STUDIES ON HASHISH I. ISOLATION & IDENTIFICATION OF CANNABINOLS AND

EFFECT OF CERTAIN FACTORS

Z. I. EL-DARAWY, M. I. ROUSHDY*, A. M. RIZK*, F. M. HAMMOUDA* & Z. M. MOBARAK

National Centre o f Social and Criminological Research, A wkaf City, Gezira Post Office, Cairo, Egypt

ABSTRACT

Fractionation of the Hashish constituents as well as the isolation of certain consti- tuents on a preparative scale was carried out using several physio-chemical techniques viz. solvent fractionation, column and thin-layer chromatographic methods. Six con- stituents viz. cannabidiolic acid, cannabidiol, cannabinot, tetrahydrocannabinol, can- nabichromene and a positive Beam component (cannabidiol isomer) were isolated and identified using chromatographic data and ultraviolet spectrophotometry. Separation of the Hashish constituents was also achieved by gas-liquid chromatography. The effect of certain factors viz. silica gel, acidity and alkalinity was studied revealing a remark- able change.

Cannabis or Hashish, one of the oldest drug plants, represents at the time being an important group of unique character in the modern growing fields of socio- and psychopharmacology (Chopra & Chopra, 1957). The abuse of this drug was, and still is, more wide spread throughout the World than the abuse of any other single drug known.

Through the centuries many chemical races of the plant have come to existance and are progressively recognized. However, the botanical origin so far seems to be namely Cannabis sativa L. and the active drug remains to be the oleo-resin obtained from the flowering tops of the unfertil ized female plant. Botanically there is only one species of Hemp namely Cannabis sativa L.; such names as C indica (Hamilton, 1913), C satipa var. indica (Merrill, 1938), C americana (Merrill, 1938; Wolf, 1949) are not botanically admis- sible and must be regarded as ' common ' names of the same calibre as Indian Hemp and Hashish.

Several vernacular names viz. Hashish, Magoun, Kif, Bangi, M a r i h u a n a . . . etc. (Watt & Breyer-Brandwijk, 1962) are known for Cannabis in the dif-

* National Research Centre, Dokki, Cairo.

Qual. Plant. Mater. Veg. XXI, 4 : 311 - 325, 1972 311

ferent countries. The numerous preparations having Hashish activity are all obtained from the Cannabinaceae famiiy.

The classical constituents of Cannabis are cannabidiol (I), cannabinol (II) and tetrahydrocannabinol (III, IV). The structure of these compounds has been confirmed by the intensive work of several authors (Cahn, 1930-1933; Bergel et al., 1943; Adams et al., 1949). Cannabidiolic acid (V) as well as another acids were also isolated from the leaves and stem tops (Krejci & Santavy, 1955; Krejci et al., 1958, 1959; Schultz & Haffner, 1958, 1959). Several other constituents e.g. cannabinolic acid, cannabigerol, can- nabichromene.., etc (VI-XVI) had been isolated from Hashish (Claussen et al., 1966, 1968; Goani & Mechoulam, 1964, 1966; Hilvely et al., 1966; Kane, 1968; Korte et al., 1965; Mechoulam et al., 1964-t969; Obata, 1966; Okamoto, 1967; Shoyama et al., 1968, i970; Spulak et al., 1968; Yamauchi et al., 1967, 1968; Vollner et al., 1969).

Thin-layer chromatography had been successively applied by several authors for the separation of cannabinols. The first application was that by Krejci (1961). Different adsorbents viz. silica gel (impregnated with dimethylformamide or AgNO3 or without) (Korte & Sieper, 1964 a,b; Turk

CH:j C.H~

H3c I1 6H ~ -CsH~ H~C C H I 5 II

CH 2 CH 3 Cannabidiol (I) Cannablnol (~)

CH 3

H3C C5Hll CH 3

z~9- ti- an s Tetr ahydr o- cannabinol (TIT)

C,H 3

O~COOH

HC- -C

CH 2

Cannab(d(ol;c ac;d ( Y }

C H 3

H3C CsH11

~B- t rans Tetrahydro- c annabinol ( ] ~ )

CH 3

O~COOH

H3C - - ~ ' 0 ~ C 5 H l t CH 3

Cannabinot(c acid

( '£I)

312

et al., 1969 a,b), alumina (Stone, 1969) or silica gel and poly (vinyl alcohol) dipped in Et2NH (Grlic, 1970) were used. The chemical detection of the chromatographed spots, which is far more superior to physical detection was carried out by spraying with a variety of reagents viz. Gibbs (1927), Beam (1911), Duquenois (1938) and tetrazotised o-dianisidine (Fietz-David & Blangey, 1949).

