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Correlation of Pigment and Flavanol Content with Antioxidant Properties in Selected Aged Regional Wines from Greece
JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2002) 15, 655–665 doi:10.1006/jfca.2002.1070 Available online at http://www.idealibrary.com on
ORIGINAL ARTICLE Correlation of Pigment and Flavanol Content with Antioxidant Properties in Selected Aged Regional Wines from Greece Anis Arnous*, Dimitris P. Makris*,w,1, and Panagiotis Kefalas*,w *Department of Food Quality Management, Mediterranean Agronomic Institute of Chania (MAICh), P.O. Box 85, 73100 Chania, Greece; and wLaboratory of Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (MAICh), P. O. Box 85, 73100 Chania, Greece Received August 14, 2001 and in revised form November 21, 2001
Selected Greek regional, aged red wines were assessed for their polyphenolic composition using well-established spectrophotometric methodology, and tested for antioxidant potential by three different assays. The total polyphenol (TP) concentration was found to vary from 1217 to 3772 mg L1 gallic acid equivalents, total anthocyanin (TA) content ranged from 44.3 to 360.1 mg L1, and total flavanol (TF) from 291.1 to 664.8 mg L1 catechin equivalents. The antiradical activity was evaluated using DPPHK, and varied from 0.73 to 1.37 mm Troloxs equivalents, while reducing power values were from 5.29 to 10.80 mm ascorbic acid equivalents, using a modified FRAP assay. The hydroxyl free radical scavenging activity was determined with the deoxyribose method, and ranged from 38.5 to 59.9% decrease in A532 relative to control. The examinations showed that TP and TF may provide a significant contribution to the overall antioxidant status of wines, but TA appears to be a less important factor in this respect. Reducing power was found to be highly associated with the antiradical activity of wines. r 2002 Elsevier Science Ltd. All rights reserved. Key Words: aged red wines; anthocyanins; antiradical activity; flavanols; hydroxyl free radical scavenging activity; reducing power.
INTRODUCTION Phenolic substances are naturally present in essentially all plant material, including food of plant origin. Thus, polyphenolic substances are ubiquitous in vegetables, cereals, fruits, nuts, but also in plant products such as wine, cider, beer, tea, cocoa, etc. Wines in particular constitute an excellent source of dietary polyphenols, as they may contain from 200–300 (white wines) to 1000–4000 (red wines) mg L 1 total phenols (Bravo, 1998). Red wine, however, appears to exhibit biological effects that are usually not seen with white wine, which may be attributed to the large amounts of flavonoids, particularly anthocyanins, flavanols, and flavanol derivatives (proanthocyanidins) (Cao and Prior, 2000). 1 To whom correspondence and reprint requests should be addressed. Tel.: +30-821-81151, ext. 559. Fax: +30-821-81154. E-mail: [email protected]
0889-1575/02/060655 + 11 $35.00/0
r 2002 Elsevier Science Ltd. All rights reserved.
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Since the claim that flavonoids could have a vitamin function, their biological effects have been mainly interpreted on the basis of their antioxidant properties, that are related to both free radical scavenging and transition metal chelating (Ursini et al., 1999). Flavonoids may be effective antioxidants in a wide range of systems, being capable of scavenging peroxyl radicals, alkyl peroxyl radicals, superoxide, hydroxyl radicals, nitric oxide and peroxynitrile, in aqueous and organic environments. Further, supporting the possibility that certain polyphenols may have antioxidant function in vivo are several rodent feeding studies indicating that phenolic-rich extracts of red wine lower oxidative products such as protein carbonyls, DNA base damage and malonaldehyde in blood and a range of tissues (Duthie et al., 2000). As regards anthocyanins, they have been proven very efficient antioxidants in a number of systems (Rice-Evans et al., 1995; van Acker et al., 1996; Wang et al., 1997, 1999; Heinonen et al., 1998; Degenhardt et al., 2000; Espı´ n et al., 2000), and in vitro LDL oxidation inhibitors (Satue´-Gracia et al., 1997), while other studies demonstrated a relationship between anthocyanin content and antioxidant activities of red grape extracts (Meyer et al., 1997), grape juices (Frankel et al., 1998) and various red wines (Sato et al., 1996; Sa´nchez-Moreno et al., 2000). Other investigations, however, indicated that anthocyanins may be less significantly correlated with the antioxidant properties of red wines (Frankel et al., 1995; Burns et al., 2000). Flavanols have also been shown to exhibit powerful antioxidant activities in different environments (Ricardo da Silva et al., 1991; Rice-Evans et al., 1995; Salah et al., 1995; Heinonen et al., 1998; Kondo et al., 1999; Bors et al., 2000), and the antioxidant abilities of red grapes (Teissedre et al., 1996; Meyer et al., 1997), juices (Frankel et al., 1998) and wines (Frankel et al., 1995; Sato et al., 1996; Teissedre et al., 1996; Simonetti et al., 1997; Burns et al., 2000) have always been correlated with the flavanol content. Greek regional wines are produced from native but also the so-called ‘international’ cultivars, including Cabernet Sauvignon, Merlot, Syrah, Grenache, etc. Most wines are produced by co-vinification of grapes deriving from more than one cultivars, whereas single-variety regional wines are of more limited occurrence in the Greek market. There is a considerable lack of information with regard to polyphenolic composition of Greek red wines and as a result the antioxidant capacity, which is related to polyphenolic content, has not been examined. The present study represents an approach with respect to determining the anthocyanin and flavanol content of selected, aged red wines, and aims at investigating correlation of these two classes of flavonoids with the in vitro antioxidant potential, as this may be illustrated by the antiradical activity, the reducing power, and the hydroxyl free radical scavenging activity.
MATERIALS AND METHODS Wines Fifteen representative, high-quality, regional wines were examined, which are produced according to standard procedures, and with defined varietal composition (Table 1). For comparison reasons, all samples examined were of 1998 vintage (3 years old). Wines cover all viticultural areas of Greece, they are available in the Greek market, and widely consumed. All samples were stored at 101C in obscurity, and analysed shortly after opening.
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TABLE 1 Origin and varietal composition of the wines tested Appellation Kritikos Topikos Macedonikos Topikos Peloponissiakos Topikos Topikos Dramas Topikos Chalcidikis Topikos Florinas Topikos Imathias Topikos Letrinon Topikos Op. Lokridos Topikos Plagion Egialias Topikos Stereas Elladas Topikos Tegeas
Cultivar(s)
Location
Kotsifali/Mandilaria/Liatiko Xinomavro Agiorgitiko/Cab. Sauv. Cab. Sauv./Cab. Franc/Merlot/Limnio Merlot Xinomavro/Merlot Syrah Refosco/Mavrodafni Cab. Sauv./Xinomavro/Limnio Cab. Sauv. Syrah/Grenache/Carignan/Cab. Sauv. Cab. Sauv./Merlot
Crete (S) Macedonia (N) Peloponese (S) Macedonia (N) Macedonia (N) Macedonia (N) Macedonia (N) Peloponese (S) Sterea Ellada (C) Peloponese (S) Sterea Ellada (C) Peloponese (S)
Note: Letters N, C, and S assign north, central and south Greece, respectively.
