The ferric reducing antioxidant power (FRAP) assay was adapted from
the method of Benzie and Strain (65). FRAP reagent contained 5 mL of 10 mM TPTZ in
40 mM HCl plus 5 mLof 20 mM FeCl3·6H2O and 50 ml of 0.3 M acetate buffer (pH
3.6) and then warmed at 37 °C before using. FRAP reagent (180 μL) was mixed with
20 μL of sample in sterile disposable 96-well microplate. The absorbance at 595 nm
was measured at 120 min in the dark condition using a microplate reader. Ethanolic
solutions of known concentration of ferrous salt, ranging from 10-100 μM (final
concentration) FeSO4·7H2O, was used for the preparation of the calibration curve.
The equivalent concentration (EC1) was defined as the concentration of antioxidant
having a ferric-TPTZ reducing ability equivalent to that of 1 mM FeSO4·7H2O. EC1
was calculated as the concentration of antioxidant giving an increase in the FRAP
assay equivalent to the theoretical absorbance value of a 1 mM concentration of
ferrous salt solution, determined by using the corresponding regression equation. (±)-
α-Tocopherol served as known antioxidants. All measurements were taken in triplicate
and the mean values were calculated.