DPPH radical scavenging activity

The capacity to scavenge the ‘‘stable’’ free radical DPPH by n-butanolic and ethyl acetate fraction was measured according to Hanato et al (1998) which is based on the reduction of methanolic solution of the coloured free radical of 1, 1- diphenyl-2-picryl hydrazyl (DPPH). A methanol DPPH solution (0.1 mM, 1 ml) was mixed with serial dilutions (10, 20, 40, 60, 80μg/ml) of the n-butanolic fraction and ethyl acetate fraction and incubated for 30 min at room temperature (250C). For each concentration the assay was run in triplicate and the absorbance was read at 517 nm using microplate reader (Powerwave XS, Biotek, USA). Ascorbic acid (Loba chiemie, India) was used as standard to compare with fractions. IC50 (the antiradical dose required to cause a 50% inhibition) for ascorbic acid, n-butanolic and ethyl acetate fraction was determined. The ability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenging effect (%) = (ADPPH – A test) / ADPPH X 100

Evaluation of total antioxidant capacity by phosphomolybdenum method

The total antioxidant capacity of fractions was evaluated by the method of Prieto et al (1999). The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH (9). 0.1 ml of fraction (100-800 μg/ml) was mixed with 1 ml of the reagent solution (28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid) and sample was incubated at 950C for 90 min. After the mixture had cooled to room temperature, the absorbance of each solution was measured at 695 nm using an UV/Vis spectrophotometer (Beckman DU-530). The values are presented as the means of triplicate analysis. The antioxidant capacity was expressed as ascorbic acid equivalent by using the standard ascorbic acid graph.