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ABSTRACT
Cannabis or Hashish, one of the oldest drug plants, represents at the time
being an important group o f 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 unfertilized 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 ' c o m m o n ' names o f 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-
CH:j C.H~
CH3 CH3
C,H3 CH 3
O~COOH O~COOH
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 CH3
H3O C ~ C5Hll
HO" ~ C s H ~i
313
OH OH
Cannablchromene t ~ )
CH3
H%lC5 H2
H3C 02C 0
~CsHII
xk .J~ CH3 H OH
H11C5~ ~ ~O ~ \
CH3 Cannabipinot
Tetr ahydr o c annabitr iot OR
CannabldioI c a r b o x y t i c acid Cannabicy¢toi
ester (~!~'1 ( ~ )
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"
CBDA
t 2
Fig. ( t )
Thin-layer Chromatogram of Purified
Hashish Re~in
Adsorbent : S i l i c a gel G.
Solvent s~'stem ; Pet, ether ( 4 0 - 6 0 ' C ) -
Ether-Ethyl acetate (go : 5 : 5)
Sgraying reagent : 1) Beam,
21Tetrazotised o-totidine.
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
= ~.max (nm)
Table 2
Column Chromatography
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 2 1 - 5 0
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 ' 6 0 - 8 0 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.
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
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 o f 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 b y 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 8
ILl
e,"
1
¢r'
0 9
I,--
£.J
ILl
I.--
LIJ
£3
10
11
| I
5 10 15 20
TIME Cminutesl
319
.... ' < ] 3 ~ ffl
.']'.
u
.~_
I
)
TNC ) B
CBJI
¢BC
® "g
...... ' 0 ,lz
o X
0
0
CBDA
I.--
u~
~g a:o
._~
v
m
a
0 ;E .c
TH~
D uJ "~
i o
CBN
CBC
CS~T
0
0
~ 0 ..... ...; E
~7-
_
q
E
,~
N
i "-
'~)0
T-
2
320
~c
~ o
¢J
(ZZ~
0
._ ~
.=
i
I o
CBO
THC o
CBN
CBC
CBO'
,.,...
,.-,, ,,..,."
CBDA
I-
~ t
O@ ~0ooo
u o
~= -~
ac: '~
Q
W
O.
i
U
2>
o .
C8D ~ o
(D ~ i
THC
CBN
CBc
% ? ,a ~ -.
CBD'
o
•- ~
" ~ ._
CSD~ o
DISCUSSION
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
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
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.
REFERENCES
323
Hanf-Pflanze und ihre Umwandlung zu Haschisch-Inhaltstoffen. Liebigs Ann.
Chem. 713 : 166.
12. Claussen, U., Spulak, F. V. & Korte, F. (1966). Zur chemischen Klassifizierung
von Pflanzen XXXI. Haschisch X. Cannabichromen, ein neuer Haschisch-Inhalts-
Stoff. Tetrahedron. 22 : 1477.
13. Ctaussen, U., Spulak, F. V. & Korte, F. (1967). Haschisch XIV. Tetrahedron. 24 :
1021.
14. Duquenois, P. & Moustafa, H. N. (1938). Identification and assay of Cannabis
indica, or. Egypt. Med. Assoc. 21 : 224.
15. Farmilo, C. G., Davis, T. W. M., Mandenheuvel, F. A. & Lane, R. (1962).
Chemical analysis of marihuana, IV. Biogenesis, paper chromatography, gas
chromatography. Proc. Can. Soc. Forensic ScL 1, paper 2.
16. Fietz-David, H. E. & Blangy, L. (1949). Fundamental process of dye chemistry.
Interscience Publisher Inc., New York.
17. Gaoni, Y. & Mechoulam, R. (1964). Hashish I i t - Isolation, structure and partial
synthesis of an active constituent of hashish. Z Amer. Chem. Soc. 86 : 1646.
18. Gaoni, Y. & Mechoutam, R. (1966). Cannabichromene, a new active principle in
hashish. Chem. Commun. (1) : 20.
19. Grlic, L. (1970). Simple thin-layer chromatography of cannabinoids by means of
silica gel sheets treated with amines. J. Chromatog. 48 : 562.
20. Gibbs, H. D. (1927). Phenol tests, I I I - The indophenol test. J. Biol. Chem. 72 :
649.
21. Hamilton, H. C., Lescohier, A.W. & Perkins, R.A. (1913). The physiological
activity of Cannabis sativa. J. Amer. Pharm. Assoc. 2 : 322.
22. Heaysman, L. T., Walker, E. A. & Lewis, D.T. (1967). The application of gas
chromatography to the examination of the constituents of Cannabis sativa.
Analyst. 92 : 450.
23. Hilvely, R. L., Mosher, W. A. & Hoffman, F. W. (1966). Isolation of trans - A6
-tetrahydrocannabinol from marijuana. J. Amer. Chem. Soc. 88 : 1832.
24. Kane, V. V. & Razdan, R. K, (1968). Constituents of hashish. A novel reaction of
olivetol with citral in the presence of pyridine. Total synthesis of d 1 cannabi-
cycol and d 1 cannabiehromene. J. Amen. Chem. Soc. 90 : 6551.
25. Korte, F., Haag, M. & Claussen, U. (1965). Zur chemischen Klassifizierung von
Pflanzen. XXVIII. Haschisch VII-Tetrahydrocannabinol-carbonsaiire, ein neuer
Haschisch-Inhaltstoff. Angew. Chem. 77 : 862.
