| Ding-Zhi-Xiao-Wan prescription(DZXW),originated from a Chinese classical prescription book"Bei-Ji-Qian-Jin-Yao-Fang",was written by Sun Si-miao in Tang Dynasty.It is composed of Ginseng Radix,Poria cocos,Polygala Radx,and AcoriTatarinowii Rhizoma according to a certain proportion(3:3:2:2).It is one of the basic prescriptions for treating neurodegenerative diseases in traditional Chinese medicine with extraordinary curative effects on strengthening the mental function.However,the lack of systematic study of its chemical groups in vivo and in vitro,,which limits the application in modem clinical.In this paper,the horizontal and vertical correlation analysis strategy was established to systematically study the pharmacodynamics material basis of DZXW in the treatment of Alzheimer's disease(AD)in vivo and in vitro,by using metabolic studies in vivo and in vitro and combining with liquid and gas chromatography-mass spectrometry technologies(LC-MS and GC-MS).Longitudinal correlation analysis strategy refers to the progressive analysis of monomer components,single medicine and prescription step by step.First,the LC-MS and GC-MS parameters,the rules of fragmentation in mass spectrometry and the metabolic rule of representative monomer components in vitro were studied and analyzed,which provided data support for acquiring mass spectrometry diagnostic ions and metabolic rule of each type of ingredients;second,based on the research data of representative monomer components as support and guidance,each single drug was systematically analyzed,and the multidimensional chemical components in vitro and in vivo were summarized to form an in-house database.Finally,guided by the multidimensional chemical group database of single herbs,the multidimensional chemical group of DZXW prescription in vitro and in vivo was systematically analyzed.Horizontal correlation analysis strategy refers to the progressive analysis of in?vitro substance group,in vitro metabolites group,in vivo metabolites group and pharmacodynamics group in vivo.First,the extracts of DZXW were systematically analyzed.The fragmentation rules of various components,retention time,molecular weight and structure information were summarized.The in vitro chemical substance group was systematically elucidated and the in-house library of in vitro chemical constituents was constructed.Then,the in vitro intestinal microflora metabolites group and in ivitro liver microsome metabolites group were established by in vitro intestinal microflora metabolism and liver microsome technologies.The research processes followed the metabolic characteristics and rules of oral drugs in vivo.Third,the metabolism of DZXW in the main organs and tissues of the rats through oral administration was detected,and the metabolites group in vivo of DZXW was established.Finally,guided by the metabolites group in vivo,the network pharmacological analysis(i.e.targeted network pharmacological analysis)was carried out to clarify the chemical components acting on the target and pathway of disease,and to clarify the pharmacodynamic substance basis and mechanism of DZXW in vivo.1.Establishment of chemical group in vitroAccording to the different chemical constituents of four single drugs in DZXW,including Ginseng Radix,Poria cocos,Polygala Radix,and Acori Tatarinowii Rhizoma,a targeted analysis strategy was established.The main component of Acori Tatarinouwii Rhizoma were volatile oils.Therefore,the extracts of Acori Tatarinowii Rhizoma were separately analyzed by GC-MS.Fifty main components of volatile oil were detected and identified.Among them,the content of P-asarone was more than 70%of the total volatile oil.For other single drugs,the ultra-high liquid chromatography tandem quadrupole-time-of-flight mass spectrometry(UHPLC-Q-TOF-MS)technology was applied to analyze the chemical constituents,and UNIFI software was used to assist the analysis.At the same time,ion mobility technology was used to separate the co-effluent from Poria cocos.A total of 59 ginsenosides,136 Polygala compounds and 121 Poria cocos triterpenoids were detected and identified.2.Establishment of metabolites group invitroThe invitro metabolites group mainly includes invitro intestinal microflora and liver micromers metabolites groups.The metabolites of Ginseng Radix,Poria cocos,Polygala Radix in vitro were established by UHPLC-Q-TOF-MS and their metabolic rules were summarized.Among them,Ginseng and Polygala compounds mainly underwent deglycosylation metabolism,while the triterpenic acids in Poria cocos were not metabolized by intestinal microflora invitro.Then the main intestinal microflora metabolites were selected and further studied by using in vitro liver microsomes incubation model.The triterpenic acids in Poria cocos under the action of liver microsomal enzyme mainly undergo four kinds of oxidative metabolism,including M+O-H2,M+O,M+O2-H2,and M+O2.The ginseng metabolites of liver microsome of were related to the position of carbohydrates in their chemical structure,and they mainly underwent two kinds of metabolic pathways:oxygenation and dehydrogenation.Only two oxygenated metabolites of tenuifoliside A were detected in Polvgala Radix.3.Establishment of metabolites in vivoThe invitro chemical substances and metabolites of each single medicine group were correlated and analyzed.The in vivo metabolites of each single drug group were established,including urine,feces,plasma,liver and brain.At the same time,the metabolism of intestinal microflora in vitro and feces in vivo,liver microsomes in vitro and liver in vivo were compared and analyzed,which further verified the reliability and rationality of the model invitro.A total of 62 components of Poria cocos compounds,113 components of Ginseng and 13 components of Polygala lenuifolia were detected and identified in plasma.Meanwhile,the P-asarone was detected in the blood and brain of rats by GC-MS.By integrating the metabolic rules and metabolites of each single drug,the metabolism of DZXW in vivo was systematically studied.A total of 150 prototype components and 50 metabolites were detected and identified in vivo.The results provided material basis for further pharmacodynamic and pharmacological studies of DZXW in the treatment of AD.The metabolism of DZXW in normal and AD model rats was further compared and analyzed,including the model of AD induced by intraperitoneal injection of LPS and injection of A?25-35 into hippocampus,which defined the material basis of DZXW in the treatment of AD.4.Establishment of pharmacodynamics group in vivoGuided by the metabolite group in vivo,the network of "compounds-targets-diseases"was constructed by using the means of targeted network pharmacology that selected all tlhe blood-entry components in ivivo.The pharmacodynamic components of DZXW(Ginsenoside Rh2,Ginsenoside Rg3,Ginsenoside Rb1,Ginsenoside Rg1,Pachymic acid,Oleanolic acid,Poricoic acid B,Poricoic acid A,and Poricoic acid C)and their corresponding targets(e.g.COX2,NR3Cl,VDR,CASP3,NFKB1,IFNG,RARG and TNFa)were identified for the treatment of AD.These results systematically clarified the pharmacodynamic basis and potential mechanism of DZXW in the treatment of AD. |
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