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Journal of Ethnobiology and

Ethnopharmacology

ISSN: 2222-1271
www.ijscience.com

In vitro Anti-Oxidant Activity, Phytochemical Screening and Amino Acids Profile of Cannabis
sativa
Esraa MM Ali1, Eltayeb Fadul2, Asaad Khalid2, Ahmed A Elgamel3, Dinna A Hassan4, Samia H Abdelrehman1 and
Khojali, SME1
1

Department of Biochemistry, Nutrition, Toxicology and Pharmacology, Central Veterinary Research Laboratory,
Khartoum, Sudan; 2Medicinal and Aromatic Plants Research Institute, National Centre for Research P.O. Box 2404
Khartoum, Sudan; 3The Poison Center, the National Ribat University, P.O. Box 22, Khartoum, Sudan; 4Department of
Molecular biology, Central Lab. Khartoum, Sudan
ABSTRACT
Since Sudan has a great diversity of indigenous (medicinal) plant species, this research investigated in vitro
antioxidant activity, phytochemical Screening and amino acid profile of Cannabis sativa.
Seeds and whole plant of C.sativa were extracted by methanol and they were subjected to investigate their antioxidant
activity via DPPH and iron chelating assays. The study of amino acids has been done to recognize, which of them
presented in this plant. Results obtained showed that the radical scavenging activity (RSA %) of seeds and whole plant
of C.sativa methanolic extract against ferrous iron and DPPH free radical were found to be (100.01, 490.01% on
C.sativa seeds) , (580.02, 450.01% on C.sativa whole plant), respectively. Phytochemical screening showed that,
C.sativa seeds contain Saponins, and Tannin in a highest concentrations but other constituents as Alkaloid,
Flavonoids, Sterols, Triterpenes, and Cumarins in trace amounts. Anthraquinone glycoside and Cyanogenic glycoside
were missing in this part. C.sativa whole plant confirmed presence of Tannin, Alkaloid, Sterols, and Triterpenes in a
high concentrations but Anthraquinone glycoside, and Cyanogenic glycoside were not Amino in attendance also.
Amino acid profile demonstrated present of 17 acid namely (Aspartic Acid, Threonine, Serine, Glutamic Acid,
Glycine, Alanine, Cystine, Valine, Methionine, Isolucine, Leucine, Tyrosine, Phenylalanine, Histidine, Lysine,
Ammonia, and Arginine) in different concentrations.
These studies show that the proximate analysis test of Cannabis sativa seeds (moisture, Dry mater, Ash, crude protein,
Ether extract, crude fiber and nitrogen free extract NFE) are higher compared to the whole plants.
Keywords: Cannabis sativa, antioxidant activity, amino acids, phytochemical screening and proximate analysis.
*

Corresponding author: Esraa MM Ali, Department of Biochemistry, Nutrition, Toxicology and Pharmacology
Central Veterinary Research laboratory, Khartoum, Sudan
How to cite this article: Ali EMM, E Fadul, A Khalid, AA Elgamel, DA Hassan, SH Abdelrehman and SME Khojali,
2013. In vitro anti-oxidant activity, phytochemical screening and amino acids profile of Cannabis sativa. J Ethnobiol.
Ethnopharmacol, 2(1): 5-8.
delays or inhibits oxidation of oxidizable substrate by
neutralizing free radicals (Antolovich et al., 2002).
Antioxidants activity is an important parameter and it is
widely determined in various plant materials. In the recent
years, the strategy of implementing the diet with
antioxidants especially deriving from natural sources is
becoming more and more convincing against oxidative
stress damage (Vertuani et al., 2002). Therefore, the
objective of the present work was to find the antioxidant
activity of Cannabis sativa.

INTRODUCTION
Cannabis sativa belongs to the family Cannabinaceae
and known by various names worldwide. It is called
Marijuana in America. Bhang, Ganja and Charas in India,
Kif in North Africa, Dogga in South Africa, Takrori in
Tunisia, Habak in Turkey, Hashish in Middle East,
Djomba or liamba in Central Africa and Brazil, Sodom,
Tampl, Gum, Guage, Stuff, Kinshasha, Swala and Whiskt
in Ghana, Grifa in Mexico and Macohna in parts of South
America (Sarpong, 1971; Sachindra and pradhan, 1977). In
Sudan, the most famous names of Cannabis is Bango and
Hashish. The name Bango in Sudan may be derived from
the Indian name Bhang.
Antioxidants have several definitions, but the
common one can be expressed as any substance that

