Introduction
Structure of cannabinols
Biogenesis of cannabinols
Seperation and isolation of cannabis constituents by various methods
Chemical and physical tests for cannabis and cannabinols
Antibacterial constituents
Author: Ljubi?a GRLIC
Pages: 29 to 38
Creation Date: 1964/01/01
Despite the great number of important contributions made in the course of the last twenty-five years to the study of the chemistry of cannabis resin, the chemical components of cannabis still remain a subject of intensive research. The greatest progress was made in 1940-1942 by American and British authors in determining the chemical structure of the closely related components of the "red oil" (cannabinol, cannabidiol and tetrahydrocannabinol), as well as in identifying the tetrahydrocannabinols (THCs) as active principles of the drug. Although THCs have been obtained not only from the hemp resin, but also prepared synthetically and semisynthetically, they have never been isolated from cannabis in a homogeneous crystalline form. This is mainly due to the fact that the structure of THC involves a great number of stereoisomers and that the hashish activity is attributed to a mixture of isomeric THCs in the cannabis resin. Some of the isomers examined show considerable variations in their physiological potency. In a series of subsequent studies, a number of active THC homologs has been prepared by synthesis, and the relationship between the structure and the biological potency of these compounds has been established.
Investigations carried out independently in Czechoslovakia and Germany over the years 1955-1960 have shown that, besides THCs exhibiting hashish activity, there are also some other components possessing different biological activity. The recently isolated cannabidiolic acid was found to be a sedative and antibacterial agent of the drug.
Chemical variations in cannabis of various origins and varieties have been studied by several authors. It has been established that, in addition to the differences in the potency of the various resins, the presence and the amount of inactive cannabinols may also be characteristic for a given type. The variations observed are now explained mainly by the differences in the progress of the gradual phytochemical conversion of cannabinols. The study of the genetic and ecological factors affecting this process is of particular interest in explaining the formation of the various types of the drug. Attempts have been made to classify cannabis into several types according to the relative content or predominance of various cannabinols in the resin. These studies have contributed to the present views on the biogenesis, formation and occurrence of cannabinols in the hemp plant.
Numerous studies recently carried out in Canada, Germany, Czechoslovakia, the United States and Yugoslavia concern new analytical methods for the detection, separation or quantitative assay of cannabinols and other constituents of cannabis. It is obvious, however, that tetrahydrocannabinols, the main biological agents, remain the centre of interest. Reproducible chromatographic methods for separation and quantitative determination of active THCs are now available. However, they have not yet been fully applied and, even in the most recent studies, many important questions dealing with the properties of the separated THC isomers and with their occurrence in various cannabis types remain unanswered. The most important problem to be solved is the possibility of the direct chemical estimation of the biological potency of cannabis. This work will obviously encounter great difficulties, as the exact interrelationship between the various biological actions of THCs still remains unknown. A close co-operation of chemists and pharmacologists is required in this field of investigation. At any rate, modern analytical methods have indicated the possibilities of clarifying even these questions.
The work on improving chemical reactions for the identification of cannabis is still in progress. This is the main objective of the research programme on cannabis established by the United Nations Narcotics Commission in 1959. It has been treated in a number of United Nations documents. The problem of the geographical identification also appears to have attracted considerable attention. It would also be of interest to examine the possibilities of working out chemical tests for detecting cannabis addiction. In addition, it seems that the presence of active components in various parts of the hemp plant also requires further study, as their occurrence is probably not limited to the flowering tops of the female plant.
From the present situation regarding the chemical investigation of cannabis, it may be expected that the work remaining to be done in the coming years will complete our present knowledge on this subject. Important contributions were made in the last few years, and the methods developed offer wide opportunities for further studies. In the present report, the development and the results of the chemical research on cannabis, made available since 1960, are reviewed.
Attempts were recently made to solve some of the remaining problems dealing with the structure of cannabinols, such as the configuration of the two asymmetric C atoms in THC and the position of the alicyclic double bond. Taylor & Strojny [51] have examined the possible stereospecific synthetic routes by the synthesis of some model compounds related to THC. The structure proposed by Adams et al. [3] , [4] in 1940 has been modified by Mechoulam [35] , [36] , who proposed a stereochemical structure for cannabidiol on the basis of the obtained NMR spectrum in deuteriochloroform. The position of the double bond in the hydro-aromatic ring was shown to be conjugated to the phenyl group or to the double bond in the side chain (figure 1). ?antavy [50] has independently reached the same conclusion on the basis of optical rotation data.
