Comprehensive screening, separation, extraction optimization, and bioactivity evaluation of xanthine oxidase inhibitors from <i>Ganoderma leucocontextum</i>

This study presents a novel approach for screening, extracting, and preparing xanthine oxidase (XOD) inhibitors from Ganoderma leucocontextum. These inhibitors have potential applications in drug and food research and development. Affinity ultrafiltration-liquid chromatography coupled with mass spectrometry, molecular docking (MD), and molecular dynamics simulations (MDS) were employed to accurately and rapidly screen bioactive compounds as XOD inhibitors. These methods allowed for structural identification and assessment of target efficacy, enabling a comprehensive analysis of the active compounds at three levels. To overcome challenges related to low content and extraction efficiency of bioactive compounds from G. leucocontextum, a complex, activity-oriented preparation method was developed, utilizing a BP neural network combined with response surface methodology and universal quasichemical functional group activity coefficient-based (UNIFAC) activity-driven consecutive-countercurrent chromatography. Optimal process conditions, such as alcohol concentration, material-to-liquid ratio, extraction time, and the number of extractions were determined, leading to theoretical predictions of triterpene yield. Seven active ingredients, including Ganoderic acid A, Ganoderic acid D, Ganodermanontriol, Ganoderiol F, Ganoderiol B, Ganoderiol A, and Ganoderal A, were successfully identified and isolated using a solvent system of n-hexane/ ethyl acetate/ methanol/ water (4.0: 8.0: 5.3: 3.0, v/v/v/v). The mechanism of action of these xanthine oxidase inhibitors was further elucidated through enzyme kinetics. These findings highlight the potential of G. leucocontextum extracts in gout prevention and treatment, advancing our understanding of the molecular mechanisms of edible fungi and offering promising prospects for discovering new therapeutic agents from natural food sources.