Isolation Of Components With Anti-angiogenesis Activity In Albizia Julibrissin And Research Of Their Action Mechanism Targeting VEGFR Signaling Pathway

The dried stem bark from Albizia julibrissin, a highly valued traditional Chinese medicalherb, has been shown to suppress the tumor growth and angiogenesis. In our previous studies,we have demonstrated that total saponins are the most bioactive components of Albiziajulibrissin extracts. In this study, a component H5was obtained from n-butanol fraction ofethanol extract of Albizia julibrissin by D101macroporous resin elution, silica gel columnchromatography and RP-18column chromatography, successively. The composition ofcomponent H5was simpler and with higher anti-angiogenesis activity on EA.hy926cells. Amain compound whose molecular weight was2171.6was found by UPLC-TOF-MStechnique, and its purity was79.15%by the HPLC analysis. Compared with the knownsaponins isolated before from Albizia julibrissin, the compound with the same molecularweight represented julibroside J5, julibroside J8, isomer of J5and isomer of J8, and the formulaof this compound was C102H162O49. Thus, we investigated the inhibition effects of componentH5on VEGF-induced angiogenesis in vitro and in vivo.During the process of angiogenesis, VEGF is the most important inducible factor, whichcan promote the proliferation of EA.hy926cells. The VEGF-induced proliferation ofEA.hy926cells was remarkably suppressed by component H5, and the value of IC50was3.12±0.77μg/mL. Meanwhile, with1.5μg/mL and3μg/mL of component H5,VEGF-mediated migration and tube formation of EA.hy926cells were inhibited in adose-dependent manner. To gain further insight into the mechanism by which component H5inhibited VEGF-induced endothelial cells proliferation, the flow cytometry was used toinvestigate the effect of component H5on cell cycle and apoptosis of EA.hy926cells. Wefound that the proportion of endothelial cells was decreased in S phase and increased inG0/G1phase with component H5-treated for48h, and with the concentration increased, theapoptosis rate of EA.hy926cells increased. Those results demonstrated that component H5could inhibit S phase entry and induce cell arrest in G0/G1phase and induced apoptosis inEA.hy926cells in a concentration-dependent manner. Hoechst33342staining showed thattypical apoptotic features such as decreased nucleus size, condensed chromatin appeared inEA.hy926cells with3μg/mL of component H5treatment. Western blot assay demonstratedthat the phosphorylation level of VEGFR2, Fak, Akt and Erk, which related to theangiogenesis modulated by VEGF/VEGFR2signaling pathway, was significantly suppressedby component H5.To further explore the inhibition effect of component H5on VEGF-induced angiogenesis,the mouse matrigel plug assay was used. After14days, compared with that of VEGF controlgroup, VEGF-induced neovascularization in matrigel plug was remarkably suppressed bycomponent H5through inhibiting endothelial cells infiltration into the plug. Meanwhile, theexpression of p-Akt and p-Erk in VEGF/VEGFR2signaling pathway was down-regulated.The anti-tumor effect of component H5was investigated in a whole animal model in vivo,and the mice bearing H22hepatoma cells transplantation tumor model was established.Compared with the control group, the tumor growth was significantly inhibited in component H5-treated group. With the concentration increased, extensive densely vesicular nuclei andsparse cytoplasm were been found, and large areas of necrosis appeared in the tumor tissue.The values of MVD and the expression level of p-VEGFR2, p-Erk and p-Akt in the treatmentgroups with component H5were signifcantly reduced. In conclusion, these results suggestthat component H5inhibits tumor neovascularization and suppresses tumor growth throughinhibition of the VEGF/VEGFR2signaling pathway.