2.8. Ferric Reducing Antioxidant Power (FRAP) Assay

The total antioxidant potential of a sample was determined using the ferric reducing ability of plasma FRAP assay [31] as a measure of antioxidant power. Briefly, the FRAP reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), a solution of 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃ at 10:1:1 (v/v/v). The reagent (3000 μL) and brew solutions (100 μL) were added to each sample and mixed thoroughly. The absorbance was taken at 593 nm after 180 min. Standard curve was prepared using different concentrations 100–1000 mM of iron sulfate II. All solutions were used on the day of preparation. FRAP unit determines the ability of the reduction of 1 mM of iron (III) to iron (II). The results were expressed as mM Fe²⁺. Analyses were performed in triplicate on each brews.