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Antioxidant activities of some Greek wines and wine phenolic extracts
ARTICLE IN PRESS Journal of Food Composition and Analysis 21 (2008) 614– 621
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Original Article
Antioxidant activities of some Greek wines and wine phenolic extracts Ioannis G. Roussis a,, Ioannis Lambropoulos a, Panagiotis Tzimas a, Anna Gkoulioti b, Vasilios Marinos b, Dimitrios Tsoupeis c, Ioannis Boutaris c a b c
Laboratory of Food Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece Ampeloeniki, 57001 Thessaloniki, Greece Ktima Kyr-Yianni, 59200 Yiannakochori Imathias, Greece
a r t i c l e in f o
a b s t r a c t
Article history: Received 30 January 2007 Received in revised form 9 February 2008 Accepted 9 February 2008
Antioxidant activities and phenolic composition of eight Greek wines were determined. Red wines scavenged 1,10 -diphenyl-2-picryl-hydrazyl radical (DPPHd) to a much greater degree than whites, in proportion to their phenolic contents. Red wines inhibited b-carotene bleaching, while white wines were almost inactive. Red wines were more active than quercetin in DPPHd and less active in b-carotene assay. Xinomavro-red must exhibited significant activities in both assays. Roditis-white must scavenged DPPHd, and exhibited some activity in b-carotene assay. Young Xinomavro and Roditis wines and their extracts were more active in scavenging DPPHd than the respective aged wine and extracts. However, young Xinomavro wine and its extracts were less active than those of aged wine in b-carotene assay. Roditis wine extracts exhibited only limited activity in b-carotene assay. Xinomavro young and aged wine extracts rich in anthocyanins and flavanols were more active than the others in both assays used. & 2008 Elsevier Inc. All rights reserved.
Keywords: Food composition Red wine White wine Greece Greek wines Scavenging capacity Antioxidant activity Phenolics Reactive oxygen species (ROS) Maceration
1. Introduction Current research has confirmed that food rich in antioxidants plays an essential role in the prevention of several diseases. On the other hand, oxidation of lipids in foods is a major cause of chemical spoilage and its products are potentially toxic. Antioxidants are widely used in many foods to prevent fat rancidity (Jadhav et al., 1996). Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used because they are effective, and cheaper than natural ones (Pokorny, 2007). However, the safety and toxicity of synthetic antioxidants have raised important concerns. Hence, considerable interest has been given to the use of natural antioxidants which may also have nutritional properties (Jadhav et al., 1996). Many phenolic antioxidants are present in wines. Wine phenolics are considered to scavenge reactive oxygen species (ROS) (Sanchez-Moreno et al., 1999; Roussis et al., 2005), to inhibit oxidation of oil systems (Sanchez-Moreno et al., 2000; Kovacevic
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Ganic et al., 2006), and to inhibit human low-density lipoprotein (LDL) oxidation (Frankel et al., 1993; Tselepis et al., 2005). Wine phenolics originate from grape juice and especially skins, and also from barrels used in winemaking (Waterhouse, 2002). Red and white wines differ in their phenolic composition due to differences in phenolic composition of red and white grapes and in the winemaking procedures. Red winemaking includes the procedure of maceration while white winemaking does not (Jackson, 1994). This is thought to be the main reason for the relative low polyphenol content and for the lower antioxidant activity of white wine in comparison to red wine (Fuhrman et al., 1998; Vinson and Hontz, 1995). Red wines are good dietary source of various phenolics, including benzoic and cinnamic acid derivatives, flavanols, flavonols and anthocyanins. White wines contained mainly hydroxycinnamates, and benzoic acids (Waterhouse, 2002). The efforts of present work were to evaluate the antioxidant activity and phenolic composition of some Greek wines and wine extracts. Wine and wine extracts examined had previously been reported to decrease heat shock protein levels and cell populations and to inhibit LDL oxidation, and red wine extracts to scavenge ROS (Roussou et al., 2004; Tselepis et al., 2005; Roussis et al., 2005).
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2. Materials and methods 2.1. Wines Eight commercial wines of the Greek vineyard were examined. They were made from the following grape varieties: (1) Agiorgitiko, dry red wine (Nemea, Boutaris); (2) Limnio, dry red wine (Limnio, Karras); (3) Syrah, dry red wine (Syrah, Ktima Kyr-Yianni); (4) Debina, dry white wine (White dry Zitsa, Union of Agricultural Cooperatives of Ioannina); (5) Athiri, dry white wine (Athiri Vounoplagias, Rhodes); (6) Robola, dry white wine (Robola Kephallonias); (7) Sauvignon blanc, dry white wine (Sauvignon blanc Grypas); and (8) Traminer, dry white wine (Traminer, Katogi Averof). Red wines examined were 2.5 years old and white ones 0.5–1.5 years old. Syrah, Sauvignon blanc and Traminer were regional wines, while the others were classed as ‘‘Appellation of Origin’’. Moreover, must, young and aged wines of Xinomavro and Roditis varieties were used. Xinomavro dry red wine of Appelation of Origin (Naoussa, Greece) and Roditis dry table white were used. In Xinomavro winemaking typical procedures for red wines were applied, including maceration. A young wine that was 6 months old and wine aged for 12 months in oak barrels were used. In Roditis winemaking, typical procedures for white wines were applied, and the free run juice was used. Young wine that was 6 months old and wine aged for 6 