Chemical methods
Biological methods
Discussion
Author: Dr. Ljubiša Grlic
Pages: 37 to 46
Creation Date: 1962/01/01
Introduction
Conversion of cannabinols. - The chemistry of narcotic resin obtained from Cannabis sativa L. has been investigated by a large number of authors. From what has been learnt so far, the most important and major part of the resin consists of a group of closely related constituents, which may be called cannabinolic compounds, or cannabinols. They seem to be converted to each other by a gradual phytochemical process, the nature and rate of which are affected by many various factors.
Cannabidiolic acid, recently isolated by Czechoslovakian [ 40] and German [ 56] authors, seems to represent the initial compound in the conversion process of cannabinols [ 58] . According to Krejci et al. [ 41] , [ 44] , it is 3-methyl-6-isopropenyl-4-n-pentil-2,6-hydroxy-1, 2, 3, 6-tetrahydrophenyl-3-carboxylic acid. A similar structure for cannabidiolic acid has been proposed by Schultz & Haffner [ 57] , differing only in the chelate form by which the carboxylic group was supposed to be bound. Cannabidiolic acid does not exhibit any physiological potency - usually called "hashish activity ". It has, however, been found to possess antibiotic [ 18] , [ 36] , [ 42] , [ 43] , [ 57] and sedative activity [ 44] , [ 56] .
Cannabidiolic acid is readily decarboxylized, and thus converted into cannabidiol [ 58] . Cannabidiol is a dihydrophenol, isolated by Adams et al. 1940 [ 1] , from freshly harvested North American hemp. This is not the active principle of cannabis either, but was found to be responsible for a widely applied cannabis reaction known as Beam test [ 11] . This reaction, besides its use for the identification of cannabis, was erroneously considered as an indication of the potency of the drug [ 33] .
By intramolecular condensation, cannabidiol may be converted into a mixture of physiologically and optically active tetrahydrocannabinols [ 4] . This seems to represent the further step of the phytochemical conversion process of cannabinols. Two isomeric tetrahydrocannabinols, differing in the position of the double bond, have been obtained in this way by Adams et al. [ 5] . Wolner et al. [ 66] have isolated a highly potent tetrahydrocannabinol from Indian charas. A number of homologues, differing in their physiological potency, have been synthesized by Adams et al. [ 6] , [ 7] , [ 8] . Although tetra-hydrocannabinols have not been obtained from cannabis resin in pure homogeneous crystalline form, it is believed that hemp resin contains a mixture of isomeric tetrahydrocannabinols, which are the active principle of the drug.
The final product of conversion is cannabinol, which was isolated in crystalline form by Wood et al. as early as 1899 [ 68] . Cannabinol is a product of spontaneous dehydrogenation of tetrahydrocannabinols. It was definitely identified by synthesis in 1940 by Adams et al. as 1-hydroxy-3-amyl-6, 6,9-trimethyl-6-dibenzopyran [ 3] . As reported by Loewe [ 45] , physiological potency of cannabinol is very low, and may be considered as practically insignificant if compared with that of tetrahydrocannabinols.
Some other minor constituents have been found in cannabis resin as well. In 1896, Wood et al. [ 67] isolated and identified the hydrocarbon n-nonacosane (C 29H 60). From the essential oil of Egyptian hashish, Simonsen & Todd isolated p-cymene, 1-methyl-4-isopropenylbenzene and humulene [ 59] . Further studies of this fraction were recently carried out by Canadian authors [ 18] , [ 49] . However, in the present study, only the content of cannabinols in various types of hemp resin has been examined and discussed.
Variations in composition and potency. - Variations in the amount of the resin producedand in the physiological activity of the drug were observed even before the first experimental studies on cannabis were initiated. Thus, it was known long ago that hemp originating from tropical countries is richer in the content of resin and physiologically stronger than the plant from temperate regions. It was also believed that varieties grown in Asia were the best producers of active resin [ 60] . Cannabis grown in Brazil was considered to be more active than marihuana grown in the United States [ 16] .
Since the work by Adams and Todd groups (1939-1942), when the chemical structure of the most important cannabis constituents was elucidated, there was some evidence that the chemical composition of the resin itself may vary according to the provenance of the drug. Thus, in a purified fraction of the resin extracted from American marihuana, 45-50% of cannabidiol was found by Adams et al. [ 2] . According to the findings reported by Todd et al. [ 35] , [ 60] , Indian charas did not contain cannabidiol, whereas in Egyptian hashish approximately equal quantities of both cannabidiol and cannabinol were found. Asahina & Shiuchi detected cannabidiol in Indian charas, but not in hemp plants grown in Japan [ 9] . Indian and Egyptian resin did not contain an alkali-soluble fraction (35, 48), whereas a large part of American marihuana was reported by Fulton to be extractable by alkali [ 20] . The results obtained by Schultz & Haffner [ 58] indicate a lower content of cannabidiolic acid in Indian cannabis than in the European one.
In addition to the variations in chemical composition, the differences in physiological potency of cannabis extracts of various origin have also been observed. By taking dog ataxia potency refered to synthetic racemic tetrahydrocannabinol as P 1.0, Tunisian hemp was found by Loewe to exhibit P 0.52, whereas American marihuana exhibited only P 0.003 [ 47] . American cannabis seems to contain tetrahydrocannabinols of a lower potency (P 6.0-9.5) than oriental hemp (P 12-14.6) [ 47] .
