Tracking antiangiogenic components from Glycyrrhiza uralensis Fisch. based on zebrafish assays using high-speed countercurrent chromatography.

Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.

Toxic effects of celastrol on embryonic development of zebrafish (Danio rerio).

Celastrol is a terpenoid purified from Tripterygium wilfordii Hook F. As a natural product with pharmacological activities, this compound is a promising candidate for drug development. To provide more information about its toxicity for clinical trials, toxicity assessment of celastrol was conducted with zebrafish model in vivo. 1hour post-fertilization (hpf) embryos were treated with various concentrations of celastrol for 120h. Developmental phenotypes were observed and survival rates were recorded. The results showed that the hatching rates of embryos treated with 1.0µM or higher celastrol were significantly lower. Embryos exposed to 1.0µM celastrol had no blood flow in trunk vessels at 48hpf with a median effect concentration (EC(50)) of 0.94µM. At 72hpf serious edema in pericardial sac was observed in the surviving larvae (hatched from embryos treated with 1.5µM celastrol). Bent tails or hook-like tails were seen as 0.5µM celastrol and the EC(50) for tail malformation was 0.66 µM at 72hpf. The lethal effect of celastrol on zebrafish embryos was dose-dependent and the LC(50) values of celastrol on 1hpf embryos were approximately 1.40µM. These results indicate that celastrol affects the normal development of zebrafish embryo in µM concentrations.