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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 1Department 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

A B S T R A C T

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.

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

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.

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

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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 ion-ferrozine 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

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.

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Table 3: Mineralsm (ppm) and proximate analysis (%) of seeds and whole plant of Cannabis sativa No K Na P Ca Mg Moisture Dry

matter Ash Crude

protein Ether extract

Crude fiber

Non free extract

Seeds 8.9 3.5 2.5 4.5 0.125 10.0 97.0 11.0 19.4 30.9 18.0 49.5 Whole plant 7.2 3.0 2.o 4.3 0.118 9.1 97.0 9.1 20.0 23.3 18.5 33.7

Table 4 and Figure 1: Amino acid profile contents of seeds plant of cannabis sativa

No Compound Name

Amount [mg/100g]

Amount [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Aspartic Acid Threonine Serine Glutamic Acid Glycine Alanine Cystine Valine Methionine Isolucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonia Arginine Total

949.293 404.940 360.196 1477.718 380.554 617.013 69.494 757.187 135.901 590.980 853.554 233.288 547.939 309.147 385.485 667.531 1462.171 10202.391

9.3 4.0 3.5

14.5 3.7 6.0 0.7 7.4 1.3 5.8 8.4 2.3 5.4 3.0 3.8 6.5

14.3 100.0

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

No Compound Name

Amount [mg/100g]

Amount [%]

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

Aspartic Acid Threonine Serine Glutamic Acid Glycine Alanine Cystine Valine Methionine Isolucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonia Arginine Total

1074.952 556.200 377.853

1317.260 552.004 671.815 76.506 845.621 99.412 704.518

1018.441 315.432 664.192 273.131 568.660 675.416 927.261

10718.673

10.0 5.2 3.5 12.3 5.1 6.3 0.7 7.9 0.9 6.6 9.5 2.9 6.2 2.5 5.3 6.3 8.7

100.0

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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