MATERIALS AND METHODS


The seeds and whole plants of Cannabis sativa were
collected from Darfor and Radoum state. The plant was
identified and authenticated by Dr. Ahmed Awad Elgamel
5

6
who is the scientific advisor of the Drug Combat General
Directorate and the manager of the Poison Center of the
National Ribat University. All plant parts were air dried,
under the shadow with good ventilation and then ground
finely in a mill to be used for extracts preparation.
Extraction was carried out according to the method
described by Harbone (1984). To prepare extracts for
screening of antioxidant and amino acids activities, 100 g
air-dried parts of plant were macerated in 500 ml of
Methanol for 3 hours at room temperature with occasional
shaking. The supernatant was decanted and clarity field by
filtration through a filter paper. After filtration, the solvent
was then removed by rotary evaporator at 55C. Each
residue was weighed and the yield percentage was
determined (percent of dry weigh) and stored at -20C for
further analysis in tightly sealed glass vial.
The 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl
stable free radical (DPPH) was determined according to
the method of Farzana et al. (2005) with some
modification. In 96-wells plate, the test samples were
allowed to react with DPPH for half an hour at 37C. The
concentration of DPPH was kept as 300M. The test
samples were dissolved in Dimethyl sulfoxide (DMSO)
while the 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl
stable free radical was prepared in ethanol. After
incubation, decrease in absorbance was measured at
517nm using multiplate reader spectrophotometer.
Percentage radical scavenging activity of samples was
determined in comparison with a DMSO treated control
group. Propyl gallate was used as standard. The change in
absorbance at 517 nm was measured to calculate DPPH
percentage radical scavenging activity by using the
following formula:
RSA% = [100- (AS/AC) x100]
Where the RSA is radical scavenging activity, AS was the
absorbance in the presence of samples and AC was the
absorbance of the control.
The iron chelating ability was determined according
to the modified method of Kexue et al. (2006). The Fe+2
was monitored by measuring the formation of ferrous ionferrozine complex. The experiment was carried out in 96
microtiter ELISA plate with a final volume of 200 l/well.
The plant extracts was mixed with FeSO4. The reaction
was initiated by adding 5mM ferrozine. The mixture was
shaken and left at room temperature for 10 min. The
absorbance was measured at 562 nm. EDTA was used as
standard, and DMSO as control. All tests and analysis
were run in triplicate.
Phytochemical screening is extremely fluent in
providing formation about the nature of constituents
potentially available in each plant sample. It was
considered important to correlate between the nature of
chemical constituents and antioxidant activity. Various
Phytochemical screening test was conduct to detect
secondary plant constituents as Saponins, Tannin,
Anthraquinone, glycoside, alkaloid, flavonoids, sterols,
triterpenes, cyanogenic, glycoside and cumarins present in
plants by described method of Harbone (1973), Trease
and Evans (1989) and Sofowara (1993).
Amino acids content of Cannabis sativa seeds and
whole plant were determined using SAYKAM amino
acids analyzer, S5200 sample injector, S.4300 amino
acids reaction module and S. 2100 solvent delivery

J Ethnobiol Ethnopharmacol, 2013, 2(1): 5-8.


system. Samples were prepared according to the method
described by Sparkman et al. (1958).
In the present work, seeds and whole plant of
Cannabis sativa were extracted by methanol and they
were subjected to investigate their antioxidant activity
using DPPH, iron chelating assays, amino acids profile
and phytochemical screening test. Minerals (Ca, K, Ns, P
and Mg) were determined according to the method of
Butrimovitz and Purdy (1977) using atomic absorption
spectrometry.
Statistical analysis
All data were presented as means S.D. Statistical
analysis for all the assays were done using Microsoft
excel program.
RESULTS
Table 1 shows the DPPH radical scavenging activity,
and ferrous iron-chelating activity of seeds and whole
plant of cannabis sativa. The free radical scavenging
ability of the C. sativa methanol seeds and whole plants
extracts exhibited 49 and 45% respectively. Similarly,
iron chelating was higher in whole plant than seeds.
Phytochemical screening test of C. sativa revealed the
presence of higher concentrations of Saponin and Tannin
in seed than whole plants. Tannin, alkaloid, steroid and
Triterpenes are higher in the whole plants. Anthraquinone
glycoside and Cyanogenic glycoside were not found in
whole plant and seeds (Table 2).
Table 1: Anti-oxidant activity of seeds and whole plant of
Cannabis sativa
No. Sample code
Percent RSA
Percent iron
chelating
1
Whole Plant
450.01
580.02
2
Seeds
490.01
100.01
Table 2: Phytochemical screening of seeds and whole plants of
C. sativa
Test
Whole plants
Seeds
Saponins
+
++
Tannin
++
++
Anthraquinone glycoside
Alkaloid
++
+
Flavonoids
+
+
Sterols
++
+
Triterpenes
++
+
Cumarins
+
+
Cyanogenic glycoside
(-): Represents no substances found in the sample; (+):
Represents the presence of substances is found in traces; (++):
The substance is clearly found