26. Korte, F. & Sieper, H. (1964 a). Zur chemischen Ktassifizierung yon Pflanzen.
XXIV. Untersuchung yon Haschisch-lnhaltstoffen dutch Dttnnschichtchromato-
graphic. J. Chromatog. 13 : 90.
27. Korte, F. & Sieper, H. (1964 b). Zur chemischen Klassifiziemng yon Pflanzen.
XXV. Bestimmung yon Haschisch-Ingaltstoffen durch Diinnschhchtchromato-
graphie. J. Chromatog. 14: 178.
28. Korte, F. & Sieper, H. (1965). Recent results of hashish analysis; cited in
'Hashish, its chemistry and pharmacology', Ciba Foundation Study Group No. 21,
Churchill, London (Eis. G. E. W. Wolstenholme and J. Knight) 15.
29. Krejci, Z. (1961). Substances with antibacterial and hashish effect in hemp.
Casopis, L ekaru Ceshy ch. 100 : 1351.
30. Krejci, Z., Horak, M. & Santavy, F. (1958). Constitution of the cannabidiolic
acid and an acid of the m.p. 133 °, isolated from Cannabis sativa. Acta Univ.
Palackianae Olomucensis. 16 : 9.
31. Krejci, Z., Horak, M. & Santavy, F. (1959). Cannabis sativa, an antibiotic medi-
324
cinal agent III. Isolation and constitution of two acids obtained from C. sativa.
Pharmazie. 14 : 349.
32. Krejci, Z. & Santavy, F. (1955). The isolation of further substances from the
leaves of Indian hemp (Cannabis sativa L. var. indiea}. Acta Univ. Palackianae
Olomucensis. 6 : 59.
33. Mechoulam, R. (1964). Cannabis. Harokeah Hawri. 10 : 96.
34. Mechoulam, R. Ben-Zvi, Z., Yagnitinskey, B. & Shani, A. (1969). New tetra-
hydrocannabinolic acid. Tetrahedron Left. 2339.
35. Mechoulam, R. & Gaoni, Y. (1965). Hashish IV, Isolation and structure of canna-
binolic, cannabidiolic and cannabigerolic acids. Tetrahedron. 21 : 1223.
36. Mechoulam, R. & Gaoni, Y. (1967). Recent advances in the chemistry of Hashish.
Fortsehr. Chem. Org. Naturstoffe. 25 : 175.
37. Morill, F. T. (1938). Marihuana, the new dangerous drug. Washington, Foreign
Policy Assoc. Inc.
38. Obata, Y. & Ishikawa, Y. (1966). Constituents of hemp plant, I I I - Isolation of
Gibbs-positive compound from Japanesehemp.Agr. Biol. Chem. (Tokyo). 30 : 6i9.
39. Okamoto, K. (1967). Compounds of Japanese Cannabis. Kagaku Keisatsu
Keukyush Kokoku. 20 : 109.
40. Schultz, O. E. & Haffner, G. (1958). A sedative principle in German hemp.
(Cannabis sativa). Arch. Pharm. Berl. 291 : 391.
41. Schultz, O. E. & Haffner, G. (1959). A sedative and antibacterial principle in
German hemp. (Cannabis sativa). Z. Naturforsch. 146 : 98.
42. Shoyama, Y., Fujita, T., Yamauchi, T. & Nishioka, I. (1968). Cannabis I I - Canna-
bichromenic acid, a genuine substance of cannabichromene. Chem. Pharm. BulL
(Tokyo). 16 : 1157.
43. Shoyama, Y., Yamauchi, T. & Nishioka, I. (1970). Cannabis V - Cannabigerolic
acid monomethylether and cannabinolic acid. Chem. Pharm. Bull (Tokyo). 18 :
1327.
44. Spulak, F. V., Claussen, U., Fehlhaber, H.W. & Korte, F. (1968). Haschisch
X I X - Tetrahydrocannabitriol-cannabidiolcarbon-s~iureester, ein neuer Haschisch-
tnhaltstoff. Tetrahedron. 24 : 5379.
45. Stone, H. M. (1969). An investigation into forensic chemical problems associated
with Cannabis. United Nations Secretariat Document ST/SoA /SER, S/18.
46. Turk, R. E., Daharir, H. I. & Forney, R. B. (1969 a). Simple chemical method to
identify marihuana. Z Forensic Soft 14 : 349.
47. Turk, R. E. Forney, R. B., King, L. G. & Ramachandram, S. (1969 b). Extraction
and chromatographic isolation, purification and identification of tetrahydrocan-
nabinol and other compounds form marihuana. J. Forensic ScL 14 : 385.
48. Vollner, L , Bieniek, D. & Korte, F. (1969). Haschisch X X - Cannabidiverin, ein
neuer Haschisch-Inhaltstoff. Tetrahedron Lett. 145.
49. Watt, J. M. & Breyer-Brandwijk, H.G. (1962). The Medicinal and poisonous
plants of Southern and Eastern Africa. E. & S. Levingston Ltd. 2nd ed.
50. Wolf, P. O. (1949). Marihuana in Latin America, the threat it constitutes',
Washington. Linacre Press.
51. Yamauchi, T., Shoyama, Y., Aramaki, H., Azuma, T. & Nishioka, I. (1967).
Tetrahydrocannabinolic acid, a genuine substance of tetrahydrocannabinol.
Chem. Pharm. Bull. (Tokyo). 15 : 1075.
52. Yamauchi, T., Shoyama, Y., Matsuo, Y. & Nishioka, I. (1968). Cannabis llI.
Cannabigerol monomethyl ether, a new component of hemp. Chem. Pharm. Bull.
(Tokyo). 16 : 1164.
325