The Study Of Establishing The Fingerprint Chromatogram And Revealing The Anti-hepatic Fibrosis Effect Of Corydalis Saxicola Bunting Based On Serum Metabonomics

Objective:1.Establish the high performance liquid chromatography?HPLC?fingerprint of Corydalis saxicola Bunting?CS?and use the multi-components analysis,to provide the basis for the recognition and evaluation of CS.2.To reveal the mechanism of anti-hepatic fibrosis of CS based on serum metabonomicsMethods:1.Using the high performance liquid chromatography?HPLC?to optimize the extraction process of CS,and evaluated the reliability.2.The similarity of CS samples from 10 different regions was evaluated by application of"Chinese medicine chromatographic fingerprint similarity evaluation system"?2004 a?.And the quantitative analysis of 4 components of CS,such as coptisine,dehydrocavidine,palmatine,berberine,was carried out in the present study.3.Healthy 40 male SD rats were randomly divided into 2 groups:control group?n=10?and treatment group?n=30?.The rats in control group were treated with olive oil,and the treatment group was given 50%carbon tetrachloride?CCl₄?olive oil solution for 2 times per week,with a total of 10 weeks.After seven weeks when treated with 50%CCl₄ olive oil solution,the rats were divided into model group,positive group,and CS group,which were administered for 4 weeks.4.Evaluate the intervention effect of CS by measuring the activities of the alanine aminotransaminase?ALT?,aspartate aminotransaminase?AST?,alkaline phosphatase?ALP?,albumin?ALB?,globulin?GLB?,total protein?TP?,malondialdehyde?MDA?,superoxide dismutase?SOD?and histopathologic examination.5.Find the difference of serum metabolites between the model group and the control group,and analyze the possible relationship between the potential biomarkers and the hepatic fibrosis,based on the metabolomics by nuclear magnetic resonance?NMR?and combined with statistical analysis and online metabolome database.Furthermore,we could characterize the changes of endogenous metabolites in the serum of rats after treatment with CS,in order to find the potential biomarker and target which may be related to the anti-hepatic fibrosis effects.Results:1.A total of 15 common peaks were found out in the HPLC fingerprint,and the similarity of the 10 batches of CS samples was more than 0.940.2.The average content of coptisine,dehydrocavidine,palmatine and berberine in CS were 0.50±0.17 mg/g,10.25±1.46 mg/g,6.02±1.78 mg/g and 2.17±0.41 mg/g,respectively.3.The levels of ALT,AST and ALP in serum were decreased significantly?P<0.05?,and the levels of ALB/GLB was increased significantly?P<0.05?after treated with CS;the level of MDA was decreased significantly?P<0.05?,and the level of SOD was increased significantly?P<0.05?after treated with CS;the pathological changes of liver in rats were improved after treated with CS.4.A total of 13 metabolites were characterized as potential biomarkers involved in CCl₄-induced hepatic fibrosis,which the levels of lactate,alanine,arginine,acetate,N-acetyl-glycoprotein,creatine and choline were elevated,and the levels of lipid,leucine,valine,isoleucine,O-acetyl-glycoprotein and glucose were declined.Those 13 metabolites were corresponding to the dysfunctions of four pathways including amino acid metabolism,carbohydrate metabolism,lipid metabolism and other metabolism.5.The levels of alanine,acetate and creatine in serum were decreased,and the levels of lipids,leucine,and O-acetyl-glycoprote were increased after treated with CS on CCl₄-induced hepatic fibrosis in rats,which indicated that CS treatment might remedy the dysregulation of valine,leucine and isoleucine metabolism,alanine,aspartate and glutamate metabolism,arginine and proline metabolism,carbohydrate metabolism disorders,and lipid peroxidation induced by CCl₄ injury.Conclusion:1.The combination of fingerprint and multi-components quantitative analysis can comprehensive reflected the quality of CS and can be regarded as a scientific basis for controlling the quality of CS.2.CS can improve the changes of biochemical indexes in serum and liver,and relieve the pathological states of liver fibrosis in rats.3.The hepatic fibrosis model induced by CCl₄ could cause significant changes of serum metabolic profiles in rats,including amino acid metabolism,carbohydrate metabolism,lipid metabolism and other metabolism,which could be regulated by CS treatment significantly.