Chemical and Physiological Identification of Indian Hemp
Author: P. DUQUÉNOIS
Pages: 30 to 33
Creation Date: 1950/01/01
Professor at the Faculty of Strasbourg, expert accredited to the courts of law
The identification of Indian hemp ( Cannabis sativa L. var. indica) or of any product containing active hemp resin (charas, hashish, kif, marihuana, etc.) no longer offers any difficulty today. The purpose of the present article is to indicate the surest methods of chemical and physiological characterization used at present in France. Several of these methods are of recent date and are not yet well known to experts.
Cannabinol, which was isolated as an impure oil by Wood and his collaborators (1896) and subsequently by Fraenkel, Czerkis and, later, Casparis, was finally obtained in the pure state by Cahn and reproduced in 1940 by the two independent syntheses of Ghosh, Todd and Wilkinson and of Adams, Baker and Wearn. This solid (melting point: 163 degrees-164 degrees C.), which corresponds to the formula 6"-hydroxy-2-2-5'-trimethyl-4"-n-amyl-dibenzopyran, and which was formerly considered to be responsible for the physiological activity, is accompanied by related derivatives, namely the tetrahydrocannabinols-the real intoxicating agents-and cannabidiol, which is physiologically inactive. In recent years it has been found possible to obtain these substances from Indian hemp resin.
All the dibenzopyranic substances we have just mentioned-cannabinol, tetrahydrocannabinol and cannabidiol-are capable of being acetylated and yield p-nitrobenzoates. They all possess at least one phenolic functional group.
It is not surprising, therefore, that Indian hemp extracts, which contain these phenolic substances, should exhibit various analytical reactions of the phenols or polyphenols. These reactions may be carried out on the residue obtained by taking the extract produced by the exhaustive extraction of Indian hemp with petroleum ether and evaporating off the solvent in a water-bath.
Ferric cation reaction
The residue from the extract is redissolved in 1 cubic centimetre of 95 per cent alcohol, and one or two drops of 1/10 Fe C1 3 solution are added to the alcoholic solution. A greyish-green coloration is produced.
This indicative reaction is of course in no way specific: many phenols give an analogous green colour (pyrocatechol, homopyrocatechol, pyrocatechoic aldehyde, caffeic acid, carvacrol and certain tannins give it, but many of these substances cannot be extracted with petroleum ether).
The petroleum ether extract gives a red coloration verging on orange with this reagent. Tyrosine, protides containing this amino-acid and many phenols give this coloration.
A few crystals of vanillin and then ten drops of officinal H 3PO 4 are added, in the evaporating dish, to the residue from the petroleum ether extract. A pink coloration is produced which disappears with the addition of alcohol. We have also obtained a pink coloration with phenol and hexylresorcinol. Resorcinol, indole and piperine give red colorations and alpha-naphthol and tryptophane give a violet coloration under the same conditions.
The extracted resin is more or less of a reducing agent, depending on the commercial varieties, the origin and the age of the sample. Reduction reactions, which are usually not pronounced, are not distinctive: neither Fehling's solution nor Nessler’s reagent(in the cold) are reduced, but with the latter a purplish coloration is formed round the sides of the dish (see Beam's reaction below). Unlike morphine, the extracted resin does not reduce iodic acid.
Silver ammonio-nitrate solution is sometimes reduced by extracts prepared from fresh samples. Cannabinol and the tetrahydrocannabinols are weak reducing agents, while cannabidiol must be responsible for the reduction observed with Tollens' reagent.
These are the reactions used by the toxicologists and chemists employed in the technical police.
The so-called Beam's reaction
This reaction was in reality discovered by Fraenkel and has been incorporated in the French Pharmacopoeia.
A few drops of a 5 per cent alcoholic solution of caustic potash are added to the petroleum ether extract. A stable violet-purple coloration is produced, which turns to blue on the addition of water.
We have reported the few substances which also give this reaction (alkyl dioxyquinones, juglone, embelin). Without being absolutely specific, this reaction is very useful, owing to its simplicity.
The reaction is not always dependable. R. Weitz and Dardanne were the first to show that it can be absent with officinal preparations obtained by means of hot alcohol. Using old samples (more than twenty years old) which are, however, still active for cold-blooded animals, we have not always succeeded in reproducing this reaction. It is now known that Beam's reaction is characteristic not of a tetrahydrocannabinol but of cannabidiol, which is physiologically inactive.
J. Bouquet6 has modified Beam's classical alkaline test.
Beam's acid reaction
Another of Beam's reactions is carried out in an acid medium. A few cubic centimetres of absolute alcohol saturated with gaseous HCl, when added to the extraction residue, gives a cherry-red colour which disappears on the addition of water. The reaction is not very practical in use. The following more convenient modification has been proposed by Mrs. Remziye Hissar :
The residue from the petroleum ether extract is dissolved in 1-2 cubic centimetres of CHCl 3, which is poured into a test-tube. One cubic centimetre of pure H 2SO 4 is added with a pipette, shaking well. After a few moments a layer of acid settles beneath the chloroform showing a mahogany coloration which slowly deepens during the next twenty-four hours.
