Integrating Metabolomics and Network Pharmacology to Identify Xanthine Oxidase Inhibitors from Raspberry Leaf:...

The rising global prevalence of hyperuricemia has intensified the search for natural xanthine oxidase (XO) inhibitors from food-grade botanicals. This study screened leaf extracts from four plant species for XO inhibitory activity and identified raspberry leaf as the most potent source. Among seven extraction and pre-treatment methods, ultrasound-assisted extraction (UAE) outperformed conventional decoction and all alternatives. Box--Behnken response surface methodology optimized UAE parameters to 70 °C, 50 min, and a solid-to-liquid ratio of 1:20 g/mL, yielding an extract with 97.77 ± 0.08% XO inhibition and an IC₅₀ of 0.3433 mg/mL-a 5.34-fold potency gain over decoction. Kinetic characterization established a reversible mixed-type inhibition mechanism (K_i = 0.147 mg/mL), alongside parallel improvements in DPPH, ABTS, and hydroxyl radical scavenging. Widely targeted UHPLC-MS/MS metabolomics identified 2987 metabolites; network pharmacology and molecular docking revealed that the origin of XO inhibition potentially involves terpenoids and coumarins-not the flavonoid and polyphenol fractions conventionally assumed-with dihydroactinidiolide, 7-hydroxy-8-methoxycoumarin, and piperlongumine as the candidate active constituents. These results position UAE-optimized raspberry leaf extract as a promising natural functional ingredient for hyperuricemia management and expand the phytochemical scope of plant-derived XO antagonists beyond flavonoids.