Objective: Angiogenesis plays a critical role in tumor growth and metastasis. Endothelial cell proliferation has been used as an in vitro model of angiogenesis. Trifolium pratense was suggested for cancer treatment in traditional medicine and scientific report. The present study was designed to investigate the effect of T. pratense hydroalcoholic extract on the human umbilical vein endothelial cell (HUVEC) apoptosis and nitric oxide (NO) secretion in vitro.

Materials and Methods: Hydroalcoholic extract of T. pratense was prepared and its different concentrations (0, 12.5, 25, 50, 100, 200, 400, 800 μg/ml) were used for cell treatment. Cell viability was assessed by trypan blue, MTT and lactate dehydrogenase methods. Furthermore, nitric oxide (NO) secretion of the cells was measured by Griess reaction, and their apoptosis was evaluated by fluorescent dyes staining. One-way ANOVA was used for data analysis.

Results: After 24, 48 and 72 hours treatments, the IC50 values were 1069.35, 174.70 and 68.07 μg/ml, respectively. T. pratense extracts exerted a significant difference between the groups treated compared to the control group (p<0.05) and increasing the dose significantly decreased cell viability. T. pratense extract significantly increased LDH in dose- and time-dependent manners in the cell culture medium (p<0.05). NO decreased in a dose-dependent manner, and cell apoptosis was increased significantly (p<0.05).

Conclusions: T. pratense exerts a cytotoxic effect on HUVEC in dose- and time-dependent manner. It can be considered as an anti-angiogenesis agent and potentially beneficial for further development of new anti-cancer agents.