Cardiovascular Toxicology

, Volume 13, Issue 3, pp 278–289

Cardioprotective Role of Syzygium cumini Against Glucose-Induced Oxidative Stress in H9C2 Cardiac Myocytes

  • Neha Atale
  • Mainak Chakraborty
  • Sujata Mohanty
  • Susinjan Bhattacharya
  • Darshika Nigam
  • Manish Sharma
  • Vibha Rani
Article

Abstract

Diabetic patients are known to have an independent risk of cardiomyopathy. Hyperglycemia leads to upregulation of reactive oxygen species (ROS) that may contribute to diabetic cardiomyopathy. Thus, agents that suppress glucose-induced intracellular ROS levels can have therapeutic potential against diabetic cardiomyopathy. Syzygium cumini is well known for its anti-diabetic potential, but its cardioprotective properties have not been evaluated yet. The aim of the present study is to analyze cardioprotective properties of methanolic seed extract (MSE) of S. cumini in diabetic in vitro conditions. ROS scavenging activity of MSE was studied in glucose-stressed H9C2 cardiac myoblasts after optimizing the safe dose of glucose and MSE by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide. 2′,7′-dichlorfluorescein diacetate staining and Fluorescence-activated cell sorting analysis confirmed the suppression of ROS production by MSE in glucose-induced cells. The intracellular NO and H2O2 radical–scavenging activity of MSE was found to be significantly high in glucose-induced cells. Exposure of glucose-stressed H9C2 cells to MSE showed decline in the activity of catalase and superoxide dismutase enzymes and collagen content. 4′,6-diamidino-2-phenylindole, propidium iodide and 10-N-nonyl-3,6-bis (dimethylamino) acridine staining revealed that MSE protects myocardial cells from glucose-induced stress. Taken together, our findings revealed that the well-known anti-diabetic S. cumini can also protect the cardiac cells from glucose-induced stress.

Keywords

Syzygium cumini Glucose Diabetic cardiomyopathy Cardiac hypertrophy Reactive oxygen species Oxidative stress Extracellular matrix remodeling 

