Cannabis as a medicament

Sections

INTRODUCTION
1. Treatment with cannabis in ancient, folk and official medicine up to the beginning of the twentieth century 2
A brief survey of the methods of isolation and the physical and chemical properties and structures of the isolated antibacterial substances
Methods and resultsof the bacteriological experiments
Survey of clinical experiencesBy Z. Krejci
Summary

Details

Author: J. Kabelik,, Z. Krejci,, F. Santavy
Pages: 5 to 23
Creation Date: 1960/01/01

Cannabis as a medicament

J. Kabelik,
Z. Krejci,
F. Santavy
The Institute of Hygiene and Epidemiology (Director: Prof. Dr. J. Kabelik) and the Institute of Medical Chemistry (Director: Prof. Dr. F. Santavy) of the Medical Faculty of the Palacky University, Olomouc, Czechoslovakia

INTRODUCTION

Within the scope of a systematic study of antibacterially effective substances [ 5] present in the flora growing in the temperate climate of Czechoslovakia, we have undertaken to investigate more than 3,000 different plant species. In this way we came upon the Indian hemp plant - Cannabis sativa var. indica - which has been investigated in detail. The subsequent finding of the antibacterial effect of the extract from Cannabis indica - direct evidence was provided some years ago at our Institute of Hygiene [ 46] - stimulated much interest in cannabis from the medical point of view, and resulted in numerous reports published from different institutes and departments [ 36] , [ 43] , [ 49] , [ 51] , [ 65] , [ 78] , [ 81] , [ 82] ). Independently from us the antibacterial effect of the extracts from Cannabis sativa was described and studied some years later, particularly by Ferenczy [ 24] , [ 25] and Drobotko [ 20] . The interest of our clinicians was aroused in this respect by the very good results achieved with the antibacterially effective substances from various plants referred to in the literature [ 22] , [ 23] , [ 29] , [ 59] , [ 73] , [ 74] , [ 86] , [ 87] 1.

The preliminary results obtained were published in 1952 in Lekarske listy [ 47] and in a compilation of communications submitted at the scientific conference held by the university in Olomouc in 1955. A survey of our work was published in the journal, Die Pharmazie, 1957-1959 [ 41] , [ 50] , [ 52] .

Our paper is divided into the following sections:

Treatment with cannabis in ancient, folk and official medicine up to the beginning of the twentieth century.
A brief survey of the methods of isolation and the physical and chemical properties and structures of the isolated antibacterial substances.
Methods and results of the bacteriological experiments.
Survey of clinical experiences.

1. Treatment with cannabis in ancient, folk and official medicine up to the beginning of the twentieth century 2

By J. Kabelik

The knowledge of narcotics and stimulants extends far back to ancient and medieval times but since then, as far as is known, no new discoveries have been made. In Europe, of course, some of them appear not to have been known until later, as was the case with the narcotics of the Old World - for example, with hashish [ 44] , [ 88] . Europeans learned of it for the first time through the Crusaders, but its deliberate use as well as abuse - according to Perrot [ 67] - came from Napoleon's soldiers, who introduced it after their return from the Egyptian campaign.

Cannabis is, however, a very old cultivated plant apparently indigenous to central Asia [ 68] . Cannabis was found by our archaeologists to have existed in central Europe in the Bylony Culture (7,000 years ago). The Chinese knew of it perhaps 4,000 years ago, certainly in the 9th century B.C., primarily as a medicinal herb and a century later as a textile plant. According to Herodot, the Scythians knew it as a plant which can be spun and as an oil-producing plant and, apparently, even as a narcotic which they made use of in their steam baths - the sauna. Perhaps the earliest accounts of the medicinal utilization of cannabis may be found in Indian medical literature. Ancient Indian surgery, according to Susrat (Samhita), used hyoscyamus and cannabis as anaesthetics [ 32] . From the Egyptian medical papyruses [ 64] , information has been gained about a plant from which cordage could be made, and it was probably cannabis which was referred to. But no records could be found on its narcotic action. The preparations made from it (in all probability from the cannabis shoots) were applied externally-namely, exclusively as antiseptics - and then perhaps even as analgetics, in the same way as in Hellenic medicine. Cannabis extracts have been employed for irrigation in diseases of the anus, and in form of compresses the drug has been applied to sore toenails. In Rhamses' papyrus, washing sore eyes with extracts from cannabis and also from some other plant is recommended. The papyrus of Berlin recommends fumigation with cannabis in some undefined disease. Cannabis has been prescribed in feverish diseases of the bladder and, even at present, in homoeopathic medicine it ranks first when cystitis is treated. Furthermore, extracts combined with honey were injected into the body of the uterus to achieve constriction of the uterus and, externally, an ointment combined with fat was applied antiseptically in the same way as was done in medieval medicine up to the present time. Cannabis shoots were well known to Galen [ 27] and to Dioscorid [ 85] . Homer's nepenthes - potion of oblivion - has been identified by some authors as a cannabis drug, but generally it is believed to be a preparation made from Hyoscyamus muticus - a plant familiar to the Egyptians. Both these authors make greater use of the seeds and of the oil extracted from them than of the cannabis shoots. It is the same in old popular and even in modem medicine. The seed pulp was a favourite dish, and from the seeds an edible, industrial and medicinal oil was obtained. In Czechoslovakia, a preparation from seed pulp was recently introduced by Širek [ 60] to act as a roborant diet in treatment of tuberculosis.

1. Comprises an extensive survey of reports by authors from the USSR.

2. The botanical-historical and technical aspects of cannabis are not dealt with in this report.

But hemp seed does not form the subject matter of this paper; it is only treatment with cannabis shoots which is discussed, and only with regard to the healing of wounds, and not to the hashish effect.

The uses mentioned in the Egyptian papyruses point fundamentally to antiseptic use. Analogous uses were known, in varying degrees, to African natives [ 28] , [ 29] , and were recorded in medical herbals. There is no information on the narcotic action. It is of interest that in Egypt they are supposed to have learned of the hashish effect only during the Middle Ages, from the Arabs. This could be explained by the fact that the Arabs were the first to import the variety producing the physiologically active resin, for at present Cannabis indica Lam. is not considered to he a species but a variety of Cannabis sativa L. and not even a particular variety, Cannabis is a very variable and plastic plant of variable height and variously membered and formed leaves; there exists a monoecious variety, too. Any cannabis plant can produce hashish under favourable climatic conditions. Vice versa, according to Pulewka [ 71] the Indian variety does not always produce the narcotic substance, not even in warm countries. Plants cultivated in 369 places in Anatolia did not produce hashish substances throughout; the occurrence was conditioned by the climate and the habitat. Likewise, the hashish effect has not been found present in Cannabis indica cultivated in north Moravia, though this variety grows exceedingly well. The stem attains a height of about 4 m, bears rich seeds, and the tops produce resins which have a very pronounced antibiotic and analgesic effect. It is possible, however, that there was no hashish-producing Variety grown in ancient Egypt, for the climate was, at that time, in all probability rather damp and subsequently much cooler than it is at present. This may be concluded from the fact that in antiquity in Italy and the Balkans snow was a more frequent phenomenon than it is nowadays, and north Africa was the granary of Rome, and not a desert.