Outline of the present study. - Inspite of all these indications, no systematic work has been undertaken so far to examine the potency and the content of cannabinolic constituents in cannabis resin of various types and provenance. This is partly due to the lack of chemical methods by which active tetrahydrocannabinols could be distinguished from other inactive cannabinols, and which could replace biological tests for the determination of physiologically active components. As is known, the most commonly used reactions for the identification of cannabis resin, such as Beam [ 11] , Duquénois & Negm [ 15] and Ghamravy test [ 22] are not necessarily connected with the active principles of the drug. The polarimetric method for the estimation of hashish activity [ 23] was considered to be inaccurate [ 50] . The colorimetric [ 50] and titrimetric [ 33] methods for the determination of the potency of hemp resin have also been worked out, but they seem to have been based on some erroneous assumptions at the time when the proper structure and properties of the active principles had not been completely elucidated. Separation of various cannabinols by column and paper chromatography was described recently by Korte & Sieper [ 38] , [ 39] , and by de Ropp [ 55] . Although this technique might offer new possibilities for the estimation of physiological strength of the drug, no comparative experimental data by these methods have since been made available.
In the studies carried out in this institute, two already known but seldom applied cannabis reactions have been found to exhibit, under certain fixed experimental conditions, the differences between reacting to active tetrahydrocannabinols and to other inactive cannabinolic compounds. The obtained differences in the reaction of cannabis resin of various activities and various stages of the conversion process have been explained by carrying out the same tests with pure cannabinolic compounds. 1On this basis, simple and rapid techniques indicating the ripeness and the approximate physiological activity of cannabis have been proposed. Some other new procedures have also contributed to elucidate the composition and the type of various samples which have been available to us.
On the basis of the experimental results obtained so far, it is assumed that the differences in the chemical composition of various types of cannabis resin are mostly affected by the state of development of the phytochemical conversion process by which cannabidiolic acid is gradually transformed to cannabidiol, tetrahydrocannabinols and cannabinol. In some previous papers, this process was called "ripening" of the resin [ 29] , [ 30] , [ 31] , [ 63] .
It seems that in varieties of cannabis plant developed under unfavourable or temperate climatic conditions the progress of the conversion process is checked. The results obtained by two analytical procedures [ 29] , [ 53] have confirmed the earlier assumption [ 63] that such unripe cannabis predominantly contains cannabidiolic acid. The same series of investigations has indicated that in plants developed in a tropical climate, the conversion process is much more advanced. Thus, a ripe type of cannabis will contain a considerable amount of active tetra-hydrocannabinols. A number of samples exhibiting intermediate properties between the two types has also been encountered and referred to as an intermediate type, containing mostly cannabidiol. Inactive cannabinol will predominate if the ripening process continues (overripe type). Analyses of samples originating from Canada have indicated an unusual chemical composition. Such cannabis which, instead of cannabinols, contains predominantly their disintegration products, is referred to as an alterated type.
According to the findings reported by Fulton [ 20] , alkali-soluble carmabinolic constituents, predominating in the fresh resin, are converted by long standing at room temperature to alkali-insoluble compounds. We have confirmed that the conversion process of cannabinols may be continued under favourable conditions even in the stored drug. Therefore, the proposed classification into chemical types does not correspond necessarily to a definite and constant characteristic of a given sample.
For the supply of reference substances, the author wishes to thank Dr. S. Loewe (College of Medicine, University of Utah, Salt Lake City), Professor Dr. F. Korte and Dr. H. Sieper (Chemical Institute, University of Bonn) and Professor Dr. O. E. Schultz (Pharmaceutical Institute, University of Kiel).
It should be born in mind that the above classification is based on a simplified explanation which probably covers only one part of the complex phytochemical processes occurring in cannabis resin. Besides climatic conditions, genetic and various ecological factors seem to influence the chemical composition of the drug.
Fifty-seven samples of cannabis, originating from 17 countries, were available for the experimental work reported in the present study. 2 As most samples originated from seizures, the country concerned did not necessarily correspond to the place where a sample was produced. The quantity of a single sample was never sufficient to carry out complete quantitative chemical or biological analyses by means of the most appropriate methods. Therefore, the conclusions regarding the composition and the type of samples have been drawn on the basis of the procedures requiring only small samples.
The starting material for the analysis was crude resin or its unevaporated petrol-ether solution. Cannabis was macerated with petrol-ether (1: 20) for 24 hours and the mixture filtered. To obtain the dry crude resin, the solvent was evaporated. Seeds were removed from samples before extraction. Extinction readings were made on a Jobin-Yvon "Maroc" spectrophotometer, using 1 cm cells. Direct light had to be avoided in all the procedures applied.
Content of Acid Fraction
Systematic determination of the acid fraction in various samples of cannabis resin has been carried out in this institute, and the results have been reported elsewhere [ 29] . The procedure adopted was based on the experience of some previous authors [ 20] , [ 58] . The extract in petrol-ether was shaken out with a solution containing 5% NaOH and 5% Na 2SO 3.The alkali extract obtained was acidified by means of diluted sulphuric acid, extracted with ether, dried in vacuum, weighed and calculated as a percentage of crude resin. In order to avoid decarboxylation of cannabidiolic acid, the procedure was gone through rapidly. Although the acid fraction obtained in this way could contain some secondary weak acids, the results were treated and discussed as if they corresponded only to the content of cannabidiolic acid, which is obviously the main constituent of this fraction.