According to the hypothesis by Todd [52] , cannabinols are formed in the plant by the condensation of a terpene derivative, menthatriene, with olivetol. The first product of such a synthesis would be cannabidiol (CBD), followed by cyclization to tetrahydrocannabinol (THC) and, finally, by loss of hydrogen, to cannabinol (CBN). After the isolation of cannabidiolic acid (CBDA) by Krejci & ?antavy [31] and by Schultz & Haffner[46] , this hypothesis was modified. In a highly interesting study including methods for the separation and quantitative assay of various cannabinols, Schultz & Haffner [47] suggested that CBD is formed in cannabis resin by decarboxylation of CBDA. In addition, some findings on the composition of various types of cannabis by Grlic [14] , [16] , [19] confirmed the view that CBDA may be considered as the initial compound in the phytochemical conversion of cannabinols. A detailed scheme of the biogenesis of cannabinols was given by Farmilo et al.[12] . In accordance with the Birch [6] acetic acid hypothesis for phenol synthesis, it is assumed that an acid form of olivetol may occur in the plant. The first step in the biosynthesis would be the condensation of an hexanoic acid with three molecules of acetic acid, yielding a cyclohexenedione acid as an intermediate, which could enolize to the dihydroxyphenolic acid (olivetolic acid). The second component would be menthadiene (limonene), instead of menthatriene, as mentioned by Todd. Olivetolic acid with menthadiene yields CBDA, which can be converted into either CBD by decarboxylation, or by cyclization to form THC-acid which itself may decarboxylate to THC and finally by dehydrogenation to form CBN (figure 2). In our opinion, the first way of conversion of CBDA is more likely to occur, or at least seems to predominate in cannabis resin.
Korte & Sieper [27] have described in detail a technique of column chromatography followed by countercurrent separation by which crystalline cannabidiol (and possibly also THC) has been isolated from cannabis grown in Germany. By applying countercurrent distribution and using the system n-hexane-methanol- water (10:9:1) they have also isolated two isomers from THC-resin, prepared synthetically according to Adams et al. [1] . The first was in the form of long colourless needles, melting at 128?C, and the second in colourless prisms melting at 62-63?C. In another publication (28), the same authors reported on a third isomer, isolated in small quantities as colourless needles melting at 86-87?C. The three isomers may be distinguished one from another by ultraviolet absorption. The first exhibits a maximum at 266 m? (log ? 4.29), the second at 273 m? (log ? 4.12) and the third at 258 m? (log ? 4.25).
After the study by Asahina, reported in the Bulletin on Narcotics in 1957 [5] , the use of paper chromatography for detecting and separating the various components of cannabis has been described by several authors.
Obata & Ishikawa [40] reported on detecting eugenol and guaiacol as well as two unknown carbonyl compounds in hemp extracts by paper chromatography. The same authors isolated piperidine as one of the components of unpleasant odour of the hemp plant [41] . It was shown to be partly associated with amino acids.
De Ropp [43] reported on the separation of eight components from the phenolic fraction of the red oil, by using the system dimethylformamide-cyclohexane (1:10). All constituents gave a yellow or orange colour with diazotized sulfanilic acid. Both the colour and the rate at which it developed varied with different compounds. By further partition chromatography on diatomaceous earth (Celite 545), the same author separated the active fraction (THC) from other components. After purifying this fraction by high vacuum distillation, a colourless resinous THC(C 21H 30O 2) was obtained, solid at room temperature and showing a specific rotation [?]25/ D - 161?. UV absorption maxima of the isolated substance were at 275 and 282 m? (log ? 3.26 and 3.28), shifting in 0.1 N sodium hydroxide to 292 m? (log ? 3.53), with a second peak at 325 m?. The product could not be crystallized.
The same method, with minor modifications, was used by Farmilo & Davis [11] . These authors have separated at least ten phenolic compounds from the petrol ether extract of cannabis, and have reported on the R F values obtained for CBN, CBD, THC and CBDA. The relative content of various cannabinols, as found in the examined samples of cannabis, is discussed in the same paper. A further modification was described by Davis et al.[9] .
