Cell Biochemistry and Biophysics

, Volume 77, Issue 1, pp 99–107 | Cite as

Superoxide Formation in Cardiac Mitochondria and Effect of Phenolic Antioxidants

  • Arina L. Dudylina
  • Marina V. Ivanova
  • Konstantin B. Shumaev
  • Enno K. RuugeEmail author
Original Paper


Since mitochondria are the main cellular source of reactive oxygen species, it is important to study the effect of dietary phenolic compounds on the level of ROS in these organelles. Using the EPR spectroscopy and TIRON probe, the ability of the investigated phenols (quercetin, rutin, caffeic acid, curcumin, and resveratrol) to scavenge superoxide anion radicals generated by isolated heart mitochondria of Wistar rats under variable oxygen partial pressure was studied. It was shown that during a 10 min incubation, caffeic acid in concentrations of 10–500 μM most effectively scavenged superoxide radicals formed in the complex III of the mitochondrial respiratory chain. A comparable antioxidant effect of rutin under these experimental conditions was observed at higher concentrations of 1–10 mM. The antioxidant activity of quercetin in the concentration range of 10–500 μM during the first minutes of incubation was higher than that of caffeic acid. Of the phenolic compounds studied, curcumin had the least effect on the superoxide radicals.


Reactive oxygen species Heart mitochondria Hypoxia Phenolic compounds Antioxidants EPR spectroscopy 



caffeic acid (3,4-dihydroxycinnamic acid)


curcumin ((1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)


lithium phthalocyanine


low density lipoprotein


superoxide anion radical


quercetin (3,3',4',5,7-pentahydroxyflavone)


reactive oxygen species


resveratrol (3,5,4'-trihydroxy-trans-stilbene)


rutin (vitamin P)




