Post-column Activity Screening Of Direct Anti-thrombin Active Ingredients In Salvia Miltiorrhiza

Objective:Thrombin is the key enzyme for anticoagulant thrombophilia research.Nowadays,the existing direct inhibitors have bleeding and many other adverse reactions.Salvia miltiorrhiza is one of the top five drugs used in blood circulation prescription mostly.Modern research shows that the water-soluble components of Salvia are mainly antioxidant and anticoagulant.However,due to themaximum absorption between water-soluble components of Salvia and the chromogenic products-nitroaniline(280nm)were similar,we can not use the traditional chromogenic substrate method for its direct screening.Therefore,this study intends to establish a new method which can screen rapidly,and determine antithrombin substances in complex extracts of traditional Chinese medicine.The water-soluble components of Salvia miltiorrhiza will be identified by HPLC-separation,identification and post-column activity screening,and the screened potential active ingredients will be verified and further explored.Method:1.Separate the water-soluble components of Salvia miltiorrhiza by HPLC,and investigate the precision,repeatability and stability of the method,to lay the foundation for the subsequent collection from HPLC post-column fractions.Identify the water-soluble components of Salvia by HPLC-DAD-MS/MS mass spectrometer,which is used as a reference for the identification of active ingredients2.Establish a new HPLC method to determine the antithrombin activity of the chemical constituents.On the basis of the chromogenic substrate method,control reaction time,reaction temperature and other factors,to extract resulting product,p-nitroaniline with ethyl acetate and analyzed by HPLC.,and to investigate the effects of different thrombin concentrations,buffer solution pH,incubation time and determination time on the method.At the same time,the reliability of the method was measured by measuring the inhibitory trend of the enzyme active drug--aga class before and after using the chromogenic substrate method.3.Identify the water-soluble components of Salvia by HPLC-separation,identification and post-column activity screening,and the screened potential active ingredients will be verified and further explored.And make a preliminary active determination of the screened potential antithrombin active ingredients.4.The ITC curve of quasi-active ingredient and thrombin was determined by isothermal titration calorimetry(ITC).The interaction between the quasi-active ingredient and thrombin was further studied by calculating the total thermodynamic parameters.Result:1.This study established the HPLC method for the determination of water-soluble components of Salvia miltiorrhiza Bunge,15 chromatographic peaks were successfully separated on HPLC.The results showed that there was no significant difference in the chromatographic peak area and retention time of the solution of the total phenolic acid solution of salvia miltiorrhiza for 10 hours at room temperature.The retention time of every peak was stable between repeated injections.The results confirmed 19 kinds of salvia phenolic compounds by combined with the two-level mass spectrometry information and the relevant reference literature between sample and the reference solution,which provided the separation and experimental basis for the follow-up post-column fractionation and identification experiments.2.It was successfully established A new method on investigate the chemical constituents which have an anti-thrombin effect by HPLC,which eliminating the absorption of the sample itself interference effectively.The optimal pH of the buffer solution was 8.3,and the reaction was started at 37 ? for 5 min,all samples must be measured within 3 h.According to the results,the inactivation rate of thrombin by Sodium Danshensu,Salvianolic acid A and Salvianolic Acid B under a given set of conditions were 3.06%,77.77%and 2.35%.3.The active ingredients of Salvia were thoroughly screened,and the inhibitory activity curve has two significant inhibitory peaks.They were salvianolic acid D,isoferuric acid and salvianolic acid A with confirmed.The inhibitory activity of each quasi-active ingredient was tested by high,medium and low concentration,the results showed that the thrombin inhibitory activity was significantly improved when the salvianolic acid D and iso-ferulic acid were mixed add into the system.And later,we found that the iso-ferulic acid can also enhance the inhibitory activity of Salvianolic acid A.4.The ITC results showed that the binding constant of Agaroban and thrombin was the largest,followed by Salvianolic acid A,the binding constant was low when Salvianolic acid D or iso-ferulic acid added alone into the thrombin solution.However,the binding constant was significantly increased when the two solutions were mixed and titrated,greater than the sum of the binding constants of the two titration added alone.And its ITC curve type has a significant change,proved that salvianolic acid D and iso-ferulic acid may act on different targets of thrombin.The Gibbs free energy ?G measured by each reaction was less than 0,proved the reaction occurs spontaneously.The ?H was less than 0 while the ?S is greater than 0,preliminary judgment that the drug and thrombin binding in the form of ion bonds.Conclusion:This study provides a reliable screening and testing method for finding antithrombin active components from complex natural drugs;a new idea for selection of active inhibitors of other enzymes.It also provides an effective method for the interaction between small molecule drugs and biological macromolecules.In addition,it makes us know more about Salvia's material basis and mechanism of action for the activating blood effect.