockholm, Sweden.
2 School of Pharmacy, American University of Beirut, Lebanon.
3 Central Military Pharmacy, Karolinska Sjukhuset, Stockholm 60.
Thin-layer chromatography was carried out on Silica Gel G plates impregnated with 20% dimethylformamide in acetone. The plates were allowed to dry a few minutes in the air before applying the sample ( [ 11] ) and were developed with 20% ether in light petroleum. The cannabinoids were detected with a spray of 0.2% Echtblausalz "Merck" in 2N aqueous NaOH ( [ 14] ).
Gas chromatography ( [ 14] ) was performed with Varian Model 204 or 2100 gas chromatographs (FID); 180 cm X 2-3 mm glass columns. Column packing : 5% SE-30 on 100/120 mesh Gas Chrom P (AW-DMCS), 5% XE-60 on 80/100 mesh Chromosorb W (AW-DMCS) and 3% JXR on 100/120 mesh Gas Chrom Q. See also table I, where retention times for different columns are shown. Injector and detector temperatures were maintained at 250-260°. Mass spectrometry of cannabinoids ( [ 17] ) was carried out with an LKB 9000 gas chromatograph-mass spectrometer.
Four different parts of each sex were compared : ( a) flowers including bracts, (sometimes including immature fruits); ( b) upper leaves (small, palmately compound leaves-surrounding the flowers); ( c) large leaves (from lower part of the stem); ( d) stems (lower part). Flowering tops: Upper 20 cm of the flowering plant.
Fresh plant material from 5-10 plants of each sex was dried at low temperature (30-40°) for 1-2 days. An aliquot (0.5 gm) was extracted twice with 15-20 ml light petroleum by grinding with a pestle in a mortar. The extract was evaporated and the residue redissolved in a known quantity (1.0 ml) of light petroleum and chromatographed by GLC and TLC.
Cannabinoids were estimated quantitatively (peak height) by GLC using known amounts of cannabidiol as standard and were identified by TLC, GLC and mass spectrometry. The figures given in table II include both the amount of a neutral phenol (e.g. cannabidiol) and the corresponding acid (cannabidiolic acid), since the acids decarboxylate in the gas chromatograph.
|
3% JXR |
5% SE-30 |
||||
|---|---|---|---|---|---|
|
Compound (internationally used abbreviations) |
205 ° |
235 ° |
225 ° |
245 ° |
5% XE-60 225 ° |
|
Cannabichromene
|
3.7 | 2.2 | 4.1 | ||
|
Cannabidiol (CBD)
|
3.4 | 1.2 | 6.9 | 2.7 | 4.0 |
|
Δ1(6)-Tetrahydrocannabinol (Δ6-THC)
|
4.4 | 3.2 | 4.4 | ||
|
Δ1-Tetrahydrocannabinol (Δ1-THC)
|
4.6 | 1.6 | 9.0 | 3.4 | 5.0 |
|
Cannabinol (CBN)
|
5.6 | 1.9 | 11.1 | 4.2 | 8.1 |
|
Cannabigerol
|
5.8 | 4.0 | 8.0 | ||
|
Cannabinoids, % of dry weight |
||||
|---|---|---|---|---|
|
Male |
Female |
|||
|
Sample and plant part |
CBD |
Δ1-THC |
CBD |
Δ1-THC |
|
Beirut 26.4.1969
|
||||
|
Flowers
|
1.6 | 0.03 | 2.6 | 0.06 |
|
Upper leaves
|
0.6 | 0.01 | 1.8 | 0.00 |
|
Large leaves
|
0.2 | 0.01 | 0.2 | 0.03 |
|
Stem
|
0.08 | 0.00 | 0.2 | 0.00 |
|
Bekaa 19.6.1969
|
||||
|
Flowers
|
1.3 | 0.1 | 2.4 | 0.5 |
|
Upper leaves
|
0.6 | 0.2 | 1.1 | 0.1 |
|
Large leaves
|
0.4 | 0.1 | 0.3 | 0.1 |
|
Stem
|
0.04 | 0.01 | 0.1 | 0.0 |
|
Bekaa 26.6.1969
|
||||
|
Flowers
|
1.6 | 0.2 | 2.8 | 0.04 |
|
Upper leaves
|
0.9 | 0.1 | 1.4 | 0.04 |
|
Large leaves
|
0.2 | 0.1 | 0.4 | 0.02 |
|
Stem
|
0.2 | 0.06 | 0.2 | 0.01 |
|
Hizzine 3.9.1969
|
||||
|
Flowers (fruits)
|
0.7 | 0.6 | 0.3 | 0.4 |
|
Upper leaves
|
0.2 | 1.2 | 1.0 | 0.7 |
|
Large leaves
|
0.1 | 0.4 | 0.2 | 0.2 |
|
Stem
|
0.02 | 0.3 | 0.1 | 0.1 |
|
Caucasus/Sweden 1.10.1969
|
||||
|
Flowers (fruits)
|
0.6 | 0.1 | 0.5 | 0.1 |
|
Upper leaves
|
0.3 | 0.1 | 0.5 | 0.1 |
|
Large leaves
|
0.4 | 0.01 | 0.1 | 0.01 |
|
Stem
|
0.05 | 0.04 | 0.02 | 0.01 |
|
Turkey/Sweden 1.10.1969
|
||||
|
Flowers (fruits)
|
0.7 | 0.01 | 0.8 | 0.02 |
|
Upper leaves
|
0.7 | 0.02 | 0.5 | 0.01 |
|
Large leaves
|
0.5 | 0.01 | 0.7 | 0.02 |
|
Stem
|
0.04 | 0.00 | 0.03 | 0.00 |
|
Bratislava/Sweden 1.10.1969
|
||||
|
Flowers (fruits)
|
0.7 | 0.01 | 0.7 | 0.02 |
|
Upper leaves
|
0.2 | 0.00 | 0.5 | 0.02 |
|
Large leaves
|
0.2 | 0.00 | 0.4 | 0.01 |
|
Stem
|
0.00 | 0.00 | 0.00 | 0.00 |
|
Morocco/Sweden 8.1969
|
||||
|
Flowering tops
|
||||
|
grown outdoors
|
0.04 | 0.4 | ||
|
grown indoors
|
0.04 | 0.1 | ||
|
Unknown/Sweden 8.1969
|
||||
|
Flowering tops
|
0.1 | 0.1 | ||
Thus, table II only gives a fair estimate of cannabinoids, also due to different response for cannabinoids in the flame ionization detector.
