4.11. XOD Inhibition Assay

The direct inhibitory effect of BLEx and its fractions on XOD activity was evaluated as the conversion of xanthine to uric acid under XOD (from bovine milk, Sigma). Uric acid, xanthine, and allopurinol (Fujifilm Wako) were dissolved in 0.1 M phosphate buffer (PB; pH 8.0). Next, 50 μL PB, 50 μL samples (appropriate concentrations of BLEx, fractions, or 1 mM allopurinol), and 100 μL of 1 mM xanthine were added to a 96-well plate (UV-star, Greiner Bio-One, Kremsmünster, Austria). Finally, XOD was added to a final concentration at 0.1 mU and reacted for 3 min at 37 °C. The production of uric acid was measured as the absorbance at 293 nm using SpectraMax (Molecular Devices, San Jose, CA, USA).