World Applied Sciences Journal 19 (7): 918-923, 2012

ISSN 1818-4952
IDOSI Publications, 2012
DOI: 10.5829/idosi.wasj.2012.19.07.1922

Estimation of Biologically Active Cannabinoids in Cannabis indica by

Gas Chromatography-mass Spectrometry (GC-MS)
1,2
Muhammad Nasimullah Qureshi, 2Farina Kanwal,
2
Muhammad Siddique, 2Inayat-ur-Rahman and 2Muhammad Akram

1
Department of Chemistry, Abdul Wali Khan University Mardan, Pakistan
2
Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar-25120, Pakistan

Abstract: Cannabis indica was collected from five different localities of Pakistan and analyzed for medicinally
active compounds by GC-MS. The biologically active chemical compounds amongst the naturally occurring
cannabinoids are delta 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) which were present in all samples
in high percentage. The highest percentage of THC present was 23.84% and that of CBD was 54.48%. Other
canabinoids quantified were: delta 9-tetrahydrocannabivarin (THCV), cannabivarin (CBV), cannabichromene
(CBC), cannabipinol (CBP), cannabigerl (CBG), hexahydrocannabinol (HHCBN), cannabinol (CBN). The main
objective of the study was to investigate biologically active compounds of Cannabis sp. from various localities.
Results from the study proved that Cannabis is not meant only for its recreational purposes but the presence
of biologically active medicinal compounds in high concentrations make it a valuable source to be used in
herbal preparation for different ailments.

Key words: Cannabis indica % GC-MS % Cannabinoids

INTRODUCTION the plants [8,9]. Approximately 61 cannabinoids are

Cannabis have a long history, dating back several
thousand years when plant was first discovered and used
by Indian and Chinese civilization for recreational,
medicinal, religious and industrial purposes [1,2]. It is
among the very oldest of economic plants, providing fiber
for the manufacture of textile and ropes, [3] and for drug
resin [4]. Medicinal properties of the plants were
recognized in China 2700 years ago for the relief of pain,
muscular spasms, convulsions, epilepsy, asthma and
rheumatism [5]. An Irish surgeon OShaughnessy
introduced cannabis to Europe in 1842 after observing its
therapeutic use [6]. His studies showed the efficacy of
Cannabis as a muscle relaxant, anti-convulsant, anti-
emetic and analgesic. However, similar to other herbal
preparations, its potency was unreliable, contributing
to decline of its therapeutic use.
The chemistry of the Cannabis is quite complex and
the isolation and extractions of the active ingredients are
difficult even today [7]. Cannabinoids are terpenophenolic
compounds unique to Cannabis. They are produced by
glandular trichomes that occur on most aerial surfaces of
known to exist, although some of these are breakdown
products or artifacts [10,11]. The most active chemical
compound of naturally occurring cannabinoids is delta 9-
tetrahydrocannabinol (THC). It was isolated and
synthesized in 1964 and is clearly the most
pharmacologically active [12]. Some of its analogues are:
cannabidiol (CBD), cannabinol (CBN) and cannabigerol
(CBG) etc. Cannabinol (CBN) and cannabidiol (CBD) are
the aromatic derivatives of THC. These compounds have
been isolated from different cannabis species [13].
Despite all its medicinal properties, Cannabis faced
an onslaught of prohibitive legislation in the early
twentieth century, leading to its elimination from
pharmacies across the globe. It was banned due to its
increasing use as recreational Cannabis among the youth.
An emphasis on the negative aspect of Cannabis as a
recreational drug, mainly based on unauthorized use of
smoked herb of indifferent quality, has made it difficult to
achieve an objective risk benefit analysis [14]. However,
in 1986 in USA tetrahydrocannabinol (THC), the main
constituent of Cannabis, was licensed as an anti-emetic
drug in cancer patients receiving chemotherapy.

