Intestinal Absorption Properties Of Zhishi-Shaoyao And Shaoyao-Gancao In Sini

The compatibility of prescription in traditional Chinese medieine(TCM) is the special feature of Chinese Materia Medica.Traditional Chinese medicine matchs is the smallest unit in the compatibility of prescription.It is composed simply,take a clearly effect,edge scientific research easily,etc.Hence,it is a favorable point to study the mechanism of the compatibility of prescription.The experts of chinese medical science have researched and explored the rule of the compatibility for a long time.However,the researches were more emphasis on compatibility of clinical application,chemistry,pharmacology,than the correlation with process of drug in vivo and therapeutic effect.Now the researchs of modern medicine demonstrated drugs of oral administration have to be absorbed by gastrointestinal tract firstly.The oral administration is the main form of Chinese herbs,so it is necessary to research absorption,metabolism and drug interaction in vivo,that active components and active priciples of the compatibility of prescription in traditional Chinese medicine.Then it would be expounded the mechanism of the compatibility of prescription.In this study,the main components of Zhishi-Shaoyao and Shaoyao-Gancao Sini contained were selected as a model medicine.Character of its absorption and metabolism were studied in a four-site perfused rat intestinal model.Sini was first found in the Treatise on Exogenous Febrile Diseaseine of Zhang Zhongjing in Han Dynasty.It is well-known to reconcile the liver and spleen,and is composed of Paeoniae Radix,Fructus Aurantii Immaturus,Bupleurum,Glycyrrhiza uralensis,including Zhishi-Shaoyao,Shaoyao-Gancao,Chaihu-Shaoyao,Chaihu-Zhishi, with the role of liver qi stagnation and in clinic it was uesd to treat the Rib epigastric abdominal pain caused by liver and spleen qiyu decoction.This study focused on the research of intestinal absorption that the main components of Zhishi-Shaoyao and Shaoyao-Gancao in Sini in perfused rat intestinal model,In order to clarify mechanism of the compatibility of Sinisan.In this thesis,firstly reviewed the researched advance of the mechanism of the compatibility of Chinese herbals,Sinisan medicine and absorption and metabolism of drugs respectively.Completed the following studies in experimental part:1.Extracts from Fructus Aurantii Immaturus,Paeoniae Radix and Glycyrrhiza uralensis were prepared.Firstly determination of UPLC for naringin,hesperidin and neohesperidin of the extract of Fructus Aurantii Immaturus were established,and compared to contents of those components of Fructus Aurantii Immaturus from different areas.In this study,we selected the moderate content in Fructus Aurantii Immaturus herbs for the experiment.The extracts of medicinal herbs were prepared respectively,naringin,hesperidin and neohesPeridin in Fructus Aurantii Immaturus,paeoniflorin in Paeoniae Radix,glycyrrhizin acid,glycyrrhizin in Glycyrrhiza uralensis as indicators,then established determination method of UPLC to ensure the content of extracts.The result was Fructus Aurantii Immaturus extract containing naringin 52.65 mg,hesperidin 9.25 mg,neohesperidin 66.25 mg;Paeoniae Radix extract containing paeoniflorin 22.41 mg;Glycyrrhiza uralensis extracts containing glycyrrhizin acid 16.35 mg,glycyrrhizin 22.38 mg per mL.2 Research on physical and chemical properties of the main components of Zhishi-Shaoyao and Shaoyao-Gancao.(1) Research on physical and chemical properties of the main components that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin of Zhishi-Shaoyao and Shaoyao-Gancao.Reference to Chinese Pharmacopoeia investigated the approximate solubility of each component;Determined the components of the oil-water partition coefficient(logP values)by shake flask method,and the results were naringin (-0.44),hesperidin(-0.39),neohesperidin(-0.31),paeoniflorin(-0.67),glycyrrhizin acid(0.78) and glycyrrhizin(0.16),and combines with prediction that physical and chemical parameters of each component by Marvin online software,to speculate absorption of the components in vivo according to the Five Rules.Only judged from the physical and chemical properties of the above components,that they may have poor absorption in vivo.(2)Study on the stability of model drugs was necessarily.Firstly chemical stability of compounds in model drugs was investigated in order to ensure there liability of results in Perfused rat intestinal model.Stability of tested drugs in hank,s balanced salt solution during temperature 37℃and PH differ from5.0 to 8.0 for 24 h was investigated.The results showed that compounds naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid, glycyrrhizin were stable in differ PH from 5.0 to 8.0 solutions.So all tested drugs were suitable for the vitro perfused test.3 Methodology ofperfused sample analysis was established.The UPLC determination method of naringin,hesperidin,neohesperidin,paeoniflorin, glycyrrhizin acid,glycyrrhizin contained in perfused rat intestinal model sample and the HPLC determination in plasma and bile samples was established.Results indicated that linearity,precision,repeatability,stability,recovery of these compounds fit the bill in this method.4 In perfused rat intestinal model to study intestinal absorption of the 20,50,100μmol.L^-1 three concentration compounds and extracts in the combination drug Zhishi-Shaoyao,Shaoyao-Gancao.The results showed that all the components and extracts were poorly absorbed in the duodenum,jejunum,ileum,colon of rat.Effective permeability coefficient of the compounds(P_eff^* values) were＜0.5.However,compared with the corresponding compounds,the absorption of extracts had increased.Through the statistical software SPSS13.0 analysis the percent absorption of naringin(20μmol.L^-1) in extracts of Fructus Aurantii Immaturus increased by 41.1%in duodenum,39.9%in jejunum,32.57%in ileum and the percent