Study On The Method Of Screening Quality Control Components Of Astragalus Based On Multi-component Metabolis

The quality evaluation and control of Traditional Chinese medicine(TCM)were important means to ensure the safety and effective of clinical medication of TCM,how to control the quality of TCM more objectively and accurately,how to identify active components of TCM,establishing a scientific and feasible quality assessment method system is the key issue in TCM development process.In this paper,base on the association in"quality,curative,effect and metabolism",Astragali Radix for the study,adopting in vitro,in situ and in vivo models combined with the technology of HPLC/UPLC-MS to investigate the multi-component intestinal absorption,blood transport,urine excretion and digestive system biotransformation of Astragali Radix,screening therapeutic basis for Astragali Radix quality evaluation.So as to provide reliable theoretical basis and methodological basis for the establishment of the association in“quality,curative,effect and metabolism" of quality evaluation system of TCM.The major experimental contents and achievements are as follows:1.Study on multi-component absorption and metabolism of Astragali Radix decoction and the quality control ingredients selection.To investigate the absorption and metabolism of the chemical constituents of Astragali Radix Decoction,everted rat gut sacs was carried out as well as the blood was taken from abdominal aorta,and all of the samples were analysised by HPLC.A total of 16 constituents were absorbed by intestinal of rat,including six components which were absorbed in prototype forms directly,in which two components were identified as calycosin-7-O-β-D-glucoside,Ononin by comparing with the standards.10 compounds were absorbed into blood in new form.On the other hand,8 chemical constituents could be absorbed into blood via analysis serum samples obtaining from aorta abdominalis of rats.The only one could be absorbed directly by intestine,while six metabolites were absorbed into blood in new form.Base on the comparative analysis of the intestinal absorption and the compsition in blood,Intestine and liver of Astragali Radix decoction has a certain effect on the chemical composition of water.The absorption and metabolism differences between intestine and liver were significant.The absorption and metabolism of Astragalus in rats were evaluated preliminary.To investigate metabolism of the chemical constituents of Astragali Radix Decoction,the blood was taken from abdominal aorta as well as collected rat urine by metabolic cage after oral administration,and all of the samples were analysised by UPLC-MS.There are fifteen known constituents identified in the decoction of Astragali Radix,including Calycosin-7-O-β-d-glucoside,Ononin,Calycosin-7-O-Glc-6"-O-malonate,Calycosin,Formononetin.eleven constituents were detected in the blood,seven metabolic components,and others are of the prototypes.There are fourteen constituents identified in the Urine sample,including nine metabolic components and five prototypes,hydroxylation,methylation,deglycosylation,glucuronidation and sulfation were the major metabolic pathways of multicomponent in Astragali Radix.Based on the result of the MS of composition in Astragali Radix,plasma and urine,ten components,those include Calycosin-7-O-glucoside,Ononin,Calycosin,Formononetin,Cosmosiin,Daidzein,3-Hydro-9,10-diMP and Isomer of 3-Hydro-9,10-diMP can be the quality control ingredients of Astragali Radix.2.Metabolic routes along digestive system of Calycosin-7-O-β-d-glucoside:Complicated Sequential Metabolism Way in Rats.This study adopted an approach to profile the sequential biotransformation process of calycosin-7-O-β-d-glucoside based on comparative analysis.In vitro incubation,in situ closed-loop and in vivo blood sampling,they were used to evaluate the relative contribution of each site to the entirety metabolism of calycosin-7-O-β-d-glucoside along the digestive system by HPLC and UPLC-MS.The results indicated that calycosin-7-O-β-d-glucoside was quite stable in digestive juice,and it was metabolized primarily by the liver,and the metabolism of intestinal flora and gut wall also paly important role of the process.And glucuronidation,sulfation,and glycosylation were main occurred in liver,the intestinal flora and gut wall were the sites of the deglycosylation reaction.3.Metabolic routes along digestive system of Astragali Radix:multicomponent sequential metabolism way in rat.This study adopted a multicomponent sequential metabolism method to profile the metabolic routes along digestive system of Astragali Radix based on comparative analysis by HPLC and UPLC-MS.The incubation experiment results indicated that RA multi-components were quite stable in digestive juice.A total of 6 prototype components and 6 metabolites were detected by comparative analysis of MS data for different plasma samples,and deglycosylation,glucuronidation,sulfation,and glycosylation were the major metabolic pathways of RA multi-components.Liver was the major metabolic organ for RA,and the metabolism of intestinal flora and gut wall also play important role of the metabolism process.Base on the comparative analysis,those include Calycosin-7-O-glucoside,Ononin,Calycosin,Formononetin,Daidzein and 3-Hydro-9,10-diMP,can be the quality control ingredients of Astragali Radix.To sum up,the absorption and metabolism of Astragali Radix in rats were evaluated by the method of everted rat gut sacs and abdominal aorta.In the experiment of multicomponent metabolism in vivo,the major metabolic pathways and the prototypes corresponding metabolites were summarized,and 9 quality control components were selected preliminary.Metabolic routes along digestive system of Calycosin-7-O-β-d-glucoside provided further evidence that the sequential metabolism method could be a general approach to describe the biological activities of these components are changed before these components are delivered to the target sites.Characterizing the multicomponent sequential metabolism of Astragali Radix in Rats evaluated the relative contribution of each site to the entirety metabolism of multicomponent in Astragali Radix the digestive system,and 6 quality control components were screened.Finally,a total of 7 components including Calycosin-7-O-β-d-glucoside,Ononin,Calycosin,Formononetin,Daidzein and 3-Hydro-9,10-diMP can be used as the standard component for quality control of Astragali Radix.The method of in vitro,in situ and in vivo combined with HPLC/UPLC-MS,screen therapeutic basis for Astragali Radix via profile the dynamic process of its multi-component metabolism.This approach provided a new idea for evaluation and quality control of TCM.