European Journal of Nutrition

, Volume 48, Issue 1, pp 54–61 | Cite as

Neuroprotective effect of cocoa flavonids on in vitro oxidative stress

  • Emma Ramiro-Puig
  • Gemma Casadesús
  • Hyoung-gon Lee
  • Xiongwei Zhu
  • Andrew McShea
  • George Perry
  • Francisco J. Pérez-Cano
  • Mark A. Smith
  • Margarida Castell
ORIGINAL CONTRIBUTION

Abstract

Background

Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways.

Aim of the study

To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line.

Methods

We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis.

Results

Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%.

Conclusions

Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation.

Keywords

cocoa c-JNK MAPK neuronal damage oxidative stress 

Abbreviations

AD

Alzheimer’s disease

ANOVA

Analysis of variance

Amyloid-β protein

DA

diacetate

DCF

2′,7′-dichlorofluorescein

DMSO

Dimethyl sulfoxide

ERK 1/2

Extracellular signal-regulated kinases 1/2

GSTPi

Glutathione-S-transferase enzyme-class Pi

H2DCF

reduced DCF

JNK

c-Jun N-terminal kinase

LDH

Lactate dehydrogenase

MAPK

Mitogen-activated protein kinases

ORAC

Oxygen radical absorbance capacity

PMSF

Phenylmethylsulfonyl fluoride

ROS

Reactive oxygen species

RT

Room temperature

SOD

Superoxide dismutase

TBS

Tris buffered saline solution

TBST

TBS with 0.1% Tween 20

Notes

Acknowledgments

E.R. is the recipient of fellowships from the Generalitat de Catalunya (2003FI 00578 and 2005BE 00709).

