2.10. Antioxidant Activity

The antioxidant activity of the sample was measured using a DPPH, ABTS⁺, and TPTZ assay. The tested samples were prepared at different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/mL). VC was used as the positive control.

The determination of DPPH free radical scavenging activity followed the previous method carried out by Zhou et al., with appropriate modification [34]. Briefly, three hundred microliters of DPPH· methanol solution (0.1 mM) were mixed with 100 μL of samples and incubated at room temperature for 30 min in the dark. Then, the absorbance was measured at 517 nm using a microplate reader (SuperMax-3100, Shanghai Flash Spectrum Biological Technology Co., Ltd., Shanghai, China). The DPPH· scavenging activity was calculated according to Equation (2):

where A₂ was the absorbance of the mixtures, with A₁ without DPPH·, and A₀ without sample. The absence was substituted by an equivalent solvent.

The determination of ABTS free radical scavenging activity followed the previous method carried out by Wang et al., with appropriate modification [35]. Briefly, the ABTS⁺ solution (7.0 mM) was mixed with an equal volume of K₂S₂O₈ solution (2.45 mM). The ABTS⁺ working solution was configured following incubation at 4 °C for 16 h in darkness and was diluted with PBS (pH = 7.4) to the absorbance value at 734 nm as 0.70 ± 0.05. Fifty microliters of sample solution were mixed with 150 μL of ABTS⁺ working fluids and incubated at room temperature for 5 min in the dark and the absorbance was measured at 734 nm. The ABTS⁺ scavenging activity was calculated according to Equation (3):

where A₂ was the absorbance of mixtures, with A₁ without ABTS⁺ and A₀ without sample. The absence was substituted by an equivalent solvent.

The FRAP of polysaccharides was determined by a T-AOC Assay Kit (Macklin Biochemical Co., Ltd., Shanghai, China) [21]. Briefly, the FRAP reagent was freshly prepared by mixing acetate buffer, TPTZ solution, and ferric chloride at the volume ratio of 7:1:1. Then, thirty microliters of sample solution and 90 μL of distilled water were added to 900 μL of the FRAP reagent. The mixture was kept at 37 °C for 10 min in the dark and the absorbance was measured at 593 nm. An FeSO₄ solution (0.003125–0.1 μM) was used for the standard curve (y = 11.119x − 0.0074, R² = 0.9988) and the FRAP value was calculated according to the Equation (4):

where x is calculated from the standard curve, and Cpr is the sample concentration.