Determination of antioxidant activity by ABTS, DPPH, and FRAP

Three antioxidant activity tests, including ABTS, DPPH, and ferric reduction ability of plasma (FRAP), were performed using slightly modified procedures described previously (Jung et al., 2013). For the ABTS assay, 7 mM ABTS was dissolved in 2.45 mM potassium persulfate solution and the sample was incubated in the dark for 1 day at room temperature to obtain a dark-blue colored solution. The solution was then diluted until the absorbance reached 0.7 ± 0.02 at 734 nm using a microplate reader (Spectronic Genesys 6, Thermo Electron, Waltham, mA, USA). Twenty microliters of sample were then mixed with 180 μL of diluted ABTS solution in 96-well plates and allowed to react for 6 min in the dark. Absorbance was measured at 734 nm using a microplate reader.

For the DPPH assay, 20 μL of sample extract was mixed with 180 μM of 0.2 mM DPPH ethanol solution in 96-well plates for 20 min at room temperature. Absorbance was measured at 515 nm using a microplate reader.

The FRAP assay was conducted with freshly prepared FRAP reagent, which was made by combining mixed acetate buffer (pH 3.6), 10 mM TPTZ (in 40 mM HCl solution), and 20 mM FeCl3•6H2O (in distilled water) in a ratio of 10:1:1, respectively. Ten microliters of each sample were then mixed with 300 μL of FRAP reagent in 96-well plates for 6 min at 37°C. The absorbance was measured at 570 nm using a microplate reader. All results were presented as the Trolox equivalent antioxidant capacity (mM), with the standard solution concentration curve ranging from 0.0078 to 1.000 mM, and all experiments were carried out in triplicate.