References

  1. 1.
    Banerjee, A., Dasgupta, N., & De, B. (2005). In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chemistry, 90, 727–733.CrossRefGoogle Scholar
  2. 2.
    Valko, M., Leibfritz, D., Moncol, J., Mark, T. D., Cronin, M. T. D., Mazur, M., et al. (2007). Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry & Cell Biology, 39, 44–84.CrossRefGoogle Scholar
  3. 3.
    Neill, S., Desikan, R., & Hancock, J. (2002). Hydrogen peroxide signaling. Current Opinion in Plant Biology, 5, 388–395.PubMedCrossRefGoogle Scholar
  4. 4.
    Kangralkar, V. A., Patil, S. D., & Bandivadekar, R. M. (2010). Oxidative stress and diabetes: A review. International Journal of Pharmaceutical Applications, 1, 38–45.Google Scholar
  5. 5.
    Laakso, M. (1999). Hyperglycemia and cardiovascular disease in type 2 diabetes. Diabetes, 48, 937–942.PubMedCrossRefGoogle Scholar
  6. 6.
    Panicker, G. K., Karnad, D. R., Salvi, V., & Kothari, S. (2012). Cardiovascular risk of oral antidiabetic drugs: Current evidence and regulatory requirements for new drugs. Journal of the Association of Physicians of India, 60, 56–61.PubMedGoogle Scholar
  7. 7.
    Devasagayam, T. P., Tilak, J. C., Boloor, K. K., Sane, K. S., Ghaskadbi, S. S., & Lele, R. D. (2004). Free radicals and antioxidants in human health: Current status and future prospects. Journal of the Association of Physicians of India, 52, 794–804.PubMedGoogle Scholar
  8. 8.
    Vardi, M., Blum, S., & Levy, A. P. (2012). Haptoglobin genotype and cardiovascular outcomes in diabetes mellitus natural history of the disease and the effect of vitamin E treatment. Meta-analysis of the medical literature. European Journal of Internal Medicine, 23, 628–632.PubMedCrossRefGoogle Scholar
  9. 9.
    Myung, S. K., Ju, W., Cho, B., Oh, S. W., Park, S. M., Koo, B. K., et al. (2013). Efficacy of vitamin and antioxidant supplements in prevention of cardiovascular disease: Systematic review and meta-analysis of randomised controlled trials. British Medical Journal, 18(346), f10.CrossRefGoogle Scholar
  10. 10.
    Saikat, S., & Raja, C. (2011). Oxidative stress: Diagnostics, prevention, and therapy in the role of antioxidants in human health (ACS symposium series) (pp. 1–37). Washington DC: American Chemical Society.Google Scholar
  11. 11.
    Hill, M. F. (2008). Emerging role for antioxidant therapy in protection against diabetic cardiac complications: Experimental and clinical evidence for utilization of classic and new antioxidants. Current Cardiology Reviews, 4, 259–268.PubMedCrossRefGoogle Scholar
  12. 12.
    Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity, 2, 270–278.PubMedCrossRefGoogle Scholar
  13. 13.
    Bopp, A., DeBona, K. S., Belle, L. P., Moresco, R. N., & Moretto, M. B. (2009). Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients. Fundamental & Clinical Pharmacology, 23, 501–507.CrossRefGoogle Scholar
  14. 14.
    Ponnusamy, S., Ravindran, R., Zinjarde, S., Bhargava, S., & Ravi Kumar, A. (2011). Evaluation of traditional Indian antidiabetic medicinal plants for human pancreatic amylase inhibitory effect in vitro. Evidence Based Complementary and Alternative Medicine,. doi:10.1155/2011/515647.PubMedGoogle Scholar
  15. 15.
    Saravanan, G., & Pari, L. (2008). Hypoglycemic and antihyperglycemic effect of Syzygium cumini bark in strptozotocin induced diabetic rats. Journal of Pharmacology and Toxicology, 3, 1–10.Google Scholar
  16. 16.
    Helmstädter, A. (2008). Syzygium cumini (L.) SKEELS (Myrtaceae) against diabetes–125 years of research. Pharmazie, 63, 91–101.PubMedGoogle Scholar
  17. 17.
    Baliga, M. S., Bhat, H. P., Baliga, B. R. V., Wilson, R., & Palatty, P. L. (2011). Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): A review. Food Research International, 44, 1776–1789.CrossRefGoogle Scholar
  18. 18.
    Ayyanar, M., & Subash-Babu, P. (2012). Syzygium cumini (L.) Skeels: A review of its phytochemical constituents and traditional uses. Asian Pacific Journal of Tropical Biomedicine, 2, 240–246.PubMedCrossRefGoogle Scholar