absorption of hesperidin(20μmol·L^-1) in the extracts significantly increased by 45.83%in duodenal,47.17%in jejunum,30.85%in ileum;neohesperidin(20μmol·L^-1)in the extracts had a significant increase in percent absorption that 53.83%in duodenal,47.61%in jejunum,32.05%in ileum and intestinal abosoption of the other concentration extracts of Fructus Aurantii Immaturus also increased significantly compared with their corresponding monomers.It is indicated that other components in of extracts in Fructus Aurantii Immaturus would promote intestinal absorption of naringin,hesperidin, neohesperidin.The percent absorption of Paeoniflorin(20μmol·L^-1) in extracts of Paeoniae Radix increased by 24.92%in jejunum,36.33%in ileum compared with monomer and the others concentration extracts also increased more than compound.All of these indicated that other components of extracts in Paeoniae Radix will also promote intestinal absorption of paeoniflorin.The percent absorption of glycyrrhizin(20μmol·L^-1) in extracts of Glycyrrhiza uralensis increased by 20.22%(p＜0.05)in ileum and the others concentration of extract had a increase compared with the same concentration of compound,while glycyrrhizin had no significantly change.It is indicated that other components of extracts in Glycyrrhiza uralensis would promote absorption of glycyrrhizin,but had no effect on glycyrrhizic acid.5 Research on Intestinal absorption of the main components and extracts of Zhishi-Shaoyao and Shaoyao-Gancao and explained their mechanism of the compatibility initially.Select the different ratio of compatibility to study the intestinal absoption of math-pair, the ratio of compounds and extracts were both 1:1,1:5,5:1.The results of compatibility in Zhishi-Shaoyao showed when the ratio was 1:1 naringin combined with paeoniflorin,paeoniflorin can promote absorption of naringin 5.94%in duodenum and 5.37%jejunum in rat while absorption of paeoniflorin decreased 8.37%in duodenum and 8.92%in jejunum.when ratio was 1:5,5:1,the absoption of naringin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 hesperidin combined with paeoniflorin,the percent absoption of hesperidin increased by 11.76%in duodenal,8.09% in jejunum,9.57%in ileum and 18.78%in colon while the percent absoption of paeoniflorin decreased by 2.89%in duodenum and 6.89%in colon,when ratio was 1:5,5:1,the absoption of hesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 neohesperidin combined with paeoniflorin,the percent absoption of neohesperidin increased by 9.23%in ileum.when the ratio was 1:5,5:1,the absoption of neohesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 5:1 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the absorption of naringin increased by 6.93%in duodenum 14.02%in jejunum.When the ratio was 1:5 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the percent absoption of paeoniflorin increased by 3.79%in duodenum 5.25%in ileum 12.94%in colon compared with the percent absoption of extracts of Paeoniae Radix.The results of compatibility in Shaoyao-Gancao showed that,with the ratio of compatibility,of glycyrrhizin acid and paeoniflorin was 1:1,compared to compounds,the percent absorption of the glycyrrhizin acid in rat decreased by 3.3%in duodenal,6.25%in jejunum,13.08%in ileum,3.18%in colon respectively;with the ratio of compatibility of glycyrrhizin acid and paeoniflorin was 1:5 and 5:1,the percent absorption of the glycyrrhizin acid in rat increased a little that by 9.53%,4.59%in the ileum,the percent absorption of the paeoniflorin in rat decreased a little that by 12.94%,11.32%in the ileum; with the ratio of compatibility of glycyrrhizin and paeoniflorin was 1:1,compared to compounds,the percent absorption of paeoniflorin in rat decreased by 6.02%in ileum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:1,compared to single extracts of Paeoniae Radix,the percent absorption of paeoniflorin in rat increased by 6.02% in duodenum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:5, compared to single extracts of Glycyrrhiza uralensis,the percent absorption of glycyrrhizin acid in rat increased by 11.34%in duodenum,9.39%in jejunum,10.71%in ileum,6.63%in colon respectively,but there is no significant difference after statistical analysis.6 Research on Intestinal absorption that naringenin(aglycone of naringin) and hesperidin(aglycone of hesperetin) preliminarilyThe principal components of Zhishi-Shaoyao and Shaoyao-Gancao are glycosides,that would be into aglycones by hydrolysis of intestinal bacteria in the human body,then absorbed or metabolized,therefore a preliminary examination of this experiment studied on intestinal absorption of naringenin(aglycone of naringin) and hesperetin(aglycone of hesperidin) in Fructus Aurantii Immaturus in rat.The results showed that effective permeability coefficient(P_eff^* value) of.naringenin and hesperidin in rat were higher in different segments of intestine of rat.It was demonstrated that P_eff^* of naringenin decreased in order as flows:3.15±0.30 in colon,2.77±0.43 in duodenum,2.39±0.30 in jejunum, 1.90±0.53 in ileum;And P_eff^* of hesperidin decreased in order as flows:3.05±0.28 in colon, 2.63±0.31 in jejunum,2.50±0.26 in duodenum,1.99±0.17 in ileum.There were two metabolic products which naringenin and hesperidin produced,but UPLC can not separate effectively.The polarity of Metabolites increased,so they were probably the same type of two-phase metabolites.In summary,the main components of Zhishi-Shaoyao and Shaoyao-Gancao that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin were poorly intestinal absorption.The compatibility of drugs had no significant effect on intestinal absorption of each component.It is a further study on the intestinal absorption in order to reveal the mechanism of the compatibility of match-pair,from the points of Multi-components/components(glycosides,aglycone),multi-angles(metabolic enzymes, intestinal bacteria,transporters).