Study The Mechanism Of The Major Constituents From Ginkgo Biloba Extract On Human Thrombin

Aims:Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease（CVD）in both eastern and western countries,but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized.Thrombin,a key serine protease regulating the blood coagulation cascade and the processes of thrombosis.Thrombin inhibitor therapy has been recognized as one the most effective therapeutic options for the prevention and treatment of cardiovascular and thrombotic diseases.This study aims to investigate the inhibitory effect of major constituents in Ginkgo biloba on human thrombin activity,and reveal the prevention and treatment mechanism of Ginkgo biloba on cardiovascular and thrombotic diseases.Methods:1.Thrombin activity inhibition assays were established by a typical incubation mixture of human thrombin and its specific fluorescence substrate probe and fluorescence intensity of human thrombin-specific fluorescence substrate probe hydrolytic metabolite was determined via a multi-mode microplate reader.2.Sixteen major constituents from Ginkgo biloba were obtained and their ability to inhibit human thrombin was carefully investigated by a fluorescence-based biochemical assay.The IC₅₀ values（concentration of inhibitor that reduces enzyme activity by50%）of each constituent were calculated by nonlinear regression using GraphPad Prism 6.0 software.3.The inhibition kinetic modes and the corresponding inhibition constant（K_i）values were investigated by performing a set of analyses in which the concentrations of both the fluorescent substrate and the inhibitor were varied.The second plot of the slopes from the Dixon and Lineweaver-Burk plots as a function of inhibitor was used to calculate the corresponding inhibition constant（K_i）value and inhibition kinetic modes.4.In order to gain deeper insights into the key interactions between inhibitors and human thrombin from the view of ligand-enzyme interactions,molecular docking simulations of inhibitors with the active site,exosite-I and exosite-II of human thrombin were performed.Results:1.Primary screen results demonstrated nine Ginkgo biloba constituents inhibited thrombin-mediated Z-GGRAMC acetate hydrolysis by 50%or more at the high concentration（100μM）.2.The IC₅₀0 values of these nine constituents were evaluated.Among all tested Ginkgo biloba constituents,four natural biflavones,including ginkgetin,isoginkgetin,bilobetin and amentoflavone displayed strong inhibitory effects on human thrombin with IC50 values ranging from 8.05μM to 17.83μM;five flavonoids displayed moderate inhibitory effects on thrombin with IC50 values ranging from 23.90μM to 82.08μM;the terpene lactones showed negligible inhibitory activity.3.The inhibition constant（Ki）value and inhibition kinetic modes of the four identified biflavones against thrombin-mediated Z-GGRAMC acetate hydrolysis were carefully characterized by performing a set of analyses with varying concentrations of both the fluorescent substrate and the inhibitor.The results demonstrated that ginkgetin,isoginkgetin,bilobetin,and amentoflavone were mixed-type inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis.The Ki values of ginkgetin,isoginkgetin,bilobetin,and amentoflavone,were evaluated as 6.32μM,11.01μM,4.12μM,and 8.06μM,respectively.4.Docking simulations demonstrated that ginkgetin,isoginkgetin,bilobetin,and amentoflavone could bind in the catalytic cavity and to the two anion binding exosites（ABE1 and ABE2）.The detailed studies on structure-activity relationships of biflavones as thrombin inhibitors were carried out.The results demonstrated that the C-7 or C-4’’’hydroxyl group from these four biflavones could strongly interact with Arg-221 or Lys-60 via a salt bridge in the active site of human thrombin,which suggested that the C-7 or C-4’’’hydroxyl group of the biflavones plays a key role in thrombin inhibition.Conclusion:The results demonstrated that four biflavones from Ginkgo biloba showed strong inhibitory effects on human thrombin.These findings provide powerful new evidence for explaining the beneficial effects of Ginkgo biloba extract on CVD,and also suggested that the biflavones in Ginkgo biloba could serve as lead compounds for the design and development of biflavone-based thrombin inhibitors.