months in oak barrels were used. 2.2. Wine phenolic extracts Total wine extract was de-alcoholated wine concentrated by rotary evaporation at 25 1C and 80 mbar. Wine added to an equal volume of distilled water was concentrated to the original volume (25 1C, 80 mbar) in order to remove the alcohol without destroying the phenolic compounds. Liquid/liquid extractions of de-alcoholated wine were performed to obtain three extracts containing different classes of polyphenol compounds (Ghiselli et al., 1998). The de-alcoholated wine (pH 2.0) was first extracted with ethyl acetate to obtain an aqueous phase containing mainly the anthocyanins and an organic phase containing mainly flavanols, phenolic acids and flavonols. This aqueous phase was the first fraction (X1 or R1). The organic phase after evaporation was redissolved in water at pH 7.0, and extracted again with ethyl acetate to obtain an organic phase containing mainly flavanols and flavonols and an aqueous phase containing mainly phenolic acids and flavonols. This organic phase was the second fraction (X2 or R2). The aqueous phase was adjusted at pH 2.0 and extracted again with ethyl acetate to collect its phenolic compounds. This organic phase was the third fraction (X3 or R3). Each of the three Xinomavro-red wine fractions was subfractionated into non-polymeric (monomeric and dimeric) and polymeric polyphenols using a Sephadex LH-20 column (Kantz and Singleton, 1990). Non-polymeric polyphenols (sub-fraction a, X1a/X2a/X3a) were desorbed by methanol/acetic acid from the gel, and polymeric polyphenols (sub-fraction b, X1b/X2b/X3b) by acetone/acetic acid. Samples in 10% ethanol were used. 2.3. Analysis of phenolics The total phenolic content was determined by the Folin–Ciocalteau method (Singleton and Rossi, 1965) using gallic acid as a standard. The absorbances at 280, 340, 360 and 520 nm were also used to estimate total phenolic content, tartaric esters, flavonols and anthocyanins, respectively (Mazza et al., 1999). The method consisted of placing 0.25 mL of sample in a test tube and adding
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0.25 mL 0.1% HCl in 95% ethanol and 4.55 mL of 2% HCl. The solution was mixed and after 15 min the absorbances at 280, 340, 360 and 520 nm were read. Standard used were gallic acid in 10% ethanol for total phenolics, caffeic acid in 10% ethanol for tartatic esters, quercetin in 95% ethanol for flavonols, and malvidin-3-glucoside in 10% ethanol for anthocyanins. The total flavanol content was estimated using the p-dimethylaminocinnamaldehyde (DMACA) method (Arnous et al., 2001). Wine appropriately diluted in methanol (0.4 mL), were mixed with 2 mL of DMACA solution (0.1% in 1 N HCl in methanol). The mixture was vortexed and allowed to react at room temperature for 10 min. The absorbance at 640 nm was then read against a blank prepared similarly without DMACA. Catechin was used as a standard. All samples were analyzed by HPLC-DAD for individual phenolic compounds. Samples were filtered using syringe filter (PTFE 0.45, Altech) prior to the injection. Waters 600E system with a 996-photodiode array detector and a 600E pump was used. Chromatograms were treated using the Millenium 32 program. The column used was a C18 reversed phase Spherisorb (4.0 250 mm2) with 5-mm packing. The mobile phases were, A: water/glacial acetic acid (98:2), B: methanol/water/glacial acetic acid (60:38:2) and C: methanol/ glacial acetic acid (98:2). The gradient used was 0–30 min 100% A at 0.20 mL/min, 30–40 min 58.3% A–41.7% B at 0.60 mL/min. 40–120 min 41.7% A–58.3% B at 0.20 mL/min, 120–155 min 25% A–75% B at 0.30 mL/min, 155–165 min 100% C at 0.60 mL/min, 165–180 min 100% C 0.90 mL/min. The purity of each classified peak was confirmed by the low peak angle and the purity threshold values and the single spectrum that corresponded to each peak. Peak identification and classification was carried out as described previously (Roussou et al., 2004). Some peaks were identified on the basis of the retention time and the UV–Vis spectra of several standards used. All peaks were classified using absorbance characteristics of the phenolic classes derived from the literature (Robards et al., 1999; Lee and Widmer, 1996) and from our observations using several standards. The absorbance wave lengths of phenolic classes were as follows: benzoic acids at 250–280 nm; hydroxycinnamic acids at 305–330 nm, and several of them also at 290–300 nm; anthocyanins at 450–560 nm and at 240–280 nm, and some of them at 315–325 nm; flavanols at 270–280 nm and around 230 nm; flavonols at 350–380 nm and at 250–270 nm, and some of them around 300 nm; flavones and isoflavones at 300–350 nm and at 245–270 nm; flavanones at 270–295 nm, and some of them at 300–320 nm. Peaks exhibited maximum absorbance at 280–305 nm expressed as unclassified 280 nm. Peaks exhibited maximum absorbance at around 230 nm and also absorbed at around 280 nm expressed as unclassified 230 nm. Subsequently, all peaks were classified into nine groups. As main phenolic peaks were taken those exhibiting high area at 280, 255, 320, 360 or 520 nm.
2.4. Antioxidant activities Antioxidant activity of samples was determined using the methods of 1,10 -diphenyl-2-picryl-hydrazyl radical (DPPHd) and bleaching of b-carotene. The ability of samples to scavenge the DPPHd was evaluated as described previously (Larrauri et al., 1998). In test tubes, 0.1 mL of sample (wine or extract in 10% ethanol) or 0.1 mL of 10% ethanol (control) was added to 3.9 mL of DPPHd solution (6 105 mol/L in methanol), and the mixture was mixed well. The absorbance at 515 nm was measured at various time intervals. A blank was prepared for each sample using methanol instead of the DPPHd solution.