Korte & Sieper [25] , [26] have reported on separating CBD, CBN and THC in the hemp extracts by descending paper chromatography, using a paper impregnated with silicone resin and the solvent system ligroinebenzene-chloroform-methanol-water (2:2:1:4:1). Spots may be developed by means of five different combinations of spray reagents, each of them yielding characteristic colours with the three components. The separated cannabinols may also be identified by measuring directly on the chromatogram their UV absorption spectra. An exact quantitative determination is apparently possible by this method only if it is carried out together with simultaneous chromatograms of pure reference substances.
A similar, easily reproducible method was developed by Schultz & Mohrmann [49] . They applied ascending chromatography and a toluene-methanol-water system (5:5:1.5) also on hydrophobic paper (Dowex silicone). Besides the above three cannabinols, these authors reported on the R F value for CBDA (as diacetate).
Another method for the separation of the various components of cannabis resin by paper chromatography was described by Kol?ek et al. [24] . They have used a paper saturated with formamide and an ascending procedure with benzene as the mobile phase. By treating separately both acid and phenolic fractions of the red oil obtained from cannabis of Yugoslav origin, it was possible to obtain a number of spots yielding various colours with di-o-anisidine-tetrazolium-chloride. The chromatograms were eluted with ethanol, treated with p-nitro-diazo-benzene-tetrafluoroborate, and the absorbance of the yellow azo-product measured after one hour at 425 m?. Cannabidiol was used as the reference substance. Quantitative data on the antibiotic potency of the separated components have been tabulated and discussed.
In addition to the components of the resin, hydrosoluble constituents of cannabis have also been subjected to separation by paper chromatography. B?zner [7] reported on the examination of acid and alkaline hydrolyzates of alcohol extracts of freshly cut and shredded hemp plants by ascending paper chromatography. This work demonstrated that it was possible to distinguish different varieties of the cannabis plant on the basis of the differences in their sugar content.
Krejci [32] reported the use of this modern analytical technique for the separation of cannabis components. The application of thin-layer chromatography indicated that CBD and CBDA are responsible for the antibiotic effect of cannabis grown in Czechoslovakia which, however, did not exhibit any hashish effect in human and animal experiments.
Large possibilities for the separation of cannabinols offered by thin-layer chromatography on silica-gel are described by Korte & Sieper 28. Silica-gel was impregnated with dimethylformamide, and cyclohexane was used as the liquid phase. For the detection of spots, the colours obtained by using a number of various reagents for phenols are described. The most useful detector was found to be di-o-anisidine-tetrazoliumchloride (Echtblausalz B). It gives with CBD, THC and CBN orange, red and violet colours respectively, so that the three components may be identified on chromatograms without using reference substances.
From the THC-resin synthetically prepared according to Adams et al. [1] from 1-methyl-cyclohexanone-3-carboxylate and olivetol, the authors [28] have isolated by means of thin-layer chromatography the same three isomers, which have been mentioned above as being isolated in crystalline form by means of countercurrent distribution. In addition, THC synthesized according to Todd et al. [13] was found to contain two other isomers, different from the products contained in THC obtained by Adams et al. [1] . THC obtained from CBD by the procedure with ethanolic HCl and by means of pyridine hydrochloride [2] has been separated by thin-layer chromatography into three and two isomers respectively, whereas the product obtained by means of p-toluene-sulfonic acid [3] could not be separated by using the technique described. THC isolated from cannabis grown in Germany (melting at 146°C), according to chromatograms obtained, was not identical with any synthetic or semi-synthetic THC.
Chromatograms reported by Korte & Sieper obtained from the extracts of cannabis of various origins are of particular interest (figure 3). Spots having the same R F values have been shown to yield with di-o-anisidine-tetrazolium chloride the same coloured products, and thus may indicate the same substances. Spots V, VI and VII are three isomers of THC. Among them, spot V (THC I) is predominant in all the extracts examined. THC III, according to its R F value and the colour of the product, is identical to the isomer previously isolated from German hemp (melting at 146°C). Spots IV and III correspond to CBN and CBD, whereas spot II (giving the same azo-product and the same Beam test as CBD) is probably, according to the opinion of the authors, an izomerization product of CBD. CBDA (spot I) remains at the starting point.
In the extracts of hashish of oriental origin (chromatograms 1 to 4) CBDA is not present, whereas all of the remaining cannabinols have been detected in various quantities. German hemp (chromatograms 5 and 6) contains a large quantity of CBD, some CBDA and THC, whereas CBN was not detected at all. Indian cannabis cultivated in Germany (chromatogram 7) containe d more THC and less CBD than the German hemp. However, in a fresh German cannabis only CBDA and CBD have been detected (chromatogram 8). These findings confirm the explanation by Grli? [17] , [19] concerning the differences in the composition of various type s of cannabis. The examination of the product of pyrolysis of CBD (chromatogram 9), exhibiting 13 spots identical with the components of cannabis extracts, also confirms previous assumptions.
