Research Article PHYTOCHEMICAL ANALYSIS AND EVALUATION OF ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF WITHANIA SOMNIFERA ROOT DIPANKAR CHAUDHURI 1 , NIKHIL BABAN GHATE 1 , RHITAJIT SARKAR 1 , NRIPENDRANATH MANDAL 1* 1 Division of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata - 700054, India; * Nripendranath Mandal Division of Molecular Medicine Bose Institute P-1/12 CIT Scheme VIIM Kolkata - 700054, India; Email: mandaln@rediffmail.com Received: 13 September 2012, Revised and Accepted: 18 October 2012 ABSTRACT Withania somnifera, also known as ǮAshwagandhaǯ, is an important medicinal plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years. In view of its varied therapeutic potential, it has also been the subject of considerable modern scientific attention. The present study was performed to evaluate the phytochemicals and in vitro antioxidant activity of 70% methanolic extract of W. somnifera roots. The extract was used to determine the antioxidant potential using different tests including total antioxidant activity; efficiencies for scavenging of hydroxyl, superoxide, nitric oxide, singlet oxygen radicals, hypochlorous acid and inhibition of lipid peroxidation. The results obtained indicate that although W. somnifera has been widely used for centuries as immunostimulants in different ailments including neurodegenerative disorders, the 70% methanolic extract of the same also has possibility as an antioxidant and free radical scavenging agent. Keywords: Phytochemical screening, reactive oxygen species, reducing power INTRODUCTION Since time immemorial, medicinal plants are of great importance to the health of individuals and communities. The medicinal plant products, which are derived from plant parts such as stem bark, leaves, fruits and seeds have been part of phytomedicine that produce a definite physiological action on the human body. The most important of these natural bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds 1 . A number of reactive molecules are generated through various biological redox reactions such as superoxide radical (O2 .- ), hydroxyl radical ( . OH), hydrogen peroxide (H2O2) and nitric oxide (NO . ) 2 which can directly react with biological macromolecules such as proteins, lipids and DNA of healthy human cells and cause cell membrane disintegration, DNA mutation and protein damage. Deregulation of these reactive oxygen species (ROS) can further create cancer, atherosclerosis, cardiovascular disease, liver injury, ageing and inflammatory disease 3 . Antioxidants act as oxygen scavengers by interrupting the oxidation process by reacting with free radicals, chelating catalytic metals 4 . Some synthetic antioxidants were developed in the past few decades but they are suspected of having some adverse effects. Therefore, in search of suitable alternative natural antioxidants has received much attention to identify and develop more potent antioxidants of natural origin to replace synthetic ones. Different kinds of plant material have already been reported as natural antioxidant 5 . Withania somnifera (L.) Dunal (Solanaceae) grows in many of the drier regions of Indian subcontinent and in tropical and subtropical zones of the Mediterranean region and northern Africa to Southwest Asia. In Ayurveda, Withania is widely claimed to have potent aphrodisiac, sedative, rejuvenative and life prolonging properties 6 . The plant was traditionally used to promote youthful vigor, endurance, strength, and health, nurturing the time elements of the body and increasing the production of vital fluids, muscle fat, blood, lymph, semen and cells. The similarity between these restorative properties and those of ginseng roots has led to ashwagandha roots being called Indian ginseng 7 . The roots are used as a nutrient and health restorative in pregnant women and old people. The decoction of the root boiled with milk and ghee is recommended for curing sterility in women. The roots are also used in constipation, senile debility, rheumatism, general debility, nervous exhaustion, loss of memory, loss of muscular energy and spermatorrhoea 8 . It has been used owing to its antioxidant property to treat rheumatism and neurodegenerative disorders 8,9 . Recently, it has been developed as an immunosuppressive agent for the inflammatory diseases 10,11 . However, the scope for the current study lies in the fact that no earlier report has been made for the antioxidant capacity of the 70% methanolic root extract of W. somnifera. MATERIALS AND METHODS Chemicals 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtained from Roche diagnostics, Mannheim, Germany. 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), 2- deoxy-2-ribose, mannitol, lipoic acid, quercetin, sodium nitroprusside, 1,10-phenanthroline and ferrozine were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Folin- ciocalteu reagent, Mercuric chloride, Potassium Iodide, Anthrone, Vanillin, 2,4-Dinitrophenylhydrazine, Thiourea and N,N-dimethyl-4- nitrosoaniline were obtained from Merck, Mumbai, India. DPPH, gallic acid, (+) catechin and curcumin were obtained from MP Biomedicals, France. Catalase, reserpine and Sodium bi carbonate were obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Evans blue, Lead acetate were purchased from BDH, England. D- glucose was procured from Qualigens Fine Chemicals, Mumbai. Diethylene-triamine-pentaacetic acid (DTPA) was obtained from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India. Plant Material and Extraction The plant material was bought from local vendor of Kolkata, West Bengal, India. The root powder (100 g) of the dried root of W. somnifera was stirred using a magnetic stirrer with 1000 ml mixture of methanol: water (7:3) for overnight; then the mixture was centrifuged at 2850 x g and the supernatant decanted. The process was repeated again with the precipitated pellet. The supernatants were collected, concentrated in a rotary evaporator and lyophilized. The dried extract was stored at -20°C until use. Double distilled or MilliQ grade water was used to dissolve the extract and for further dilution during the experiments. Animals The Animal Ethical Clearance Committee of the Institute (Registration number: 95/1999/ CPCSEA) approved the use of adult male Swiss Albino mice (Mus musculus), weighing 20-25 gm for experimentation. Each polypropylene cage contained 4 mice at a time; supplied with ad libitum laboratory diet and water. The mice were kept at 25 ± 2°C and 60 ± 5% humidity and normal photo cycle (12 h dark/12 h light). Phytochemical Analysis Qualitative tests Phytochemical analysis of 70% methanol extract of W. somnifera root extract was carried out using standard qualitative methods as Asian Journal of Pharmaceutical and Clinical Research Vol 5, Suppl 4, 2012 ISSN - 0974-2441 Academic Sciences