Ferrous sulphate

The ferric reducing antioxidant power (FRAP) assay was carried out according to the technique of Surin et al. [31 ]. The FRAP reagent was prepared by mixing 25 mL of 0.3 M acetate buffer (pH 3.6), 2.5 mL of 4,6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich, Darmstadt, Germany) solution in 40 mM HCl (RCI Labscan, Bangkok, Thailand), and 2.5 mL of 20 mM ferrous sulphate (Loba Chemie, Mumbai, India). Then, 50 µL of sample (5 mg/mL) was mixed with 950 µL of FRAP reagent and incubated in the dark for 30 min. Absorbance was measured at 593 nm in a 96-well microplate. The ferric reducing antioxidant power of the sample was determined based on ferrous sulphate (Merck KGaA, Darmstadt, Germany). Results were expressed as ferrous sulphate equivalent antioxidant capacity (FEAC) with µmol Fe²⁺/g sample.