4,5-dihydroxybenzene-1,3-disulfonate, disodium salt



This work was supported by the Russian Foundation for Basic Research (grants 15-04-05211 and 18-015-00125).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Visioli, F., De La Lastra, C. A., Andres-Lacueva, C., Aviram, M., Calhau, C., Cassano, A., D’Archivio, M., Faria, A., Favé, G., Fogliano, V., Llorach, R., Vitaglione, P., Zoratti, M., & Edeas, M. (2011). Polyphenols and human health: a prospectus. Critical Reviews in Food Science and Nutrition, 51, 524–546.CrossRefGoogle Scholar
  2. 2.
    Gibellini, L., Bianchini, E., De Biasi, S., Nasi, M., Cossarizza, A., & Pinti, M. (2015). Natural compounds modulating mitochondrial functions. Evidence-Based Complementary and Alternative Medicine, 2015, 527209.CrossRefGoogle Scholar
  3. 3.
    Jia, Z., Zhu, H., Misra, B. R., Mahaney, J. E., Li, Y., & Misra, H. P. (2008). EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol. Molecular and Cellular Biochemistry, 313, 187–194.CrossRefGoogle Scholar
  4. 4.
    Ksenzenko, M., Konstantinov, A. A., Khomutov, G. B., Tikhonov, A. N., & Ruuge, E. K. (1983). Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain. FEBS Letters, 155, 19–24.CrossRefGoogle Scholar
  5. 5.
    Moukette, B. M., Pieme, C. A., Njimou, J. R., Nya Biapa, C. P., Marco, B., & Ngogang, J. Y. (2015). In vitro antioxidant properties, free radicals scavenging activities of extracts and polyphenol composition of a non-timber forest product used as spice: Monodora myristica. Biological Research, 48, 15.CrossRefGoogle Scholar
  6. 6.
    Benedec, D., Vlase, L., Oniga, I., Mot, A. C., Damian, G., Hanganu, D., Duma, M., & Silaghi-Dumitrescu, R. (2013). Polyphenolic composition, antioxidant and antibacterial activities for two romanian subspecies of Achillea distans Waldst. et Kit. ex Willd. Molecules, 18, 8725–8739.CrossRefGoogle Scholar
  7. 7.
    Gorlach, S., Fichna, J., & Lewandowska, U. (2015). Polyphenols as mitochondria-targeted anticancer drugs. Cancer Letters, 366, 141–149.CrossRefGoogle Scholar
  8. 8.
    Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their reaction mechanisms. RSC Advances, 5, 27986–28006.CrossRefGoogle Scholar
  9. 9.
    Pietraforte, D., Turco, L., Azzini, E., & Minetti, M. (2002). On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein. Biochimica et Biophysica Acta, 1583, 176–184.CrossRefGoogle Scholar
  10. 10.
    Khan, F. A., Maalik, A., & Murtaza, G. (2016). Inhibitory mechanism against oxidative stress of caffeic acid. Journal of Food and Drug Analysis, 24, 695–702.CrossRefGoogle Scholar
  11. 11.
    Villano, D., Fernandez-Pachon, M. S., Troncoso, A. M., & Garcıa-Parrilla, M. C. (2005). Comparison of antioxidant activity of wine phenolic compounds and metabolites in vitro. Analytica Chimica Acta, 538, 391–398.CrossRefGoogle Scholar
  12. 12.
    Laranjinha, J., & Cadenas, E. (1999). Redox cycles of caffeic acid, α-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation. Life, 48, 57–65.Google Scholar
  13. 13.
    Bors, W., & Michel, C. (2002). Chemistry of the antioxidant effect of polyphenols. Annals of the New York Academy of Sciences, 957, 57–69.CrossRefGoogle Scholar
  14. 14.
    Hou, H., Grinberg, O. Y., Taie, S., Leichtweis, S., Miyake, M., Grinberg, S., Xie, H., Csete, M., & Swartz, H. M. (2003). Electron paramagnetic resonance assessment of brain. tissue oxygen tension in anesthetized rats. Anesthesia and Analgesia, 96, 1467–1472.CrossRefGoogle Scholar
  15. 15.
    James, P. E., Grinberg, O. Y., & Swartz, H. M. (1998). Superoxide production by phagocytosing macrophages in relation to the intracellular distribution of oxygen. Journal of Leukocyte Biology, 64, 78–84.CrossRefGoogle Scholar
  16. 16.
    Greenstock, C. L., & Miller, R. W. (1975). The oxidation of Tiron by superoxide anion: kinetics of the reaction in aqueous solution and in chloroplasts. Biochimica et Biophysica Acta, 396, 11–16.CrossRefGoogle Scholar
  17. 17.
    Otto, M., Stach, J., Kirmse, R., & Werner, G. (1981). The Tiron radical as indicator substance in catalytic determination of trace metals. Talanta, 28, 345–347.CrossRefGoogle Scholar
  18. 18.
    Ledenev, A. N., Konstantinov, A. A., Popova, E. Y., & Ruuge, E. K. (1986). A simple assay of the superoxide generation rate with Tiron as an EPR-visible radical scavenger. Biochemistry International, 13, 391–396.Google Scholar
  19. 19.
    Korkina, O. V., & Ruuge, E. K. (2000). Generation of superoxide radicals by heart mitochondria: a study by the method of spin trapping under conditions of constant oxygenation. Biofizika, 45, 695–699.Google Scholar
  20. 20.
    Bors, W., Saran, M., & Michel, C. (1979). Pulse-radiolytic investigations of catechols and catecholamines. II. Reactions of Tiron with oxygen radical species. Biochimica et Biophysica Acta, 582, 537–542.CrossRefGoogle Scholar
  21. 21.
    Pietta, P. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63, 1035–1042.CrossRefGoogle Scholar
  22. 22.
    Mura, F., Silva, T., Castro, C., Borges, F., Zuniga, M. C., Morales, J., & Olea-Azar, C. (2014). New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: Spectroscopic, electrochemistry, and cellular studies. Free Radical Research, 48, 1473–1484.CrossRefGoogle Scholar
  23. 23.
    Fernandez-Panchon, M. S., Villano, D., Troncoso, A. M., & Garcia-Parrilla, M. C. (2008). Antioxidant activity of phenolic compounds: from in vitro results to in vivo evidence. Critical Reviews in Food Science and Nutrition, 48, 649–671.CrossRefGoogle Scholar
  24. 24.
    Ghorbani, A. (2017). Mechanisms of antidiabetic effects of flavonoid rutin. Biomedicine & Pharmacotherapy, 96, 305–312.CrossRefGoogle Scholar
  25. 25.
    Karlsson, J., Emgard, M., Brundin, P., & Burkitt, M. J. (2000). trans-Resveratrol protects embryonic mesencephalic cells from tert-butyl hydroperoxide: electron paramagnetic resonance spin trapping evidence for a radical scavenging mechanism. Journal of Neurochemistry, 75, 141–150.CrossRefGoogle Scholar
  26. 26.
    Das, K. C., & Das, C. K. (2002). Curcumin (diferuloylmethane), a singlet oxygen (1O2) quencher. Biochemical and Biophysical Research Communications, 295, 62–66.CrossRefGoogle Scholar
  27. 27.
    Priyadarsini, K. I. (2013). Chemical and Structural Features Influencing the Biological Activity of Curcumin. Current Pharmaceutical Design, 11, 2093–2100.Google Scholar
  28. 28.
    Choudhury, A. K., Raja, S., Mahapatra, S., Nagabhushanam, K., & Majeed, M. (2015). Synthesis and evaluation of the anti-oxidant capacity of curcumin glucuronides, the major curcumin metabolites. Antioxidants, 4, 750–767.CrossRefGoogle Scholar
  29. 29.
    Sirota, R., Gibson, D. B., & Kohen, R. (2015). The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines. Redox Biology, 4, 48–59.CrossRefGoogle Scholar
  30. 30.
    Chouchani, E. T., Methner, C., Nadtochiy, S. M., Logan, A., Pell, V. R., Ding, S., James, A. M., Cochemé, H. M., Reinhold, J., Lilley, K. S., Partridge, L., Fearnley, I. M., Robinson, A. J., Hartley, R. C., Smith, R. A., Krieg, T., Brookes, P. S., & Murphy, M. P. (2013). Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I. Nature Medicine, 19, 753–759.CrossRefGoogle Scholar
  31. 31.
    Potapovich, A., & Kostyuk, V. A. (2003). Comparative study of antioxidant properties and cytoprotective activity of flavonoids. Biochemistry (Moscow), 68, 514–519.CrossRefGoogle Scholar
  32. 32.
    Sandoval-Acuna, C., Ferreira, J., & Speisky, H. (2014). Polyphenols and mitochondria: an update on their increasingly emerging ROS-scavenging independent actions. Archives of Biochemistry and Biophysics, 559, 75–90.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of PhysicsLomonosov Moscow State UniversityMoscowRussia
  2. 2.National Medical Research Centre for CardiologyMoscowRussia
  3. 3.Bach Institute of Biochemistry, Research Centre of BiotechnologyRussian Academy of SciencesMoscowRussia

Personalised recommendations