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World Applied Sciences Journal 19 (7): 918-923, 2012
ISSN 1818-4952
© IDOSI Publications, 2012
DOI: 10.5829/idosi.wasj.2012.19.07.1922
Corresponding Author: Dr. Muhammad Nasimullah Qureshi, Department of Chemistry,
Abdul Wali Khan University Mardan, Pakistan. Tel: +92-937-929122.
918
Estimation of Biologically Active Cannabinoids in Cannabis indica by
Gas Chromatography-mass Spectrometry (GC-MS)
Muhammad Nasimullah Qureshi, Farina Kanwal,
1,2 2
Muhammad Siddique, Inayat-ur-Rahman and Muhammad Akram
2 2 2
Department of Chemistry, Abdul Wali Khan University Mardan, Pakistan
1
Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar-25120, Pakistan
2
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 severalproducts or artifacts [10,11]. The most active chemical
thousand years when plant was first discovered and usedcompound of naturally occurring cannabinoids is delta 9-
by Indian and Chinese civilization for recreational,tetrahydrocannabinol (THC). It was isolated and
medicinal, religious and industrial purposes [1,2]. It issynthesized in 1964 and is clearly the most
among the very oldest of economic plants, providing fiber pharmacologically active [12]. Some of its analogues are:
for the manufacture of textile and ropes, [3] and for drugcannabidiol (CBD), cannabinol (CBN) and cannabigerol
resin [4]. Medicinal properties of the plants were(CBG) etc. Cannabinol (CBN) and cannabidiol (CBD) are
recognized in China 2700 years ago for the relief of pain,the aromatic derivatives of THC. These compounds have
muscular spasms, convulsions, epilepsy, asthma andbeen isolated from different cannabis species [13].
rheumatism [5]. An Irish surgeon O’ShaughnessyDespite all its medicinal properties, Cannabis faced
introduced cannabis to Europe in 1842 after observing its an onslaught of prohibitive legislation in the early
therapeutic use [6]. His studies showed the efficacy oftwentieth century, leading to its elimination from
Cannabis as a muscle relaxant, anti-convulsant, anti-pharmacies across the globe. It was banned due to its
emetic and analgesic. However, similar to other herbal increasing use as recreational Cannabis among the youth.
preparations, its potency was unreliable, contributingAn emphasis on the negative aspect of Cannabis as a
to decline of its therapeutic use. recreational drug, mainly based on unauthorized use of
The chemistry of the Cannabis is quite complex andsmoked herb of indifferent quality, has made it difficult to
the isolation and extractions of the active ingredients areachieve an objective risk benefit analysis [14]. However,
difficult even today [7]. Cannabinoids are terpenophenolic in 1986 in USA tetrahydrocannabinol (THC), the main
compounds unique to Cannabis. They are produced byconstituent of Cannabis, was licensed as an anti-emetic
glandular trichomes that occur on most aerial surfaces ofdrug in cancer patients receiving chemotherapy.
known to exist, although some of these are breakdown
World Appl. Sci. J., 19 (7): 918-923, 2012
919
In addition, in clinical studies THC has shown significant Instrumentation: A gas chromatograph from Shimadzu
stimulation of appetite and increase of body weight in HIV hyphenated to a mass spectrometer QP 2010 plus
positive and cancer patients. As this plant is abundantly(Tokyo, Japan) equipped with an auto-sampler (AOC-20S)
available in Pakistan, the present study is aimed toand auto-injector (AOC-20i) was used. Helium was used
analyze the species of Cannabis indigenous to Pakistan,as carrier gas. All chromatographic separations were
especially Khyber Pukhtoonkhwa for its most importantperformed on a capillary column (DB-5ms; Technokroma)
class of natural phytocannabinoids i.e. THC, CBD andhaving specifications: length; 30 m, i.d.; 0.25 mm,
CBN. Cannabis species found in North West region ofthickness; 0.25 µm. Other GC-MS conditions are: ion
Pakistan are high in THC contents with a widely varyingsource temperature (EI); 280 °C, interface temperature;
ratio of cannabidiol content. In reference to the content of 280°C, solvent cut time; 2 min. 1 µL of sample and
THC and CBD, it is possible to distinguish between drugstandard were injected into the GC column. Injector
hemp and fiber hemp. The phenotypes of Cannabis aretemperature was 300°C and Injector was operated in split
characterized by the ratio of THC and CBD. For drugmode. The column temperature program started at 100 °C
hemp this ratio is greater than 1.0 and for fiber hemp it isfor 2 min and changed to 300°C at the rate of 10°C/min.
