Study On The Chemical Profiling In Vivo And Pharmacokinetic Of Traditional Chinese Medicine Formula Dan Zhi Tablet

Traditional Chinese medicine formula Dan Zhi Tablet?DZT?is a TCM formula that consists of Astragalus membranaceus?Huangqi?,Salvia miltiorrhiza?Danshen?,Whitmania pigra?Shuizhi?,Ligusticum chuanxiong?Chuanxiong?and Pheretima aspergillum?Dilong?in a ratio of 15:12:6:3:10.DZT has been estimated to show significant therapeutic effects on increasing energy,unblocking collaterals,activating blood and relieving pains.It has been used in clinic for easing the symptoms of convalescence of cerebral infraction disease,and many years clinical application confirms that DZT has remarkable curative effect.No report has been published on the metabolism of DZT,and its serum pharmacochemistry study and the dynamic changes of bioactive components in vivo were still not clear,which hindered interpreting the potential bioactive constituents and studying integrative mechanisms.In the present study,DZT was chosen as the research topic.A selective and simple UPLC-QTOF/MS method combined with intelligent automated analysis software was developed to detect the constituents in rat plasma after oral administration of DZT,including prototypes and metabolites.Furthermore,the components which could penetrate Blood-Brain-Barrier?BBB?have also been detected in rat cerebrospinal fluid?CSF?.Finally,pharmacokinetic study of DZT was successfully established to clarify the dynamic change process of the bioactive components.Therefore,the pharmacodynamic material basis was clear,which could provide scientific basis for clinic.The specific research contents are as follows:1.Serum pharmacochemistry of DZTBased on the methods of metabonomics and guided by serum pharmacochemistry theory,the method of UPLC-QTOF/MS combined with UNIFI and multivariate statistical analysis software was established.The prototype components and metabolites in rats plasma after oral administration of DZT were analyzed and identified.Firstly,an in-house database of the chemical constituents from DZT in vitro,was established and then directly imported into UNIFI library.The data of the prototype constituents in vivo was screened by matching them with database,and further confirmed.Secondly,another library containing adequate information on biotransformation rules of absorbed prototype constituents by UNIFI software was established for screening the metabolites of DZT in vivo.And then,the data of candidate metabolites were displayed in UNIFI software for further matching analysis.Thirdly,OPLS-DA analysis?n=6?conducted by Progenesis QI and Ezinfo software was processed to differentiate potential constituents from endogenous matrix,yielding unmatched prototype and metabolite constituents rapidly.Fourthly,a comparison test of the characteristic fragment ions between DZT-dosed group and each single herb group was processed to distinguish some overlapped chromatographic peaks and assign the potential compounds from various sources.Using this strategy,a total of 170 compounds,including 51 prototype constituents and 119 metabolites were unambiguously or tentatively identified in rat plasma.According to the structure types,they can be divided into flavonoids,tanshinones,phthalides,phenolic acids,drilodefensins and others.It is the first systematic metabolic study of DZT in vivo and some metabolites were also reported for the first time,which could provide a scientific basis for explaining the multiple functions of DZT.More importantly,the integrated strategy also shows promising perspectives in the identification of the metabolites in TCM from a complicated biological matrix.2.Cerebrospinal fluid pharmacochemistry of DZTBased on the results of prototype constituents and identified metabolites in plasma and guided by the theory of cerebrospinal fluid pharmacochemistry,the method of UPLC-QTOF/MS was established.This chapter made a comprehensive qualitative analysis of the components which could pass through the BBB after oral administration of DZT in rats.By extracting ion chromatography?EIC?,the chemical constituents of blank cerebrospinal fluid,drug-containing cerebrospinal fluid and drug-containing plasma were compared.Finally,17 prototype constituents,as well as 14 metabolites,were tentatively characterized and confirmed by their retention times,MS^E spectra.From the results obtained,four types of constituents have been found to be absorbed in brain and thus played an indispensable role in curing the disease,including flavonoids,tanshinones,phthalides and furan sulfonic acids.In this study,a sensitive,accurate and applicable analytical method for the determination of components in vivo was established.It is the first time to identified the components of DZT in CSF,which provide data support for network pharmacological studies and pharmacokinetic studies.3.A comparative study of pharmacokinetics of DZT in rats of sham-operated and cerebral infarction modelOn the basis of the above research,nine target analytes with definite pharmacological activity were selected and the middle cerebral artery occlusion?MCAO?model was used to explore the difference of pharmacokinetic parameters between MCAO model group and sham operation group rats after oral administration of DZT?4 g/kg rat weight?.Acetonitrile precipitated protein was used for plasma samples,and gradient elution was performed based on positive and negative multiple reaction monitoring?MRM?acquisition modes.Using aloe emodin as internal standard under positive ion mode,including three flavonoids?ononin,calycosin,formononetin?,one phthalide?senkyunolide I?and four tanshinones?dihydrotanshinone I,tanshinone I,cryptotanshinone,tanshinone IIA?were determined in 10min,respectively.Isofraxidin was selected as internal standard under negative ion mode to determine the5-ethyl-2-hexyfuran-3-sulfonic acid in 5 min.The method is specific and sensitive.Based on the non-atrioventricular model,PK parameters were calculated by DAS 2.0software.The results showed that some pharmacokinetic parameters of nine bioactive components were significantly different between MCAO model group and sham-operated group.Compared with the sham-operated group,the AUC_0-t and AUC_0-?of MCAO group were higher in ononin,dihydrotanshinone I and tanshinone I,while the AUC_0-t and AUC_0-?of cryptotanshinone and tanshinone IIA were lower in MCAO group;the C_max and T_max of senkyunolide I were larger in MCAO group;the AUC_0-t and AUC_0-?of 5-ethyl-2-hexyfuran-3-sulfonic acid were lower in MCAO group;and there was no significant difference in formononetin and calycosin between the two groups.It is the first to establish the method to determine the serum concentration of nine active ingredients in MCAO and sham-operated rats after oral administration of DZT,which provides a scientific basis for guiding rational use in clinic and improving drug efficacy.In conclusion,this paper established a method to systematically characterize the components in plasma and CSF of rats after oral administration of DZT,and compared the pharmacokinetic characteristics of the active components in vivo between model group and sham-operated group.It provided theoretical basis for clinical medication safety and drug monitoring,and also provided strategies and methods for the study of other TCMs in complex biological matrix.