FIGURE 1, Cannabis sativa var. indica

All the information obtained from European folk medicine with regard to treatment with cannabis shows clearly that there do not appear to be any narcotic substances in it, or if there are then only in a negligible amount. Instead of that, emphasis has been laid on the antiseptic effect, hence on the antibiotic and to a small extent even on the analgetic effect, which has been upheld in official medicine up to the beginning of this century. In Austria, up to World War I, a salicylate collodion combined with Extractum Cannabis was prescribed for application to corns. Unna's green salve, which was used as a remedy for lupus, contained: Acid salicyl., Liq. Stib. chlorati aa 2.0, Extr. Cannabis ind., Creosoti aa 4.0 , Adeps lanae 8.0. It may be noticed that the substances contained in cannabis have a powerful antibiotic effect upon Myc. tuberculosis, as will be discussed later. On the basis of the results obtained from our investigations, it is suggested that in many analogous cases it would be advantageous to return to cannabis preparations again.

The ancient herbals [ 17] , [ 63] , [ 85] and those of the Middle Ages mentioned medical use of the seed, the roots (emoelients) and of the tops containing resinous substances. As previously stated, only the latter will be discussed here in detail. The leaves and the juice extracted from them or the macerated leaves were used as a vermifuge for horses, and fisherman soaked the ground with a liquid prepared from them to force up the dew-worms, which they used as bait. Tabernaemontanus [ 85] and Kramerarius [ 63] recommended kneading the dried leaves with butter and application of them in form of an ointment to burns. We have obtained positive results with extracts from cannabis in treatment of burns. Ruellius cit. [ 63] , [ 85] advised the use of cannabis extract as ear-drops in the treatment of ear ache, and also for treatment of wounds and ulcers. Women stooping due to a disease of the uterus were said to stand up straight again after having inhaled the smoke of burning cannabis. In cystitis and in urinary diseases, a decoction of hemp shoots with wine and water was recommended; the steam was allowed to rise as hot as could be endured against the perineum, after which the patient was advised to urinate. This use has more to do with the analgetic than the antiseptic effect. As previously mentioned, homoeopaths value highly both the teep cannabis D ₂ ? 0.25 g (teep is the fresh drug ground with lactose) and the tinctura Cannabis indicae D ₃ up to D ₄ which are employed in cases of cycstitis and of urethritis. The homoeopathic utilization of cannabis is fundamentally based on its effect on the central nervous system - i.e., in migraine as discussed in Schoeler's Kompendium [ 76] and by Auster & Schafer [ 9] . The homoeopaths do not otherwise utilize the antiseptic effect when they employ it internally; it is only the centrally sedative action they make use of in the same way as the allopathists did formerly - i.e., in gastralgias and the like. In these cases of internal application the antiseptic effect is, however, doubtful save in the case when the intestinal flora is concerned.

So far the cannabis preparations - hashish preparations - have been frequently investigated therapeutically, particularly in neurology and in psychiatry, but they were abandoned because the results achieved were not uniform. Nevertheless it would be advantageous to utilize the analgetically sedative effect without the narcotic action of the hashish. Burroughs Wellcome & Co. manufacture a special product: Cannabine Tannate - cannabis combined with tan, Hydrastis canadensis and Secale cornutum - which has a sedative effect in metrorrhagias and in dysmenorrhagias. From earlier times, otorhinolaryngologists have preserved a prescription against tinnitus aurium: ZnO, Extr. Valerianae, Extr. Hyoscyami, Extr. Cannabis aa 1.6 M.f. pill No. 60, D.S. 3-5 pills daily per os. Finally, some years ago, in the American Journal of Pharmacy, vol. II (cit. Dinand [ 18] ), the following treatment was recommended in migraine: before meals take for a fortnight conscientiously and daily 0.015 g Extr. Cannabis indicae, the third week 0.02 g, the fourth week 0.03 g, to be continued for some months. In all these cases it is the action on the central nervous system which makes its influence felt. Of rather particular interest is the frequent combination of cannabis with tan both for internal and external application. We have obtained very good results in stomatitis aphtosa, gingivitis, and in paradentoses with a mouth wash of the following composition: Tinct. Cannabis 20.0, Tinct. Salviae, Tinct. Chamomillae, Tinct. Gemmarum populi (or another tan - for example, Tinct. Gallarum) aa 10.0, to be applied in the form of sprays or liniments to the inside of the mouth. The use of cannabis as an analgetic but not as an antibiotic in stomatology has been also briefly mentioned by Hegi [ 34] .

FIGURE 2, Cannabis sativa - engraving from the Czech edition of Mathioli's herbal [ 63] from the year 1596

In folk medicine particular use is made of the seed. But we also come across the utilization of the shoots for antibiotic and repellent purposes. Around cabbage plants, cannabis plants are grown to repel pieris (caterpillars), and twigs of cannabis are hung in bedrooms to repel gnats and flies.

During the Middle Ages cannabis decoctions were given to cattle for diarrhoea [ 63] , [ 85] . In Argentina cannabis is considered a real panacea for tetanus, melancholia, colic, gastralgia, swelling of the liver, gonorrhoea, sterility, impotency, abortion, tuberculosis of the lungs and asthma. In Argentina [ 58] even the root-bark has been collected in spring, and employed as a febrifuge, tonic, for treatment of dysentery and gastralgia, either pulverized or in form of decoctions. The root when ground and applied to burns is said to relieve pain. Oil from the seeds has been frequently used even in treatment of cancer; we have also come across this application in European folk medicine. Also in Argentina, in folk medicine, hemp shoots extracted with butter ( Extr. Cannabis ind. pingue) are supposed to have a powerful hashish effect, it is believed already, in an amount of 0.1 g; it is employed as a remedy in the Basedow disease. The ethereal extract is less active, and in Argentina it is administered for headache, neuralgia, gout, rheumatism, chorea, melancholia, hysteria, delirium, gastralgia and anorexia. The aqueous macerated product has no narcotic effect at all, and is employed for treatment of tuberculosis of the lungs and as a hypnotic for children and to relieve spastic constipation. An infusion of the leaves is considered to possess a diuretic and a diaphoretic effect. In Europe we also come across many of these uses. Thus Graemer (cit. Dinand [ 18] ) recommends the following for treatment of gastralgia: 0.75 g Extr. Cannabis ind., 10 g ether; 10 drops daily on sugar. For rheumatism a decoction of leaves (15-20 g/0.5 1) is taken internally, and externally poultices prepared of seeds and packings of shreds or tow are used. In Brazil [ 58] hemp is considered to be a sedative, hypnotic and antiasthmatic remedy. A pronounced antibiotic effect has been observed in South America, where fresh leaves after being ground are used as a poultice for furuncles, and in folk medicine in Europe for treatment of erysipelas (Dinand [ 18] ). Even seed pulp is applied in such cases, but as there are no antibiotics in the seeds we must assume that there is another therapeutic factor involved. In the popular treatment of headache, the plant is preserved in vinegar together with juniper, and the extract is used in form of compresses. Githens [ 28] and also Watt & Breyer-Brandwijk [ 89] report on the utilization of cannabis (dagga) in South Africa. There it is smoked because of its narcotic action, but it is also used medicinally. Next to the effect upon the central nervous system we find a considerable use as an antibiotic. For example, the Xosa tribe employs it for treatment of inflammation of the feet. In Southern Rhodesia it is a remedy for anthrax, sepsis, dysentery, malaria and for tropical quinine-malarial haemoglobinuria. The Suto tribe fumigates the parturient woman to relieve pain. These analgetic, 3 sedative and antibiotic properties of cannabis in internal and external application are well known to African tribes.

