Studies On The Chemical Constituents And Their Hepatoprotective Activity Of Dalbergia Odorifera And Hypericum Japonicum

The discovery of small molecules from medicinal plants with therapeutic effects for diseases has been an efficient approach to drug discovery.Several hepatoprotective drugs commonly used in clinical practice have been discovered from plants,such as oxymatrine and silybin,etc.However,the emergence of drug resistance and adverse reaction of long-term use in clinical practice make it particularly important and necessary to discover new small molecules with hepatoprotective activity.In this thesis,two kinds of medicinal plants,Dalbergia odorifera and Hypericum japonicum,which belong to the liver meridian in TCM,have been studied on their chemical compositions and hepatoprotective activity,as well as the mechanisms of action.Twenty-seven pure compounds,including flavonoids,sesquiterpenes,arylbenzofurans,phenolics and other types of compounds,were purified by solvent extraction,normal-phase and reversed-phase chromatographic separation,and identified by nuclear magnetic resonance(NMR),mass spectrometry(MS),infrared spectroscopy(IR),ultraviolet spectroscopy(UV),optical rotation(OR),circular dichroism(CD),and computational chemistry methods.The hepatoprotective activities of the isolated compounds were evaluated by MTT method using D-Gal N induced AML12 cell injury model,and several active components with hepatoprotective effects were identified.The preliminary mechanism of hepatoprotective effects was further investigated for compound 20,which has better activity than silybin.In the first part,twelve compounds,including three flavonoids(1-3),five sesquiterpenes(4-8),and four other compounds(9-12),were isolated from D.odorifera.The structure of compound 9,a new arylbenzofuran,was identified by spectroscopic and computational chemistry methods.The hepatoprotective activity was evaluated by MTT method using D-Gal N induced AML12 cell injury model,and compounds 4,6,7,and 9 were found to have strong protective effects at 30 ?M(P < 0.05),among which the hepatoprotective activity of 4 and 9was comparable to that of silybin.In the second part,fifteen known compounds,including 11 flavonoids(13-23)and other compounds(24-27),were isolated from H.japonicum,a TCM plant once was marketed as an injection for the treatment of hepatitis.Among the isolated constituents,compounds 16,26,and27 were discovered from this plant for the first time.Pharmacodynamic screening test by MTT method revealed that compound 20(taxifolin 7-rhamnoside)had a strong protective effect(P <0.01)against D-Gal N induced AML12 cells damage and was stronger than silybin,which lead us for further study of the action mechanism of compound 20.The levels of cellular liver function enzymes(ALT,AST,LDH)and changes in the content of oxidative stress related factors(SOD,GSH,MDA)were measured using detection kits.The anti-apoptotic effect of compound 20 was detected by flow cytometry,and the expression levels of Bax and Bcl-2proteins were detected by Western Blotting method.The results showed that compound 20 significantly reduced the cell damage by D-Gal N(P < 0.01)and increased the SOD activity in cells,with statistically significant levels at 40 ?M and 80 ?M doses(P < 0.05).It also attenuated the depletion of GSH and decreased the MDA content in cells.It exhibited anti-apoptotic effects by down-regulating Bax and up-regulating Bcl-2 protein levels.The results showed that compound 20 can significantly reduce the toxic effects of D-Gal N on AML12 cells,and its mechanisms of action were related to repairing liver function,reducing oxidative damage,and up-regulating Bcl-2 and down-regulating Bax protein levels.In summary,compounds 4,6,7,9,and 20 from D.odorifera and H.japonicum showed hepatoprotective activity,and the hepatoprotective activity of 20 was better than that of silybin.Both compound 20 and silybin have the basic structure of dihydroflavonol.Further mechanism of action studies revealed that the therapeutic effects of 20 related to repairing liver function,attenuating oxidative damage and apoptosis by up-regulating Bcl-2 and down-regulating Bax protein levels.The polarity of hydroxyl and glycosyl flavonoids from H.japonicum are mostly larger than those from D.odorifera,which may play a certain role in their liver protection effects.The sesquiterpenes(4,6,7)and aryl benzofurans(9)isolated from D.odorifera showed promising hepatoprotective activity.The present study provides a certain basis for clarifying the hepatoprotective ingredients of H.japonicum and discovering of small molecules with hepatoprotective activity.