| Based on the review of the kinds of ginseng,the research progress of wild ginseng,and the research technology for ginseng chemical composition,various methods were combined to deeply study the chemical constituents with small molecular weights in wild ginseng,the similarities and differences of chemical composition in wild ginseng and cultivated ginseng,the effect and mechanisms of wild ginseng on COPD.The innovative results were displayed below:1.Study on the chemical constituents of wild ginseng(1)Isolation and identification of chemical constituents of wild ginsengA total of 55 compounds were isolated from 95% ethanol extract of 20-year-old wild ginseng by using silica gel column chromatography,macroporous resin chromatography,sephadex column chromatography,ODS column chromatography and HPLC.The structures of 55 compounds,including 47 triterpenes,2polyacetylenes,4 steroids and 2 hydrocarbon,were identified by analysis of physical properties and chemical properties,NMR and HR-MS.Among them,compounds 1~4 were new compounds,and compounds 5~16 were isolated from ginseng for the first time.The study lays a foundation and provides scientific data for elucidating the chemical constituents of wild ginseng.(2)Analysis and identification of chemical constituents of wild ginseng by LC-MSUPLC-Q/TOF-MS technology combined with the UNIFI platform of natural product analysis was used to quickly analyze and identify the constituents with the molecular weight in the range of 100~1500 Da from 80% methanol extracts of30-year-old wild ginseng for the first time.The results showed that 80% methanol extract of wild ginseng was rich in the chemical constituents with various structure types.A total of 101 compounds including triterpenes(the main component),organic acids and organic acid esters,sterols and alkynyl alcohols,amino acids,aldehydes and ketones had been identified by comparing with reference substances,or by analyzing accurate molecular weight and typical fragments.The research provides new ideas and theoretical basis for clarifying the chemical constituents of wild ginseng.(3)Study on fingerprints and chemical pattern recognition of wild ginseng root and rhizomeHPLC fingerprints of the root and rhizome of 30-year-old wild ginseng were established for the first time.A total of 19 common peaks were screened out,and 12 of them were identified.The similarities of 40 batches of wild ginseng root and rhizome samples ranged from 0.714 to 0.892.The results of CA and PCA analysis showed that 40 batches of samples were divided into two categories,namely wild ginseng roots and wild ginseng rhizomes.The analysis of OPLS-DA showed that ginsenosides Rg1,Re,Rb1,Rc,and panaxydol were the main constituents that caused the differences in chemical composition of roots and rhizomes.The study provides a theoretical basis for the selection of indexes for the quality evaluation of wild ginseng.(4)Determination and analysis of ginsenosides in roots,stems,leaves and seeds of wild ginsengThe contents of total saponins and 12 monomer saponins in roots,stems,leaves and seeds of 20-year-old wild ginseng were determined for the first time.The determination by using UV-visible spectrophotometric method showed that the content of total saponins showed in leaves was the highest(20.3%),followed by roots(6.8%),stems(5.0%),and seeds(3.8%).The determination by HPLC-UV method showed that the contents of monomer saponins in each part were quite different:ginsenosides in roots were mainly Rg1,Rb1,Rc,Re,and Rd;ginsenosides in stems were mainly Re,Rb1,Rb3,Rd and PPT;ginsenosides in the leaves were mainly Re,Rd,Rg1,Rb3,Rc,and Rb2;ginsenosides in seeds were mainly Re,Rg1 and Rc.The results provide a reference for the quality evaluation of various parts of wild ginseng,and also provide a scientific basis for the development and utilization of the aerial parts of wild ginseng.(5)Analysis of volatile components in roots,stems,leaves and seeds of wild ginsengBased on HS-SPME combined with GC-MS,the volatile components of roots,stems,leaves and seeds of 20-year-old wild ginseng were determined for the first time.A total of 184 volatile components were identified.Among them,54 components including hydrocarbons(23.4%),alcohols/phenols(21.4%),esters(16.3%)and aldehydes(6.8%)were identified in roots;84 components including hydrocarbons(80.5%),alcohols/phenols(4.0%)and esters(4.8%)were in stems;68 components including hydrocarbons(86.5%),aldehydes(3.7%),ketones(2.0%)and esters(2.2%)were in leaves;81 components including hydrocarbons(81.6%),esters(4.5%),alcohols/phenols(3.5%)and