Study On The Mining Of Quality Markers Of Xinkeshu Tablets And Its Application In Quality Evaluation

Xinkeshu tablets(XKST),as a common Chinese patent medicine for the treatment of cardiovascular diseases,has been used in the clinic for nearly 30 years,and its clinical effect is quite good.However,like most Chinese patent medicines,the chemical composition of this drug is very complex and the target of its action is numerous.It cannot accurately and completely reflect chemical substance basis with therapeutic effects for quality control only according to the index ingredients in Chinese Pharmacopeia.At the same time,conventional quality control methods are difficult to meet the increasing demand for high quality and high consumption.Therefore,it is a key problem to find out the quality related components of this drug and establish the quality analysis methodology system conforming to the characteristics of the complex system of traditional Chinese medicine.In this paper,"Quality marker(Q-marker)" was taken as the breakthrough point,and a variety of research methods were used to explore Q-marker of Xinkeshu tablets.Based on this,near infrared spectroscopy(NIRS)technology is used to achieve rapid quality evaluation of XKST.The main contents and results of this study were listed as follows:(1)Chemical composition identification and serum pharmacochemistry study of XKST.The UHPLC-Q-Exactive HRMS technique was used to analyze XKST,its source materials and serum of rats after intragastric administration.According to the fragment ion information and retention time of secondary mass spectrometry in positive and negative ion mode,109 compounds of XKST were identified by comparison of standard substances,literature review and database search.Among them,40 components were derived from Salvia miltiorrhiza Bunge(Danshen),42 were derived from Pueraria montana var.lobata Willd.(pueraria),11 were derived from Crataegus pinnatifida Bunge(hawthorn),15 from Panax notoginseng(Burkill)F.H.Chen(panax notoginseng)and 2 were derived from Aucklandia costus Falc(Radix Aucklandiae).On this basis,through the serum pharmacochemistry study,referring to the possible phase I and phase II metabolic activities in the organism,a total of 30 absorbed blood prototype components and 23 metabolites were inferred from the serum of rats given Xinkeshu tablets by gavage.By tracing the source of metabolites,we obtained 5 source components of metabolites that did not enter blood in the form of prototype components,and combined with the previous 30 prototype components into blood,a total of 35 potential active components of XKST were screened out.(2)Study on metabolomics and network pharmacology of XKST.The model of myocardial ischemia-reperfusion injury(MIRI)in rats was established by operation,and Xinkeshu tablets had a good therapeutic effect on MIRI through general observation,pharmacodynamic index investigation and pathological tissue section observation.Subsequently,based on the plasma metabolomics study,the effects of MIRI on endogenous metabolites in rat plasma and the corresponding regulatory effects of XKST were discussed.Combined with multivariate statistical analysis,301 pathologic biomarkers of MIRI were found,and 166 biomarkers in the XKST treatment group were found to be reversible,in which eight of the biomarkers showed statistical significance.Based on the reversible biomarkers and Metabo Analyst metabolic pathway analysis,the related metabolic pathways of XKST for the treatment of MIRI disease were established.Based on MIRI pathological biomarkers,combined with KEGG database and Metabo Analyst analysis of metabolic pathways,metabolic network analysis was carried out to find the disease targets of MIRI.On this basis,targets of three other representative cardiovascular diseases,including coronary heart disease,myocardial infarction and arrhythmia,were retrieved from the database.Combined with the 35 potential active components of Xinkeshu tablets identified earlier,the relationship between ingredient-target-disease was mined through network pharmacology,and a total of 29 pharmacodynamic components of XKST were found by using reverse target mapping.Endothelin-1(EDN1),angiotensin converting enzyme(ACE)and transthyretin(TTR)were speculated to be the common core targets of XKST in the treatment of these four groups of diseases,and HIF-signaling pathway,cocaine addiction and serotonergic synapse is its important mechanism.(3)Study on spectrum-effect relationship and pharmacodynamic verification based on zebrafish pharmacodynamic model.Three HPLC fingerprint determination methods of XKST were established on the basis of the preliminary research of our research group and the 2020 edition of Chinese Pharmacopeia.By comparing with standard substances and qualitative results of UHPLC-Q-Exactive HRMS analysis,components corresponding to HPLC fingerprints common peaks of 16 batches of XKST were identified,and these components satisfy the measurability principle.Subsequently,the anti-arrhythmic efficacy of 16 batches of XKST was evaluated using the heart rate recovery rate of zebrafish larva.Combined with HPLC fingerprints of 16 batches of XKST,the spectrum-effect relationship between the HPLC fingerprint and the heart rate recovery rate of zebrafish larva was established by orthogonal signal correction and partial least squares regression(OSC-PLSR)analysis,and the antiarrhythmic components of XKST were deduced.The pharmacodynamic components identified in the early stage were screened based on the principle of testability,and other index components on the characteristic map of XKST appeared in the 2020 edition of the Chinese Pharmacopoeia were added,and a total of 11 components were selected.Three zebrafish pharmacodynamics models,which including arrhythmia,thrombus and myocardial injury,were used to verify the efficacy of those 11 components.Combined with the efficacy verification results,based on the five elements of Q-marker principle,Danshensu,puerarin,daidzein,salvianolic acid B,daidzein,salvianolic acid A,protocatechual and 3’-methoxyl puerarin were preliminary determined as Q-markers of XKST.