The acid content ranged from 3.8 to 41.7% in the examined samples of the resin. It was lowest in overripe and ripe cannabis originating in tropical areas, whereas it was highest in unripe samples from central European countries. Most of the samples from temperate regions showed a tendency to intermediate values.
The author is indebted to the Division of Narcotic Drugs of the United Nations, to Professor Décio Parreiras, Faculty of Medicine, Rio de Janeiro, and to Mr. Henry L. Giordiano, acting Commissioner of Narcotics, Washington, D.C., for the supply of cannabis samples.
The results obtained may confirm that cannabidiolic acid is the initial compound in the phytochemical conversion of cannabinols. It was concluded that the acid fraction content may be used as an indication of the progress of the ripening process occurring in cannabis resin.
Indophenol Test
The indophenol reaction described by Gibbs [ 24] on phenols with a free para position, yielding coloured products with quinone chlorimides, was applied for the characterization of cannabis resin. Under certain fixed experimental conditions, stable reaction products were obtained with various types of the drug, showing a large scale of colours. Two absorption constants were proposed, indicating both the stage of the conversion process and the presence of disintegration products of cannabinols in the resin examined [ 30] .
The reaction was performed in the following way. To 2 ml of a 0.05% ethanolic solution of the crude resin, 0.5 ml of a freshly prepared 0.03% ethanolic solution of 2,6-dichloroquinonechlorimide were added. The mixture was rapidly transfered into 5 ml of a 0.1 N NaOH solution containing 0.6% of crystalline Na 2 SO 3and shaken immediately thereafter. The extinction of the coloured product was measured at 420, 525 and 630 m µ, using ethanol as a blank.
The colour of the product obtained can range from pink through violet and blue to bluish green. It was shown to be mostly dependent on the stage of the phytochemical conversion process of cannabinolic constituents. The results were explained by means of the products obtained under the same conditions with pure cannabidiol (pink product) and synthetic hexyltetrahydrocannabinol (blue product). The absorption curves exhibited by various types of resin were mostly attributed to the additive absorption of the two products. In addition, the presence of cannabinol and some inactive interfering substances seemed to influence the increase of the absorption at about 420 m µ(yellow), yielding as a result a bluish green product of reaction.
The absorption curves of the reaction products as obtained for three characteristic samples of cannabis resin are given in figure 1.
" Ripening value" and " disintegration value ". - Two absorption characteristics are proposed for the examination of cannabis resin. The ratio E 630/E 520indicated directly the stage of the transformation process of cannabinols, and was called the "ripening value ". On the other hand, an increased E 420/E 520 ratio (" disintegration value ") is attributed to the content of cannabinol or of decomposition products in a drug developed or stored under unfavourable conditions. Both constants for the samples analysed are given in table 2.
As may be seen, the following results were obtained for various groups of samples. The unripe type exhibited a ripening value not higher than 0.55 and a pink colour of the product. Experimentally grown samples from central Europe belonged to this group. The intermediate type showed a violet or indigo reaction product with a rather wide range of ripening value (0.55-1.5) (samples from Mediterranean area). Ripe cannabis exhibited a strong blue reaction with a high ripening value (1.5-1.8), but an unincreased disintegration value ( 1.1) (mostly tropical samples). The overripe type showed a bluish green colour, a high ripening value, together with an increased disintegration value (> 1.1). Finally, the alterated type did not exhibit any distinct colouration with the indophenol test. It mostly showed a low ripening value and a particularly high disintegration value (1.2-2.5).
It will be noted that, by applying the two constants based on the indophenol test, cannabis resin of various composition may be clearly and easily distinguished. The procedure is very simple and rapid, requiring only minute samples (a few mg of resin) and not more than15 minutes for one analysis. The reproducibility of the results is very good. A more detailed description and discussion of this method are given elsewhere [ 30] .
Ultraviolet Absorption Characteristics
Ultraviolet absorption spectra of cannabis extracts have been studied by Biggs [ 12] , Farmilo [ 17] , Asahina [ 10] and Bradford & Bracket [ 14] . These studies have been reviewed and discussed in a recent document by the United Nations Secretariat [ 63] . It was found that the ultraviolet absorption spectrum of a crude cannabis extract may provide interesting information on the composition of the drug. Samples of various stages of the conversion process have exhibited characteristic differences in their absorption curves. The results have been explained by the differences observed in the absorption characteristics of various cannabinols. The peak over the region of 300 mµ was attributed to the presence of cannabidiolic acid, while the absorption maximum over the region of 260-280 mµ is due in general to all cannabinolic constituents. However, according to the data reported [ 38] , the maximum of cannabinol is situated over a somewhat higher wavelength than that of its precursors. Consequently, it was concluded that the gradual ripening of the resin will result in a bathochromic shift of the main absorption maximum over the region 260-280 mµ, and in the loss of the secondary maximum over 300 mµ.
Typical ultraviolet absorption spectra of diluted ethanolic extracts of cannabis resin are shown in figure 2. According to the explanation given above, curve 1, exhibiting a marked secondary maximum over the region of 300 mµ corresponds to the unripe cannabis, containing predominantly cannabidiolic acid. Curve 2 represents an intermediate type, while curve 3 corresponds to the ripe type of the drug.