Although several methods for separation of the cannabinols by gas-liquid chromatography, have been described in the literature, all have had certain limitations. No success was obtained with the relatively non-polar columns of methyl, silicone gum (Claussen et al., 1966; Heaysman et al., 1967; Okamoto, 1967) and cyanosilicone gum (Caddy et al., 1967); while the somewhat polar stationary phase (Carbowax 20 M) (Heaysman et al., 1967) gave better separation of the cannabinols. On the other hand, the high temperature 230°C required for tile column is liable to limit the efficient life of the column. The best resolution obtained by Stone (1969) using neopentyl glycol adipate plus trimer acid column, still needs high column temperature of 220°C.

c H3

O~ COOH

i- O- v ~CBHI 1 C H 3

~9- ¢r an s -Tetrahydro- cannablno[ic acid (~)

C. H3

H3 O C ~

~c" -o- y -%H. COOH

A 1 -Tetrahydro-

cannablno{ic acid B ( T'X" }

CH 3

O~j~ oo H

,,c..;. ) . . IL H3C" "O" ~ "CsHI1

~ T e t r a h y d r o -

cannablnolic acid A ( ~n-r )

C5Hll

Can~ab;gerol (X)

Cannabigero|ie acid ( ~ )

CH2= C.CH 3 ( CH2~3C.CH3=CH

HO" ~Cs H ~i

Cannabig • rol monomethyi ether ( ~ )

313

OH OH

C, H2-CH csH, c %~2c °C,.CH2.CHrI..0/%j-.% H1, CH CH 3 CH 3 II C..,

C'~3 CH 3 Cannabichromenlc acid

Cannablchromene t ~ )

CH3

H%lC5 H 2

H3C 02C 0 ~CsHII x k .J~ CH3 H OH

H11C5 ~ ~ ~O ~ \ CH3 Cannabipinot

Tetr ahydr o c annabitr iot OR

CannabldioI ca rboxy t i c acid Cannabicy¢toi

ester (~!~'1 ( ~ )

Adulteration of Hashish by mixing with different herbs or drugs, makes its detection and identification somewhat difficult. This work deals with the fractionation of Hashish constituents using several physico-chemical tech- niques involving solvent fractionation, thin-layer and column chromato- graphy of certain constituents on a preparative scale.

EXPERIMENTAL AND RESULTS

Preparation of Hashish Resin

10 g of powdered Hashish were macerated twice with methanol (25 ml each) for 24 h, in dark, at room temp. Methanol was evaporated in vacuo at 40°C and the residue was taken with petroleum ether (40-60°C) - ether (80:20) and filtered from the insoluble matter. The filtrate was passed through a column packed with silica gel and elution was carried out using the same solvent mixture.

Thin-Layer Chromatography

Plates coated with silica gel G, using different solvents were tried. The solvent petroleum ether (40-60°C) - ether - ethyl acetate (90:5:5) proved to be the best revealing the presence of 11 components, upon spraying with tetrazotised o-tolidine (Fig. 1).

314

& & c,o THC

C ~ CBN CBC

0 0 :BO"

t 2

CBDA

Fig. ( t )

Thin- layer Chromatogram of Puri f ied

Hashish Re~in

Adsorbent :S i l i ca gel G.

Solvent s~'stem ; Pet, ether ( 4 0 - 6 0 ' C ) -

E ther -E thy l acetate ( g o : 5 : 5 )

Sgraying reagent : 1) Beam,

2 1 T e t r a z o t i s e d o-totidine.

Isolation o f Cannabidiolic A cid

Cannabidiolic acid (CBDA) was separated from the purified Hashish resin as follows: The petroleum ether - ether etuate was shaken with three successive portions (20 ml each) of aqueous solution of sodium carbonate (5%) and sodium sulphite (5%) (I : 1). The combined aqueous solution, after washing with petroleum ether-ether was rendered acidic with H2 SO4 (3%) and the liberated CBDA was extracted with ether. The ether extract was dehydrated over anhydrous Na2 SO4 and the solvent was removed in vacuo. TLC revealed the presence of CBDA which was further purified by prepara- tive TLC using petroleum ether (100-140°C) - acetone (1:1). The isolated CBDA was found to possess the same Rf as the authentic using different solvents systems and had the same U.V. absorption. (Tab. 1)

315

Table 1

Absorption maxima {nm} o f Cannabinols

= ~.max (nm)

Cannabinols Hexane Methanol

Cannabidiolic acid 225, 270, 311 224, 266, 304 Cannabidiol 212, 282 213, 283 Tetrahydrocannabinot 212, 234, 283 213, 234, 284 Cannabinol 219, 285 220, 285

Table 2

The cannabinot components in the fractions collected from the column chromatography

