Study On Quality Standard And Pharmacokinetics Of Calculus Bovis Sativus Based On Bile Acids

Purpose:Calculus Bovis?C.Bovis,CB?is a kind of valuable traditional Chinese medicine,mainly used in the treatment of coma,delirium,epileptic seizures,throat abscess,carbuncle,boils and other diseases,modern research also confirmed its diversified pharmacological effects.The pharmacological effects of Calculus Bovis Sativus?in vitro cultured Calculus Bovis,CBS?is considered to be equivalent to natural CB,and is used as an ideal alternative to natural CB because of the high price and the low quality drug source of natural CB.At present,bile acid?BAs?and bilirubin is considered to play the main pharmacological effects in CB.Cholic acid?CA?and bilirubin are used as quality control of CB and CBS in Chinese pharmacopoeia.However,the composition of CB is complex,only use CA and bilirubin as quality control cannot represent internal standards of CB and only two components cannot explain its various pharmacological effects.Therefore,the determination of various components in CB for quality control and subsequently clarify its pharmacological effects are of great significance.In this study,CBS and natural CB were used as the main research object.The contents of taurine?Tau?and12 kinds of BAs in CBS and natural CB were determined by liquid chromatography coupled with tandem mass spectrometry?LC-MS/MS?.The detection method of Tau and BAs in whole blood and tissue samples was also established and their pharmacokinetic process and distribution in the tissues were compared.This may provide a pharmacokinetic thinking for their material basis study.Methods:A rapid and simple LC-MS/MS method is developed for identification of Tau and 12 kinds of BAs.The CBS and natural CB were ground to fine powders and filtrated with 20 mesh sieves.The contents of 13 components in 15 batches of CB were determined and compared.LC-MS/MS methods for simultaneous determination of Tau and 12 BAs in blood and tissue samples of mice were also established.The whole blood and tissues of mice were collected at different time points after CB and CBS treated.The concentration of Tau and BAs in serum and tissues were determined by LC-MS/MS.Results:1.A LC-MS/MS analysis method for simultaneous detection of Tau and 12BAs was successfully established.The method is simple and rapid with high sensitivity and good specificity.2.The characteristics of BAs and Tau contents in CBS and natural CB were analyzed and compared.The results showed that CA/DCA in CBS was almost twice as high as that in natural CB,and that non-binding BAs/bindingBAs in natural CB was higher than that in CBS.Therefore,the combination of CA/DCA,non-binding BAs/binding BAs and the chromatographic quantitative data of each BA can be used as a specific indicator to identify CBS and natural CB.Besides,the Tau/?TCA+TDCA+TCDCA?in CBS and natural CB was compared and found no significant difference between CBS and natural CB.3.LC-MS/MS methods were built to simultaneously detect Tau and 12 BAs in the biological samples of mice.The precision,accuracy,stability and other conditions of this method were in line with the requirements of the determination method of bioequivalence experiment and this method can be applied to the pharmacokinetic and tissue distribution study in mice.4.CA,DCA,TCA,TDCA,CDCA and Tau were both detected in the blood of CBS group and natural CB group.Besides,GCA was also detected in blood of CBS group.The AUC_?0-t?of CA,DCA,TCA,CDCA,Tau and GCA were calculated by using the DAS2.1.1 pharmacokinetic program.There were no significantly differences in AUC_?0-t?of CA,DCA,TCA,CDCA,Tau and GCA between the two groups.In CBS group,Tau and BAs were well distributed in heart and lung.TCA and TDCA were also distributed in the spleen and brain tissues.The distribution of GCA and Tau in lung was better than that in other tissues.CDCA was distributed at all time points in each tissue.In natural CB group,the distributions of several BAs in this group were not as good as that in CBS group.CA was distributed in the heart,lung and kidney.The GCA was not measured in the tissues of CB group.The Tau distribution was less than that in the CBS group,while the distribution of CDCA,TCA and TDCA showed the same distribution trend in natural CB group and CBS group.Conclusion:In this study,a LC-MS/MS method was developed to detect Tau and 12kinds of BAs,and the methods for determining the concentration of Tau and 12 kinds of BAs in biological samples of mice were also established.These methods have high sensitivity and specificity.It is suitable for CB Component analysis,quality control and authenticity identification.In addition,it was successfully applied to the study of the pharmacokinetic characteristics of Tau and BAs of CBS and natural CB in mice.This study may provide a reference for the future study of CB,as well as other traditional Chinese medicine.