Study On Quality Control And In Vivo Process Of Bazhen Yizhi Prescription

Bazhen Yizhi prescription is composed of Longan Arillus,Alpiniae Oxyphyllae Fructus,Citri Reticulatae Pericarpium,Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle,Poria,Macrocephalae Rhizoma,Nelumbinis Semen,Dioscoreae Rhizoma,It has the functions of enhancing intelligence,delaying aging,and improving memory.In recent years,Bazhen Yizhi Prescription has made certain progress in extraction,preparation technology and pharmacological research,but its quality control research and in vivo process research are very few.This experiment intends to establish analytical methods for thin-layer chromatography research,characteristic chromatogram research,and content determination for quality control research,in order to provide experimental basis for the establishment of Bazhen Yizhi Prescription quality standards,and through the intestinal absorption research,tissue distribution research,metabolite analysis,excretion research and pharmacokinetic research perspectives to analyze the in vivo process of Bazhen Yizhi Prescription.Chapter one,Literature researchThis chapter systematically summarizes the research status of Bazhen Yizhi Prescription and the research progress of the main chemical components,pharmacological effects and in vivo processes of each medicinal material.According to the literature,in addition to the large amount of polysaccharides in the water extract of Bazhen Yizhi Prescription,Alpiniae Oxyphyllae Fructus and Macrocephalae Rhizoma also contain relatively rich volatile oil components.these volatile components can also play the pharmacological effects of treating memory loss,delaying aging and preventing Alzheimer’s disease.Does the volatile oil exert pharmacological effects on other components at the same time?Therefore,this experiment is based on the question of whether the composition of volatile oil will affect the process in vivo.Chapter two,Research on the Quality Control of Bazhen Yizhi PrescriptionSection 1,TLC identification of Bazhen Yizhi PrescriptionTo explore the TLC identification method of each single medicinal material in the water extract of Bazhen Yizhi prescription by screening and optimizing a variety of extraction methods,developing agents and chromogenic agents.It was found that Longan Arillus,Alpiniae Oxyphyllae Fructus,Poria,Macrocephalae Rhizoma,Nelumbinis Semen,Dioscoreae Rhizoma could not show the same color spots in the same position as the control.Finally,a TLC identification method of Citri Reticulatae Pericarpium,Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in Bazhen Yizhi prescription was established.Section 2,Establishment of characteristic chromatogram of Bazhen Yizhi prescription.The characteristic chromatogram of different batches of Bazhen Yizhi prescription were established by ultra-high performance liquid chromatography(UPLC),and the quality of each batch.of Bazhen Yizhi prescription was controled by using the characteristic chromatogram.A total of 19 common peaks were identified in the characteristic chromatogram,and 15 of them were identified by ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry(UPLC-QTOF/MS).The similarity of each batch of samples was more than 0.936.Section 3,Establishment of a method for the determination of Chemical components in Bazhen Yizhi prescriptionUPLC technology was used to quantitatively analyze the 3,4-Dihydroxybenzoic acid,liquiritin,hesperidin,isoliquiritin,tangeretin,nobiletin,glycyrrhizic acid in each batch of Bazhen Yizhi Prescription,except for liquiritin and hesperidin.Chapter 3,Research on the in vivo process of Bazhen Yizhi PrescriptionThe metabolites were analyzed by ultra high performance liquid chromatographylinear ion trap-electrostatic orbital trap tandem mass spectrometry(UPLC-LTQOrbitrap-MS).The pharmacokinetics,tissue distribution and excretion of Bazhen Yizhi prescription were studied by ultra high performance liquid chromatography-triple quadrupole-compound linear ion trap mass spectrometry(UPLC-QTRAP-MS).At the same time,the effects of volatile oil on the in vivo process of Bazhen Yizhi prescription were compared by grouping administration(water extract group:NEO and water extract group:NEO+EO).Section 1,Study on the intestinal absorption of Bazhen Yizhi PrescriptionBased on the experiment of one-way intestinal perfusion in rats to explore the intestinal absorption characteristics of Bazhen Yizhi Prescription.Investigate different intestinal segments.The results showed that the main absorption sites of glycyrrhizic acid were the colon and ileum;the main absorption sites of nobiletin,tangeretin,isoliquiritin,and hesperidin were the duodenum;the main absorption