Corresponding Author: Dr. Muhammad Nasimullah Qureshi, Department of Chemistry,

Abdul Wali Khan University Mardan, Pakistan. Tel: +92-937-929122.
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In addition, in clinical studies THC has shown significant
stimulation of appetite and increase of body weight in HIV
positive and cancer patients. As this plant is abundantly
available in Pakistan, the present study is aimed to
analyze the species of Cannabis indigenous to Pakistan,
especially Khyber Pukhtoonkhwa for its most important
class of natural phytocannabinoids i.e. THC, CBD and
CBN. Cannabis species found in North West region of
Pakistan are high in THC contents with a widely varying
ratio of cannabidiol content. In reference to the content of
THC and CBD, it is possible to distinguish between drug
hemp and fiber hemp. The phenotypes of Cannabis are
characterized by the ratio of THC and CBD. For drug
hemp this ratio is greater than 1.0 and for fiber hemp it is
less than 1.0. So by calculating the Cannabis phenotype
ratio fiber and drug hemp can be differentiated.
Experimental
Extraction: Five samples of Cannabis resin were collected
from Teerah Valley of Khyber Pukhtoonkhwa, Pakistan
and authenticated at the herbarium of PCSIR Laboratories
Complex Peshawar. 10 g resin from each sample was
ground and extracted with 200 ml of n-hexane under
reflux maintaining the temperature between 40-50C for
75 minutes. The extract was then evaporated under
vacuum and stored in refrigerator.
GC-MS Analysis of Extract
Sample Preparation: From the above extract 0.2 g sample
was taken and stirred in 15 ml of n-hexane. 30 ml of
acetonitrile saturated with n-hexane was added and mixed
in separating funnel. Acetonitrile layer was collected and
600 ml of aqueous solution containing 2% NaCl and 100
ml of n-hexane was added. The mixture was thoroughly
mixed and organic layer was collected. Water drops
present in organic layer were removed by the addition of
anhydrous Na2SO4 and solution was filtered and
evaporated to dryness. The residue was reconstituted in
1 ml of cyclohexane. Solution was filtered and 1 l of the
filtrate was injected into the GC-MS.
Instrumentation: A gas chromatograph from Shimadzu
hyphenated to a mass spectrometer QP 2010 plus
(Tokyo, Japan) equipped with an auto-sampler (AOC-20S)
and auto-injector (AOC-20i) was used. Helium was used
as carrier gas. All chromatographic separations were
performed on a capillary column (DB-5ms; Technokroma)
having specifications: length; 30 m, i.d.; 0.25 mm,
thickness; 0.25 m. Other GC-MS conditions are: ion
source temperature (EI); 280 C, interface temperature;
280C, solvent cut time; 2 min. 1 L of sample and
standard were injected into the GC column. Injector
temperature was 300C and Injector was operated in split
mode. The column temperature program started at 100 C
for 2 min and changed to 300C at the rate of 10C/min.
The temperature was hold for 10 minutes. Total elution
time was 42 minutes. MS scanning was performed from
m/z 85 to m/z 380. GC-MS solutions software provided by
the supplier was used to control the system and to
acquire the data. Identification of the compounds was
carried out by comparing the mass spectra obtained with
those of standard mass spectra from the NIST library
(NIST 05).
RESULTS AND DISCUSSION
There are wide variations in the relative amounts of
cannabinoids in Cannabis [15]. Many factors have been
considered responsible for these variations. These
include the genetic characteristics, environment, maturity,
sex, part of harvested plant and the time which has
elapsed between harvesting and chemical analysis, as well
as the conditions of storage of the plant. The chemical
composition of different parts of the plant also varies.
In present study plant resins were collected because
delta 9-tetrahydrocannabinol is found in plants flowering
or fruit tops, leaves and resin. It has been reported that
Cannabis resin has a higher THC contents than rest of
the plant material.[16] Table 1 shows percentage
concentrations of different canabinoids found in five
samples of Canabis indica collected from various points

Table 1: Percentage concentration of different canabinoids in Canabis indica from various localities
Sample Storage time THCV CBV CBD CBC CBP CBG HHCBN THC CBN THC+CBN/CBD
CN-1 1 month 2.03 0.72 53.41 - - 0.36 0.52 23.84 16.51 0.76
CN-2 6 month 1.65 0.65 54.48 1.29 - 0.32 0.47 23.60 16.89 0.74
CN-3 1 year 1.44 0.65 49.18 4.51 1.84 0.70 0.28 24.27 15.07 0.80
CN-4 2.5 years 0.11 5.14 39.98 - - - 0.12 2.29 52.0 1.36
CN-5 4 years 0.03 1.92 26.60 1.77 1.59 0.11 0.82 1.03 67.40 2.57