Chemicals Ascorbic acid, catechin, p-dimethylaminocinnamaldehyde (DMACA), 2,2-diphenylb-picrylhydrazyl (DPPHK) radical, EDTA (disodium salt), gallic acid, 2,4,6tripyridyl-s-triazine (TPTZ), and sodium metabisulphite were from Sigma Chemical Co. (St Louis, MO). 2-Deoxyribose was from Fluka. Citric acid, ferric chloride hexahydrate (FeCl3 ? 6H2O), Folin-Ciocalteu reagent, hydrogen peroxide, iron ammonium sulphate, thiobarbituric acid (TBA), and Troloxs were from Aldrich (Steinheim, Germany). Trichloroacetic acid (TCA) was from Riedel-de Hae¨n (Germany). Determination of Total Polyphenols (TP) Total polyphenol content of wines was determined using the Folin-Ciocalteu method (Waterman and Mole, 1994), adapted to a micro scale. In a 1.5-mL Eppendorf tube, 0.79 mL distilled water, 0.01 mL sample appropriately diluted, and 0.05 mL FolinCiocalteu reagent were added and vortexed. After exactly 1 min, 0.15 mL of sodium carbonate (20%) was added, and the mixture was vortexed and allowed to stand at room temperature in obscurity, for 120 min. The absorbance was read at 750 nm, and the total polyphenol concentration was calculated from a calibration curve (r2 = 0.9990), using gallic acid as standard (50–800 mg L1). Results were expressed as mg L1 gallic acid equivalents (GAE). Determination of Total and Coloured Anthocyanins Measurements were performed using well-established spectrophotometric methodology (Somers and Evans, 1977; Zoecklein et al., 1990). Analytically, wine sample was placed in a 0.2-cm path length quartz cuvette, and the absorbance was measured at 520 nm (A520). Following this, 0.02 mL of a 20% sodium metabisulphite solution was added, the sample was mixed well, and after 1 min the absorbance was read at 2 520 nm ASO 520 : A 12% ethanolic solution was used as blank. All measurements were corrected to a 1.0-cm path length. Further, wine (0.02 mL) was mixed with 0.98 mL 1 n HCl solution (dilution 1:50) in a 1.5-mL Eppendorf tube, vortexed, and allowed to stand for 180 min at room temperature. The absorbance was read at 520 nm
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ðAHCl 520 Þ; using a 1.0-cm path length cuvette. For the blank, 0.02 mL of a 12% ethanolic solution was used instead of wine. The concentration of total anthocyanins (TA) and coloured (ionized) anthocyanins (CA) was calculated as follows: SO2 TA ðmg L1 Þ¼ 20 ½AHCl 520 ð5=3Þ A520 ; 2 CA ðmg L1 Þ¼ 20 ðA520 ASO 520 Þ:
Determination of Total Flavanols (TF) The total flavanol content was estimated using the p-dimethylaminocinnamaldehyde (DMACA) method (Vivas et al., 1994; Li et al., 1996; McMorrough et al., 1996). This method has a great advantage over the widely used vanillin assay, since there is no interference by anthocyanins. Further, it provides higher sensitivity and specificity (Li et al., 1996). Wine (0.2 mL), diluted 1:100 with MeOH, was introduced into a 1.5-mL Eppendorf tube and added 1 mL DMACA solution (0.1% in 1 n HCl in MeOH). The mixture was vortexed and allowed to react at room temperature for 10 min. Following this, the absorbance at 640 nm was read against blank prepared similarly without DMACA. The concentration of TF was estimated from a calibration curve, constructed by plotting known solutions of catechin (1–16 mg L1) against A640 (r2 = 0.9987). Results were expressed as catechin equivalents (mg L1 CTE). Measurement of the Antiradical Activity (AAR) All samples were diluted 1:10 with MeOH immediately before the analysis. An aliquot of 0.025 mL of diluted sample was added to 0.975 mL DPPHK solution (60 mm in MeOH), vortexed, and the absorbance was read at t = 0 and 30 min. Results were expressed as Troloxs equivalents (mm TRE) using the following equation: AAR ðmm TREÞ ¼ 0:018 %A515 þ 0:017 as determined from linear regression, after plotting %DA515 of known solutions of Troloxs against concentration (0.08–1.28 mM, r2 = 0.9935), where %DA515 = [(A515(0) – A515(30))/A515(0)] 100. Measurement of the Hydroxyl Free Radical Scavenging Activity (SAHFR) For the determination of the hydroxyl free radical scavenging capacity of wines, the deoxyribose method was used, as described by Ghiselli et al. (1998), slightly modified. Wine sample (0.05 mL) was mixed with 0.345 mL of 10 mm phosphate buffer, pH 7.4, containing 2.5 mm 2-deoxyribose. An aliquot of 0.05 mL of 2 mm iron ammonium sulphate premixed with 2.08 mm EDTA was added, the mixture was vortexed, and the reaction was initiated by adding 0.05 mL 1 mm ascorbic acid and 0.02 mL 1.5 mm hydrogen peroxide. Samples were maintained at 371C for 30 min, and then added 0.5 mL 30% TCA and 0.5 mL 1% TBA (in 0.05 n NaOH). Samples were heated at 901C for 20 min, cooled, and the absorbance at 532 nm was measured. Control sample was prepared by adding distilled water instead of wine. Hydroxyl free radical scavenging activity (SAHFR) was calculated as the % reduction in A532 relative to control. All wine samples were diluted 1:200 with buffer just before the analysis, to achieve a mean total phenol concentration of approximately 10 mg L1 GAE.
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Measurement of the Reducing Power (PR) For the determination of the reducing ability of wines a protocol based on the ferric reducing/antioxidant power (FRAP) assay was developed, as described previously (Benzie and Strain, 1996; Pulido et al., 2000), with modifications. Wine (0.05 mL), diluted 1:10 with distilled water, and 0.05 mL of ferric chloride (3 mm in 5 mm citric acid) were mixed well in a 1.5-mL Eppendorf tube, and incubated for 30 min in a water bath at 371C. Following this, the mixture was added to 0.90 mL of 1 mm TPTZ solution in 0.05 m HCl, and vortexed. After exactly 10 min the absorbance was read at 620 nm. PR was calculated from a calibration curve, established by plotting known amounts of ascorbic acid against A620. Results were expressed as ascorbic acid equivalents (mm AAE) using the following equation: PR ðmm AAEÞ ¼ ð0:679 A620 0:008Þ FD ; where FD is the dilution factor. For the blanks, distilled water was added instead of ferric chloride/citric acid. One blank was prepared for each wine tested. For all measurements, a computer-controlled HP 8452A diode-array spectrophotometer was used. Statistics In all cases analyses were performed in triplicate, unless elsewhere specified, and values averaged. The standard deviation (s.d.) was also calculated. Correlation between AAR, PR and SAHFR with TA, TF, and TP was established using regression analysis at a 99% significance level. RESULTS Antiradical Activity (AAR) The percent decrease in A515 of a methanolic DPPHK solution has been found to be linear in response to increasing amounts of Troloxs, the water-soluble analogue of a-tocopherol. On this basis, AAR of the wines that have been appropriately diluted, could be expressed in terms of Troloxs equivalents (TRE), which appears to be a more descriptive expression, compared with methodologies that calculate only the per cent decrease in A515. Moreover, this approach might be useful in comparing AAR values deriving from other methods using known antioxidants as reference standards. A similar procedure has been used in the estimation of free radical quenching of leaf pigment extracts (Arnao et al., 2001). AAR of the wines tested was shown to vary from 0.73 to 1.37 mm TRE, the average being 0.99 mm TRE (Table 2). The highest AAR was found for wine No. 10, which also had the highest TP, TA, and TF contents. By contrast, the lowest AAR was observed for wine No. 4 (0.73 mm TRE), which had the lowest TP content. When AAR values were plotted against TP content, a r2 = 0.7082 was calculated (Table 3), indicating that the antiradical efficiency of the wines may be linked to their TP concentration. However, correlation of TF with AAR was even higher (r2 = 0.7287) but poor with TA content (r2 = 0.2473), although correlation with CA was more prominent (r2 = 0.3723). This finding provided evidence that AAR might be significantly associated with flavanols (catechin, epicatechin, proanthocyanidins).
