Ferric-Reducing Antioxidant Power Assay

FRAP assay is a novel method for assessing antioxidant power where the ferric-reducing ability of a sample extract is tested. Ferric to ferrous ion reduction at low pH causes a colored ferrous–tripyridyltriazine complex to form. FRAP values are obtained by comparing the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions in known concentration (Benzie and Strain, 1999). The ferric-reducing antioxidant power (FRAP) assay measures the antioxidant potential of antioxidants to reduce the Fe³⁺/tripyridyl-s-triazine complex present in stoichiometric excess to the blue ferrous form (Hauck, 2018). FRAP reagent was freshly prepared by mixing together 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) and 20 mM ferric chloride in 0.25 M acetate buffer, pH 3.6 in proportions of 1:1:10 (v/v), respectively. Pearl millet extract (50 μl) was added to 1.5 ml of FRAP reagent. The absorbance was read at 593 nm after 4-min incubation at ambient temperature against distilled water as a blank in a plate reader. A calibration curve of ascorbic acid concentration (100–1,000 μmol/L) versus absorbance was used to calculate values, and results are expressed in mg/ml ascorbic equivalents/g dry weight for plant total extracts from three determinations.