Determination of antioxidant activity

DPPH radical scavenging activity of phenolics was assessed by measuring the ability to bleach a black colored methanol solution of DPPH radicals as described by previous method [6, 16]. One milliliter phenolic extract was mixed with 5 mL 60 µM DPPH dissolved in methanol. The absorbance was measured at 517 nm against a solvent blank. The scavenging rate of DPPH radicals was expressed as the scavenging rate of one gram of dried samples and was calculated using Eq. (1).

where A_control is the absorbance of the control solution, A_sample is the absorbance in the presence of phenolic extracts in DPPH solution, and A_{error correcting}, which is the absorbance of the extract solution without DPPH used for error correction. V_final represents the final volume of methanolic extract, V_test represents the volume used for activity test, and W_sample represents the total weight of sample DW.

Hydroxyl radical scavenging activity (HRSA) was assessed according to Mäkynen et al. [17] with some modifications. The reaction mixture was generated by adding 30 µL 2-deoxy-2-ribose (17 mM), 30 µL extract, 30 µL 1.2 mM EDTA, 60 µL 0.3 mM FeCl₃, 30 µL 34 mM hydrogen peroxide (H₂O₂), and 60 µL 0.6 mM ascorbic acid. The reaction was performed at 37 °C for 1 h. Thereafter, 150 µL 1 % (w/v) thiobarbituric acid (TBA) and 300 µL 2.8 % (w/v) trichloroacetic acid (TCA) were added to the mixture, which was subsequently incubated at 100 °C for 15 min. The absorbance was measured at 532 nm against a blank containing deoxyribose and buffer. The HRSA values were expressed as scavenging rate per gram of dried samples and were calculated using Eq. (2).

where Abs_contol is the absorbance of the control solution (30 µL distilled water instead of extract) and Abs_sample is the absorbance in the presence of phenolic extracts. V_final is the final volume of the extract, V_test is the volume used for activity test, and W_sample is the total weight of sample DW.

Ferric reducing antioxidant power (FRAP) assay was performed according to previous report [16, 17]. Briefly, a FRAP solution was mixed with 10 mL 0.3 M sodium acetate buffer solution (pH3.6), 1mL 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) in 40 mM HCl, and 1mL 20 mM FeCl₃. The FRAP reagent was warmed to 37 °C in a water bath. Next, 0.1 mL phenolic extract was mixed with 1.8 mL FRAP reagent and 3.1 mL ultrapure water. The absorption of the reaction mixture was measured at 593 nm after incubation for 30 min at room temperature. FRAP values were calculated from a standard curve prepared using FeSO₄. FRAP values were expressed as µmol FeSO₄ per gram of dried samples.