Assessment of the Combination of Anthocyanin and Quercetin as an Angiogenic Inhibitor through a Chick Embryo Chorioallantoic Membrane Assay
Alexandria Marie Sombiro1, Daphne Di Valdestamon1, Kyle Dominic Aguirre1 and Harold Mediodia1
1Philippine Science High School Western Visayas Campus – Bito-on, Jaro, Iloilo City 5000, Department of Science and Technology, Philippines
Angiogenesis is a normal physiological process where new capillary branches sprout from preexisting blood vessels. Excessive angiogenesis can lead to the development of cancer, atherosclerosis, rheumatoid arthritis, etc. Anthocyanin is known for inhibiting angiogenesis in human endothelial cells. Quercetin has also shown an inhibitory effect when it was administered to the chorioallantoic membrane of chicken eggs. However, it has been proven that single flavonoids are less effective compared to when they are combined. This study aimed to assess the combined effect of anthocyanin and quercetin in inhibiting blood vessel growth in a chick embryo chorioallantoic membrane. Anthocyanin, quercetin, and their combination were each prepared with a concentration of 15 mg/L. The treatments were administered to seven-day-old chicken eggs and dimethyl sulfoxide served as the positive control. The appearance of each chrorioallantoic membrane was evaluated using the software ImageJ through calculation of fractal dimension values three days after the treatments were introduced. The values were then statistically analyzed through Kruskal-Wallis where it was found out that there was no significant difference between all treatments when compared to the controls. Of the three flavonoid treatments, the combination of anthocyanin and quercetin appeared to be the most effective in reducing blood vessel growth. This shows that the combination of anthocyanin and quercetin is more efficient in inhibiting angiogenesis. This treatment could be a novel source of treating pathological angiogenesis.
- Abid, M.R., Kachra, Z., Spokes, K.C. and Aird , W.C., (2000). ”NADPH oxidase activity is required for endothelial cell proliferation and migration”FEBS Lett. 486, 252-256.
- Burrell K, and Zadeh G, (2012). Molecular mechanisms of tumor angiogenesis. In: Tumor Angiogenesis.p.277-296
- Cho, M., Hunt, T.K. and Hussain, M.Z., (2012). Hydrogen peroxide stimulates macrophage vascu lar endothelial growth factor release, Am. J. Physiol. Heart Circ. Physiol. 280, H2357-H2363.”
- Davis, J.N., Singh, B., Bhuiyan, M., and Sarkar, F. H., (1998). ”Genistein-induced upregulation of p21 WAF1, downregulation of cyclin B and induction of apoptosis in prostate cancer cells”. Nutrition and Cancer, 32, 123-131.
- Gacche R.N., Shegokar H.D., Gond D.S., Yang Z., and Jadhav A.D., (2011). ”Evaluation of Selected Flavonoids as Antiangiogenic, Anticancer, and Radical Scavenging Agents: An Experimental and In Silico Analysis.”61(3):651-663.
- Han, Y.J., Kwon, Y.G., Chung, H.T., Lee, S.K., Simmons, R.L., Billiar, T.R. and Kim, Y.M., (2001). Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kB activation: role of H2O2 and nitric oxide in inducible nitric oxide synthase expression in macrophages, Nitric Oxide 5, 504-513.
- Hidalgo, M., Sanchez-Moreno, C., and de PascualTeresa, S., (2010). ”Flavonoid-flavonoid interaction and its effect on their antioxidant activity”. 121(3):691-696.
- Huang, C., Li, J., Song, L., Zhang, D., Tong, Q., Ding, M., Bowman, L., Aziz, R., and Stoner, G. D.,(2006). ”Black Raspberry Extracts Inhibit Benzo ( a ) Pyrene Diol-Epoxide Induced Activator Protein 1 Activation and VEGF Transcripti on by Targeting the Phosphotidylinositol 3-Kinase / Akt Pathway”, (1), 581 588. https://doi.org/10.1158/0008-5472.CAN-05-1951
- Johnson, K.E., and Wilgus, T.A., (2014). ”Vascular Endothelial Growth Factor and Angiogenesis in the Regulation of Cutaneous Wound Repair”.3(10):647 661.
