Ligand Fishing for Xanthine Oxidase Inhibitors From Brassica rapa L. via Bioaffinity Ultrafiltration‐HPLC‐MS/MS: Mechanistic Insights and In Vitro Anti‐Hyperuricemic Activity

Abstract

Hyperuricemia is a prevalent metabolic disorder arising from dysregulated purine metabolism, wherein xanthine oxidase (XOD) serves as the key rate‑limiting enzyme catalyzing the final oxidation of hypoxanthine and xanthine to uric acid. Although Brassica rapa L. (chamagu) has been previously reported to exert antihyperuricemic effects, the specific bioactive constituents responsible for XOD inhibition and their underlying mechanisms remain elusive. In this study, UF‑HPLC‑MS/MS was employed to screen 183 potential XOD inhibitors from the water extract of chamagu. Through combined molecular docking and in vitro activity assays, psoralen was identified as a potent XOD inhibitor (IC 50 = 0.237 ± 0.003 mg/mL) and selected for further characterization. Mechanistically, psoralen binds spontaneously to XOD at a 1:1 stoichiometric ratio, primarily driven by van der Waals forces and hydrogen bonding, which induce local conformational perturbations in the protein microenvironment and consequently suppress enzymatic activity. In cellular models, psoralen significantly reduced XOD activity and uric acid levels in xanthine‑stimulated BRL‑3A hepatocytes, while concurrently alleviating oxidative stress as evidenced by decreased superoxide anion (O 2 · − ) levels and restored total antioxidant capacity. Moreover, in uric acid‑treated HK‑2 renal tubular cells, psoralen mitigated oxidative stress (decreased MDA levels and increased SOD activity) and inflammatory responses (reduced IL‑1β, IL‑6, and TNF‑α levels, and increased IL‑10 levels). Collectively, these findings elucidate the molecular basis of the uric acid‑lowering action of chamagu via XOD targeting, providing experimental rationale for its further development and utilization as a functional food.