| Objective:Kai-Xin-San was first recorded in “Volume 14 Small intestine of Essential Recipes for Emergent Use Worth A Thousand Gold”.The prescription was composed of four medicinal herbs,such as ginseng,poria,polygala tenuifolia and acorus tatarinowii.The ratio of the four medicinal herbs used in this experiment was 3:3:2:2.Modern studies have shown that Kai-Xin-San has a good therapeutic effect on neurodegenerative diseases such as Alzheimer’s disease,but the material basis for its efficacy is not clear.Therefore,this study identified the group of chemical substances outside Kai-Xin-San、Metabolic characteristics of Kai-Xin-San incubated in intestinal tract in vitro and Comparison of blood components between normal rats and AD model rats to provide the basis for the study on the pharmacodynamic substance basis of Kai-Xin-San against AD.Methods:1.UPLC-LTQ-Orbitrap-MS was used to collect the positive and negative ion modes of alcohol extracts from Kai-Xin-San.The chemical components in Kai-XinSan were identified and inferred by combining with standard substances and literature,and the representative components in Kai-Xin-San were analyzed by mass spectrometry.2.In the established in vitro rat intestinal incubation system,UPLC-LTQOrbitrap-MS technology combined with Compound Discoverer software was used to find the metabolites of representative compounds of Kai-Xin-San under the action of intestinal flora,and the metabolic characteristics were summarized.Then the metabolites of intestinal bacterial solution of Kai-Xin-San were identified.3.The rat model of AD is established by intraparitoneal injection of D-galactose and bilateral injection of oligo-Aβ25-35 into hippocampus.Under the guidance of serum pharmacochemistry theory and multivariate statistics,UPLC-LTQ-Orbitrap-MS technique is adopted in SIEVE and SIMCA for data preprocessing.Combined with the self-established Kai-Xin-San Compound database and the previously identified chemical compounds in vitro,the blood prototype components were identified,the metabolites were explored by Compound Discoverer software,and the blood components were compared between normal rats and AD model rats.4.The key targets of KXS against AD were discovered by using target prediction,GO enrichment,KEGG pathway analysis and other technologies.The main action pathways and biological processes were analyzed,and then the network relationship between KXS components,target sites and pathways was discussed.Finally,the molecular docking technology was used to verify.It lays a foundation for further study of its anti-AD mechanism Results:1.In this study,269 chemical constituents were identified from the alcohol extract of Kai-Xin-San,including 7 substances,and 55 compounds were identified from ginseng,mainly ginsenosides.32 compounds were identified from Poria,mainly triterpenoids.A total of 127 compounds were identified,including saponins,xanthones and glycoesters.Fifty-five compounds,including phenylpropanes and alkaloids,were identified from acorus tatarinowii.2.Kai-Xin-San and its representative components were metabolized in rat intestinal flora in vitro,among which ginseng compounds mainly underwent oxidation,dehydrogenation,dehydration,hydration,desugar,glycosylation and complex reactions.Deglycosylation,oxidation,ester hydrolysis,methylation and compound reaction occurred in polygala tenuifolia.The main reaction of poria was oxidation,dehydrogenation,deoxygenation,methylation,hydration,deacylation and compound reaction.Oxidation and demethylation of acorus tatarinowii compounds occur mainly.According to the metabolic rules of the representative components in the intestinal bacteria solution in vitro,a total of 79 metabolites were identified in the Kai-Xin-San incubation solution.3.After administration of Kai-Xin-San extract,37 Kai-Xin-San related components were identified in the serum samples of rats in the normal administration group,of which 24 prototype components(including ginsenosides,poria triterpene acids,polygonate saponins,polygonate ketones,polygonate esters)and 13 metabolites(including ginsenosides desugaration,hydration and oxidation products)were identified.poria triterpene acids hydroxylation,deoxygenation,demethylation,desaturation,glycine binding,and complex reaction products);Twenty components related to Kai-Xin-San,including 11 prototype components and 9 metabolites,were detected in serum samples of rats treated with AD model.There were 12 components in normal group and model group,including 9 prototype components and 3 metabolic components.4.Network pharmacology and molecular docking analysis showed that The components of KXS into blood may be involved in regulating signal pathways such as PPAR,IL-17,HIF-1,Relaxin and VEGF through 140 AD-related targets such as MAPK3,HSP90AA1,STAT3,AKT1 and JUN,thus participating in the regulation of intracellular receptor-mediated signal transduction and positive regulation of inflammation by cytoplasmic calcium ion concentration.Lipopolysaccharide plays an anti-AD role by regulating isoreaction. |
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