A similar technique for thin-layer chromatography was described recently in the Bulletin on Narcotics by Miras et al. [37] . The method was used to compare the composition of ordinary cannabis extract with the sublimate of smoked cannabis. Four main constituents of the resin (CBDA, CBD, CBN and THC) were separated from the cannabis extract. However, the spot corresponding to CBDA (with the R F value of 0.06) was not detected in the chromatogram of the sublimate.
In another recent study [29] , Korte & Sieper described the possibilities for the quantitative spectrophotometric determination of various cannabinols after separation by thin-layer chromatography. On account of the low absorptivity of cannabinols in the ultraviolet region, it was not possible to prepare sufficiently concentrated eluates on which to carry out direct measurements of the ultraviolet absorption of the isolated compounds. Therefore, the authors have converted the separated components into coloured azo-products of high absorptivity over the visible region of the spectrum. As in the previously described technique [28] , chromatograms have been developed by means of cyclohexane on silica-gel impregnated with dimethylformamide. A freshly prepared solution of di-o-anisidine-tetrazolium chloride in N/10 NaOH was used as reagent. CBDA remains at the starting point as an orange spot, followed (with the increase of R F) by CBD (orange), CBN (dark violet), THC I (scarlet), THC II (light violet) and THC III (pink). The spots have been extracted by means of a mixture of glacial acetic acid and methanol (1:1). The range of applicability of the Lambert-Beer law has been experimentally established, and empirical extinction co-efficients for four coloured products (corresponding to CBD, CBN, THC I and THC II) have been calculated. Amounts of THC III have been too small for quantitative assay, whereas CBDA, remaining at the starting point together with other impurities, could not be separately determined. The determination of four cannabinols in cannabis extracts by the proposed technique takes 4 to 5 hours. The results of the analysis of six samples of c annabis, examined by the proposed method, are reproduced in table 1. It is of interest to compare the amounts obtained for the various cannabinols with the values of the various constants tabulated for the same samples by Grli? [17] . Thus, a high content of CBN in the first two samples corresponds to their classification as "overripe" cannabis [17] . Other data reported in the two papers are also in fairly close agreement.
| % in dry cannabis | ||||
|---|---|---|---|---|
| Sample | CBD | THC I | THC II | CBN |
Nigeria UNC 59
| -
| 0.079 | 0.019 | 0.481 |
Brazil UNC 61
| -
| 0.190 | trace
| 0.410 |
Cyprus UNC 33
| -
| 0.230 | 0.150 | 0.060 |
Morocco UNC 21
| 0.129 | 0.096 | trace
| 0.123 |
Geneva UNC 51
| 0.129 | 0.059 | 0.028 | 0.017 |
Canada UNC 37
| 0.103 | trace
| trace
| -
|
The significance of the work by Korte & Sieper is in making available a simple and reproducible analytical method for quantitative determination of various cannabinols and particularly for the separation of THC isomers. This appears to offer wide possibilities for further detailed studies of the most important constituents of cannabis.
Kingston & Kirk [23] have separated six fractions from a petrol-ether extract of cannabis, by passing it through a silicone rubber on Chromosorb W. The tem- perature applied was 250°C, with a helium flow rate of 40 ml/min. Twelve fractions have been detected by collecting each fraction from several runs and re-chromatographing them at 225°C. UV absorption characteristics were reported for seven of the fractions. Among those, CBN and two THC isomers have been detected, whereas CBD was not present in the extract examined. The reaction of various fractions obtained with the Duqu?nois-Negm test have been studied and reported. On the basis of the results obtained, the authors supposed that gas chromatography would enable the determination of the origin of the drug.
In a preliminary study, Scaringelli [45] has obtained, by a similar technique of gas chromatography, five distinct peaks from cannabis extracts. Although these fractions have not been positively identified, on the basis of the results obtained the author supposed that further studies on the application of this technique would enable the determination of the biological activity of cannabis.