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Fig. 1: Continued

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Fig. 1: GC chromatograms of canabinoids quantified in Canabis indica

while chromatograms obtained from GC-MS analyses of
Canabis indica are shown in the figure 1. The highest
percentage of THC is 23.84% in CN-1 while CN-5 shows
lowest percentage i.e.1.03%.There are several reasons
for this variation. THC is usually present in Cannabis
plant as a mixture of monocarboxylic acids, which is
readily and efficiently decarboxylated upon heating [17].
THC decomposes when exposed to air, heat and light.
Storage conditions of plant material are also very
important because THC binds readily to glass and plastic,
reducing recoveries during analysis [18]. Besides these
the other important reason is that with the passage of
time, THC content in plant material gradually decreases as
a result of oxidation to cannabinol (CBN). Cannabinol is
a chemical degradation product and its relative abundance
increases with the aging of the sample [19]. Our results
show the same trend as sample 1 which is the fresh
sample analyzed with in a month, delivered highest
concentration of THC and lowest concentration of CBN
while sample 5 (analyzed after 4 years) as its THC
contents are lowest and CBN contents are highest.
Tetrahydrocannabivarin (THCV) on storage gives rise to
cannabivarin (CBV) [20]. These two products CBN and
CBV are present in high concentration in old samples and
are in low concentration in fresh samples.
Cannabidiol CBD is an anti psychotic cannabinoid
found in hemp plant. It is a major constituent of the plant,
representing up to 40% in its extracts [21]. In table sample
I show percentage of CBD as 53.41% while lowest
percentage is present in sample 5 i.e.26.60%. Cannabidiol

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is not psychoactive and was thought not to affect
the.psychoactivity of THC [22]. However recent
evidence shows that users of cannabis with a high
CBD/THC ratio were less likely to experience
schizophrenia like symptoms [23]. It has been studied that
the presence of cannabidiol ameliorates the psychoactive
effect of THC [24] therefore, patients prefer to use milder
forms of Cannabis that have a significant percentage of
CBD. The reason for this milder effect is that CBD block
the metabolism of delta 9-THC to 11-Hydroxy-THC, which
is more psychoactive than THC and may produce
dysphoria. CBD has a number of potentially therapeutic
effects including anti-inflammatory,anti-convulsant,
muscle relaxant, analgesic, anti-emetic and anxiolytic
properties [25].
On the basis of cannabidiol to tetrahydrocannabinol
ratio Cannabis has been classified into drug type and
fibre type [26]. Fetterman et al proposed that
Cannabis should be classified as drug type if the ratio
(THC% + CBN%)/CBD% was greater then 1.0 and fiber
type if the ratio is less than 1.0 [27]. Small and Beckstead
also supported this theory [28]. It is clear from the table
that most of the samples have this ratio near to 1 or more
than 1; so they are classified as drug type Cannabis.
Other canabinoids quantified as shown in the table were:
cannabichromene (CBC), cannabipinol (CBP), cannabigerl
(CBG), hexahydrocannabinol (HHCBN).
CONCLUSION
The significance advances in cannabinoid research
has opened new frontiers and are leading to a better
understanding of cannabis effect in humans.Medicinal
Cannabis must be of consistent quality, because
Cannabis based medicines are more complex, containing
a mixture of cannabinoids, terpens and flavonoids.
These compounds interact synergistically with one
another [29]. A medicine must be safe and effective for its
purpose. For the development of herbal medicine, the
plant material chosen must be thoroughly investigated.
Current study showed the percentage of different
cannabinoids present in Cannabis collected from
North-West area of Pakistan. The plants under study
showed high percentages of CBD and THC which are the
most important active constituents of Cannabis. For the
development of Cannabis based medicines for specific
indications e.g. migraine, antiemetic, epilepsy etc, it is
necessary to investigate the specific ratios of
cannabinoids in plant materials used for such
preparations.
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ACKNOWLEDGEMENT
This research was supposrted and financed by the
PCSIR Laboratories Complex, Peshawar.
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