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For each sample concentration tested, the reaction kinetics was plotted. Moreover, the EC50 values of each wine, i.e. the concentration of total phenolics (mg/L) required to lower the initial DPPHd concentration by 50%, was also calculated (linear equation). The exact DPPHd concentration was calculated from a calibration curve with the equation C ¼ 9.5 A, where C the concentration of DPPHd (mg/L) and A the absorbance at 515 nm. EC50 value of quercetin was also determined (linear equation). The ability of samples to inhibit the coupled oxidation of b-carotene and linoleic acid was evaluated as described previously (Gazzani et al., 1998). In test tubes, 0.1 mL of sample (wine or extract in 10% ethanol) or 0.1 mL of 10% ethanol (control) was added to 3.0 mL of b-carotene and linoleic acid emulsion. The mixture was mixed well and the tubes were put in a water bath at 50 1C. The absorbance at 470 nm was measured after 0, 20, 40, 60, 80, 100 and 120 min of incubation. The b-carotene and linoleic acid emulsion was prepared by adding 22.2 mL linoleic acid and 185 mL Tween 40 in 1 mL of b-carotene solution (200 mg/L) in chloroform. Chloroform was evaporated to dryness by rotary evaporation at 25 1C and 80 mbar. Then, 50 mL aerated distilled water were added and the mixture was shaken. Each sample was read against an emulsion prepared as described but without b-carotene (blank). For each sample concentration tested, the reaction kinetics was plotted. Moreover, the EC50 value of each sample, i.e. the concentration of total phenolics (mg/L) required to lower the initial absorbance at 470 nm by 50%, was calculated (logarithmic equation). EC50 value of quercetin was also determined (logarithmic equation). 2.5. Statistical analysis Each experiment was repeated three times and the results reported are the means of the three trials. The one way analysis of variance (ANOVA), using the Duncan test at a level of significance Po0.05 was used for the statistical analysis (SPSS 11.5).
3. Results and discussion
Robola exhibited higher tartaric esters than the others followed by Debina. Similar total phenolics of red wines (1000–4000 mg/L) and of white wines (some hundreds mg/L), and similar values for the determined phenolic classes have also been reported by others (Sanchez-Moreno et al., 2000; De Beer et al., 2003; Minussi et al., 2003; Fernadez-Pachon et al., 2004). All red and white wines were analyzed by HPLC for their phenolic composition. Red wines contained various phenolics (benzoic acids, cinnamic acids, flavanols, flavonols, flavanones, flavones, anthocyanins, unclassifieds at 230 nm, and unclassified at 280 nm). On the other hand, white wines contained mainly benzoic and cinnamic acids. These phenolic classes for red and white wines have also been reported by others (Landrault et al., 2001; Waterhouse, 2002). The main phenolic peaks in chromatograms of each wine are presented in Table 2. As it can be seen some wines, such as Debina, Robola, Limnio and Syrah, exhibited one very high peak of benzoic or cinnamic acid each. All wines scavenged DPPHd, and their scavenging activities decreased by dilution. Red wines were more active than whites. Red wines diluted 10 or more times exhibited comparable activity to undiluted white ones. This may be attributed to their higher phenolic content. Moreover, most red wines exhibited lower EC50 values at 5 min (initial scavenging capacity) and at 40 min (total scavenging capacity) than most white wines indicating the high potency of red wine phenolics (Table 3). However, Limnio red Table 2 Peaks of phenolic compounds with high area in the chromatograms of wines Wine
Major peaks of phenolics and their area
Agiorgitiko
Cinnamic acid, 77.0a; flavanol, 57.1; cinnamic acid, 42.8; gallic acid, 31.3; unclassified 230 nm, 6.7; anthocyanin, 3.4; anthocyanin, 1.0; anthocyanin, 0.3 Cinnamic acid, 152.9; cinnamic acid, 40.9; cinnamic acid, 38.1; gallic acid, 28.0; flavanol, 20.4; unclassified 230 nm, 5.9; anthocyanin, 0.5; anthocyanin, 0.2; anthocyanin, 0.1 Cinnamic acid, 71.8; flavanol, 24.8; cinnamic acid, 21.7; gallic acid, 18.9; cinnamic acid, 15.3; cinnamic acid, 11.0; unclassified 230 nm, 7.6; anthocyanin, 5.9; flavonol, 3.2; anthocyanin, 1.2 Benzoic acid, 652.4; benzoic acid, 132.2; cinnamic acid, 55.6 Cinnamic acid, 28.2; cinnamic acid, 9.4; cinnamic acid, 8.4; feroulic acid, 7.3; benzoic acid, 6.9; flavonol, 5.0 Cinnamic acid, 126.2; flavone, 19.9; cinnamic acid, 14.3; caffeic acid, 8.6; cinnamic acid, 7.2; benzoic acid, 5.3; benzoic acid, 3.9 Cinnamic acid, 28.1; benzoic acid, 8.8; caffeic acid, 7.2; benzoic acid, 4.7 Cinnamic acid, 12.8; tyrosol, 5.3; caffeic acid, 4.5; benzoic acid, 3.4; p-coumaric acid, 2.8; benzoic acid, 2.7; benzoic acid, 2.1; flavone, 1.8
Limnio
Syrah
Debina Athiri
3.1. Antioxidant activities and phenolic composition of various wines Total phenolics, tartaric esters, flavonols, anthocyanins, and flavanols of various red and white wines of the Greek vineyard are presented in Table 1. As expected, red wines contained much higher phenolics than whites. Among red wines, Syrah exhibited the higher total phenolics, tartaric esters, flavonols, anthocyanins, and flavanols. All white wines exhibited similar total phenolics, while they did not contain anthocyanins and flavanols. However,
Robola
Sauvignon blanc Traminer
a
106, is the area of the peak.
Table 1 Total phenolics (Folin and A280 nm), tartaric esters, flavonols, anthocyanins and flavanols of red and white wines of the Greek vineyard Wine
Total phenolics (mg/L gallic acid)
A280 nm (mg/L gallic acid)
A320 nm (mg/L caffeic acid)
A360 nm (mg/L quercetin)
A520 nm (mg/L malvidin3-glucoside)
Flavanols (mg/L catechin)
Agiorgitiko Limnio Syrah Debina Athiri Robola Sauvingnon blanc Traminer
2082b738 2704c7145 3184d7169 261a710 213a79 258a710 272a719 277a76
1138c725 1450d734 1700e733 314b79 206a76 219a717 212a77 208a79
208d719 279e713 308f716 62b75 40a75 96c76 48ab76 57ab77
144b710 174c712 219d713 35a74 24a73 52a76 28a74 28a75
335c721 224b714 523d714 0a70 0a70 0a70 0a70 0a70
230.8b77.5 338.5c721.5 527.1d77.5 0.6a70.1 0.7a70.1 0.3a70.0 0.7a70.1 1.4a70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at Po0.05. Standard deviation.