Martin et al. [34] have examined by gas chromatography more than 30 samples of essential oils obtained by steam distillation of cannabis of different origins. A helium carrier gas-flow was used with a rate 75 ml/min. through a column packed with " Apiezon M " grease supported on Chromosorb W heated to approximately 220°C. Separated fractions were dissolved in CCI 4 for IR spectrophotometry, and after evaporating the solvent, the residue was dissolved in ethanol for colour tests and UV spectrophotometry. Eighteen constituents were found in the oil samples obtained from fresh green plants. Colour tests for cannabis (Beam, Duqu?nois and Ghamravy) were carried out on a few drops of the ethanolic solution of each fraction. No positive Beam test was obtained with any of the fractions separated, whereas a positive Duqu?nois reaction was obtained with both ? and ?-caryophyllene. RRT values and percentages of each fraction, as found in four of the examined samples, were separately tabulated. Some of the peaks on the chromatograms were considered to be particularly characteristic for the essential oil of cannabis. Chromatograms of oils from dried cannabis, hashish and charas showed distinct differences from those of the fresh oils, and may be identified on this basis. As is seen from figure 4, myrcene, limonene, ? and ?-caryophyllene have been detected in the fractions examined.
Farmilo et al. [11] , [12] have used a methyl-silicone gum rubber on Chromosorb W column for the separation of the cannabis constituents. The packing material was activated at 225°C for 12 hours in an argon gas stream and then at 325°C for 24 hours without the gas flow. The conditions for chromatography have varied considerably (temperature from 160 to 194°C, gas flow rate from 66 to 104 ml/min., detector beta or flame ionisation). The way of preparing samples of essential oils, cannabis extracts and pure cannabinols for analysis is described in detail [12] . Pure cannabinols gave good chromatograms by using the quantities of 0.5 to 1.0 ?g. The obtained RT and RRT values for CBD, THC, CBN and pyrahexyl are reported, while CBDA-diacetate did not chromatograph under the conditions tested, even after 40 minutes. The presence of CBD was indicated in a steam distillate of fresh cannabis [12] , although in a previous work [34] no positive Beam test was obtained with any of the fractions separated by gas chromatography from the essential oil of cannabis. The number of constituents separated in various samples of the resin of seized cannabis varied from seven to one. As the analysis of cannabis samples was not carried out under identical conditions as the examination of pure cannabinol-standards, the results could not be compared directly. In a further paper by Davis et al. [9] , additional results obtained by this technique were reported and discussed, including quantitative data on the content of CBD, THC and CBN in cannabis samples of different origins.
A rapid and simple method for determining simultaneously CBDA, CBD, THC and CBN was developed recently by Lerner [33] . By treating the petrol-ether extract of cannabis with diazomethane, CBDA was converted into its methyl ester, and thus made amenable to gas chromatography. Other cannabinols are not affected by methylation; they may be recovered unchanged after gas chromatography, as has been confirmed by the IR spectra of the eluted material. The product of diazomethane treatment was dissolved in ether and subjected to gas chromatography in an argon ionization gas chromatograph operated at 180°C, in which the flow of argon was 80 ml/min. A cyanoethyl silicone gum in 0.5 % concentration on a 120-mesh silanized Chromosorb W support was used as the column. The retention times were established for CBD, THC, CBDA-methyl ester and CBN by using standard substances of high purity. In addition, RT values for four unknown peaks were also given, and relative contents of altogether eight components in various samples of cannabis were tabulated. Quantitative variations of the major constituents of the resin in various samples were quite considerable.
In a document by the United Nations Secretariat [53] , the specificity of the three most commonly used reactions for the identification of cannabis has been studied. Eleven samples of cannabis and 120 other plant species (belonging to 28 botanical families) were investigated. Table 2 reproduces the results obtained with cannabis samples and with plant species showing a tendency to react positively with at least one of the three tests. As may be seen, all the cannabis samples gave a strongly positive Duqu?nois-Negm test, whereas their behaviour with the Beam and Ghamravy tests varied from sample to sample. On the other hand, a number of other plant species showed tendencies to react with the tests for cannabis, particularly with the Ghamravy reaction. These were mostly plants containing volatile oils. For this reason, the relationship between certain plant constituents and the reactions obtained with corresponding plant extracts have also been studied and discussed [53] , [55] . Altogether 48 pure substances of vegetable origin (mostly constituents of volatile oils) were tested with the three reactions for cannabis. Results for the positively reacting substances are recorded in table 3. The results obtained indicated that some of the substances tested exhibited identical or very similar reactions as cannabis resin, particularly with Duqu?nois-Negm and Ghamravy tests. These investigations, which have been carried out in the United Nations narcotics laboratory, have enabled conclusions to be drawn regarding the specificity, sensitivity and usefulness of the three most important tests for cannabis identification. The Beam test proved to be more specific, whereas the Duqu?nois-Negm test was the more sensitive one. The Ghamravy test was of limited value, and was recommended only if used in combination with two other tests. Among 120 plant species examined, only one ( Salvia officinalis) showed a tendency to react in all three tests. Consequently, it is considered that a parallel application of all three tests offers a relatively high probability (but not a complete assurance) of the successful identification of the drug.
