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

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Antiangiogenesis, Zebrafish, VEGF


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.


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How to Cite

Tambe, K. B., Karande, V. V., & Gatne, M. M. (2019). Determination of Lethal Concentrations and Anti-Angiogenic Potential of Some Herbs in Zebrafish Model. Toxicology International, 25(2), 100–108.



Original Research
Received 2018-10-14
Accepted 2018-10-17
Published 2019-07-19



Auerbach R, Lewis R, Shinners B, Kubai NA. Angiogenesis assays: A critical overview. Clinical Chemistry.2003; 49(1):32–40. PMid: 12507958.

Folkman J. Angiogenesis in cancer vascular, rheumatoid and other disease. Nature Medicine. 1995; 1:27–31. PMid: 7584949.

Kobayashi H, Lin PC. Angiogenesis links chronic inflammation with cancer. Methods in Molecular Biology. 2009; 511:185–91. PMid: 19347298.

Kurebayashi J. Expression of Vascular Endothelial Growth Factor (VEGF) family members in breast cáncer. Jpn J Cancer Research. 1999; 90:977–81. PMid: 10551327 PMCid: PMC5926164.

Shaheen RM. Anti-angiogenic therapy targeting the tyrosine kinase receptor for Vascular Endothelial Growth Factor Receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis.Cancer Research.1999; 59:5412–6. PMid: 10554007.

Yoshiji H. KDR/Flk-1 is a major regulator of Vascular Endothelial Growth Factor induced tumor development and angiogenesis in murine hepatocellular carcinoma cells.Hepatology. 1999; 30:1179–86. PMid: 10534339.

Droller MJ. Vascular Endothelial Growth Factor is a predictor of relapse and stage progression in superficial bladder cancer. J Urology. 1998: 160(5):1932.8. Kitamura M. Concentrations of Vascular Endothelial Growth Factor in the sera of gastric cancer patients.Oncology Receptor. 1998; 5:1419–24.

Balbay M. Highly metastatic human prostate cancer growing within the prostate of athymic mice over expresses Vascular Endothelial Growth Factor. Clinical Cancer Research. 1999; 5:783–9. PMid: 10213213.

Dong X, Han Z, Yang A. Angiogenesis and anti-angiogenic therapy in hematologic malignancies. Critical Reviews in Oncology/Hematology. 2007; 62:105–18. PMid: 17188504.

Quinn TP, Peters KG, De Vries, Ferrara N, Williams LT. Fetal liver kinase 1 is a receptor for Vascular Endothelial Growth Factor and is selectively expressed in vascular endothelium.

Proc Natl Acad Sci USA. 1993; 90:7533–7. PMid: 8356051.

Serbedzija GN, Flynn E. Willett CE. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis. 1999; 3:353– 9.

Zon LI, Peterson RT. In vivo drug discovery in the zebrafish.Nature Reviews Drug Discovery. 2005; 4:35–44. PMid: 15688071.

Norrby K. In vivo models of angiogenesis. Journal of Cellular and Molecular Medicine. 2006; 10:588– 612. PMid: 16989723 PMCid: PMC3933145.

Vogel A. Weinstein B. Studying vascular development in the zebrafish. Trends in Cardiovascular Medicine.2000; 10(8):352–60.

Isogai S, Horiguchi M, Weinstein BM.T he vascular anatomy of the developing zebrafish: An atlas of embryonic and early larval development. Dev Biol. 2001; 230:278–301.

Westerfield M. The Zebrafish Book. Guide for the Laboratory use of zebrafish (Danio rerio) (University of Oregon Press, Eugene). 4th Ed. 2000. p. 122–8.

Reed LJ, Muench H. Simple method of estimating fifty percent endpoints. The American Journal of Hygiene. 1938; 27:493–7.

Thamilarashi AN, Mangalagowri A, Gurumoorthi P. Antiangiogenic activity of myricetin through VEGF - A downregulation in zebrafish (Danio rerio) in vivo. International Journal of Scientific and Engineering Research. 2013; 4:9.

Mahapatra AK, Nisha Kumari O, Abhimanyu K. Prospective role of Indian medicinal plants in inhibiting Vascular Endothelial Growth Factor (VEGF) mediated Pathological Angiogenesis. J Homeop Ayurv Med. 2013; 2:121. http://

Makker N, Nangia P, Larry T, Malathy PV, Shekhar EP, Victor H, et al. Inhibition of breast tumor growth and angiogenesis by a medicinal herb: Ocimum gratissimum. Int J Cancer.2007; 121:884–94. PMid: 17437270 PMCid: PMC3613994.

Bhardwaj LK, Patil KS, Juwatkar PV, Shukla VK. Plant products potential as anti- angiogenic and in cancer management.IJRAP. 2010; 1(2):339–49.

Mathur R., Gupta SK, Singh N, Mathur S, KochupillaiV, Thirumurthy V. Evaluation of the effect of Withanias omnifera root extracts on cell cycle and angiogenesis. Journal of Ethnopharmacology. 2006; 105:336–41. PMid: 16412596.

Umadevi M, Sampath KP, Bhowmik D, Duraivel S. Traditionally used anticancer herbs in India. Journal of Medicinal Plants Studies. 2013; 1(3):56–74.

Moon EJ, You ML, Ok-Hee L, Myoung-Jin L, Seung-Ki L, Myung-Hee al. A novel angiogenic factor derived from Aloe vera gel: β-sitosterol, a plant sterol. Angiogenesis.1999; 3(2):117–23.

Chobotova K, Vernallis A, Fadzilah AAM. Bromelain's activity and potential as an anti-cancer agent - Current evidence and perspective. Cancer Lett. 2010; 290:(2):148–56.