It may be concluded that ancient and folk medicine have utilized cannabis as an antibiotic and analgesic externally and later as a sedative internally. Narcotic effect has been observed in hot climates only.

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 [ 1] , [ 11] , [ 42] , [ 45] of the substances contained in the plant, Cannabis sativa. The following substances have been isolated: cannabinol [ 5] , [ 12] , [ 15] , [ 93] , [ 94] , cannabidiol [ 5] , [ 12] , [ 38] , tetrahydrocannabinol [ 92] , quebrachitol ( l-inositolmonomethylether) [ 5] , p-cymen [ 79] , 1-methyl-4-isopro-pylbenzen [ 79] , the so-called cannabol [ 37] 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 [ 90] . The quantitative changes of carotene found present in Cannabis sativa have been investigated by Lebedev [ 54] .

3. It is perhaps also of interest that cannabis greatly increases the hypnotic effect of barbiturates.

Moreover there exists a series of communications [ 10] , [ 15] , [ 26b] , [ 31b] , [ 57b] , [ 65b] 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. [ 57b] . 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.

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 [ 1] , [ 11] , [ 42] , [ 45] .

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 [ 46] , [ 47] found that there are substances contained in it which show an antibiotic effect upon some micro-organisms.

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 [ 71] . 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 [ 60] , [ 61] ) 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.

Full size image: 108 kB, FIGURE 3, Cannabis sativa var. indica - plant shootsA - Flowering shoots of the pistillate plant (rich in resins).B - Flowering shoots of the staminate plant.

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 4 with an aqueous NaHCO ₃ solution with which the acids, particularly the cannabidiolic acid, were extracted to yield a slightly yellow product. By shaking with NaHCO ₃ 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.

TABLE 1

Output and biological activity of the single fractions isolated from cannabis

	Output a (grammes)	Biological activity
Petrol ether fraction .	59.0	+++
Ether fraction	0.6	+
Chloroform fraction	0.2	-
Ethanol-chloroform fraction	0.05	-
Aqueous residue	6.3	-
Phenolic constituent		+++
Acetylated phenolic constituent		++
Acid constituent (cannabidiolic acid)		++++
Acetylated cannabidiolic acid		+++
Acetylated and hydrogenated cannabidiolic acid		++++
Cannabidiol		+++
Trans-cinnamic acid		-

^00p000

^a 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 C ₂₂H ₃₀O ₄'[α] _D ²⁴-95° ± 8° (c = 1.00 in chloroform); [α] _D ²⁴-115° ± 4° (c = 1.05 in ethanol). The substance crystallized readily after acetylation, m.p. 80-100°/127-128°, 5 [α] _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 [ 77] .

From some of the crops the acetylester of the acid, m.p. 95-100/ 110-115° 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.

$Full size image: 29 kB, FIGURE 4, Rate of inhibition of the acid and phenolic fraction of the extract from cannabis upon the inoculated staphylococcus (meat peptone broth)1 - Acetylated fraction from which the cannabi[...]$

Determination of the structure

As previously mentioned, the biologically active acid isolated from Cannabis sativa has the empirical formula C ₂₂H ₃₀O _4, and yields, when acetylated, a diacetyl derivative C ₂₆H ₃₄O ₆ 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 [ 49] , [ 59] .

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 [ 49] 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 [ 51] , [ 52] .

On comparing the optical rotation of the cannabidiolic acid, its acetyl derivative and its tetrahydro derivative with the cannabidiol [ 2] , [ 3] derivatives we found a remarkable correspondence. An analogous agreement exists [ 4] 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.

TABLE 2

Values of the optical rotation of the single substances (in ethanol) (Degrees)


Substance	[L]D	D
Cannabidiolic acid and its derivatives
Cannabidiolic acid	-115	-412
Diacetyl derivative	-114	-504
Tetrahydrodiacetyl derivative	-22	-98
Cannabidiol and its derivatives .
Cannabidiol (2)	-125	-393
3,5-dinitrobenzoate (2)	-76 *	-513
Dimethylether (2)	-133	-455
Monomethylether (2)	-118	-373
Tetrahydrodimethylether (3)	-28	89
Tetrahydrodiacetyl derivative (3)	-30	-104
Tetrahydrocannabinol and its derivatives (l b, 4)
Isomer I	-267	-834
Isomer II	-130	-406
Hexahydrocannabinol	-70	-220

* Acetone

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 [ 94] , 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). 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).

6. 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.

TABLE 3

IR-frequency values within the carbonyl range


	Concentrated Solution	Diluted solution	Dioxan
	In chloroform
Acetylester of the cannabi-diolic acid	1698 cm-1	1700 cm-1	1728 cm-1
Hydrogenated acetylester of the cannabidiolic acid	1698 cm-1	1700 cm-1	1728 cm-1

FIGURE 5, UV-spectra of the cannabidiol derivatives (in ethanol). A - Acetylester of the cannabidiolic acid. B - Hydrogenated acetylester of the cannabidiolic acid. For comparison the curves of the dimethylether of the cannabidiol (C) and of the dimethylether of the dihydrocannabidiol (D) have been taken from the literature [ 6] .

Full size image: 26 kB, FIGURE 5, UV-spectra of the cannabidiol derivatives (in ethanol)

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.

Full size image: 40 kB, 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-1belongs to the aromatic vibrations and the frequencies at 900 and 1650 cm-1which 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 [ 7] which has been found present in numerous plants [ 8] . 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 (FeC1 _3,CHC1 ₃+ 10% KOH, Vanillin + HCl) to be found in both groups of these substances.

Independently from us (1955) Schulz & Haffner [ 77] (1958) isolated and described the above-mentioned cannabidiolic acic. The determination of the constitution was carried out [ 77b] (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 [ 77c] 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.

