A brief survey of the methods of isolation and the physical and chemical properties and structures of the isolated antibacterial substances
By F. Santavy & Z. Krejci
During the last two decades, attention has been drawn to the isolation and toxicology of the substances contained in the plant, Cannabis sativa. The following substances have been isolated: cannabinol, cannabidiol, tetrahydrocannabinol, quebrachitol (l-inositolmonomethylether), 1-methyl-4-isopro-pylbenzen, the so-called cannabol of phenolic character which yields a readily crystallizing ester with the chloride of the azobenzencarbonic acid and an optically active and volatile material which was not studied in more detail. In addition, from the individual parts of the plant a series of less important substances has been isolated and listed in detail in Wehmer's compendium. The quantitative changes of carotene found present in Cannabis sativa have been investigated by Lebedev.
It is perhaps also of interest that cannabis greatly increases the hypnotic effect of barbiturates.
Moreover there exists a series of communications concerned with the isolation and study of substances contained in cannabis which can be extracted from the petrol extract due to its solubility in alkaline lye or in sodium carbonate. The most detailed work in this line was carried out by Todd et al. But none of the authors succeeded in isolating from that portion any acid in crystalline form,-i.e., either an acid itself or its derivative - designating it or determining its constitution. Probably it was the amorphous substance isolated by us and the cannabidiolic acid which was studied further that Todd et al. had in hand (see below) but they conclude: "... it is concluded that the alkali-soluble portion of the resin contains esters of cannabidiol and cannabinol with a phenolic acid ".
Among the isolated and identified substances mentioned above, cannabinol, cannabidiol and tetrahydrocannabinol proved to be pharmacologically interesting with regard to the hashish effect; a specific hashish-effect is produced only by tetrahydrocannabinol. The formulas of these three substances are:
All these compounds have already been prepared synthetically and, in addition, a series of homologous derivatives has been prepared for the purpose of establishing the relationships between the constitution and the biological effectiveness.
At this stage of the development of the investigation of substances contained in Cannabis sativa we began a new line of investigation when one of us found that there are substances contained in it which show an antibiotic effect upon some micro-organisms.
1. Starting material
Fresh or dried pistillate flowers of the cannabis plant (Cannabis sativa var. indica - fig. 3) were used in order to extract the active substances. A plant was used which was not cultivated in a tropical climate, but in the temperate climate of Czechoslovakia for more than five years. We know perfectly well that this factor had undoubtedly a considerable influence both upon the quantitative production of the effective substances or, respectively, upon the composition of the substances contained in the cannabis, and this has been also confirmed by Pulewka. Consequently, it may be assumed that this inconstant "variety indica" has become closely similar to Cannabis sativa, a plant grown for industrial purposes. Positive results have also been obtained when investigating effective substances contained in Cannabis sativa L., a plant commonly cultivated particularly in Slovakia for the industrial utilization of the hemp fibre. We succeeded in showing that the drug from this plant contains antibacterially effective substances. Unfortunately, the drug from the warmer, tropical or subtropical regions was not available for comparison. The drug obtained from Romania and Yugoslavia did not vary fundamentally in comparison with the raw material available in Slovakia. It must be born in mind that the various cannabis species cultivated in the same climate differ considerably with regard to the quantity of the biologically active substances present; the quantity of the substance amounted from about 0.2 g to 25 g/kg. Likewise, the varying quality of the period of vegetation (dry, rainy, warm and cool weather and also the manure) produces a considerable effect upon the production of the resin and, subsequently, of the biologically active substances. The best, and most valuable raw material yielding antibacterial substances and containing on the average 1-1.5% effective resin substances has been found at the stage when the seed is mature.
FIGURE 3, Cannabis sativa var. indica - plant shoots. A - Flowering shoots of the pistillate plant (rich in resins). B - Flowering shoots of the staminate plant.
2. Method of isolation
The preliminary experiments showed that the biologically effective substances (46-50) of the resin from Cannabis sativa may be easily extracted with ether; the paper chromatography revealed that highly polar substances were being dealt with. After having carried out a series of preliminary experiments, our method of isolation was as follows.
Fresh and rapidly dried tops and leaves of Cannabis sativa were extracted in the cold in a percolator with benzene or, preferably, with petrol ether. After partial distillation of the solvents under reduced pressure at 30°C, the residue was carefully extracted with 4% natrium hydroxide, into which both the acids and the substances of phenolic character could be taken up. They were extracted with chloroform after acidification.
First of all the oily residue was shaken in a nitrogen atmosphere with an aqueous NaHCO3 solution with which the acids, particularly the cannabidiolic acid, were extracted to yield a slightly yellow product. By shaking with NaHCO3 an oily residue was obtained which was shown to consist in the main of substances of phenolic character. Both the acid and the phenolic fractions produced an antibacterial effect upon some micro-organisms.
