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EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Abstract

Resveratrol (3,4′,5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.

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Abbreviations

DEPMPO:

5-(Diethoxyphosphoryl)-5-methylpyrroline-N-oxide

DEPMPO-OH:

DEPMPO-hydroxyl adduct

DEPMPO-OOH:

DEPMPO-superoxide adduct

DMF:

Dimethylformamide

EPR:

Electron paramagnetic resonance

IC50:

The concentration eliciting 50% of the maximum inhibition

DTPA:

Diethylenetriaminepentaacetic acid

KO2 :

Potassium superoxide

PBS:

Phosphate-buffered saline

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

References

  1. Fremont L (2000) Biological effects of resveratrol. Life Sci 66:663–673

    PubMed  Article  CAS  Google Scholar 

  2. Bradamante S, Barenghi L, Villa A (2004) Cardiovascular protective effects of resveratrol. Cardiovasc Drug Rev 22:169–188

    PubMed  CAS  Google Scholar 

  3. Gusman J, Malonne H, Atassi G (2001) A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis 22:1111–1117

    PubMed  Article  CAS  Google Scholar 

  4. Virgili M, Contestabile A (2000) Partial neuroprotection of in vivo excitotoxic brain damage by chronic administration of the red wine antioxidant agent, trans-resveratrol in rats. Neurosci Lett 281:123–126

    PubMed  Article  CAS  Google Scholar 

  5. Jang M, Pezzuto JM (1998) Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin. Cancer Lett 134:81–89

    PubMed  Article  CAS  Google Scholar 

  6. Cao Z, Li Y (2004) Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury. Eur J Pharmacol 489:39–48

    PubMed  Article  CAS  Google Scholar 

  7. Freeman BA, Crapo JD (1982) Biology of disease: free radicals and tissue injury. Lab Invest 47:412–426

    PubMed  CAS  Google Scholar 

  8. Fantone JC, Ward PA (1985) Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. Hum Pathol 16:973–978

    PubMed  CAS  Article  Google Scholar 

  9. Kong LD, Wolfender JL, Cheng CH, Hostettmann K, Tan RX (1999) Xanthine oxidase inhibitors from Brandisia hancei. Planta Med 65:744–746

    PubMed  Article  CAS  Google Scholar 

  10. Floyd RA (1990) Role of oxygen free radicals in carcinogenesis and brain ischemia. Faseb J 4:2587–2597

    PubMed  CAS  Google Scholar 

  11. Shigematsu S, Ishida S, Hara M, Takahashi N, Yoshimatsu H, Sakata T, Korthuis RJ (2003) Resveratrol, a red wine constituent polyphenol, prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion, platelet-activating factor, or oxidants. Free Radic Biol Med 34:810–817

    PubMed  Article  CAS  Google Scholar 

  12. Leonard SS, Xia C, Jiang BH, Stinefelt B, Klandorf H, Harris GK, Shi X (2003) Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem Biophys Res Commun 309:1017–1026

    PubMed  Article  CAS  Google Scholar 

  13. Reiter CD, Teng RJ, Beckman JS (2000) Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 275:32460–32466

    PubMed  Article  CAS  Google Scholar 

  14. Li Y, Kuppusamy P, Zweir JL, Trush MA (1996) Role of Cu/Zn-superoxide dismutase in xenobiotic activation II. Biological effects resulting from the Cu/Zn-superoxide dismutase-accelerated oxidation of the benzene metabolite 1,4-hydroquinone. Mol Pharmacol 49:412–421

    PubMed  CAS  Google Scholar 

  15. Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier JL, Trush MA (1998) Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 273:2015–2023

    PubMed  Article  CAS  Google Scholar 

  16. Fridovich I (1970) Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem 245:4053–4057

    PubMed  CAS  Google Scholar 

  17. Frejaville C, Karoui H, Tuccio B, Le Moigne F, Culcasi M, Pietri S, Lauricella R, Tordo P (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals. J Med Chem 38:258–265