Fraction No. Cannabinols

1 - 5 THC and CBC 6 - 10 THC, CBC and CBN

11 - 15 THC, CBN and CBD 16 - 20 CBN and CBD 21 - 50 CBD and CBD isomer (CBD') 51 - 70 CBD

Isolation o f the Other Cannabinols

The other cannabinols viz. tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), cannabichrome (CBC) and another component (CBD') (giving positive Beam reaction) were isolated from the petroleum

ether-ether eluate, after removing of the cannabidiolic acid, by application of cotumn and preparative TLC

Column Chromatography

Silica gel, impregnated wi th dimethylformamide was used (80 g silica gel '40 mesh' treated with dimethylformamide '40 m l ' , filtered and the acetone was removed by heating the silica gel at about 35°C till it .smelled no acetone). One g of the.cannabidiolic acid-free Hashish resin was frac- tionated on the impregnated silica. Elution was carried out using cyclo-

316

hexane saturated with dimethylformamide and the course of the chromato- graphic fractionation was followed on plates coated with silica gel G (using petroleum ether - ether ethyl acetate 90:5:5) and the results obtained are shown in Table 2. Fractions 1-5, 6-10, 11-15, 16-20 and 21-50 were further fractionated into their cannabinols by preparative TLC.

Preparative TLC

The fractions obtained from the column were fractionated using plates coated with silica gel G (1 mm thick). The plates, after developing with petroleum ether - ether - ethyl acetate (90:5:5) were air dried and the outer strips (about 2 cm width) were sprayed (after covering the central part with a glass plate ) with tetrazotised o-tolidine. The zones containing the cannabinols, from the unsprayed central portion were scrapped off and separately extracted with ether. By applying this technique CBD, CBN, THC, CBC and CBD' (probably a cannabidiol isomer) (Korte & Sieper, 1965) were isolated.

The identity of the isolated cannabinols was proved by Rf, using dif- ferent solvents, and by UV. (Table 1).

Gas-Liquid Chromatography

The purified Hashish resin was investigated by GLC using the following conditions: Apparatus: F. & M Model 500, programmed high temperature gas

chromatograph equipped with Model 1609 Flame ionization detector attachment.

Column dimensions: Copper column 2 meters length and 4 mm diameter. Solid support: Chromosorb '60-80 mesh' (acid washed), Stationary phase: Apiezon L 20%. Carrier gas : Nitrogen. Temperature: 170°C. Radio-Flowmeter Reading: H2 4.5; air 11.7; N2 2.7.

The results obtained are shown in Fig. 2 and Table 3. The characteristics ,of the Hashish resin chromatogram are compared with the chromatograms for standard constituents at the same working conditions.

Effect of HCl, Ammonia and Silica Gel on Hashish Constituents,

The effect of these factors on Hashish resin were preliminary studied and the resulting compounds were detected by two-dimensional TLC. The plates (20 x 20 cm) were marked by a needle to make two strips each about 5 cm

317

Table 3

Major components in hashish extract by gasliquid chromatography

Peak No. Retention Identification relative time (RT) contents minutes

1 3.6 unknown small 2 4.0 unknown medium 3 5.0 unknown medium 4 5.6 from CBD large 5 7.0 unknown small 6 8.0 from CBD large 7 9.0 unknown medium 8 9.8 unknown large 9 13.6 unknown large

10 14.2 THC medium 11 19.0 CBN medium

wide at the right hand side as well as at the upper part. Hashish resin was applied on the lower left corner of the plate as well as on each of the strips to serve as reference for the corresponding development. The plates were developed with the solvent (petroleum ether - ether - ethyl acetate 90:5:5) in the first direction to a height of about 14cm, removed from the chromatographic tanc and air dried. For studying the effect of HC1 or ammonia vapours, the plate in each case after drying was placed in a jar saturated with HC1 or ammonia vapours (attained by placing a beaker con- taining conc. HC1 or ammonia in the jar) for one hour, then redeveloped in the second direction with the same solvent system. In order to study the effect of silica gel adsorbent, the plate after the first development was kept in dark for 45 hours, then developed in the second direction.

The results obtained (Fig. 3 - 6 ) revealed a remarkable change.

318

LLI L.J Z 0 n l..O ILl e,"

¢r' 0 I,-- £.J ILl I.-- LIJ £3

8

1 | I

5 10

9

10

11

15 20

T I M E Cminutesl

F ig . ( 2 ) G a s l i q u i d C h r o m a t o g r a m of P u r i f i e d

H a s h i s h R e s i n .

319

TNC

CBJI

¢BC

CBDA

TH~ CBN CBC

CS~T

3 2 0

.']'.