Output and biological activity of the single fractions isolated from cannabis
Output (grammes*) Biological activity Petrolether fraction
Acetylated phenolic constituent
Acid constituent (cannabidiolic acid)
Acetylated cannabidiolic acid
Acetylated and hydrogenated cannabidiolic acid
* Values referred to 1 kg of dried material.
After three reprecipitations the acid fraction was allowed to dry over a long period under water pump vacuum at 20°C to yield a colourless glassy substance, the empirical formula of which is C22H30O4 '[&alpha]D24 -95° ± 8° (c = 1.00 in chloroform); [&alpha]D24 -115° ± 4° (c = 1.05 in ethanol). The substance crystallized readily after acetylation, m.p. 80-100°/127-128°, [&alpha]D -114° ± 4° (c = 1 in ethanol).
Repeated isolations showed that the antibiotically effective cannabidiolic acid from Cannabis sativa is very sensitive to atmospheric oxygen (particularly in a warm atmosphere) and, furthermore, that the acid undergoes changes due to higher temperature and a simultaneously reduced pressure.
Modification according to Schultz & Haffner.
From some of the crops the acetylester of the acid, m.p. 95-100/ 110-15° was obtained. According to our assumption, there are either two isomers present whose occurrence is conditioned by the climate, or these substances are interconvertible.
FIGURE 4, Rate of inhibition of the acid and phenolic fraction of the extract from cannabis upon the inoculated staphylococcus (meat peptone broth) 1 - Acetylated fraction from which the cannabidiol acid crystallized. 2 - The acetylated phenolic residue.
3. Determination of the structure
As previously mentioned, the biologically active acid isolated from Cannabis sativa has the empirical formula C22H30O4, and yields, when acetylated, a diacetyl derivative C26H34O6 whose extinction curve in the UV-range closely resembles that of the cannabidiol derivatives. At the beginning of our experimental work, therefore, the acid obtained by us was already called cannabidiolic acid:
By boiling with alkali, the acetylated cannabidiolic acid is saponified and partially decarboxylated, which results in precipitation of a substance of phenolic character from the solution, particularly after its saturation with carbon dioxide. That the substance undergoes decarboxylation was best seen when we tried to purify the proper acid isolated by us or the acid liberated from its acetylester by the help of sublimation. The process gives rise to cannabidiol, a substance characterized by the positive Beam-reaction and as 3,5-dinitrobenzoate. This supports our former view that, regarding the structure, the antibacterially effective acid, isolated from Cannabis sativa, is closely related to cannabidiol.
The position of the carboxy1 group in the molecule of the cannabidiolic acid was studied along the physical-chemical pathway.
On comparing the optical rotation of the cannabidiolic acid, its acetyl derivative and its tetrahydro derivative with the cannabidiol, derivatives we found a remarkable correspondence. An analogous agreement exists between the derivatives of the tetrahydrocannabinol and hexahydrocannabinol; acetylation and methylation do not bring about a significant change in the degree of the optical rotation. The percentage rate of the molecular optical rotation of the hydrogenated and the unhydrogenated derivatives is, as far as these three groups are concerned, practically the same. The optical rotation changes only with the varying position of the double bond of the B-ring and because of the formation of an ethereal bridge due to conversion of cannabidiol into tetrahydrocannabinol. From the above it may be concluded that the carboxyl group of the cannabidiolic acid does not seem to be attached to some carbon of the isocyclic nucleus where it could lead to the formation of a further optically active centre, but that it is attached either to the aromatic nucleus or to some other carbon where an optically active centre cannot be formed. The ready decarboxylation excludes its position on the carbons 7, 9 and 15.
Values of the optical rotation of the single substances (in ethanol) (Degrees)
Cannabidiolic acid and its derivatives
Cannabidiol and its derivatives
89 Tetrahydrodiacetyl derivative (3)
Tetrahydrocannabinol and its derivatives (l b, 4)
In addition, we have tried to determine the position of the carboxyl spectroscopically.
The acetyl derivative of the cannabidiolic acid shows an inflexion in the UV-range, or a maximum at the same wavelength as cannabidiol, or its derivatives (fig. 5). Analogously, the same maxima are produced by the diacetylester of the hydrogenated cannabidiolic acid.
Evidently, therefore, the carboxyl group, of the cannabidiolic acid is not conjugated with the double bond of the nucleus B.
Still more striking is the behaviour of the carboxyl group in IR-spectroscopy (fig. 6). We attribute the frequency at 1770 cm -1 to both phenoacetoxy groups. The frequency at 1698 cm -1 to the free carboxy group, or to its carbonyl, respectively. This frequency does not vary, either with the acetylester of the cannabidiolic acid or with its hydrogenated product (cf. table 3).
