This study presents a comprehensive screening and identification of bioactive components from Gastrodia elata, focusing on their potential as inhibitors of xanthine oxidase (XOD) in the treatment of hyperuricemia and gout. Key compounds, including gastrodin, p-hydroxybenzyl alcohol, and parishins A, B, and E, were found to exhibit significant inhibitory effects on XOD. Through a combination of high-throughput in vitro and in vivo screening, activity-oriented online extraction and separation techniques (ultrasonic-assisted centrifugal extraction coupled on line with a solvent concentrator, a countercurrent chromatography, and a semi preparative liquid chromatography, UACE-OSC-CCC-SPLC), these compounds were highly purified and demonstrated strong binding affinities to XOD via hydrogen bonding, van der Waals forces, and hydrophobic interactions. A novel matrix analysis method was developed for the first time, enabling a comprehensive and systematic evaluation of these active ingredients. Among them, parishin A showed the strongest XOD inhibition (IC50 of 3.11 μg/mL), nearing the potency of allopurinol (IC50 of 0.79 μg/mL). Gastrodin and p-hydroxybenzyl alcohol exhibited mixed inhibition, while parishins A, B, and E displayed competitive inhibition. Target prediction and protein-protein interaction (PPI) network analyses indicated that these compounds may modulate inflammatory and oxidative stress pathways, particularly through proteins such as TNF and PTGS2. In vivo experiments confirmed that G. elata extracts significantly reduced uric acid levels, inhibited XOD activity, demonstrated anti-inflammatory effects, and preserved renal function. These findings highlight G. elata as a promising natural XOD inhibitor and offer a novel therapeutic strategy for gout treatment, paving the way for the future development of G. elata-based therapeutic agents.

Gastrodia elata, Computational Simulations, in Vivo Experiments, High-Throughput Screening, Chromatography