The FRAP assay is a method of measuring the ability of reductants (antioxidants) to reduce Fe3+–Fe2+. The formation of blue coloured Fe2+–TPTZ complex (Fe2+ tripyridyltriazine) increases the absorbance at 593 nm. The method of Kubola and Siriamornpun
(2008) was used with some modifications. The FRAP reagent was freshly prepared by mixing 100 mL of acetate buffer (300 mM, pH 3.6), 10 mL TPTZ solution (10 mM TPTZ in 40 mM/HCl), 10 mL
FeCl36H2O (20 nM) in a ratio of 10:1:1 and 12 mL distilled water, at 37 C. To perform the assay, 1.8 mL of FRAP reagent, 180 lL Milli-Q water and 60 lL sample, standard or blank were then added to the same test tubes, and incubated at 37 C for 4 min;
absorbance was measured at 593 nm, using the FRAP working solution as a blank. The reading of relative absorbance should be within the range 0–2.0; otherwise, the sample should be diluted.
In the FRAP assay, the antioxidant potential of sample was determined from a standard curve plotted using the FeSO47H2O linear regression equation to calculate the FRAP values of the sample.