Study On Quality Control Method And In Vivo Process Of Fufang-Xiyi Powder

The Fufang-Xiyi Powder is composed of 9 medicinal materials:Lizard,Astragali Radix,Bletillae Rhizoma,Dioscoreae Rhizoma,Notoginseng Radix,Margarita,Ostreae Concha,Cervi Cornus Colla,Vinegar-prepared Corydalis yanhusuo.This Chinese medicine compound has remarkable curative effect in the treatment of gastrointestinal diseases such as gastric ulcer,atrophic gastritis and gastric cancer,and has been proved to be non-toxic by modern pharmacological experiments.In this article,the various medicinal materials in the Fufang-Xiyi Powder were identified by microscopy and thin layer.The method of compound characteristic spectrum analysis was established At the same time,the content of multi-index components was determined.Based on the chemical constituents in vitro,the whole process of the formulation in vivo was systematically studied,and the effects of the addition of volatile components and herbal medicine containing no volatile components were also discussed.It is expected to provide a research basis for the establishment of the quality standard of Fufang-Xiyi Powder,and guide its more reasonable and safe application in clinical practice.Chapter one Literature researchThe current research situation of Fufang-Xiyi Powder was summarized,and the main chemical components,related pharmacological effects and research progress in vivo process of each single medicinal material are reviewed.Literature studies have shown that the current research on Fufang-Xiyi Powder is mostly focused on pharmacodynamics,and there are no reports on the quality control and in vivo process of compound prescriptions.In addition to the common chemical components,the medicinal materials such as Astragali Radix,Vinegar-prepared Corydalis yanhusuo,Notoginseng Radix also contain volatile components that bring special odors.The medicinal materials such as Lizards,Cervi Cornus Colla,and Margarita contain a lot of amino acids,proteins and peptides.This chapter lays a theoretical foundation for the design of experimental scheme and the succeed conduct of subsequent experiments.Chapter two Research on the quality control method of Fufang-Xiyi PowderSection 1 Microscopic and thin-layer identification of Fufang-Xiyi PowderThe 9 medicinal materials in the Fufang-Xiyi Powder were microscopically identified.The compound powder was made into water-filled tablets or permeabilized tablets and observed under a microscope.The microscopic identification methods of cork cells and fiber bundles in the Astragali Radix,epidermal cells and mucous cells in Bletillae Rhizoma,thick-walled cells in Vinegar-prepared Corydalis yanhusuo,resin canals and cork cells in Notoginseng Radix were established.The Astragali Radix,Dioscoreae Rhizoma,Bletillae Rhizoma,Vinegar-prepared Corydalis yanhusuo,Notoginseng Radix in the compound prescriptions were identified by thin layer.The control medicinal materials,the single medicinal materials in the prescription and the whole Fufang-Xiyi Powder were in the same position consistent spots appeared,and the negative control solution of each medicine in the whole prescription did not appear spots in the same position.Section 2 Establishment of characteristic spectrum analysis method for chemical constituents of Fufang-Xiyi PowderThe chemical components of 10 batches of Fufang-Xiyi Powder were analyzed by the formic acid water-hexanitrile system and the phosphate buffer system,and two characteristic spectrum analysis methods were established.The established methods have good stability,repeatability and precision.The two analysis methods matched 25 and 17 common peaks respectively,and the similarity of 10 batches of samples was good.Section 3 HPLC content determination of multiple index components in FufangXiyi PowderTwo analytical systems were used to determine the content of 10 chemical components in the compound,including flavonoids,saponins,benzyl alcohol ester glycosides and alkaloids,at four wavelengths.The linearity of each component was good within a certain range,and the r value was All are greater than 0.9967.The established method has good precision,repeatability,stability and specificity,and the sample recovery rate is less than 106.35%.Chapter three Research on the internal process of Fufang-Xiyi PowderUPLC-LTQ-Orbitrap-MS/MS technology was used to identify the metabolites of compound lizard scattered in rat plasma,bile,urine and feces.And using ultra-high performance liquid chromatography(UPLC)and triple quadrupole-linear ion trap(QTRAP)technology to establish a method for the simultaneous determination of Formononetin,Ononin,Calycosin,Calycosin-7-glucoside,Corydaline,Corydalis and Palmatine in Fufang-Xiyi Powder,and to study the dynamic changes of the chemical components in rats.The established method’s specificity,precision,accuracy,extraction recovery rate and sample stability all meet the requirements.Based on the in-vivo process of the Fufang-Xiyi Powder prescription,the experiment was divided into three groups,namely the whole prescription group(A group),the volatile component removal group(U