Table 3 showed that the mineral contents which are


high in the seeds than the whole plant and also the
proximate analysis of C. sativa in seeds (moisture, dry
mater, ash, crude protein, ether extract, crude fiber and
nitrogen free extract) are higher compared to the whole
plant.
Table 4 and 5 show the amino profile in seeds and
whole plant. Glutamic acid and arginine in seeds are
higher compare than the whole plants.

J Ethnobiol Ethnopharmacol, 2013, 2(1): 5-8.

Table 3: Mineralsm (ppm) and proximate analysis (%) of seeds and whole plant of Cannabis sativa
No
K
Na
P
Ca
Mg
Moisture Dry
Ash
Crude
matter
protein
Seeds
8.9
3.5
2.5
4.5
0.125
10.0
97.0
11.0
19.4
Whole plant 7.2
3.0
2.o
4.3
0.118
9.1
97.0
9.1
20.0
Table 4 and Figure 1: Amino acid profile contents of seeds
plant of cannabis sativa
No Compound
Amount
Amount
Name
[mg/100g]
[%]
9.3
949.293
Aspartic Acid
1
4.0
404.940
Threonine
2
3.5
360.196
Serine
3
14.5
1477.718
Glutamic Acid
4
3.7
380.554
Glycine
5
6.0
617.013
Alanine
6
0.7
69.494
Cystine
7
7.4
757.187
Valine
8
1.3
135.901
Methionine
9
5.8
590.980
10 Isolucine
8.4
853.554
11 Leucine
2.3
233.288
12 Tyrosine
5.4
547.939
13 Phenylalanine
3.0
309.147
14 Histidine
3.8
385.485
15 Lysine
6.5
667.531
16 Ammonia
14.3
1462.171
17 Arginine
100.0
10202.391
Total

DISCUSSION
The methanolic extracts of Cannabis sativa had an
antioxidant activity and protective effect against
freeradicals. Many epidemiologic studies have addressed
the possible preventive effects of antioxidants in disease
causation and progression (Peter Mller and Steffen Loft,
2003).
Reactive oxygen species, while leading to aging, also
serve an important function of white blood cells by
removing bacteria and other foreign material. The human
body has evolved the ability to produce enzymes and
antioxidants which limit the damage that might be caused
by these free radicals. During times of high oxidative
stress, the body's natural antioxidants are not sufficient to
control these free radicals and excess damage can occur
(Pinnell, 2003).
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Butrimovitz, G.P. and Purdy. W.C. 1977. The
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Ether
extract
30.9
23.3

Crude
fiber
18.0
18.5

Non free
extract
49.5
33.7

Table 5 and Figure 2: Amino acid profile contents of whole


plant of cannabis sativa
No Compound
Amount
Amount
Name
[mg/100g]
[%]
10.0
1074.952
1 Aspartic Acid
5.2
556.200
2 Threonine
3.5
377.853
3 Serine
12.3
1317.260
4 Glutamic Acid
5.1
552.004
5 Glycine
6.3
671.815
6 Alanine
0.7
76.506
7 Cystine
7.9
845.621
8 Valine
0.9
99.412
9 Methionine
6.6
704.518
10 Isolucine
9.5
1018.441
11 Leucine
2.9
315.432
12 Tyrosine
6.2
664.192
13 Phenylalanine
2.5
273.131
14 Histidine
5.3
568.660
15 Lysine
6.3
675.416
16 Ammonia
8.7
927.261
17 Arginine
100.0
10718.673
Total

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