Study On The Cholagogic Mechanism Of Saikosaponins From Bupleuri Radix Based On Fxr Receptor

Background:In recent years,studies have shown that Bupleuri Radix has a variety of pharmacological activities such as cholagogic and liver-protective.There have been many studies on the cholagogic of Bupleuri Radix and Chinese herb compound containing Bupleuri Radix,but the study of mechanism was less.It has been proved that saikosaponin was the main component of the pharmacological activity in Bupleuri Radix,but the specific active monomer composition was not clear and only the research of saikosaponin a and d has been found.The previous study of our group has made clear that the saikosaponins in the alcohol extraction are more than the water decoction in Bupleuri Radix,and the main components were the saikosaponin a,b₂ and d.But if the Bupleuri Radix alcohol extraction had cholagogic effect,which saikosaponins have the effect,what is the mechanism and its dynamic changes in the body are all not clear.Therefore,it is necessary to conducted a more comprehensive and in-depth study of the cholagogic mechanism of saikosaponin and Bupleuri Radix alcohol extraction,and further explored its main active ingredient.Objective:To study the cholagogue effect of saikosaponins,discuss the cholecystasis mechanism in the level of molecular and investigate the main activity components.To study the pharmacokinetic characteristics of the drug.To provide the reasonable administration for clinical and the new drug development in Bupleuri Radix.Methods:1.30 mice of C57BL/6 were randomly divided into 5 groups:the blank group?K?,saikosaponin a?SSa?,saikosaponin b₂?SSb₂?,saikosaponin d?SSd?and Bupleuri Radix alcohol extraction?ESS?.After administration Bupleuri Radix alcohol extraction and saikosaponins,biochemical indicators in liver and serum of mice were analyzed for alanine aminotransferase?ALT?,aspartate transaminase?AST?,alkaline phosphatase?ALP?,total cholesterol?TC?and triglyceride?TG?.The automatic biochemical analyzer was used for the determination.The bile acids measured was according to the kit.The total bile acid?TBA?was measured by the enzyme colorimetric microplate method.2.Established UPLC-MS method for quantitative metabolomic analysis of bile acids in mouse liver,bile and content of cecum.Chromatographic was performed with a Waters HSS T3 column?1.8?m,2.1×100 mm?as the analytical column at a column temperature of 45°C and a flow rate of 0.3 mL/min at full scan.An elution system consisting 0.1%formic acid water?A?and acetonitrile?B?was used as the mobile phase?0-4 min,20%-20%B;4-15 minutes,20%-40%B;15-20 minutes,40-60%B;20-22 minutes,60-60%B;22-22.5 minutes,60-90%B;22.5-23.5 minutes,90-90%B;23.5-25 min,90-20%B;25-26 min,20-20%B?.Mass spectrometry was selected Full Scan mode.Mass spectrometry was in Target and the both positive and negative ESI modes at a range of m/z 50–1200.Xcalibur 3.0 software was used to perform primary-level mass spectrometry data collection on biological samples.The spray voltage?+?was 3.0 KV and spray voltage?-?was 2.7 KV.The capillary,capillary and probe heater temperature were set as 320,300 and 300°C.The sheath and aux gas pressure were 35 and 10arb.The resolution of full scan and dd-MS2 were 70000 and17500,while NCE stepped were 12.5,25 and 37.5 eV.3.Used the q-PCR technology to analyse the Fxr and related genes mRNA expression in liver and ileum after administration SSa,SSb₂,SSd and ESS,and to explore the cholagogic mechanism of saikosaponin,investigate the main activity component of Bupleurum.4.The UPLC-MS method was established to simultaneous determination content of SSa,SSb₁,SSb₂,SSc and SSd,and used for pharmacokinetics study after administration Bupleuri Radix alcohol extraction and different saikosaponins.40 mice of C57BL/6 were randomly divided into 4 groups:saikosaponin a?SSa?,saikosaponin b₂?SSb₂?,saikosaponin d?SSd?and Bupleuri Radix alcohol extraction?ESS?.Blood was collected at different time.The above method was used to determined the drug concentration in blood,and the curve of drug-time could be gained at the same time.Pharmacokinetic parameters were carried out by DAS software.Chromatographic was performed with a Waters BEH C18 column?100 mm x 2.1 mm,1.8 m?as the analytical column at a column temperature of 40°C and a flow rate of 0.3 mL/min at full scan.An elution system consisting 0.1%formic acid water?A?and acetonitrile?B?was used as the mobile phase?0-13 min,33%B;13-14.5 