aldehydes(2.0%)were in seeds.The kinds and contents of volatile components in roots,stems,leaves and seeds of wild ginseng were quite different: there were 27,37,19,and 35 characteristic components respectively,and only 9 shared components.This study can not only provide data support for the research of the chemical composition of each part of wild ginseng,but also provide a reference for the further development and reasonable utilization of each part.2.Comparative study on chemical composition of wild ginseng and cultivated ginseng(1)The metabolomics study of wild ginseng and cultivated ginsengBased on UPLC-Q/TOF-MS combined with multivariate statistical analysis,the untargeted metabolomics study for 30-year-old wild ginseng and 5-year-old cultivated ginseng was conducted for the first time.It was found that there was a significant difference in chemical composition between two kinds of ginseng.A total of 14 potential chemical markers were identified by comparing with the reference substances,or analyzing accurate molecular weight and typical fragment.The markers with higher contents in wild ginseng were ginsenosides Rg1,Re2,Rf,Rg4,gypenosides IX,XVII,and panaxydol.Most of them were the rare saponins with the changed side chain except Rg1 and Rf,the characteristic component in ginseng.The markers with higher contents in cultivated ginseng were ginsenosides Re,Rb3,Rd,notoginsenoside R1,ginsenoside L10,12,13,15-trihydroxy-9-octadecenoic acid,(E,E)-9-Hydroxyoctadecyl-10,12-dienoic acid and n-pentadecanal.Most of them were the common saponins and alkanes.The research could provide scientific basis for establishing wild ginseng’s quality standard which is different from cultivated ginseng.(2)Chemical pattern recognition of the monomer composition in wild ginsengThe chemical pattern recognition and analysis of the monomer component in30-year old wild ginseng and 5-year-old cultivated ginseng were carried out based on the HPLC-UV for the first time.The detection wavelength was 203 nm.The ratios of any two chromatographic peak areas were calculated.With the analysis of CA,PCA and OPLS-DA,6 pairs of composition,of which the ratios were markedly different between wild ginseng and cultivated ginseng,were recognized as follows:panaxydol/oleanolic acid,panaxynol/oleanolic acid,panaxynol/Re,panaxynol/Rd,panaxydol/Re and Rf/Rd.The research results could provide new ideas and method for identifying the characteristic components of wild ginseng.(3)Comparative Study on volatile components between wild ginseng and cultivated ginsengBased on the HS-SPME combined with GC-MS,a comparative study of the volatile components between 30-year-old wild ginseng and 5-year-old cultivated ginseng was performed for the first time.A total of 69 volatile components,including53 sesquiterpenes,8 monoterpenes,3 aldehydes,2 esters,1 acid,1 ketone,1 ether were identified.Among them,50 components,including(E)-β-farnesene(23.12%),terpineol(12.22%)and β-elemene(11.98%),were identified from cultivated ginseng.While 38 components,including paeonia oleifera(19.95%),α-neurosyringene(12.54%)and α-guaiacene(10.47%),were identified from wild ginseng.There were12 shared components in two kinds of ginseng.At the same time,the kinds and contents of volatile components in these two kinds of ginseng were quite differernt.There were 17 characteristic components accounting for 29.91% of the total volatile components in cultivated ginseng.Among these components,the content of(E)-β-farnesene(23.12%)was the highest.There were 15 characteristic components accounting for 19.35% of the total volatile components in wild ginseng.Among these components,the content of 4,11,11-trimethyl-8-methylene-[1R-(1R*,4Z,9S*)]-bicyclo[7,2,0] undec-4-ene was the highest.3.Study on bioactivity and mechanisms of wild ginseng against chronic obstructive pulmonary disease(COPD)(1)Effects of different extracts of wild ginseng on inflammatory damage induced by cigarette smoke extract in A549 cellsHuman lung cancer epithelial cell line A549 cells were stimulated by exogenous cigarette smoke extract to establish cigarette smoke injury model in vitro.The effects of petroleum ether,ethyl acetate,and n-butanol extract of wild ginseng on inflammatory damage induced by cigarette smoke extract were evaluated in A549 cells for the first time.The results showed that the levels of TNF-α,IL-1β and IL-6 in the supernatant of CSE-induced A549 cells were significantly reduced by n-butanol extract.It could be concluded that n-butanol extract had the protective effect against the inflammatory damage in CSE-induced A549 cells.