References

  1. 1.
    Andrés-Lacueva C, Lamuela-Raventós RM, Jáuregui O (2000) An LC method for the analysis of cocoa phenolics. LC-GC Eur 12:902–905Google Scholar
  2. 2.
    Canas N, Valero T, Villarroya M, Montell E, Verges J, Garcia AG, Lopez MG (2007) Chondrotitin sulfate protects SH-SY5Y cells from oxidative stress. J Pharmacol Exp Ther 323:946–953CrossRefGoogle Scholar
  3. 3.
    Casadesús G, Ramiro-Puig E, Webber KM, Atwood CS, Castell Escuer M, Bowen RL, Perry G, Smith MA (2006) Targeting gonadotropins: an alternative option for Alzheimer disease treatment. J Biomed Biotechnol 39508:1–8CrossRefGoogle Scholar
  4. 4.
    Cho ES, Lee KW, Lee HJ (2008) Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK. Mutat Res 640:123–130Google Scholar
  5. 5.
    Commenges D, Scotet V, Renaud S, Jacqmin-Gadda H, Barberger-Gateau P, Dartigues J-F (2000) Intake of flavonoids and risk of dementia. Eur J Epidemiol 16:357–363CrossRefGoogle Scholar
  6. 6.
    Cooper KA, Campos-Giménez E, Jiménez Alvarez D, Nagy K, Donovan JL, Williamson G (2007) Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentrations in chocolate. J Agric Food Chem 55:2841–2847CrossRefGoogle Scholar
  7. 7.
    Cooper KA, Donovan JL, Waterhouse AL, Williamson G (2008) Cocoa and health: a decade of research. Br J Nutr 99:1–11CrossRefGoogle Scholar
  8. 8.
    Crews WD Jr, Harrison DW, Wright JW (2008) A double-blind, placebo-controlled, randomized trial of the effects of dark chocolate and cocoa on variables associated with neuropsychological functioning and cardiovascular health: clinical findings from a sample of healthy, cognitively intact older adults. Am J Clin Nutr 87:872–880Google Scholar
  9. 9.
    Dai Q, Borenstein AR, Wu Y, Jackson JC, Larson EB (2006) Fruit and vegetable juices and Alzheimer’s disease: the Kame project. Am J Med 119:751–759CrossRefGoogle Scholar
  10. 10.
    Datla KP, Zbarsky V, Rai D, Parkar S, Osakabe N, Aruoma OI, Dexter DT (2007) Short-term supplementation with plant extracts rich in flavonoids protect nigrostriatal dopaminergic neurons in a rat model of Parkinson’s disease. J Am Coll Nutr 26:341–349Google Scholar
  11. 11.
    de Boer VC, Dihal AA, van der Woude H, Arts IC, Wolffram S, Alink GM, Rietjens IM, Keijer J, Hollman PC (2005) Tissue distribution of quercetin in rats and pigs. J Nutr 135:1718–1725Google Scholar
  12. 12.
    Erlejman AG, Fraga CG, Oteiza PI (2006) Procyanidins protect caco-2 cells from bile acid- and oxidant-induced damage. Free Radic Biol Med 41:1247–1256CrossRefGoogle Scholar
  13. 13.
    Granado-Serrano AB, Martin MA, Bravo L, Goya L, Ramos S (2006) Quercetin induces apoptosis via caspase activation, regulation of bcl-2, and inhibition of PI-3-kinase/Akt and ERK pathways in a human hepatoma cell line (HepG2). J Nutr 136:2715–2721Google Scholar
  14. 14.
    Granado-Serrano AB, Martin MA, Izquierdo-Pulido M, Goya L, Bravo L, Ramos S (2007) Molecular mechanisms of (-)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line. J Agric Food Chem 55:2020–2027CrossRefGoogle Scholar
  15. 15.
    Heo HJ, Lee CY (2005) Epicatechin and catechin in cocoa inhibit amyloid beta protein induced apoptosis. J Agric Food Chem 53:1445–1448CrossRefGoogle Scholar
  16. 16.
    Kamata H, Hirata H (1999) Redox regulation of cellular signalling. Cell Signal 11:1–14CrossRefGoogle Scholar
  17. 17.
    Lecumberri E, Goya L, Mateos R, Alía M, Ramos S, Izquierdo-Pulido M, Bravo L (2007) A diet rich in dietary fiber from cocoa improves lipid profile and reduces malondialdehyde in hypercholesterolemic rats. Nutrition 23:332–341CrossRefGoogle Scholar
  18. 18.
    McEligot AJ, Yang S, Meyskens FL Jr (2005) Redox regulation by intrinsic species and extrinsic nutrients in normal and cancer cells. Annu Rev Nutr 25:261–295CrossRefGoogle Scholar
  19. 19.
    McShea A, Ramiro-Puig E, Munro SB, Casadesus G, Castell M, Smith MA (2008) Clinical benefit and preservation of flavonols in dark chocolate manufacturing. Nutr Rev 66:630–641CrossRefGoogle Scholar
  20. 20.
    Miller KB, Stuart DA, Smith NL, Lee CY, McHale NL, Flanagan JA, Ou B, Hurst WJ (2006) Antioxidant activity and polyphenol and procyanidin contents of selected commercially available cocoa-containing and chocolate products in the United States. J Agric Food Chem 54:4062–4068CrossRefGoogle Scholar
  21. 21.
    Natsume M, Osakabe N, Yasuda A, Baba S, Tokunaga T, Kondo K, Osawa T, Terao J (2004) In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic Res 38:1341–1348CrossRefGoogle Scholar
  22. 22.
    Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, Van Norren K, Van Leeuwen PAM (2001) Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr 74:418–425Google Scholar
  23. 23.
    Pensalfini A, Cecchi C, Zampagni M, Becatti M, Favilli F, Poli P, Catarzi S, Bagnoli S, Nacmias B, Sorbi S, Liguri G (2008) Protective effects of new derivatives against amyloid-induced oxidative stress. Free Radic Biol Med 44:1624–1636CrossRefGoogle Scholar
  24. 24.