group),and herbal medicine containing volatile components group(O group).In order to explore the effects of the addition of volatile components and non-volatile components on the processes of other components in vivo.Section 1 Study on the intestinal absorption of Fufang-Xiyi Powder in ratsThrough the rat in vivo intestinal perfusion model,the absorption of the seven components measured in different intestinal segments was investigated,and the absorption differences of each component in different intestinal segments of different groups were compared at the same time.The results showed that each component was absorbed in the entire intestinal segment,and the ingredients are well absorbed.But the absorption parameters of the three groups of A、U、O in the jejunum segment showed significant differences.The Papp value of Formononetin in the O group was significantly higher than other two groups,while the-Papp value of Formononetin and Calycosin-7-glucoside was higher in the A group.The order of Papp value of Corydaline,Corydalis and Palmatine is:O group>A group>U group.Section 2 Study on the tissue distribution of Fufang-Xiyi Powder in miceThe tissue distribution of the seven components in the Fufang-Xiyi Powder in the heart,liver,spleen,lung,kidney,stomach,and intestine was studied.At the same time,the effects of the addition of volatile components and non-volatile components on the distribution of other chemical components in different tissues were investigated.The results showed that the content of Formononetin in the liver is the highest,the content of Calycosin-7-glucoside is higher in the kidney,and Ononin,Calycosin-7-glucoside and alkaloids are more distributed in the stomach and intestines.For comparison between different groups,Formononetin has a higher content in liver and intestine tissues in group O,and a higher content in lung and kidney tissues in group U;Ononin has a higher content in liver and spleen in group O,and it was significantly higher than the other two groups;the content of Palmatine in the U group in the heart and spleen was higher than other two groups,but the content in the O group in the lung and intestines was higher.Section 3 Study on the metabolism of Fufang-Xiyi Powder in ratsUPLC-LTQ-Orbitrap-MS/MS technology and quality loss strategy were used to identify the metabolites in rat plasma,bile,urine,and feces after gavage of FufangXiyi Powder.34 metabolites of the ingredients including,Formononetin,Calycosin,Ononin,Calycosin-7-glucoside,Corydalis,Corydalis,Tetrahydroberberine,Ferulic acid,Notoginsenoside R1,Gastrodin were identified.Flavonoids mainly undergo methylation,demethylation,hydroxylation,dehydroxylation and other phase Ⅰreactions in rats,as well as sulfation,glucuronidation and other phase Ⅱ reactions;alkaloids mainly undergo demethylation Grouping and binding reactions;phenolic acids mainly undergo methylation and glucuronidation reactions;glycosides are mainly metabolized by removing sugars in the body.Section 4 Study on excretion of Fufang-Xiyi Powder in ratsThe study on the excretion of 7 components in Fufang-Xiyi Powder in rat bile,urine and feces showed that all the tested components were excreted through bile,urine and feces.For Formononetin and Calycosin of the flavonoid aglycones,they are mainly excreted in urine,and the excretion shows a rapid increase trend within 0-8h,and the law is similar in the three groups of AOU,and the cumulative excretion is:Group A=Group O>Group U.The Ononin was mainly excreted through bile.Compared with the other two groups,the amount of excretion in the O group was significantly reduced.Calycosin-7-glucoside was mainly excreted in urine,and the excretion of group U was significantly higher than that of the other two groups.The Corydaline is mainly excreted through urine,while Corydalis and Palmatine are mainly excreted through feces.The excretion time is relatively lagging behind the flavonoids,and the excretion of the three components in the bile shows significant differences.The excretion of group O is the largest and shows an increasing trend.Section 5 Study on the pharmacokinetics of Fufang-Xiyi Powder in ratsAfter the rats were given the three groups of compound prescriptions,the fundus venous blood was taken at 14 time points within 5min-24h.After the samples were pre-processed and measured by the instrument,the relevant pharmacokinetic parameters were fitted by DAS software.The pharmacokinetic parameters of the three experiments were compared.The results showed that for the flavonoids:Calycosin-7glucoside are converted into Calycosin in the body,and there is hepato-enteric circulation.The AUC of Formononetin and Calycosin was higher,and the AUC and Cmax values in group A were lower than those of the other two groups and showed significant differences.The t1/2 of Formononetin in group A was significantly lower than that of the other two groups.For alkaloids:the tmax of Corydaline and Corydalis in group A was significantly higher than that of the other two groups,and the Cmax of group O was the largest,but for the compound Palmatine,the AUC and Cmax of group U were larger.