Non-targeted Metabolomics Study Of Quality Control Of Pulsatilla Adans And Mechanism Of Pulsatilla Decoction In The Treatment Of Acute Ulcerative Colitis

Objective:Based on non-targeted metabolomics,UHPLC-MS technology was used to establish the quality control platform of Pulsatilla Adans?PSA?,and the mechanism of action of Pulsatilla Decoction in the treatment of ulcerative colitis was comprehensively described.Methods:At first,full-scan-data-dependent MS/MS?FS-ddMS²?acquisition and selective ion monitoring?SIM?modes for parent ion scan and daughter ions scan was conducted based on UHPLC/Q-Orbitrap-MS.The fragmentation patterns of ten major triterpenoid saponins were studied.Then the clevage rules were summarized.TraceFinder^TM 4.1 was used to establish a nontargeted bi-directional screen screening strategy to identify the chemical constituents of pentacyclic triterpenoid saponins in four Pulsatilla Adans herbs.Multivariate statistical analysis was used to distinguish the Pulsatilla Adans herbs.Secondly,UHPLC-Q-TOF-MS/MS technique was employed to acquire exact MS and MS/MS datas.The data acquired were analyzed and identified by MetabolitePilot^TM 2.0 software.According to the biotransformation knowledge and fragmentation pattern of parent drug,the metabolites of betulin were deduced and Clog P values were applied to distinguish isomers.At last,a mouse model of ulcerative colitis was established.The experimental animals were divided into model group and control group,positive control group,low-dose treatment group,medium-dose treatment group and high-dose treatment group?n=8?,and the model was verified by HE staining.Metabolomics testing was performed using LC-MS.Raw data was processed and analyzed by Progenesis QI and SIMCA-P 14.1 software.Using the T-test to obtain the p-value and establish the orthogonal partial least squares discriminant analysis?OPLS-DA?model to obtain the VIP value,while the components satisfying the P<0.05,FC?1.2 or FC?0.8,CV<30%?QC?and the VIP>1 are considered to be differential compounds.Compound identification was conducted by comparing the mass spectrometry information of different compounds with the online database?HMDB,METLIN?.The receiver operating characteristic?ROC?curve was applied to verify the accuracy of the potential diagnostic biomarkers,and the online software MetaboAnalyst 4.0 was performed to biomarker enrichment and metabolic pathway analysis.Results:Based on UHPLC/Q-Orbitrap-MS technology,a total of 157triterpenoid saponins were screened from Pulsatilla Adans herbs,and 34constituents were reported in Pulsatilla Adans herbs for the first time.Twelve differential compounds were obtained by multivariate statistical analysis?the ROC curve analysis showed good accuracy,AUC=1?.It is used to distinguish four kinds of Pulsatilla Adans herbs.Based on UHPLC-Q-TOF-MS/MS technology,a total of 64 metabolites were detected in the plasma,urine,feces,bile and intestinal bacteria biotransformation samples after oral administration of betulin to rats.Includes 56?phase metabolites,and 8?phase metabolites.In the metabonomics study of intestinal bacteria and feces,the original data were screened and identified by the multivariate statistical analysis and online database matching,twenty-three potentially differential metabolites were screened out and confirmed,including endogenous small molecular substances such as lipids,fatty acids and sterols.These biomarkers are mainly involved in biotin metabolism,steroid hormone biosynthesis,biosynthesis of unsaturated fatty acids,retinol metabolism,sphingolipid metabolism,glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,linoleic acid,phenylalanine and?-linolenic acid metabolism,steroid biosynthesis,porphyrin and chlorophyll metabolism,niacin and nicotinamide metabolism,tryptophan metabolism,arachidonic acid metabolism,etc.Conclusion:This study used UHPLC/Q-Orbitrap-MS detection technology to establish a comprehensive chemometrics analysis strategy platform for the characterization and classification of four kinds of PSA.This method provides promising perspectives in the analysis and identification of the ingredients of Traditional Chinese Medicine?TCM?,and the identification of similar herbs from the same species.Based on UHPLC-Q-TOF-MS/MS method,the metabolites of in vitro and in vivo biological samples after oral administration of betulin to rats were successfully identified,which laid a foundation for the clarification of the pharmacological mechanism of betulin.After that,the non-targeted metabolomics technique based on systems biology was established to analyze the endogenous substances of biotransformation and fecal samples in mice with ulcerative colitis under the action of low-dose Pulsatillae decoction.It was finally discovered that Pulsatilla decoction can interfere with changes in the biological multiple metabolic pathways.This experiment provides ideas and guidance for the molecular mechanism of Pulsatillae decoction in the treatment of ulcerative colitis.