Anti-angiogenesis activity

HUVECs (KeyGEN BioTECH Co., Ltd, Jiangsu, China) were cultured in ECM (endothelial cell medium) with 5% (v/v) FBS, 1% (v/v) ECGS, and 1% penicillin-streptomycin (w/v) at 37°C in a cell incubator. The MTT assay was employed to assess the anti-angiogenic effect of the isolated compounds against HUVECs as described previously with mild modification (Liang et al., 2015; Nguyen et al., 2015). Briefly, HUVECs were seeded into a 96-well plate and incubated. Different concentrations of compounds 1–14 were added onto the cells after 4 h, and continued to be co-incubated for 48 h. Next, the cells were co-incubated with MTT (Sigma, St. Louis, MO, USA) to a final concentration of 0.05% for another 4 h. Then, replacing the MTT solution with 150 mL DMSO, and kept the plates being shaken for 3 min. The optical density of each well was measured on a microplate reader at λ = 570 nm.