Anti-angiogenic activity of Gracilaria coronopifolia J.G. Agardh extract by lowering the levels of trace metals (iron, zinc and copper) in duck chorioallantoic membrane and in vitro activation of AMP-kinase

  • Oliver B. VillafloresEmail author
  • Katrin Mae M. Ortega
  • Analin Empaynado-Porto
  • Stephen Lirio
  • Hwa-Kwang Yak
  • Dharmatov Rahula Albano
  • Mary Jho-Anne T. Corpuz
Original Article


AMP-activated protein kinase (AMPK) is an intracellular energy sensor important in metabolic regulation, cell growth, and survival. However, the specific role of AMPK signaling pathway in the inhibition of angiogenesis remains unclear. The study highlights the activity on AMP activated protein kinase signaling pathways of a marine algae, Gracilaria coronopifolia, and its effects on angiogenesis. It was found that the most potent extract, GCD, inhibited angiogenesis significantly in the duck chorioallantoic membrane assay and also activated the enzyme AMP-kinase, in vitro. The dichloromethane extract was found most active in inhibiting angiogenesis in the duck chorioallantoic membrane (IC50 = 1.21 μg/mL) followed by GCH (IC50 = 3.08 μg/mL) (p = 0.479) and GCM (IC50 = 8.93 μg/mL) (p = 0.042). Benferroni post hoc analysis revealed that there was no significant difference between the percent inhibitions of GCH and GCM extracts (p = 0.479). Consequently, angiogenic inhibition caused lowering of iron, zinc, and copper levels in the duck CAM. Thin layer chromatography and gas chromatography–mass spectrometry revealed the components of each extracts. Notably, this is the first report on the kinase activity of a red algae G. coronopifolia extracts and a colorimetric-based quantification of angiogenesis based on metal content of CAM. Our data also suggest a novel therapeutic approach for inhibiting angiogenesis through the AMPK pathway.


Gracilaria coronopifolia Angiogenesis AMP-activated protein kinase CAM assay 



This work was funded by Philippine Department of Science and Technology—Science Education Institute (DOST-SEI). The experiments were done at the University of Santo Tomas Research Center for Natural and Applied Science (UST-RCNAS) and Chung Yuan Christian University (CYCU).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.


  1. 1.
    Pan S, Deng X et al (2018) Black tea affects obesity by reducing nutrient intake and activating AMP-activated protein kinase in mice. Mol Biol Rep 45:689–697. Google Scholar
  2. 2.
    Soraya H, Esfahanian N et al (2012) Anti-angiogenic effects of metformin, an AMPK activator, on human umbilical vein endothelial cells and on granulation tissue in rat. Iran J Basic Med Sci 15(6):1202–1209Google Scholar
  3. 3.
    Han F, Li CF et al (2018) The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance. Nat Commun 9(1):4728. Google Scholar
  4. 4.
    Peyton KJ, Liu X et al (2012) Activation of AMP-activated protein kinase inhibits the proliferation of human endothelial cells. J Pharmacol Exp Ther 342(3):827–834. Google Scholar
  5. 5.
    Jeon SM (2016) Regulation and function of AMPK in physiology and diseases. Exp Mol Med 48(7):e245. Google Scholar
  6. 6.
    Showkat M, Beigh MA et al (2014) mTOR signaling in protein translation regulation: implications in cancer genesis and therapeutic interventions. Mol Biol Int 14:686984. Google Scholar
  7. 7.
    Goodman M, Liu Z et al (2014) AMPK activators as a drug for diabetes, cancer and cardiovascular disease. Pharm Reg Affairs 3(2):118. Google Scholar
  8. 8.
    Cunha SI, Pietras K (2011) ALK1 as an emerging target for antiangiogenic therapy of cancer. Blood 117(26):6999–7006. Google Scholar
  9. 9.
    Saghiri MA, Asatourian A et al (2015) Functional role of inorganic trace elements in angiogenesis—part I: N, Fe, Se, P, Au, and Ca. Crit Rev Oncol Hematol 96(1):129–142. Google Scholar
  10. 10.
    Saghiri MA, Asatourian A et al (2015) Functional role of inorganic trace elements in angiogenesis—part II: Cr, Si, Zn, Cu, and S. Crit Rev Oncol Hematol 96(1):143–155. Google Scholar
  11. 11.
    Merrill, JF, Thomson DM et al (2012) Iron deficiency causes a shift in AMP-activated protein kinase (AMPK) subunit composition in rat skeletal muscle. Nutr Metab (Lond). 9(1): 104.
  12. 12.
    Gybina AA, Prohaska JR (2008) Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase. Exp Biol Med (Maywood) 233(10):1262–1270. Google Scholar
  13. 13.
    Gybina AA, Prohaska JR (2008) Copper deficiency results in AMP-activated protein kinase activation and acetylCoA carboxylase phosphorylation in rat cerebellum. Brain Res 1204:69–76. Google Scholar
  14. 14.
    Wiggins HL, Wymant JM et al (2015) Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells. Biochem Pharmacol 93(3):332–342. Google Scholar
  15. 15.
    Filomeni G, Picirillo S et al (2009) The isatin-Schiff base copper(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis. Carcinogenesis 30(7):1115–1124. Google Scholar
  16. 16.
    Roldan MJ, Chin TY et al (2018) Cytotoxic and angiosuppresive potentials of Zehneria japonica (Thund. Ex. Murray) S.K. Chen (Cucurbitaceae) crude leaf extracts. Phil J Health Res Dev 22(1):43–52Google Scholar
  17. 17.
    Covarubias LA, Macabeo A et al (2015) Antibacterial activity and concentration dependent modulation of angiogenesis of the saponins of Schefflera luzoniensis. Res J Pharm Biol Chem Sci 6:418–422Google Scholar
  18. 18.
    Zarcinas BA, Cartwright B et al (1987) Nitric acid digestion and multi-element analysis of plant material by inductively coupled plasma spectrometry. Commun Soil Sci Plan 18(1):131–146. Google Scholar
  19. 19.
    Ivsic AG, Tamhina B (2003) Extraction and formation of iron(III) thiocyanate complexes: application for spectrophotometric determination of iron. Croat Chem Acta 76(4):323–328Google Scholar
  20. 20.
    Karipcin F, Arabali F, Karatas I (2003) Synthesis and characterization of 4-(alkylaminoisonitrosoacetyl)biphenyls and their complexes. Russ J Coord Chem 32:109–115. Google Scholar
  21. 21.
    Karipcin F, Kabalcilar E (2007) Spectroscopic and thermal studies on solid complexes of 4-(2-pyridylazo)resorcinol with some transition metals. Acta Chim Slov 54:242–247Google Scholar
  22. 22.
    Hunt JB, Neece SH, Ginsburg A (1985) The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase. Anal Biochem 146(1):150–157Google Scholar
  23. 23.
    Smirnova SV, Samarina TO et al (2015) Solubilization of 4-(2-pyridylazo)resorcinol in hydrophobic–hydrophilic ionic liquids and extraction of heavy metal ions from aqueous solutions. Moscow Univ Chem Bull 70(5):229–233. Google Scholar
  24. 24.
    Villaflores OB, Macabeo A et al (2010) Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis. Pharmacogn Mag 6(24):339–344. Google Scholar
  25. 25.
    Rosas C, Sinning M et al (2014) Celecoxib decreases growth and angiogenesis and promotes apoptosis in a tumor cell line resistant to chemotherapy. Biol Res 47(1):27. Google Scholar
  26. 26.
    Montanari RM, Barbosa LCA et al (2011) Chemical composition and antibacterial activity of essential oils from verbenaceae species: alternative sources of (E)-caryophyllene and germacrene-D. Quím Nova 34:1550–1555Google Scholar
  27. 27.
    Prabakaran R, Kirutheka E (2018) GCMS, phytochemicals and antioxidant activities of in vitro callus extracts of Strobilanthes kunthiana (Nees) T. Anderson ex Benth: an endemic plant of acanthaceae. Braz J Biol Sci 5(10):359–372. Google Scholar
  28. 28.
    Elaiyaraja A, Chandramohan G (2016) Comparative phytochemical profile of Indoneesiella echioides (L) nees leaves. J Pharmacogn Phytochem 5(9):383–389Google Scholar
  29. 29.
    Abubakar MN, Majinda RRT (2016) GC-MS analysis and preliminary antimicrobial activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines (Basel). Google Scholar
  30. 30.