(4)Study on molecular interaction of XKST and its Q-marker with proteins related to drug natures and tastes.In combination with the theory of drug natures and tastes,molecular docking technique was used to predict the binding effects between representative target proteins of functional taste and oral taste and eight Q-markers of XKST.The molecular docking ability of eight Q-markers in XKST with three functional taste target proteins,including EDN1,ACE and TTR,is different,which reflects the multi-component and multi-target characteristics of traditional Chinese medicine(TCM).In the molecular docking with taste receptor type 2 member 10(TA2R10)corresponding to bitter taste and olfactory receptor 7D4(OR7D4)corresponding to pungent taste,salvianolic acid A,Danshensu and protocatechualdehyde showed better performance.Subsequently,based on biolayer interferometry(BLI)technology,ForteBio Octet K2 molecular interaction instrument was used to investigate and verify the molecular dynamics parameters of eight Qmarkers and extract of XKST with representative taste target proteins OR7D4,TAS2R10 and ACE.The results showed that among the eight quality markers of XKST,salvianolic acid A had the smallest the molecular dynamics parameter KD value binding with the three target proteins,which showed the strongest molecular interaction force.The converted KD values of XKST extracts binding to these three target proteins were roughly between salvanolic acid A and other Q-markers.Partial least squares regression was used to analyze the correlation between the contents of eight Q-markers in XKST and the KD value of the binding between the extract of XKST and ACE target proteins.The results showed that salvanolic acid A,Danshensu and protocatechualdehyde contributed the most to the binding between the whole component of XKST and ACE target proteins,and the results were statistically significant.On this basis,combined with the performance of monomer components binding to representative target proteins of oral taste and functional taste,it is speculated that the binding of the target proteins related to drug natures and tastes with the overall chemical composition of XKST is mainly the effect of salvianolic acid A,Danshensu and protocatechualdehyde play the role of deduction,and other Q-markers were partially involved.And these three main components are all from the royal medicine of of XKST,ie.Danshen,which is in line with the principle of compound compatibility of traditional Chinese medicine.The results of this experiment verified the rationality of the Q-marker of XKST in the prescription compatibility of TCM from the perspective of drug natures and tastes.(5)Construction of NIRS quality evaluation system of XKST and its model transfer study.A rapid quantitative analysis model the KD value of XKST binding to the efficacy target protein ACE was established by by NIRS technology combined with relevant chemometric methods.Through the model parameters,it can be determined that NIRS can be used for rapid and approximate quantitative prediction of the binding ability of XKST to the efficacy target protein ACE.On the other hand,after comparing and optimizing different pretreatment methods,the NIRS quantitative analysis models of eight Q-markers in XKST under two systems of intact tablets and uncoated tablets were established respectively.It can be seen from the modeling results that compared with the intact tablets model,the uncoated tablets model had a stronger ability to predict the contents of most Q-markers in XKST.In order to improve the prediction accuracy of the intact tablet model,we used improved principal component analysis(IPCA)method,which proposed earlier by our research group,taking puerarin as an example,to transfer model from the intact tablets to coated tablets.The results showed that the prediction accuracy of the intact tablets model was improved after the transfer,and the method could be used to achieve a more accurate and rapid non-destructive determination of Q-marker contents in XKST.Taking XKST as the research object,this study established a methodological system for rapid drug quality assessment featuring discovery,verification and application of Q-marker.That is,Q-marker was found by means of overall chemical composition characterization,serum pharmacochemistry,metabolomics,spectrumeffect relationship study,network pharmacology and other pharmacodynamic substances identification methods,and then its effectiveness and the rationality of the formula compatibility were verified by combining with zebrafish pharmacodynamics and molecular interaction study.Based on KD of binding to the efficacy target protein and Q-markers contents,a NIRS quality rapid evaluation system was established for efficacy and component evaluation.In this way,drugs can be more scientific and reasonable rapid non-destructive quality evaluation.