Chemical and biological evaluation of the resin of hemp grown for seed in the central districts of the European part of the USSR

Abstract

Details

Author: E. A. Kechatov
Pages: 5 to 9
Creation Date: 1959/01/01

Chemical and biological evaluation of the resin of hemp grown for seed in the central districts of the European part of the USSR

E. A. Kechatov Department of the technology of medicaments and galenical preparations, Piatigorsk Pharmaceutical Institute, USSR

Since remote times Russia has been a country with a highly developed hemp-growing industry. The products from the processing of hemp - hemp seed oil and fibres-were used both within the country and as important articles of trade on the international market. By the end of the nineteenth century Russia was exporting three million poods * of hemp and one million poods of oil-cake ( [ 1] ).

Under the Soviet regime, hemp growing has been further developed. The considerable extension of the crop areas, the use of modern agrotechnical methods, selection work and its widespread adoption in practice, led to the Soviet Union's occupying first place in the world for extent of crop areas and output of hemp products.

However, hemp was grown in Russia exclusively for technical purposes. The use of hemp products as narcotics was unknown, although work was carried out with a view to ascertaining whether hemp (grown for seed) could be used as a substitute for Indian hemp. ** Thus, Professor Petrov, in describing the use of hemp grown for seed in the popular medicine of Russia and other countries, proposed that it be tested in hospitals for the purpose of determining its suitability as a substitute for opium ( [ 2] ). A very interesting report was that by Zinin & Pelikan on the investigation of hemp samples collected in the Orenburg province by Captain Lukomski. The latter, in doing research work on hashish, prepared, from the tops of male and female hemp plants grown in the Kharkov province, aqueous and alcohol extracts and tested them on himself. He found that they produced a weaker effect than did similar preparations made from Indian hemp. According to Lukomski, preparations made from wild hemp growing in the Orenburg province produced a distinctly narcotic effect.

Zinin & Pelikan tested Lukomski's preparations at the Academy of Medicine and Surgery and came to the conclusion that Orenburg hemp contained a narcotic substance, the effect of which was ten times weaker than that of preparations made from oriental hashish. Moreover, in most cases, instead of a cheerful effect these preparations produced apathy, melancholy and restlessness ( [ 3] ). In subsequent works Shenfeld ( [ 4] ) and Sokolov ( [ 5] ) expressed the assumption that the resin of hemp grown for seed had the same components as the resin of Indian hemp, but its content of them was very small, and for this reason hemp grown for seed did not have a narcotic effect. However, no clear answer to the question whether or not the resinous secretions of hemp grown for seed contain narcotic substances is to be found in the literature on the subject. So far, no data were available on the composition of the resinous secretions of that hemp. This fact made it necessary for us to investigate its resinous secretions and their content of narcotic substances. As a result of our research work on varieties of hemp grown for seed in the Northern Caucasus, it was established that their resinous secretions gave positive results with cannabinol compound reagents, but at the same time they did not have the physiological effect peculiar to the resin of Indian hemp ( [ 7] ).

*

1 pood = 36 lb. avoirdupois.

**

By the expression "Indian hemp ", the author means the Indian variety of the plant and products obtained from it, such as hashish.

Experimental part

At present, the analysis of the resinous secretions of hemp, containing narcotic substances, is carried out in three ways.

First - by means of qualitative reactions, on the functional groups contained in the active substances (Hydroxyl group) and by a series of special reactions usually named after their inventors.

Second - by separating the component parts of the resin in its pure state (cannabinol, cannabidol).

Third - by means of biological evaluation on animals of preparations containing resinous secretions of hemp. The Soviet scientists Sarguin, V. V. Vasilieva, Pershin, Zverev and others have done work in this field.

As we had no opportunity of carrying out the methods of separating the component parts of the resin in its pure state, we confined ourselves in the given work to qualitative reactions and the biological evaluation of preparations containing resinous secretions of hemp grown for seed. For the purpose of research, samples were taken of the tops of female plants of the main varieties of hemp grown for seed in the central districts of the European part of the USSR - the traditional areas of hemp-growing in Russia. The samples were collected mainly in the plantations of the All-Union Scientific Research Institute for Fibre Crops at Glukhov, Sumskaya district. The raw material was gathered at the end of the flowering period when the plants were beginning to bear fruit and the greatest secretion of resin was observed. Thick extracts were prepared from the collected material by the maceration method according to the State Pharmacopoeia, eighth edition: extracting agent - 90° alcohol. For the purpose of comparison, an officinal thick extract of Indian hemp (Extractum Cannabis indicae spissum) was used, the date of its preparation being unknown to us.

