Xanthine oxidase inhibitory potentials of flavonoid aglycones of Tribulus terrestris: in vivo, in silico and in vitro studies

Background: Despite the ongoing safety-driven spate of flavonoid xanthine oxidase (XOD) inhibition investigations, there is a lack of flavonoid-based uricostatic antihyperuricemic agents in clinical medicine. The poor pharmacokinetic profiles of glycosides (the natural form of existence of most flavonoids) relative to their aglycones could be largely responsible for this paradox. This investigation was aimed at providing both functional and molecular bases for the possible discovery of XOD inhibitory (or uricostatic) anti-hyperuricemic flavonoid aglycones from the leaves of a flavonoid-rich medicinal plant, Tribulus terrestris. To this end, the flavonoid aglycone fraction of T. terrestris leaf extract (FATT) was evaluated in vivo for antihyperuricemic activity in ethanol-induced hyperuricemic mice, monitoring serum and liver uric acid levels. Molecular docking and molecular dynamics simulation studies were carried out on the three major flavonoid aglycones of T. terrestris (isorhamnetin, quercetin and kaempferol) against an inhibitor conformation XOD model. The three flavonoids were also subjected to in vitro XOD activity assay, comparing their IC50 to that of allopurinol, a standard uricostatic antihyperuricemic drug.Results: FATT significantly lowered serum uric acid (p < 0.0001) and liver uric acid (p < 0.05) levels of the experimental animals, implying anti-hyperuricemic activity with uricostatic action mechanism allusions. Molecular docking studies revealed high binding affinity values (- 7.8, - 8.1, - 8.2 kcal/mol) for the aglycones (isorhamnetin, quercetin and kaempferol, respectively). Radius of gyration and RMSD analyses of the molecular dynamics simulation trajectories of the three aglycone-XOD complexes revealed substantial stability, the highest stability being demonstrated by the kaempferol-XOD complex. In vitro XOD activity assay showed kaempferol (IC50: 8.2 +/- 0.9 mu g/ml), quercetin (IC50: 20.4 +/- 1.3 mu g/ml) and isorhamnetin (IC50: 22.2 +/- 2.1 mu g/ml) to be more potent than allopurinol (IC50: 30.1 +/- 3.0 mu g/ml).Conclusion: This work provides a scientific basis for the use of T. terrestris in the treatment of hyperuricemia-related (e.g. kidney stone and gout) disorders. It also provides the molecular basis for a focussed screening of the flavonoid aglycones chemical space for the possible discovery of flavonoid-based uricostatic anti-hyperuricemic drugs or drug templates.