Ferric Reducing Antioxidant Power Assay

Ferric reducing power (FRAP) was also measured to estimate the AA [38 (link)]. The diluted extract (1 mL) was placed in a 15 mL test tube and 2.5 mL of phosphate buffer at pH 6.6 and 2.5 mL of a 1.0% potassium ferrocyanide solution were added. The mixture was shaken and incubated at 50 °C for 20 min, then 2.5 mL of 10% trichloroacetic acid with 2.5 mL of water and 0.5 mL of 1% FeCl₃ were added. The mixture was homogenized in a vortex (Mistral Multi-Mixer, Melrose Park, IL, USA). The solution was rested for 30 min in the dark and a green complex (ferrous chloride–potassium ferrocyanide) was formed. The absorbance was measured at 700 nm using a UV-VIS spectrophotometer, model 2600 (Shimadzu, Kyoto, Japan). The AA and results were estimated as for the ABTS method.