A universal fluorescent sensing platform based on “signal switching induced by enzyme catalyzed substrate hydrolysis” for high-throughput discovery of xanthine oxidase inhibitors from traditional Chinese medicine

Introduction: Traditional Chinese medicine (TCM) serves as the treasure house for discovering lead compounds in drug development, demonstrating significant potential in the creation of anti-gout agents. However, given the complexity of TCM, conventional detection methods not only were susceptible to interference from metabolites but also suffered from low throughput, thereby hindering the efficient screening of inhibitors. Methods: This study developed a universal fluorescent sensing platform based on the principle of enzyme specific substrate recognition for the high-throughput screening of xanthine oxidase (XOD) inhibitors. Results: This approach offered two advances: (1) Interference-resistant detection: for screening in the complex system of TCM, its high emission wavelength (λem = 690 nm > 680 nm) and larger Stokes shift (Δλ = 148 nm) further enhanced anti-interference capability. (2) High-throughput screening capacity: by integrating HPLC-fully automatic partial fraction collector (HPLC-FC) and UHPLC-Q-TOF/MS, we established an "isolation-detection-identification" platform, screening and identifying four XOD inhibitors (Quercetin-3-sambubioside, Hyperoside, Isoquercitrin and Astragalin) from Nelumbinis Folium (NF) extract, with their combined inhibitory contribution accounting for 61.22% of the total inhibition activity. Discussion: Therefore, this strategy provided a powerful methodological tool for the high-throughput discovery of next-generation XOD inhibitors from TCM. And it offers a universal paradigm for the high-throughput discovery of bioactive metabolites from TCM targeting different diseases.