3.5. Superoxide Anion Radical (•O2–)-Scavenging Assay

The superoxide anion radical (•O₂^–)-scavenging assay method was developed in our laboratory [42]. Briefly, the sample was dissolved in ethanol at 5 mg/mL. The sample solution (x μL, where x = 2–10 μL) was mixed with (980–x) μL Tris-HCl buffer (0.05 M, pH 7.4) containing the disodium salt of ethylenediaminetetraacetic acid (1 mM). After 20 μL pyrogallol (60 mM in 1 mM HCl) had been added, the mixture was shaken at room temperature immediately. The absorbance of the mixture at 325 nm was measured (Unico 2100, Shanghai, China) against Tris-HCl buffer as a blank every 30 s for 5 min. The •O₂^– scavenging ability was calculated as

where ΔA_{325nm,control} is the increase in A_325nm of the mixture without the sample, and ΔA_325nm,sample is that with the sample; T = 5 min.