Toxicology International (Formerly Indian Journal of Toxicology)

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Determination of Lethal Concentrations and Anti-Angiogenic Potential of Some Herbs in Zebrafish Model


Affiliations
1 Department of Veterinary Pharmacology and Toxicology, Bombay Veterinary College, Parel, Mumbai – 400012, Maharashtra, India
     

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Plants 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 μg/ml, 408.06 μg/ml and 500 μg/ml respectively. W. somnifera extract showed reduced RBCs in DA, but not in the ISV and DLAV region at a concentration of 100 μg/ml, whereas embryos revealed slight inhibition of ISV at concentration 200 μg/ml. At concentration of 250 μg/ml there was yolk sac oedema. For O. sanctum there was inhibition of ISV, DA and DLAV at the dose rate of 300 μg/ml and at dose 200 μg/ml there was slight inhibition of ISVs showing anti-angiogenic effect. A. sativus showed vessel inhibition particularly in ISV region at dose 400 μg/ml. At concentration 500 μg/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.

Keywords

Antiangiogenesis, Zebrafish, VEGF.
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  • Determination of Lethal Concentrations and Anti-Angiogenic Potential of Some Herbs in Zebrafish Model

Abstract Views: 313  |  PDF Views: 0

Authors

K. B. Tambe
Department of Veterinary Pharmacology and Toxicology, Bombay Veterinary College, Parel, Mumbai – 400012, Maharashtra, India
V. V. Karande
Department of Veterinary Pharmacology and Toxicology, Bombay Veterinary College, Parel, Mumbai – 400012, Maharashtra, India
M. M. Gatne
Department of Veterinary Pharmacology and Toxicology, Bombay Veterinary College, Parel, Mumbai – 400012, Maharashtra, India

Abstract


Plants 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 μg/ml, 408.06 μg/ml and 500 μg/ml respectively. W. somnifera extract showed reduced RBCs in DA, but not in the ISV and DLAV region at a concentration of 100 μg/ml, whereas embryos revealed slight inhibition of ISV at concentration 200 μg/ml. At concentration of 250 μg/ml there was yolk sac oedema. For O. sanctum there was inhibition of ISV, DA and DLAV at the dose rate of 300 μg/ml and at dose 200 μg/ml there was slight inhibition of ISVs showing anti-angiogenic effect. A. sativus showed vessel inhibition particularly in ISV region at dose 400 μg/ml. At concentration 500 μg/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.

Keywords


Antiangiogenesis, Zebrafish, VEGF.

References





DOI: https://doi.org/10.18311/ti%2F2018%2Fv25i2%2F22497