Study On Chemical Constituents And Antitumor Activity Of Tibetan Medicine Oxytropis Falcata Bunge

In order to clarify the active constituents of anti-tumor effects of Oxytropis falcata Bunge,constituent isolation and purification wwer carried out by column chromatography from the active site—dichloromethane part.Five compounds(1-5)were elucidated as ?-Sitosterol(1),7-methoxy flavanone(2),2'-hydroxy-4'-methoxy chalcone(3),2',4'-dihydroxy chalcone(4)and N-2-phenylethylcinnamamide(5).Among these compounds,compound 5 was isolated from this genus for the first time.Compound 2,4 and 5 isolated from dichloromethane part were screened on human hepatocellular SMMC-7721 cells,human mammary adenocarcinoma MDA-MB-231,HepG2 human hepatoma cells,Huh7 human hepatocellular cells and mouse melanoma B16F10 cells to search for anti-tumor drugs.2',4'-dihydroxy chalcone was found to be much effective to HepG2 human hepatoma cells.We examined anti-tumor effect and possible mechanism of 2',4'-dihydroxy chalcone in HepG2 human hepatoma cells.Moreover,we studied inhibitory effect of 2',4'-dihydroxy chalcone on proliferation of HepG2 human hepatoma cells with different concentration.And we also studied apoptosis of HepG2 cells induced by 2',4'-dihydroxy chalcone and further investigated its molecular mechanisms.Hoechst 33258 staining was performed to evaluate the inhibitory and apoptotic effects of 2',4'-dihydroxy chalcone on HepG2 cells.Cell cycle,apoptotic rate,mitochondrial membrane potential and the concentration of ROS were analyzed by flow cytometry(FCM).The concentration of Ca2+ was analyzed by fluorescence microscope.The Western blotting assay was performed to evaluate the expressions of Bcl-2,Bax,Cyt-c,Caspase-9 and Caspase-3 on HepG2 cells.Results showed the growth inhibition of 2',4'-dihydroxy chalcone on HepG2 cells was dose-dependent and the IC50 value of 2',4'-dihydroxy chalcone at 24 h was 66.50 ?mol/L.2',4'-Dihydroxy chalcone was proved to cause apoptosis of HepG2 cells and many typical apoptotic morphological features were found,such as shrunk cells,the apoptotic bodies and the crescent or piece like cell nucleuses.Apoptotic rate of HepG2 human hepatoma cells was increased along with the increase of 2',4'-dihydroxy chalcone concentration.When treated with 2',4'-dihydroxy chalcone for 24 h,HepG2 cells were arrested at G2/M phase.Meanwhile,2',4'-dihydroxy chalcone decreased the expression of antiapoptotic protein Bcl-2 but increased the expressions of pro-apoptotic proteins,such as:Bax?Cyt-c?caspase-3 and Caspase-9 in a dose-dependent manner.2',4'-dihydroxy chalcone also increased the ratio of Bax/Bcl-2 in HepG2 cells,which suggested 2',4'-dihydroxy chalcone induced apoptosis through the mitochondrial dependant pathway in HepG2 cells.