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wine exhibited EC50 values at 5 min comparable to those of white wines. On the contrary, Sauvignon blanc white wine exhibited EC50 values at 5 min and at 40 min comparable to those of red wines. Quercetin appeared to be more active (lower EC50) than red wines. Quercetin activity was dose dependent in the range 0–5.0 mg/L. DPPHd scavenging by wines and also a correlation between antiradical efficiency and total phenolic content or flavanols has been reported by others (Frankel et al., 1995; Soleas et al., 1997; Sanchez-Moreno et al., 1999; De Beer et al., 2003; Katalinic et al., 2004; Fernandez-Pachon et al., 2006; Kovacevic Ganic et al., 2006). Red wines inhibited the coupled oxidation of b-carotene and linoleic acid, and their activities decreased by dilution. The EC50 values of red wines (Table 4) indicate that their phenolics were more active or equivalent to quercetin. Quercetin activity was
Table 3 EC50 values (mg/L) of red and white wines of the Greek vineyard and quercetin for the scavenging of DPPHd Wine
EC50 at 5 min
EC50 at 40 min
Agiorgitiko Limnio Syrah Debina Athiri Robola Sauvingnon blanc Traminer Quercetin
6.7b70.3
3.1b70.0 4.0cd70.1 3.5bc70.1 6.1e70.4 5.6e70.8 4.4d70.3 4.2d70.1 5.8e70.5 1.6a70.2
10.4d70.1 6.1b70.6 12.2e70.5 12.7e70.5 11.5e70.7 7.8c70.8 10.7d70.6 2.1a70.3
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
Table 4 EC50 values (mg/L) of red wines of the Greek vineyard and quercetin for the inhibition of coupled oxidation of b-carotene and linoleic acid Sample
EC50 at 20 min
EC50 at 120 min
Agiorgitiko Limnio Syrah Quercetin
5.8 e17b73.8 e17 1.6 e27a71.0 e27 4.6 e27a72.3 e27 1.7 e17b70.9 e17
0.15b70.05 0.25b70.06 0.01a70.01 0.26b70.05
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
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dose dependent in the range 0–6.5 mg/L. White wines exhibited only limited activity. Twenty-fold diluted white wines exhibited some activity, while less diluted white wines were inactive or prooxidants. Inhibition of b-carotene bleaching by wines and also a correlation between antioxidant activity and anthocyanins and catechins has been reported by others (Katalinic et al., 2004). Other researchers have reported that in b-carotene assay anthocyanins and flavonols are very active followed by flavanols, while phenolic acids are less active (Fukumoto and Mazza, 2000). 3.2. Antioxidant activities and phenolic composition of Xinomavrored and Roditis-white must and wine Total phenolics, tartaric esters, flavonols and anthocyanins of Xinomavro-red and Roditis-white must, young and aged wines are presented in Table 5. Xinomavro young wine exhibited much higher phenolics than must, which may be attributed to phenolic extraction during maceration, and aged wine higher than young wine, which may be due to migration from oak barrel (Waterhouse, 2002). However, aged wine exhibited lower anthocyanin content than young wine, as determined by the absorbance at 520 nm. Similar results have also been reported by others and have attributed to the transformation of monomeric anthocyanins to more stable oligomeric forms which absorb at wavelength lower than 520 nm (Gomez-Plaza et al., 2000). Roditis must exhibited higher total phenolics and also tartaric esters and flavonols than young and aged wines. This may be due to precipitation of some phenolics during winemaking. The amount of anthocyanins of Roditis must may be attributed to the light pink color of Roditis grape. Young and aged wines exhibited similar phenolic levels. HPLC chromatograms of Xinomavro-red and Roditis-white must, young and aged wines are presented in Figs. 1 and 2, respectively. In chromatogram of Xinomavro wine, as well as in chromatograms of all the other red wines analysed, an increase of baseline at 50–85 min was observed. Similar behavior during red wine analysis was observed by others, and had been attributed to polymeric compounds exhibiting the same size and polarity (Waterhouse et al., 1999). Phenolic levels and composition of Xinomavro and Roditis wines were similar to those reported for red and white wine, respectively (Sanchez-Moreno et al., 2000; Landrault et al., 2001; De Beer et al., 2003; Minussi et al., 2003; Fernadez-Pachon et al., 2004). Xinomavro and Roditis musts exhibited scavenging activity of DPPHd, which decreased by dilution. Similarly, antiradical activity of commercial grape juices has been reported by others (Davalos et al., 2005). However, activity of each must was much lower than young and aged wines. Moreover, Xinomavro and Roditis musts
Table 5 Total phenolics (Folin and A280 nm), tartaric esters, flavonols and anthocyanins of Xinomavro-red and Roditis-white must, young and aged wines Total phenolics, Folin (mg/L gallic acid)
Total phenolics (A280 nm, mg/L gallic acid)
Tartaric esters (A320 nm, mg/L caffeic acid)
Flavonols (A360 nm, mg/ L quercetin)
Anthocyanins (A520 nm, mg/L malvidin-3-glucoside)
Xinomavro Must Young wine Aged wine
1790a759 3476b771 3802c736
832a720 1652b725 1916c716
201a713 263b72 315c77
155a79 193b76 225c76
306a716 361b75 325a78
Roditis Must Young wine Aged wine
371b713 291a75 304a78
264b713 210a76 248b74
83b74 73a71 75a71
79b73 39a72 40a72
23b73 1a70 3a71
Sample
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at Po0.05. Standard deviation.
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11
0 200.
Must, 280 nm 17
AU
0 150.
16 18
0 100.
15
23
19 20 21
0 050. 1
4
5
6
7
0 000. 0 015.
14 12 13
2223
40 39
24 25 26 27
28
8 9 10
29
30
31
33 34
32
36
35
Must, 520 nm
AU
0 010. 0 005. 37 38
0 000. 2223 24 25
1 000. 14
0 800.
