Determination of Lethal Concentrations and Anti-Angiogenic Potential of Some Herbs in Zebrafish Model
Keywords:Antiangiogenesis, Zebrafish, VEGF
AbstractPlants have always been used in various traditional medicinal systems, VEGF inhibitors from plant sources are matter of great interest in recent times. The present study aimed to validate the model for anti-angiogenesis in zebrafish using some herbal compounds. Zebrafish larvae, 24 hpf were exposed to different concentrations of test compound, positive and negative controls were selected on basis of data from laboratory experiments. The positive controls included SU5416 and 0.1%DMSO taken as negative control. The test compounds were Withania somnifera, Ocimum sanctum and Ananas sativus. DMSO was used as a vehicle control (except in Ananas sativus it was water). The concentrations to be tested were determined on the basis of a prior assay carried out to determine the median lethal concentration (LC50) of the drug by exposing 6 hpf embryos to different concentrations of the drugs over a significant range. The anti-angiogenic assay was run on zebrafish larvae, 72 hpf, after fixation with 4% paraformaldehyde and staining with o-Dianisidine. Both vessel inhibition and morphological structure were observed under 10X power of inverted microscope. During the LC50 and anti-angiogenic assays, gross morphological abnormalities, if any, were observed for and randomly selected larvae were processed for staining. Withania somnifera, Ocimum sanctum and Ananas sativus.s showed inhibition of ISV, DA and DLAV when compared with positive and negative control. The LC50 for W. somnifera, O. sanctum and A. sativus was 300 Î¼g/ml, 408.06 Î¼g/ml and 500 Î¼g/ml respectively. W. somnifera extract showed reduced RBCs in DA, but not in the ISV and DLAV region at a concentration of 100 Î¼g/ml, whereas embryos revealed slight inhibition of ISV at concentration 200 Î¼g/ml. At concentration of 250 Î¼g/ml there was yolk sac oedema. For O. sanctum there was inhibition of ISV, DA and DLAV at the dose rate of 300 Î¼g/ml and at dose 200 Î¼g/ml there was slight inhibition of ISVs showing anti-angiogenic effect. A. sativus showed vessel inhibition particularly in ISV region at dose 400 Î¼g/ml. At concentration 500 Î¼g/ml showed developmental disorder was evident. Zebrafish model was judged to be practical in a preclinical set up. However further studies are required to establish correlation between zebrafish and mammalian models of angiogenesis.
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