Study On The Material Basis And Mechanism Of Hepatoprotective Effect Of Gentiana Rigescens Franch.Ex Hemsl.

Liver injury poses a serious threat to human life and health,modern pharmacological studies have shown that many traditional Chinese medicines have significant protective effects on the liver,with minor toxic and side effects and have become a good resource for developing new drugs to prevent and treat liver injury.Gentiana rigescens Franch.Ex Hemsl.is a perennial herb of Gentianaceae,which has a long history of folk medicine and has functions such as liver protection,anti-inflammatory,analgesic as well as anti-tumor,etc.The pharmacological effects of gentiopicroside and swertiamarine,which are the main components of G.rigescens have been intensely studied,but the development and utilization of other active ingredients are slightly inadequate.Therefore,it is necessary to dig more deeply into the chemical constituents and pharmacological effects of G.rigescens.This study used a heated reflux extraction method to obtain the crude extract of G.rigescens.L02 normal human hepatocytes were selected to screen for its anti-liver injury activity in vitro and obtain the liver protective active site VII.The active site VII was isolated and purified through column chromatography,Sephadex LH-20 and other methods to obtain monomer compounds.The structure of the monomer compound was identified by NMR,LC-MS and other spectroscopic techniques as well as reference literature.The in vitro hepatoprotective activity of some monomeric compounds was then determined using 2 in vitro liver injury models of carbon tetrachloride（CCl₄）and acetaminophen（APAP）.Based on in vitro liver protective activity and network pharmacological prediction results,loganic acid（LA）was screened as the research object to explore LA’s protective effect and mechanism on CCl₄-induced acute liver injury in mice.The research results of this article will provide a theoretical basis for further developing G.rigescens.The main results of this study are as follows:（1）The crude powder of G.rigescens was extracted by heating reflux with 90%ethanol3 times,2 h each time,and the extract was condensed by a rotary evaporator under reduced pressure to obtain the total extraction of G.rigescens.And the total extraction was gradient eluted with CH₂Cl₂-CH₃OH（12:1-1:4）.The same fractions were combined after TLC detection,and a total of 7 sites（Ⅰ～Ⅶ）were obtained.To establish CCl₄-induced and APAP-induced models of liver injury in vitro as well as screen the active liver protective sites of G.rigescens（Ⅰ～Ⅶ）.The experimental results showed that site VII had the best liver protection effect among the two kinds of in vitro models of liver injury,and certified that site VII was the active site for liver protection.（2）The chemical constituents of hepatoprotective active fraction VII of G.rigescens were separated by forward silica gel column chromatography and Sephadex LH-20 column chromatography,and the structures were identified in NMR,LC-MS and other techniques and reference literature.18 compounds were isolated and identified from the liver protective active site VII of G.rigescens,including 7 hydrocarbon compounds:n-tetracosane（1）,n-triacontane（3）,n-pentadecane（4）,n-heptacosane（5）,n-octadecane（6）,n-decane（10）and n-hentetracontane（17）;3 iridoid compounds:loganic acid（12）,6’-O-β-D-glucosylloganic acid（13）and gentiopicroside（15）;3 fatty acid compounds:ethyl laurate（11）,n-triacontanoic acid（14）and methyl eicosanoate（16）;1 ketone compounds:4-hydroxy-4-methyl-2-pentanone（2）;4 other compounds:4-O-n-butyl-α,α-D-trehalose（7）,α-nigerose（8）,9-hexadecen-1-ol（9）and gentianose（18）.In this study,18 compounds were isolated and identified,and 15 compounds were isolated from G.rigescens for the first time,including compounds 1-11,14 and 16-18.By establishing CC1₄-induced and APAP-induced hepatocyte injury models in vitro,some monomeric compounds were screened for anti-liver injury activity.It was found that compounds 12,13,15 and 18 had certain liver protective activities,among which compounds12 and 15 had significant liver protective activities.（3）Based on the network pharmacology approach,LA’s molecular mechanism of action for liver injury was comprehensively analyzed by“multi-component,multi-target and multi-pathway”.At the same time,Gene Ontology（GO）analysis was performed on the targets.The results indicated that LA might regulate the expression of 90 targets such as GAPDH,IL2,IL6,STAT3,CASP3,DNMT1 and other targets through biological processes such as proteolysis,response to drugs,apoptotic process,response to ethanol and inflammatory response.Thus,it regulates cancer pathways,apoptosis,Rap1 signaling pathway,hepatitis B,TNF signaling pathway,micro RNAs in cancer,Fox O signaling pathway,necrosis and metabolic associated fatty liver disease signaling pathways to exert anti-liver injury effects.According to the network pharmacology prediction,LA’s mechanism in treating liver injury may be related to regulating inflammatory reactions,oxidative stress,and cell apoptosis.To verify the results of network pharmacology,this chapter will explore the protective effect of LA on CCl₄-induced acute liver injury in mice and its related mechanisms.Studies have shown that LA pretreatment can reduce the activities of alanine transaminase（ALT）and aspartate transaminase（AST）in CCl₄-induced acute liver injury in serum of mice and inhibit pathological liver damage.Anti-inflammatory effect:LA pretreatment can inhibit the activities of serum inflammatory cytokines tumor necrosis-α（TNF-α）,interleukin-6（IL-6）and Interleukin-1β（IL-1β）induced by CCl₄,while LA pretreatment can reduce the m RNA expression levels of TNF-α,IL-6,IL-1βand nuclear factor-kappa B（NF-κB）in mice liver tissue.Inhibition of oxidative stress:LA pretreatment can increase the contents of glutathione（GSH）,Superoxide dismutase（SOD）,and catalase（CAT）in mice with acute liver injury and reduce malondiadehyde（MDA）and cytochrome P450 2E1（CYP2E1）m RNA levels in liver tissue.At the same time,LA pretreatment can increase the expression levels of nuclear factor erythroid 2 related factor（Nrf2）and its downstream genes glutamate-cysteine ligase catalytic subunit（GCLC）,haem oxygenase-1（HO-1）,and quinone oxidoreductase 1（NQO1）in CCl₄liver injury mice,thereby restoring the balance between the oxidation and antioxidant system in the organism.Inhibition of apoptosis:TUNEL results demonstrated that apoptotic cells significantly decreased after LA pretreatment.Moreover,LA pretreatment can reduce the activity of cysteinyl aspartate specific proteinase 3（Caspase-3）and cysteinyl aspartate specific proteinase 9（Caspase-9）in the liver induced by CCl₄,reduce the expression of the proapoptotic gene Bcl-2-associated X protein（Bax）,and increase the transcription level of the anti-apoptotic gene B cell iymphoma-2（Bcl-2）.The experimental results have indicated that LA plays an anti-liver injury role through three aspects:anti-inflammatory,regulated the Nrf2pathway inhibited oxidative stress and restrained cell apoptosis.This study revealed that the material basis for G.rigescens to play a protective role in the liver is mainly the iridoids and conducted preliminary research on its protective effect against liver injury and its mechanism.To lay a theoretical foundation for the rational development and utilization of G.rigescens.