Champagne Wine Polyphenols Protect Primary Cortical Neurons against Peroxynitrite-Induced Injury DAVID VAUZOUR, ² KATERINA VAFEIADOU, ² GIULIA CORONA, ‡ SUSAN E. POLLARD, ² XENOFON TZOUNIS, ² AND JEREMY P. E. SPENCER* ,² Molecular Nutrition Group, School of Chemistry, Food and Pharmacy, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, United Kingdom, and Dipartimento di Biologia Sperimentale, Sez. Patologia Sperimentale, Universita ` degli Studi di Cagliari, 09042 Monserrato, Italy White wines are generally low in polyphenol content as compared to red wines. However, Champagne wines have been shown to contain relatively high amounts of phenolic acids that may exert protective cellular actions in vivo. In this study, we have investigated the potential neuroprotective effects of Champagne wine extracts, and individual phenolics present in these extracts, against peroxynitrite- induced injury. Organic and aqueous Champagne wine extracts exhibited potent neuroprotective activity against peroxynitrite-induced injury at low concentrations (0.1 µg/mL). This protection appeared to be in part due to the cellular actions of individual components found in the organic extracts, notably tyrosol, caffeic acid, and gallic acid. These phenolics were observed to exert potent neuroprotection at concentrations between 0.1 and 10 µM. Together, these data suggest that polyphenols present in Champagne wine may induce a neuroprotective effect against oxidative neuronal injury. KEYWORDS: Cortical neurons; protective effect; Champagne wine; peroxynitrite; phenolics INTRODUCTION Oxidative insults to neuronal cells have been implicated in the pathogenesis of neurodegenerative disorders such as Alzhe- imer’s and Parkinson’s diseases (1, 2). The generation of reactive oxygen species, such as superoxide (O 2 •- ) and hydrogen peroxide (H 2 O 2 ), in mitochondria or via the autoxidation of catecholamines has been proposed to contribute to neuronal injury (3). Furthermore, the colocalization of nitrogen monoxide (NO) and superoxide (O 2 •- ) within neurons may lead to the formation of peroxynitrite (ONOO - )(4), which may induce the oxidation of DNA, lipids, and protein sulfhydryls or the nitration of DNA and phenolic compounds such as tyrosine (5). Indeed, levels of 3-nitrotyrosine have been shown to be elevated in a variety of neurodegenerative diseases (6) and has been found in brain tissue from Parkinson’s disease patients (7), suggesting that peroxynitrite and other reactive nitrogen species may play a role in the pathophysiology of these diseases. There has been much recent interest in the potential of plant- derived polyphenols to protect against neuronal injury. Regular moderate consumption of red wine has been shown to be beneficial by counteracting cerebral aging (8) and by inducing cardioprotective effects (9). Flavonoids have been observed to protect against both age-related cognitive and motor decline (10) and against 6-hydroxydopamine neurotoxicity and MPTP le- sioning of the nigrostriatal tract (11). Although polyphenols such as flavonoids are powerful hydrogen-donating antioxidants and scavengers of reactive nitrogen species in vitro (12), recent findings demonstrate a role for specific flavonoids in interacting selectively within signaling cascades that regulate neuronal survival following exposure to oxidative stress (13). For example, accumulating evidence suggests that flavonoids might exert neuroprotective effects at nanomolar concentrations through the selective modulation of both protein kinase and lipid kinase signaling cascades, such as tyrosine kinase, PI 3-kinase/ Akt, PKC, and mitogen-activated protein (MAP) kinase path- ways (14). This, along with evidence that dietary polyphenols can cross the blood brain barrier (15), suggests that such dietary components have the potential to act within the central nervous system. Champagne wine has been shown to contain relatively high amounts of phenolics such as tyrosol and caffeic acid (16). Such phenolics have not been thoroughly investigated for their ability to modulate oxidative stress-induced cellular injury. In this study, we initially investigated the potential neuroprotective effects of organic vs aqueous extracts of Champagne wine in order to gain an insight into which individual components hold the most neuroprotective potential. These major polyphenolic constituents, notably tyrosol, caffeic acid, and gallic acid, were then investigated for their ability to protect against peroxynitrite- induced neuronal injury. MATERIALS AND METHODS Chemical and Reagents. Sodium nitrite, manganese dioxide, hydrogen peroxide solution (30%, wt/vol), gallic acid, p-coumaric acid, and caffeic acid were from Sigma Chemical Co. (Poole, United * To whom correspondence should be addressed. Tel: +44 118 378 8724. Fax: +44118 931 0080. E-mail: j.p.e.spencer@reading.ac.uk. ² The University of Reading. ‡ Universita ` degli Studi di Cagliari. 2854 J. Agric. Food Chem. 2007, 55, 2854-2860 10.1021/jf063304z CCC: $37.00 © 2007 American Chemical Society Published on Web 03/24/2007