4.9. Oxygen Radical Absorbance Capacity (ORAC) Assay

ORAC assay was performed according to Davalos et al. [27] method using a 96-well format (Costar, 96-well clear round bottom plate, Corning). Assay final volume of 200µL was carried out in 75 mM phosphate buffer (pH 7.4). The solutions of the antioxidant (20 µL) and fluorescein (120 μL; 70 nm, final concentration) were prepared in the well of the microplate and preincubated for 15 min at 37 °C 60 µL (12 mM, final concentration) of 2,2′-Azobis-(2-methylpropionamidine) dihydrochloride (AAPH) solution was added and placed immediately in a microplate reader (Infinite M200 Tecan, Männedorf, Switzerland), and the fluorescein was recorded (excitation and emission wavelengths of 485 and 527 nm, respectively) every minute for 1 h. Three dilutions of samples were used in each assay and for every sample, three replications were used. We used a blank of fluorescein and AAPH in each assay. Diminution curves of fluorescence (intensity vs. time) were registered, followed by the calculation of area between the two Diminution curves (with or without antioxidant). Subtracting the blank amount from that of the sample or standard was acquired for the pure area under the curve. A standard curve of Trolox solution using different concentrations (1–6 μM) was used in every assay. Final ORAC values were represented as μmol Trolox equivalents (TE) mL⁻¹ of extract.