The Effect Component Of Kudiezi Injection Based On Cell Biochromatography To Protect Endothelial Cells From Hypoxia Injury

Kudiezi injection has been playing an important role in treatment of cerebrovascular and cardiovascular diseases,which is applied widely in clinic.However,it is not clear about their effective constituents,owing to the low isolation and sensitivity of traditional separation methods.A lot of time had been spend in the separation of ineffective or non-active ingredients in traditional Chinese medicine(TCM),which also leads to the loss of active ingredients during the separation process.Nowadays,the interaction of drugs with target cells has been successfully employed as a screen for bioactive components in TCMs.In this study,the effective method has been employed as a tool for screening active components in Kudiezi injection by using human umbilical vein endothelial cells(HUVECs)extraction and sensitive UHPLC-HR-MSn method.The potential bioactive components in Kudiezi injection could be selectively bound to the cells so that the unbound components were washed away by the phosphate buffer saline(PBS),while the cell combound components in the extract of denatured cells were determined by UHPLC-LTQ-Orbitrap analysis.Finally,verify experiments will take for the cell combound components.The application of HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis is a rapid,simple and reliable method for screening immunological active components from TCMs.ObjectiveA rapid and sensitive UHPLC-HR-MSn method was used for the identification of Kudiezi injection and its main metabolites in rat plasma;a effective method has been employed as a tool for screening active components in Kudiezi injection by using HUVECs extraction and sensitive UHPLC-HR-MSn method;to study the effect of potential bioactive components in Kudiezi injection after hypoxia in HUVECs.Methods1.Investigate the in vivo metabolism of Kudiezi injection following intravenously administered.ACQUITY UHPLC BEH C18(1.7?m,2.1x100 mm)was used with 0.1%formic acid-acetonitrile solution as the mobile phase in gradient elution.The Kudiezi injection and plasma were detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode.2.The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs so that the unbound components were washed away by the PBS,while the cell combound components in the extract of denatured cells were determined by UHPLC-LTQ-Orbitrap analysis.3.Set up Na2S2O4 induction of HUVEC anoxic damage model,and apply NaHS and potential bioactive components to intervene.Cell Counting Kit-8 assay(CCK-8)was chosen to measure the cell viability.The content of LDH and SOD were detected in supernatants and cells,respectively.Results1.Based on the accurate mass measurements,the retention time and mass fragmentation patterns,in total,53 compounds were tentatively identified and characterized.Furthermore,metabolites in rat plasma after intravenous administration of Kudiezi injection were also analyzed.A total of 38 compounds were identified,including 27 prototypes and 11 metabolites through metabolic pathways of methylation,glucuronide conjugation,sulfate conjugation,and hydrolysis.2.Based on the accurate mass measurements and the retention time,in total,9 compounds were tentatively identified and characterized,including 3 phenolic acids,2 flavonoids and 4 sesquiterpene lactones.Furthermore,9 cell combound components are included in the 38 chemical constituents which found in rat plasma.3.The statistical data determined by CCK-8 assay showed that NaHS and the 3 potential bioactive components have obvious protective effect on cells(P<0.01 or P<0.05),no obvious difference between the two groups(P<0.01 or P<0.05).The statistical data showed that NaHS and the 3 potential bioactive components can decrease the content of LDH in cells and increase the content of SOD in supernatants,respectively(P<0.01 or P<0.05).Conclusion1.These results are very helpful for the better comprehension of the metabolism and very useful for further study of pharmacodynamic substance from Kudiezi injection.2.HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis provides a rapid and efficient method for the identification of potential bioactive components in TCMs.3.A simple,stable as well as reliable modle of hypoxia on HUVECs was established successfully.The application of HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis is a rapid,simple and reliable method for screening active components from TCMs.