The above experience has been completed by introducing extinction ratios, which have been already successfully applied for the characterization of a number of vegetable extracts by means of the technique of direct ultraviolet spectrophotometry [ 25] , [ 26] , [ 27] , [ 28] , [ 32] .
The following procedure was used. The resin was dissolved in 95% ethanol, the solution filtered and diluted with the same solvent until at 260 mµ an extinction value between 0.3 and 0.4 was reached. The extinction was measured against the solvent at 260, 280, 300 and 310 mµ, and the ratios E 260/E 280 and E 300/E 310 were calculated. The reproducibility of both the constants was very good.
The ratios obtained for the samples examined (table 2) were shown to be related to the ripeness of cannabis resin. As the table shows, the ratio E 260/E 280 was the highest (> 1.45) in the experimental samples from Germany, which definitely belonged to the unripe type. The lowest values ( 1) were exhibited by ripe and overripe samples from tropical countries. This ratio decreased by the gradual bathochromic shift of the absorption curve, caused by the progress of the conversion process of cannabinols.
The value E 300/E 310 seemed to be in reverse relation to the content of cannabidiolic acid, on account of the increase of the absorption curve of cannabidiolic acid over the range 300-320 mµ. It was found to be high in tropical samples (> 1.6), while rather low in cannabis from temperate regions. Particularly fresh samples from temperate regions seemed to exhibit very low values in this ratio.
Peroxide-Sulphuric Acid Test
Duquénois & Negm [ 15] have described a very sensitive reaction for the identification of cannabis resin, using perhydrol and sulphuric acid. However, the reaction was reported as being less specific than the vanillin-acetaldehyde test on cannabis, which was proposed in the same paper, and applied on a large scale for the identification of cannabis resin.
The possibilities of applying the peroxide-sulphuric acid test for the characterization of cannabis resin have been studied in this institute, and the results reported elsewhere [ 31] . The following procedure was used. 0.2 ml of the unevaporated petrol-ether extract of the drug were left to evaporate in a porcelain dish. To the residue, 2 drops of a 20% H 2O 2 and 0.5 ml of concentrated H 2SO 4 were added and the dish was rotated gently for 1 minute. The colour of the liquid was observed after 5 minutes. The colour obtained for most of the samples ranged from pink to brown or greenish brown. In addition, some of the samples did not exhibit any colour at all.
In order to elucidate the differences obtained, the same test was performed with some pure cannabinolic compounds. Cannabidiol yielded a pink product, which in higher concentrations appeared as blood-red. Tetrahydrocannabidiol exhibited a violet colour. Six various synthetic tetrahydrocannabinol homologues showed a strong brown reaction. Synthetic cannabinol at first exhibited a green colour, changing quickly to greenish brown. Cannabidiolic acid acetate reacted in yielding a colour ranging in various concentrations from orange to pink.
This reaction does not appear to be quantitatively measurable, as it was not possible to stabilize the colour obtained, which was formed within a few minutes and faded thereafter. In spite of this inconvenience, the colour of the reaction product was found to be characteristic for certain types or me resin.Reactions obtained for the samples examined are given in table 2.
Samples exhibiting a pink colour obviously correspond to an unripe or intermediate type, containing mostly cannabidiolic acid or cannabidiol. Samples in which the phytochemical conversion of cannabinols was more advanced (ripe cannabis) yielded a brown reaction product. The reaction was greenish brown for overripe samples, while a negative reaction was exhibited by the alterated type of cannabis.
As may be noted, the peroxide-sulphuric acid test seems to be applicable as a rough indication of the progress of the ripening process in hemp resin. In addition, the test seems to be suitable for rapid and rough orientation on the potency of the drug. A highly potent cannabis will exhibit a strong brown colour clue to the presence of tetrahydrocannabinols. Cannabis yielding an orange, pink or red reaction contains precursors of physiologically active components, and may be considered as potentially active, being converted into active form under favourable conditions. A greenish brown colour or a negative reaction indicates the loss of physiological activity.
Consequently, the proper use of the peroxide-sulphuric acid test does not appear to lie in its possible identification of the drug, but to distinguish rapidly cannabis resin of various compositions and potency.
Beam Test
The reaction originally described in 1911 by Beam [ 11] consists in the violet colour shown by cannabis resin if treated with 5% ethanolic KOH. The test was widely applied for the identification of cannabis, and a large number of various modifications have been proposed. However, samples of cannabis have often been found which failed to work with the test. A considerable number of studies on the suitability of the Beam test for the identification of cannabis resin have beer published, expressing sometimes quite opposite views ant findings [ 13] , [ 15] , [ 51] , [ 52] , [ 54] , [ 61] , [ 62] , [ 65] .
It is known today that among cannabinols only cannabidic exhibits a positive Beam test. Therefore, the reaction expected to given only by cannabis containing cannabidic and also cannabidiolic acid, which is known to be readily converted to cannabidiol. Consequently, ripe cannabis should be expected to yield a negative or a weakly positive reaction while the overripe type of the resin will mostly give a negative result.
In our experiments, the following procedure was used. 5 the evaporated residue of 0.2 ml of petrol-ether extract, 2 drops of 5% ethanolic KOH were added. A positive reaction was indicated by a violet colour developing after a five minutes.
