Inhibition of Xanthine Oxidase by Dietary Flavonoids: SwissModel-Based Docking, In Vitro, In Vivo Structure-Activity Investigations and Gut Microbiota-Based Systematic Toxicological Evaluation

As a prevalent global metabolic disorder, hyperuricemia necessitates innovative mechanistic studies to overcome the severe side effects associated with current xanthine oxidase (XOD) inhibitors. In this study, through in vitro activity experiments, the Quantitative Structure-Activity Relationship (QSAR) of 20 dietary flavonoids on XOD inhibitory activity was systematically analyzed. The results showed that compared with the functional groups on the A and C rings of dietary flavonoids, diosmetin with one methoxy group and one hydroxyl group on the B ring (IC50 = 0.3966 ug/mL) and quercetin with two hydroxyl groups on the B ring (IC50 = 0.3555 ug/mL) are the most potent candidates. Enzyme kinetic analyses confirmed both diosmetin and quercetin as mixed-type inhibition mechanism. Further, swissmodel-based docking analyses proved that the key active sites of diosmetin were Arg958 and Gly935 and the key active sites of quercetin were Pro1248 and Thr1290. In vivo validation demonstrated that both diosmetin and quercetin significantly reduced serum uric acid (SUA) levels, inhibit XOD activity, and ameliorate renal pathological damage in hyperuricemic mice. The analysis by 16S rRNA sequencing showed that both diosmetin and quercetin supplementation increased the abundance of beneficial bacteria such as Lactobacillus johnsonii, and reduce the abundance of harmful bacteria such as Desulfovibrio fairfieldensis. Notably, diosmetin exhibited lower hepatotoxicity than quercetin, as evidenced by reduced ALT and AST levels and higher molecular docking scores with the p38MAPK protein. These findings provide a robust theoretical foundation for developing natural therapeutics with enhanced efficacy and safety. The translational potential of flavonoid-based interventions underscores their promise for clinical application in metabolic disease management.