Methods and results of the bacteriological experiments

By Z. Krejci

I. - PRELIMINARY BACTERIOLOGICAL EXPERIMENTS WITH EXTRACTS FROM Cannabis sat. var. indica

Preparation of extract for the preliminary microbiological evaluation

It was clear from the beginning of our experimental work that the antibiotic effect of the drug must stand in close relationship with the content of the resin substances contained in the leaves and, particularly, in the shoots of the pistillate plant (fig. 3). The ground drug was extracted by the method devised by Carlson (13, 14). The different parts of the plant (roots, stems, leaves, shoots and seeds) were extracted at higher temperature, normal room temperature, and even at 1°C, at the ratio of 1:5, with ethylalcohol, ethylether, a physiological solution, 1.5% sulphuric acid, and 1% sodium hydrocarbonate. The extraction was carried out to advantage with organic solutions, particularly with petrolether, benzene and ethylalcohol.

FIGURE 7, Rate of the inhibitory effect of the raw extract from cannabis upon Staphylococcus aureus (modified Oxford method). One drop of the extract has been used for carrying out the test. I - Extract in 70 per cent ethyl alcohol . II - Extract in 96 per cent ethyl alcohol. III - Diethyl ether extract. IV - Control test with 96 per cent ethyl alcohol .

Full size image: 28 kB, FIGURE 7, Rate of the inhibitory effect of the raw extract from cannabis upon Staphylococcus aureus (modified Oxford method)

The bacteriological technique

The extracts prepared in various ways and by using various kinds of solvents were tested for the presence of antibacterially effective substances by the help of the modified Oxford method. On an Agar culture medium contaminated with the examined microbe, a filter paper 10 mm in diameter, saturated with the antibiotic, was applied. The inhibition zone around the butts found present after 24 hours of incubation was used for the evaluation of the effectiveness of the substance. In order to ascertain the maintenance of a given set of conditions, particularly the same amount of microbes, most of the tests were carried out in bacteriological culture mediums with inoculated microbes (fig. 7).

The other and more detailed investigations (the rapidity of the effect, the inhibition factors and so on) were carried out routinely in the fluid medium of peptone water or, with the aid of Sauton culture mediums (BK), respectively.

FIGURE 8 Chromatography illustrating the inhibitory effect upon the growth of the staphylococcus around the antibiotically active zone; undimensional ascending paper chromatography. b shows two zones running into each other (cf. details in the text).

Full size image: 118 kB, FIGURE 8 Chromatography illustrating the inhibitory effect upon the growth of the staphylococcus around the antibiotically active zone; undimensional ascending paper chromatography

The preliminary experiments concerning the isolation of biologically active substances were performed by the help of the ascending chromatography or the capillary analysis. The antibacterially effective fractions were detected by placing the chromatograms into inoculated culture mediums. The zone which inhibited the growth of the microbes was considered to be biologically active (fig. 8a, b).

An analogous method based on the capillary analysis and used for the isolation of effective substances is illustrated in fig. 9. The terminally situated zone in the ring chromatogram when placed into an Agar medium inoculated by staphylococcus produces a considerable inhibitory effect upon the growth of the above-mentioned micro-organisms. Further work showed that the' chlorophyll contained in the plant extract does not produce any direct antibiotic effect [ 66] , [ 84] .

Spectrum of bacteria sensitive to active substances obtained from cannabis

The investigations were carried out with specimens of gram-positive micro-organisms - i.e., Staphylococcus pyogenes aureus - and from the gram-negative series the Escheria coli were used. The extracts produced a remarkable bactericide effect upon Staphylococcus aureus, whilst E. coli showed to be resistant. On the basis of these preliminary findings we have aimed our work in this direction. Proof could be furnished that the cannabis extracts produce a very satisfactory antibacterial effect upon the following microbes: Staphylococcus pyogenes aureus, Streptococcus alpha haemolyticus, Streptococcus beta haemolyticus, Enterococcus, Diplococcus pneumoniae, B. subtilis, B. anthracis, Corynebacterium diphtheriae and Corynebacterium cutis - i.e., all of them gram-positive micro-organism. Note-worthy is the effect upon Staphylococcus aureus strains, which are resistant to penicillin and to other antibiotics (fig. 10).

That was one of the peculiar properties of cannabis which was found to be most attractive. We saw the possibility of utilizing the antibiotic locally without any danger of producing resistant strains to other antibiotics administered at the same time throughout treatment. We must also remember the very good effect of substances from cannabis upon Staphylococcus aureus, particularly nowadays, when a high percentage of staphylococcus diseases offers resistance to penicillin. It is interesting that staphylococci manifesting various degrees of resistance to one or more antibiotics (erythromycin included) are sensitive to the antibiotics from cannabis in the same degree throughout. So far, we have not observed that any resistance of the staphylococcus strains to these substances would arise. The degree of the artificially produced resistance to these substances, as shown by Martinec [ 62] , corresponds with the origin and the degree of resistance to substances of the phenolic type and to other disinfectants.

FIGURE 9 Inhibitory effect upon staphylococcus when placing the circular paper chromatotogram - capillary analysis - into inoculated Agar. The light centre and the spots around the periphery of the Agar plate indicate the boundary of the inhibitory zone produced by the active substance. It appears that there are two biologically active substances present whose partition factors show to be different; evidence in this respect could be brought forward chemically.

Full size image: 29 kB, FIGURE 9 Inhibitory effect upon staphylococcus when placing the circular paper chromatotogram - capillary analysis - into inoculated Agar

FIGURE 10 A parallel drawn between the rate of the inhibitory effect of the extract from Cannabis indica and of penicillin upon penicillin resistant strains of Staphylococcus aureus. I - 2% acidum carbolicum liq. II - 1,000 OU of penicillin. III - Hemp extract in 70% ethyl alcohol. IV - Hemp extract in 96% ethyl alcohol.

Full size image: 51 kB, FIGURE 10 A parallel drawn between the rate of the inhibitory effect of the extract from Cannabis indica and of penicillin upon penicillin resistant strains of Staphylococcus aureus

FIGURE 11 Preliminary titration of the IRC-effect upon Staphylococcus aureus (Oxford method). Concentrations: 2%, 1%, ?%, 1 : 1,000, 1 : 10,000, 1 : 100,000

Full size image: 27 kB, FIGURE 11 Preliminary titration of the IRC-effect upon Staphylococcus aureus (Oxford method). Concentrations: 2%, 1%, ?%, 1 : 1,000, 1 : 10,000, 1 : 100,000

The tests carried out with other micro-organisms, particularly with gram-negative strains, just as well as with fungi and yeast, gave negative results (table 4). On the contrary our work evidenced the positive effect of the isolated substances upon Mycobacterium tuberculosis.

