Surface plasmon resonance-based ligand fishing drives the identification of xanthine oxidase inhibitors from Terminalia chebula for hyperuricemia treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried fruit of Terminalia chebula Retz. (Combretaceae) has been traditionally used for gout and hyperuricemia (HUA) across Tibetan, Ayurvedic, and Chinese medicine. Although its extracts have demonstrated anti-hyperuricemic effects, the specific bioactive constituents targeting xanthine oxidase (XOD), the key enzyme in uric acid biosynthesis, have not been identified. AIM OF THE STUDY: This study aimed to identify the specific XOD inhibitory constituents from T. chebula fruit, characterize their inhibitory mechanisms at the molecular level, and validate their anti-hyperuricemic efficacy in vivo. MATERIALS AND METHODS: A SPR-based ligand fishing strategy was employed to capture XOD-binding compounds from the bioactive ethyl acetate fraction of T. chebula (TCEB). Captured ligands were tentatively identified by UPLC-QTOF-MS and validated through enzymatic assays, enzyme kinetics, multi-spectroscopic analysis, and molecular dynamics simulations. The in vivo anti-HUA efficacy was evaluated in a mouse model. RESULTS: Phlorizin and eurycarpin A, captured from TCEB by SPR-based ligand fishing, exhibited potent XOD inhibitory activity with IC50 values of 0.76 ± 0.04 and 0.57 ± 0.03 μM, respectively. Both compounds acted as reversible mixed-type inhibitors, binding competitively within the XOD active site. In a HUA mouse model, phlorizin significantly lowered serum UA levels, suppressed hepatic XOD activity and expression, and reduced hepatic and renal damage with no apparent acute toxicity. CONCLUSIONS: These findings provide a molecular pharmacological basis for the traditional use of T. chebula in managing gout and HUA and highlight SPR-based ligand fishing as an effective strategy for discovering bioactive constituents from ethnopharmacological resources.