Introduction Hyperuricemia is the key risk factor for gout, in which the elevated uric acid is attributed to the oxidation of hypoxanthine and xanthine to uric acid by xanthine oxidase (XO). Adverse effects of the current treatments lead to an urgent need for safer and more effective alternative from natural resources. Objective To compare the metabolite profile of Chrysanthemum morifolium flower fraction with that of its detannified fraction in relation to XO inhibitory activity using a rapid and effective metabolomics approach. Methods Proton nuclear magnetic resonance (1H‐NMR)‐based metabolomics approach coupled with multivariate data analysis was utilised to characterise the XO inhibitors related to the antioxidant properties, total phenolic, and total flavonoid contents of the C. morifolium dried flowers. Results The highest XO inhibitory activity, 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) radical scavenging activity, total phenolic and flavonoid content with strong positive correlation between them were observed in the ethyl acetate (EtOAc) fraction. Detannified EtOAc showed higher XO inhibitory activity than non‐detannified EtOAc fraction. A total of 17 metabolites were tentatively identified, of which three namely kaempferol, 4‐hydroxybenzoic acid and apigenin, could be suggested to be responsible for the strong XO inhibitory activity. Additive interaction between 4‐hydroxybenzoic acid and apigenin (or kaempferol) in XO inhibition was demonstrated in the interaction assay conducted. Conclusion Chrysanthemum morifolium dried flower‐part could be further explored as a natural XO inhibitor for its anti‐hyperuricemic potential. Metabolomics approach served as an effective classification of plant metabolites responsible for XO inhibitory activity, and demonstrated that multiple active compounds can work additively in giving combined inhibitory effects.

Xanthine oxidase (XO) is a biological enzyme that takes part in purine catabolism. It catalyses the conversion of hypoxanthine to xanthine and eventually xanthine to uric acid. The catabolism reaction increases the level of uric acid and subsequently leads to hyperuricemia. Allopurinol is a XO inhibitor that is used clinically to prevent purine catabolism. Although it is an effective XO inhibitor, it causes some side effects. Therefore, a more effective inhibitor with fewer side effects is in an urgent need. Phenolic compounds have been identified as effective XO inhibitors in many studies. In vitro and in silico study were conducted to investigate the interaction between apigenin, kaempferol and 4-hydroxybenzoic acid in XO inhibition. Apigenin was found to be the most effective XO inhibitor among the compounds tested with the best docking score of -8.2 kcal/mol as demonstrated in the molecular docking simulation which indicated its favourable interaction with XO enzyme. Additive interactions between compounds namely apigenin-kaempferol, apigenin-4-hydroxybenzoic acid and 4-hydroxybenzoic acid-kaempferol were demonstrated in both in vitro and in silico studies. The results showed that 4-hydroxybenzoic acid- apigenin (-7.4 kcal/mol) was the most stable ligands combination docked to XO. The multiple ligands docking simulation showed independent ligands bound to the XO active site at non-interfering regional location. In conclusion, the combination of these three compounds can be explored further for their additive interaction in XO inhibition, which could be beneficial in terms of the enhanced effectiveness and lower side effects when each is used at lower dose to give the same effect.