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ARNOUS ET AL. TABLE 2 Polyphenolic composition and antioxidant properties of the wines tested1
Wine code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 AV
TP(a) (mg L1 GAE)
TA(b) (mg L1)
CA(c) (mg L1)
TF(d) (mg L1 CTE)
AAR(e) (mm TRE)
SAHFR(f) (%)
PR(g) (mm AAE)
1939796 16587199 1709734 12177292 2091757 17807181 24397278 13287235 21877231 37727284 23547223 22437245 32877351 19437274 19957256 2129
111.1711.9 53.670.4 44.373.1 109.7720.1 143.570.7 270.377.7 332.475.9 271.2718.5 186.8721.4 360.179.9 207.275.1 72.674.7 121.773.8 58.577.3 232.5715.4 174.3
6.770.3 4.770.2 2.370.8 9.070.2 13.570.1 17.970.3 16.670.2 19.670.1 7.171.5 37.271.1 12.170.2 6.070.3 18.870.2 5.970.2 21.270.7 13.2
291.1710.4 309.773.1 444.7711.2 339.176.1 442.973.8 346.3711.2 650.4719.6 424.477.4 546.877.3 664.879.5 352.272.4 435.973.0 643.676.0 380.2710.8 383.0710.1 443.7
0.7770.00 0.8570.01 0.9770.02 0.7370.02 0.8770.01 1.0470.03 1.1470.05 0.8870.01 1.0870.02 1.3770.05 0.9170.05 1.0370.02 1.2270.01 0.9470.02 0.9970.02 0.99
38.573.8 41.373.9 44.272.8 42.272.0 27.072.3 59.974.0 46.673.8 42.273.1 48.573.8 57.873.0 48.572.2 41.875.3 51.572.8 43.374.7 42.073.0 45.0
5.2970.04 6.5170.05 8.1470.10 5.3670.04 7.1170.07 8.1870.22 8.5770.11 5.4970.09 7.8370.06 10.8070.18 6.6370.01 7.3970.02 8.3570.14 6.2370.01 7.1270.02 7.27
1 For the antiradical activity measurements, samples were diluted 1:10. For the hydroxyl free radical scavenging activity, samples were diluted 1:200. Note: Values represent means of triplicate determination (n = 3)7 s.d., except for SAHFR determination where n = 5. (a) Total phenols expressed as gallic acid equivalents; (b) total anthocyanins; (c) coloured (ionised) anthocyanins; (d) total flavanols expressed as catechin equivalents; (e) antiradical activity expressed as Trolox equivalents; (f) hydroxyl free radical scavenging activity; (g) reducing power expressed as ascorbic acid equivalents. 1, 2, 3: Kritikos Topikos; 4: Macedonikos Topikos; 5, 6: Peloponissiakos Topikos; 7: Topikos Dramas; 8: Topikos Chalkidikis; 9: Topikos Florinas; 10: Topikos Imathias; 11: Topikos Letrinon; 12: Topikos Op. Lokridos; 13: Topikos Plagion Egialias; 14: Topikos Stereas Elladas; 15: Topikos Tegeas.
TABLE 3 Statistical values calculated after correlation of antioxidant parameters with individual polyphenol groups, as determined using regression analysis
TA CA TF TP
AAR
SAHFR
PR
0.2473(a) 5.60(b) 0.034(c) 0.3723 9.30 0.009 0.7287 38.61 3.15 105 0.7082 34.98 5.11 105
0.185 4.19 0.061 0.1682 3.83 0.072 0.0750 2.14 0.168 0.1497 3.46 0.085
0.1942 4.37 0.057 0.2944 6.84 0.021 0.5761 20.03 6.25 104 0.5870 20.90 5.0 104
Note: (a) Adjusted value of the square correlation coefficient (r2); (b) F-value; (c) significance of F-value.
Hydroxyl Free Radical Scavenging Activity (SAHFR) For the measurement of SAHFR, all samples were diluted 1:200 with buffer, in order to achieve a concentration of approximately 10 mg L1 of total phenols, expressed as
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GAE. The analyses were carried out immediately after dilution, in order to avoid potential oxidation of phenolics due to increased pH. The inhibition of deoxyribose damage by the addition of wines varied from 27.0 to 59.9% decrease in A532 relative to control (mean 45%). The regression analyses showed a rather low correlation of % decrease in A532 with TP (r2 = 0.1497) (Table 3), but correlation with TF and TA was even lower (r2 = 0.0750), whereas TA and CA appeared to exert a rather stronger effect (r2 = 0.1865 and 0.1682, respectively). Reducing Power (PR) The method originally described by Benzie and Strain (1996) was modified for its application to the measurement of the reducing power of wines. For comparison reasons ascorbic acid was used as the calibration standard, because it is a wellcharacterized natural reducing agent. PR thus determined ranged from 5.29 to 10.80 mm ascorbic acid equivalents (AAE), the mean value being 7.27 mm AAE. Once again, wine No. 10 was the most efficient in reducing Fe3+, whereas wine No. 1, which had the lowest TF content, exhibited the weakest reducing ability (Table 2). The r2 found for TP and PR was 0.5870, while the corresponding values for TA and TF were 0.1942 and 0.5761. With respect to establishing a link of the reducing ability of wines with their antiradical and hydroxyl free radical scavenging activities, PR values were plotted against AAR and SAHFR. It was shown that PR is likely to reflect the antiradical capacity of the wines (r2 = 0.8455, F = 77.00), rather than their hydroxyl free radical ability (r2 = 0.4020, F = 10.41). DISCUSSION Due to the strong epidemiological evidence that red wine consumption may result in suppressed rates of cardiovascular disorders, there has been, in recent years, a vast amount of examinations concerning the in vitro antioxidant properties of red wines. It has been highlighted that the antioxidant properties of red wines may be linked, to some extent, with particular classes of flavonoids. On the other hand, recent studies indicate that the well-investigated antioxidant flavonols are likely to have negligible impact on the overall antioxidant status of a given wine (Gardner et al., 1999), and it appears, therefore, that the antioxidant activity of other polyphenols may be of greater importance in contributing to the reputed health benefits of moderate wine consumption. On the basis of those considerations, selected Greek regional wines were subjected to some representative antioxidants tests, and attempts were made to distinguish whether or not the anthocyanin and flavanol content account for their antioxidant efficiency. The antiradical activity (AAR) was found to be associated to a considerable degree with the TP content (r2 = 0.7082), as demonstrated for a number of relevant studies on red wines (Frankel et al., 1995; Sato et al., 1996; Simonetti et al., 1997; Fogliano et al., 1999; Sa´nchez-Moreno et al., 1999, 2000; Burns et al., 2000), but the correlation was higher with TF (r2 = 0.7287). In contrast, SAHFR was mainly correlated with the TP content (r2 = 0.1497), whereas TF appeared to play a less significant role (r2 = 0.0750). The antioxidant potential of red wines is believed to depend to a great extent on flavanol content (Simonetti et al., 1997; Burns et al., 2000; Sa´nchez-Moreno et al., 2000), but also influenced by the relative amounts of the individual flavanols. This hypothesis is supported by the fact that the antioxidant activity of proanthocyanidins is in part dictated by oligomer chain length. Flavanol monomers and dimers were found to inhibit more efficiently LDL oxidation than
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hexamers (Lotito et al., 2000), and the inhibition of the OK tended to increase in 2 the order of polymerization (Saint-Cricq de Gaulejac et al., 1999). Esterification in the 3-position with gallic acid was another important determinant with regard to scavenging hydroxyl free radical by grape seed proanthocyanidins (Ricardo da Silva et al., 1991). Therefore, the differences in AAR and SAHFR observed among the wines tested may reflect differences in flavanol and proanthocyanidin composition. Both AAR and SAHFR were less significantly related with TA content (r2 = 0.2473 and 0.1856, respectively), something that contrasts with previous results (Ghiselli et al., 1998). At this point, the fact that the anthocyanin content determined with spectrophotometry does not provide an accurate estimation of the actual amount of monomeric anthocyanins should be underlined. It was indicated (Somers and Evans, 1977) that the methodology is applicable to current vintage wines, since as wines age the formation of pigments with unpredictable responses to SO2induced bleaching and pH changes may account for doubtful results and false estimations of colour characteristics. This was unequivocally demonstrated by Bakker et al. (1986), who observed that the differences in TA content determined spectrophotometrically and by HPLC vary largely with the age of a wine. Thus, it would be reasonable to presume that the poor correlation of TA with AAR and SAHFR might be attributed to the fact that TA values represent also polymeric and other types of pigments, which may not possess similar antioxidant characteristics with