2.8. Ferric Reducing Antioxidant Power (FRAP) Assay

The antioxidant activity of MAJ was estimated according to the procedure described by Benzie and Strain [37] with some modifications. Briefly, 150 μL of FRAP reagent, prepared freshly and warmed at 37°C, was mixed with 50 μL of MAJ; distilled water was used as the reagent blank. The FRAP reagent contained 1 mL of 10 mM TPTZ solution in 40 mM HCl plus 1 mL of 20 mM FeCl₃·6H₂O and 10 mL of 0.3 M acetate buffer (pH 3.6). Absorbance at 593 nm was measured in a microplate reader (SpectraMax M2). Aqueous solutions of known Fe (II) concentrations in the range of 15.6-125 μM (FeSO₄·7H₂O) were used for calibration (r²= 0.999). Ascorbic acid was used as antioxidant standard and positive control. The absorbance of the samples was compared to a FeSO₄ standard curve and the FRAP values were expressed as μM of ferrous equivalent.