Effect of Principal Polyphenolic Components in Relation to Antioxidant Characteristics of Aged Red Wines Anis Arnous, † Dimitris P. Makris,* ,†,‡ and Panagiotis Kefalas †,‡ Department of Food Quality Management and Laboratory of Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (MAICh), P.O. Box 85, 73100 Chania, Greece Aged red wines possess significantly different polyphenolic composition compared with young ones, mainly due not only to formation of polymeric compounds but also because of oxidation, hydrolysis, and other transformations that may occur in native grape phenolics during aging. Representative Greek, single-variety, aged red wines were examined for total phenol, total flavanol, and total anthocyanin content using spectrophotometry, and attempts were made to establish correlations with the antiradical, reducing, and hydroxyl free radical scavenging activity. In addition, HPLC analyses were carried out, to ascertain whether individual polyphenols are actually responsible for the antioxidant effects of aged red wines. It was found that total flavanols are the class of polyphenols that account for hydroxyl free radical scavenging efficacy and to a lesser extent for antiradical and reducing ability, whereas there was a less significant link between the antioxidant properties and the total phenolics and only a weak relationship to total anthocyanin content. The correlation of the antioxidant properties with the principal polyphenols showed that individual compounds are weakly associated with all the antioxidant parameters, suggesting that the expression of antioxidant activity in aged red wines is rather a consequence of synergism between various phenolics, and it is not simply attributed to specific constituents. Keywords: Aged red wines; anthocyanins; antiradical activity; benzoic acids; hydroxycinnamates; hydroxyl free radical scavenging activity; flavanols; flavonols; polyphenols; reducing power INTRODUCTION In recent years much attention has been devoted to nutritional antioxidants and their association with suppressed rates of cardiovascular disorders and other health benefits. Many of these studies have focused on dietary phenolics and the epidemiological evidence that polyphenol-containing plant foods and products possess health-promoting effects have been based, to some extent, on their antioxidant properties. Red wine is an excellent source of various classes of polyphenols and may contain 1000-4000 mg L -1 of various phenols (1), which have different biological activities, includ- ing benzoic and cinnamic acid derivatives, flavanols, flavonols, and anthocyanins. Much effort has been expended on the analysis of red wine polyphenols and the relationship between polyphenol content and antioxidant capacity. The anti- oxidant properties of red wines have been correlated with their content of flavanols (2, 3), anthocyanins (4, 5), and tannic acid (6), although it is believed that the antioxidant properties of red wines are linked with the total polyphenol concentration (7, 8), rather than indi- vidual polyphenols. It should be noted, however, that maturation of red wines is a process that involves extensive transformation of native grape polyphenols, and aged wines contain a particularly complex mixture of simple and polymeric polyphenols, the nature of the latter being largely unknown. Nevertheless, there have been very few studies concerning the polyphenolic composition of aged red wines, and thus there is a lack of knowledge concerning the composition of polyphenolic antioxidants in wines having undergone aging. This is of prime importance considering that only a small number of red wines are consumed young, whereas the vast majority are aged for at least 12 months in oak barrels and 12 months in the bottle. Thus wines are available for consumption after they have reached the appropriate level of maturity. The present investigation was undertaken to deter- mine the polyphenolic composition of selected, Greek aged wines and aimed at examining the relationship of principal polyphenolic groups and individual poly- phenols to the in vitro antioxidant properties. For com- parison reasons, wines were from the same vintage, and each was made from single, unblended grape variety. MATERIALS AND METHODS Wines. Ten representative, high-quality, single-variety red wines from the 1998 vintage were tested. All wines examined were produced according to standard procedures and with defined varietal composition (Table 1). Samples covered some of the most important viticultural areas in Greece, are avail- able in the Greek market, and are widely consumed. All samples were stored at 10 °C in the dark and analyzed shortly after opening. Chemicals. Water used for HPLC analyses was Nanopure. Acetonitrile (MeCN) was of HPLC grade. Luminol (3-amino- phthalhydrazine), gallic acid, protocatechuic acid, p-hydroxy- benzoic acid, syringic acid, catechin, p-dimethylaminocinnam- aldehyde (DMACA), epicatechin, Folin-Ciocalteu reagent, rutin (quercetin 3-O-rutinoside), quercitrin (quercetin 3-O-rhamno- * Author to whom correspondence should be addressed (telephone +30 821 81151, ext 559; fax +30 821 81154; e-mail Dimitris@maich.gr). † Department of Food Quality Management. ‡ Laboratory of Chemistry of Natural Products. 5736 J. Agric. Food Chem. 2001, 49, 5736-5742 10.1021/jf010827s CCC: $20.00 © 2001 American Chemical Society Published on Web 12/17/2001