monomeric anthocyanins. Furthermore, when AAR values were plotted against CA content, a higher correlation was observed (r2 = 0.3723) compared with TA, suggesting that the ionization state of anthocyanins may be of importance with respect to their antiradical efficiency. This is corroborated by the finding that pseudo-base and quinoidal-base of malvidin 3-O-glucoside, generated respectively at pH 4.0 and 7.0, had different antioxidant behaviour (Lapidot et al., 1999). The FRAP assay has been used frequently as an additional source of information regarding the antioxidant capacity of human serum (Cao and Prior, 1998), phytoestrogens (Mitchell et al., 1998), teas (Benzie and Szeto, 1999), plant extracts (Deighton et al., 2000; Gao et al., 2000) and dietary polyphenols (Pulido et al., 2000). However, it has never been employed, as far as it is known, for measuring the ferricreducing ability of wines. As one-dimensional methodologies for evaluating the antioxidant activity of foods have been subjected to a certain criticism for their validity (Frankel and Meyer, 2000), it appears that the well-established and widely used FRAP assay may be of importance in obtaining additional information about the antioxidant properties of wines. The reducing power (PR) determined using a modified FRAP assay correlated to a higher degree with TF (r2 = 0.5761) and TP (r2 = 0.5870) than with TA content (r2 = 0.1942), while CA content was proven to have a more significant association (r2 = 0.2944) compared with TA. These results suggest that, as for AAR, the TP and TF are the main factors concerned with the reducing ability of wines, whereas the participation of anthocyanins seems to be of lesser importance. However, due to the complexity of pigment structure in aged wines, no clear conclusions could be drawn about the reducing potential and the actual contribution of monomeric anthocyanins. It is worth mentioning that PR was highly correlated with AAR (r2 = 0.8455), but did less so with SAHFR (r2 = 0.4020), indicating that there is a close relation between the antiradical efficiency and the reducing ability of wines. The two assays are based on very different principles and represent different aspects of the antioxidant potential, so any connection between PR and AAR would not be normally expected, as demonstrated by similar studies on human serum (Cao and Prior, 1998). However, the fact that there is also a correlation with SAHFR indicates that PR may
PIGMENT AND FLAVANOL CONTENT IN GREEK WINES
663
actually reflect part of the antioxidant potential of wines. It appears that the antioxidant activity might be ascribed to particular class(es) of polyphenolic antioxidants, which are able to donate hydrogen atoms, scavenge hydroxyl radicals and also participate in redox reactions. Since monomeric (catechin, epicatechin) and polymeric (proanthocyanidins) flavanols account for a great part of red wine polyphenols, it might be supported that their content is a major factor affecting antioxidant properties.
CONCLUSIONS The antioxidant properties of aged red wines appear to be governed by the total phenol and total flavanol content. The weak correlation of all three antioxidant parameters with total anthocyanins indicates that monomeric and presumably polymeric pigments are likely to have a low impact in the overall antioxidant status. Furthermore, as no data are available in the literature concerning the bioavailability and biological significance of such polymeric components, it would be unwise to make any assumption about their role in the health benefits of moderate red wine consumption. However, evidence suggests that polymeric flavanols (proanthocyanidins) may deserve a greater attention as dietary antioxidants.
ACKNOWLEDGEMENTS The authors would like to thank Mr Tasos Drosiadis (Antonopoulos Wineries, Achaia) for his kind donation of some of the wines examined.
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Duthie, G. G., Duthie, S. J., and Kyle, J. A. M. (2000). Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr. Res. Rev. 13, 79–106. Espı´ n, J. C., Soler-Rivas, C., Wichers, H. J., and Garcı´ a-Viguera, C. (2000). Anthocyanin-based natural colorants: a new source of antiradical activity for foodstuff. J. Agric. Food Chem. 48, 1588–1592. Fogliano, V., Verde, V., Randazzo, G., and Ritieni, A. (1999). Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem. 47, 1035–1040. Frankel, E. N., Bosanek, C. A., Meyer, A. S., Silliman, K., and Kirk, L. L. (1998). Commercial grape juices inhibit the in vitro oxidation of human low-density lipoproteins. J. Agric. Food Chem. 46, 834–838. Frankel, E. N. and Meyer, A. S. (2000). The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric. 80, 1925–1941. Frankel, E. N., Waterhouse, A. L., and Teissedre, P. L. (1995). Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins. J. Agric. Food Chem. 43, 890–894. Gao, X., Bjo¨rk, L., Trajkovski, V., and Uggla, M. (2000). Evaluation of antioxidant activities of rosehip ethanol extracts in different test systems. J. Sci. Food Agric. 80, 2021–2027. Gardner, P. T., McPhail, D. B., Crozier, A., and Duthie, G. G. (1999). Electron spin resonance (ESR) spectroscopic assessment of the contribution of quercetin and other flavonols to the antioxidant capacity of red wines. J. Sci. Food Agric. 79, 1011–1014. Ghiselli, A., Nardini, M., Baldi, A., and Scaccini, C. (1998). Antioxidant activity of different phenolic fractions separated from an Italian red wine. J. Agric. Food Chem. 46(2), 361–367. Heinonen, M., Rein, D., Satue´-Gracia, M. T., Huang, S.-W., German, J. B., and Frankel, E. N. (1998). Effect of protein on the antioxidant activity of phenolic compounds in a lecithin–liposome oxidation system. J. Agric. Food Chem. 46, 917–922. Kondo, Y., Ohnishi, M., and Kawaguchi, M. (1999). Detection of lipid peroxidation catalyzed by chelated iron and measurement of the antioxidant activity in wine by a chemiluminescence analyzer. J. Agric. Food Chem. 47, 1781–1785. Lapidot, T., Harel, S., Akiri, B., Granit, R., and Kanner, J. (1999). pH-Dependent forms of red wine anthocyanins as antioxidants. J. Agric. Food Chem. 47, 67–70. Li, Y.-G., Tanner, G. and Larkin, P. (1996). The DMACA-HCl protocol and the threshold proanthocyanidin content for bloat safety in forage legumes. J. Sci. Food Agric. 70, 89–101. Lotito, S. B., Actis-Goretta, L., Renart, M. L., Caligiuri, M., Rein, D., Schmitz, H. H., Steinberg, F. M., Keen, C. L., and Fraga, C. G. (2000). Influence of oligomer chain length on the antioxidant activity of procyanidins. Biochem. Biophys. Res. Commun. 276, 945–951. McMorrough, I., Madigan, D., and Smyth, M. R. (1996). Semipreparative chromatographic procedure for the isolation of dimeric and trimeric proanthocyanidins from barley. J. Agric. Food Chem. 44, 1731–1735. Meyer, A. S., Yi, O.-S., Pearson, D. A., Waterhouse, A. L., and Frankel, E. N. (1997). Inhibition of human low-density lipoprotein oxidation in relation to composition of phenolic antioxidants in grapes (Vitis vinifera). J. Agric. Food Chem. 45, 1638–1643. Mitchell, J. H., Gardner, P. T., McPhail, D. B., Morrice, P. C., Collins, A. R., and Duthie, G. G. (1998). Antioxidant efficacy of phytoestrogens in chemical and biological model systems. Arch. Biochem. Biophys. 360(1), 142–148. Pulido, R., Bravo, L., and Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food Chem. 48(8), 3396–3402. Ricardo da Silva, J. M., Darmon, N., Fernandez, Y., and Mitjavila, S. (1991). Oxygen free radical scavenger capacity in aqueous models of different procyanidins from grape seeds. J. Agric. Food Chem. 39, 1549–1552. Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M., and Pridham, J. B. (1995). The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Rad. Res. 22(4), 375–383. Saint-Cricq de Gaulejac, N., Provost, C., and Vivas, N. (1999). Comparative study of polyphenol scavenging activities assessed by different methods. J. Agric. Food Chem. 47, 425–431. Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, G. P., and Rice-Evans, C. (1995). Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain breaking antioxidants. Arch. Biochem. Biophys. 322(2), 339–346. Sa´nchez-Moreno, C., Larrauri, J. A., and Saura-Calixto, F. (1999). Free radical scavenging capacity of selected red, rose´ and white wines. J. Sci. Food Agric. 79, 1301–1304.
