Quantitative Characterization Of Bear Bile Powder Cholic Acid Group And Its Regulatory Effect On Cholic Acid Group In Rats

Bear bile is the dried gallbladder of the black bear or brown bear.It was originally published in the Tang materia medica and listed as the top grade.It is a traditional and precious medicinal material commonly used in clinic.It has a bitter cold taste,and it has the effect of "sealing the veins,preventing rot,growing muscles,and improving eyesight".Modern clinical researches showed that bile acids,such as tauroursodeoxycholic acid(TUDCA),ursodeoxycholic acid(UDCA),chenodeoxycholic acid(CDCA),cholesterol and amino acids,etc.,contained in bear bile had obvious therapeutic effects on the treatment of hepatobiliary diseases and related diseases caused by hepatobiliary disorders.Therefore,it is important to clarify chemical profiles and action mechanism of bear bile powders(BBP).Based on serially coupled reversed phase-hydrophilic interaction liquid chromatography-tailored multiple reaction monitoring,our team carried out comprehensively quantitative metabolome profiling of BBP and more than ten medicinal bile.These medicinal bile were primarily consisted of amino acids,bile acids and arachidonic acids,and preliminary results indicated that the significant differences not only occurred among bile samples originated from different species,but also within BBP from different vendors,and bile acids served as the primary indicative cluster in either case.Based on preliminary researches,this study is aim to focus on bile acids.Firstly,1H-NMR technology was used to preliminarily clarify the chemical composition of BBP and other medicinal bile.The results indicated that bile acids were not only main chemical composition but also diagnostic components of BBP and other medicinal bile.Furthermore,the method for detecting bile acid pool was established using LC-MS,and it was applied to quantitatively characterize the bile acid pool of BBP and other medicinal bile to find the diagnostic biomarkers.Combined with metabolomics method,the regulatory effect of BBP on bile acid pool in rats was carried out.The detailed research contents and results are as follows:1.Literature review:Based on domestic and foreign literature,the research progress of the chemical composition,metabolism and the role in cholestasis and liver protection of bear bile powders were systematically reviewed.The chemical profiles in BBP are various.Bile acids are main compositions in BBP,there are also bile pigments,mucins and amino acids,a small amount of lipids and trace elements in the BBP.BBP and some bile acids have significant curative effect in protecting gall bladder and protecting liver.However,in view of the medicinal substances of BBP are the same as or similar to the endogenous components,it brought great difficulties to the in-depth explanation of the medicinal substances and action mechanism of BBP,so it is urgent to systematically study the metabolism of BBP in vivo to provide a theoretical basis for the medicinal use of BBP.2.Construction of bile acid pool detection method based on the LC-MS platform:Firstly,online parameter optimization of the existing bile acid reference substances were established to obtain the corresponding optimal collision energy(OCE)and CE50(that is termed as the collision energy at 50%survival yield of the protonated or deprotonated molecular ion)to find the differences in mass spectrum performance of isomers,and then combined with quantum chemical calculation,the quantitative correlation between collision energy and chemical structure was summarized.In addition,universal metabolome standard(UMS)solution that was believed to contain all bile acids information was constructed by mixing extracts of more than ten kinds of bile medicinal materials with biological samples of rats.Automated MS/MS was applied on IT-TOF-MS to provide high-resolution precursor and fragment ion species,and diverse survey experiments including predictive multiple reaction monitoring mode(pMRM),precursor ion scanning(Prec),neutral loss(NL),enhanced mass spectrometry scan(EMS)and enhancer ion scan(EPI)were utilized on Qtrap-MS for searching BAs as more as possible.According to information provided by mass spectrometry,retention time matching,combined with reference substances and database retrieval,a total of 120 bile acid derivatives were observed and identified in UMS,including 27 free BAs,14 glycine-conjugated BAs,30 taurine-conjugated BAs,19 BA sulfonates and 30 BA glucuronides.At the same time,a method for simultaneously detection of 120 bile acids based on the LC-MS platform was established.It provided a detection method for the qualitative and quantitative analysis of bile acids in BBP and its effect on bile acid pool in vivo.3.Quantitatively characterization of the bile acid profiles in BBP:In view of the NMR can quickly and comprehensively provide qualitative and quantitative information of traditional Chinese medicines(TCMs)with non-polar and non-structural discrimination,this study used 1H-NMR to universally analyze and compare the chemical profiles of bear bile and other bile-class TCMs to find the differences.After extraction,1H-NMR spectrums were recorded in parallel,seven bile acids were identified from the representative spectrum with the assistance of literature and reference compounds.Multivariate statistical analysis results indicated that BBP was richer in UDCA and TUDCA.In order to overcome the technical problems of seriously stacked signals of similarly structured compounds in the NMR spectrum and difficult identification of trace components,the bile acids in BBP were further analyzed using LC-MS platform.A total of 63 bile acids were detected,including 20 free BAs,6 glycine-conjugated BAs,27 taurine-conjugated BAs,10 BA glucuronides.Calibration curve was constructed for each detected identity by preparing and serially diluting the UMS to acquire the quasi-contents.Among the detected BAs,10 bile acids were accurately qualified by reference standards.Afterwards,the quasi-content dataset containing the quantitative information of all bile samples was subsequently introduced for principal component analysis,the results indicated that bile acid profiles were significantly different from other medicinal bile samples,and the primary biomarkers including TUDCA,TCDCA and their isomers and glucuronidation ect.Based on NMR and LC-MS technology,the bile acids profiles in BBP and the differences with other bile-class TCMs were clarified,providing references for quality control and alternative resources development.4.The regulating effect of BBP on the bile acid profiles in rats:The bile acids in the plasma of rats after administration of BBP and its main components UDCA and TUDCA were detected by the validated method respectively.Subsequently,the quantitative program was implemented to establish the time courses of BA pool fluctuations in BBP-,UDCA-,TUDCA-and vehicle-treated rats in order to clarify the regulating effect of BBP on the bile acid profiles in rats.Mild variations were observed for quantitative pattern of the BA pool from the vehicle group within 72 h.Furthermore,BBP resulted in most significant migrations for the BA pools,indicating BBP most significantly regulated the bile acid profiles in rats.And migrations especially occurred during the period of 8?20 h postdose.Significant increments occurred for the contents of not only the downstream BAs,but those upstream molecules in the BA metabolic network.The migration distances of BAs were primarily governed by their distances away from BBP,UDCA or TUDCA.Through comprehensive quantitative analysis of the bile acids pool in rats,the regulatory effect of BBP on the bile acid group of rats was clarified,the biomarkers were found,providing guiding significance for finding drug targets and clinical applications.