The results obtained are shown in table 2. As it may seen, variations have been observed in reactions of samples exhibiting various stages of the conversion process, and results fully correspond to the above interpretation. The capability of reacting obviously decreases together with ripening of the drug.
As only one inactive constituent of the resin reacts with the Beam test (cannabidiol), possibilities for classifying cannabis resin on the basis of this reaction may be considered to be more limited than those offered by the indophenol and the peroxide-sulphuric acid tests.
Ferric Chloride Test
The procedure applied was based on the FeC1 3reaction for cannabis as described by Fulton [ 20] . To 10 ml of a 0.05% solution of resin in absolute methanol, 0.1 ml of a freshly prepared 1% FeC1 3solution in absolute methanol was added, and the colour developed (A) was recorded. Thereafter, the liquid was divided into two equal parts. To the first part, 1 ml of water was added, and the colour (B) observed. To the second part, 1 ml of a 1% ammonium acetate in absolute methanol was added at once, quickly mixed up, and the colour (C) observed immediately. Corresponding numerical values for the three colours developed were recorded for examined samples of cannabis according to the following table. (The total sum of the three values has been expressed as" FeCl 3value ".)
|
Colour A |
Colour B |
Colour C |
Value |
|---|---|---|---|
|
Violet or blue
|
Clearly violet, indigo or blue
|
Purplish red
|
3 |
|
Clearly green or bluish green
|
Weakly blue or weakly indigo
|
Transient red
|
2 |
|
Greyish green
|
Greyish indigo
|
Transient brownish red
|
1 |
|
Yellowish green
|
Yellow or brown to green
|
Immediately brown or yellow
|
0 |
Results for the samples examined are shown in table 2. As will be seen, FeCl 3value in various samples of cannabis can range from 0 to 9. The highest values (7 to 9) were obtained mostly for samples which were classified as belonging to the unripe type. On the contrary, lowest values (0 to 2) were shown by ripe and overripe cannabis. Alterated and decomposed samples also exhibited a low FeCl 3value. Values from 3 to 6 generally indicated the intermediate type of the drug.
The results obtained were in accordance with the explanation given by Fulton (20), and are correlated with results obtained for the same samples by means of other methods. Fulton obtained a positive FeCl 3test for alkali-soluble constituents of the resin which occurred mostly in fresh cannabis. This has been confirmed with our tests carried out with pure cannabinolic constituents. From the substances available, cannabidiolic acid only exhibited a positive FeCl 3test.
Among chemical constants studied, the ratio E 300/E 310as obtained by the ultraviolet absorption method was shown to be in the best correlation with FeCl 3values. Samples showing a high E 300/E 310ratio exhibited a low FeCl 3value, and vice versa. Only Canadian samples were excepted from this rule, due to alterated chemical composition, which has been observed earlier.
On the basis of the results obtained, it was concluded that FeCl 3value is closely correlated with the content of cannabidiolic acid in cannabis resin, and that it may be used as an indication of freshness of a sample belonging to a certain type. This constant obviously decreases in a stored drug.
Antibiotic Activity
Antibacterial activity of cannabis has been investigated by several authors [ 19] , [ 42] , [ 43] , [ 44] , [ 57] . Krejci et al. have obtained two antibiotic fractions from cannabis resin, the acid and the phenolic [ 44] . According to the work done by Czechoslovakian [ 37] and German [ 57] authors, cannabidiolic acid seems to represent the main antibiotic agent in cannabis resin. It inhibits the growth of Gram-positive micro-organisms. However, among the large number of antibiotic agents isolated recently from various materials, antibacterial constituents of cannabis resin do not seem to offer particular perspectives or advantages for modem therapy [ 64] .
On the basis of the results reported in the literature, it was assumed that the unripe type of cannabis, containing the largest amount of cannabidiolic acid, might exhibit the strongest antibiotic activity. Consequently, it was supposed that the ripeness and the type of a drug might be roughly indicated also by its antibacterial potency. No comparative data were available in the literature dealing with the antibiotic activity of various types of cannabis resin.
The above assumption has been confirmed by microbiological assays carried out in this institute [ 53] . The diffusion method was used on nutrient agar medium, pH 6.0, with B. subtilis strain NCTC No. 8236 as test organism. Filter-paper disc diameter was 12 mm and the incubation time 18 hours at 37°C. A solution of penicilline G sodium in phosphate buffer at pH 6.0, in concentrations 0.5 to 6.0 I.U. in 1 ml was used as standard. The dosage-response curves for ethanolic solutions of various samples of the resin have been worked out in preliminary assays, and accordingly the concentration of 60 mg in 1 ml was adopted for the procedure applied. Each result reported corresponds to the calculated mean value of 10 zone diameter readings, which have been expressed in corresponding concentrations of penicilline units according to the dosage-response curve obtained simultaneously for the standard preparation.
The purpose of this work was to examine the differences between the potency of various samples, and not to determine the real activity of cannabis as related to penicilline. Therefore, the simplified technique was adopted, using only one single set of tests, without changing the dilution in cases when the readings of the inhibition zone were out of the reliable range.
The assays were carried out in duplicate on different days, and the data presented correspond to the average value of several experiments. The reproducibility of the results was very good.
