Research On Quality Standard Of Dahuang Zhechong Pill Based On The Correlation Of Ingredients And Efficacy

The determination of index components is the key for the study of quality standard of traditional Chinese medicine(TCM).At present,most of the index components are usually those with high content and wide application,but there is little correlation with safety and effectiveness,which are difficult to reflect the clinical quality of TCM.Therefore,it is urgent to establish a quality evaluation system in line with the characteristics of traditional Chinese medicine,which can reflect the clinical efficacy and the evaluation method of effective components.As a classic prescription,Dahuang Zhechong Pill(DHZCP)is widely used in clinic.However,until now,the Pharmacopoeia of P.R.China only focuses on emodin and chrysophanol in rhubarb,and lacks the determination of components related to pharmacodynamic.It is difficult to reflect the traditional efficacy of DHZCP in promoting blood circulation and removing blood stasis and evaluate its internal quality.Based on the traditional efficacy and the modern medicine indexes of promoting blood circulation and removing blood stasis,the project used the method of spectrum effect relationship to screen the quality markers that can characterize the efficacy of DHZCP,and carried out the research on the quality standard of DHZCP based on the component-efficacy correlation analysis.First of all,in order to show the effect of DHZCP on promoting blood circulation and removing blood stasis,we established a liver cirrhosis carcinogenesis model in the liver rich in blood vessels.The development process of this disease is similar to that of vascular abnormality,so we can evaluate the effect of DHZCP on inducing vascular normalization.The model of liver cirrhosis and liver cancer in rats was induced by DEN.After different doses DHZCP were given,the pathological changes of liver were observed by HE and Masson staining;the fenestra structure of hepatic sinusoidal endothelial cells was observed by scanning electron microscope;the effects of DHZCP on angiogenesis and maturation were evaluated by immunofluorescence and Western blot.The results showed that DHZCP could improve the fenestra structure of hepatic sinusoidal endothelial cells,reduce immature abnormal angiogenesis by decreasing the content of HIF-1α,VEGF,Ang2,Tie2,induce vascular normalization,promote blood circulation and remove blood stasis,and inhibit the canceration of liver cirrhosis.Secondly,according to the research method of spectrum effect relationship,the active components of DHZCP were screened by combining the blood components and pharmacodynamic indexes.Serum pharmacochemistry method and UPLC-QTOF-MS/MS technology were used to identify and analyze the blood migration components.Finally,103 components were detected,including 56 prototypes and 47 metabolites.Meanwhile,the factors(HIF-1α,VEGF,Ang2,Tie2)related to the mechanism of liver cancer and vascular abnormality were selected as the pharmacodynamic indexes.And the blood migration component map was effectively correlated with pharmacodynamic indexes by partial least squares(PLS)method,so as to preliminarily determine the effective constituents of DHZCP in inducing the vascular normalization.On this basis,the validation experiments were carried out from the cellular level to confirm the effectiveness of the pharmacodynamic substances,and further optimize the quality markers of DHZCP related to component-efficacy correlation analysis.Finally,26 quality markers of DHZCP were determined:allantoin,hypoxanthine,salidroside,hydroxypaeoniflorin,liquiritin,isoliquiritin,baicalin,P-methoxyphenylacetic acid,wogonin,cinnamic acid,apigenin,naringenin,norwogonin,aloeemodin,rhein,chrysin,emodin,glutamic acid,valine,leucine,tryptophan,linoleic acid,oleic acid,stearic acid,lactiflorin and 8,11,14-eicosatrienoic acid.Finally,to explore and optimize the detection method of DHZCP,the 26 quality markers are divided into three categories:4 kinds of amino acids,4 kinds of fatty acids and 18 kinds of other components.The following extraction and detection methods were established.Amino acids:hydrolyzed with 6M hydrochloric acid at 100℃ for 6 hours and determined by PITC pre-column derivatization HPLC method;fatty acids:n-hexane ultrasonic extraction,acid-base combined methyl esterification method followed by GC-MS determination;other ingredients:determined by HPLC-UV method after methanol extraction.In the end,a total of 24 quality markers except for lactiflorin and 8,11,14-tricosatrienoic acid were detected,including 4 amino acids,3 fatty acids and 17 other ingredients.And using this method,the content of 10 batches of DHZCP from different sources and different dosage forms was detected and quantified,and finally the quality evaluation standard for DHZCP based on component pharmacodynamic correlation was drawn up.In conclusion,based on the effect of DHZCP on promoting blood circulation and removing blood stasis and inducing vascular normalization,this paper uses spectrum effect correlation and cell verification to screen quality markers and establish the pharmacodynamic quality evaluation method of DHZCP,so as to provide reference for improving the quality standard of Pharmacopoeia and provide theoretical basis for its related research.