| Liver fibrosis was the change of liver structure and function caused by liver injury.It was an important pathological process from chronic hepatitis to cirrhosis and liver cancer.Once liver fibrosis developed into liver cirrhosis and liver cancer,the cure rate was very low and seriously threatened the lives of patients.However,the treatment of liver fibrosis could effectively prevent its futher deterioration and increase the cure rate.Therefore,the research of efficient,safe,and small side effects drugs to treat liver fibrosis has become an urgent problem to be solved.Swertia chirayita was a commonly used"Dida" medicinal herbs used in the treatment of liver diseases included in the masterpiece "Jing zhu ben cao" of Tibetan medicine,and it contained a variety of chemical components such as xanthones.Literature studies have shown that xanthone compounds are a series of chemical components with a wide range of biological activities,which have anti-inflammatory,liver-protecting,antioxidant,hypoglycemic,anti-leukemia,anti-fungal,and anti-tumor effect.The previous research of our group showed that Swertia chirayita has anti-liver fibrosis effects,and xanthone compounds were one of its main chemical components.In order to confirm that xanthones are the main pharmacological substances against liver fibrosis,this study firstly used UPLC-ESI-Q-TOF MS technology to characterize xanthones in Swertia chirayita,and determined the composition of xanthone.Then a method for determining the content of xanthones in one measurement and multiple evaluations was established for purification process research.Then the xanthones were separated and purified using polyamide resin to obtain high-purity xanthones.Finally,the anti-hepatic fibrosis activity of xanthones was studied,and its mechanism of action was explored by metabolomics methods.The specific research contents included the following five parts:1.UPLC-ESI-Q-TOF MS analysis of xanthones from Swertia chirayitaIn order to study the material basis of xanthones in Swertia chirayita,the gradient elution was carried out on an Acquity HSS T3 column(100 mm×2.1 mm,1.8μm)with 0.1%formic acid in water and 0.1%formic acid in acetonitrile as mobile phase.The flow rate was 0.4 mL/min,and the column temperature was 40℃.Mass spectrometry data of alcohol extracts of Swertia were collected under positive and negative ion modes.It was used to analyze the data in combination with mass spectrometry data,reference material information and literatures related to Swertia.The results showed that a total of 25 xanthones were identified in Swertia chirayita,and 4 of them were found for first time in Swertia.They were swerpunilactone A,swerpunilactone B,and 7-O-β-D-xylopyranosyl-1,8-dihydroxy-3-methoxy-xanthone,8-0[β-D-xylopyranose(1→6)-β-D-glucopyranose]-1,7-dihydroxy-3-methoxyxanthone.The results of this study showed that the xanthone was a kind of main component contained in Swertia chirayita,and its structure was richer,which laid a solid foundation for further purification and preparation of effective parts.2.The content of 8 main xanthones in Swertia chirayita was determined by quantitative analysis of multi-components by single markerTo establish a one-test and multiple-evaluation method for the determination of eight xanthones in Swertia chirayita,the relative correction factors of seven xanthones(1-hydroxy-3,7,8-trimethoxyxanthone,1-hydroxy-3,5-dimethoxyxanthone,1,3,5,8-tetrahydroxyxanthone,1,8-dihydroxy-3,5-dimethoxyxanthone,Bellidifolin,SwerpunilactoneA,1,8dihydroxy-3,7-dimethoxyxanthone)were determined by HPLC with the mangiferin as the internal standard,which were to calculate the content of each component in Swertia chirayita.8 xanthones were determined by external standard method in 12 batches of Swertia chirayita.The results showed that the correction factors had good repeatability and the established method was accurate,which could be applied to the process purification,content determination and quality evaluation of Swertia chirayita.3.Preparation of xanthonesIn order to optimize the purification process of the xanthones in Swertia chirayita,in this study,L9(34)orthogonal experiment was first used to investigate the effects of solid-liquid ratio,ethanol concentration and extraction times on the extraction efficiency of xanthones,and the optimal extraction process parameters were determined.Then the single factor parallel test method was used to optimize the type of purified resin,to