- Koizumi, K., Tsutsumi, Y., Yoshioka, Y., Watanabe, M., Okamoto, T., Mukai, Y., Nakagawa, S., and Mayumi, T., (2003). ”Anti-angiogenic effects of dimethyl sulfoxide on endothelial cells”. Biological Pharmaceutical Bulletin, 26(9), 1295-1298. https://doi.org/10.1 248/bpb.26.1295
- Krenn, L., and Paper, D.H., (2009). ”Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.). 16(12):1083-8.
- Lian, F., Bhuiyan, M., Li, Y.M., Wall, N., Kraut, M., and Sarkar, F. H., (1998). ”Genistein-induced G2- M arrest, p21WAF1 upregulation and apoptosis in a nonsmall-cell lung cancer cell line. Nutrition and Cancer, 31, 184-191.
- Lin, M.T., Yen, M.L., Lin, C.Y. and Kuo, M.L., (2003). Inhibition of vascular endothelial growth factor induced angiogenesis by resveratrol through interruption of src-dependent vascular endothelial cadherin tyrosine phosphorylation, Mol. Pharmacol. 64, 1029 1036.
- Maniago, K.G.N., Mari, C.G.S, and Pareja, M.C., (2014). ”Angiogenic effect of Curcuma longa Linn. (turmeric) tea powder on the chorioallantoic membrane of 10-day old Annas luzonica (duck) eggs”.5(4): 32-37.
- Matsukawa, Y., Mami, N., Sakai, T., Satomi, Y., Yoshida, M., Matsumoto, K., Nishino, H., and Aoike, A., (1993).”Genistein arrests cell cycle progression at G2- M.” Cancer Research, 53, 1328 1331.
- Nobosse P., (2016). Methods for anthocyanin. ResearchGate.
- Polytarchou, C., and Papadimitriou, E. , (2004). ”Antioxidants inhibit angiogenesis in vivo through down-regulation of nitric oxide synthase expression and activity.” Free Radic Res, 38(5), 501 508.https://doi.org/10.1080/10715760410001684621
- Ribatti, D., Nico, B., Vacca, A., and Presta, M., (2006). ”The gelatin sponge-chorioallantoic membrane assay. 2006;1(1):85 91.
- Romero, I., Paez, A., Femelo, A., Lujan, M., and Berenguer, A., (2002). ”Polyphenols in red wine inhibit the proliferation and induce apoptosis of LNCaP cells.” British Journal of Urology International, 89, 950 954.
- Sagar, S.M., Yance, D., and Wong, R.K., (2006). ”Natural health products t hat inhibit angiogenesis: A potential source for investigational new agents to treat cancerPart 2.”13(3).
- Tantiado, R.G., (2016). Incubation of Eggs and CAM Assay
- Tantiado, R. G., and Tan, P., (2015). ”Evaluation of the Angiosuppresive Activity of Tinospora rumphii Boerl. Stem Extract Using the Chorio-allantoic Membrane Assay in Anas platyrhynchos Embryos.” International Journal of Bio-Science and Bio-Technology 4, no. 2 (June 2012): 93-102. The Angiogenesis Foundation. Angiogenesis. Cambridge, M. Available from: https://www.angio.org/learn/angiogenesis/
- Tan, W., Lin, L., Li, M., Zhang, Y., and Tong, Y., (2003). Quercetin , a dietary-derived flavonoid , possesses antiangiogenic potential, 459, 255-262. https://doi.org/10.1016/S0014-2999(02)02848-0
- Ucuzian, A.A., Gassman, A.A., East, A.T., and Greisler, H.P., (2010). Molecular mediators of angiogenesis. 2010;31(1):158-175.
- Ushio-Fukai, M., Tang, Y., Fukai, T., Dikalov, S.I., Ma, Y., Fujimoto, M., Quinn, M.T., Pagano, P.J., Johnson, C. and Alexander, R.W., (2002). Novel role of gp91phox-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis, Circ. Res. 91, 1160-1167.
- Wang, S. Y., and Jiao, H., (2000). ”Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen.” Journal of Agricultural and Food Chemistry, 48(11), 5677
- Wang, Z., Dabrosin, C., Yin, X., Fuster, M.M., Arreola, A., Rathmell, W.K., Generali, D., Nagaraju, G.P., El-Rayes, B., Ribatti, D., et al., (2015).Broad targeting of angiogenesis for cancer prevention and therapy.