Studies on non enzymatic antioxidants of Phyllanhus niruri L

Introduction Oxygen derived free radicals have played a major role in the pathogenesis of a number of degenerative diseases. These free radicals molecules are released during the normal metabolic process of oxidation and thus can lead even to serious diseases like cancer, cardiac associated disorders (CAD), acceleration of the aging process and contribute to arthritis (Cross 1987). Natural foods and food-derived antioxidants such as vitamins, phenolics and phytochemicals have received growing attention because they are known to function as chemo-preventive agents against oxidative damages (Carrasco-Pancorbo et al, 2005; Perez-Bonilla et al, 2006; Valavanidis et al, 2004). Plants constitute an important source of active natural products that differ widely in terms of their structure and their biological properties. In recent years, the prevention of cancer and CAD has been associated with the intake of fresh fruits, vegetables, herbal teas that are rich in natural antioxidants (Virgili et al, 2001). The protective effects of plant products are due to the presence of several components that have distinct mechanisms of action; some are enzymes and proteins, while others are low molecular weight compounds such as vitamins, flavonoids, anthocyanins and phenolic compounds (Amro et al, 2002; Park et al, 2005). Different antioxidant compounds act through different mechanism, no single method can fully evaluate the antioxidant activity. These assays have presented distinct challenges in evaluation of purified individual compound, mixed extracts, fractions, herbs, supplementation of vitamins and other chemicals/medicines. Thus Optimization of right method/s of application and evaluation is the need of time (Pellegrini et al, 1999) Thus there are many different antioxidant components in plants and it is relatively difficult to measure each antioxidant component, separately. Owing to the complexity of the oxidation and anti-oxidation processes, no single testing method was found to be capable of providing a comprehensive picture of the antioxidant profile. Therefore, in the present investigation a multi-method approach was necessary to assess the antioxidant activity of biological samples (Koleva et al, 2002) P. niruri commonly known as Stonebreaker because of its antilithic property. Various bioactivities such as antidiabetic (Okoli et al, 2011), anti-hepatotoxicity, (Ravikumar et al, 2011) antilithic, anti-hypertensive, antiplatelet aggregation activity (Kamal et al, 2012), antimicrobial activity (Mathur et al, 2012), anti-HIV and anti-hepatitis B (Bagalkotkar et al, 2011; Naik and Juvekar , 2003) have been reported. Materials and Methods Chemicals Vitamin A, C and E, the stable free radical DPPH 3-(2pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4-triazine (Ferrozine), TPTZ (2, 4, 6-tris (2pyridyl)-s-triazine) were obtained from Sigma chemicals (Sigma-Aldrich GmbH, Sternheim, Germany). Tissue Culture MS medium (Murashige and Skoog, 1962) was used for initiation and maintenance cultures of P. niruri . Nodal segments were surface sterilized with HgCl2 solution, rinsed thrice with sterile distilled water; Explants were inoculated on Laminar flow hood fitted with ultraviolet light lamp, in the flasks containing culture medium aseptically. Cultured flasks were incubated in culture chamber with temperature maintained at 25 ± 1°C and 1200 lux light was provided for 16 h. The callus culture was established by using various treatment doses of hormones. These calli were maintained for about six months with frequent subculturings at time interval of 4-6 weeks. Growth index (GI) was calculated after 2, 4, 6 and 8 weeks of fresh sub culturing to record the growth pattern. GI= Final wt of the tissue – initial wt of the tissue/ initial wt of the tissue All the plant parts collected were cleaned and oven dried at 100oC for 10 min to deactivate the enzymes and then at 25-28 0 C till constant weight was achieved and then powdered. The voucher specimen of experimental plant was deposited in Herbarium of Department of Botany, University of Rajasthan, Jaipur (RUBL No. 30247).