Studies on cannabis obtained from hemp plants grown in Japan

Sections

INTRODUCTION
EXTRACTION OF HEMP TOPS
COLOUR REACTION OF CANNABIS EXTRACT
DETECTION OF CANNABIS COMPONENTS BY PAPER CHROMATOGRAPHY
SPECTROPHOTOMETRY OF CANNABIS RESIN
FUTURE WORK

Details

Author: Haruyo Asahina
Pages: 17 to 20
Creation Date: 1957/01/01

Studies on cannabis obtained from hemp plants grown in Japan

Haruyo Asahina Narcotic Section, National Hygienic Laboratory, Tokyo, Japan

INTRODUCTION

Hemp plants grown in Japan have been demonstrated to be toxic by physiological tests on goldfish or dogs, but the physiologically active principle, presumably tetrahydrocannabinol, has not yet been isolated from it, and little is known to us about its content of active resin, although it is said to be less potent than the hemp grown in some other countries.

Among the identification tests usually employed for the identification of cannabis samples seized in the illicit traffic, the best-known chemical test, Beam's colour reaction, is not based on tetrahydrocannabinol, and the hemp of Japanese culture that we have examined so far gave a negative test with Beam's reagent.

A reliable forensic test merely to identify cannabis could be based on any chemically active compound uniquely characteristic of hemp; but any test to identify potent hemp or to assay the cannabis drug for potency still awaits discovery, and must be one given only by physiologically active components.

For the purpose of separating the active principles from cannabis of domestic origin and clarifying their properties, we have undertaken basic research on cannabis.

This investigation was supported in part by a grant in 1956 from the Welfare and Health Ministry.

The following topics were studied in this fiscal year: (1) Extraction of hemp tops; (2) Colour reactions of hemp extract; (3)Detection of cannabis components by paper chromatography; (4) Spectrophotometry of cannabis resin.

EXTRACTION OF HEMP TOPS

At the time of flowering of the domestic hemp plants cultivated in 1956 at the Minami Oshihara Experimental Station of the Tochigi Prefectural Farm, .the plants were cut one foot from the top, and the twigs and leaves were airdried. They had no narcotic odour.

The extraction of the dried tops was carried out by courtesy of Dr. Nishizawa at the Tokyo Factory of the Takeda Pharmaceutical Industries, Ltd., according to the procedure devised by Mr. Takeuchi, chemist of that factory.

When 1 g. of the air-dried tops was cut into small pieces and dried at 105°C to constant weight, the loss of weight was 11.9%.

The principle of this extraction is to absorb the active components by charcoal from petroleum ether solution.

The same procedure was employed by Mr. Takeuchi in 1953 and 1954 for the extraction of cannabis, in studies of the physiological activity under grants of the Agricultural Ministry.

In 1940, Remziye Hisar and Sazile Edesen reported their study of the absorption of the cannabis resin in petroleum ether solution by animal charcoal.

The method of extraction is shown below:

Full size image: 22 kB

The amounts of extractables in each fraction, No. 1 to No. 4, separated by this procedure from 15 kg of cannabis plants, were as follows:

Fraction

Amount Grammes

Yield Per cent

Character

1 30.0 0.20
Brownish black, half solid
2 50.0 0.33
Yellowish brown, jellied with fine crystals
3 145.5 28.5
Dark reddish brown, viscous
4 0.96 0.19
Dark brown, viscous

COLOUR REACTION OF CANNABIS EXTRACT

A number of colour reactions observed by various researchers with cannabis resin were examined. A few of these are:

  1. Beam's reaction (alcoholic potassium hydroxide);

  2. Ghamrawy's reaction (p-dimethylaminobenzaldehyde);

  3. Duquenois' reaction (vanillin, acetaldehyde);

  4. Diazo reaction (sulfanilic acid, sodium nitrite);

  5. Gibbs' reaction (2,6-dibromoquinone-4-chlorimide).

  1. Beam's Reaction

    This reaction is the best-known for the identification of cannabis, and is characteristic of cannabidiol, which is physiologically inactive.

    This component is absent from the extract of hemp plants grown in Japan that we tested, and no spot could be detected by the Beam reagent on filter paper in the chromatography of that extract.

    Pure cannabidiol, however, gives a very distinct colour reaction, and can be easily detected on filter paper by this reagent, in the manner described in Duquenois' article.

    Therefore, it is concluded that our domestic hemp at flowering contains no cannabidiol, or certainly very little.

    It is pointed out in the literature that Beam's test on resins from some varieties of cannabis gave negative results.

