Study On The Purification Of Amarogentin From Swertia Mussotii Franch And The Antifibrotic Mechanism

1 BackgroundLiver fibrosis is a necessary stage from chronic liver disease to cirrhosis.It’s a hub of hepatitis,liver cirrhosis,and liver cancer.The effective treatment for cirrhosis is limited to liver transplantation.But due to the lack of organ,recurrence of the primary disease and other factors,liver transplantation therapy is limited.Blocking liver fibrosis has become a key problem in the treatment of chronic liver disease.Researching on prevention liver fibrosis has become a hot topic over the world.But efficient anti-fibrotic drugs with few side effects have not been discovered.Swertia mussotii Franch is widely used for liver disease in traditional Chinese medicine of the whole herb.But little study was reported on the mechanism of main effective component.Amarogentin（AG）is a secoiridoid glycoside that extracted from Swertia mussotii Franch.And there is no standard substance to sell in the domestic market.Many studies have shown that AG is the active components of the work.But no reports have found whether it has the antifibrotic effect.On the basis of optimization extraction and purification process of AG,the present study was designed to evaluate the hepatoprotective effects and the underlying mechanism of AG in vivo and vitro.At the same time,by observing the change of metabolic profiles in serum,we discussed the possible antifibrotic mechanism of AG from the perspective of the influencing metabolic pathways.2 Methods2.1 Extraction,separation and identification of AG:Taking Swertia mussotii Franch powder into the neutral phosphate buffer,fully mixed,freezing and thawing,centrifugal to remove impurities.Finally,we got the filtrate.After removing the water from filtrate,joined in the saturated ammonium sulfate solution,sedimentation,raw AG were isolated after centrifuging.Column chromatography silica gel mixed with CHCl₃ and CH₃OH,sedimentation,silica gel column chromatogram was made.Dissolved the crude extract with a small amount of ethanol,added the silicone,removed of the solvent,added to the silica gel column chromatography.Then purify it by silica gel column chromatography,CHCl₃ and CH₃OH（9:1）elution.The standard AG was tested as a control,tracking monitored with TLC,collection of eluent when the AG in a single spot.Distilled the solvent off from aforementioned collection to get purified AG.We identified the chemical structure by spectroscopic methods and physical data.Pilot-scale study of AG was also verified for three times.2.2 Protective effect of AG on TGF-β₁-induced liver fibrosis in HSC-T6:HSC-T6 were cultured in vitro and were separated as the following five groups:normal group,TGF-β₁ group,and AG groups（0.01,0.1,1.0 mg/mL）.The following parameters were tested:（1）Effect of TGF-β₁ on HSC proliferation was evaluated by CCK-8 assay.（2）Effect of AG on TGF-β₁-induced HSC proliferation was evaluated by CCK-8 assay.（3）The levels of inflammatory factors in the serum were measured by ELISA.（4）Effect of AG on TGF-β₁-induced hepatic accumulation ofα-SMA and MAPK signal pathway was detected by Western Blot.2.3 Protective effect of AG on CCl₄-induced liver fibrosis in mice:The C57BL/6J mice were randomly divided into the following six groups:normal group,model group,colchicine group,and AG groups（25,50,100 mg/kg）.Animals except the control group were administered 20%CCl₄ dissolved in olive oil（6 mL/kg）via subcutaneous injection twice per week for seven consecutive weeks to establish the liver fibrosis model.The control group was administered 6 mL/kg olive oil at the same time points.After one week of CCl₄ administration,the control and model groups received equal quantities of 0.5%sodium carboxymethylcellulose solution;colchicine（0.1 mg/kg）and AG（25,50,100 mg/kg）were suspended in a 0.5%sodium carboxymethylcellulose solution,respectively,and administered by oral gavage every day for six consecutive weeks.After the experiment,we used the serum and liver tissue to detect the change of biochemical indicators of ALT、AST、Alb、MDA、SOD、GSH-Px and Hyp.The paraffin-embeded liver tissues were stained with HE,Masson and Sirius Red to observe the level of liver fibrosis.Immunohistochemical and Western blot was used to detect the expression ofα-SMA.Western blot was also used to detect the protein expression of TGF-β₁/Smad pathway