Samples examined:
Beirut 26.4.1969 (growing wild on American University of Beirut campus); Bekaa 19.6.1969; Bekaa 26.6.1969 (cultivated in the Bekaa) and Hizzine 3.9.1969 (cultivated, from the village of Hizzine) were all collected in Lebanon on the dates given.
Caucasus/Sweden 1.10.1969 was grown in a green-house in Stockholm in the summer of 1969 from a Cannabis sample containing seeds collected in the Caucasus about 60 km south of Pyatigorsk. Plants harvested 1.10.1969.
Turkey/Sweden 1.10.1969, seeds obtained from Turkish Cannabis sample collected from a cultivation near Afyon in Asia Minor. Bratislava/Sweden 1.10.1969, seeds obtained from the Medicinal Plants garden, Faculty of Pharmacy, Bratislava, Czechoslovakia (Doz. M. Felklova), grown in a green-house in Stockholm. These three samples were grown simultaneously under identical conditions.
Morocco/Sweden 8.1969, seeds of Moroccan origin were grown outdoors in a garden outside Stockholm and the flowering tops collected by the end of August. Some seeds were also simultaneously grown indoors in pots.
Unknown/Sweden 8.1969, seeds of unknown origin grown in a city park in Sweden in the summer of 1969.
The results of the analyses of fresh Cannabis materials are presented in table II and figs. 1-2.
Our data show that cannabinoids are present in all parts of the plants. Calculated on dry weight, the cannabinoids are most abundant in flowering tops and the young, small leaves surrounding the flowers. Analysis of different parts of the plant suggest that there is usually little variation in the relative amounts of cannabidiol/Δ1-tetrahydrocannabinol although we have encountered some minor deviations (fig. 1), mainly in cannabinoid content of stem and large leaves.
A comparison (table II) between male and female Cannabis shows that both sexes contain roughly similar amounts of cannabinoids in similar ratios. Unpublished data ( [ 15] ) on Cannabis grown in Mississippi, USA are in general agreement with this conclusion.
Our GLC data of the nine investigated samples further shows that fresh Cannabis material contains essentially no cannabinol ( 1% of the total amount of cannabinoids) indicating that this compound, known to be present in large amounts in old Cannabis samples, may entirely be an artefact. None of the two samples (Hizzine, Morocco/Sweden) rich in Δl-tetrahydrocannabino1 contained detectable amounts of Δ6-tetrahydrocannabinol ( 1-2% Δ1-THC content).
The samples Morocco/Sweden and Unknown/Sweden show that it is possible to grow in Sweden marihuana rich in the psychotomimetically active Δl-tetrahydrocannabinol, provided one has the proper seed material.
Thus, seeds of a "Δl-tetrahydrocannabinol producing Cannabis strain" can produce Δl-tetrahydrocannabinol in cooler (Sweden) as well as warmer (Morocco) climates. The plausible implication of this fact is, that the type of cannabinoid produced by the plant is dependent upon the inherited properties of the seed and that the influence of the climate is limited.
Knowing that "good marihuana quality" Cannabis seeds have been shipped from one country to another, and will continue to do so, for illegal cultivation, and considering the point just made above, it is evident, that there is no valid basis for attempts to correlate the cannabinoid content with country of origin for a cannabis sample.
It is also evident (fig. 2) that in nature there is a variety of "chemotypes" of C. sativa from one extreme, producing almost exclusively cannabidiol, over intermediate forms, to forms producing predominantly Δl-tetrahydrocannabinol.
Authentic cannabinoids were provided by Drs. R. Mechoulam, Jerusalem; L. Nilsson, Stockholm and T. Petrzilka, Zürich. Mass spectrometric facilities were kindly provided by Drs. J. E. Lindgren and B. Holmstedt as well as S. Strömberg and T. Norin, Stockholm. The valuable assistance of Mr. R. Hessling, Stockholm, is gratefully acknowledged. This study was supported by the Swedish Medical Research Council.
R. E. Schultes, in Symposium on Botany and Chemistry of Cannabis and its Derivatives, Churchill, London, in press.
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