Ultrafiltration Affinity-liquid Mass Spectrometry Technology To Screen The Active Ingredients Of Traditional Chinese Medicine Antithrombin

Objective:Thrombin is an important target for anticoagulation,and a new oral anticoagulant directly acts on thrombin to exert anticoagulant effects.The new oral anticoagulant has serious adverse reactions such as hemorrhage,lack of specific antagonists,and serious complications.Salvia miltiorrhiza is the most commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis.At present,it has been shown that its ingredients have strong anticoagulant effect.Angelica sinensis is often used as a blood-activating anticoagulant in combination with Danshen.Therefore,Danshen and Angelica are selected as ultrafiltration affinity screening targets.This paper intends to establish a screening method for ultrafiltration affinity thrombin inhibitors to rapidly and high-throughput screening of anti thrombin components in traditional Chinese medicine.The components of Salvia miltiorrhiza and Angelica were subjected to ultrafiltration screening after thrombin binding,and the antithrombin active components were determined by mass spectrometry identification and anticoagulant activity assay.The mechanism of antithrombin activity in Salvia miltiorrhiza and Angelica sinensis was studied by activity verification and isothermal titration calorimetry to confirm its anticoagulant activity and explore its antithrombin mechanism.Methods:1.To establish an anti-thrombin active ingredient screening method based on ultrafiltration affinity technology,to investigate the effects of different incubation time and incubation temperature on the method,and to investigate the solvent tolerance of thrombin.2.The fingerprints of Danshen and Angelica sinensis L.and water extracts were established by UPLC method.The components of Salvia miltiorrhiza and Angelica were identified by UPLC-LTQ-Obitrap-MSn mass spectrometer.The anti-thrombin active components were screened by Danshen and Angelica by the established ultrafiltration affinity screening method.4.The antithrombin activity of the selected components was verified by the chromogenic substrate method and the titration curve of the potential antithrombin active ingredient and thrombin was determined by an isothermal titration calorimeter(ITC)to analyze the thermodynamic parameters.In order to explore the potential mechanism of action of the active ingredient.Results:Results:1.This paper successfully established a screening method for ultrafiltration affinity antithrombin active ingredients.After investigation,the incubation conditions were determined to be 40 min at 37℃.Inactivation of thrombin has no binding to salvianolic acid A,demonstrating that although thrombin does not have non-specific binding to the ligand,specific binding plays a major role.Thrombin has good solvent tolerance and is stable for 50 min in 30%DMSO PBS and 60%methanol.The UPLC separation method of water extract and alcohol extract of Salvia miltiorrhiza and water extract and alcohol extract of Angelica sinensis was established.The results showed that all the separations were good,and there was no significant difference in the peak area and retention time of the sample solution,and the repeatability was good..In this paper,9,9,4,and 7 potential antithrombin active ingredients were screened in Danshen water extract,Danshen alcohol extract,Angelica water extract and Angelica extract.Based on the comparison of standard products,reayxs database,ETCM database and related references,26 components of Danshen aqueous extract,22 components of Danshen alcohol extract,24 components of Angelica sinensis extract,and Angelica extract were identified.The 45 components provide the basis for the determination of components after ultrafiltration affinity screening,and provide a new basis for the study of Danshen and Angelica.4.According to the activity verification experiment,it was confirmed that Danshensu,β arbutin,salvianolic acid D,dihydrotanshinone I,tanshinone IIA,Oujiang lactone,and Isoproterolide I have antithrombin activity,a bear Fruit glycosides do not have antithrombin activity,confirming the reliability of the method.Isothermal titration calorimetry determines the binding of salvianolic acid D to thrombin as an ionic bond,revealing the possible mode of action of thrombin and potential active ingredients.Conclusion:This study provides a reliable screening method for finding antithrombin active ingredients from complex natural medicines.This study provides new ideas for the screening of inhibitors of other enzymes.This study provides a new idea for ultrafiltration affinity screening methods for water-insoluble compounds.This study provides an effective method for the study of the interaction between small molecule drugs and biological macromolecules such as enzymes.At the same time,this study provides a reference and basis for the material basic research of Danshen and Angelica antithrombin activity.