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Sa´nchez-Moreno, C., Satue´-Gracia, M. T., and Frankel, E. N. (2000). Antioxidant activity of selected Spanish wines in corn oil emulsions. J. Agric. Food Chem. 48, 5581–5587. Sato, M., Ramarathnam, N., Suzuki, Y., Ohkubo, T., Takeuchi, M., and Ochi, H. (1996). Varietal differences in the phenolic content and superoxide radical scavenging potential of wines from different sources. J. Agric. Food Chem. 44, 37–41. Satue´-Gracia, M. T., Heinonen, M., and Frankel, E. N. (1997). Anthocyanins as antioxidants on human low-density lipoprotein and lecithin–liposome systems. J. Agric. Food Chem. 45, 3362–3367. Simonetti, P., Pietta, P., and Testolin, G. (1997). Polyphenol content and total antioxidant potential of selected Italian wines. J. Agric. Food Chem. 45, 1152–1155. Somers, T. C. and Evans, M. E. (1977). Spectral evaluation of young red wines: anthocyanin equilibria, total phenolics, free and molecular SO2, ‘‘chemical age’’. J. Sci. Food Agric. 28, 279–287. Teissedre, P. L., Frankel, E. N., Waterhouse, A. L., Peleg, H., and German, J. B. (1996). Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines. J. Sci. Food Agric. 70, 55–61. Ursini, F., Tubaro, F., Rong, J., and Sevanian, A. (1999). Optimization of nutrition: Polyphenols and vascular protection. Nutr. Rev. 57(8), 241–249. van Acker, S. A. B. E., van de Berg, D.-J., Tromp, M. N. J. L., Griffioen, D. H., van Bennekom, W., van der Vijgh, W. J. F., and Bast, A. (1996). Structural aspects of antioxidant activity of flavonoids. Free Rad. Biol. Med. 20(3), 331–342. Vivas, N., Glories, Y., Lagune, L., Saucier, C., and Augustin, M. (1994). Estimation du degre´ de polymerisation des procyanidins du raisin et du vin par la me´thode au p-dimethylaminocinnamalde´hyde. J. Int. Sci. Vigne Vin 28(4), 319–336. Wang, H., Cao, G., and Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins. J. Agric. Food Chem. 45, 304–309. Wang, H., Nair, M. G., Strasburg, G. M., Chang, Y.-C., Booren, A. M., Gray, J. I., and DeWitt, D. L. (1999). Antioxidant and anti-inflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. J. Nat. Prod. 62, 294–296. Waterman, P. G. and Mole, S. (1994). Analysis of Phenolic Plant Metabolites, pp. 83–91. Blackwell Scientific Publ., Oxford. Zoecklein, B. W., Fugelsang, K. C., Gump, B. H., and Nury, F. S. (1990). Production Wine Analysis, pp. 129–168. Van Nostrand Reinhold Publ., New York.
ORIGINAL ARTICLE Correlation of Pigment and Flavanol Content with Antioxidant Properties in Selected Aged Regional Wines from Greece Anis Arnous*, Dimitris P. Makris*,w,1, and Panagiotis Kefalas*,w *Department of Food Quality Management, Mediterranean Agronomic Institute of Chania (MAICh), P.O. Box 85, 73100 Chania, Greece; and wLaboratory of Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (MAICh), P. O. Box 85, 73100 Chania, Greece Received August 14, 2001 and in revised form November 21, 2001
Selected Greek regional, aged red wines were assessed for their polyphenolic composition using well-established spectrophotometric methodology, and tested for antioxidant potential by three different assays. The total polyphenol (TP) concentration was found to vary from 1217 to 3772 mg L1 gallic acid equivalents, total anthocyanin (TA) content ranged from 44.3 to 360.1 mg L1, and total flavanol (TF) from 291.1 to 664.8 mg L1 catechin equivalents. The antiradical activity was evaluated using DPPHK, and varied from 0.73 to 1.37 mm Troloxs equivalents, while reducing power values were from 5.29 to 10.80 mm ascorbic acid equivalents, using a modified FRAP assay. The hydroxyl free radical scavenging activity was determined with the deoxyribose method, and ranged from 38.5 to 59.9% decrease in A532 relative to control. The examinations showed that TP and TF may provide a significant contribution to the overall antioxidant status of wines, but TA appears to be a less important factor in this respect. Reducing power was found to be highly associated with the antiradical activity of wines. r 2002 Elsevier Science Ltd. All rights reserved. Key Words: aged red wines; anthocyanins; antiradical activity; flavanols; hydroxyl free radical scavenging activity; reducing power.
INTRODUCTION Phenolic substances are naturally present in essentially all plant material, including food of plant origin. Thus, polyphenolic substances are ubiquitous in vegetables, cereals, fruits, nuts, but also in plant products such as wine, cider, beer, tea, cocoa, etc. Wines in particular constitute an excellent source of dietary polyphenols, as they may contain from 200–300 (white wines) to 1000–4000 (red wines) mg L 1 total phenols (Bravo, 1998). Red wine, however, appears to exhibit biological effects that are usually not seen with white wine, which may be attributed to the large amounts of flavonoids, particularly anthocyanins, flavanols, and flavanol derivatives (proanthocyanidins) (Cao and Prior, 2000). 1 To whom correspondence and reprint requests should be addressed. Tel.: +30-821-81151, ext. 559. Fax: +30-821-81154. E-mail: [email protected]
0889-1575/02/060655 + 11 $35.00/0
r 2002 Elsevier Science Ltd. All rights reserved.