TABLE 4

Effect of the Cannabis indica resin upon some common pathogene micro-organisms


Investigated strain	Effect
1. Micrococcus albus	Positive
2. Staphylococcus pyogen. aureus haemolyt. sensitive to penicillin	Positive
3. Staphylococcus pyogen. aureus haemolyt. resistant to penicillin	Positive
4. Streptococcus alpha haemolyt.	Positive
5. Streptococcus beta haemolyt.	Positive
6. Enterococcus	Positive
7. Diplococcus pneumoniae	Positive
8. Erysipelothrix rhusiopath	Positive
9. Sarcina lutea	Positive
10. Corynebact. diphtheriae	Positive
11. Corynebact. Cutis	Positive
12. Bac. anthracis	Positive
13. Bac. Subtilis	Positive
14. Bac. mesentericus	Positive
15. Clostridium perfringens	Positive
16. Escherichia coli	Negative
17. Salmonella typhi	Negative
18. S. paratyphi B	Negative
19. Sh. Shigae (Sh. Flexneri, Sh. Kruse Sonnei)	Negative
20. Pseudomonas aeruginosa	Negative
21. Proteus vulgaris	Negative
22. Mycobacterium tuberculosis	Positive

II. - BACTERIOLOGICAL EXPERIMENTS CARRIED OUT WITH ISOLATED AND PURIFIED RESIN FROM CANNABIS

As previously mentioned in the section on chemistry, both the perfect extraction of the effective substance and the method of isolation of the resins have been elaborated and patented [ 53] . Whilst the preliminary investigations were carried out with hemp extracts obtained by the help of some organic solvents (ethylalcohol, ethylether, petrolether, benzene), the proper 'work and study of the antibacterial effect were accomplished throughout by the help of the purified substance of resin nature, further on designated as the IRC (Isolated Resin from Cannabis), from the viewpoint of chemistry a mixture of acid and phenolic fractions; hence, we have used not the crystalline forms which have been shown to be less effective but the mixture mentioned above - the resin. The crystalline forms have been found particularly useful for the evaluation of the chemical structure, the determination of the chemical and physical properties and the constitution of the effective substances. Consequently, all the results mentioned below will refer to the IRC, tested upon Staphylococcus aureus haemolyticus.

TABLE 5

Velocity of the bactericide effect of various IRC-concentrations upon the investigated micro-organisms

IRC-concentration	Velocity of the effect when adding antibiotics
1 : 100	Immediately sterile
1 : 1,000	Sterile after 15 to 30 minutes
1 : 10,000	Sterile after 3 hours
1 : 100,000	Sterile after 8 hours
1 : 1,000,000	After 24 hours remarkably inhibited growth
Control without IRC	Massive growth and opacity

Determination of the magnitude of the bactericide effect

It could be demonstrated by us that the IRS produces a pronounced bactericide effect in vitro; in lower concentrations the growth and reproduction, of sensitive bacteria are but slowed down. The rate of the effect in vitro has been tested both by the help of the modified Oxford method on solid culture mediums and in fluid mediums of peptone water. The Oxford method was shown to be by far the least sensitive. There were shown to be many factors involved which have no connexion with the antibiosis as, for example, diffusion, the number of inoculated microbes and so forth. It can be utilized with advantage only for purposes of orientation (fig. 11). Whilst the maximum of the effect of the IRC-dilution was found to be with a concentration of about 1:10,000 when the Oxford method was used, the tests carried out in the fluid medium of peptone water evidenced a bactericide effect still in a dilution of 1: 100,000 after inoculation within 24 hours, and a significant inhibition of the growth in a dilution of 1: 1,000,000.

FIGURE 12 A tridimensional graphic representation of the velocity of the bactericide effect of various IRC-concentrations upon staphylococcus aureus in fluid media in vitro

Full size image: 41 kB, FIGURE 12 A tridimensional graphic representation of the velocity of the bactericide effect of various IRC-concentrations upon staphylococcus aureus in fluid media in vitro

The antibacterial magnitude of the IRC-effect in ethylenglycol upon the BK H/37 RV strain in a Sauton fluid medium was found by titration up to a dilution of 1: 100,000. In detailed work carried out in the Laboratory for Bacteriology of the Sanatorium at Vyšné Hágy (High Tatra) where independent tests for control were carried out, the limit of the effect upon the same strain of Mycobacterium tuberculosis was determined to be present up to a dilution of 1: 150,000.

FIGURE 13 Graphic representation of the decrease of the number of micro-organisms after various time-intervals and in different IRC-concentrations

Full size image: 26 kB, FIGURE 13 Graphic representation of the decrease of the number of micro-organisms after various time-intervals and in different IRC-concentrations

TABLE 6

Velocity of the bactericide IRC-effect upon Staphylococcus aureus

	Number of colonies
Time control	IRC 1:100	IRC 1:1,000	IRC 1:10,000	IRC 1:100,000	Control
Immediately after application of antibiotics	-	5	8,640	20,600	25,000
After 15 minutes	- -	3	560	20,500
After 30 minutes	-	-	37	16,000
After 45 minutes	- -	-	-	20,300	Proportional in- crease of the number of the micro- organisms
After 1 hour	-	-	29	18,800
After 2 hours	-	-	12	1,760
After 3 hours	-	-	14	278
After 4 hours	-	-	-	240
After 5 hours	-	-	-	32
After 6 hours	-	-	-	21
After 8 hours	-	-	-	4
After 12 hours	-	-	-	-
After 24 hours	-	-	-	-	Massive growth

Determination of the velocity of the bactericide effect of the various IRC-concentrations upon test

The velocity of the bactericide effect was determined with the aid of the routinely used method in the medium of pep-tone water infected with staphylococcus (100,000,000 bacteria in 5 ml). We recorded the time within which the infected peptone water became sterile due to the appropriate IRC concentration. The results obtained were shown to be uniform (table 5, and diagram in fig. 12).

FIGURE 14 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in solid culture media

Full size image: 48 kB, FIGURE 14 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in solid culture media

Resin isolated from Cannabis indica	Agar	Agar plasma	Blood-Agar
10/ ₀₀ aqueous solution	20	15	11
1% alcohol solution	26	16	14
1% aqueous solution	27	19	17

FIGURE 15 Effect of the inactivating substances in vitro upon the decrease of the antibacterial IRC-effect in: 1 - Meat-peptone Agar. 2 - Agar with 10% plasma.

Full size image: 39 kB, FIGURE 15 Effect of the inactivating substances in vitro upon the decrease of the antibacterial IRC-effect in:1 - Meat-peptone Agar.2 - Agar with 10% plasma.

FIGURE 16 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in fluid media; evaluated after a period of 24 hours

Full size image: 44 kB, FIGURE 16 Graphic representation of the influence of the inactivating substances in vitro upon the decrease of the IRC-effect in fluid media; evaluated after a period of 24 hours

FIGURE 17 Graphic representation of the pH-effect upon the activity of streptomycin (Stoll) and of the IRC when staphylococcus species are concerned

Full size image: 31 kB, FIGURE 17 Graphic representation of the pH-effect upon the activity of streptomycin (Stoll) and of the IRC when staphylococcus species are concerned

Analogously we followed the decreasing number of bacteria. Fifty cc of sterile peptone water were infected with a diluted 18-hour-old Staphylococcus aureus culture which, according to the control, contained 30,000 micro-organisms in 1 cc. Into each of the individual flasks the IRC was added, progressively diluted from 1:100 up to 1:100,000 and, with the aid of the usual method of cultivation, and after definite time intervals, the decreasing number of bacteria in 1 cc was recorded (table 6, and diagram in fig. 13).