Our thanks are due to Dr. Horak, Institute of Chemistry, Academy of Sciences, Prague, for the measuring of the infra-red spectra and their evaluation.
IR-frequency values within the carbonyl range
Acetylester of the cannabidiolic acid
Hydrogenated acetylester of the cannabidiolic acid
With regard to the fact that the frequencies of the carbonyl of both acetoxy compounds (i.e., of the saturated and of the unsaturated) are found to be the same, it may be assumed that it is not a carboxyl situated in position 4, 5 and 9 which is being dealt with, for the disrupture of the conjugation due to reduction would be followed by a shift of the frequency to higher values. On the contrary, the frequencies observed in chloroform (1698 cm-1) and in dioxane (1728 cm-1) evidence again that it is not the alicyclic acid (i.e., in position 1,2,3,6) - namely, one whose carbonyl is not conjugated - for the frequencies of such acids have been found to appear in the higher frequency region (in chloroform at about 1715, in dioxane at about 1740 cm-1). But there was a very good agreement to be seen with regard to the frequency of benzoic acid (in chloroform 1694, in dioxane 1724 cm-1).
FIGURE 6 IR-spectra I - Acetylester of the hydrogenated cannabidiolic acid. II - Acetylester of the cannabidiolic acid. Both compounds have been solved in chloroform.
If we take it for granted that there is a carboxyl attached to the aromatic nucleus, it is only conceivable in position 3 (or 5, respectively).
The frequency at 1615 cm-1 belongs to the aromatic vibrations and the frequencies at 900 and 1650 cm-1 which disappear during hydrogenation must be attributed to methylene in isopropylene.
On the basis of the organically preparatory experiments carried out so far, comparison of the optical rotation and of the UV- and the IR-spectra, the formula for cannabidiolic acid suggested by us is as follows:
(3-methyl-6-isopropenyl-4'-n-pentyl-2',6'- dihydroxy-l,2,3,6-tetrahydrodiphenyl-3'-carboxylic acid)
The arrangement of the substituents of the nucleus A of the cannabidiolic acid, as formulated by us, does not appear to be exceptional among the natural substances obtained from plants. If, in the case of the cannabidiolic acid, we do not take into consideration the partially aromatic nucleus B, we obtain the olivetolic acid which has been found present in numerous plants. This substance is also readily decarboxylized and thus converted into olivetol which forms the aromatic moiety of cannabidiol. There are also some deeply related reactions (FeC13,CHC13+ 10% KOH, Vanillin + HCl) to be found in both groups of these substances.
Independently from us (1955) Schulz & Haffner (1958) isolated and described the above-mentioned cannabidiolic acic. The determination of the constitution was carried out (1959) by means of the organic-preparative methods which led them to the same results as those obtained by us (1958). In their communication the authors conclude that the cannabinols occur in the plant due to decarboxylation of the aforementioned cannabidiolic acid.
In addition to the cannabidiolic acid a further acid, m.p. 131-133°, has been isolated from Cannabis sativa, and identified by us as trans-cinnamic acid.
Our study of the Mideuropean flora with regard to its contents of substances producing antibacterial effects comprehends 3,000 species from which the Indian hemp - Cannabis indica - grown in Czechoslovakia has been selected for elaborate investigation. A preliminary method of isolation accomplished by paper chromatography with the disclosure of an effective zone in the biological way has been described.
The most advantageous methods of extraction were determined, and the bactericide effect of the hemp substances experimentally proved in vitro on Gram-positive microorganisms: Staphylococcus pyogenes autreus haemolyticus, Staphylococcus aureus - resistant to penicillin, Streptococcus beta haemolyticus, Streptococcus viridans, Pneumococcus Cornyebacterium diphteriae, and Bacillus anthracis.
Gram-negative microorganisms of the typhus-coli group remain resistent, as well as Pseudomonas aeruginosa and Proteus vulgaris. An excellent antibacterial effect on Mycobacterium in vitro even in a dilution 1:150,000 could be ascertained.
A parallel between the bactericide effect of isolated, amorphous, and crystal substances, and a comparison of the sensibility of the two applied bacterial methods, both the modified Oxford method and the tests in a liquid medium was made in detail. The limit of efficacy in the maximal dilution of biologically active substance (1:100,000) and the velocity of their effect in various dilutions were determined. The influence of inactivating factors has been studied in detail. Blood, plasma, and serum partly inactivate them and reduce their antibacterial effect.
As a conclusion, a comparison of the efficacy of these active substance[s] with penicillin and streptomycin at various pH was worked out, and a summary of hemp preparations manufactured for the purpose of clinical application in stomatology, oto-rhino-laryngology, dermatology and phthisiology has been given.