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Since the claim that flavonoids could have a vitamin function, their biological effects have been mainly interpreted on the basis of their antioxidant properties, that are related to both free radical scavenging and transition metal chelating (Ursini et al., 1999). Flavonoids may be effective antioxidants in a wide range of systems, being capable of scavenging peroxyl radicals, alkyl peroxyl radicals, superoxide, hydroxyl radicals, nitric oxide and peroxynitrile, in aqueous and organic environments. Further, supporting the possibility that certain polyphenols may have antioxidant function in vivo are several rodent feeding studies indicating that phenolic-rich extracts of red wine lower oxidative products such as protein carbonyls, DNA base damage and malonaldehyde in blood and a range of tissues (Duthie et al., 2000). As regards anthocyanins, they have been proven very efficient antioxidants in a number of systems (Rice-Evans et al., 1995; van Acker et al., 1996; Wang et al., 1997, 1999; Heinonen et al., 1998; Degenhardt et al., 2000; Espı´ n et al., 2000), and in vitro LDL oxidation inhibitors (Satue´-Gracia et al., 1997), while other studies demonstrated a relationship between anthocyanin content and antioxidant activities of red grape extracts (Meyer et al., 1997), grape juices (Frankel et al., 1998) and various red wines (Sato et al., 1996; Sa´nchez-Moreno et al., 2000). Other investigations, however, indicated that anthocyanins may be less significantly correlated with the antioxidant properties of red wines (Frankel et al., 1995; Burns et al., 2000). Flavanols have also been shown to exhibit powerful antioxidant activities in different environments (Ricardo da Silva et al., 1991; Rice-Evans et al., 1995; Salah et al., 1995; Heinonen et al., 1998; Kondo et al., 1999; Bors et al., 2000), and the antioxidant abilities of red grapes (Teissedre et al., 1996; Meyer et al., 1997), juices (Frankel et al., 1998) and wines (Frankel et al., 1995; Sato et al., 1996; Teissedre et al., 1996; Simonetti et al., 1997; Burns et al., 2000) have always been correlated with the flavanol content. Greek regional wines are produced from native but also the so-called ‘international’ cultivars, including Cabernet Sauvignon, Merlot, Syrah, Grenache, etc. Most wines are produced by co-vinification of grapes deriving from more than one cultivars, whereas single-variety regional wines are of more limited occurrence in the Greek market. There is a considerable lack of information with regard to polyphenolic composition of Greek red wines and as a result the antioxidant capacity, which is related to polyphenolic content, has not been examined. The present study represents an approach with respect to determining the anthocyanin and flavanol content of selected, aged red wines, and aims at investigating correlation of these two classes of flavonoids with the in vitro antioxidant potential, as this may be illustrated by the antiradical activity, the reducing power, and the hydroxyl free radical scavenging activity.
MATERIALS AND METHODS Wines Fifteen representative, high-quality, regional wines were examined, which are produced according to standard procedures, and with defined varietal composition (Table 1). For comparison reasons, all samples examined were of 1998 vintage (3 years old). Wines cover all viticultural areas of Greece, they are available in the Greek market, and widely consumed. All samples were stored at 101C in obscurity, and analysed shortly after opening.
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TABLE 1 Origin and varietal composition of the wines tested Appellation Kritikos Topikos Macedonikos Topikos Peloponissiakos Topikos Topikos Dramas Topikos Chalcidikis Topikos Florinas Topikos Imathias Topikos Letrinon Topikos Op. Lokridos Topikos Plagion Egialias Topikos Stereas Elladas Topikos Tegeas
Cultivar(s)
Location
Kotsifali/Mandilaria/Liatiko Xinomavro Agiorgitiko/Cab. Sauv. Cab. Sauv./Cab. Franc/Merlot/Limnio Merlot Xinomavro/Merlot Syrah Refosco/Mavrodafni Cab. Sauv./Xinomavro/Limnio Cab. Sauv. Syrah/Grenache/Carignan/Cab. Sauv. Cab. Sauv./Merlot
Crete (S) Macedonia (N) Peloponese (S) Macedonia (N) Macedonia (N) Macedonia (N) Macedonia (N) Peloponese (S) Sterea Ellada (C) Peloponese (S) Sterea Ellada (C) Peloponese (S)
Note: Letters N, C, and S assign north, central and south Greece, respectively.
Chemicals Ascorbic acid, catechin, p-dimethylaminocinnamaldehyde (DMACA), 2,2-diphenylb-picrylhydrazyl (DPPHK) radical, EDTA (disodium salt), gallic acid, 2,4,6tripyridyl-s-triazine (TPTZ), and sodium metabisulphite were from Sigma Chemical Co. (St Louis, MO). 2-Deoxyribose was from Fluka. Citric acid, ferric chloride hexahydrate (FeCl3 ? 6H2O), Folin-Ciocalteu reagent, hydrogen peroxide, iron ammonium sulphate, thiobarbituric acid (TBA), and Troloxs were from Aldrich (Steinheim, Germany). Trichloroacetic acid (TCA) was from Riedel-de Hae¨n (Germany). Determination of Total Polyphenols (TP) Total polyphenol content of wines was determined using the Folin-Ciocalteu method (Waterman and Mole, 1994), adapted to a micro scale. In a 1.5-mL Eppendorf tube, 0.79 mL distilled water, 0.01 mL sample appropriately diluted, and 0.05 mL FolinCiocalteu reagent were added and vortexed. After exactly 1 min, 0.15 mL of sodium carbonate (20%) was added, and the mixture was vortexed and allowed to stand at room temperature in obscurity, for 120 min. The absorbance was read at 750 nm, and the total polyphenol concentration was calculated from a calibration curve (r2 = 0.9990), using gallic acid as standard (50–800 mg L1). Results were expressed as mg L1 gallic acid equivalents (GAE). Determination of Total and Coloured Anthocyanins Measurements were performed using well-established spectrophotometric methodology (Somers and Evans, 1977; Zoecklein et al., 1990). Analytically, wine sample was placed in a 0.2-cm path length quartz cuvette, and the absorbance was measured at 520 nm (A520). Following this, 0.02 mL of a 20% sodium metabisulphite solution was added, the sample was mixed well, and after 1 min the absorbance was read at 2 520 nm ASO 520 : A 12% ethanolic solution was used as blank. All measurements were corrected to a 1.0-cm path length. Further, wine (0.02 mL) was mixed with 0.98 mL 1 n HCl solution (dilution 1:50) in a 1.5-mL Eppendorf tube, vortexed, and allowed to stand for 180 min at room temperature. The absorbance was read at 520 nm
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ðAHCl 520 Þ; using a 1.0-cm path length cuvette. For the blank, 0.02 mL of a 12% ethanolic solution was used instead of wine. The concentration of total anthocyanins (TA) and coloured (ionized) anthocyanins (CA) was calculated as follows: SO2 TA ðmg L1 Þ¼ 20 ½AHCl 520 ð5=3Þ A520 ; 2 CA ðmg L1 Þ¼ 20 ðA520 ASO 520 Þ:
Determination of Total Flavanols (TF) The total flavanol content was estimated using the p-dimethylaminocinnamaldehyde (DMACA) method (Vivas et al., 1994; Li et al., 1996; McMorrough et al., 1996). This method has a great advantage over the widely used vanillin assay, since there is no interference by anthocyanins. Further, it provides higher sensitivity and specificity (Li et al., 1996). Wine (0.2 mL), diluted 1:100 with MeOH, was introduced into a 1.5-mL Eppendorf tube and added 1 mL DMACA solution (0.1% in 1 n HCl in MeOH). The mixture was vortexed and allowed to react at room temperature for 10 min. Following this, the absorbance at 640 nm was read against blank prepared similarly without DMACA. The concentration of TF was estimated from a calibration curve, constructed by plotting known solutions of catechin (1–16 mg L1) against A640 (r2 = 0.9987). Results were expressed as catechin equivalents (mg L1 CTE). Measurement of the Antiradical Activity (AAR) All samples were diluted 1:10 with MeOH immediately before the analysis. An aliquot of 0.025 mL of diluted sample was added to 0.975 mL DPPHK solution (60 mm in MeOH), vortexed, and the absorbance was read at t = 0 and 30 min. Results were expressed as Troloxs equivalents (mm TRE) using the following equation: AAR ðmm TREÞ ¼ 0:018 %A515 þ 0:017 as determined from linear regression, after plotting %DA515 of known solutions of Troloxs against concentration (0.08–1.28 mM, r2 = 0.9935), where %DA515 = [(A515(0) – A515(30))/A515(0)] 100. Measurement of the Hydroxyl Free Radical Scavenging Activity (SAHFR) For the determination of the hydroxyl free radical scavenging capacity of wines, the deoxyribose method was used, as described by Ghiselli et al. (1998), slightly modified. Wine sample (0.05 mL) was mixed with 0.345 mL of 10 mm phosphate buffer, pH 7.4, containing 2.5 mm 2-deoxyribose. An aliquot of 0.05 mL of 2 mm iron ammonium sulphate premixed with 2.08 mm EDTA was added, the mixture was vortexed, and the reaction was initiated by adding 0.05 mL 1 mm ascorbic acid and 0.02 mL 1.5 mm hydrogen peroxide. Samples were maintained at 371C for 30 min, and then added 0.5 mL 30% TCA and 0.5 mL 1% TBA (in 0.05 n NaOH). Samples were heated at 901C for 20 min, cooled, and the absorbance at 532 nm was measured. Control sample was prepared by adding distilled water instead of wine. Hydroxyl free radical scavenging activity (SAHFR) was calculated as the % reduction in A532 relative to control. All wine samples were diluted 1:200 with buffer just before the analysis, to achieve a mean total phenol concentration of approximately 10 mg L1 GAE.