less than 1.0. So by calculating the Cannabis phenotypeThe temperature was hold for 10 minutes. Total elution
ratio fiber and drug hemp can be differentiated. time was 42 minutes. MS scanning was performed from
Experimental the supplier was used to control the system and to
Extraction: Five samples of Cannabis resin were collectedacquire the data. Identification of the compounds was
from Teerah Valley of Khyber Pukhtoonkhwa, Pakistancarried out by comparing the mass spectra obtained with
and authenticated at the herbarium of PCSIR Laboratories those of standard mass spectra from the NIST library
Complex Peshawar. 10 g resin from each sample was(NIST 05).
ground and extracted with 200 ml of n-hexane under
reflux maintaining the temperature between 40-50°C for RESULTS AND DISCUSSION
75 minutes. The extract was then evaporated under
vacuum and stored in refrigerator. There are wide variations in the relative amounts of
GC-MS Analysis of Extract considered responsible for these variations. These
Sample Preparation: From the above extract 0.2 g sampleinclude the genetic characteristics, environment, maturity,
was taken and stirred in 15 ml of n-hexane. 30 ml ofsex, part of harvested plant and the time which has
acetonitrile saturated with n-hexane was added and mixed elapsed between harvesting and chemical analysis, as well
in separating funnel. Acetonitrile layer was collected andas the conditions of storage of the plant. The chemical
600 ml of aqueous solution containing 2% NaCl and 100composition of different parts of the plant also varies.
ml of n-hexane was added. The mixture was thoroughlyIn present study plant resins were collected because
mixed and organic layer was collected. Water dropsdelta 9-tetrahydrocannabinol is found in plant’s flowering
present in organic layer were removed by the addition ofor fruit tops, leaves and resin. It has been reported that
anhydrous Na SO and solution was filtered andCannabis resin has a higher THC contents than rest of
2 4
evaporated to dryness. The residue was reconstituted inthe plant material.[16] Table 1 shows percentage
1 ml of cyclohexane. Solution was filtered and 1 µl of the concentrations of different canabinoids found in five
filtrate was injected into the GC-MS. samples of Canabis indica collected from various points
m/z 85 to m/z 380. GC-MS solutions software provided by
cannabinoids in Cannabis [15]. Many factors have been
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
World Appl. Sci. J., 19 (7): 918-923, 2012
920
Fig. 1: Continued
World Appl. Sci. J., 19 (7): 918-923, 2012
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Fig. 1: GC chromatograms of canabinoids quantified in Canabis indica
while chromatograms obtained from GC-MS analyses ofincreases with the aging of the sample [19]. Our results
Canabis indica are shown in the figure 1. The highestshow the same trend as sample 1 which is the fresh
percentage of THC is 23.84% in CN-1 while CN-5 showssample analyzed with in a month, delivered highest
lowest percentage i.e.1.03%.There are several reasons concentration of THC and lowest concentration of CBN
for this variation. THC is usually present in Cannabiswhile sample 5 (analyzed after 4 years) as its THC
plant as a mixture of monocarboxylic acids, which iscontents are lowest and CBN contents are highest.
readily and efficiently decarboxylated upon heating [17].Tetrahydrocannabivarin (THCV) on storage gives rise to
THC decomposes when exposed to air, heat and light.cannabivarin (CBV) [20]. These two products CBN and
Storage conditions of plant material are also veryCBV are present in high concentration in old samples and
important because THC binds readily to glass and plastic, are in low concentration in fresh samples.
reducing recoveries during analysis [18]. Besides theseCannabidiol CBD is an anti psychotic cannabinoid
the other important reason is that with the passage of found in hemp plant. It is a major constituent of the plant,
time, THC content in plant material gradually decreases as representing up to 40% in its extracts [21]. In table sample
a result of oxidation to cannabinol (CBN). Cannabinol isI show percentage of CBD as 53.41% while lowest
a chemical degradation product and its relative abundance percentage is present in sample 5 i.e.26.60%. Cannabidiol
World Appl. Sci. J., 19 (7): 918-923, 2012
922
is not psychoactive and was thought not to affectACKNOWLEDGEMENT
the.psychoactivity of THC [22]. However recent
evidence shows that users of cannabis with a highThis research was supposrted and financed by the
CBD/THC ratio were less likely to experiencePCSIR Laboratories Complex, Peshawar.
schizophrenia like symptoms [23]. It has been studied that
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