Separation Of The Antiangiogenic Effective Component From Albizia And Study Of Its Mechanism

The growth and migration of tumors rely on tumor angiogenesis providing the nutritionand path, thus inhibiting tumor angiogenesis became a kind of important method to thetreatment of tumor. As the vascular endothelium cells play an important role in angiogenesis,it could restrain tumor angiogenesis by inhibiting vascular endothelial cells’ proliferation. Inthis paper, the HMEC-1(human microvascular endothelial cells) has been used as a drugactivity screening model, the active substances has been separated from the cortex albiziaewhich can inhibit endothelial cell growth, and the mechanism of the active substances in thecell has been discussed for further study.Taking Albizia ethanol backflow-butanol extraction phase(component1) as raw material,and the products(component2,3) was obtained through eluenting by the mixture of20%HP-20macroporous resin and70%ethanol gradient, then the Molish reaction and Fehlingresponse were took to identify the physicochemical properties of the component1,2and3.Reaction results showed that component1,2,3were all glycosides and hemolytic reactionshowed that component1and3were the saponins. Then the total glycosides was hydrolysisby acid to obtain corresponding aglycones substance(component4,5and6) in the followingprocess, the all6componets were screened by the HMEC-1activity and LDH vitality, theevaluation of toxic effect was obtained. IC50value of component1is3.49±0.03μg/mL,12.5μg/mL in no cytotoxicity, IC50value of component3is2.17±0.13μg/mL,5μg/mL in nocytotoxicity.EGF has a promotion effect on the cell’s growth in vitro,and component1and3caninhibit the cell’s growth, immigration and angiopoiesis when induced by EGF, and adose-dependent manner was significant. What’s more, by testing the protein expression ofp-Akt and p-Erk1/2in PI3K/AKT and Ras/Raf/MEK/ERK showed that component1,3inhibited p-Akt expression, with a dose-dependent manner. And component1had a stronginhibitory effect on the p-Erk1/2expression at5μg/mL while component3at2.5μg/mL.Moreover,there is an effect of the active substance by orally.At last, trypan blue staining method and Hoechst33342were used to test the necrosisand apoptosis, the result showed that cell necrosis did not happen with5μg/mL in component1while with2.5μg/mL in component3; Flow cytometry detection showed the component1,3had no obvious effect on cell cycle but an obvious effect on cell apoptosis. The Caspase-3incell apoptosis was studied by Western blot and the result showed that with the increasing ofcomponent concentration, the Caspase-3protein was activated which greatly strengthen theresult. In addition, component3was much more effective than component1under the sameconcentration.