21
AU
9
20
0 600. 0 400.
1
15 11
0 200.
3 5 2 4 6 7 8
0 000.
10 1213
26
Young wine, 280 nm
28 29 30 27 31 36 37 32 35 3334
19 16 18 17
38
39 40
43 44 45 47 49 50
48
Young wine, 520 nm
0.020
AU
42
51 52
46
0 010.
53 41
0 000. 17
0 800.
21 19
AU
0 600.
22
23 24 25
20
27
26
18
0 400. 31 32
11 57
0 200.
3 12 4 6 8
0 000.
8
15 10 12 13 14 16
28
Aged wine, 280 nm
29 30 33 34 35 37
36 38 39 40
41
4243 44
46
47
Aged wine, 520 nm
0. 008
50 49
51
AU
0. 006 0.004
45
48
0. 002 0. 000 0
20
40
60
80
100
120
140
160
180
Minutes Fig. 1. HPLC chromatograms of Xinomavro-red must, young and aged wines at 280 and 520 nm. Must: 1–4, unclassifieds 230 nm; 5, 6 benzoic acids; 7, gallic acid; 8, flavanol; 9, cinnamic acid; 10, unclassified 280 nm; 11, cinnamic acid; 12, unclassified 280 nm; 13, cinnamic acid; 14, flavanone; 15, cinnamic acid; 16, flavanol; 17, flavanone; 18, 19, flavanols; 20, flavanone; 21, benzoic acid; 22, 23, cinnamic acids; 24, unclassified 280 nm; 25–27, flavanols; 28–30, unclassifieds 280 nm; 31, 32, flavonols; 33, rutin; 34, flavonol; 35, anthocyanin; 36, unclassified 280 nm; 37, 38, anthocyanins; 39, benzoic acid; 40, flavanol. Young wine: 1, 2, unclassifieds 230 nm; 3, 4, cinnamic acids; 5–7, benzoic acids; 8, unclassified 280 nm; 9, gallic acid; 10, benzoic acid; 11, cinnamic acid; 12, unclassified 280 nm; 13, benzoic acid; 14, 15, cinnamic acids; 16, flavanone; 17, cinnamic acid; 18, flavanone; 19, flavanol; 20, 21, flavanones; 22–29, flavanols; 30, unclassified 280 nm; 31, cinnamic acid; 32, flavanol; 33, cinnamic acid; 34, benzoic acid; 35, and 36, flavanols; 37, 38, unclassifieds 280 nm; 39, 40, benzoic acids; 41, anthocyanin; 42, flavonol; 43, cinnamic acid; 44, flavonol; 45, rutin; 46, anthocyanin; 47, flavonol; 48, anthocyanin; 49, 50, flavonols; 51–53, anthocyanins. Aged wine: 1–3, unclassifieds 230 nm; 4, cinnamic acid; 5, benzoic acid; 6, cinnamic acid; 7–9, benzoic acids; 10, flavone; 11, gallic acid; 12, unclassified 280 nm; 13, benzoic acid; 14–16, flavanols; 17, cinnamic acid; 18–22, flavanols; 23, flavone; 24–32, flavanols; 33, 34, unclassifieds 280 nm; 35, flavanone; 36, 37, flavanols; 38, syringic acid; 39, flavone; 40, cinnamic acid; 41, p-coumaric acid; 42, cinnamic acid; 43, 44, unclassifieds 280 nm; 45, anthocyanin; 46, unclassified 280 nm; 47, rutin; 48, 49, anthocyanins; 50, benzoic acid; and 51, anthocyanin.
exhibited higher EC50 values at 5 min (initial scavenging activity), and also at 60 min (total scavenging activity) than young and aged wines (Table 6). Consequently, the lower antiradical potency of must may be attributed to its lower phenolic content or to the lower potency of must phenolics. Both young wines exhibited
lower EC50 values at 5 min than the respective aged wine, indicating the potency of their phenolics. Xinomavro-red must exhibited significant antioxidant activity in the b-carotene assay, which decreased by dilution. Xinomavro must diluted 10 times exhibited total antioxidant activity
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2
0.120
619
Must
3 25 24
0.080
26
23
AU
4 5 8
0.040
12 11
6 7 9
1
0.000 0.600
13
10
29
27
16 19 18 2122 15 17 20
28 31 33 34 32 35
14
37
43 44
39
42
38
45
40 41
30
Young wine
4 1 6
0.400
30
AU
23 11
22
5 9
7
0.200
12 8
2
0.000
3
10
17
32
28
35
24 21
18
14
37 33
19
16 13
26 25 27
20
15
29
31
43 36
38 39
34
40 41
42
AU
47 46 48
4
6
0.400 5
19
7 10
1
8 9 3
22 21
11 18
13
0.000
44
Aged wine
2
0.800
45
15 14
16
27
23
20
17
24
26 25
33 34 36 29 32 28 35 37 31 38
12
20
46
39 40
48 45 47
42 43 44
41
30
40
60
80
100
120
140
160
180
Minutes Fig. 2. HPLC chromatograms of Roditis-white must, young and aged wines at 280 nm. Must: 1–3, unclassifieds 230 nm; 4–6, cinnamic acids; 7, 8, benzoic acids; 9, cinnamic acid; 10, benzoic acid; 11, flavonol; 12, flavanol; 13, cinnamic acid; 14, flavone; 15, cinnamic acid; 16, flavanol; 17, benzoic acid; 18, cinnamic acid; 19, benzoic acid; 20–22, cinnamic acids; 23, benzoic acid; 24, unclassified 280 nm; 25, 26, benzoic acids; 27, 28, cinnamic acids; 29, benzoic acid; 30, unclassified 230 nm; 31, 32, unclassifieds 280 nm; 33–35, benzoic acids; 36, cinnamic acid; 37, flavanol; 38, benzoic acid; 39, unclassified 280 nm; 40, rutin; 41, flavonol; 42, benzoic acid; 43–45, flavanols. Young wine: 1–3, unclassifieds 230 nm; 4, 5, flavanones; 6, flavone; 7, 8, benzoic acids; 9, cinnamic acid; 10–12, benzoic acids; 13, unclassified 280 nm; 14, flavanone; 15, 16, benzoic acids; 17–21, cinnamic acids; 22, flavanone; 23, cinnamic acid; 24, flavone; 25, flavanol; 26, benzoic acid; 27, cinnamic acid; 28, benzoic acid; 29, unclassified 230 nm; 30, caffeic acid; 31, cinnamic acid; 32, 33, benzoic acids; 34, flavone; 35, unclassified 280 nm; 36, benzoic acid; 37, p-coumaric acid; 38, cinnamic acid; 39, flavanol; 40, benzoic acid; 41, unclassified 280 nm; 42, benzoic acid; 43–45, cinnamic acids; 46, benzoic acid; 47, unclassified 280 nm; 48, flavanol. Aged wine: 1–3, unclassifieds 230 nm; 4–5, cinnamic acids; 6–12, benzoic acids; 13, unclassified 280 nm; 14, benzoic acid; 15, unclassified 280 nm; 16–18, cinnamic acids; 19, benzoic acid; 20, flavanone; 21, flavone; 22–24, flavanones; 25, benzoic acid; 26, caffeic acid; 27, cinnamic acid; 28, flavanone; 29, cinnamic acid; 30, unclassified 280 nm; 31–34, benzoic acids; 35, flavanone; 36, cinnamic acid; 37, 38, cinnamic acids; 39, unclassified 280 nm; 40, benzoic acid; 41, 42, unclassifieds 280 nm; 43–45, cinnamic acids; 46, benzoic acid; and 47, 48, flavanols.