  19. 19.
    Shoba, B. (2012). Antibacterial, phytochemical analysis of water extract of Syzygium cumini and analytical study by HPLC. Asian Journal of Experimental Biological Sciences, 3, 320–324.Google Scholar
  20. 20.
    Bhowmik, D., Gopinath, H., Pragati Kumar, B., Duraivel, S., Aravind, G., & Sampath Kumar, K. P. (2012). Traditional and medicinal uses of Indian black berry. Journal of Pharmacognosy Phytochemistry, 1, 37–42.Google Scholar
  21. 21.
    Moresco, R. N., Sperotto, R. L., Bernardi, A. S., Cardoso, R. F., & Gomes, P. (2007). Effect of the aqueous extract of Syzygium cumini on carbon tetrachloride-induced hepatotoxicity in rats. Phytotherapy Research, 21, 793–795.PubMedCrossRefGoogle Scholar
  22. 22.
    Atale, N., Jaiswal, A., Chhabra, A., Malhotra, U., Kohli, S., Mohanty, S., et al. (2011). Phytochemical and antioxidant screening of Syzygium cumini seed extracts: A comparative study. Journal of Pharmaceutical Research, 4, 4530–4532.Google Scholar
  23. 23.
    Mastan, S. K., Chaitanya, G., Bhavya Latha, T., Srikanth, A., Sumalatha, G., & Eswar Kumar, K. (2009). Cardioprotective effect of methanolic extract of Syzygium cumini seeds on isoproterenol-induced myocardial infarction in rats. Der Pharmacia Lettre, 1, 143–149.Google Scholar
  24. 24.
    Sreejit, P., Kumar, S., & Verma, R. S. (2008). An improved protocol for primary culture of cardiomyocyte from neonatal mice. In Vitro Cellular and Developmental Biology Animal, 44, 45–50.PubMedCrossRefGoogle Scholar
  25. 25.
    Brownlee, M. (2001). Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813–820.PubMedCrossRefGoogle Scholar
  26. 26.
    Green, K., Brand, M. D., & Murphy, M. P. (2004). Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes, 53, S110–S118.PubMedCrossRefGoogle Scholar
  27. 27.
    Ferrari, M., Fornasiero, M. C., & Isetta, A. M. (1990). MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. Journal of Immunological Methods, 131, 165–172.PubMedCrossRefGoogle Scholar
  28. 28.
    Hescheler, J., Meyer, R., Plant, S., Krautwurst, D., Rosenthal, W., & Schultz, G. (1991). Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9C2) line from rat heart. Circulation Research, 69, 1476–1486.PubMedCrossRefGoogle Scholar
  29. 29.
    Barcia, J. J. (2007). The giemsa stain: Its history and applications. International Journal of Surgical Pathology, 15, 292–296.PubMedCrossRefGoogle Scholar
  30. 30.
    Wang, H., & Joseph, J. A. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine, 27, 612–616.PubMedCrossRefGoogle Scholar
  31. 31.
    Marotta, M., & Martino, G. (1985). Sensitive spectrophotomeric method for the quantitative estimation of collagen. Analytical Biochemistry, 150, 86–90.PubMedCrossRefGoogle Scholar
  32. 32.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.PubMedCrossRefGoogle Scholar
  33. 33.
    Marcocci, L., Magguire, J. J., Droy-Lefaix, M. T., & Packer, L. (1994). The nitric oxide-scavenger properties of Ginkgo biloba extract EGB 761. Biochemical and Biophysical Research Communications, 15, 462–475.Google Scholar
  34. 34.
    Ruch, R. J., Cheng, S. J., & Klaunig, J. E. (1989). Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10, 1003–1008.PubMedCrossRefGoogle Scholar
  35. 35.
    Aebi, H. E. (1983). Catalase. In H. U. Bergmeyer (Ed.), Methods of enzymatic analysis (pp. 273–286). Weinheim: Verlag Chemie.Google Scholar
  36. 36.
    Beauchamp, C., & Fridovich, I. (1971). Super oxide dismutase: Improved assays and assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.PubMedCrossRefGoogle Scholar
  37. 37.
    Kapuscinski, J., & Skoczylas, B. (1978). Fluorescent complexes of DNA with DAPI (4′,6-diamidine-2-phenyl indole dihydrochloride) or DCI (4′,6- dicarboxyamide-2-phenyl indole). Nucleic Acids Research, 5, 3775–3799.PubMedCrossRefGoogle Scholar
  38. 38.
    Brana, C., Benham, C., & Sundstrom, L. (2002). A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry. Brain Research Protocols, 10, 109–114.PubMedCrossRefGoogle Scholar
  39. 39.
