3.4.4. Peroxyl Radical Scavenging Capacity (PSC)

The PSC assay was using the method described by Adom and Liu [43] with slight modification. Briefly, 80 µL of DCFH-DA (2.48 mM) was hydrolyzed with 900 µL of KOH (1.0 mM) for 3–5 min to remove the diacetate (DA) moiety and then diluted to 6 mL total volume with phosphate buffer (75 mM, pH 7.4). An amount of 150 µL of fluorescein (50 mM) was mixed with 25 µL of sample and incubated at 37 °C for 30min. An amount of 25 µL samples were mixed with 150 µL of DCFH and incubated at 37 °C for 30 s. After that, 25 µL of AAPH (50 mM) was added to each well and fluorescence measurements were taken over a 40 min time. The fluorescence was set at 485 nm as the excitation wavelength and 538 nm as the emission wavelength. To build the blank decay curve and Trolox standard decay curve, 25 µL of phosphate buffer solution or Trolox standard solution was added instead of the sample solution. The areas under the average fluorescence–reaction time kinetic curve (AUC) for both controls and samples were integrated and used as the basis for calculating antioxidant activity, which was calculated by Equation (4)

where SA is AUC for sample or standard dilution and CA is AUC for the control reaction using only buffer. The median effective concentration (IC50) was defined as the dose required to cause a 50 % inhibition (PSC unit = 0.5). The PSC values were expressed as µM of ascorbic acid equivalents (AEE) per 100 g of dried sample.