Luteolin inhibited ex vivo angiogenesis by CAM and matrigel plug assay and in vitro angiogenesis by rat aortic ring assay.

(a) Luteolin inhibits ex vivo angiogenesis in CAM assay. Fertile leghorn chicken eggs were candled on embryonic day 8; a small opening was made at the top of the live eggs. Luteolin for treatment was mixed with 0.5% methyl cellulose in water and gently placed on the CAM. The eggs were incubated for 48 h and photographed. Blood vessels density was quantified by Image J software and represented as a bar diagram. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from 20 and 40 µmole/L luteolin. (b) Luteolin inhibits ex vivo angiogenesis in matrigel plug assay. Matrigel plug containg VEGF and luteolin were implanted into the CAM at day 9 of fertilized chicken eggs. After 96 h of incubation, the matrigel plugs were taken out and dispersed in PBS and incubated at 4°C overnight. Hemoglobin levels were determined using Drabkin’s reagent according to manufacturer instructions. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from VEGF control. (c) Luteolin inhibits microvessel outgrowth from the rat aortic ring. Dorsal aorta from a freshly sacrificed Sprague–Dawley rat was cut into ∼1 mm long pieces using surgical blade. Each ring was placed in a collagen pre-coated 96-well plate. VEGF, with or without different dilutions of luteolin, was added to the wells. On day 6, the rings were analyzed by phase-contrast microscopy and microvessel outgrowths were quantified and photographed. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls.