In vitro, in vivo , and computational evaluation of xanthine oxidase inhibitory and antihyperuricemic activities of Piper betle L. ethyl acetate fraction

Abstract Objectives Gout is a metabolic disorder characterized by hyperuricemia and urate crystal deposition. This study evaluated the xanthine oxidase inhibitory, antihyperuricemic, anti-inflammatory, analgesic, and in silico target-binding properties of an EtOAc fraction of Piper betle L. aerial parts. Methods P. betle aerial parts were extracted with ethanol and partitioned to obtain an EtOAc fraction. Xanthine oxidase inhibition was evaluated spectrophotometrically, and antihyperuricemic activity was examined in potassium oxonate-treated mice given the fraction (200 or 600 mg/kg). Carrageenan-induced paw edema and acetic acid-induced writhing were used to assess anti-inflammatory and analgesic effects. Hydroxychavicol was docked with xanthine oxidase, cyclooxygenase-2, and transient receptor potential vanilloid 1, followed by molecular dynamics simulations. Results The ethyl acetate fraction inhibited xanthine oxidase with an IC 50 of 4.28 ± 0.15 μg/mL, whereas allopurinol showed an IC 50 of 1.06 ± 0.07 μg/mL. In hyperuricemic mice, serum uric acid showed a decreasing trend, and urinary uric acid was significantly reduced at 600 mg/kg. Paw edema was reduced at 600 mg/kg, whereas writhing responses were not significantly inhibited. Docking predicted favorable hydroxychavicol binding to the three targets, but molecular dynamics indicated weaker ligand retention in the xanthine oxidase complex than in the cyclooxygenase-2 and transient receptor potential vanilloid 1 complexes. Conclusions The ethyl acetate fraction of P. betle demonstrated preclinical xanthine oxidase inhibitory, urate-lowering, and anti-inflammatory potential. Stronger chemical standardization and mechanistic validation are required before therapeutic relevance can be inferred.