A Box-Behnken design-optimized preparative strategy for anti-hyperuricemic flavonoids from Humulus lupulus L.: from extraction to bioactivity evaluation.

This study established a standardized preparative platform for bioactive flavonoids from Humulus lupulus L., integrating process optimization, phytochemical isolation, and bioactivity evaluation. Ultrasound-assisted extraction was optimized via a Box-Behnken design (BBD), yielding a robust protocol (60% ethanol, 22:1 solvent-to-solid ratio, 42 min) for high-yield production of hops total flavonoid extract (HTF). Targeted chromatographic isolation from the optimized HTF led to the identification of fifteen compounds, predominantly prenylated flavonoids. In vitro screening revealed potent xanthine oxidase (XOD) inhibitory activity, with xanthohumol D being most active (IC50 = 18.08 μg/mL). Critically, this preparatively defined HTF exhibited a dual in vivo mechanism in a hyperuricemic mouse model: reducing serum uric acid via XOD inhibition and providing renoprotection. Thus, the strategy not only provides a standardized preparative method but also positions HTF as a promising multi-targeted phytotherapeutic candidate.