Table 6 EC50 values (mg/L) of Xinomavro-red and Roditis-white must, young and aged wines for the scavenging of DPPHd
Table 7 EC50 values (mg/L) of Xinomavro-red must, young and aged wines for the inhibition of coupled oxidation of b-carotene and linoleic acid
Sample
EC50 at 5 min
EC50 at 60 min
Sample
EC50 at 20 min
EC50 at 120 min
Xinomavro Must Young wine Aged wine
5.0c70.2 2.4a70.1 3.6b70.3
2.3b70.2 1.9a70.1 1.7a70.2
Must Young wine Aged wine
6.4 e6a71.8 e6 30.4 e6b72.6 e6 3.6 e6a71.8 e6
1.6b70.3 2.5c70.5 0.4a70.2
Roditis Must Young wine Aged wine
9.8b71.8 4.1a70.3 4.9a70.4
3.8a70.2 2.1b70.1 2.5c70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
comparable to young wine diluted 10 times and aged wine diluted 20 times, eventhough it contained much lower total phenolics than those. Moreover, EC50 value of Xinomavro must was lower
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
than that of young wine (Table 7). Xinomavro aged wine appeared to be more active and exhibited lower EC50 value than young wine and must. Increased antioxidant capacity with aging of red wines has been reported by others (Echeverry et al., 2005). Roditis-white must exhibited some activity in the b-carotene assay. However, its activity was much lower than Xinomavro-red must, even when tested at the same total phenolic content. Young and aged Roditis
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wines exhibited only limited activity. They exhibited prooxidant activity or none activity, respectively, at 20 min (initial antioxidant activity). At 120 min (total antioxidant activity), 8 or 4-fold diluted wines exhibited some activity while less diluted not. 3.3. Antioxidant activities and phenolic composition of Xinomavrored and Roditis-white wine extracts Young and aged Xinomavro-red and Roditis-white wines fractionated into three fractions each by extraction. All these phenolic fractions were examined for scavenging DPPHd and inhibiting b-carotene bleaching. HPLC chromatograms revealed that Xinomavro young or aged wine fractions 1 contained mainly anthocyanins and flavanols, fractions 2 flavanols, flavonols and tyrosol, and fractions 3 cinnamic acids, benzoic acids and flavonols. Chromatographic analysis also indicated that Roditis young or aged wine fractions 1 contained mainly unclassified peaks with max. 230 nm and some phenolic acids, fractions 2 phenolic acids and tyrosol, and fractions 3 phenolic acids. All wine extracts exhibited antiradical potency in a dosedependent manner, indicating that many red and white wine phenolics may be active (Table 8). Similarly, scavenging of DPPHd by various wine phenolics has been reported by others (Frankel et al., 1995; Soleas et al., 1997). Young wine fractions exhibited lower EC50 values than the respective of aged wine at 5 min (initial scavenging activity) and also at 60 min (total scavenging activity). This indicates the higher antiradical potency of all classes of young compared to aged red and white wine phenolics. In Xinomavro, no systematic differences were observed among young wine fractions. However, among aged wine fractions, fraction 1 exhibited lower EC50 values at 5 min than the others, indicating the possible potency of anthocyanins and flavanols. Similarly, flavanols exhibited best correlation among wine phenolics for DPPHd scavenging (Arnous et al., 2002; De Beer et al., 2003), while red wine extract containing anthocyanins and flavanols exhibited best DPPHd scavenging among the other extracts (Ghiselli et al., 1998; Burns et al., 2001; Fernadez-Pachon et al., 2004). In Roditis, young and aged wine fractions 3 exhibited lower EC50 values than the others, indicating the possible potency of cinnamic and benzoic acids. Each of the three Xinomavro-red aged wine fractions was sub-fractionated into non-polymeric (monomeric and dimeric) and polymeric polyphenols. All sub-fractions exhibited antiradical Table 8 EC50 values (mg/L) of Xinomavro-red young and aged wine phenolic fractions for the scavenging of DPPHd Fraction
EC50 at 5 min
EC50 at 60 min
Xinomavro 1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
1.8a70.1 2.0a70.1 2.3a70.1 3.9b70.2 5.0c70.2 4.6c70.3
1.4a70.1 1.6b70.1 1.4a70.1 2.1cd70.2 2.5d70.3 1.8bc70.2
Roditis 1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
3.2c70.1 1.9b70.1 1.2a70.1 6.4d70.5 3.2c70.1 3.3c70.4
1.6b70.1 1.4b70.1 0.9a70.1 3.3d70.3 2.0c70.1 2.0c70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
Table 9 EC50 values (mg/L) of Xinomavro-red young and aged wine phenolic fractions for the inhibition of coupled oxidation of b-carotene and linoleic acid Fraction
EC50 at 20 min
EC50 at 120 min
1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
1.5 e3a70.3e3 1.7 e3ab70.2e3 2.2 e3b70.3e3 0.2 e3c70.2e3 0.4 e3c70.1 e3 1.9 e3b70.1 e3
1.1c70.1 1.1c70.1 1.2d70.1 0.6a70.1 0.9b70.1 0.9b70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