Pinene, terpene, terpineol, guaiac, incense, gum benzoin, jalap, sage, rosemary, lavender and tobacco give more or less similar red colorations.
One cubic centimetre of a reagent composed of: p-dimethylaminobenzaldehyde, 1 gr.; concentrated sulphuric acid, 5 cc.; distilled water, 1 cc.-is allowed to act on the petroleum ether extract. After a minute on the water-bath there appears a reddish-brown colour, which turns to purplish-red on cooling. On the addition of a few drops of water, a blue or indigo coloration appears and persists after dilution.
The specificity is fairly good. Similar colorations are obtained with some resins or essences (phenolic, terpenic), but these are not absolutely identical with the successive colorations that appear in the case of hashish.
The sensitivity is very high. Even 1/2 milligram of resin is detected. Old samples generally show this reaction better than Beam's reaction.
The reaction of P. Duquénois and H. Negm (acetaldehyde-vanillin)
Two cubic centimetres of the reagent: vanillin, 0.40; acetaldehyde, 0.06 (6 drops); 95 per cent alcohol, 20 cubic centimetres, are poured onto the petroleum ether extract (preferably still warm) in an evaporating dish.
The mixture is stirred, and when solution is complete 2 cubic centimetres of concentrated HCl are added. A circular movement is imparted to the dish and a succession of colors appears: sea-green, slate, followed by indigo within 10 minutes. Within half an hour the color turns to violet and within an hour to intense violet.
This reaction is specific if one considers the succession of tints. Phloroglucinol gives a violet coloration in the end, but starts with red, not green. Moreover, phloroglucinol is insoluble in petroleum ether.
Sensitivity: down to 1/2 milligram of resin.
Dependability: equal to that of Ghamrawy's reaction and therefore better than that of Beam's reaction.
With H. Negm, we have reported another reaction. The residue from the petroleum ether extract, when acted on by 2-3 drops of 100-volume hydrogen peroxide and then by 10 drops of concentrated sulphuric acid, gives an intense blood-red coloration.
The sensitivity is also of the order of 1/2 milligram.
The test is not specific. Aspirin, diaminophenol and brucine also give a bright red coloration, turning yellow in the case of brucine. Phenacetin turns brownish-red.
Like the following reaction, it does not occur with old samples, the physiological action of which is weakened.
The United States Pharmacopoeia mentions the inebriating and convulsant action of Indian hemp on dogs. The method is somewhat insensitive since large doses are required to obtain marked symptoms. Using white mice weighing between 15 and 20 grammes each, Mrs. Remziye Hissar had to use approximately 5 milligrams of resin to obtain definite recognizable effects. Miss Jeanne Levy has shown that rabbits, which are exceptionally tolerant, are poor test animals.
We prefer the use of the poikilothermals which provide a more sensitive method of identifying the drug.
Tadpoles of the red frog (Rana fusca)
The tadpole of the Rana fusca is more sensitive than the adult frog and can be used to demonstrate the strong toxic action of the resin. But this animal, which has the additional disadvantage of being seasonal, does not permit of clear differentiation between the resin and other poisons. A phase of excitation (whirling movements) quickly appears (after a few minutes) followed by a depressive phase with small contractions and spontaneous convulsions (with strychnine, the convulsions are induced rather than spontaneous) and then a fatal asphyxic phase with vasoconstriction and discoloration of the head, the median part of the body retaining its pigmentation.
If the tadpoles are placed in fresh water before death occurs, no sequelae are observed (paralysis with strychnine).
Minnows (Phoxinus laevis)
Using minnows in ordinary water to which an aqueous maceration of Indian hemp had been added, H. Negm of our laboratory, observed a phase of excitation (sudden movement, rapid swimming, circular movements, respiratory difficulty, convulsive fits) followed by a depressive period (motor sluggishness, slow breathing, which becomes panting and loss of balance); the minnow falls to the bottom of the aquarium, lies on its side and dies after a time which is proportional to the concentration of poison. We have carried out experiments on gold fish, Carassius auratus, weighing 1.5 grammes and placed in 80 cubic centimetres of water with resin added, which show that, with 1 milligram of resin, loss of balance begins to appear in 20 minutes followed by strong convulsions in 40 minutes and death after 1 hour 40 minutes on an average.