FIGURE 18 Graphic representation of the pH-effect upon the activity of penicillin (Stoll) and upon the acetyl derivative of the acid from cannabis

Full size image: 14 kB, FIGURE 18 Graphic representation of the pH-effect upon the activity of penicillin (Stoll) and upon the acetyl derivative of the acid from cannabis

Substances inactivating the antibacterial effect of the IRC

A detailed study has been carried out in vitro on solid and in fluid culture mediums with regard to the effect of blood, serum, cysteine, thiosulfate, meat peptone broth and Sauton medium upon the bactericide magnitude of the IRC. All the substances mentioned above, save for cysteine and thiosulfate, showed a decrease in the biological effect to a larger or smaller extent (fig. 14, 15). Whilst in the standard peptone water there could be observed a bactericide effect upon the staphylococcus up to a concentration of 1:100,000, under the same conditions after addition of an amount of 10 per cent blood or plasma the concentration showed itself active only up to a dilution of 1:1,000. It is interesting to notice that bloods of various origin, and even of different groups, do not produce the same inactivating effect. In the same way, in comparison with the medium of peptone water, the peptone broth showed a decrease in the bactericide effect by one degree (fig. 16).

Effect of the pH-value upon the antibacterial effect of the IRC

Stoll et al (83) published a report dealing with the problem of the dependence of the magnitude of the antibacterial effect upon the pH-value with regard to penicillin and streptomycin. They found that at lower pH-values the anti-biotics of acid character produce a more pronounced effect than at the pH-value within the alkaline range (penicillin). On the contrary, the effectiveness of streptomycin rises steeply with the rising pH. In our experiments the same results were obtained. We followed on solid culture mediums, and at pH-values that varied between 5 and 8, the rate of the inhibiting effect upon Staphylococcus aureus. The various concentrations tested were as follows: ( a) natrium salts of the IRC in alkaline aqueous solutions, ( b) the crystalline acetyl derivative of the isolated cannabidiolic acid.

FIGURE 19 pH-effect upon the IRC-action upon Staphylococcus aureus in meat-peptone Agar a - 1 per cent IRC (sodium-salt) in alcohol (5 drops placed into a depression in the Agar-surface). b - 1 per cent IRC (sodium-salt) in water (5 drops placed into a depression in the Agar-surface). (one drop on filter paper applied to the Agar-surface). c - 1 per cent IRC (sodium-salt).

Full size image: 43 kB, FIGURE 19 pH-effect upon the IRC-action upon Staphylococcus aureus in meat-peptone Agar a - 1 per cent IRC (sodium-salt) in alcohol (5 drops placed into a depression in the Agar-surface)

In the first case the increasing effectiveness of the solution along with the rising pH-values bore analogy to the one of streptomycin (fig. 17).

On the contrary, in the second case, the effectiveness of the acid tested showed an increase on the acid side, which was analogous with the effect of the pH-value upon the antibiotic effect of penicillin (fig. 18).

As may be seen from both diagrams, there is a noticeable difference to be found with regard to the size of the inhibition zones under standard conditions and varying pH. The maximum of sodium salts in water lies at about pH 7.5, subsequently approximately at the pH of the plasma. Less advantageous is the considerable decrease of the effect in an acid medium - for example, of a suppurant wound. The photograph of fig. 19 portrays the considerable disparity as to the effectiveness of the isolated substances at varying pH under conditions which are otherwise maintained. The same concentrations of active substances have been tested in parallel line in an Agar culture medium once at pH 5.5, and the second time at pH 7.5.

On the basis of the dependence mentioned above, conclusions should be drawn as to the practical utilization of the preparations with antibacterial substances from cannabis at varying pH of some pathological processes as, for example, in suppurant wounds, furuncles, otitis, sinusitis and the like.

Survey of clinical experiences By Z. Krejci

Some chemical and physical properties of the isolated effective substances (IRC) particularly with regard to their stability

The antibiotically active and effective substance (IRC) of resinous appearance and nature, utilized at present for the preparation of the antibacterial preparations, does not appear to be a chemical entity, but a mixture of two substances. It is of acid nature conditioned by the presence of phenolic and carboxyl substances. These acid substances retain their antibacterial properties even after acetylation and following division in the neutral portion (acetylated phenols) and the acid one (acetylated-phenol-acid).

Going deeper into the matter showed that the biological effectiveness of the active amorphous substances is not so stable as was previously assumed. On the contrary, their effectiveness decreases due to the oxidizing action of atmospheric oxygen, particularly in a warm atmosphere. In an oxygen-free medium, even at higher temperatures (in the autoclave) they are comparatively stable.

The effective substances are all readily soluble in common organic solvents - namely, in ethylalcohol, ethylether, chloroform, benzene, petrolether and so on. At pH 7 they are slightly soluble in water and in physiological solutions. This property is a certain drawback in these substances both for clinical utilization and precise pharmacological evaluation. Because of their acid nature they are readily soluble in aqueous solu-tions of alkalis - i.e., in sodium and potassium hydroxide, sodium carbonate, sodium hydrocarbonate and so on. They can be easily emulsified, and as emulsions they are comparatively stable.

The pharmacodynamic effect of the antibacterial substances from Cannabis sativa var. indica

The pharmacological evaluation was carried out at the Institute for Pharmacology of the Medical Faculty of the Palacky University. Klabusay & Lenfeld [ 43] succeeded in showing some well-defined effects of the isolated amorphous substances (IRC): analgetic, anticonvulsant, and, when locally applied, the initially irritative and later on anaesthetic effects.

The hashish effect due to tetrahydrocannabinol could not be found in substances obtained from plants cultivated in the moderate climate of Czechoslovakia - i.e., it could be evidenced neither in animal experiments nor in experiments carried out on man ( per os).

The analgetic action was studied in white rats by the help of the method of mechanical irritation according to Levy and Buchel in the modification according to Votava and in guinea pigs by the method according to Regnier. The IRC was administered perorally in emulsions with gum arabic in doses from 100 to 50 mg/kg; and also subcutaneously and intraperitoneally in ethylenglycol up to 500 mg/kg weight. From these experiments it could be seen that the IRC produces an analgetic action when administered perorally in doses of 100 mg, subcutaneously and intraperitoneally - i.e., from 50 mg/kg onward. Higher doses (500 mg/kg) of the IRC result in intensified analgesia lasting for some hours and complete inhibition of the activity of the central nervous system or sleep.