Qualitative (Cannabinol) reactions of resinous substances

The test was according to the State Pharmacopoeia of the USSR, eighth edition. From the extracts and the control, solutions in concentrations of 1 : 10 were prepared with 90°, alcohol. Five drops of the alcoholic solution were mixed with one drop of 1 % alcoholic solution of furfurole and the mixture was stratified with 5 ml of strong sulphuric acid. In all cases there was observed a brown coloration turning to violet-brown. The reaction was not specific.

Test with Millon's reagent. As the main substances to which narcotic effects are attributed (tetrahydrocannabinol, cannabinol, cannnabidiol) contain a phenolic group, they give positive phenol- hydroxyl reactions. A test with Mfflon's reagent and a number of others may serve as an example of this. When Million's reagent was added to the alcoholic solutions of the extracts and of the control and when these were heated in a water-bath, there appeared a slowly settling characteristic flaky deposit of a brownish-pink colour. Here again the reaction was not specific, but Goldberg & Babayev used it in the forensic chemical expertise of anash (hashish) ( [ 8] ).

Reaction of diazotization ( [ 9] ). A solution of sulphanilic acid (1.5 g of sulphanilic acid in a mixture of 490 ml of water and 10 ml of concentrated hydrochloric acid) and sodium nitrite acted on the alcoholic solutions of the abstracts under study and the control. After the addition of the axcess of bicarbonate there appeared a yellow tint, turning to orange-red. On subsequent acidification (excess) with sulphuric acid the tint became bright red in all the samples.

Beam's test ( [ 10] ). A drop of the solution of the petroleum ether extract of the samples under study and of the control was placed on chromatographic paper. The stain formed in drying was processed with a 3 n alcoholic solution of potai- sium hydroxide. Al violet coloration appeared. A number of authors consider this reaction specific for Cannabidiol, which has no narcotic effect ( [ 9] , [ 11] ). Several variants of Beam's reaction (with chloroform and sulphuric acid) according. to Hissar, with hydrogen peroxide and copper sulphate, also gave positive results.

Ghamrawy's test ( [ 9] , [ 11] ). Petroleum extracts from the test extracts and the control were evaporated in an evaporating dish. To the residue was added 1 ml of Vazitski's reagent (3 g.of paradimethylaniinobenzaldehyde in a mixture of 85 ml of concentrated sulphuric acid and 15 ml of water). There appeared a reddish-brown tint, turning to violet-reld on cooling. When a few drops of water were added the tint turned to blue. This reaction has been incorporated in the French Pharmacopoeia for the identification of Indian hemp. However, as is evident, it could not be regarded as specific. It must be pointed out that Girard & Pepier used this reaction in analysing French hemp grown for seed ( [ 12] ) and obtained the same. results as -we did.

Duquénois and Negm's test ( [ 9] , [ 11] ). The extracts under study (and the control) were processed with petroleum ether. petroleum extracts were evaporated in an evaporating dish and an 8 % solution of vanillin in alcohol and 2-3 drops of 2 % aqueous solution of acetaldehyde were added to the residue. Finally, 2 ml of concentrated hydrochloric acid were added. Immediately there appeared a pink tint, quickly turning to violet, which soon became more intense. In the end the tint of the control sample became dark brown; and in the samples under study, dark green. Duquénois asserted that the initial tint was green, but Fulton observed the formation of a pink tint. This difference is explained by the different origin of the resin.

In the literature and in forensic-chemical expertise the last two reactions are considered specific for resins producing narcotic effects (mixtures of tetrahydrocannabinols), but, as our tests showed, they give positive results also with resinous secretions of hemp grown for seed (see table 1).

Biological evaluation of resinous secretions of hemp growm for seed

In order to be quite sure. of the presence of active component substances in the resin it is necessary to carry out a biological evaluation of resinous secretions. In the Soviet Union the most widely adopted method was that of Schtrub- Gaier ( [ 13] ), based on the fact that when solutions of Indian hemp preparations are introduced into the blood of rabbits, a general anacstghesia develops and the corneal reflex is lost. Sarguin & Vasilieva ( [ 14] ) modified this method and suggested introducing alcoholic instead of acetone solutions of preparations. Pershin ( [ 15] ) used an aqueous emulsion of a thick extract of Indian hemp and obtained a quicker, more lasting stronger effect than with the use of acetone solutions. We too used the latter method for our research work.

