2.4.2. DPPH and ABTS Radical Scavenging Activity and Ferric-Reducing Antioxidant Power (FRAP)

Free radical scavenging activity was determined using the DPPH radical assay of Surin et al. [25]. The sample solution (2 mL) was mixed with 2 mL of 0.2-mmol/L DPPH solution. After incubation in the dark at 30 °C for 30 min, the absorbance was measured at 517 nm. The percentage of inhibition of the DPPH radical (n = 3) was calculated. The results were expressed as IC₅₀ values, the lowest concentration of the sample required to inhibit 50% of the radicals.

For the ABTS radical scavenging assay of Chaiwong et al. [26], 100 µL of the sample from 0.5–5.0 mg/mL was mixed with 900 µL of 7-mM ABTS reagent. After incubation in the dark at 30 °C for 6 min, the absorbance was measured at 734 nm. The percentage of inhibition of the ABTS radical was calculated. The results (n = 3) were expressed as the IC₅₀ values.

The reducing power was determined using the FRAP assay of Surin et al. [27], with some modifications. Briefly, 100 µL of the sample (100 mg/mL) was mixed with 1900 µL of FRAP reagent, which consisted of 2.5 mL of 10-mM TPTZ in 40-mM HCl, 2.5 mL of 20-mM FeCl₃ and 25 mL of 0.3-M acetate buffer (pH 3.6). The absorption was measured at 595 nm, and aqueous FeSO₄ solutions were used to prepare the calibration curve. The measurements were done in triplicate (n = 3). The untreated ultrasonication sample was used as the control.