| 0 | Negative
|
+
| Trace
|
++
| Weakly positive
|
+++
| Positive
|
++++
| Strongly positive
|
| species | Beam | Ghamravy | Duquenois-Negm |
|---|---|---|---|
UNC 1A
| ++
| ++++
| ++++
|
UNC 1B
| ++++
| ++++
| ++++
|
UNC 1C
| +++
| ++++
| ++++
|
UNC 1D
| ++++
| ++++
| ++++
|
UNC 1E
| ++++
| ++
| ++++
|
UNC 1F
| ++
| +++
| ++++
|
UNC 2
| ++
| +
| ++++
|
UNC 3
| ++
| +
| ++++
|
UNC 4
| ++++
| ++++
| ++++
|
UNC 5
| 0 | ++++
| ++++
|
UNC 6
| ++
| +++
| ++++
|
| species | Beam | Ghamravy | Duquenois-Negm |
|---|---|---|---|
Salvia officinalis L.
| +
| +++
| ++
|
Thymus vulgaris L.
| 0 | +++
| ++
|
Rosmarinus officinalis L.
| ++
| +++
| 0 |
Satureja hortensis L.
| 0 | ++
| +++
|
Lavandula officinalis Chaix
| 0 | +++
| 0 |
Eucalyptus Globulus Labill.
| 0 | +++
| ++
|
Arthemisia Absynthium L.
| 0 | +++
| 0 |
Arthemisia Dracunculus L.
| 0 | ++
| ++
|
Cinnamomum Camphora Nees
| 0 | ++
| 0 |
Laurus nobilis L.
| 0 | ++
| 0 |
Angelica Archangelica L.
| 0 | ++
| 0 |
Lepidium sativum L.
| 0 | +++
| 0 |
Armoracia lapathifolia Gilib
| 0 | +
| 0 |
Ficus carica L.
| 0 | 0 | +
|
Ficus elastica Roxb.
| 0 | ++
| 0 |
Pelargonium capitatum (L.) Ait
| 0 | ++
| +
|
Filipendula ulmaria (L.) Maxim
| 0 | +++
| 0 |
Papaver somniferum L.
| 0 | +
| 0 |
Rhamnus Frangula L.
| 0 | +
| +
|
Nicotiana Tabacum L.
| 0 | +
| 0 |
Atropa Belladona L.
| 0 | +
| 0 |
Datura Stramonium L .
| 0 | +
| 0 |
Hyoscyamus niger L.
| 0 | +
| 0 |
Juniperus oxycedrus L.
| 0 | +
| 0 |
Juniperus Sabina L.
| 0 | +
| 0 |
Juniperus communis L.
| 0 | +
| 0 |
Larix decidua Mill.
| 0 | +
| 0 |
Novak et al. [38] , [39] have described the results obtained for various samples of cannabis using both the alkaline and the acid Beam test, tests of Ghamravy and Duqu?nois-Negm, a modified test of the British Pharmacopoeia Codex, reaction of Rathenasinkam, reaction of Viehoever and the determination of Beam-C1 value. Extracts of Hungarian and Indian cannabis have shown distinct differences in response to all these tests (except the Viehoever reaction). It was supposed that a positive response to most of the reactions tested was to be attributed to physiologically inactive components of cannabis. On the basis of the results obtained, it was concluded that not more than traces of active components were present in the hemp extracts of Hungarian origin.