We dissolved the thick extracts in 90° alcohol (1 : 1) and prepared 5 % and 10 % emulsions (emulsifier-gelatosc;). From the control, thick extract of Indian hemp, we prepared 1-2% emulsions. Under the guidance of Prof S. D. Sokolov we introduced these emulsions intravenously into rabbits and observed the change in the sensitivity of the cornea by means of producing the normal reflex on stimulation. In order to create identical conditions for all the animals, we always introduced the same volume of emulsion (0.25 ml to 1 kg of weight), changing only the concentration of the emulsion. The results of the tests are given in table 2.

Pershin considers that the minimum quantity of the pre-paration producing extinction of the corneal reflex in rabbits (corneal unit of action or corneal dose) for the Indiam hemp extract is 0.001 g to 1 kg of weight. Our samples in the case of 5% emulsion in 0.25 ml contained 0.012 g of extract to 1 kg of weight - that is, 12 times as much; in the case of 10 % emulsion in 0.25 ml they contained 0.025 g to 1 kg of weight - that is, 25 times as much. However, our samples did not produce extinction of the corneal reflex in rabbits. Sarguin & Vasilieva determined the corneal dose for 0.75 % of alcoholic solution as 0.25 ml of solution with a resin content of 0.001875 g. Our samples exceeded this corneal dose 6.3 times (5% emulsion) and 13.1 times (10 % emulsion), but no extinction of the corneal reflex was observed.

TABLE 1. - Qualitative (Cannabinol) reactions of resinous substances of hemp grown for seed

No.

Variety of hemp

Cultivation area

Place of picking

Date of picking

Test according to Slate Pharmacopocia, eight edition

Test with Million's reagent

Test with diazotization method

Beam's test

Test with para-dimethyl-amino-benzaldehyde and sulphuric acid

Test with vanillin, aldehyde and hydrochloric acid

1
Almetiev
Tatar Autonomous SSR
Glukhov town
1958
Violet-brown coloration
Brownish-pink residue
Yellow coloration turning to orange-red; after acidification, bright red coloration
Violet coloration
Reddish-brown coloration turning to violet-red; after adding water, blue
Pink coloration turning to violet, then dark green
2
Bashkir
Bashkir Autonomous SSR
Glukhov town
"
"
"
"
"
"
"
3
Bashkir
"
Bashkir Autonomous SSR, Bielebaev distr.
"
"
"
"
"
"
"
4
Glukhov I
Central prov. Of European Russia
Glukhov town
"
"
"
"
"
"
"
5
Glukhov 3
"
"
"
"
"
"
"
"
"
6
Glukhov 6
"
"
"
"
"
"
"
"
"
7
Improved Staro-Oskolskaya
Koursk, Bielgorod, Orlov, Penza province
"
"
"
"
"
"
"
"
8
Novgorod-Severskaya
Chernigov province
"
"
"
"
"
"
"
"
9
Perevozskaya
Gorky province
"
"
"
"
"
"
"
"
10
Trubchevskaya
Briansk, Orlov province
"
"
"
"
"
"
"
"
11
South maturing SM-84
Poltava province
Zolotonosha
"
"
"
"
"
"
"
12
South maturing SM-84
"
Glukhov town
"
"
"
"
"
"
"
13
Zolotonosha No.1
"
Zolotonosha
"
"
"
"
"
"
"
14
Cherkas
Ukrainian SSR, Cherkas prov.
Glukhov town
"
"
"
"
"
"
"
15
Pavlograd
Dniepropetrovsk, Odessa, Nikolaev province
"
"
"
"
"
"
"
"
16
Bolshe-Pisarev
Penza, Sumy provience
"
"
"
"
"
"
"
"
17
Monoecious Central Russian
Central prov. Of European Russia
"
"
"
"
"
"
"
"
18
Monoecious Bernbwigskaya
"
"
"
"
"
"
"
"
"
19
Monoecious Iegevskaya
"
"
"
"
"
"
"
"
"
 
Control: thick extract of Indian hemp
     
Violet-brown coloration
Brownish-pink residue
Yellow coloration turning to orange-red; after acidification - bright red
Violet coloration
Reddish-brownb coloration turning to violet-red; after adding water - blue
Pink coloration turning to violet; then dark green

TABLE 2. - Biological evaluation of resinous substances of hemp grown for seed

 

Series No. 1

Series No. 2

No.

