| Quality control is one of the key issues for the modernization of traditional Chinese medicines(TCMs).Nowadays,its research methods based on traditional morphological and histological identification have already developed into those based on modern chromatographic and mass spectrometric analysis.Most of the analytical methods have been limited to select one or several marker components with high content in TCMs.However,they are usually not effective conponents of TCMs,and thus it is difficult to efficiently control the quality of TCMs.Therefore,in order to clarify the key issue,Kudiezi injection was adopted as the model drug in the present study,which has reliable clinical effects in treating cerebral infarction.The methods for quality control and in vivo metabolism were established,and pharmacokinetics and pharmacodynamics(PK-PD)model based on the effect compartment drug concentration of multiple components has been constructed for the first time.Eventually,the study will provide new idea and methodology based on multi information fusion of HPLC fingerprint-chemical constituents-multi-component quantification-multi-component PK-PD study for TCMs quality control system.The main contents and contributions were summarized as follows:1.Research on chemical constituents:A HPLC-ESI-MSn method was first to be developed for the screening and identification of the constituents in the Kudiezi injection.According to the proposal fragmentation pathways,a total of 33 constituents attributed to nucleosides,phenolic acids,flavonoids and sesquiterpene lactones were quickly identified from Ixeris sonchifolia and Kudiezi injection.Among them,10 constituents including guanosine,1-CQA(caffeoylquinic acid),3-CQA,4-CQA,3-pCoQA(p-cinnamylquinic acid),5-pCoQA,3-FQA(ferulicquinic acid),4-FQA,3,4-DiCQA(dicaffeoylquinic acid)and 3,5-DiCQA were reported from Ixeris sonchifolia for the first time.2.In vivo metabolism:Firstly,the prime components and metabolites in rat plasma after intravenous administration of Kudiezi injection were identified using UPLC-MS/MS coupled with three different scan modes of full-MS,MRM and neutral loss scan.As a result,29 metabolites were screened and identified,which including 25 prime components and 4 phase? metabolites,respectively.The phase ? metabolites were deduced as CQA methyl conjugates and luteolin-7-O-?-D-glucoside sulfate conjugates.Secondly,the established UPLC-MS/MS method was successfully applied to simultaneous quantification of 8 phenols in the rat plasma including 3-CQA,5-CQA,4-CQA,3,4-DiCQA,3,5-DiCQA,4,5-DiCQA,LG(luteolin-7-O-?-D-glucoside)and AG(apigenin-7-O-?-D-glucoside)from Kudiezi injection.As a result,they all showed good linearity(R>0.9958)in the given concentration ranges of 2.6-238.0,5.6-258.0,4.6-210.0,2.4-110.0,3.6-166.0,2.5-116.0,0.7-134.0 and 0.4-65.2 ngˇmL-1,respectively.Meanwhile,the method was also successfully applied to the pharmacokinetic study of 8 phenols after intravenous administration of Kudiezi injection and co-administration of Xueshuantong freeze-dried powder.Then,their respective pharmacokinetic characteristics have been explored using the software of WinNonlin.The pharmacokinetic parameters of polyphenols including AUC,CLZ,Cmax,and so on,were significantly different between groups with high and low dose(P<0.05).When combining with Xueshuantong,the pharmacokinetic parameters of polyphenols also were significantly different(P<0.05).Thirdly,the UPLC-MS/MS method was subsequently adopted to study the tissue distributions of phenols from Kudiezi injection.Owing to the wide distribution and being quantified of the LG,a quantitative method was subsequently developed and validated to explore its dynamic distribution in 10 kinds of rat tissues.The result showed that the concentrations of LG in various tissues were as follows:at 15 min,kidney>spleen>liver>small bowel>muscle>heart>lungs>stomach>cerebellum>cerebrum;at 30 min,kidney>liver>spleen>small bowel>muscle>cerebrum>lung>heart>stomach>cerebellum;at 60 min,kidney>small bowel>liver>cerebrum>spleen>heart>lung>muscle>stomach;at 120 min,stomach>small bowel>liver>cerebrum>spleen>muscle>lung>stomach>heart.At last,multivariate statistical methods were introduced to clarify the relationship between the elimination rate of certain polyphenol and concentrations of polyphenols in rat plasma.In the end,the kinetics models of component-component interaction have been constructed among three groups of high dose,low dose and co-administration of Xueshuangtong freeze-dried powder.And the correlation coefficients(R)of 24 models were no less than 0.85.Meanwhile,the results showed that the elimination rate of phenols was not only related to their concentration,but also associated with the concentration of the other constituents in rat plasma.3.Research on quality control based on PK-PD model:Firstly,the hypothetical effect compartment was introduced to perform the "flexible connection" between the PK model and the antioxidation PD model of phenols in Kudiezi injection.Meanwhile,the PD model based on effect compartment drug concentration has been constructed.Finally,LG,5-CQA and 4,5-DiCQA in Kudiezi injection were preliminarily confirmed to be relevant to activities of quantitative change of SOD,which would be added to the quality control standards of Kudiezi injection.Secondly,the HPLC fingerprint of Kudiezi injection with 14 common peaks has been established,including the above three active components.And the similarity among various batches of Kudiezi injection was no less than 0.94.Moreover,the principal component analysis and cluster analysis were applied to the quality control and evaluation of Kudiezi injection from different manufacturers,and they were divided into two categories in the end.Thirdly,a simple and rapid UPLC-MS/MS method for simultaneous determination of 10 phenols including 3-CQA,5-CQA,4-CQA,chicoric acid,LG,3,4-DiCQA,3,5-DiCQA,4,5-DiCQA,luteolin and caffeic acid has been developed and validated using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry(UPLC-ESI-MS/MS).The established method was validated in terms of good linearity(R>0.9995).And very low limits of detection(LODs)of 0.002-0.63 ngˇmL-1 and limits of quantitation(LOQs)of 0.0050-2.93 ngˇmL-1 of the ten components were achieved.Furthermore,the analysis was accomplished within 9 minutes and all of the 3-CQA isomers and 3-DiCQA isomers could reach their respective baseline separation.In conclusion,the study has established the novel research mode of HPLC fingerprint-chemical constituents-multi-component quantification-multi-component PK-PD,and screened and confirmed the quality control components which were related to the antioxidant effect of Kudiezi injection,which can provide new ideas and methodology to construct TCMs quality control system based on effective components. |
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