The potency obtained for 11 samples examined is given in table 2. It is therein shown that the lowest values were exhibited by ripe samples from Brazil. A low potency was also shown by samples from Greece, Yugoslavia and Canada. Samples from Spain and Cyprus were much stronger, while the highest antibiotic activity was exhibited by the unripe sample from Germany, known to contain a large amount of cannabidiolic acid.
The results of microbiological assays were in close agreement with those obtained by chemical procedures. As figure 3 shows, there is obviously an inverse relationship between the antibiotic activity and the ripeness of the resin as estimated by chemical methods. In addition, the results confirm the findings obtained by previous authors according to which the antibiotic activity of cannabis resin is to be mainly attributed to the presence of cannabidiolic acid.
Corneal Areflexia in Rabbits
The two chief pharmacological methods used to determine physiological activity of cannabis resin are the ataxia test in dogs, and the corneal areflexia test in rabbits. The dog ataxia test seems to be closely correlated to the psychic action in man, and has been applied on a large scale by Loewe [ 47] for quantitative assays of various tetrahydrocannabinol homologues. The corneal areflexia test in rabbits, as proposed by Gayer [ 21] , was found to be inappropriate for quantitative purposes on account of great inter- and intra-individual variations, but was improved later [ 46] .
Two types of areflexia producing agent are supposed to occur in cannabis resin. Besides tetrahydrocannabinols, exhibiting both ataxia and areflexia activity, some other unknown constituents have been considered to be responsible for a positive Gayer test. They are readily destroyed by oxidation, possessing little or no ataxia activity, but a marked areflexia potency [ 47] .
For the purpose of a rough comparison between physiological potency of various types of sample, the corneal areflexia test in rabbits has been applied in this institute [ 34] . Five characteristic samples, belonging to various types of cannabis, were selected for bioassays. The incidence of lid responses to 100 touches of central cornea by means of a calibrated hair was observed after intravenous administration of a 1% solution of the resin in propylene glycol (1 ml pro kg). Altogether, 48 animals were tested.
The results obtained have confirmed great individual variations, as reported by Loewe [ 46] , thus being inapplicable for exact quantitative evaluation. However, the differences between the response obtained for 5 samples examined have been estimated as being evident and distinct enough to permit a rough qualitative comparison. Thus, sample UNC 28 (unripe) showed a completely negative response. The reaction for sample UNC 21 (considered by chemical analysis to lie between unripe and intermediate) was faintly positive. A clearly positive response was obtained for sample KZ 102 (ripe type), and somewhat stronger was the response for UNC 1A (situated between intermediate and ripe). UNC 47 (intermediate type) was the strongest of the samples examined.
In figure 4, to illustrate the relationship between the are-flexia potency and the ripeness of the samples examined, the approximate intensity of reaction was plotted against the ripeness of samples as estimated by the results of chemical methods. According to the curve obtained, the content of areflexia-active agents does not run parallel with hashish-active tetrahydrocannabinols. Areflexia potency appears to reach its maximum in the intermediate type (containing predominantly cannabidiol), while it decreases together with the further ripening of the resin (i.e., with the increase of tetrahydrocannabinol content).
|
Indophenol test |
U.V. absorption |
||||||||
|---|---|---|---|---|---|---|---|---|---|
|
Type of resin |
E 630/E 520 |
E 420/E 520 |
E 260 |
E 310 |
Peroxide-sulphuric acid test a |
Beam test b |
FeCl 3value |
Antibiotic activity (I.U. of penicilline to 60 mg of resin |
Biological potency (response to areflexia test in rabbits) |
|
Unripe
|
0.55
|
1.0
|
1.5-1.7
|
1.0-1.3
|
3 | 3 |
7-9
|
5-10
|
Negative
|
|
Intermediate
|
0.55-1.5
|
1.0
|
1.0-1.5
|
1.3-1.6
|
2 | 2 |
3-6
|
1-5
|
Very strong
|
|
Ripe .
|
1.5 -1.8
|
1.1
|
0.7-1.0
|
1.6-1.8
|
1 |
0-1
|
0-2
|
1
|
Positive
|
|
Overripe
|
1.6 -2.2
|
> 1.1
|
0.6-1.1
|
1.5-1.9
|
0-1
|
0 | 0 | 0 |
-
|
|
Alterated
|
-
|
> 1.2
|
1.0-1.7
|
1.1-1.4
|
0 |
0-1
|
0-2
|
2
|
-
|
a Key for peroxide-sulphuric test: 0-negative, 1-brown, 2-reddish brown, 3-pink.
b Key for Beam test: 0-negative, 1-weakly positive, 2-positive, 3-strongly positive.
It is hoped that these assays, which are still in progress, might contribute to a certain extent to the elucidation of the nature of unknown areflexia-active constituents in cannabis resin. There are some indications that these substances occur mostly in the intermediate type of cannabis. Consequently, constituents accompanying cannabidiol or closely related to this compound might be considered as affecting corneal areflexia in rabbits. By further ripening of the resin, these constituents may be readily converted to ataxia-active tetrahydrocannabinols, which agrees with the findings by Loewe [ 47] . On account of the small number of samples and animals tested, the above results and assumptions should be regarded only as preliminary.
For the final classification of the samples examined, table 1 indicates ranges of values by various methods and corresponding ripeness (type) of the resin.The type of sample as estimated by the methods described is recorded in the last column of table 2.
