Experimental Study Of Angelica Pubescens And Osthole Isolated From Angelica Pubescens Inhibiting Angiogenesis

Objectives:This experiment aims to study the effect and the mechnism of ethanol extracts from Angelica pubescens and osthole on anti-angiogenesis in vitro and in vivo.Methods:.MTT assay was used to observe the effect of ethanol extracts from Angelica pubescens and osthole on the proliferation of human stomach cancer cell MKN-45,BGC-823,human lung cancer cell A549,human breast cancer MCF-7 cell and human colon carcinoma cancer cell LOVO.And the most sensitive tumor cell to the medicine had been screened.Tumor cell which was most sensitive to the drug was used as control, the inhibition effects of ethanol extracts from Angelica pubescens and osthole on proliferation of human umbilical vein endothelial cells (HUVECs) were measured by MTT assay. Transwell migration assay and tube formation assay were used to observe the impact of ethanol extracts from Angelica pubescens and osthole on cell migration and tube forming ability of HUVECs. The rate of ethanol extracts from Angelica pubescens and osthole inducing HUVEC apoptosis and the effect of ethanol extracts from Angelica pubescens and osthole on cell cycle of HUVEC was calculated by f low cytometry. The Chick chorio allantoic membrane (CAM) model was used to observe the antiangiogenic effect of ethanol extracts from Angelica pubescens and osthole in vivo.Results:Osthole had inhibiting effect on the multiplication of MKN-45,BGC-823, A549, MCF-7 cell and LOVO cells and the inhibiting effect was dependent on concentration.IC50 of MKN-45,BGC-823, A549, MCF-7 cell and LOVO cells were 43.299μg/ml,56.790μg/ml,37.444μg/ml,38.397μg/ml and 36.2311μg/ml,respectively; Under the same conditions,ethanol extracts from Angelica pubescens had weaker inhibiting effect than othole,IC50 of five kinds of cell lines were 50.336mg/ml,198.265mg/ml,41.456mg/ml,32.544mg/ml and 31.28mg/ml, respectively.So the most sensitive cell to ethanol extracts from Angelica pubescens and osthole were both LOVO cell. Ethanol extracts from Angelica pubescens and osthole(3.75-30μg/mL)which both inhibited cell proliferations of LOVO was much lower than that of HUVEC, and the difference was significant (P<0.05). Moreover,Ethanol extracts from Angelica pubescensand osthole (3.75-30μg/mL) inhibited the proliferation of HUVECs (5.16%～10.15% and22.64%～65.56%,respectively) after 48 h treatment.The inhibiting effects were both dependent on concentration.When HUVECs were incubated with osthole(3.75-30μg/mL), the number of tubules was reduced and the lumen lost its integrity after 24 h;The inhibition rate of migration was-2.16%～8.00% when treated with ethanol extracts from Angelica pubescens(3.75-30μg/mL),The inhibition rate of migration was 13.70%～63.04% when treated with Osthole(3.75-30μg/mL). The rate of HUVEC apoptosis after treat with ethanol extracts from Angelica pubescens(3.75-30μg/mL) for 48h was 6.1%～14.4%.The rate of HUVEC apoptosis after treat with osthole(3.75-30μg /mL) for 48h was 18.8%～89.5%. Ethanol extracts from Angelica pubescens(3.75-30μg /mL) and osthole (3.75-30μg/mL) may block the HUVEC cell cycle and arrest at G0-G1 phase after 24h,and the effect of the latter better than the former.It was observed that osthole suppressed the neovascularization of CAM in vivo with the inhibition rate of 10.24%～59.45% when treated with osthole(3.75-30μg/mL),the inhibition rate of the neovascularization of CAM in vivo when treated with ethanol extracts from Angelica pubescens(3.75-30μg/mL) was lower than the inhibition of osthole.Conclusion:The effect of ethanol extracts from Angelica pubescens and osthole on anti-angiogenesis in vitro and in vivo is better than the effect of ethanol extracts from Angelica pubescens,it may conclude that the main antiangiogenic constitution of Angelica pubescens is osthole.Osthole effectively suppresses angiogenesis in vitro and vivo,which may be through inhibiting cell proliferation, migration and tube-formation, inducing apoptos is and suppres s ng cell cycle of HUVECs.