min,33-40%B;14.5-15.5min,40-33%B;15.5-17 min,33%-40%B;17-24 min,40%-60%B;24-25 min,60%-33%B?.Mass spectrometry was in positive ion mode and multi-response monitoring mode?MRM?at ESI source.The spray voltage was 5500 V.The gas curtain,atomization and dry gas pressure were 35,50 and 50 psi of N₂.The ion source temperature and gas flow rate were 500°C and 12 L/min.Results:1.Administration different saikosaponins 14 days,the rate of liver and body weight as well as the determination of biochemical index in serum and live were compared.The results showed that the ratio of liver weight to body weight was decreased in different groups,and the ratio of SSa and SSd group were significantly lower than that in the blank group.Compared with the blank group,the serum TBA of SSa and SSb₂ was significantly higher than that in the blank group,and the TBA of SSb₂ and ESS in the liver was prominently reduced.In serum,the SSa,SSb₂ and ESS groups significantly decreased of TC,and the TC content of SSd group decreased prominently in the liver.The TG in liver decreased significantly in SSa and SSb₂group,and serum TG increased prominently in SSd group.The ALP in serum of SSd group was significantly decreased,and there was little difference in AST between different administration groups,and the ALT in ESS group increased significantly.The study indicated that different groups have little impact on liver function and could promote bile acid excretion in liver.2.UPLC-MS analysis was performed on 16 kinds of bile acids in the liver,bile and content of cecum of the mice after administration saikosaponin.In the determination of bile acid content in liver,the content of?-MCA,?-MCA and T-CDCA in SSa group was significantly higher than that in the blank.The content of HDCA,DCA,T-HDCA and T-DCA were significantly reduced in SSb₂ group.In SSd group,there ware a significant increase in?-MCA and T-CDCA,while DCA was significantly reduced.In ESS group,there was a significant increase in?-MCA,?-MCA and T-CDCA,while T-?-MCA,TCA,T-UDCA,T-HDCA and total bile acid were dramatically reduced.In bile,there were a significant increase in?-MCA and T-?-MCA in SSb₂ group.The T-DCA of SSd group was significantly increased.In ESS group,?-MCA was decreased significantly.The results showed that HDCA in SSb₂ was significantly reduced in the content of cecum.UDCA and HDCA in ESS group were significantly reduced.The results were basically similar to TBA,and different saikosaponins had different regulatory effects on bile acids.3.Analyzed Fxr related genes mRNA expression in the liver and the ileum in different groups and used the q-PCR technology.The results showed that the liver of SSa group Fxr,Shp,Cyp7a1,Ntcp,Abcc4 and Cyp27a1 mRNA expression level increased significantly.The expression level of Shp mRNA was significantly increased in SSb₂ group.The expression of Cyp7a1,Abcc4,Cyp27a1 and Hmgcr mRNA in SSd group were significantly increased.The expression of Bsep mRNA in ESS group was significantly decreased.Abcc4,Cyp27a1 and Hmgcr expression were significantly increased.In ileum,the expression of Mdr1 mRNA in SSa group was significantly higher than blank group.The Ibabp,Osta,Ostb and Mdr1 in SSd group were significantly increased.The expression of Asbt,Osta,Ostb and Mdr1 in ESS increased significantly.The mRNA expression of Mdr1 in SSa,SSd and ESS groups were significantly increased.4.In pharmacokinetic study,the content determination method of saikosaponin indexes composition were established by UPLC-MS,and gained the concentration of drug,curve of drug-time,as well as pharmacokinetic parameters in different compositions.The result showed that Tmax was at 0.47 h,t1/2 was at 0.90 h after administration SSa and at the same time metabolite M was detected and the Tmax was at 1.47 h,t1/2 at about 3.16 h.Tmax of SSd at about 0.48 h,t1/2 was at 0.68 h.Tmax in SSb₂ was 0.78 h,t1/2was at 2.74 h.In ESS group,there was no saikosaponin index could be detected.Conclusion:After 14 days,the mice had almost no liver damage.In liver,SSa,SSd and ESS via induced Abcc4 mRNA expression to transport bile from the liver cells into systemic circulation and reduced the content of bile acid.In ileum,SSa,SSd and ESS via induced expression of Mdr1 mRNA to adjust the content of bile acid.SSa and SSd were the main active compositions for ESS.It had the characteristics of fast eliminate and absorption and wide distribution in vivo.The synergistic effects of multicomponent and multi-target of Chinese medicine were suggested.