    PubMed  Article  CAS  Google Scholar 

  18. Zweier JL (1988) Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem 263:1353–1357

    PubMed  CAS  Google Scholar 

  19. Wattanapitayakul SK, Bauer JA (2001) Oxidative pathways in cardiovascular disease: roles, mechanisms, and therapeutic implications. Pharmacol Ther 89:187–206

    PubMed  Article  CAS  Google Scholar 

  20. Griendling KK, FitzGerald GA (2003) Oxidative stress and cardiovascular injury: Part II: animal and human studies. Circulation 108:2034–2040

    PubMed  Article  Google Scholar 

  21. Molavi B, Mehta JL (2004) Oxidative stress in cardiovascular disease: molecular basis of its deleterious effects, its detection, and therapeutic considerations. Curr Opin Cardiol 19:488–493

    PubMed  Article  Google Scholar 

  22. Kehrer JP (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 23:21–48

    PubMed  Article  CAS  Google Scholar 

  23. Cai L, Kang YJ (2001) Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovasc Toxicol 1:181–193

    PubMed  Article  CAS  Google Scholar 

  24. Datta K, Sinha S, Chattopadhyay P (2000) Reactive oxygen species in health and disease. Natl Med J India 13:304–310

    PubMed  CAS  Google Scholar 

  25. Storey KB (1996) Oxidative stress: animal adaptations in nature. Braz J Med Biol Res 29:1715–1733

    PubMed  CAS  Google Scholar 

  26. Das S, Falchi M, Bertelli A, Maulik N, Das DK (2006) Attenuation of ischemia/reperfusion injury in rats by the anti-inflammatory action of resveratrol. Arzneimittelforschung 56:700–706

    PubMed  CAS  Google Scholar 

  27. Valentine JS, Curtis AB (1975) A convenient preparation of solutions of superoxide anion and the reaction of superoxide anion with a copper(II) complex. J Am Chem Soc 97:224–226

    PubMed  Article  CAS  Google Scholar 

  28. Timmins GS, Liu KJ, Bechara EJ, Kotake Y, Swartz HM (1999) Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*. Free Radic Biol Med 27:329–333

    PubMed  Article  CAS  Google Scholar 

  29. Delmas-Beauvieux MC, Peuchant E, Thomas MJ, Dubourg L, Pinto AP, Clerc M, Gin H (1998) The place of electron spin resonance methods in the detection of oxidative stress in type 2 diabetes with poor glycemic control. Clin Biochem 31:221–228

    PubMed  Article  CAS  Google Scholar 

  30. Lubrano V, Di Cecco P, Zucchelli GC (2006) Role of superoxide dismutase in vascular inflammation and in coronary artery disease. Clin Exp Med 6:84–88

    PubMed  Article  CAS  Google Scholar 

  31. Darley-Usmar V, White R (1997) Disruption of vascular signalling by the reaction of nitric oxide with superoxide: implications for cardiovascular disease. Exp Physiol 82:305–316

    PubMed  CAS  Google Scholar 

  32. Heunks LM, Vina J, van Herwaarden CL, Folgering HT, Gimeno A, Dekhuijzen PN (1999) Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease. Am J Physiol 277:R1697–R1704

    PubMed  CAS  Google Scholar 

  33. Battelli MG, Musiani S, Valgimigli M, Gramantieri L, Tomassoni F, Bolondi L, Stirpe F (2001) Serum xanthine oxidase in human liver disease. Am J Gastroenterol 96:1194–1199

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by NIH R01 HL71190 (YL) and a grant from Harvey Peters Foundation (HM).

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Correspondence to Hara P. Misra.

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Jia, Z., Zhu, H., Misra, B.R. et al. EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol. Mol Cell Biochem 313, 187 (2008). https://doi.org/10.1007/s11010-008-9756-y

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  • DOI: https://doi.org/10.1007/s11010-008-9756-y

Keywords

  • EPR
  • Resveratrol
  • Superoxide
  • Xanthine oxidase
  • Oxygen consumption