In a previous study some differences in the toxicity between the sublimate from smoked cannabis and resins from ordinary cannabis of the same origin have been found 1.
Author: C. MIRAS & , S. SIMOS, J. KIBURIS
Pages: 13 to 15
Creation Date: 1964/01/01
In a previous study some differences in the toxicity between the sublimate from smoked cannabis and resins from ordinary cannabis of the same origin have been found [ 1] .
This observation is of interest, since among the cannabis addicts very few cannabis eaters exist, while most of them are cannabis smokers.
Since many differences exist among the constituents of resins derived from cannabis of different origins, we thought it would be interesting to find out whether smoking affects the active cannabinols, and to what extent.
For the above reasons, in the present study, cannabis was smoked as described previously and the constituents found in the extract of the sublimate were analysed by the technique of thin-layer chromatography and compared with those extracted from the ordinary cannabis of the same origin. 1
To obtain the resin constituents from ordinary cannabis, the sample was treated as follows: 10 g of the cannabis sample was pulverized in a mortar, and extracted with methanol at room temperature. Thin-layer chromatography was applied to check complete extraction. The extract was filtered and the solvent removed under reduced pressure, at 40°C. The residue was taken up in methanol, and the solution was kept under nitrogen.
The sublimate from smoked cannabis was prepared according to the method described elsewhere [ 1] .
The resin constituents obtained from 10 g of cannabis after smoking and extraction of sublimate, or by simple extraction of ordinary cannabis were dissolved in the same volume of methanol. Thus, 1.9 g of resin constituents derived from the sublimate after smoking 10 g of cannabis was dissolved in methanol, to a volume of 35.5 ml (sample A). Also, 3.55 g of resin constituents obtained by extraction of 10 g of ordinary cannabis was dissolved in methanol to a volume of 35.5 ml (sample B).
1 From a sample of 200 g of the same origin (Syria) made available to the authors by the Narcotics Department of the Greek Police.
Comparative studies have been carried out on both samples by column chromatography and thin-layer chromatography.
Column chromatography. - Aluminium oxide (Fluka type 507) was used at pH 7.0 4 ± 0.5 in columns of internal diameter of 10 mm and height of 9 cm. The flow rate was 1.5 ml/minute. The elution systems were ( a) methanol, ( b) 5% acetic acid in methanol.
Usually 0.5 ml of each sample (A and B) were chromatographed on this column, cannabidiol, cannabinol and tetrahydrocannabinol were eluted quantitatively with 60 - 70 ml methanol (the completion of the elution was checked by thin - layer chromatography).
Methanol was evaporated under reduced pressure, and the residue dried in a current of nitrogen. The dry residue was dissolved in 0.5 ml methanol and chromate - graphed on thin - layer chromatography.
Thin-layer chromatography. - Standard methods were used for thin - layer chromatography. The impregnation of the plates was achieved with 30 % acetone in dimethyl - formamide, or in a system of CHCI 3 - dimethylformamide 40 : 60.
The plates were dried in air for 15 to 20 minutes before application of the spots. The saturation of the chamber atmosphere was accomplished with dimethylformamide saturated with cyclohexane. Cyclohexane saturated with dimethylformamide were used for the development of chromatograms at 24°C [ 2] .
For the staining of the separated spots the following reagents were used: ( a) Beam's, ( b) Pauly's, and ( c) 2.6 dichlorochinonechlorimide [ 3] [ 4] .
In all cases reference substance (standards 2) were used in the same chromatograms. The quantitative comparison of cannabinols was performed with the aid of a spectrophotometer Zeiss model PMQ II.