Variety of hemp

Place of picking

Date of picking

Weight of rabbit in kg

Concentration of emulsion

Dose in ml

Time taken in minutes for positive effect to set in

Duration of action

Weight of rabbit in kg

Concentration of emulsion

Dose in ml

Time taken in minutes for positive effect to set in

Duration of positive action

1
Almetiev
Glukhov
1958 3.3
5%
0.82
Positive effect
-
4.4
10%
1.1
Positive effect
-
        4.4   0.6
not observed
  3.0   0.75
not observed
 
2
Bashkir
Glukhov
"
2.0
5%
0.5
Not observed
-
3.4
10%
0.85
Not observed
-
        3.9   0.97     3.0   0.75    
3
Bashkir
Bashkir Aut
"
2.4
5%
0.6
Not observed
-
2.0
10%
0.5
Not observed
-
   
SSR., Bielibaev district
  3.9   0.97     3.0   0.82    
4
Glukhov 1
Glukhov
"
2.5
5%
0.62
Not observed
-
3.0
10%
0.75
Not observed
-
        3.6   0.9     2.4   0.6    
5
Glukhov 3
Glukhov
"
2.8
5%
0.7
Not observed
-
3.9
10%
0.97
Not observed
-
        2.3   0.57     2.6   0.65    
6
Glukhov 6
"
"
2.9
5%
0.72
Not observed
-
2.7
10%
0.67
Not observed
-
        2.4   0.6     3.7   0.91    
7
Improved Staro-
"
"
1.9
5%
0.47
Not observed
-
3.4
10%
0.85
Effect
-
 
Oskolskaya
    3.3   0.82     2.0   0.5  
not observed
8
Novgorod-
"
"
4.4
5%
0.1
Not observed
-
4.4
10%
1.1
Not observed
-
 
Severskaya
    3.0   0.75     2.7   0.67    
9
Perevozskaya
"
"
3.5
5%
0.87
Not observed
-
4.0
10%
1.0
Not observed
-
        2.5   0.62     3.1   0.77    
10
Trubchevskaya
"
"
3.1
5%
0.77
Not observed
-
2.0
10%
0.5
Not observed
-
        3.5   0.87     3.3   0.82    
11
South maturing
Zolotonosha
"
3.9
5 %
0.97
Effect not
-
3.4
10%
0.85
Effect not
-
 
SM - 84
    2.0   0.5
observed
  3.1   0.77
observed
 
12
South maturing
Glukhov
"
3.1
5%
0.77
Not observed
-
3.3
10%
0.82
Effect not
-
 
SM - 84
    2.5   0.62     2.4   0.6
observed
 
13
Zolotonosha
Zolotonosha
"
2.4
5%
0.6
Not observed
-
4.4
10%
1.1
Not observed
-
        3.4   0.85     3.0   0.75    
14
Cherkas
Glukhov
"
4.4
5 %
1.1
Not observed
-
3.5
10%
0.85
Not observed
-
        2.5   0.62     2.5   0.62    
15
Pavlograd
Glukhov
  3.0
5%
0.75
Not observed
-
3.9
10%
0.97
Not observed
-
        2.6   0.65     2.0   0.5    
16
Bolshe -Pisarev-
Glukhov
  3.9
5 %
0.97
Not observed
-
2.9
10%
0.72
Not observed
-
 
skaya
    2.0   0.5     3.1   0.77    
17
Monoecious
Glukhov
  2.7
5%
0.67
Not observed
-
4.0
10%
1.0
Not observed
-
 
Central Russian
    3.3   0.82     2.6   0.65    
18
Monoecious
Glukhov
  1.9
5 %
0.47
Not observed
-
3.6
10%
0.9
Not observed
-
 
Bernburgskaya
    3.5   0.87     2.4   0.6    
19
Monoecious
Glukhov
  2.8
5 %
0.7
Not observed
-
3.0
10%
0.75
Not observed
-
 
Iegevskaya
    4.0   1.0     2.7   0.67    
 
Control :
                       
 
Control :
                       
20
Emulsion of
    3.0
1%
0.75
After 20 minutes
35 3.2
2%
0.77 5 minutes 48
 
thick extract of
    4.4   1.1
lessening of the
minutes
2.0   0.5 19 minutes
minutes
 
Indian hemp
         
sensitivity of the
           
             
cornea was observ-
           
             
ed (delayed reflex)
           
21
Alcoholic solution
     
0.75%
Ranging
20-25 minutes
30  
10%
Ranging
10-20
1 hour
 
of Indian hemp
     
alcoholic
from
 
min-
 
alcoholic
from
minutes
 
 
resin (according to
     
solution
0.5  
utes
 
solution
0.48    
 
Sarguin-Vasilieva)
     
of resin
to 0.65
     
of resin
to 0.63
   

The thick extract of Indian hemp used by us as the control and introduced in the form of 1% emulsion (0.25 ml with a content of 0.0025 g. of extract to 1 kg of weight) produced a weakening and slowing-down of file corneal reflex, but did not take it away altogether. The use of 2 % emulsion (0.25 ml with a content of 0.001 g extract to 1 kg of weight) produced a distinct loss of the corneal reflex.