    Petit, J. M., Maftah, A., Ratinaud, M. H., & Julien, R. (1992). 10 N-Nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria. European Journal of Biochemistry, 209(267–273), 40.Google Scholar
  40. 40.
    Perelman, A., Wachtel, C., Cohen, M., Haupt, S., Shapiro, H., & Tzur, A. (2012). JC-1: Alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death and Disease, 3. doi:10.1038/cddis.2012.171.
  41. 41.
    Tao, L. S., Mackenzie, C. R., & Charlson, M. E. (2008). Predictors of postoperative complications in the patient with diabetes mellitus. Journal of Diabetes and Its Complications, 22, 24–28.PubMedCrossRefGoogle Scholar
  42. 42.
    Lamblin, N., Fertin, M., De-Groote, P., & Bauters, C. (2012). Cardiac remodeling and heart failure after a first anterior myocardial infarction in patients with diabetes mellitus. Journal of Cardiovascular Medicine, 13, 353–359.PubMedCrossRefGoogle Scholar
  43. 43.
    Ku, P. M., Chen, L. J., Liang, J. R., Cheng, K. C., Li, Y. A., & Cheng, J. T. (2011). Molecular role of GATA binding protein 4 (GATA-4) in hyperglycemia-induced reduction of cardiac contractility. Cardiovascular Diabetology, 10, 1–15.CrossRefGoogle Scholar
  44. 44.
    Ansley, D. M., & Wang, B. (2013). Oxidative stress and myocardial injury in the diabetic heart. Journal of Pathology, 229, 232–241.PubMedCrossRefGoogle Scholar
  45. 45.
    Ahuja, P., Sdek, P., & MacLellan, W. R. (2007). Cardiacmyocyte cell cycle control in development, disease and regeneration. Physiological Reviews, 87, 521–544.PubMedCrossRefGoogle Scholar
  46. 46.
    Feng, B., Chen, S., Chiu, J., George, B., & Chakrabarti, S. (2008). Regulation of cardiomyocyte hypertrophy in diabetes at the transcriptional level. American Journal of Physiology Endocrinology and Metabolism, 294, E1119–E1126.PubMedCrossRefGoogle Scholar
  47. 47.
    Watkins, S. J., Borthwick, G. M., & Arthur, H. M. (2011). The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. In vitro Cellular Developmental Biology Animal, 47, 125–131.PubMedCrossRefGoogle Scholar
  48. 48.
    Seddon, M., Looi, Y. H., & Shah, A. M. (2007). Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. Heart, 93, 903–907.PubMedCrossRefGoogle Scholar
  49. 49.
    Umadevi, S., Gopi, V., Simna, S. P., Parthasarathy, A., Yousuf, S. M. J., & Elangovan, V. (2012). Studies on the cardio protective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2–1) cells. Cardiovascular Toxicology, 12, 304–311.PubMedCrossRefGoogle Scholar
  50. 50.
    Tsutsui, H., Kinugawa, S., & Matsushima, S. (2009). Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovascular Research, 81, 449–456.PubMedCrossRefGoogle Scholar
  51. 51.
    Glucose in cell structure. Available from http://www.sigmaaldrich.com/ life-science/cell-culture/learning-center/media expert/glucose.html.
  52. 52.
    Farhangkhoee, H., Khan, Z. A., Chen, S., & Chakrabarti, S. (2006). Differential effects of curcumin on vasoactive factors in the diabetic rat heart. Nutrition and Metabolism, 3, 1–18.CrossRefGoogle Scholar
  53. 53.
    Zhang, G. X., Kimura, S., Murao, K., Obata, K., Matsuyoshi, H., & Takaki, M. (2010). Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat. American Journal of Physiology Heart and Circulatory Physiology, 298, H433–H439.PubMedCrossRefGoogle Scholar
  54. 54.
    Caglayan, E., Stauber, B., Collins, A. R., Lyon, C. J., Yin, F., Liu, J., et al. (2008). Differential roles of cardiomyocyte and macrophage peroxisome proliferators-activated receptor gamma in cardiac fibrosis. Diabetes, 57, 2470–2479.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Neha Atale
    • 1
  • Mainak Chakraborty
    • 1
  • Sujata Mohanty
    • 1
  • Susinjan Bhattacharya
    • 1
  • Darshika Nigam
    • 2
  • Manish Sharma
    • 3
  • Vibha Rani
    • 1
  1. 1.Department of BiotechnologyJaypee Institute of Information TechnologyNoidaIndia
  2. 2.Department of Biochemistry, School of Life SciencesDr. B. R. Ambedkar UniversityAgraIndia
  3. 3.Peptide and Proteomics DivisionDIPAS, DRDONew DelhiIndia

Personalised recommendations