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Measurement of the Reducing Power (PR) For the determination of the reducing ability of wines a protocol based on the ferric reducing/antioxidant power (FRAP) assay was developed, as described previously (Benzie and Strain, 1996; Pulido et al., 2000), with modifications. Wine (0.05 mL), diluted 1:10 with distilled water, and 0.05 mL of ferric chloride (3 mm in 5 mm citric acid) were mixed well in a 1.5-mL Eppendorf tube, and incubated for 30 min in a water bath at 371C. Following this, the mixture was added to 0.90 mL of 1 mm TPTZ solution in 0.05 m HCl, and vortexed. After exactly 10 min the absorbance was read at 620 nm. PR was calculated from a calibration curve, established by plotting known amounts of ascorbic acid against A620. Results were expressed as ascorbic acid equivalents (mm AAE) using the following equation: PR ðmm AAEÞ ¼ ð0:679 A620 0:008Þ FD ; where FD is the dilution factor. For the blanks, distilled water was added instead of ferric chloride/citric acid. One blank was prepared for each wine tested. For all measurements, a computer-controlled HP 8452A diode-array spectrophotometer was used. Statistics In all cases analyses were performed in triplicate, unless elsewhere specified, and values averaged. The standard deviation (s.d.) was also calculated. Correlation between AAR, PR and SAHFR with TA, TF, and TP was established using regression analysis at a 99% significance level. RESULTS Antiradical Activity (AAR) The percent decrease in A515 of a methanolic DPPHK solution has been found to be linear in response to increasing amounts of Troloxs, the water-soluble analogue of a-tocopherol. On this basis, AAR of the wines that have been appropriately diluted, could be expressed in terms of Troloxs equivalents (TRE), which appears to be a more descriptive expression, compared with methodologies that calculate only the per cent decrease in A515. Moreover, this approach might be useful in comparing AAR values deriving from other methods using known antioxidants as reference standards. A similar procedure has been used in the estimation of free radical quenching of leaf pigment extracts (Arnao et al., 2001). AAR of the wines tested was shown to vary from 0.73 to 1.37 mm TRE, the average being 0.99 mm TRE (Table 2). The highest AAR was found for wine No. 10, which also had the highest TP, TA, and TF contents. By contrast, the lowest AAR was observed for wine No. 4 (0.73 mm TRE), which had the lowest TP content. When AAR values were plotted against TP content, a r2 = 0.7082 was calculated (Table 3), indicating that the antiradical efficiency of the wines may be linked to their TP concentration. However, correlation of TF with AAR was even higher (r2 = 0.7287) but poor with TA content (r2 = 0.2473), although correlation with CA was more prominent (r2 = 0.3723). This finding provided evidence that AAR might be significantly associated with flavanols (catechin, epicatechin, proanthocyanidins).
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ARNOUS ET AL. TABLE 2 Polyphenolic composition and antioxidant properties of the wines tested1
Wine code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 AV
TP(a) (mg L1 GAE)
TA(b) (mg L1)
CA(c) (mg L1)
TF(d) (mg L1 CTE)
AAR(e) (mm TRE)
SAHFR(f) (%)
PR(g) (mm AAE)
1939796 16587199 1709734 12177292 2091757 17807181 24397278 13287235 21877231 37727284 23547223 22437245 32877351 19437274 19957256 2129
111.1711.9 53.670.4 44.373.1 109.7720.1 143.570.7 270.377.7 332.475.9 271.2718.5 186.8721.4 360.179.9 207.275.1 72.674.7 121.773.8 58.577.3 232.5715.4 174.3
6.770.3 4.770.2 2.370.8 9.070.2 13.570.1 17.970.3 16.670.2 19.670.1 7.171.5 37.271.1 12.170.2 6.070.3 18.870.2 5.970.2 21.270.7 13.2
291.1710.4 309.773.1 444.7711.2 339.176.1 442.973.8 346.3711.2 650.4719.6 424.477.4 546.877.3 664.879.5 352.272.4 435.973.0 643.676.0 380.2710.8 383.0710.1 443.7
0.7770.00 0.8570.01 0.9770.02 0.7370.02 0.8770.01 1.0470.03 1.1470.05 0.8870.01 1.0870.02 1.3770.05 0.9170.05 1.0370.02 1.2270.01 0.9470.02 0.9970.02 0.99
38.573.8 41.373.9 44.272.8 42.272.0 27.072.3 59.974.0 46.673.8 42.273.1 48.573.8 57.873.0 48.572.2 41.875.3 51.572.8 43.374.7 42.073.0 45.0
5.2970.04 6.5170.05 8.1470.10 5.3670.04 7.1170.07 8.1870.22 8.5770.11 5.4970.09 7.8370.06 10.8070.18 6.6370.01 7.3970.02 8.3570.14 6.2370.01 7.1270.02 7.27
1 For the antiradical activity measurements, samples were diluted 1:10. For the hydroxyl free radical scavenging activity, samples were diluted 1:200. Note: Values represent means of triplicate determination (n = 3)7 s.d., except for SAHFR determination where n = 5. (a) Total phenols expressed as gallic acid equivalents; (b) total anthocyanins; (c) coloured (ionised) anthocyanins; (d) total flavanols expressed as catechin equivalents; (e) antiradical activity expressed as Trolox equivalents; (f) hydroxyl free radical scavenging activity; (g) reducing power expressed as ascorbic acid equivalents. 1, 2, 3: Kritikos Topikos; 4: Macedonikos Topikos; 5, 6: Peloponissiakos Topikos; 7: Topikos Dramas; 8: Topikos Chalkidikis; 9: Topikos Florinas; 10: Topikos Imathias; 11: Topikos Letrinon; 12: Topikos Op. Lokridos; 13: Topikos Plagion Egialias; 14: Topikos Stereas Elladas; 15: Topikos Tegeas.
TABLE 3 Statistical values calculated after correlation of antioxidant parameters with individual polyphenol groups, as determined using regression analysis
TA CA TF TP
AAR
SAHFR
PR
0.2473(a) 5.60(b) 0.034(c) 0.3723 9.30 0.009 0.7287 38.61 3.15 105 0.7082 34.98 5.11 105
0.185 4.19 0.061 0.1682 3.83 0.072 0.0750 2.14 0.168 0.1497 3.46 0.085
0.1942 4.37 0.057 0.2944 6.84 0.021 0.5761 20.03 6.25 104 0.5870 20.90 5.0 104
Note: (a) Adjusted value of the square correlation coefficient (r2); (b) F-value; (c) significance of F-value.
