Oxygen Radical Absorbance Capacity Assay

The AA was determined by the oxygen radical absorbance capacity (ORAC) method described previously [15 (link)]. Briefly, the reaction was performed at 37 °C in 75 mM phosphate buffer at pH 7.4. The reaction mixture (200 μL) contained 180 μL of 70 nM fluorescein, 90 μL of 12 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 30 μL of diluted sample or standard (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox, Hoffman-LaRoche, basel, Switzerland) at concentrations ranging from 1 to 160 μM. Reaction mixtures were placed in a black 96-well plate (Fisher Scientific, Hampton, VA, USA) and the fluorescence was read in a Synergy HT microplate reader (BioTek Instruments, Winowski, VT, USA) every minute at excitation and emission wavelengths of 485 and 520 nm, respectively. The equipment was controlled by Gen5™ software, version 1.1 (BioTek Instruments). Results were expressed as μmol Trolox equivalents (TE)/g dw.