Mechanistic evaluation of a polyphenol-rich cell-free extract from Bacillus sp. DO-R5: In vitro xanthine oxidase inhibition, in silico docking, and in vivo anti-gout efficacy

Gout is a metabolic disorder characterized by hyperuricemia and the deposition of urate crystals in joints, leading to acute inflammation and severe pain. This study evaluated the therapeutic potential of an o ptimized e xtract derived from the cell-free supernatant of Bacillus sp. DO-R5 (hereafter designated as oDO-R5e). This polyphenol- and flavonoid-rich extract was produced by an endophytic bacterium isolated from the roots of Dillenia ovata . A comprehensive approach integrating in vitro , in silico , and in vivo models was employed. In vitro , oDO-R5e exhibited potent xanthine oxidase (XO) inhibition (IC 50 = 6.66 ± 0.09 μg/mL) compared to the allopurinol standard (IC 50 = 1.36 ± 0.03 μg/mL), significantly outperforming its crude counterpart and many plant-derived extracts. Supporting this, in silico molecular docking revealed that its key constituents, notably chlorogenic acid, exhibited robust binding affinities to the XO active site (−8.0 kcal/mol), surpassing the standard drug allopurinol. Corroborating these findings, in vivo murine models showed that oral administration of oDO-R5e (200 and 400 mg/kg) dose-dependently reduced serum uric acid levels, achieving an efficacy statistically comparable to allopurinol ( p > 0.05) in potassium oxonate-induced hyperuricemia. Furthermore, the extract significantly attenuated oxidative stress by decreasing hepatic and renal MDA levels while restoring GSH content. oDO-R5e also exhibited notable acute anti-inflammatory and peripheral analgesic activities by reducing carrageenan-induced paw edema (comparable to diclofenac) and markedly decreasing acetic acid-induced writhing episodes. These findings demonstrate that oDO-R5e exerts multitarget effects, comprising urate-lowering, antioxidant, anti-inflammatory, and analgesic activities driven by strong XO inhibition. Owing to its sustainable microbial origin and comprehensive efficacy, oDO-R5e represents a highly promising natural candidate for gout management.