The anticonvulsive effect was evaluated in white mice and in frogs. The convulsions were produced in mice by pentamethylentetramine (0.1 g/kg intraperitoneally), in frogs by strychnine by administering doses of 40 mg/kg subcutaneously. The effective dose of the IRC preventing convulsions due to pentazole in 50% of mice was established as 0.61 g/kg per os; for strychnine an effective dose of 0.83 g/kg when applied subcutaneously.

The determination of the local anaesthetic effect was carried out by evaluation of the surface anaesthesia according to the method of Lebduška & Vrba using the cornea of rabbits and guinea pigs, and by the evaluation of the anaesthesia according to the method of infiltration of Bulbringe & Wayda, using the skin of guinea pigs. The IRC was applied in ethylenglycol. The IRC was effective from the concentration of 5% and up, whereas a total local anaesthetic effect could be observed only by using a 10% concentration; it lasted for about 45 to 90 minutes.

The toxicity was determined in mice by administering an emulsion with gum arabic, and the death rate recorded within 48 hours. By the Burn method, LD ₅₀ was established as 1.83 g/kg per os. Smaller doses did not produce any remarkable changes in mice; doses of 1 g/kg (IRC) were followed, after some hours, by a slight inhibition of the motor function with apathy and immobility. Larger doses resulted in a total inhibition of the activity of the central nervous system, sleep and exitus within 24 hours.

The local compatibility was evaluated on the cornea of rabbits by administering a solution in drops and by application of the solutions of the tested substances into the subcutaneous area of the auricle of the rabbit. The effect of the IRC upon the mucous membranes was locally irritating. On local application to the skin no clinical evidence of acute irritation was found. The intramuscular application resulted in an inflammatory reaction with the exudation of the liquid.

Clinical experiences

In recent years a series of pharmaceutical preparations have been prepared and clinically examined particularly with regard to the needs of stomatology, otorhinolaryngology, gynaecology, dermatology and the like. The preparations have been locally applied throughout but parenteral administration has not been made use of, particularly because of the poor solubility of the examined substances in physiological solutions. Besides, some of the properties such as the pharmacological one (irritation) and the bacteriological one (inactivation with blood and serum) of the active substances which have been isolated do not allow a suitable utilization of these preparations by means of injections.

For the needs of stomatology, an aseptic dentin powder was prepared, impregnated with 2 to 5% of biologically active substances (IRC) from cannabis. This dentin powder containing IRC was successfully used for the indirect covering (in about 300 patients) and for the direct covering of the pulp (70 cases), in beginning pulpitis and irritation of the pulp. Even in cases like that we notice the obvious advantage of the antibacterial effect of the locally anaesthetic action. Save in cases of massively infected pulps, Soldan [ 81] , [ 82] did not record any failures.

Somewhat less uniform results were obtained with this preparation by Šimek [ 78] , who applied dentin combined with IRC or a special salve containing IRC. In uncomplicated Caries profunda, a remarkable and spontaneous effect could be achieved in 64% of the patients; in Caries profunda, with an incidental opening of the vital pulp, in 38.4% of the patients. In Pulpitis partialis, the results of this therapy were positive in 41% of the investigated cases. The authors have, on the whole, acknowledged the anaesthetic effect of these preparations.

Very advantageous is the application of the effective substances from cannabis in otorhinolaryngology. Hubacek [ 36] reports on the very good results obtained particularly after application of 1% of an alcoholic solution of the IRC and of a dusting powder (boric acid with 2 to 5% of the IRC) in acute and chronic otitis, furunculosis of the outer part of the nostrils and of the external auditory meatus, and even in some cases of bilateral sinusitis, the latter having been treated without success with a series of punctions and penicillin irrigations. He describes an interesting case of bilateral chronic Sinusitis maxillaris, lasting for about three years and treated without success with a total of about 30 punctions. After application of our IRC preparations, one side was treated as a control with penicillin, but with negative result, and the other side with three IRC punctions and irrigations. The side treated with negative effect with penicillin was easily and successfully treated with cannabis preparations. In the same way Navratil [ 65] illustrates in his small table of chronic otitis a considerable improvement in 13 of a total of 18 cases. All the cases under investigation were also controlled bacteriologically. In clinical practice the very good results achieved with the preparations mentioned above-i.e., with the alcoholic solution and the dusting powder-stand in support of the good results obtained in vitro and justify the suggestion to have both these preparations made available for pharmaceutical production.

The manifold utilization of these substances particularly in the form of alcoholic solutions with glycerine has been confirmed by the results obtained after application of this preparation in obstetrics in order to prevent staphylococcic mastitis in the treatment of rhagades and fissures on the nipples of nursing women. Of great importance is the locally anaesthetic component together with the antibacterial effect, particularly upon Staphylococcus aureus, which is considered to be the causing agent of the postpartum inflammation of the mamma. Heczko & Krejci [ 33] evidenced staphylococcus in 84% of the total of 160 parturient women. It is significant that in 89% of cases the staphylococcus was resistant to penicillin, in 18% to streptomycin, and in some cases resistant to penicillin, streptomycin, aureomycin and terramycin and sensitive only to chloromycetin and to the IRC.

The publication of the results obtained from these investigations evoked a lively interest among obstetricians who up to the present have tried to find means of substitution both for the inadequate and still used gentian violet, and the local inefficacious application of some antibiotics as, for example, aureomycin, which has been recommended by some authors.

FIGURE 20 Comparison of the inhibitory effect of some antibiotics in combination with active substances from cannabis upon Staphylococcus aureus P - penicillin 100 OU; S - streptomycin 1 mg; T - terramycin 5 mg; A - aureomycin 5 mg; CH - chloramphenicol 5 mg; Ci - tincture cannabis Zone in the centre of the plate - 2 per cent of IRC.

Full size image: 44 kB, FIGURE 20 Comparison of the inhibitory effect of some antibiotics in combination with active substances from cannabis upon Staphylococcus aureus P - penicillin 100 OU; S - streptomycin 1 mg; T [...]

For the purpose of dermatology, a salve containing 2% of effective substances has been prepared and found very useful in pyodermia of staphylococcic etiology, infected burns, and particularly in decubitus of immobile patients in rehabilitation centres. Of great interest was the follow-up of a physician and pathologist, who was treated with the IRC for a severe infection of the thumb of the right hand, an injury he suffered in the dissecting room. The severe condition, threatening amputation, and the absolute resistance of the microflora to available antibiotics were overcome by substances from cannabis.

The results obtained in treatment of specific tuberculous fistulas and published by Procek [ 70] as well as the up to now unpublished results of the investigations carried out at Vyšné Hágy must be considered as preliminary ones even though the antituberculosis effect in vitro is very satisfactory.

In the cases mentioned above, no extraordinary allergy of the organism to effective substances from cannabis or any particular ability of the IRC to sensitize were observed.

