Study On The Chemical Constituents,Anti-COPD Effect Of Wild-simulated American Ginseng And The Pharmacokinetics Of New Active Compound

Based on the research progress of chemical constituents and biological activities of wild American ginseng,wild-simulated American ginseng（Ws AG）and field-grown American ginseng（Fg AG）,and the review of the prevention and treatment of chronic obstructive pulmonary disease（COPD）with traditional Chinese medicine,the studies of the chemical constituents of Ws AG,the anti-COPD activity in vitro and in vivo of total saponins of Ws AG（TSW）and the preliminary pharmacokinetic evaluation of a new active component in rats was carried out.The results systematically expounded the chemical composition of Ws AG,clarified the anti-COPD pharmacological effects of TSW and the active component（Quinquengenin W,QW）,and evaluated the pharmacokinetic parameters of QW,which filled the research blank in the study of chemical constituents and biological activities of Ws AG,provided scientific data for expanding the medicinal range of Ws AG,and also found a new candidate drug with good anti-COPD activity and good pharmacokinetic properties.The innovative results are displayed as follows:1.Study on the chemical constituents of Ws AG1.1 Analysis of chemical constituents of Ws AG by UPLC-QTOF-MS1.1.1 Rapid analysis and identification of chemical constituents of Ws AGA total of 128 common components had been identified from the main root（MR）,branch root（BR）,rhizome（RH）,adventitious root（AR）,and fibrous root（FR）of Ws AG using UPLC-QTOF-MS technology combined with UNIFI platform of natural product analysis.The results showed that the chemical composition of each part of Ws AG was same,and the secondary metabolites in MR,BR,RH,AR and FR of Ws AG were diverse in structural types,but mainly rich in triterpenoid saponins.1.1.2 Non-targeted metabolomics of Ws AG（1）Based on UPLC-QTOF-MS combined with multivariate statistical analysis,the untargeted metabolomics study for Ws AG underground parts（MR,BR,RH,AR,and FR）was conducted.A total of 31 potential differential components were identified to distinguish MR and BR,RH and AR,FR parts.The results revealed the content differences of secondary metabolites in underground different parts of Ws AG,and could provide evidence for the rational application of different parts of Ws AG.（2）Based on same method as above,the untargeted metabolomics study for Ws AG and Fg AG was conducted for the first time.A total of 22 potential differential components were identified to distinguish Ws AG from Fg AG.This study illustrated the differences between Ws AG and Fg AG,and provided a basis for explaining the effect of different growth environments on secondary metabolites.1.2 Isolation and identification of chemical constituents of Ws AG based on classical separation method and molecular network technologyA total of 59 compounds were isolated from 95%ethanol extract of Ws AG by using classical and chemical-guided separation technology,and identified by analysis of physical and chemical properties,NMR and HR-MS.Among of them,compounds 1～4 were new compounds,named quinquengenin W,quinquenoside W₁,W₂and W₃,respectively.And compounds 6～8,16,21,23～28,30,37～41,44～46 and 50～52 were isolated from the underground part of American ginseng for the first time.1.3 Determination of ginsenosides in Ws AG based on HPLC-ELSD technologyThe contents of total saponins and 22 monomer saponins in Ws AG were determined by using UV-visible spectrophotometric method and HPLC-ELSD technology,respectively.The results showed that the content of total saponins in Ws AG was slightly higher than that in Fg AG.As for monomer saponins,the ratios of Rg1/Re and（Rg1+Re）/Rd in Ws AG were larger than that in Fg AG,which could be used as potential characteristic markers to distinguish Ws AG from Fg AG.1.4 Analysis of volatile components in Ws AG based on GC-MS technologyBased on HS-SPME combined with GC-MS method,it was determined that the volatile components of Ws AG and Fg AG mainly contained sesquiterpenoids,and the content of（E）-β-farnesene was the highest.There were 25 common components in Ws AG and Fg AG.In addition,a total of 29 characteristic components were identified in Ws AG.And 35characteristic components were identified in Fg AG.1.5 Semi-synthetic preparation of new compound QWQW was prepared by semi-synthesis with protopanaxadiol as the raw material,with a yield of 30.7%.This experiment realized the directional synthesis of trace natural products,which laid the raw material foundation for the subsequent study.2.Study on the anti-COPD effect of Ws AG2.1 Effects of TSW and new compounds on inflammatory damage in A549 cells.The effects of TSW,total saponins of field-cultivated American ginseng（TSF）and 4new compounds（quinquenosides W₁,W₂,W₃and QW）isolated from Ws AG on A549 cell inflammatory injury model induced by cigarette smoke extract were evaluated for the first time.The results showed that TSW,TSF and 4 new compounds had different degrees of improvement on the inflammatory injury in A549 cells.TSW showed a better effect on IL-6and TNF-αthan TSF.For the new compounds,QW had the best biological activity.2.2 Intervention effects of TSW and QW on COPD model miceThe CS-induced COPD mice were treated with TSW,TSF and QW,respectively,aiming at evaluating the effects of TSW,TSF and QW on COPD.The results showed that TSW（20,40 mg/kg）,TSF（20,40 mg/kg）and QW（10,20 mg/kg）had good anti-COPD effects.And TSW showed better anti-COPD activity than TSF.2.3 Integrating metabolomics and network pharmacology to explore the anti-COPD mechanism of TSWBy integrating metabolomics and network pharmacology method,there were 16metabolites,9 key targets and 5 metabolic pathways closely related to the anti-COPD pharmacological effects of TSW were identified.A total of 6 key components were screened out as the key components with anti-COPD effect,which was helpful to explain that the anti-COPD effect of TSW was stronger than that of TSF.2.4 Validation of 6 key components contributing to the anti-COPD effect of TSWMolecular docking was used to analyze the binding patterns of the 6 key components and 2 key targets,TNF-αand SOD.The results showed that the binding energy of the 6 key components with the two key targets was-10.2～-6.7 kcal/mol with some hydrogen bond interactions,which indicated that the ligands had a good binding effect with receptors.A549 cell inflammatory injury model was used to validate and evaluate the activity of 6key components.It was confirmed that these 6 key components were the material basis that TSW showed stronger effect on COPD than Fg AG.3.Study on the pharmacokinetics of QW intragastrically administered in ratsThe plasma concentration-time curves of QW（10.0,20.0,40.0 mg/kg）after oral administration and QW（2.0 mg/kg）in rats after intravenous injection at different time points were determined by the established UPLC-MS/MS method.The results showed that the absorption and the elimination of QW in vivo was slow,and the circulation time was long.And the the kinetic process of QW in vivo was a linear process and had the same trend as the dose dependence in pharmacodynamic studies.Taking the AUC_（0-t）of intravenous administration of QW as a reference,the absolute bioavailability of intragastric administered QW was 19.61%.In addition,UPLC-QTOF-MS technology combined with UNIFI metabolite analysis platform was used to analyze and identify the main metabolites in plasma,urine and feces of rats after intragastric administration of QW.A total of 12metabolites were identified,including 3 phase I metabolites and 9 phase II metabolites.In conclusion,in this dissertation,the chemical constituents of Ws AG were elucidated for the first time,and 4 new compounds were isolated from Ws AG,which filled the blank of the research on the chemical constituents of Ws AG;the anti-COPD effects of TSW and TSF were studied for the first time,which provided a theoretical basis for finding new anti-COPD natural drugs,and expanded the medicinal use of American ginseng and filled the blank of biological activity research of Ws AG;an innovative candidate drug"Quinquengenin W"with good anti-COPD activity and good pharmacokinetic properties was screened out.