    Pereda J, Sabater L, Cassinello N, Gómez-Cambronero L, Closa D, Folch-Puy E, Aparisi L, Calvete J, Cerda M, Lledo S, Vina J, Sastre J (2004) Effect of simultaneous inhibition of TNF-alpha production and xanthine oxidase in experimental acute pancreatitis: the role of mitogen activated protein kinases. Ann Surg 240:108–116CrossRefGoogle Scholar
  25. 25.
    Porter LJ, Ma Z, Chan BG (1991) Cacao procyanidins: major flavanoids and identification of some minor metabolites. Phytochemistry 30:1657–1663CrossRefGoogle Scholar
  26. 26.
    Pura Naik J (2001) Improved high-performance liquid chromatography method to determine theobromine and caffeine in cocoa and cocoa products. J Agric Food Chem 49:3579–3583CrossRefGoogle Scholar
  27. 27.
    Ramiro E, Franch A, Castellote C, Andrés-Lacueva C, Izquierdo-Pulido M, Castell M (2005) Effect of theobroma cacao flavonoids on immune activation of a lymphoid cell line. Br J Nutr 93:859–866CrossRefGoogle Scholar
  28. 28.
    Ramiro E, Franch A, Castellote C, Pérez-Cano FJ, Permanyer J, Izquierdo-Pulido M, Castell M (2005) Flavonoids from Theobroma cacao down-regulate inflammatory mediators. J Agric Food Chem 53:8506–8511CrossRefGoogle Scholar
  29. 29.
    Ramiro-Puig E, Urpi-Sarda M, Pérez-Cano FJ, Franch A, Castellote C, Andres-Lacueva C, Izquierdo-Pulido M, Castell M (2007) Cocoa-enriched diet enhances antioxidant enzyme activity and modulates lymphocyte composition in thymus from young rats. J Agric Food Chem 55:6431–6438CrossRefGoogle Scholar
  30. 30.
    Ramos S, Alia M, Bravo L, Goya L (2005) Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2). J Agric Food Chem 53:1271–1280CrossRefGoogle Scholar
  31. 31.
    Raza H, John A (2005) Green tea polyphenol epigallocatechin-3-gallate differentially modulates oxidative stress in PC12 cell compartments. Toxicol Appl Pharmacol 207:212–220Google Scholar
  32. 32.
    Ruffels J, Griffin M, Dickenson JM (2004) Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death. Eur J Pharmacol 483:163–173CrossRefGoogle Scholar
  33. 33.
    Sánchez-Rabaneda F, Jáuregui O, Casals I, Andrés-Lacueva C, Izquierdo-Pulido M, Lamuela-Raventos RM (2003) Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao). J Mass Spectrom 38:35–42CrossRefGoogle Scholar
  34. 34.
    Schauss AG, Wu X, Prior RL, Ou B, Huang D, Owens J, Agarwal A, Jensen GS, Hart AN, Shanbrom E (2006) Antioxidant capacity and other bioactivities of the freeze-dried Amazonian palm berry, Euterpe oleraceae mart. (acai). J Agric Food Chem 54:8604–8610CrossRefGoogle Scholar
  35. 35.
    Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, Tabaton M, Perry G (1998) Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem 70:2212–2215Google Scholar
  36. 36.
    Spencer JP, Abd-el-Mohsen MM, Rice-Evans C (2004) Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch Biochem Biophys 423:148–161CrossRefGoogle Scholar
  37. 37.
    Tomas-Barberan FA, Cienfuegos-Jovellanos E, Marín A, Muguerza B, Gil-Izquierdo A, Cerda B, Zafrilla P, Morillas J, Mulero J, Ibarra A, Pasamar MA, Ramón D, Espín JC (2007) A new process to develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy humans. J Agric Food Chem 55:3926–3935CrossRefGoogle Scholar
  38. 38.
    Uberti D, Piccioni L, Colzi A, Bravi D, Canonico PL, Memo M (2002) Pergolide protects SH-SY5Y cells against neurodegeneration induced by H2O2. Eur J Pharmacol 434:17–20CrossRefGoogle Scholar
  39. 39.
    Waetzig V, Herdegen T (2005) Context-specific inhibition of JNKs: overcoming the dilemma of protection and damage. Trends Pharmacol Sci 26:455–461Google Scholar
  40. 40.
    Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616CrossRefGoogle Scholar
  41. 41.
    Wollgast J, Anklam E (2000) Review on polyphenols in Theobroma cacao: changes in composition during the manufacture of chocolate and methodology for identification and quantification. Food Res Int 33:423–447CrossRefGoogle Scholar
  42. 42.
    Yilmaz Y, Toledo RT (2004) Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid. J Agric Food Chem 52:255–260CrossRefGoogle Scholar
  43. 43.
    Youdim KA, Dobbie MS, Kuhnle G, Proteggente AR, Abbott NJ, Rice-Evans C (2003) Interaction between flavonoids and the blood-brain barrier: in vitro studies. J Neurochem 85:180–192CrossRefGoogle Scholar

Copyright information

© Spinger 2008

Authors and Affiliations

  • Emma Ramiro-Puig
    • 1
  • Gemma Casadesús
    • 2
  • Hyoung-gon Lee
    • 3
  • Xiongwei Zhu
    • 3
  • Andrew McShea
    • 4
  • George Perry
    • 3
    • 5
  • Francisco J. Pérez-Cano
    • 1
  • Mark A. Smith
    • 3
  • Margarida Castell
    • 1
  1. 1.Dept. de FisiologiaFacultat de Farmàcia (Universitat de Barcelona)BarcelonaSpain
  2. 2.Dept. of NeuroscienceCase Western Reserve UniversityClevelandUSA
  3. 3.Dept. of PathologyCase Western Reserve UniversityClevelandUSA
  4. 4.Research and DevelopmentTheo ChocolateSeattleUSA
  5. 5.College of SciencesUniversity of Texas at San AntonioSan AntonioUSA

Personalised recommendations