  2. Ghamrawy's Reaction

  3. Duquenois' Reaction

  4. Diazo Reaction

    These three reactions are also well known for the identification of hemp. All fractions of hemp extract that we examined gave positive reactions.

  5. Gibbs' Reaction

    Phenols with no substituent in the para position to the hydroxyl produce this reaction. Todd employed this reaction in his chemical elucidation of cannabinol, cannabidiol, and tetrahydrocannabinol, and reported that it is positive with these compounds.

    The hemp plants grown in Japan and all fractions of hemp extracts that we tested gave this reaction, and it was possible to detect the reactive spot from hemp extract which was chromatographed on filter paper, as described in detail elsewhere in this report.

    Of the fractions of hemp extracts, No. 3 and No. 4 gave deeper colour tests than No. 1 or No. 2, both by a colour test on the extract or on the paper chromatogram.

    It is concluded from the colour reactions that our domestic hemp contains the same chemically active component or components responsible for these tests as cannabis from the hemp of other countries.

DETECTION OF CANNABIS COMPONENTS BY PAPER CHROMATOGRAPHY

In 1954, Duquenois reported his microchromatographic study on the identification of cannabis using Beam's reagent as colour developing reagent and ammoniacal acetone as solvent.

As described above, there is no component found in the extract of Japanese hemp which is detectable by Beam's reagent, so we used Gibbs' reagent as the developing reagent in the chromatography.

In our experiments, ammoniacal acetone which Duquenois used gave a spot nearly at the top of solvent, and so was not very suitable. Many solvents were tried experimentally to find a suitable developing solvent, but they were all unsatisfactory in terms of shape of the spot, Rf value, and trailing, on an ordinary filter paper.

Then we used an alumina-impregnated filter paper. Of the solvents tried on this paper, 95% methanol gave fairly good results.

Paper Chromatography

Filter paper; Toyo Roshi No. 50, 2.5 ? 40 cm, impregnated with alumina.

Impregnation of filter paper with alumina: Filter paper is dipped in 30% solution of aluminium sulfate at 60-65°C for three minutes and hung in ammoniacal vapour for one hour in an air-tight container. Then it is washed with running water for six hours, and air-dried.

Container for chromatography: Toyo Roshi Type C.

Procedure: A base line is drawn 5 cm from the lower edge of the filter paper, paralleling that edge. On the middle point of this line, the solution of extract of hemp to be chromatographed is spotted.

Developing solvent: 95% methanol.

Height of development: approximately 25 cm above the base-line.

Movement of solvent: ascending.

Spot developing reagent: Preliminary trials with a number of reagents giving colour reactions with cannabis led to the selection of Gibbs' reagent as the most sensitive. A few of these are given below.

  1. Gibb's Reagent

    1. 5% sodium acetate solution;

    2. saturated alcoholic solution of 2,6-dibromoquinone-4-chlorimide.

The filter paper is sprayed with solution (i), then with solution (ii).

  1. Ghamrawy's Reagent

Two cc of 10% hydrochloric acid added to 100 cc of 3% alcoholic solution of p-dimethylaminobenzaldehyde. The filter paper is sprayed with this reagent and dried at 60°C.

  1. Saturated Ligroin Solution of Iodine

The Rf values and colorations thus far obtained by this procedure using Gibbs' reagent were as follows:

Sample

Rf value

Colouration by Gibbs’ reagent

Fraction 1
0.62
blue
Fraction 2
0.62
blue
Fraction 3
0.61
blue
Fraction 4
0.63
blue
Cannabidiol
0.71
purple
Pyrahexyl (Abbott)
0.58
bluish violet

Pyrahexyl is a synthetic analogue of tetrahydrocannabinol, and its chemical name is 1-hydroxy-3-n-hexyl-6,6,9-trimethyl-7,8,9,10-tetrahydro-6-dibenzopyran.

The minimum quantities detectable of the fractions No. 2, 3, and 4, by Gibbs' reagent in the chromatographic procedure were 600 γ, 50 γ, and 50 γ, respectively. The spot of cannabidiol, the Rf value of which is 0.71, can also be developed by Beam's reagent. But the spots of the hemp extracts No. 1 to No. 4 were not shown by this reagent.