(2)Effects of monomer ginsenoside in wild ginseng on inflammatory damage induced by cigarette smoke extract in A549 cellsThe protective effects of four new ginsenosides(Rm1,Rm2,Rm3 and Rm4)and three known analogues(Rb2,Rd,and Rg3)isolated from n-butanol extract against COPD induced by cigarette smoke extract were evaluated in A549 cells for the first time.The results showed that the levels of TNF-α,IL-1β and IL-6 in the supernatant of CSE-induced A549 cells were reduced to various degrees,and ameliorated the inflammatory response.Among these seven compounds,ginsenosides Rg3 and Rb2 had the strongest effect.The HDAC2 pathway might be involved in the protective effect against the CSE-mediated inflammatory response in A549 cells.(3)Intervention effect of n-butanol extract of wild ginseng on COPD model miceThe CSE-induced COPD mice model was established by nasal smoking method.The COPD mice were intragastrically administrated with n-butanol extract of wild ginseng(BEWG).The effect of BEWG on COPD model mice was investigated for the first time.The results showed that BEWG high-dose group(40 mg/kg/d)and medium-dose group(20 mg/kg/d)could increase the weights of COPD mice,increase the forced expiratory volume(FEV100/FVC),reduce static compliance(Cchord)and airway resistance(RI),reduce the levels of pro-inflammatory factors such as TNF-α,IL-1β and IL-6,increase the content of SOD,reduce the content of MDA,and improve lung tissue pathological damage.It had been proved that BEWG could improve lung function,reduce inflammatory response and oxidative damage,and enhance anti-oxidative stress in a dose-dependent manner in model mice.It could be concluded that wild ginseng had a good anti-COPD effect.(4)Study on serum pharmacochemistry and network pharmacology of wild ginseng with anti-COPD effectBased on UPLC-Q/TOF-MS combined with multivariate statistical analysis such as PCA and OPLS-DA,a study on the components of BEWG migrating to serum of COPD mice was performed for the first time.By comparing with the references or analyzing the extract molecular weight and the typical fragments,a total of 17 migration components,including prototypes or metabolites,were identified.The migration components were ginsenosides Rg1,Re,Rf,Rb1,Rc,Ro,Rh1,Rd,Rg3,Rh2,Rs3,CK,protopanaxatriol,vinaginsenoside R4,3-O-(β-D-glucopyranosyl)-oleanolic acid-28-O-(β-D-glucopyranosyl),panaxynol and panaxydol.Taking the above 17 migration components as “candidate compounds”,the interaction network of “components-COPD targets-pathways” was established for the first time by applying network pharmacology.The potential key protein targets of wild ginseng against COPD were predicted such as IL6,IL1 B,TNF,MMP9,and MAPK1,and the first three protein targets had been verified in the pharmacological activity study.It is also predicted that wild ginseng might play the anti-COPD role by regulating signal pathways such as Pathways in cancer,TNF,PI3K-Akt and metabolic pathways such as arachidonic acid metabolism,linoleic acid metabolism,steroid hormone biosynthesis.The research provides a scientific basis for further discussion on the mechanism of anti-COPD effects of wild ginseng.(5)Metabonomics study of wild ginseng against COPDThe effects of BEWG on endogenous metabolites and related metabolic pathways in CSE-induced COPD mice were studied for the first time by using UPLC-Q/TOF-MS-based metabolomics.The results showed that the contents of many endogenous metabolites in COPD model mice had changed significantly compared with normal mice.After intervention with BEWG,the levels of 20 endogenous metabolites such as L-tryptophan,arachidonic acid,linoleic acid,lecithin and leukotriene A4 could be significantly re-regulated.It was then inferred that wild ginseng exerted anti-COPD activity by intervening 7 metabolic pathways such as linoleic acid metabolism,arachidonic acid metabolism,steroid hormone biosynthesis,retinol metabolism,ether lipid metabolism,glycerophospholipid metabolism and tryptophan metabolism.This part of the study also validated the three metabolic pathways predicted by network pharmacology.In summary,this dissertation had conducted in-depth research on the chemical composition and pharmacological activity of wild ginseng.The study could provide scientific basis for clarifying wild ginseng’s chemical composition and differences from cultivated ginseng,and could also provide theoretical support for expanding the medicinal scope of wild ginseng... |
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