sites of liquiritin were the colon and jejunum.The NEO+EO group and the NEO group’s absorption status in different intestinal segments were investigated.The results showed that compared with the NEO group,volatile oil(EO)can significantly increase the absorption of tangeretin in the four intestinal segments.The absorption of hesperidin and glycyrrhizic acid in the duodenum and the absorption of nobiletin in the duodenum,jejunum,and colon;while the absorption of liquiritin in the jejunum,ileum,and colon is reduced;the absorption of isoliquiritin in the colon is reduced.Section 2,Research on the Organizational Distribution of Bazhen Yizhi PrescriptionBased on the tissue distribution experiment,the distribution of Bazhen Yizhi prescription in heart,liver,spleen,lung,kidney and brain was studied,and whether the volatile oil affected the distribution of Bazhen Yizhi prescription in each tissue was explored.The results showed that the content of isoliquiritin in all tissues was lower than the detection limit.nobiletin and liquiritin,are mainly distributed in the liver;tangeretin are mainly distributed in the spleen and liver;hesperidin and glycyrrhizic acid are mainly distributed in the liver and lungs,and both can be detected in brain tissue.tangeretin was rapidly distributed in liver and spleen within 1 hour after administration,while hesperidin was mainly distributed in liver and lung,and the content in liver decreased slowly.In addition,compared with the EO group,the content of hesperidin in the liver increased;the content of nobiletin increased in the kidneys,but the distribution in the lungs decreased;the distribution of tangeretin decreased in the liver and brain,The content of glycyrrhizic acid in the heart,lungs,and kidneys all increase,and the distribution of liquiritin in the lung first increased and then decreased.Section 3,Metabolism Study of Bazhen Yizhi PrescriptionBased on the metabolic experiment,the metabolites and possible metabolic pathways of isoliquiritin,chrysin,tangeretin,nobiletin,hesperetin,Poricoic Acid A in Bazhen Yizhi prescription were analyzed.44 metabolites were identified in plasma,bile,urine and feces of rats.Among them,isoliquiritin can be converted into isoliquiritigenin,and the latter can undergo a more extensive phase Ⅱ binding reaction.Polymethoxy compounds tangeretin and nobiletin can convert methoxy to hydroxyl groups through the catalysis of phase I metabolic enzymes to carry out phase II metabolic reactions such as sulfation and glucuronidation.Section 4,study on the excretion of Bazhen Yrzhi PrescriptionBased on the excretion experiment,study the excretion of hesperidin,nobiletin,tangeretin,glycyrrhizic acid,liquiritin,and isoliquiritin in Bazhen Yizhi Prescription,and explore the effect of volatile oil on the excretion of Bazhen Yizhi Prescription.These 6 components can be detected in bile,urine,and feces.Among them,bile excretion is the main excretion pathway of glycyrrhizic acid,and liquiritin,tangeretin and nobiletin are mainly excreted in urine and in stool samples.The highest content of hesperidin and isoliquiritin were detected.Compared with the EO group,in the bile samples,the cumulative excretion of nobiletin and isoliquiritin was reduced,and there were differences in the contents of other components,but there was no significant difference.Although the content of each component in the urine sample has changed,there is no significant difference.In the stool sample,the trend of the two groups was consistent and slower in the first 8 hours.The excretion volume increased during the 8-12 h period,and the cumulative excretion volume of each component within 12-48 h continued to be higher than that of the NEO+EO group,but there was no significant Sexual differences.Section 5,Pharmacokinetic Study of Bazhen Yizhi PrescriptionBased on the pharmacokinetic experiment,the pharmacokinetics of hesperidin,nobiletin,tangeretin,glycyrrhizic acid,liquiritin and isoliquiritin in rats were studied,and whether the volatile oil(EO)had an effect on the pharmacokinetics of Bazhen Yizhi prescription was studied.The results showed that hesperidin and glycyrrhizic acid showed double peaks in the plasma concentration-time curve,while AUC0-t and Cmax were higher.The concentration-time curves of the six components showed the same trend in NEO+EO and NEO groups.Compared with the group added with volatile oil,the Cmax of hesperidin and nobiletin decreased,while the AUC0-t and Tmax of liquiritin and isoliquiritin increased significantly.It shows that the addition of volatile oil to the water extract of Bazhen Yizhi prescription will have an effect on the pharmacokinetics of the prototype components in the water extract in plasma.