| Gentiana macrophylla Pall.,whose genus and famlily belong to Gentianaceae and Gentiana,in which the root of this medicinal plants is a Traditional Chinese Medicine(TCM)and is being used to treat rheumatism arthralgia and damp-heat jaundice,etc.In this paper,the chemical constituents in the root of Gentiana macrophylla Pall.from the region of Qinling Mountains in Shaanxi Province were isolated and identified;the quality of this medicinal plant from this region was evaluated;the effect of the root of Gentiana macrophylla Pall.ethanol extract on non-alcoholic fatty liver disease(NAFLD)was explored,and its pharmacodynamic substances were investigated by the spectrum-effect relationship analysis method;based on the spectrum-effect relationship analysis,the effect of the key active ingredient was further assessed.(1)Chemical constituents of Gentiana macrophylla Pall.In this section,33 compounds were obtained from the root ethanol extract of Gentiana macrophylla Pall.by the methods of atmospheric pressure column chromatography,preparative liquid chromatography and crystallization,in which 33 compounds were elucidated by spectroscopy methods with MS and NMR as well as reference substances,as follows:(1)roburic acid,(2)1,2-benzenedicarboxylic acid-bis-(2-methyl-propyl)ester,(3)palmitic acid,(4)dibutyl phthalate,(5)N-pentacosyl-2-carboxy-benzoyl amide,(6)erythrocentaurin,(7)?-sitosterol,(8)uvaol,(9)ursolic acid,(10)oleanolic acid,(11)maslinic acid,(12)stigmasterol,(13)3,4-Dihydroxybenzaldehyde,(14)daucosterol,(15)sucrose,(16)isoorientin,(17)isointigenin,(18)gentiopicroside,(19)sweroside,(20)swertiamarin,(21)loganic acid,(22)6'-O-?-D-glucosylgentiopicroside,(23)gentimacroside,(24)6'-O-?-D-glucosylsweroside,(25)5-formylisochromen-1-one,(26)2'-(2,3-dihydroxybenzoyl)-gentiopicroside,(27)N-heneicosanoic acid,(28)ethyl palmitate(29)6'-O-?-D-glucopyranosylloganic acid,(30)stearic acid,(31)6?-O-acetylgentiopicroside,(32)1?,2?,3?,24-tetrahydroxyolean-12-en-28-oic acid,(33)gentianose,in which 3,4,8,11,13,25,26,27,28,29,30 and 32 were isolated and identified from the root of Gentiana macrophylla Pall.for the first time,and 25,27 and 28 were isolated and identified from Gentiana for the first time.(2)Study on the fingerprint and content analysis of main chemical constituents in root of Gentiana macrophylla Pall.Qinling region in Shaanxi Province is one of the main cultivation areas of Gentiana macrophylla Pall.In order to evaluate the quality of the medicinal plant produced in this areas,an HPLC-ELSD method was established for the simultaneous determination of gentiopicroside,swertiamarin,6'-O-?-D-glucosylgentiopicroside and loganic acid in the root of Gentiana macrophylla Pall.from the region of Qinling Mountains in Shaanxi Province.The results showed that these four active ingredients in the root of Gentiana macrophylla Pall.were abundant,which indicated that this medicinal materials produced in Qinling area has good clinical application value and potential value.On the basis of the above analysis,10 batches of chromatography of the medicinal plant were determined and obtained by using the method of HPLC-ELSD after optimization of the chromatographic conditions.Then,the fingerprint of Gentiana macrophylla Pall.was established after similarity evaluation by the similarity evaluation system for chromatographic fingerprint of TCM,and eight common characteristic chromatographic peaks in fingerprints were confirmed based on the study of the chemical constituents of Gentiana macrophylla Pall.,in which there were gentiopicroside,swertiamarin,loganic acid,6'-O-?-D-glucosylgentiopicroside,roburic acid,stigmasterol,?-sitosterol and oleanolic acid,respectively.Thus,a more reliable analysis method was provided for the quality control of the medicinal plant,and the work was provided a basis for the investigation the of the pharmacodynamic substances in the following study.(3)Effects of the root crude extracts of Gentiana macrophylla Pall.on NAFLDIn this section,the effect of the root crude extract of Gentiana macrophylla Pall.on the NAFLD was evaluated in the fructose-fed mice.At the end of the experiment,administration of the root crude extract exerted antiobesity and protective effect against metabolism disorder in fructose-fed mice.Liver histological examinations indicated that administration of the root crude extract obviously reduced hepatic lipid accumulation.The decreases of serum insulin and glucose levels by the root crude extract treatment suggested the improvement of body insulin resistance.In addition.administration of the root crude extract mitigated liver insulinresistance(P-IRS1)and inhibited the development of inflammation(phospho-JNK1/2/3,phospho-NF-?B p65,phospho-IKK alpha/beta,XBP1,TNF-? and IL-6).Moreover,the root crude extract treatment ameliorated hepatic oxidative stress(MDA,GSH,NO,SOD,GSH-px and CAT).These results demonstrated that the root crude extract showed protective effects against NAFLD-related metabolic diseases in fructose-fed mice.(4)Study on the spectrum-effect relationship between the fingerprint of Gentiana macrophylla Pall.and its effect on NAFLDIn order to further investigate the pharmacodynamic substances of the crude extract for NAFLD,the spectrum-effect relationship,which was a correlation analysis between the effect of the crude extract on the reduction of serum and liver triglyceride content and eight common chromatographic peaks in the fingerprint,was analyzed by the method of gray correlation analysis.According to the gray sequence,gentiopicroside might be the key effective component in the crude extract.(5)Effects of gentiopicroside on NAFLDBased on the result of the spectrum-effect relationship analysis,the effect of gentiopicroside on the NAFLD was further evaluated in fructose-fed mice.At the end of the experiment,fructose-fed mice administrated with gentiopicroside showed antiobesity and protective effect against serum metabolic parameters disorder.Histological examinations suggested the alleviation of hepatic ballooning degeneration and steatosis by gentiopicroside treatment.Moreover,gentiopicroside administration mitigated hepatic oxidative stress along with decreases of hepatic pro-inflammation cytokines,which was associated with decrease of hepatic XO activity and enhancements of anti-oxidant defense system enzymes(SOD,GSH-px,CAT and Nrf2).In addition,gentiopicroside reduced liver fat production by down-regulating SREBP-1,FAS and ACC1 expressions.The present study demonstrated that gentiopicroside alleviated NAFLD and metabolic alterations in fructose-fed mice.Thus,gentiopicroside was confirmed as the effective ingredient of the crude extract of Gentiana macrophylla Pall.on the prevention and treatment of NAFLD and its complications.In conclusion,in this study,some of chemical constituents in the ethanol extract of Gentiana macrophylla Pall.have been revealed.It was found that Gentiana macrophylla Pall.from the region of Qinling Mountains in Shaanxi Province has good clinical application value and potential value.Moreover,the fingerprint of Gentiana macrophylla Pall.was established by using the method of HPLC-ELSD.In addition,the crude extract of Gentiana macrophylla Pall.has been found that it showed an improvement effect on NAFLD in mice,in which the spectrum-effect relationship analysis indicated that gentiopicroside might be the key pharmacodynamic substance in the crude extract of Gentiana macrophylla Pall.Based on the spectrum-effect relationship analysis,effect of gentiopicroside on NAFLD was further confirmed in fructose-fed mice,where the results showed that gentiopicroside alleviated NAFLD and metabolic alterations in fructose-fed mice. |
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