As table 2 shows, various methods mostly yield results which are in close agreement. The most striking and characteristic values have been obtained by all the methods for two extreme groups of samples: the unripe experimentally grown cannabis from central Europe (samples UNC 23-28), and the ripe cannabis from tropical regions (UNC 8-19 and KZ 101-109). However, in samples classified as intermediate, certain results sometimes do not run parallel. For example, the ultraviolet ratio E 260/E 280 was relatively low (corresponding to the ripe cannabis) in the group of samples from Greece (UNC 1A-1F), whereas other values have mostly indicated an intermediate type. Such differences are obviously due to complexity of phytochemical processes in hemp resin. They might be of use for a more detailed differentiation of certain particular types of the drug. Such possibilities may be seen from a "scattes diagram" (figure 5), where the ultraviolet ratio E 260/E 280 irplotted against the ripening value (the ratio is E 630/E 520 by the indophenol test).
|
Indophenol test |
U.V. absorption |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
Sample |
E 630/E 520 |
E 420/E 520 |
E 260/E 280 |
E 300/E 310 |
Peroxide sulphuric acid test |
Beam test |
FeCl3 value |
Antibiotic activity (I.U. of penicilline to 60 mg of resin) |
Biological potency (response to areflexia test in rabbits) |
Type (ripe-ness) |
|
|
Greece:
|
UNC 1 A
|
1.32 | 0.93 | 0.78 | 1.34 |
1-2
|
1 | 3 | 3 |
I-R
|
|
| 1 B | 0.78 | 0.84 | 0.93 | 1.59 | 1 | 3 | 3 |
I-R
|
|||
| 1 C | 1.25 | 0.89 | 0.88 | 1.53 | 1 | 1 | 1 | 0.85 |
I-R
|
||
| 1 D | 0.71 | 0.69 | 0.91 | 1.52 |
1-2
|
3 | 3 |
I
|
|||
| 1 E | 0.89 | 0.68 | 0.80 | 1.60 | 1 | 2 | 2 | 1.54 |
I-R
|
||
| 1 F | 1.12 | 0.87 | 0.82 | 1.68 |
1-2
|
1 | 0 | 0.91 |
R
|
||
|
Cyprus:
|
4 | 0.76 | 0.70 | 0.91 | 1.36 | 2 | 3 | 5 |
I
|
||
|
Burma:
|
5
|
1.88 | 1.56 | 0.77 | 1.68 | 1 | 0 | 0 |
O
|
||
|
Cyprus:
|
6 | 0.69 | 0.78 | 1.46 | 1.20 | 3 | 3 | 7 |
U-I
|
||
|
Sweden:
|
7 | 0.96 | 0.80 | 1.41 | 1.10 | 3 | 3 | 9 |
U-I
|
||
|
Costa Rica:
|
8 | 1.80 | 1.08 | 0.78 | 1.69 | 1 | 0 | 0 |
R
|
||
|
Brazil:
|
11 | 1.74 | 0.89 | 0.71 | 1.69 | 2 | 1 | 0 |
R
|
||
| 12 | 1.72 | 0.90 | 0.74 | 1.69 | 1 | 0 | 0 |
R
|
|||
| 14 | 1.78 | 0.83 | 0.79 | 1.60 | 1 | 0 | 0 |
R
|
|||
| 16 | 1.83 | 1.06 | 1.07 | 1.50 | 2 | 0 | 2 |
R
|
|||
| 17 | 1.80 | 1.00 | 0.76 | 1.71 | 1 | 0 | 0 |
R
|
|||
| 19 | 1.68 | 0.82 | 0.71 | 1.76 | 1 | 0 | 0 |
R
|
|||
|
Morocco:
|
21 | 1.13 | 0.91 | 1.48 | 1.22 | 3 | 2 | 6 | 1 |
U-I
|
|
|
United Kingdom:
|
22 | 1.77 | 1.10 | 1.51 | 1.36 | 0 | 0 | 3 |
O-A
|
||
|
Germany:
|
23 | 0.52 | 0.69 | 1.48 | 1.20 | 3 | 3 | 7 |
U
|
||
| 24 | 0.52 | 0.71 | 1.57 | 1.25 | 3 | 3 | 7 |
U
|
|||
| 25 | 0.48 | 0.69 | 1.69 | 1.28 | 3 | 3 | 6 |
U
|
|||
| 26 | 0.49 | 0.64 | 1.46 | 1.26 | 3 | 3 | 8 | 10.0 |
U
|
||
| 27 | 0.52 | 0.70 | 1.60 | 1.28 | 3 | 3 | 5 |
U
|
|||
|
Switzerland:
|
28 | 0.52 | 0.83 | 1.49 | 1.17 | 3 | 2 | 7 | 0 |
U
|
|
|
Cyprus:
|
32 | 0.74 | 0.78 | 1.35 | 1.29 |
2-3
|
3 | 3 | 5.60 |
U-I
|
|
| 33 | 1.60 | 1.46 | 1.10 | 1.44 | 0 | 0 | 0 |
O
|
|||
| 34 | 0.56 | 1.07 | 1.56 | 1.14 | 3 | 2 | 7 |
U
|
|||
|
Canada:
|
36 | 0.63 | 1.48 | 1.40 | 1.24 | 0 | 1 | 2 |
A
|
||
| 37 | 0.57 | 1.38 | 1.07 | 1.21 | 0 | 1 | 0 |
A
|
|||
| 38 | 0.63 | 1.70 | 1.52 | 1.38 | 0 | 0 | 0 |
A
|
|||
| 39 | 0.61 | 1.76 | 1.43 | 1.34 | 0 | 1 | 0 |
A
|
|||
| 40 | 0.59 | 2.48 | 1.20 | 1.30 | 0 | 0 | 0 |
A
|
|||
| 41 | 0.61 | 2.13 | 1.39 | 1.20 | 0 | 1 | 0 | 1.00 |
