Studies On Isolation And Pharmacokinetics In Vivo Of Triterpenoid Saponins From Pulsatilla Chinensis

Pulsatilla chinensis is the dried root of Pulsatilla chinensis(Bunge)Rege.Triterpenoid saponins is the main composition of Pulsatilla chinensis.Researches show that Pulsatilla saponin has a series of pharmacological activity such as anti-tumor,anti-inflammatory,antibacterial and relaxing blood vessels.In this research,we will focus on optimization of extraction and separation process of Pulsatilla saponin.The composition of Pulsatilla saponin will be preliminary analyzed and identified by using HPLC-MS/MS-Q/TOF which has the capability of high resolution.We will have pharmacokinetics studies in rats after oral administration in vivo.All above will lay the foundation for research of the mechanism of Pulsatilla saponin and development of modern Chinese medicine.The following results were achived:1 · The method of extraction and purification of Pulsatilla saponin from crude drugs was established.The extraction and purification factors such as solvent,extraction time,number of times and macroporous resin types were investigated.The method of extraction and purification of Pulsatilla saponin was confirmed at the end of the experiment.The extraction solvent is 70%ethanol,the liquid ratio is 8:1,extracting for 3 times and 1.5 hours for each time.Using D101 type macroporous resin for separation.40%ethanol was used for purification and collect 90%ethanol elution fraction.And then the Pulsatilla saponin was obtained.2.The analysis of Pulsatilla saponin componentsThe composition of Pulsatilla saponin will be analyzed and identified by using HPLC-MS/MS-Q/TOF.Summarize the fragmentation patterns of existing reference sample of Pulsatilla saponin.After a series of reactions of hydrolysis,parent nucleus ion fragments at m/z 473 is yielded,and ions 437 were obtained by loss of two H2O units.By analyzing the MS/MS fragmentation ions and comparing with the reference,the other 17 known triterpene saponins were tentatively proposed.3.The pharmacokinetic of Pulsatilla saponinsThe quantitative result in Pulsatilla saponin showed that Anemoside B4(1)>Anemoside A3(0.032)>Anemosapogenin(2.3*10-4).In vivo,3 analytes were founded in portal vein plasma after oral administration of Pulsatilla saponin,and the order of AUC in portal vein plasma was that Anemosapogenin(1)>Anemoside B4(0.34)>Anemoside A3(0.0058);Anemoside A3 was not detected in liver,and the order of AUC in liver was that Anemosapogenin>Anemoside B4;the order of AUC in systemic plasma was that Anemosapogenin(1)>Anemoside B4(0.86)>Anemoside A3(0.012).Combining with the primary hepatocytes metabolism,Intestinal Bacteria Metabolism and pharmacokinetic data of Pulsatilla saponin,it was deduced that after oral administration of Pulsatilla saponin,under the effect of intestinal Bacteria,Anemoside B4 is metabolized to Anemoside A3 and Anemosapogenin.most of Anemoside B4 came into systemic plasma,few of it came into liver and cannot be metabolized.With the action of intestinal Bacteria,Anemoside A3 is metabolized to Anemosapogenin.And it may be excreted in liver.After Anemosapogenin passing portal vein plasma,it may come into systemic plasma and accumulate in liver,some of it can be metabolized or excreted in liver.with the primary hepatocytes metabolism test,metabolization of Anemosapogenin in liver can be proved.