Smooth-tailed stickleback (Gasterosteus Leiurus)
One use of the Portier and Lopez-Lomba method on this species shows that the stickleback is the ideal animal for the investigation of the toxic potency of Indian hemp. For details of the method used and the results obtained, we refer the reader to our paper pub- lished in 1939. The small weight of the fish used as reagent, and its sensitivity which will show the presence of 0.3 to 0.4 milligram of resin extracted by petrolium ether, permit of the confirmation of the chemical analysis. Nevertheless, the biological method lacks the specificity of the chemical reaction using acetaldehyde-vanillin. The effects are similar to those we have observed with other convulsant poisons (e.g., narcotine) in considerably larger doses.
Loss of balance begins to appear after approximately 10 minutes with 1 milligram in 80 cubic centimetres of water, after approximately 25 minutes with 0.5 milligram and after approximately 30 minutes with 0.3. milligram.
Strong spontaneous convulsions which throw the stickleback out of the water appear after approximately 11 minutes with 1 milligram and after 30 to 45 minutes with 0.5 milligram in 80 cubic centimetres of water.
Rotatory movements about the longitudinal axis of the body begin after 20 minutes with a dose of 1 milligram in 80 cubic centimetres. With a dose of 0.3 milligram, the subject turns about its axis with no sense of orientation or balance after 75 minutes.
The speed with which the symptoms appear therefore depends on the dose of the active substance. The order of their appearance is fairly constant.
Earthworms and other helminths
In a chemical paper prepared in 1938 in collaboration with H. Negm we showed that all the colour reactions obtained with Indian hemp resin are akin to those obtained with santolactone, terpenic essences (chenopodium, thyme, eucalyptus), rotenone and embelin, phloroglucinol, the derivatives of which constitute the filicic group, and hexyl resorcinol, all of which are anthelminthic substances. Indian hemp seeds were used for their vermifugal properties by the ancient Chinese, and H. Negm has studied this action for the first time on the earthworm, using the Sollmann method.
Earthworms in a solution of resin wriggle, twist and show excitation which subsequently diminishes. The animal extrudes earth through the anal segment after which the contractions cease in that portion. Paralysis gradually extends to the lower two thirds of the body. Only the head continues to move. Finally, the animal dies, the segments being relaxed and flaccid and the clitellum swollen.
The oily extract is much more active than the aqueous extract. The anthelminthic action can also be observed with Ascaris lombricoides.
Indian hemp, the most dreaded social poison of modern times in America and in some countries of the Near East, can be identified by numerous methods.
Of the chemical methods, the Ghamrawy reaction and the acetaldeyde-vanillin reaction we proposed in 1937 in collaboration with H. Negm, are the best. The latter method, capable of coloricetric application, was adopted by the League of Nations Sub-Committee on Cannabis in 1939.
The presence of active resin can be confirmed without difficulty by experiments on small fishes, particularly sticklebacks.
All the colour reactions discovered before the constituents of Cannabis sativa L. var. indica were known, can now be explained. They are for the most part due to cannabidiol, a derivative of resorcinol. In relatively fresh drugs, cannabidiol always predominates in the resin. However, it may disappear in galenic treatment or during prolonged storage, while the tetrahydrocannabinols derived from it may still be present and make the preparations active.
Certain special reactions, common to terpenic substances, show the structural relationship of cannabinol, cannabidiol and the tetrahydrocannabinols to paracymene and methyl-4-isopropylbenzene, which can be deduced from their formulas.
The intensity of the colour reactions is in many cases related to the activity of the drug on poikilothermals and probably also in homeothermals but it will be readily understood that the relationship is not always proportional: the inactive cannabidiol, which in many cases determines the results of the chemical tests, is no more than the precursor of the highly active tetrahydrocannabinols in the drug.
Cahn, Journ. Chem. Soc., 986, 1930; 630, 1931; 1400, 1933.02
Ghosh, R., Todd, A. R., Wilkinson, Journ. Chem. Soc ., 1933, 1940.03
Adams, Baker, Wearn, Journ. Amer. Chem. Soc ., 62, 2204, 1940.04
Weitz, R., Dardanne, A., Journ. Pharm. Chim ., 29, 457, 1924.05
Duquénois, P., H. Negm M., Annales M?d. L?gale , 18?me ann. 485, 1938.06
Bouquet, J., Arch., Inst. Pasteur Tunis , 26, 288, 1937.07
Hissar, R., Annales de Chimie (Istanbul) 3, 167, 1938.08
Duquénois, P., H. Negm M., Journ. of the Egypt. Med. Assoc. 21, 23, 1938.09
Duquénois, P., H. Negm M., Bull. Sc. Pharmacol . 45, 203, 1938.10
H. Negm M., Contribution ? l'tude du hachich et de sa prohibition en Egypte, Thèse Doct. Univ. (Pharm.), Strasbourg, 1938.11
Duquénois, P., Bull. Sc. Pharmacol . 46, 222, 1939.