Study On Pharmacokinetics And Tissue Distribution Of Saikosaponins In Radix Bupleurum

Background:Bupleurum,which has been used for thousands of years in clinical,mainly used to treat stagnation of liver Qi,fever with cold,irregular menstruation disorders,and so on.It belongs to Heart,lung and gallbladder meridian in Chinese medicine theory.In recent years,more and more in-depth studies on saikosaponins showed that saikosaponins has a variety of pharmacological activity:hepatoprotective,antipyretic,anti-inflammatory,sedative,cough,and so on.However,saikosaponins are not stable and extremely easy to transform,and are relatively complex and diverse,and has low bioavailability in vivo.So more comprehensive qualitative analysis of saikosaponins in Radix Bupleurum were studied in this study.Although there have been many research on saikosaponins pharmacokinetics and metabolism,most of them just for individual saponin compound,and lacked in the study of tissue distribution except for saikosaponin d,So the aim of this study was a more comprehensive and in-depth research for pharmacokinetics and tissue distribution of Bupleurum saikosaponins.With the development of traditional Chinese medicine,the use of alcohol extraction is more and more,but the water extraction is commonly used in traditional Chinese medicine history,then effect of the two different extraction methods for Bupleurum chinense in composition is not clear.Therefor,this study aimed to compare the differences of composition species and number between alcohol extraction and water extraction for Radix Bupleurum,and explore pharmacokinetic of saikosaponins in the two extraction methods.Combination of medicinal herbs are more popular than single herb in Traditional Chinese medicinal,Radix Bupleurum has been widely used in prescriptions such as Xiaoyaosan,Chaihu-shugansan and so on,in order to compare the effects of combination for Radix Bupleurum in vivo and vitro,the most representative of Chaihu-Baishao pair were chose to explore pharmacokinetics and tissue distribution before and after the combination.The aim of the series of studies was to provide the theory evidence for the clinical treatment and drug development.Objective:At first,a more comprehensive analysis of Radix Bupleurum were conducted,and the comparison of composition pecies and quantity differences in three different kind Radix Bupleurum samples(alcohol extract of Radix Bupleurum,water extraction of Radix Bupleurum,water extraction of Radix Bupleurum and Paeoniae Radix Alba)were carried out.Secondly,the quantitative analysis of main saponin components in Bupleurum and comparison of three different samples were explored.Thirdly,looking for the index components of Radix Bupleurum in vivo and studying pharmacokinetics of index components,and exploring influence on pharmacokinetics of different extraction methods and compatibility.Finally,the tissue distribution of saikosaponins and the effect of different extraction methods and compatibility in vivo were explored,providing a scientific basis for use of drug in clinical.Methods:1.UPLC-MS method of chemical qualitative analysis in Radix Bupleurum were established,and a more detailed mass spectrum pyrolysis regular analysis of saikosaponins were studied,and used to alcohol extract of Radix Bupleurum,water extraction of Radix Bupleurum,water extraction of Radix Bupleurum and Paeoniae Radix Alba.Chromatographic conditions:BEH C18 column(100 mm x 2.1 mm,1.7 m).The mobile phase was 0.1%formic acid in water(A)-acetonitrile(B)with gradient elution:0～30 min,10%～60%B;30～40 min and 60%～90%B;40～45 min,90%B.Flow rate:0.3 mL/min,the temperature of column:35℃.Mass spectrometry conditions:ESI mode with + 3.0 KV/-2.7 KV.Sheath Gas Pressure:35arb;Aux Gas Pressure:10arb;Capillary Temp:300℃;Heater Temp:300℃;Scan mode:Full MS(Resolution 70000)and dd-MS2(Resolution 17500,NCE 25,Stepped NCE 50%);Scan range:m/z 150-2000.2.Determination analysis method of main components SSa,SSc,SSd,SSb1 and SSb2 in Bupleurum by UPLC were established,and applied to alcohol extract of Bupleurum,water extraction of Bupleurum,water extraction of Bupleurum and Paeoniae Radix Alba.Chromatographic conditions:BEH C18 column(100 mm x 2.1 mm,1.7 m).The mobile phase was 0.1%formic acid in water(A)-acetonitrile(B)with gradient elution:0～6 min,30%B;6～10 min,30～37%B;10～17 min,37%B;17～23 min,37%～90%B;23～25 min,90%～90%B.Flow rate:0.3 mL/min,the temperature of column:35℃,wavelength:210nm,254nm.3.To explore index components of Bupleurum in vivo through metabonomics analysis after oral administration of alcohol extraction of Bupleurum in rat plasma,and determination UPLC-MS method of index components