Isolation And Purification Of Effective Compounds From Cowherb Seed To Inhibit Endothelial Cells And Evaluate Its Pharmacodynamic Function

Background and objective: tumor angiogenesis play a vital role in tumor growth and metastasis through providing the nutrition and channels. It has become more and more interesting that anti-angiogenesis therapy as a novel strategy for tumor biological control. At present, dozens of anti-angiogenesis agents that designed on the molecular mechanism of tumor angiogenesis have been evaluated in clinical trial. Human microvascular endothelial cells play an important role in angiogenesis. Its formation depends on the differentiation of bone marrow progenitor cells, the proceeding of differentiation is extremely complex and under the regulation of lots of factors. Recently, the mechanism of differentiation regulation is still unclear. This study is aimed at screening a traditional Chinese medicine depending on endothelial cells modal to anti-angiogenesis. To evaluate the anti-angiogenesis effect of cowherb seed extracts by CAM and matrigel plug in vivo. Isolation and purification of unity composition from cowherb seed extracts to inhibit the proliferation of endothelial cells. To study the inhibit function on human microvascular endothelial cells migration and adhesion by cowherb seed unity composition in vitro, and observe the changes of cell and cell nucleus morphous.Methods: Isolation and purification of total saponin from the cowherb seed extracts in macroporous resin, gradient elution with the Alcohol of diversity concentration. Collecting the elution respectively and to verificate the cytoactive on endothelial cells, then the utility composition have been isolated by AKTA resource column. Collecting the elution depend on ultraviolet absorption. Meanwhile, detecting the elution by ELSD and to choose the fraction of high activity,abundance and reconstituent simply to further segregation. Finally, separation and obtain the unity chemical compound by C18 column. Optimize the Isolation condition through changing the pH of elution phase. Determine the IC50 of group E by SRB; to control the time of medicine effect for investigating the relationship of time-effect dependent ; wound migration assay to investigate the cell migration; to evaluate the effect of extract on cell adhesion in vitro matrigel; HE staining to observe the change of cell morphous.Results: Cowherb seed extracts have the effect to suppress the proliferation of endothelial cells and the drug serum has the identical function. CAM and matrigel plug indicated that the cowherb seed extracts have the effect to anti-angiogenesis. Meanwhile, the growth and metastasis of solid tumors have been suppressed in vivo trial. Cowherb seed extracts have been isolated by macroporous resin, 50%~90% Alcohol concentration elution have the activity to inhibit the endothelial cells. Seven groups (0~6) can be obtained by the segregation of AKTA resource column, and then the 3~6 samples have the capability to inhibit the target cells. We choice the sample 4 to further separate and obtain five groups(A-E) by C18 column. The groupB are the main peak with obvious ultraviolet absorption, but have no inhibition on endothelial cells. Group D and group E have the activity in ECs, the Group D is a unity peak identified by ELSD. The IC50 of groupE is 3.60μg/mL with SRB, The relationship of time and effect indicate that it needs 4 hours for the extracts to inhibit cell growth since it had been add in the 96 wells. the cell migration of test group have been inhibited after 48 hours, the migrate rate of test is 40.33%, the control is77.68%(P<0.05).Cell adhesion assay indicate that the extract can suppress endothelial cell adhesion in concentration-dependent.Conclusions: the unity chemical compound had been discovered to inhibit proliferation and growth of endothelial cells. The compound has a potential value for tumor therapy. The cowherb seed unity composition can suppress the endothelial cells proliferation,migration and adhesion in vivo. Meanwhile, it seems to be a potential material for inhibiting the tumor angiogenesis.