3.6. FRAP Assay

The FRAP assay was adapted from Benzie and Strain FRAP [99]. Briefly, the FRAP reagent was freshly prepared by mixing 10 mM TPTZ, 20 mM FeCl₃, and 0.25 M pH 3.6 acetate buffer at 1:1:10 (volume ratio). The test sample (x = 0–10 μL, 0.5 mg/mL) was added to (20 − x) μL of 95% ethanol followed by 80 μL of FRAP reagent. The absorbance was measured at 595 nm after a 30 min incubation at 37 °C using distilled water as the blank. The relative reducing power of the sample compared to the maximum absorbance was calculated using the formula of Section 3.5.