For determination of FRAP response, 20 μL of extract diluted appropriately in DMSO was mixed
with 180 μL FRAP reagent in wells of a 96-well plate, left for 6 minutes, and the absorbance measured
at 595 nm in a Multiskan Ascent plate-reader. FRAP reagent was prepared freshly by mixing 300 mM
acetate buffer pH 3.6, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3.6H20 in the volume ratio
10:1:1. Appropriate blanks of plant extract and of FRAP reagent lacking TPTZ (to correct for color of
the extracts) were run, together with quercetin (in DMSO) and FeSO4 as a standard. FRAP activity was
calculated as Ferrous Equivalents (FE), the concentration of extract/quercetin which produced an
absorbance value equal to that of 1 mM FeSO4. Once again, extracts were first tested at a single
concentration of 4 mg/mL, and those showing good evidence of antioxidant activity were tested over a
range of concentrations to establish the FE. This method follows closely that used by previous
workers [4–7].