LC-MS-based Multivariate Statistical Analysis For The Screening Of Potential Thrombin/factor Xa Inhibitors From Danshen And Chuanxiong

Thrombin（THR）and factor Xa（FXa）play a key role in the coagulation cascade.Their inhibitors are important therapeutic drugs for thrombotic diseases.Salvia miltiorrhiza（Danshen,DS）and Ligusticum chuanxiong（Chuanxiong,CX）are commonly used traditional Chinese medicines for promoting blood circulation and removing blood stasis.Therefore,it is of great significance to screen THR and FXa inhibitors from DS and CX.In this thesis,LC-MS detection combined with multivariate statistical analysis was used to screen the active components in DS and CX that have the inhibitory activity on THR and FXa,and the interactions between the active components and enzymes were analyzed by molecular docking.This thesis mainly includes four parts（chapters）.The first part is the literature review.Firstly,the main chemical components and pharmacological effects of DS and CX were introduced.Secondly,the research progress of THR and FXa inhibitors was summarized,and the applications of multivariate statistical analysis in the study of natural products was summarized.Finally,the main contents and significance of this study were proposed.In the second part,the THR and FXa inhibitors in DS were characterized by LC-MS and multivariate statistical analysis.Firstly,the inhibitory activities of different polar extracts[ethyl acetate（EA）,butanol（BA）and remained extract（RE）from 75%ethanol extract,and water extract（WE）]of DS on THR and FXa were determined,and five fractions of SC1～SC5 were separated by silica gel column chromatography from the EA extract with the strongest activity.Secondly,the fractions（SC1～SC5）were analyzed by LC-MS and their inhibitory activities on THR and FXa were measured.Principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）were used to analyze the data of LC-MS analysis and THR/FXa activity assays,and four differential marker compounds,which were cryptotanshinone,tanshinone I,dihydrotanshinone I and tanshinone IIA,were identified by LC-MS/MS analysis.Finally,by molecular docking analysis,it was found that these four tanshinones were bound to the active sites of THR and FXa,and could interact with ALA190,ASP189,GLY216,GLY219 and other amino acid residues,and their binding energy were lower than-7 kcal·mol^-1.The third part is LC-MS detection combined with multivariate statistical analysis to characterize THR and FXa inhibitors in CX.The method used is the same as that in the second part.Firstly,the THR and FXa inhibitory activities of different polar extracts（EA,BA,RE and WE extract）of CX were measured.The EA extract had the best THR inhibitory activity,while the BA extract had the best inhibitory activity on FXa.Therefore,five fractions（EA-SC1 to EA-SC5,BA-SC1 to BA-SC5）were obtained by silica gel column chromatography,respectively.Based on the data of LC-MS analysis and enzyme activity assays of each fraction,PCA and OPLS-DA models were fitted,respectively.Among them,senkyunolide A,Z-ligustilide,ferulic acid and senkyunolide I(IC₅₀ was determined as 768.84μM)with potential THR inhibitory activity,as well as isochlorogenic acid A with FXa inhibitory activity were screened out.It was found that the four components could interact with ALA190,ASP189,GLY216,GLY219 and other amino acid residues around the active site of THR,and the binding energy was lower than-5 kcal·mol^-1.Isochlorogenic acid A were bound to the active sites of FXa,and the binding energy was-9.39 kcal·mol^-1.The IC₅₀ was determined as 558.55μM.The fourth part is the conclusion and prospect of this study.This thesis mainly used the multivariate statistical analysis method based on LC-MS analysis to screen the active components of inhibiting THR and FXa in DS and CX,and the interactions of these potential active components with THR/FXa were investigated by molecular docking analysis.The results of this study provide reference for supplementing the anticoagulant mechanisms of DS and CX.Compared with the activity-guided phytochemical separation method,the method used in this study is time-saving and reagent-conserving.It can be further applied to the prediction of THR or FXa inhibitory active components in other traditional Chinese medicines,and it is helpful in providing a clue for the discovery of new active THR and/or FXa inhibitors.