in plasma were established,applied it to pharmacokinetics of alcohol extract of Bupleurum,water extraction of Bupleurum,water extraction of Bupleurum and Paeoniae Radix Alba.In order to attain a comprehensive behavior of Bupleurum in vivo,other prototype components in vivo were semi-quantified.SD male rats were randomly assigned to three groups,and were oral administration alcohol extract of Bupleurum,water extraction of Bupleurum,water extraction of Bupleurum and Paeoniae Radix Alba,respectively.0.5 mL blood samples were collected before and after oral administration 0.083 h,0.25 h,0.5 h,0.75 h,1H,1.5 h,3 h,5 h,8 h,12 h.The determination of plasma concentration were conducted by method above,and applied to drug time curve.pharmacokinetic parameters were carried out by DAS 3.0.4.Determination of SSa,SSc,SSd,SSb1 and SSb2 after oral administration drug in rat tissues by UPLC-MS,and applied it to the study of tissue distribution.SD rats were randomly assigned to three groups(alcohol extract of Bupleurum,water extraction of Bupleurum,water extraction of Bupleurum and Paeoniae Radix Alba),heart,liver,spleen,lung and kidney tissue samples were collected after administration of 0.25 h,0.5 h,0.75 h,3 h,5h,study on tissue distribution were carried out by the method above after pretreatment of tissue samples.Results:1.Through the analysis of bupleurum alcohol extraction using UPLC-MS,23 species saikosaponin components were identified,including 4 kinds:saikosaponins,acetyl of saikosaponins,Bupleurum prosapogenin and propionyl saikosaponins.Compare the three samples,the results showed that the species in alcohol extraction were more than water decoction,but SSbl and water SSb2 in water decoction slightly more than alcohol extraction.2.Established UPLC determination method of 5 kinds saponins,and applied to the three Bupleurum samples.The result showed that SSa and SSd contents in alcohol extraction were significantly higher than that in water decoction,especially SSd,but SSb1 and SSb2 contents in water decoction were significantly increased.The method is stable,rapid,convenient and easy to popularize,which provides the basis for the quality control of Bupleurum.3.5 kinds of index components including SSa,SSc,SSd,SSb1 and SSb2,which had larger VIP value,were selected through plasma metabolism after oral administration Bupleurum alcohol extraction by UPLC-MS.In pharmacokinetic study,blood drug concentration,drug time curve and pharmacokinetics parameters were attained.The results showed that all Tmax were about 0.5 h,t1/2 were about 1.5 h,Vz/F were far bigger than 0.67 L/kg(fluid volume of rat),suggested that saikosaponins has characteristics of absorbed and eliminated quickly,wide distribution and Low bioavailability.Compare Bupleurum Decoction and alcohol extraction,the saikosaponins in Decoction were eliminated significantly faster,maybe carbohydrate components in Decoction increased and lead to faster energy metabolism,thus the elimination were sped up.when compatibility with Radix Paeoniae Alba,saikosaponins absorbed slower,but eliminated quickly,maybe it is related with the convergence effect of Paeoniae Radix Alba.4.The tissue distribution of 5 saikosaponins has similarities in the distribution experiment.When Intragastric administration of Bupleurum Decoction,SSa,SSc,SSb1,SSb2 had stronger affinity with lung and heart,especially lung tissue,the contents of SSc and SSb1 in lung tissue were significantly higher than other tissues,suggested that saikosaponins in Bupleurum Decoction has a trend to lung tissue when in vivo.When Intragastric administration of Bupleurum alcohol extraction,the contents of SSa,SSd,SSc,SSbl,SSb2 in lung and liver of rats were large,showed the high affinity with lung and liver.Compared Decoction group and alcohol extraction group,lung tissue distribution trend were similar,but ecoction group tend to distributed heart tissue,while alcohol extraction group tend to distributed liver tissue,maybe the differences of coexisting ingredients in two extraction lead to this phenomenon.Conclusion:In this study,23 saponins were identified in Bupleurum by UPLC-MS analysis,and the main saponins were quantified.The established UPLC-MS method was sensitive,rapid and reliable,and successfully applied to pharmacokinetics and tissue distribution studies of Bupleurum,attained pharmacokinetic parameters of saikosaponins and tissue distribution features.The results showed that contents of saikosaponins were higher in alcohol extraction in vitro,but saikosaponins in water decoction were more easily absorbed.One corns have two sides,the use of them should be based on the specific situation in clinical.