and MAPK pathway.Finally,we discussed the possible antifibrotic mechanism of AG.2.4 Serum metabonomics study of the hepatoprotective effect of AG on CCl₄-induced liver fibrosis in mice by GC-TOF-MS analysis:GC-TOF-MS was used to analysis the metabolic patterns of the serum samples collecting from the control,model and 100 mg/kg AG group.The pretreatment data were analyzed by the multivariate statistical analysis of PCA and OPLS-DA using the SIMCA-P 14.1.Relevant potential biomarkers screening was performed according to the VIP value（VIP>1.0）and significant test（P<0.05）from the OPLS-DA model.In addition,commercial databases including KEGG and NIST were utilized to search for the pathways of metabolites,and a free and web-based tool,MetaboAnalyst,which use the high-quality KEGG metabolic pathway as the backend knowledgebase,for pathway analysis.We used this approach to explore the possible anti-fibrosis mechanism of AG from the perspective of influential metabolic pathways.3 Results3.1 Extraction,separation and identification of AG:The content of AG was measured by HPLC,mobile phase:CH₃OH and H₂O（45:55,v/v）.The retention time of purified AG was 7.146 min.The solubility is minimum in chloroform,then water in the room temperature,water in 65°C,ethyl acetate,alcohol,and methyl alcohol.We had identified the chemical structure by spectroscopic methods and physical data.The result was basically consistent with the literatures.Pilot scale study found that the extraction rate of AG was 0.093%（3batches）.The purity of AG was 97.4%（3 batches）,RSD=3.23%.Results showed that this process was stable and reliable and meets the requirements.It’s applicable to industrial production of AG.3.2 Protective effect of AG on TGF-β₁-induced liver fibrosis in HSC-T6:CCK-8 assay showed AG could significantly,dose-dependently,suppress TGF-β₁-induced HSC proliferation compared with the model group（P<0.05）.AG could significantly inhibit the release of inflammatory factors after TGF-β₁ stimulation.Western Blot showed that the levels of phosphorylated JNK,ERK and p38 increased in the model group.After the treatment of AG,we found that the above phenomenons were ameliorated in different degrees.3.3 Protective effect of AG on CCl₄-induced liver fibrosis in mice:Biochemical assays and histopathological investigations showed that AG delayed the formation of liver fibrosis,decreased ALT,AST,MDA,Hyp levels,and increased Alb,GSH-Px,and SOD levels.Moreover,AG exhibited downregulation ofα-SMA levels in immunohistochemical and Western blot analyses.The levels of phosphorylated ERK,JNK,p38,Smad2 and Smad3 were also significantly reduced in all AG-treated groups in a dose-dependent manner.3.4 Serum metabonomics study of the hepatoprotective effect of AG on CCl₄-induced liver fibrosis in mice by GC-TOF-MS analysis:With pattern recognition analysis of PCA and OPLS-DA,a clear separation of control group,model group and AG group were achieved,and AG treatment group was located much closer to the control group than the model group.Meanwhile,data indicated that AG had improvement effect on liver fibrosis.Based on these analyses,nine metabolites relating to the anti-fibrotic effect of AG including isoleucine,threonine,beta-Alanine,adipic acid,3-hydroxy-3-methylglutaric acid,phenylalanine,indolelactate,5-Hydroxyindole-3-acetic acid and arachidonic acid were identified as potential biomarkers,which may be related with pathways of amino acids metabolism and fatty acid metabolism.4 ConclusionsOn the basis of optimization extraction and purification process of AG,we knew the hepatoprotective effects and the underlying mechanism of AG in vivo and vitro.Research found that the protective effect of AG on TGF-β₁-induced liver fibrosis in HSC.AG could significantly inhibit the release of inflammatory factors,decrease the expression ofα-SMA and inhibit MAPK pathway after TGF-β₁ stimulation.We also found the protective effect of AG on CCl₄-induced liver fibrosis in mice.AG delayed the formation of liver fibrosis,decreased ALT,AST,MDA,Hyp levels,and increased Alb,GSH-Px,and SOD levels.The possible mechanism may be by decreasing the expression ofα-SMA and suppressing the MAPK and TGF-β₁/Smad signalling pathway.Moreover,nine potential biomarkers were identified to elucidate the drug mechanism of AG,which may be related with pathways of amino acids metabolism and fatty acid metabolism.