    Saravanan P, Chandramohan G et al (2014) GC–MS analysis of phytochemical constituents in ethanolic bark extract of Ficus religiosa. J Pharm Pharm Sci 6:457–460Google Scholar
  31. 31.
    Mohamed AM, Quisenberry SS, Moellenbeck DJ (1992) 6,10,14-trimethylpentadecan-2-one: a Bermuda grass phagostimulant to fall armyworm (lepidoptera: noctuidae). J Chem Ecol 18(4):673–682. Google Scholar
  32. 32.
    Chandrasekaran M, Kannathasan K, Venkatesalu V (2008) Antimicrobial activity of fatty acid methyl esters of some members of chenopodiaceae. Z Naturforsch 63(2008):331–336. Google Scholar
  33. 33.
    Cunha LC, De Morais SA et al (2013) Chemical composition, cytotoxic and antimicrobial activity of essential oils from Cassia bakeriana Craib. against aerobic and anaerobic oral pathogens. Molecules 18(4):4588–4598. Google Scholar
  34. 34.
    Ravi L, Krishnan K (2017) Cytotoxic potential of N-hexadecanoic acid extracted from Kigelia pinnata leaves. Asian J Cell Biol 12(1):20–27Google Scholar
  35. 35.
    Belakhdar G, Benjouad A, Abdennebi EH (2015) Determination of some bioactive chemical constituents from Thesium humile Vahl. J Mater Environ Sci 6:2778–2783Google Scholar
  36. 36.
    Abubakar MN, Majinda RRT (2016) GC–MS analysis and preliminary antimicrobial activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines (Basel, Switzerland) 3(1):3Google Scholar
  37. 37.
    Cragg GM, Newman DJ (2013) Natural products: a continuing source of novel drug leads. Biochim Biophys Acta 1830(6):3670–3695. Google Scholar
  38. 38.
    Kim I, He YY (2013) Targeting the AMP-activated protein kinase for cancer prevention and therapy. Front Oncol 3:175. Google Scholar
  39. 39.
    Zulato E, Bergamo F et al (2014) Prognostic significance of AMPK activation in advanced stage colorectal cancer treated with chemotherapy plus bevacizumab. Br J Cancer 111(1):25–32. Google Scholar
  40. 40.
    Yen CN, Cho YS, Kwon HJ (2017) The effect of indatraline on angiogenesis suppression through HIF-1alpha-mediated VEGF inhibition. Biochem Biophys Res Commun 485(2):349–354. Google Scholar
  41. 41.
    Grahame HD (2016) Regulation of AMP-activated protein kinase by natural and synthetic activators. Acta Pharm Sin B 6(1):1–19. Google Scholar
  42. 42.
    Song Y, Oh GH et al (2017) Fucosterol inhibits adipogenesis through the activation of AMPK and Wnt/β-catenin signaling pathways. Food Sci Biotechnol 26(2):489–494. Google Scholar
  43. 43.
    Kyadari M et al (2013) Evaluation of antiangiogenic and antiproliferative potential of the organic extract of green algae Chlorella pyrenoidosa. Indian J Pharmacol 45(6):569–574. Google Scholar
  44. 44.
    Namvar F, Mohammad R et al (2013) Antioxidant, antiproliferative, and antiangiogenesis effects of polyphenol-rich seaweed (Sargassum muticum). Biomed Res Int 2013:9. Google Scholar
  45. 45.
    Merrill RD, Shamin AA et al (2012) High prevalence of anemia with lack of iron deficiency among women in rural Bangladesh: a role for thalassemia and iron in groundwater. Asia Pac J Clin Nutr 21(3):416–424Google Scholar
  46. 46.
    Wu K, Huang C et al (2016) Role and mechanism of the AMPK pathway in waterborne Zn exposure influencing the hepatic energy metabolism of Synechogobius hasta. Sci Rep 6:38716. Google Scholar
  47. 47.
    Filomeni G, Cardaci S et al (2011) Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment. Biochem J 437(3):443–453. Google Scholar
  48. 48.
    Ishida S, Andreux P et al (2013) Bioavailable copper modulates oxidative phosphorylation and growth of tumors. Proc Natl Acad Sci USA 110(48):19507–19512. Google Scholar
  49. 49.
    Shibuya M (2014) VEGF-VEGFR signals in health and disease. Biomol Ther 22(1):1–9. Google Scholar
  50. 50.
    Gu Z, Shan K et al (2015) n-3 polyunsaturated fatty acids and their role in cancer chemoprevention. Cur Pharm Rep 1(5):283–294. Google Scholar
  51. 51.
    Stephenson JA, Al-Taan O et al (2013) The multifaceted effects of omega-3 polyunsaturated fatty acids on the hallmarks of cancer. J Lipids 2013:261247. Google Scholar
  52. 52.
    Massaro M, Martinelli R et al (2015) Transcriptome-based identification of new anti-inflammatory and vasodilating properties of the n-3 fatty acid docosahexaenoic acid in vascular endothelial cell under proinflammatory conditions [corrected]. PLoS ONE 10(6):e0129652. Google Scholar
  53. 53.
    Ibrahim A, Mbodji K et al (2011) Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturated fatty acids in intestinal microvascular endothelium. Clin Nutr 30(5):678–687. Google Scholar
  54. 54.
    Park KR, Nam D et al (2011) β-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3 K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation. Cancer Lett 312(2):178–188. Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Oliver B. Villaflores
    • 1
    • 3
    • 4
    Email author
  • Katrin Mae M. Ortega
    • 1
    • 5
    • 6
  • Analin Empaynado-Porto
    • 1
    • 5
  • Stephen Lirio
    • 2
  • Hwa-Kwang Yak
    • 2
  • Dharmatov Rahula Albano
    • 1
    • 3
    • 4
  • Mary Jho-Anne T. Corpuz
    • 1
    • 3
    • 5
  1. 1.The Graduate SchoolUniversity of Santo TomasManilaPhilippines
  2. 2.Department of ChemistryChung Yuan Christian UniversityChung-LiTaiwan
  3. 3.Research Center for the Natural and Applied SciencesUniversity of Santo TomasManilaPhilippines
  4. 4.Department of Biochemistry, Faculty of PharmacyUniversity of Santo TomasManilaPhilippines
  5. 5.Faculty of PharmacyUniversity of Santo TomasManilaPhilippines
  6. 6.Our Lady of Fatima UniversityValenzuelaPhilippines

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