potency (data not shown). It has been reported that polymeric phenolics of red wines contribute to 50% of its scavenging capacity (Fernadez-Pachon et al., 2004). All Xinomavro-red young and aged wine fractions inhibited b-carotene bleaching (Table 9), indicating that many wine phenolics may be active. Similarly, inhibition of b-carotene bleaching by various phenolic compounds has been reported by others (Fukumoto and Mazza, 2000). Most aged wine fractions exhibited lower EC50 values than the respective of young wine. This indicates the higher antioxidant potency of aged compared to young wine phenolics. Fractions 1 of young and aged Xinomavro wines exhibited lower EC50 values than the other fractions, indicating the possible high potency of anthocyanins and flavanols. Similarly, anthocyanins and catechins exhibited best correlation among wine phenolics for inhibition of b-carotene bleaching (Katalinic et al., 2004) and anthocyanins and flavanols for inhibition of corn oil emulsion (Sanchez-Moreno et al., 2000). All Xinomavro-red aged wine sub-fractions inhibited b-carotene bleaching (data not shown), indicating the potency of both nonpolymeric and polymeric phenolics. All Roditis-white wine extracts were examined for their antioxidant potency in b-carotene bleaching at various dilutions, i.e. at total phenolic range 0.5–10 mg/L. Among them, extracts 2 and 3 exhibited only limited antioxidant activity, and extracts 1 were inactive. The most active extract, extract 2 of aged wine, exhibited % inhibition up to 75%, while the others up to 50%. These results indicate possible low effectiveness of phenolic acids in b-carotene bleaching, since extracts 2 and 3 were rich in phenolic acids. Present results indicate that red and white must, especially red one, exhibit antioxidant activities in scavenging of DPPHd and in bleaching of b-carotene. Red wines are also active in these two assays. White wines scavenge DPPHd, while they are not active in b-carotene assay. Results indicate that many red and white wine phenolics may be active in scavenging DPPHd and inhibiting b-carotene bleaching. Moreover, they indicate that anthocyanins and flavanols may be among the most active phenolics. The absence of those phenolics (anthocyanins and flavanols) in white wines may be explaining the lower potency (EC50 values) of white wine phenolics versus those of reds. In present study, white wines and white wine extracts were inactive or exhibited limited activity in b-carotene bleaching. However, white wines and extracts exhibited significant activity in inhibiting LDL oxidation (Tselepis et al., 2005). Their differences in these two assays may be attributed to poor distribution of phenolic acids (the main phenolics of white wines and extracts) between the two phases of the emulsion in the case of b-carotene assay. Results show that the order of antioxidant activities of wines and wine extracts change according to the method used, DPPHd scavenging and b-carotene bleaching. This may be attributed to
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the chemistry of these antioxidant capacity assays. DPPHd scavenging was believed to involve hydrogen atom transfer reaction. However, a recent work based on the kinetic analysis of the reaction between phenols and DPPHd shows that the reaction in fact behaves like a single electron transfer reactionbased assay (Foti et al., 2004). On the other hand, the coupled oxidation of linoleic acid and b-carotene, like other linoleic acid oxidation assays, is a hydrogen atom transfer reaction-based assay (Huang et al., 2005). Consequently, the differences observed in the two assays may be attributed to differences in electron and hydrogen transfer abilities of their phenolics. Moreover, in the case of b-carotene assay the distribution of the antioxidants (phenolics) between the two phases of the emulsion can be critical.
4. Conclusions Present results indicate that must of red and white grapes, especially red, is active in scavenging DPPHd and inhibiting bcarotene bleaching. Red wines of the Greek vineyard exhibit higher activities in these two assays than white wines. Results indicate that wine aging may affect their antioxidant activities, while anthocyanins and flavanols may be among the most active phenolics.
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Original Article
Antioxidant activities of some Greek wines and wine phenolic extracts Ioannis G. Roussis a,, Ioannis Lambropoulos a, Panagiotis Tzimas a, Anna Gkoulioti b, Vasilios Marinos b, Dimitrios Tsoupeis c, Ioannis Boutaris c a b c
Laboratory of Food Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece Ampeloeniki, 57001 Thessaloniki, Greece Ktima Kyr-Yianni, 59200 Yiannakochori Imathias, Greece
a r t i c l e in f o
a b s t r a c t
Article history: Received 30 January 2007 Received in revised form 9 February 2008 Accepted 9 February 2008
Antioxidant activities and phenolic composition of eight Greek wines were determined. Red wines scavenged 1,10 -diphenyl-2-picryl-hydrazyl radical (DPPHd) to a much greater degree than whites, in proportion to their phenolic contents. Red wines inhibited b-carotene bleaching, while white wines were almost inactive. Red wines were more active than quercetin in DPPHd and less active in b-carotene assay. Xinomavro-red must exhibited significant activities in both assays. Roditis-white must scavenged DPPHd, and exhibited some activity in b-carotene assay. Young Xinomavro and Roditis wines and their extracts were more active in scavenging DPPHd than the respective aged wine and extracts. However, young Xinomavro wine and its extracts were less active than those of aged wine in b-carotene assay. Roditis wine extracts exhibited only limited activity in b-carotene assay. Xinomavro young and aged wine extracts rich in anthocyanins and flavanols were more active than the others in both assays used. & 2008 Elsevier Inc. All rights reserved.
