3.4. Xanthine Oxidase Inhibitory Assay

The xanthine oxidase inhibitory activities of DH and DS samples were determined by xanthine oxidase inhibitory activity assay in vitro [25], which was based on the increase in absorbance at 295 nm due to the production of uric acid from xanthine. In brief, 50 µL of DH or DS samples, 30 µL of phosphate buffer (70 Mm, pH 7.5), and 40 µL of xanthine oxidase (0.05 U/mL) were added into a 96-well plate. After preincubation at 25 °C for 8 min, 60 µL of xanthine (300 µM) was added. After incubation at 25 °C for 15 min, the reaction was stopped by adding 20 µL of HCl (1.0 M). The absorbance of the mixture solution was recorded at 295 nm by a microplate reader. Allopurinol was used as a positive control.

The control sample was prepared by adding PBS instead of tested sample. The background sample was prepared by replacing tested sample with the same volume of PBS. The blank sample was prepared by adding PBS instead of xanthine oxidase solution. All experiments were repeated three times. The inhibition ratio of xanthine oxidase was calculated as follows:

DH and DS samples were prepared in a series of concentrations. The xanthine oxidase inhibitory activities of DH and DS samples were evaluated by IC₅₀ values (the concentration of the sample inhibited 50% the activity of the xanthine oxidase, and the IC₅₀ values were calculated by a logarithmic regression curve. As DH and DS samples were mixtures of multiple constituents, the molar concentrations were uncertain. Thus, the IC₅₀ values of anti-xanthine oxidase activities of DH and DS samples were expressed as mg/mL raw drug equivalents.