Determination of total antioxidant activity
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was done as described by Benzie and Strain (1996) and Huang et al. (2005). The oxidant in the FRAP assay was prepared by mixing TPTZ (2.5 ml, 10 mM in 40 mM HCl), 25 ml of 300 mM acetate buffer, and 2.5 ml of FeCl3 •H2O (20 mM). The combination of all these reagents was referred to as “FRAP reagent”. An amount of 1.8 ml freshly prepared FRAP reagent was taken in a test tube and incubated at 30°C in water bath for 10 min. Then, absorbance was taken at 0 min (t0). Immediately, 100 μl of sample extract or standard and 100 μl of distilled water was added to the test tube, mixed and incubated at 30°C for 30 min. Then, the absorbance was taken at 593 nm (t30). Ferrous sulfate was used as standard. The antioxidant potential of the sample extract was determined against a standard curve of ferrous sulphate and the FRAP value was expressed as μM Fe2+ equivalents per gram of extract and calculated using the following equation: