Study On Preparation,Chemical Composition And Anti-acute Liver Injury Effect Of Black Ginseng Total Saponins

Black ginseng is a processed product that made from ginseng by repeated steaming and drying.It has many pharmacological activities,including liver and kidney protection,nerve protection,anti-cancer,anti-aging and so on.Saponins are the main active substances of black ginseng.Different from white ginseng and red ginseng,the contents of rare saponins,such as ginsenoside Rg₃,（20R）-Rg₃,Rs₃,（20R）-Rs₃,RK₁,Rg₅,Rh₂,（20R）-Rh₂and CK,in black ginseng are greatly increased.The studies on the chemical compositions of black ginseng from domestic and abroad mainly focused on the identification and contents assay of monomer saponins,while the studies on the biological activities mainly focused on the evaluation of the pharmacological effects of water/alcohol extracts or monomer ginsenosides Rg₃,Rg₅and Rk₁.The preparation of total saponins was easier than that of monomeric saponins.At present,it had been confirmed that the total saponins of Panax ginseng,Panax quinquefolium,and Panax notoginseng had the advantages of rich sources,clarified chemical components,explicit pharmacological effects and the multi-targets and multi-pathways mechanisms.Therefore,the studies of the preparation,the chemical constituents and anti-acute liver injury activity of black ginseng total saponins（BGTS）were carried out for the first time in this thesis.And the research progress of chemical constituents and biological activities of black ginseng,and the establishment of acute liver injury model were both reviewed.The various methods and technologies,such as rapid identification of chemical composition,characteristic chromatogram,contents assay,metabolomics and network pharmacology,were comprehensively used in this study.The achieved innovative results were displayed as follows:1.Study on the preparation of BGTSThe extraction and purification parameters of total saponins were optimized by single factor test and response surface methodology.The preparation process was determined as follows:The black ginseng was crushed and sieved with No.2 sieve,and extracted with 20 times the volume of 70%ethanol for 3.5 h by reflux.After filtered,the filtrates were concentrated and added to AB-8 macroporous resin column.The resin column was firstly eluted with water until colorless,then eluted with 80%ethanol.The ethanol eluent was concentrated,dried to dryness.Thus,the BGTS was obtained.A total of 10 batches of BGTS were prepared by the above process,and the contents of total saponins were 76.91%～82.29%.The results showed that the preparation process established was simple,and the content of total saponin was high.2.Study on the chemical composition and characteristic chromatogram of BGTS（1）Rapid analysis and identification of the chemical constituents of BGTSUPLC-Q/TOF-MS technology combined with UNIFI platform of natural product analysis was used to rapidly analyze and identify the chemical constituents in BGTS.A total of 61 components had been identified by comparing with standards,or by analyzing accurate molecular weight and typical fragments,and it was found that the BGTS was mainly rich in triterpenoid saponins.What’s more,there were 17components identified from black ginseng for the first time,including ginsenoside Ro,F₅,F₃,Rg₈,Re₁,Re₂;isoginsenoside Rh₃;pseudo-ginsenoside RT₄;majoroside F₆,F₅,F₂;notoginoside R₁,R₂,（20R）-R₂,Fe;20-O-β-D-glucosyl-ginsenoside Rf and oleanolic acid-28-O-β-D-glucopyranoside glycosides.The chemical research of black ginseng was enriched by the identified chemical constituents in this thesis.（2）Establishment of the characteristic chromatogram of BGTSThe characteristic chromatogram of BGTS was established with good precision,repeatability and stability by using HPLC-UV method.A total of 9characteristic peaks were identified,including ginsenosides Rb₃,Rg₆,Rk₃,Rg₃,（20R）-Rg₃,Rk₁,Rg₅,CK and protopanaxadiol（PPD）.Taking the peak of ginsenoside Rk₁as the S peak,the relative retention times of other characteristic peaks were calculated,and the values should be within±5%of the specified values.The specified values were 0.77（peak 1）,0.84（peak 2）,0.88（peak 3）,0.95（peak 4）,0.96（peak 5）,1.00（peak 6）,1.01（peak 7）,1.02（peak 8）and 1.10（peak 9）,respectively.The established characteristic chromatogram could provide scientific data and reference for the quality control and evaluation of BGTS.（3）The content assay of characteristic components in BGTSThe content assay method of 9 characteristic components using HPLC-UV was established with good linear range,precision,repeatability and recovery.The results showed that the content ranges of each characteristic component in 10 batches of BGTS were as follows:Rg₅（14.68%～26.35%）,Rk₁（12.82%～28.31%）,PPD（2.28%～4.61%）,CK（1.12%～4.30%）,Rg₃（1.14%～3.08%）,（20R）-Rg₃（0.52%～1.60%）,Rg₆（0.40%～0.85%）,Rk₃（0.12%～0.49%）and Rb₃（0.17%～0.41%）.The content ranges of each characteristic component in the BGTSs were clarified by content assay.3.Study on the effect of BGTS against acute liver injury in mice（1）Effects of BGTS on CCl₄-induced acute liver injury in miceAfter being intragastrically administered with BGTS（15,30,60 mg/kg）for 5days,the mice were injected intraperitoneally with CCl₄olive oil dilution to establish the models of acute liver injury.Then,after 3 h and 12 h,the mice were intragastrically administered with BGTS.At the moment of 24 h after injected CCl₄,the blood was collected for assay.The data showed that the levels of ALT,AST,MDA,ROS,and IL-6 in serum could be effectively decreased,the activities of SOD and GSH-PX in serum could be effectively increased,and the extent of liver lesions and the degree of inflammatory cell infiltration could be effectively reduced with the intervention of BGTS（30,60 mg/kg）.The results showed that BGTS could ameliorate liver tissue damage in mice by reducing the inflammatory response and enhancing the antioxidant capacity.（2）Integrated metabonomics and network pharmacology to explore the anti-acute liver injury mechanism of BGTSFirstly,based on the metabolomics study using the UPLC-Q/TOF-MS technology,some metabolites such as tryptophan,which were closely related to the effect of BGTS on acute liver injury,and the metabolic pathways,such as arachidonic acid metabolism involved in these metabolites were screened out.Then,based on the network pharmacology,the key components（ginsenosides Rk₁,Rg₅,Rh₃,Rh₄,Rk₃）,the key targets（STAT3,IL-6,etc.）and the important metabolic pathways（arachidonic acid metabolism,PI3K-Akt signaling pathway,etc.）contributing to the effect of BGTS on acute liver injury were screened out.Finally,based on the integration analysis of metabolomics and network pharmacology,a total of 4 metabolites（L-tryptophan,arachidonic acid,riboflavin and leukotriene A4）,4 key targets（IL-6,STAT3,MAPK1,EGFR）,3 metabolic pathways（arachidonic acid metabolism,riboflavin metabolism,tryptophan metabolism）and 5 key components（ginsenosides Rk₁,Rg₅,Rh₃,Rh₄and Rk₃）closely related to the anti-acute liver injury effect of BGTS were identified.（3）Molecular docking between key components and key targetsThe five key components（ginsenosides Rk₁,Rg₅,Rh₃,Rh₄and Rk₃）were docked with four key targets（IL-6、STAT3、MAPK1、EGFR）by molecular docking method.The results showed that stable hydrogen bonds could be formed between the 5 saponins and 4 key targets,with binding energies ranging from-10.0～-7.0kcal/mol,which indicated that the 5 saponins and the 4 key targets had the good binding patterns.In conclusion,the following works had been carried out and completed in this thesis:（1）The preparation process of BGTS was established,and 10 batches of BGTS were prepared with the purity greater than 75%;（2）A total of 61 components were identified,including 17 components identified from black ginseng for the first time;（3）The characteristic chromatogram of BGTS was established,and the contents of 9 characteristic peaks were assayed;（4）The pharmacological effects of BGTS against acute liver injury were evaluated,and the mechanism was preliminarily discussed.There 4 metabolites,4 key targets and 3 metabolic pathways were predicted as closely related to the anti-acute liver injury effect of BGTS.（5）A total of 5 key components of BGTS were screened out.This study provided scientific data and theoretical support for the further research and development of BGTS.