I )

)

0

D

0 0

. . . . ' < ] 3 ~

...... ' 0

0

0

~g

~ 0 ......... . . . ;

' ~ ) 0

2

.~_

B

o X

I.--

o a: ._~

m

; E . c

E q ~ 7 - ,~

_ E N

T-

f f l

u

® "g , l z

u~

v

a

uJ "~ i o

i " -

b

CBO

THC

CBN CBC

CBO'

CBDA

C8D

THC

CBN

CBc

CBD'

CSD~

~c ~ o ¢J

(ZZ~

0

,.,... ,.-,, ,,..,."

O@ ~0ooo

~ t

Q

2> %

o

. _ ~

. =

i I o

o

I -

u o

~= -~ ac: ' ~

W O. i

U o .

~ o

(D ~ i

? ,a ~ - .

o

• - ~

" ~ ._ o

DISCUSSION

The results obtained from the column chromatographic technique using different adsorbents and different solvents for elution were unsatisfactory. Though the column packed with silica gel impregnated with dimethyl- formamide, using cyclohexane as eluting solvent and collecting fractions each of 10 ml, gave the best result of these trials, yet only CBD was the only constituent which obtained sole in some fractions; the other fractions; were further fractionated by preparative TLC. The latter procedure proved to be the best.

Of the eleven cannabinols detected in the Hashish resin, six (CBD, CBN, THC, CBDA, CBD', CBN) were obtained in a pure form and could be identified. Probably some of the unidentified constituents are isomeric forms of different cannabinols, particularly tetrahydrocannabinol of which more isomers were detected and reported to occur in Hashish. The presence of these isomers was attributed to several factors. A phenolic compound reported (Korte & Sieper, 1965) to have Rf between CBDA and CBN giving the same colour reaction as that of CBD and was considered as an isomer of CBD, probably resulting from the migration of the double bond in the limonene ring. This compound is reported to exist in considerable amount if pure CBD is left standing in methanolic solution at room temperature. In the present work Hashish was macerated with methanol at room tem- perature for about 24 h and therefore the possibility of the presence of such isomer is standing. Other factors either genetic or environmental involving acid, heat, atmospheric moisture, oxygen or light are also responsible for the formation of tetrahydrocannabinol isomers (Korte & Sieper, 1965).

The use of Apiezon L. as a stationary liquid phase in GLC (not tried before for cannabinols) has the advantage of using lower temperature and of being able to obtain better separation. The fact that CBD was the first to come off the column followed by THC, then CBN, is in agreement with that in the literature using other columns. The GLC of CBD shows two peaks; the same observation was obtained by Farmilo et al. (1963) who stated that these are probably degradation products. The components given by the standard CBD are represented in the Hashish extract whose chromatogram shows 11 peaks.

ZUSAMMENFASSUNG

Die Trennung von Haschisch Bestandteilen sowie i_hre Isolierung dutch physikalisch- chernische Methoden z.B. L6sungsmittel Verteitung, Saulen-und Dtinnschicht-Chroma- tographie wurde untersucht. Sechs Komponenten: Cannabidiol~ure, Cannabidiol, Cannabinol, Tetrahydrocannabinol, Cannabichromene und eine positive Beam Kom-

322

ponente (Cannabidiol isomer) wurden getrennt und durch Chromatographie und UV-Spectrophotometrie nachgewiesen. Die Trennung der Haschisch-Komponenten gelang auch durch GLC. Der Einfluss einiger Faktoren wie Silica Gel, S//ure und Alkali wurde studiert.

RI~SUME

La s@aration des constituants du Hashish et l'isolement de certains constituants par une m6thode preparative a ~t~ effectu~ utilisant plusieurs techniques physico- chimiques viz. s~paration au solvent, chromatographie sur colmne et sur couche mince. Six constituants viz. acide cannabidiolic, cannabidiol, cannabinol, t6trahydrocanna- binol, cannabichromene et un produit positif au r6actif de Beam (isom~re du canna- bidiol) ont 6t~ isold et identifi~ selon les r~sultats obtenus par chromatographie et par spectrophotom~trie ultra-viotette. La s~paration des constituants du Hashish a aussi gt~ effectu~ par la chromatographie gas liquide. L'effect de certains facteurs viz. le gel de silica, l'acidit~ et la basicit~ ont gt~ ~tudi~ rfiv~lant un changement assez notoire.

ACKNOWLEDGEMENT

The authords are deeply indebted to Late Prof. Z. F. Ahmed for his very kind help and fruitful suggestions. Thanks are to the Central Narcotic Division, Cairo for kindly supplying Hashish and to Prof. F. Korte and Dr. D. Bieniek (Bonn) for kindly supplying the authentic references.

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