Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.