Xantine oxidase

HEPES, TRIS, succinate, ADP, rotenone, oligomycin, FCCP, mannitol, bovine serum albumin (BSA) fatty acid free, safranine O, equine citochrome c, NADPH, tert-Butyl hydroperoxide (T-BHP), horseradish peroxidase, Triton X-100, H₂O₂, superoxide dismutase (SOD), glutathione, glutathione reductase (GR) and xantine oxidase were obtained from Sigma-Aldrich (St. Louis, MO); 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was from ThermoFisher Scientific (Waltham, MA). Other reagents were of the highest available purity. Sources of antibodies for immunohistochemistry and immunofluorescence will be described in S1 File.

Hyperoside with the purity higher than 98% was purchased from Zelang Biological Technology Company (Nanjing, China) and suspended in 1% carboxymethyl cellulose (CMC) solution for animal experiments. Hyperoside (CAS#482360), N-acetyl-L-cysteine (NAC), Xanthine and xantine oxidase used for cell experiments were from Sigma Corporation (Hong Kong, China) and dissolved in 0.1% DMSO. YCG063 was bought from Calbiochem (San Diego, CA, USA) and NADH from Roche (Roche Diagnostics, Indianapolis, IN, USA).

A reaction mixture with a final volume of 632 μL/Eppendorf was prepared with 50 mM phosphate buffer (pH 7.5) containing EDTA (0.05 mM), hypoxanthine (0.2 mM), 63 μL NBT (1 mM; Sigma-Aldrich, St. Louis, MO, USA), 63 μL of aqueous or ethanolic extract (distilled water for the control), and 63 μL of xantine oxidase (1.2 U/μL; Sigma); the xanthine oxidase was added last. For each sample, a blank was carried out. The subsequent rate of NBT reduction was determined based on sequential spectrophotometric determinations of absorbance at 590 nm. The solutions were prepared daily and kept from light. The results are expressed as the percentage inhibition of the NBT reduction with respect to the reaction mixture without the sample (buffer only). Superoxide anion scavenging activity was calculated as ((1−(sample O.D.− blank O.D.)/(control O.D. − blank O.D.)) × 100); IC₅₀ was defined as the concentrations at which 50% of NBT/superoxide anions were scavenged. Allopurinol was used as the positive control.