A
|
||
| 42 | 0.76 | 1.53 | 1.48 | 1.29 | 0 | 1 | 1 |
A
|
|||
| 43 | 0.61 | 2.07 | 1.46 | 1.23 | 0 | 0 | 0 |
A
|
|||
|
Spain:
|
44 | 1.47 | 0.91 | 1.29 | 1.40 | 2 | 1 | 7 |
I
|
||
| 45 | 1.32 | 0.86 | 1.35 | 1.31 | 3 | 3 | 8 | 4.12 |
U-I
|
||
| 46 | 1.36 | 0.85 | 1.60 | 1.24 | 3 | 2 | 7 |
U-I
|
|||
| 47 | 1.31 | 0.95 | 1.39 | 1.29 | 3 | 2 | 7 | 4 |
I
|
||
|
Ghana:
|
48 | 2.03 | 1.25 | 0.67 | 1.61 | 1 | 0 | 0 |
O
|
||
| 49 | 2.00 | 1.25 | 0.64 | 1.62 | 1 | 0 | 1 |
O
|
|||
|
Nigeria:
|
59 | 2.11 | 1.18 | 0.69 | 1.76 | 1 | 0 | 0 |
O
|
||
|
Venezuela:
|
60 | 1.84 | 0.85 | 1.55 | 1.20 | 3 | 0 | 7 |
U-O
|
||
|
Brazil:
|
61 | 2.13 | 1.10 | 0.75 | 1.75 | 1 | 0 | 0 |
O
|
||
| 62 | 2.08 | 1.07 | 0.76 | 1.73 | 1 | 0 | 0 |
O
|
|||
|
KZ 101
|
1.74 | 1.00 | 0.77 | 1.60 | 1 | 1 | 2 | 0.56 |
R
|
||
| 102 | 1.75 | 1.01 | 0.78 | 1.63 | 2 | 1 | 1 | 0.68 | 2 |
R
|
|
| 103 | 1.80 | 0.99 | 0.78 | 1.64 | 2 | 1 | 1 | 0.54 |
R
|
||
| 104 | 1.58 | 1.06 | 0.88 | 1.59 | 2 | 0 | 1 |
R
|
|||
| 105 | 1.63 | 1.04 | 0.76 | 1.68 | 1 | 0 | 0 |
R
|
|||
| 106 | 1.70 | 0.99 | 0.92 | 1.58 | 2 | 0 | 2 |
I-R
|
|||
| 107 | 1.75 | 1.07 | 0.80 | 1.62 | 1 | 0 | 0 |
R
|
|||
| 108 | 1.68 | 1.05 | 0.84 | 1.64 | 1 | 0 | 0 |
R
|
|||
| 109 | 1.69 | 0.95 | 0.92 | 1.68 | 1 | 0 | 2 |
R
|
|||
|
Yugoslavia:
|
110 | 0.85 | 0.81 | 0.81 | 1.57 | 2 | 3 | 0 | 0.81 |
I-R
|
|
|
U.S.A.:
|
120 | 0.65 | 0.96 | 1.05 | 1.29 | 2 | 3 | 4 |
I
|
||
Abbreviations used:
Peroxide-sulphuric acid test: 0-negative; 1-brown; 2-reddish brown; 3-pink. Beam test: 0-negative; 1-weakly positive; 2-positive; 3-strongly positive.
Biological response: 0-negative; 1-faintly positive; 2-positive; 3-strongly positive; 4-very strong.
Type (ripeness): U-unripe; 1-intermediate; R-ripe; O-overripe; A-alterated.
From figure 5 it may be seen that certain geographical groups of samples examined are separated by plotting the values obtained by the ultraviolet method and the indophenol test. However, as we do not know to what extent the properties of the examined groups of samples are typical for the area concerned, we are not able to draw any conclusions on the possibilities of identifying the area of origin of a sample by means of the methods applied. Moreover, because of great complexity, variability and instability of cannabinols, any attempt to approach the problem of origin determination of cannabis by laboratory methods will obviously encounter great difficulties. The ripeness of a sample is obviously affected by various factors not necessarily connected with its geographical provenance. Original constituents of plant resin may be also altered by preparation or storage.
Nevertheless, the results obtained in this study may confirm that the phytochemical conversion process of cannabinols occurs in the way described in the introduction to this paper, and that variations in chemical composition of various samples are mostly due to the differences in the stage of the ripening process. It was shown that the progress of the conversion process is easily detectable by means of the methods developed. It has been confirmed by several analytical procedures that this process is generally more advanced in cannabis resin from tropical areas than in the resin developed in a temperate climate.
Acknowledgement. - The author wishes to thank Mrs. A. Andrec, Mrs. L. Holik-Klaric, Mrs. M. Kupinic, Miss A. Radoševic and Mr. N. Tomic for their assistance in this study.
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