Several analytical methods indicating the composition and the type of cannabis resin have been developed by Grlic et al., such as the determination of the content of acid fraction [19] , indophenol spectrophotometric method [14] , peroxide-sulphuric acid test [15] and the determination of the "FeCl 3 value" [20] . The results obtained with some of the methods proposed are expressed arithmetically in terms of various constants, which proved to be particularly useful for characterizing various types of cannabis. These methods and the results obtained with a number of cannabis samples have been reviewed in detail, and the reader is referred to a previous paper in the Bulletin on Narcotics[14] . By using the same methods, changes in chemical properties, resulting when samples of various origins are grown in a place with mid-European climate (Zagreb), have been studied and reported [18] . It has been shown in the course of these studies that the differences in the chemical composition exhibited by various types of cannabis may be explained by the stage of development of the phytochemical process (called "ripening of the resin ") by which CBDA is gradually converted to CBD, THC and finally to CBN.1 Thus, cannabis in which CBDA (the initial substance in the conversion of cannabinols) was predominant was classified as " unripe ". The resin containing mostly physiologically active THCs was referred to as " ripe ". The " intermediate " type contained predominantly CBD, whereas the resin containing mostly inactive CBN, the final conversion product, corresponded to the " overripe " cannabis. Although the " ripening " process seemed to be affected by various factors, it was generally more advanced in cannabis from tropical areas (" ripe ") than in cannabis developed in a temperate climate (" unripe " or " intermediate "). Consequently, tropical cannabis is distinguished not only by its high resin content, but also by a high content of the active THCs in the resin itself.
It should be noted that the proposed term " ripening " of the resin and the corresponding classification into types are not necessarily related to the physiological ripeness of the hemp plant, as it appears to be misunderstood by some authors when quoting the above work. These expressions have been used in order to abbreviate the terminology and to facilitate the notion of a natural transformation process and of the variations depending on its gradual progress.
Miras et al. [37] reported the disappearance of CBDA and a relative increase of the THC content in the sublimate of smoked cannabis when compared with the ordinary hemp extract. One could explain the findings of these authors also by a " ripening " of cannabinols occurring in the smoking of the drug. Such an explanation may be of great significance, as it leads to the conclusion that the smoke of " unripe " cannabis (grown in a moderate climate) probably contains a higher quantity of toxic agents than the extracts of the same type of drug.
Possibilities for the examination of cannabis by direct ultraviolet spectrophotometry have also been re-examined in recent years and described in detail [16] , [54] . In comparison with the extracts of some aromatic plants, the directly measured ultraviolet absorption of cannabis extracts was not specific enough to be used as a basis for the identification of the drug. However, this method provides useful information about the chemical composition of cannabis resin. The absorption curves of diluted ethanolic solutions of the resin were found to be characteristic for various types of cannabis. All samples exhibited a marked absorption peak between 266 and 280 m? (mainly attributed to the additive absorption of various cannabinols) and a minimum between 246 and 250 m?. "Ripe" samples did not exhibit other absorption maxima. "Unripe" samples showed a secondary peak at 303-304 m? (explained by the predominance of CBDA), while the "intermediate" type showed only a slight convexity over the same region. For the characterization of cannabis resin, two extinction ratios have been used (E 260/E 280 and E 300/E 3l0). The reproducibility of these constants was very good, and they were found to be characteristic for the groups of samples examined. Results reported by Novak [39] , obtained by the same technique, have confirmed the above findings. Miras et al.[37] have reported on the differences between the UV absorption spectra of cannabis extract and the extract of the sublimate of smoked cannabis. The peak over the region of 300 m? (attributed to CBDA) was lacking in the extract of sublimate. Scaringelli [44] developed an ultraviolet spectrophotometric method based on the bathochromic shift shown by cannabinols when examined in acid and alkaline media. According to the experimental data reported, the use of differential values for absorption in acid and basic solutions seems to increase the specificity of the spectrophotometric identification of cannabis. This technique was reported to be useful for detecting the presence of cannabinols in adulterated material [8] .