Conclusions

  1. Resinous secretions of hemp grown for seed in the central districts of the European part of the USSR give positive reactions with the reagents adopted for determining cannabinol compounds in Indian hemp resin.

  2. A biological test of resinous secretions of hemp grown for seed showed that these substances do not produce the physiological effect peculiar to the resinous substances of Indian hemp.

  3. Resinous secretions of hemp grown for seed in the central districts of the European part of the USSR, as well as of the variety of hemp grown for seed in the Northern Caucasus, are pharmacologically inactive.

  4. As the cannabinol reactions are not specific for the narcotic substances of hemp, it is necessary in the forensicchemical expertise of hemp preparations to carry out these reactions in conjunction with biological evaluation.

Literature

001

V. K. VARLIKH: Russkiye lekarstvennye rasteniya (Russian medicinal plants). St. Petersburg, p. 345, 1901.

002

Y. V. PETROV : O Konopeli. Vseobstchii Zhurnal vrachebnoi nauki (Hemp. Universal Journal of Medical Science), No. 6, pp. 86-91, 1813.

003

N. ZININ & E. PELIKAN : Orenburgskii gashish (iz dikoi konopli) Voennomeditsinskii Zhurnal (Orenburg Hashish, Army Medical Journal), part 68, No. 2, pp. 69-74, 1856.

004

B. SHENFELD: Gashish (Cannabis indica) v meditsinskom otnoshenii, Dissertatsiya. (Hashish ( Cannabis indica) in relation to medicine. Thesis). S. Orlov Printing House, Moscow, 1863.

005

N. SOKOLOV : Farmakognosticheskoye issledovaniye indiiskoi konopli. Dissertatsiya. (Pharmacognostic research on Indian hemp. Thesis). St. Petersburg, 1879.

006

STATE PHARMACOPOEIA OF USSR, eighth edition, p. 174, 1952.

007

E. A. KECHATOV: Kannabinolnye reaktsii i biologicheskaya otsenka smolistikh vydelenii konopli, posevnoi i sornoi, proizrastayustchikh na severnom Kavkaze. Uchenye zapiski Piatigorskogo Farmatsevticheskogo Instituta (Cannabinol reactions and biological evaluation of resinous secretions of wild and seed hemp growing in the Northern Caucasus. Scientific notes of the Piatigorsk Pharmaceutical Institute), vol. 11, 171, 1957.

008

A. Z. BABAYEV & I. K. GOLDBERG : Materialy k sudebnokhimicheskomu opredeleniyu anashi. Doklady A.N. Azerb. SSR. (Material relating to the forensic-chemical determination of anash. Reports of the Analytical Office of the Azerbayan SSR), vol. 12, No. 10, pp. 749-752.

009

Charles C. FULTON : Analytical classes of cannabinol compounds in Marihuana resin. Industrial and Engineering Chemistry, analytical edition, vol. 14, No. 5, 1942.

010

M. P. DUQUÉNOIS: Essai d'identification du hachisch et du chanvre à l'aide de la microchromatographie. Annales de médecine légale et criminologie, vol. 34, No. 5, pp. 224-225, Paris, 1954.

011

DUQUÉNOIS: Chemical and physiological identification of Indian hemp. Bulletin on Narcotics, vol. 2, No. 3, pp. 30-33, 1950.

012

GIRARD & REGNIER : Issledovaniye smoly raznykh obraztsov indiiskoi konopli. Zamechaniya pro povody eyo farmakopeinogo opredeleniya. Referativny Zhurnal "Khimiya" (Investigation of the resin of various samples of Indian hemp. Remarks regarding its pharmacopoeic determination. Reference journal Chemistry), 21, No. 71758, pp. 342-343, 1958.

013

K. D. SARGUIN : Biologicheskaya otsenka lekarstvennogo syrya i farmasevticheskykh preparatov. Nauchnoye Khimiko-teknicheskoye izdatelstvo (Biological evaluation of medicinal raw material and pharmaceutical preparations. Scientific chemico-technological edition). Leningrad, pp. 217-221, 1929.

014

K. D. SARGUIN & V. V. VASILIEVA : K voprosu biologicheskoi otsenki preparatov indiiskoi konopli. Farmakologiya i toksikologiya (Concerning the biological evaluation of Indian hemp preparations. Pharmacology and Toxicology), vol. 2, No. 5, p. 63, 1939.

015

G. N. PERSHIN : K voprosu o farmakologii indiiskoi konopli. Farmakologiya i toksikologiya (Concerning the pharmacology of Indian hemp. Pharmacology and Toxicology), vol. 12, No. 4, p. 37, 1949.