Hydroxyl Free Radical Scavenging Activity (SAHFR) For the measurement of SAHFR, all samples were diluted 1:200 with buffer, in order to achieve a concentration of approximately 10 mg L1 of total phenols, expressed as
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GAE. The analyses were carried out immediately after dilution, in order to avoid potential oxidation of phenolics due to increased pH. The inhibition of deoxyribose damage by the addition of wines varied from 27.0 to 59.9% decrease in A532 relative to control (mean 45%). The regression analyses showed a rather low correlation of % decrease in A532 with TP (r2 = 0.1497) (Table 3), but correlation with TF and TA was even lower (r2 = 0.0750), whereas TA and CA appeared to exert a rather stronger effect (r2 = 0.1865 and 0.1682, respectively). Reducing Power (PR) The method originally described by Benzie and Strain (1996) was modified for its application to the measurement of the reducing power of wines. For comparison reasons ascorbic acid was used as the calibration standard, because it is a wellcharacterized natural reducing agent. PR thus determined ranged from 5.29 to 10.80 mm ascorbic acid equivalents (AAE), the mean value being 7.27 mm AAE. Once again, wine No. 10 was the most efficient in reducing Fe3+, whereas wine No. 1, which had the lowest TF content, exhibited the weakest reducing ability (Table 2). The r2 found for TP and PR was 0.5870, while the corresponding values for TA and TF were 0.1942 and 0.5761. With respect to establishing a link of the reducing ability of wines with their antiradical and hydroxyl free radical scavenging activities, PR values were plotted against AAR and SAHFR. It was shown that PR is likely to reflect the antiradical capacity of the wines (r2 = 0.8455, F = 77.00), rather than their hydroxyl free radical ability (r2 = 0.4020, F = 10.41). DISCUSSION Due to the strong epidemiological evidence that red wine consumption may result in suppressed rates of cardiovascular disorders, there has been, in recent years, a vast amount of examinations concerning the in vitro antioxidant properties of red wines. It has been highlighted that the antioxidant properties of red wines may be linked, to some extent, with particular classes of flavonoids. On the other hand, recent studies indicate that the well-investigated antioxidant flavonols are likely to have negligible impact on the overall antioxidant status of a given wine (Gardner et al., 1999), and it appears, therefore, that the antioxidant activity of other polyphenols may be of greater importance in contributing to the reputed health benefits of moderate wine consumption. On the basis of those considerations, selected Greek regional wines were subjected to some representative antioxidants tests, and attempts were made to distinguish whether or not the anthocyanin and flavanol content account for their antioxidant efficiency. The antiradical activity (AAR) was found to be associated to a considerable degree with the TP content (r2 = 0.7082), as demonstrated for a number of relevant studies on red wines (Frankel et al., 1995; Sato et al., 1996; Simonetti et al., 1997; Fogliano et al., 1999; Sa´nchez-Moreno et al., 1999, 2000; Burns et al., 2000), but the correlation was higher with TF (r2 = 0.7287). In contrast, SAHFR was mainly correlated with the TP content (r2 = 0.1497), whereas TF appeared to play a less significant role (r2 = 0.0750). The antioxidant potential of red wines is believed to depend to a great extent on flavanol content (Simonetti et al., 1997; Burns et al., 2000; Sa´nchez-Moreno et al., 2000), but also influenced by the relative amounts of the individual flavanols. This hypothesis is supported by the fact that the antioxidant activity of proanthocyanidins is in part dictated by oligomer chain length. Flavanol monomers and dimers were found to inhibit more efficiently LDL oxidation than
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hexamers (Lotito et al., 2000), and the inhibition of the OK tended to increase in 2 the order of polymerization (Saint-Cricq de Gaulejac et al., 1999). Esterification in the 3-position with gallic acid was another important determinant with regard to scavenging hydroxyl free radical by grape seed proanthocyanidins (Ricardo da Silva et al., 1991). Therefore, the differences in AAR and SAHFR observed among the wines tested may reflect differences in flavanol and proanthocyanidin composition. Both AAR and SAHFR were less significantly related with TA content (r2 = 0.2473 and 0.1856, respectively), something that contrasts with previous results (Ghiselli et al., 1998). At this point, the fact that the anthocyanin content determined with spectrophotometry does not provide an accurate estimation of the actual amount of monomeric anthocyanins should be underlined. It was indicated (Somers and Evans, 1977) that the methodology is applicable to current vintage wines, since as wines age the formation of pigments with unpredictable responses to SO2induced bleaching and pH changes may account for doubtful results and false estimations of colour characteristics. This was unequivocally demonstrated by Bakker et al. (1986), who observed that the differences in TA content determined spectrophotometrically and by HPLC vary largely with the age of a wine. Thus, it would be reasonable to presume that the poor correlation of TA with AAR and SAHFR might be attributed to the fact that TA values represent also polymeric and other types of pigments, which may not possess similar antioxidant characteristics with monomeric anthocyanins. Furthermore, when AAR values were plotted against CA content, a higher correlation was observed (r2 = 0.3723) compared with TA, suggesting that the ionization state of anthocyanins may be of importance with respect to their antiradical efficiency. This is corroborated by the finding that pseudo-base and quinoidal-base of malvidin 3-O-glucoside, generated respectively at pH 4.0 and 7.0, had different antioxidant behaviour (Lapidot et al., 1999). The FRAP assay has been used frequently as an additional source of information regarding the antioxidant capacity of human serum (Cao and Prior, 1998), phytoestrogens (Mitchell et al., 1998), teas (Benzie and Szeto, 1999), plant extracts (Deighton et al., 2000; Gao et al., 2000) and dietary polyphenols (Pulido et al., 2000). However, it has never been employed, as far as it is known, for measuring the ferricreducing ability of wines. As one-dimensional methodologies for evaluating the antioxidant activity of foods have been subjected to a certain criticism for their validity (Frankel and Meyer, 2000), it appears that the well-established and widely used FRAP assay may be of importance in obtaining additional information about the antioxidant properties of wines. The reducing power (PR) determined using a modified FRAP assay correlated to a higher degree with TF (r2 = 0.5761) and TP (r2 = 0.5870) than with TA content (r2 = 0.1942), while CA content was proven to have a more significant association (r2 = 0.2944) compared with TA. These results suggest that, as for AAR, the TP and TF are the main factors concerned with the reducing ability of wines, whereas the participation of anthocyanins seems to be of lesser importance. However, due to the complexity of pigment structure in aged wines, no clear conclusions could be drawn about the reducing potential and the actual contribution of monomeric anthocyanins. It is worth mentioning that PR was highly correlated with AAR (r2 = 0.8455), but did less so with SAHFR (r2 = 0.4020), indicating that there is a close relation between the antiradical efficiency and the reducing ability of wines. The two assays are based on very different principles and represent different aspects of the antioxidant potential, so any connection between PR and AAR would not be normally expected, as demonstrated by similar studies on human serum (Cao and Prior, 1998). However, the fact that there is also a correlation with SAHFR indicates that PR may
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actually reflect part of the antioxidant potential of wines. It appears that the antioxidant activity might be ascribed to particular class(es) of polyphenolic antioxidants, which are able to donate hydrogen atoms, scavenge hydroxyl radicals and also participate in redox reactions. Since monomeric (catechin, epicatechin) and polymeric (proanthocyanidins) flavanols account for a great part of red wine polyphenols, it might be supported that their content is a major factor affecting antioxidant properties.
CONCLUSIONS The antioxidant properties of aged red wines appear to be governed by the total phenol and total flavanol content. The weak correlation of all three antioxidant parameters with total anthocyanins indicates that monomeric and presumably polymeric pigments are likely to have a low impact in the overall antioxidant status. Furthermore, as no data are available in the literature concerning the bioavailability and biological significance of such polymeric components, it would be unwise to make any assumption about their role in the health benefits of moderate red wine consumption. However, evidence suggests that polymeric flavanols (proanthocyanidins) may deserve a greater attention as dietary antioxidants.
ACKNOWLEDGEMENTS The authors would like to thank Mr Tasos Drosiadis (Antonopoulos Wineries, Achaia) for his kind donation of some of the wines examined.
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