Development and optimization of Fe₃O₄@SiO₂-xanthine oxidase magnetic nanoparticles for rapid screening of XOD inhibitors from Trichosanthes anguina (snake gourd) with molecular docking approach.

Xanthine oxidase (XOD) inhibitors are central to hyperuricemia and gout management, but efficient, reusable enzyme-based screening platforms remain limited. In this study, XOD immobilized Fe₃O₄@SiO₂- nanoparticles (Fe₃O₄@SiO₂-XOD NPs) were developed and optimized as a reusable screening platform and applied to identify bioactive XOD inhibitors from Trichosanthes anguina (Snake Gourd). Fe₃O₄@SiO₂-XOD NPs were synthesized via glutaraldehyde-mediated crosslinking and characterized by FTIR, XRD, and SEM. Optimal immobilization was achieved at 15% glutaraldehyde, pH 7.5, 25 °C, and 1 h incubation. The immobilized enzyme retained over 65% of its activity after eight reuse cycles and exhibited high specificity toward active ligands as validated using allopurinol. Using this platform, the ethanol extract of Trichosanthes anguina inhibited XOD with an IC₅₀ of 383.50 ± 11.98 μg/mL, while the ligand-fished fraction showed significantly enhanced inhibition (IC₅₀ = 77.17 ± 6.80 μg/mL). UPLC-Q-TOF-MS/MS analysis identified eleven flavonoids, among six as potential XOD inhibitors, which was supported by molecular docking showing strong binding affinities (-6.6 to -8.3 kcal/mol), involving multiple hydrogen bonds and hydrophobic interactions with key XOD active-site residues. Furthermore, in an HK-2 cell-based HUA model induced by XOD and adenosine, the ligand-fished fraction significantly and dose-dependently reduced UA production at non-cytotoxic concentrations, with effects comparable to allopurinol. Overall, the Fe₃O₄@SiO₂-XOD NPs platform provides an efficient and reusable strategy for screening natural XOD inhibitors. Trichosanthes anguina was confirmed as a rich source of anti-hyperuricemic flavonoids, supporting its further exploration for gout management.