investigate the static adsorption time,adsorption kinetics,maximum sample load,and eluent ratio.According to the determined process parameters,a pilot scale-up study was carried out.The results showed that the optimal extraction process for Swertia chirayita was to use 80%ethanol,with a material-to-liquid ratio of 1:12 for three extractions for 0.5 h each time;polyamide was the most adsorbed and purified xanthones in Swertia chirayita.The adsorption was two-stage kinetic adsorption,the maximum sample amount was 2 g of Swertia chirayita extract per gram of polyamide adsorption,adsorption for 7 h to reach the saturated adsorption.The elution process is to elute with 5 times the amount of water first and discard the eluent,then elute with 30%ethanol and collect the eluent,then elute with 50%ethanol and discard the eluent,finally elute with 60%ethanol and collect the eluent.The 30%ethanol eluent and 60%ethanol eluent were combined,ethanol was recovered by decompression,and then dried to obtain xanthone parts.The results of this study laid the foundation for further drug efficacy evaluation and mechanism research.4.Study on the anti-liver fibrosis effect of xanthonesIn order to study the anti-fibrosis effect of xanthones,CCl4-induced rat liver fibrosis model was used to study the drug activity.Fifty male SD rats were randomly divided into 5 groups,namely the control group,the model group,the positive drug group,the low-dose xanthone group,and the high-dose group.Except for the control group,rats in the other groups were intraperitoneally injected with 40%CCl4-olive oil solution for liver fibrosis modeling.After 8 weeks,the general condition of the animals was observed,the organ coefficients were measured,and the liver tissues were subjected to HE,Masson and Sirius Red staining,and the contents of liver fibrosis related indexes in the serum of the rats were determined.The results showed that compared with the model group,the xanthone administration group could improve the mental state of rats,reduce the activities of ALP,ALT,AST,TNF-α,IL-6(p<0.05),and reduce the content of PC Ⅲ,Col-IV,HA,and LN(p<0.05).The pathological section found that the liver cells were arranged more neatly,collagen accumulation was reduced,and liver fibrosis was improved to varying degrees.The results of the study showed that the xanthone has anti-liver fibrosis activity and it was the main medicinal components in Swertia chirayita.5.Study on the serum metabolomics mechanism of xanthone against liver fibrosisIn order to explore the anti-liver fibrosis mechanism of xanthones,this study used UPLC-Q-TOF/MS technology on the basis of pharmacodynamics to analyze the changes of endogenous small molecules in serum of rats with liver fibrosis,which look for possible metabolic markers,studyed the regulatory effects of xanthones on abnormal liver fibrosis metabolism,and explored the mechanism of rat serum metabolism.Markerlynx 4.1 software was used for data processing,and PCA and OPLS-DA analysis were performed to screen out the pharmacodynamic biomarkers of VIP>1.Finally,KEGG analysis,hierarchical clustering analysis and metabolic pathway analysis were performed on the differential metabolites.Results showed that a total of 15 metabolites related to liver metabolism were identified,including model group compared with control group,Isovalerylcarnitine,LysoPC(14:0),Trihydroxycoprostanoic acid,Oleic acid,L-Palmitoylcarnitine,Linoleic acid decreased,and Glycocholic acid,Taurocholic acid,Taurodeoxycholic acid,Chenodeoxycholic acid glycine conjugate,Glycoursodeoxycholic acid,L-Carnitine,Eicosadienoic acid,Pregnenolone,Campesterol increased(p<0.05).After the intervention of xanthone,15 metabolites tended to approach the level of the control group,and linoleic acid metabolism and sphingolipid metabolism were the main metabolites.The results of this study clarified the metabonomic mechanism of the xanthones from Swertia chirayita in the treatment of liver fibrosis,and provided a basis for clinical application.To sum up,this subject analyzed the composition of the xanthones in Swertia chirayita,established a one-test and multi-evaluation method,prepared the parts of the xanthones with higher purity,which confirmed the anti-hepatic fibrosis activity,clarified the mechanism of metabonomics,and provided a basis for the in-depth development of Swertia chirayita,and provided a new way for the treatment of liver fibrosis. |
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