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

Abstract

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 optimized extract 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 (IC₅₀ = 6.66 ± 0.09 μg/mL) compared to the allopurinol standard (IC₅₀ = 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.