Table 1 shows the percentage yield of the resin soluble in methanol after the smoking of different amounts of cannabis taken from the same sample and under the same smoking conditions.
|
Grammes of cannabis which was smoked each time |
Grammes of methanol extractable resin constituents |
Yield (%) |
|---|---|---|
| 36 | 7.8 | 21.6 |
| 10 | 1.9 | 19 |
| 5 | 0.965 | 19.3 |
Table 2 shows the Rf values and the colours of CBD, CBN, THC, after spraying with 2.6 dichlorochinonechlorimide, which have been found to be the same for the samples A and B. The spot with Rf 0.06 in the chromatograms was obtained only in the extract of ordinary cannabis (sample B) and was missing from the sample A (figure 1). This spot has a fluorescence under 2,537 ?. Further examination of this spot suggests that it is identical with that of cannabidiolic acid.
|
Substance |
R f values |
Following spray with 2.6 dichlorchinonchlorimid (4) |
|---|---|---|
|
CBD
|
0.12 - 0.13
|
Pink
|
|
CBN
|
0.38 - 0.41
|
Bluish-green
|
|
THC
|
0.59 - 0.62
|
Blue
|
( a) The comparative study of the spectra of the samples A and B (figure 2) shows a characteristic difference at 300 mµ. The peak over the region of 300 m?, which is attributed to the presence of cannabidiolic acid, is lacking in the extract of sublimate of smoked cannabis.
2 For this purpose cannabidiolic acid acetate, cannabidiol, tetrahydrocannabinol and cannabinol, kindly offered by Professor Hoffman, University of Delaware, were used.
( b) Treatment of an ether solution of the resin of ordinary cannabis with 5 % KOH containing 2 % sodium bisulphite [ 5] keeps off from the ether phase the cannabidiolic acid. Chromatography of the ether layer shows that the spot with Rf 0.06 is missing, as in chromatograms of the sublimate of the smoked cannabis.
( c) The fraction of resin from ordinary cannabis eluted from the aluminium oxide column with 5 % acetic acid in methanol shows a spectrum identical with that of cannabidiolic acid. This fraction shows on thin-layer chromatograms a spot with Rf 0.06, which is stained with l % FeC1 3 in absolute methanol [ 6] . Sublimate of smoked cannabis failed to show any of the above indications.
The cannabinols have also been studied from both samples (A and B) in thin-layer chromatography simultaneously with standards. The area of silicic acid of the samples corresponding to the stained spots of tetrahydrocannabinols were scraped off, eluted with methanol, and the absorption was measured in U.V. light against a blank, of a spot-free eluted area of silicic acid.
In both samples (smoked and non-smoked) the same absorption at ? max 274 m? was measured, indicating that the same quantity of tetrahydrocannabinols exists although during the smoking process a drastic reduction (40 %) of total resin constituents of the ordinary cannabis was found.
From the present data it can be deduced that during the smoking process about 40 % of the resin constituents of the ordinary cannabis disappear, most of them probably because of burning.
Furthermore, the findings of the present experiments strongly suggest that under the described conditions of smoking, cannabidiolic acid disappears from the sublimate, probably because of decarboxylation. This decarboxylation has been described also by Schultz & Haffner [ 7] during heating of cannabidiolic acid at 100°C and pressure of 0.1 mm Hg, and Davis & Farmilo [ 5] , when they analysed the resin constituents by gas liquid chromatography.
The most interesting finding was that tetrahydrocannabinols within the limits of our experimental conditions are trapped quantitatively in the smoking machine and remaining almost unchanged.
Therefore, it can be concluded that the "hashish activity" in man is not affected generally by the smoking process although 40 % of the total resin constituents of the ordinary cannabis disappear.
A comparative study of cannabis before and after smoking is described in this paper.
It was found that during smoking approximately 40% of the resin constituents of ordinary cannabis disappear, probably because of burning.
Cannabidiolic acid is missing from the sublimate of smoked cannabis.
Tetrahydrocannabinol, the active constituent of cannabis, remains unchanged, within the limits of experimental conditions, after smoking of cannabis.
A thin-layer chromatography method for cannabis is also described.
Joachimoglou, J. & Miras, C.: Bulletin on Narcotics , XV: 3-4, p. 7.
De Ropp, R. S.: J. Am. Pharm. Assoc. Sci Ed. , 49, 756 (1960).
Korte, F. & Sieper, H.: Tetrahedron, 10, 158 (1960).
Grlic, L.: Bulletin on Narcotics , XIV: 3, 39 (1962).
Davis, T. W. M. & Farmilo, C. G.: Analytical Chemistry , 35:751 (1963).
Grlic, L.: Bulletin on Narcotics , XIV: 3, 42 (1962).
Schultz, O. E. & Haffner, C.: Arch. Pharm ., 291:63 (1958).
