In vitro and in vivo inhibition of xanthine oxidase by the flowers of Chrysanthemum morifolium Ramat.

Abstract

Background: Xanthine oxidase (XO) plays an important role in human’s purine degradation. Excessive uric acid formation results in hyperuricemia and gout. The study aimed to determine the XO inhibitory potential of Chrysanthemum morifolium Ramat. dried flower and its effect on XO gene expression in animal models.Methods: In vitro XO inhibitory assay was employed to investigate the XO inhibitory potential of C. morifolium flowers. The bioactive sub-fraction was investigated further to give additional insight on its uric acid lowering potential via animal study and XO gene expression analysis. HPLC-Q-TOF-MS/MS was utilized to identify the putative compounds in the sub-fraction.Results: Among the fractions, EtOAc fraction exhibited the highest in vitro XO inhibitory potency (51.77 ± 0.98%; IC50 = 10.64 ± 0.51 µg/mL) and it was further fractionated into 15 sub-fractions through open column chromatography. EtOAc F7, F8, F9, F10, and F11 possessed >75% XO inhibition. F9 and F10 exhibited high in vitro XO inhibitory activity, cellular pro-proliferative effect and intracellular antioxidant activity among the sub-fractions tested. These two sub-fractions were also non-cytotoxic at the concentration range of 0.1 – 10 µg/mL. F10 was shown to be very effective in both serum and urine uric acid lowering properties in rats model upon oral consumption. It was subjected to further fractionation and a total of 11 sub-fractions were obtained. F10-4, F10-8, F10-9, and F10-10 possessed >90% XO inhibition. These sub-fractions were subjected to HPLC-Q-TOF-MS/MS analysis. A total of nine known compounds have been identified and 26 unknown compounds were detected. Conclusions: The possible mechanisms contributed to the anti-hyperuricemic effect of F10 were suggested to be non-competitive inhibition of XO enzyme, XO gene expression down regulation, and enhancement of uric acid excretion. Structure elucidation of the unknown compounds and the evaluation of the XO inhibitory activity of a single or a mixture of these compounds are recommended to identify possible synergism between them.