Table 1

Sample

Solvent

Max (mµ)

Absorbance

Min (mµ)

Absorbance

Concentration (g/l)

1. Petroleum ether extract
Alcohol
280 0.610 251 0.410 0.0520
2. Fraction 2
Hexane
-
 
-
 
0.0352
3. Fraction 3
Alcohol
282 0.559 251 0.178 0.0290
4. Fraction 3
Hexane
279 0.520 251 0.175 0.0283
5. Fraction 4
Methanol
281 0.332 253 0.176 0.0256
6. Pyrahexyl
Alcohol
276 0.741 249 0.224 0.0218
 
 
229 1.700
 
 
 
7. Pyrahexyl
Hexane
275 0.800 248 0.276 0.0239
 
 
226 2.000
 
 
 
8. Pyrahexyl
Methanol
275 0.742 248 0.245 0.0216
 
 
228 1.750
 
 
 

The absorbance value was transformed into extinction value E 1 % 1 cm

Table 2

Sample

Solvent

Max (mµ)

E1 %1 cm

Min (mµ)

E1 %1 cm

1. Petroleum ether extract Alcohol
Alcohol
280 117.5 251 78.8
2. Fraction 2
Hexane
 
-
-
 
3. Fraction 3
Alcohol
282 192.9 251 61.4
4. Fraction 3
Hexane
279 183.6 251 61.8
5. Fraction 4
Methanol
281 129.8 253 68.8
6. Pyrahexyl
Alcohol
276 340.1 249 102.8
 
 
229 780.3
 
 
7. Pyrahexyl
Hexane
275 335.2 248 115.6
 
 
226 838.0
 
 
8. Pyrahexyl
Methanol
275 343.5 248 113.4
 
 
228 810.3
 
 

E 1 % 1 cm is plotted against wavelength. The curves are shown in Figs. 1, 2 and 1.

From this result and the Rf value, it is concluded that the spot of the extract of hemp grown in Japan detected by Gibbs' reagent on filter paper is produced by some other compound than cannabidiol.

SPECTROPHOTOMETRY OF CANNABIS RESIN

The ultraviolet absorptions of cannabinol, cannabidiol, tetrahydro-cannabinol, and analogous compounds were measured by Adams and Todd in their studies of the chemical structure of cannabis components.

Biggs and Farmilo applied ultraviolet spectrophotometry of a petroleum ether extract to the identification of the cannabis plant.

It is also reported by Adams, by Todd, and by Farmilo that although some variations occur in the absorption curve of isomers of tetrahydrocannabinols, these compounds can be identified with good success by the measurements of ultraviolet spectrophotometry.

The ultraviolet absorptions of the extracts from our domestic hemp and of the synthetic analogous compound, pyrahexyl, at 216 mμ to 320 mμ in methanol, and in hexane, and at 220 mµ to 320 mµ in alcohol, were measured by the Beck- man DU spectrophotometer for the petroleum ether extract, and by the Hitachi EPU-2 instrument for other measurements.

The absorbances measured at the maximum and minimum are listed in Table 1.

Fig. 1. - Absorption curves (in methanol) of I: pyrahexyl; II: fraction 4

Full size image: 9 kB, Fig. 1. - Absorption curves (in methanol) of I: pyrahexyl; II: fraction 4

Fig. 2. - Absorption curves (in alcohol) of I: pyrahexyl; II: fraction 3; III : petroleum ether extract

Full size image: 9 kB, Fig. 2. - Absorption curves (in alcohol) of I: pyrahexyl; II: fraction 3; III : petroleum ether extract

The absorption curve shows there is no absorbing material in fraction 2. The spectra of fractions 3 and 4 have maxima at 279-282, minima at 251-253 mµ, and are nearly analogous to the spectrum of pyrahexyl.

A corresponding maximum for pyrahexyl is at 275-276 mµ and minimum is at 248-249 mµ. Pyrahexyl has another maximum at 226-229 mµ.

No variation of the absorption curve was observed in different solvents.

From the spectrophotometric studies of our domestic hemp, it is concluded that it contains tetrahydrocannabinol, or an analogous compound; and in general the hemp plants grown in Japan give a resin more similar to the Malayan resin reported by Biggs than to the Canadian resin.

Fig. 3. - Absorption curves (in hexane) of I: pyrahexyl; II: fraction 3; III: fraction 2

Full size image: 9 kB, Fig. 3. - Absorption curves (in hexane) of I: pyrahexyl; II: fraction 3; III: fraction 2

FUTURE WORK

Research on the following items is planned for the future: ( a) separation and purification of each fraction by means of column chromatography; ( b) physiological activity of each fraction; and ( c) comparison of cannabis from the hemp plants grown in Japan, with cannabis of other countries, as to the content of active resin.