| Reaction | |||
|---|---|---|---|
| Substance | Beam | Duquenois-Negm | Ghamravy |
Anethole
| 0 | 0 | weak violet
|
Anisole
| 0 | 0 | pink
|
l-Borneol
| 0 | 0 | strong violet
|
d-Camphene
| 0 | 0 | strong violet
|
Cannabidiol
| strong violet
| strong violet
| violet-blue to blue
|
Carvacrol
| 0 | bluish-violet
| weak violet-red
|
l-Carvone
| Brown
| 0 | pinkish-violet
|
Caryophyllene
| 0 | weak violet
| violet blue to blue
|
Cineole
| 0 | weak violet-pink
| brown
|
Cinnamic aldehyde
| Yellow
| 0 | strong violet
|
Citral
| Brownish-yellow
| reddish-violet
| violet brown
|
Citronellal
| 0 | weak violet
| strong bluish-violet
|
Citronellol
| 0 | bluish-violet
| bluish-violet
|
p-Cymene
| 0 | weak brownish-pink
| violet-blue to blue
|
Eugenole
| 0 | 0 | brown
|
Farnesol
| 0 | bluish-violet
| strong violet
|
Geraniol
| 0 | bluish-violet
| strong violet
|
Guaiazulene
| 0 | pinkish-red
| green
|
Furfurol
| 0 | weak violet-gray
| green
|
Isoeugenol methyl ether
| 0 | 0 | reddish-brown
|
Isosafrole
| 0 | 0 | orange-red
|
Juglone
| violet-brown
| 0 | green
|
d-Limonene
| 0 | 0 | strong bluish-violet
|
l-Limonene
| 0 | 0 | strong bluish-violet
|
Linalool
| 0 | bluish-violet
| strong violet
|
Menthol
| 0 | 0 | brownish-violet to bluish-violet
|
Nerol
| 0 | violet
| violet
|
?-Phellandrene
| 0 | violet
| strong bluish-violet
|
?-Pinene
| 0 | 0 | bluish-violet
|
?-Pinene
| 0 | 0 | indigo-blue
|
Pulegone
| 0 | 0 | strong sky-blue
|
Pyrrole
| 0 | 0 | strong-violet
|
Resorcinol
| brownish-green
| pinkish-violet
| strong pinkish-red
|
Safrole
| 0 | 0 | reddish-brown
|
Squalene
| 0 | 0 | violet
|
Thujone
| 0 | 0 | violet
|
Terpineol
| 0 | 0 | strong bluish-violet
|
Thymol
| 0 | violet
| strong violet
|
A large study dealing with the chemistry of cannabis and containing the data obtained by means of a number of available methods was made by Farmilo et al.[12] . The authors have examined the variations in the chemical composition of cannabis from various geographical origins, and discussed the possibilities for determining the provenance of the drug by chemical means.
In addition to the studies of various methods and tests for cannabis and cannabinols, the behaviour of certain chemical reagents with cannabis constituents has been described and discussed in a number of papers. Most of these reactions have been applied to the spot detection in chromatographic methods. Thus, the application has been reported of the reagent of Duqu?nois-Negm [9] , [23] , [28] , [34] , Beam [9] , [12] , [26] , [27] , [28] , [34] , Ghamravy [28] , [34] , Gibbs [11] , [12] , [14] , [26] , [28] , Blackie [28] , Pauly [25] , [26] , [27] , [28] , [43] , etc. One of the most sensitive and useful reagents for cannabinols was reported to be di-o-anisidine-tetrazolium chloride (Echtblausaltz B) [24] , [28] , exhibiting different colours with various cannabinols.
Much work has been done in the last ten years on the investigation of the antibacterial constituents of cannabis. As is known, cannabidiolic acid was identified as the main antibiotic agent by Krejci et al.[30] and by Schultz & Haffner [48] . In some recent studies, the antibacterial substances and their effect have been examined by Kol?ek et al. [24] , Krejci [32] and Doktorov [10] . The chemistry and the effect of the antibacterial substances of cannabis were described in detail in the Bulletin on Narcotics by Kabelik et al. [22] . However, according to the view of the WHO [56] , there is not enough evidence in favour of making cannabis available for the extraction of antibiotic substances. Among a large number of known antibiotic agents, the antibacterial constituents of cannabis do not appear to offer particular advantages in medicine. Consequently, according to the opinion quoted [56] , cannabis preparations remain obsolete, and there is no justification for their medical use. Nevertheless, as shown by Rado?evic et al.[42] , the determination of the antibiotic potency may be of use in providing interesting information about the chemical composition of cannabis resin.
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035Mechoulam, R.: Israel J. of Chemistry, 1, 37 (1963).
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037Miras, S., Simos, S. & Kiburis, J.: Bull. Narcotics, 16, No. 1, 13 (1964).
038Nov?k, I, Buz?s, G. & T?th, L.: Pharmazie, 17, 166 (1962).
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040Obata, Y. & Ishikawa, Y.: Bull. Agr. Chem. Soc. Japan, 24, 667 (1960).
041Obata, Y., Ishikawa, Y. & Kitazawa, R.: Bull. Agr. Chem. Soc. Japan, 24, 670 (1960).
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