In conclusion we may still consider the interesting results Širek [ 80] achieved. He mentions his experiences for some years past with hemp seeds in the therapy of tuberculosis. The hemp seed was ground and extracted with milk at a temperature of 60-80°C. It represented a protein-rich moiety of the curing diet for tuberculous children, in whom a remarkable improvement due to this diet was observed by the author. But the assumed antituberculotic action of cannabis seeds could not be evidenced in experiments on guinea pigs. Therefore, in the cases reported by Širek, the therapeutic effect can be only explained by the healing nutritious diet in which the specially prepared seed is of great importance. 8

In human therapy the best results have been obtained with the following medicaments combined with substances derived from cannabis: dusting powder together with boric acid (otitis), ointment (staphylococcus infected wounds, staphylodermia and so on), ear drops (otitis chron.), alcohol solutions with glycerine (treatment of rhagades on the nipples of nursing women - prevention of staphylococcic mastitis), aqueous emulsions (sinusitis), dentin powder with the IRC (caries). The preparations mentioned above have been already tested clinically, and will eventually be made available for production.

The high yield of effective substances (average 1.5 % of the IRC in the dried drug), the considerable amount of raw material available (1.5 million hectares of cannabis plant culture in the world), and the subsequent low price both of the raw material and of the effective substances allow us to utilize the antibacterially effective substances from cannabis in veterinary medicine. Such use, which is analogical to human medicine, is comparatively wide: infected wounds (dusting powder), panaris of sheep and cattle (dusting powder), otitis in dogs (ear drops, dusting powder), mastitis in cattle, postpartum trauma, lacerated wounds and so on. Especially veterinary workers, workers concerned with cattle breeding, employees in slaughter houses, cutters and so on consider the preparations containing biologically active substances to be an important contribution to the prevenion of tuberculosis, anthrax, swine erysipelas and brucellosis in various streptococcic and staphylococcic infections of the hands and the like. The results achieved in this field, but unpublished as yet, justify the greatest optimism.

8. A considerably higher resistance against virus pneumonia has been observed in mice fed with hemp seeds.

In view of the results obtained with antibacterially effective substances isolated from cannabis, it may be concluded that they are superior and have numerous advantages as antibiotics.

The results obtained in clinical practice must receive, for the most part, a positive evaluation. In many cases satisfactory and in some cases even surprisingly good results have been obtained, particularly where a microflora resistant to a series of other antibiotics was concerned. Naturally drawbacks had to be recorded. Further clinical experiences and practice will be necessary for a definite evaluation of this antibacterially effective substance.

Summary

A systematic investigation of the mid-European flora has been carried out in order to establish the presence of antibacterially effective substances. A high content of remarkably active substances has been found present in Cannabis sativa var. indica and in Cannabis sativa, a plant cultivated for the industrial utilization of the hemp fibre. It has been investigated in detail from the bacteriological and the chemical viewpoint and from the standpoint of the eventual applicability of the effective substances in clinical practice.

In old herbals and in folk medicine in Europe, Africa and America, mention has been made of the utilization of cannabis as an antibiotic and analgetic medicament particularly for external use. The hashish effect has been known only to nations with a warm climate. In Europe, hashish made its appearance as a narcotic at the time of Napoleon's campaign in Egypt, but contrary to morphine and cocaine, its use was not widespread. In spite of this, from the pharmacological viewpoint, official medicine has investigated the hashish effect on the central nervous system whilst the analgetical and antiseptic effects have been unjustifiably forgotten.

In the chemical section of this work, the most suitable methods of extraction have been described and the technique for the isolation of effective substances developed. At the beginning of the procedure two antibacterially effective fractions were obtained: the acid and the phenolic. Then from the acid fraction a biologically active crystalline acid could be isolated. On the basis of the elementary analysis, the optical rotation, the UV- and the IR-spectroscopy, the following constitution has been established: 3-methyl-6-isopropenyl-4?n-pentyl-2?-6?-dihydroxy-1,2,3,6-tetrahydrodiphenyl-3?-carboxylic acid. Because of its close relationship to cannabidiol, it has been named cannabidiolic acid. The second-antibacterially ineffective acid has been identified as trans-cinnamic acid. In addition, the following unsaponified constituents have been isolated: paraffin (n-nonacosane) and higher alcohols.

Experimentally we could evidence the bactericide effect of the substances from cannabis in vitro upon the following gram-positive micro-organisms: Staphylococcus aureus haemolyt, Staphylococcus aureus-resistant to penicillin, Streptococcus alpha, Streptococcus beta haemolyticus, Pneumococcus, Enterococcus, Corynebacterium diphtheriae, Bacillus anthracis, Erysipelothrix rhusiopath. A significant antibacterial effect upon the Mycobacterium tuberculosis in vitro could be observed up to a dilution of 1:150,000.

The gram-negative micro-organisms of the coli-typhus group, as well as the Pseudomonas aeruginosa, Proteus vulg. and, in addition, the yeast and the fungi, proved to be resistant.

The limit of the effectiveness of the maximal dilution of the biologically active substances (1:100,000) and the velocity of the effect of various concentrations could be established.

The effect of the inactivating factor has been studied in detail. Blood, plasma and serum partially inactivate and decrease the antibacterial effect.

A comparison between the effectiveness of the active substances from cannabis and that of penicillin and streptomycin has been carried out at various pH.

Finally, the experiments made in clinical practice, particularly in stomatology, otorhinolaryngology, gynecology, dermatology, phthisiology, with some pharmaceutical preparations containing antibacterial substances from cannabis have been reported. Attention has been drawn to the advantageous utilization of the active substances from cannabis in veterinary medicine, and particularly in preventive medicine for anthropozoonoses.

^00p000

^a Values referred to 1 kg of dried material.

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Only those works referred to in our paper are listed, for at that time the Bulletin on Narcotics was not available to us. In the Bulletin there are two reports to be found (Chopra, J. C.: vol. 9, No. 1, 5-32 (1957), and Ahmed Benabud: vol. 9, No. 4, 1-17 (1957)) which, in discussing the history of the utilization of cannabis in popular medicine in India and Morocco, refer to the analgesic and antibacterial effects of cannabis (erysipelas, dysentery, gonorrheoa, cholera, etc.). Some other papers are particularly concerned with the analysis, the chemistry, and the narcotic effect of cannabis - viz., Duquénois, P.: vol. 2, No. 3, 30 (1950); Bouquet, J.: vol. 2, No. 4, 14-31 and vol. 3, No. 1, 22-46 (1951); Cordeiro de Farias, R.: vol. 7, No. 2, 6 (1955); Asahina, H.: vol 9, No. 4, 18 (1957); Joachimoglu, G.: vol. 11, No. 3, 5 (1959).

In vol. 3, No. 1, 62-81 and No. 2, 47-54 (1951) of the above-mentioned journal a comprehensive literature on cannabis and its utilization is given. In the bibliography of Bullettin, there have also been listed the periodical publications on cannabis up to 1958.