Studies On Characteristic And Analysis Of Chemical Components Of Aster Tataricus And Inhibitory Effect Of Friedelan-3beta-ol On Cytochrome P450s In Human Liver Microsome

Aster tataricus rhizoma?Ziwan in Chinese?,the dried rhizome of Aster tataricus L.f.,is a well-known traditional Chinese medicinal herb officially listed in the Chinese Pharmacopoeia.In clinical applications of traditional Chinese medicine?TCM?,this herb has been proved to be effective in treating sputum and cough diseases.For that reason,A.tataricus works as an important component of many common Chinese medicine curing cough and asthma.Phytochemical studies on A.tataricus reveal that it contains triterpenes,flavonoids,coumarins,phenolic acids,sterols and volatile oils,etc.Modern pharmacological studies have shown that the triterpenoids shionone and friedelan-3beta-ol in A.tataricus have significant expectorant,antitussive effect,and shionone is the feature component of A.tataricus.Recently it was discovered that A.tataricus extract had a number of bioactivities,including antitumor activity,antibacterial activity and antioxidant activity.In addition,existing clinical reports confirm that A.tataricus can be used as diuretic and laxative.In the present study,UHPLC-MS/MS for the quantitative and qualitative analysis of constituents in A.tataricus were developed.The incubation system of human liver microsome was established.The metabolite selective probe substrate was detected to calculate the value of IC₅0 and it was used to assess the inhibitory potency of friedelan-3beta-ol on CYP450 enzymes in human liver microsome.Part one The characterization of constituents in A.tataricus byUHPLC-Q-TOF-MS/MSObjective: To develop a practical method using high performance liquid chromatography with quadruple time-of-flight mass spectrometry?HPLC-Q-TOF-MS/MS?technology for the rapid separation and identification of triterpenes,flavonoids and coumarins in A.tataricus.Methods: First,we studied the mass fragmentation patterns of 11 standards of A.tataricus in the positive and negative ion mode by full scan,product ion scan and precursor ion scan,and summarized the fragmentation rules.Then,on the basis of the summarized rules,A.tataricus sample was analyzed by UHPLC-Q-TOF-MS/MS in the scanning mode of TOF-MS-IDA-8MS/MS to acquire exact mass and fragment ions of the unknown compounds.Then,the combination use of Peak View tools including “MasterView” were chosen to identify the constituents of A.tataricus and deduce its fragmentation rule.Chromatographic experiments were performed on an Agilent Poroshell 120 EC-C18 column?100 mm×2.1mm,2.6 ?m?with solvent A?water containing 0.1% formic acid?and solvent B?acetonitrile containing 0.1% formic acid?as the mobile phases in gradient elution.The flow rate of mobile phase was set at 0.4 mL/min and the injection volume was 2 ?L.Results: The fragmentation rules of 11 standards?4 triterpenes,5flavonoids and 2 coumarins?were summarized by studying the mass fragmentation patterns of 11 standards in the positive ion mode by full scan,product ion scan and precursor ion scan.A total of 15 compounds?4triterpenes,8 flavonoids and 3 coumarins?in crude extract of A.tataricus were characterized by UHPLC-Q-TOFMS/MS according to the summarized fragmentation rules,accurate molecular weights and characteristic fragment ions.11 of which were identified by comparing with the standards,and the other 4 compounds were identified by analyzing the information provided by MasterView,the error tolerance is ±5 ppm.Conclusion: UHPLC-Q-TOF-MS/MS was high sensitive,accurate,rapid and simple for A.tataricus identification.And it provides help for the research of quality control.Objective: To develop an UPLC-MS/MS method for the determination of shionone,luteolin,quercetin,kaempferol and ferulic acid in A.tataricus.Methods: Analysis was performed on a Waters BEH C18?5 mm×2.1 mm,1.7 ?m?eluted with acetonitrile?0.1% formic acid?and water?0.1% formic acid?in a gradient program.The flow rate was 0.3 mL/min,the injection volume was 10 ?L,and the column temperature was 40?.The multiple-reaction monitoring scanning was employed for the quantification with switching electrospray ion source polarity in positive and negative mode.Results: The regression equations showing linear relationships between peak areas and contents of each compound were obtained.The average recoveries of the compounds ranged from 97.32% to 102.0% and the precision in terms of RSD was in the range of 1.51%-2.99%.The contents of shionone,luteolin,quercetin,kaempferol and ferulic acid in 6 batches of A.tataricus were 2.69-4.64,2.22×10-2-1.04×10-1,3.68×10-4-2.08×10-3,0.12-0.30 and5.16×10-3-2.18×10-2 mg/g,repectively.Conclusion: The established method is simple,accurate,highly reproducible and sensitive,which can be used for simultaneous determination of five components in A.tataricus.Part three Inhibitory effect of friedelan-3beta-ol on CYP3A4,2C9 and2C19 enzymes in human liver microsomeObjective: A probe substrate was used to assess the inhibitory potency of friedelan-3beta-ol on CYP3A4,2C9 and 2C19 enzymes in human liver microsome.Methods: The probe substrate of CYP3A4,2C9 and 2C19 were incubated in human liver microsome with friedelan-3beta-ol of different concentrations.A UHPLC-MS/MS method was established to detect the metabolites of selective probe substrates.The IC₅0 values were determined.Results: The IC₅0 values of friedelan-3beta-ol for CYP3A4,2C9 and2C19 in human liver microsome were 113.8,1.336 and 340.5 ?mol/L,repectively.Namely that friedelan-3beta-ol is a weak inhibitor for CYP3A4 and 2C19,moderate inhibitor for 2C9.Part two Simultaneous determination of five components in Astertataricus by UPLC-MSConclusion: This probe substrate method can be utilized for the determination of the inhibition potential of friedelan-3beta-ol on CYP3A4,2C9 and 2C19 in human liver microsome.The research can provide scientific instruction for study on drug-drug interaction and rational use of drug in clinical practice.