Keywords: Food composition Red wine White wine Greece Greek wines Scavenging capacity Antioxidant activity Phenolics Reactive oxygen species (ROS) Maceration
1. Introduction Current research has confirmed that food rich in antioxidants plays an essential role in the prevention of several diseases. On the other hand, oxidation of lipids in foods is a major cause of chemical spoilage and its products are potentially toxic. Antioxidants are widely used in many foods to prevent fat rancidity (Jadhav et al., 1996). Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used because they are effective, and cheaper than natural ones (Pokorny, 2007). However, the safety and toxicity of synthetic antioxidants have raised important concerns. Hence, considerable interest has been given to the use of natural antioxidants which may also have nutritional properties (Jadhav et al., 1996). Many phenolic antioxidants are present in wines. Wine phenolics are considered to scavenge reactive oxygen species (ROS) (Sanchez-Moreno et al., 1999; Roussis et al., 2005), to inhibit oxidation of oil systems (Sanchez-Moreno et al., 2000; Kovacevic
Corresponding author. Tel.: +30 26510 98344; fax: +30 26510 98782.
E-mail address: [email protected] (I.G. Roussis). 0889-1575/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.02.011
Ganic et al., 2006), and to inhibit human low-density lipoprotein (LDL) oxidation (Frankel et al., 1993; Tselepis et al., 2005). Wine phenolics originate from grape juice and especially skins, and also from barrels used in winemaking (Waterhouse, 2002). Red and white wines differ in their phenolic composition due to differences in phenolic composition of red and white grapes and in the winemaking procedures. Red winemaking includes the procedure of maceration while white winemaking does not (Jackson, 1994). This is thought to be the main reason for the relative low polyphenol content and for the lower antioxidant activity of white wine in comparison to red wine (Fuhrman et al., 1998; Vinson and Hontz, 1995). Red wines are good dietary source of various phenolics, including benzoic and cinnamic acid derivatives, flavanols, flavonols and anthocyanins. White wines contained mainly hydroxycinnamates, and benzoic acids (Waterhouse, 2002). The efforts of present work were to evaluate the antioxidant activity and phenolic composition of some Greek wines and wine extracts. Wine and wine extracts examined had previously been reported to decrease heat shock protein levels and cell populations and to inhibit LDL oxidation, and red wine extracts to scavenge ROS (Roussou et al., 2004; Tselepis et al., 2005; Roussis et al., 2005).
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2. Materials and methods 2.1. Wines Eight commercial wines of the Greek vineyard were examined. They were made from the following grape varieties: (1) Agiorgitiko, dry red wine (Nemea, Boutaris); (2) Limnio, dry red wine (Limnio, Karras); (3) Syrah, dry red wine (Syrah, Ktima Kyr-Yianni); (4) Debina, dry white wine (White dry Zitsa, Union of Agricultural Cooperatives of Ioannina); (5) Athiri, dry white wine (Athiri Vounoplagias, Rhodes); (6) Robola, dry white wine (Robola Kephallonias); (7) Sauvignon blanc, dry white wine (Sauvignon blanc Grypas); and (8) Traminer, dry white wine (Traminer, Katogi Averof). Red wines examined were 2.5 years old and white ones 0.5–1.5 years old. Syrah, Sauvignon blanc and Traminer were regional wines, while the others were classed as ‘‘Appellation of Origin’’. Moreover, must, young and aged wines of Xinomavro and Roditis varieties were used. Xinomavro dry red wine of Appelation of Origin (Naoussa, Greece) and Roditis dry table white were used. In Xinomavro winemaking typical procedures for red wines were applied, including maceration. A young wine that was 6 months old and wine aged for 12 months in oak barrels were used. In Roditis winemaking, typical procedures for white wines were applied, and the free run juice was used. Young wine that was 6 months old and wine aged for 6 months in oak barrels were used. 2.2. Wine phenolic extracts Total wine extract was de-alcoholated wine concentrated by rotary evaporation at 25 1C and 80 mbar. Wine added to an equal volume of distilled water was concentrated to the original volume (25 1C, 80 mbar) in order to remove the alcohol without destroying the phenolic compounds. Liquid/liquid extractions of de-alcoholated wine were performed to obtain three extracts containing different classes of polyphenol compounds (Ghiselli et al., 1998). The de-alcoholated wine (pH 2.0) was first extracted with ethyl acetate to obtain an aqueous phase containing mainly the anthocyanins and an organic phase containing mainly flavanols, phenolic acids and flavonols. This aqueous phase was the first fraction (X1 or R1). The organic phase after evaporation was redissolved in water at pH 7.0, and extracted again with ethyl acetate to obtain an organic phase containing mainly flavanols and flavonols and an aqueous phase containing mainly phenolic acids and flavonols. This organic phase was the second fraction (X2 or R2). The aqueous phase was adjusted at pH 2.0 and extracted again with ethyl acetate to collect its phenolic compounds. This organic phase was the third fraction (X3 or R3). Each of the three Xinomavro-red wine fractions was subfractionated into non-polymeric (monomeric and dimeric) and polymeric polyphenols using a Sephadex LH-20 column (Kantz and Singleton, 1990). Non-polymeric polyphenols (sub-fraction a, X1a/X2a/X3a) were desorbed by methanol/acetic acid from the gel, and polymeric polyphenols (sub-fraction b, X1b/X2b/X3b) by acetone/acetic acid. Samples in 10% ethanol were used. 2.3. Analysis of phenolics The total phenolic content was determined by the Folin–Ciocalteau method (Singleton and Rossi, 1965) using gallic acid as a standard. The absorbances at 280, 340, 360 and 520 nm were also used to estimate total phenolic content, tartaric esters, flavonols and anthocyanins, respectively (Mazza et al., 1999). The method consisted of placing 0.25 mL of sample in a test tube and adding
615
0.25 mL 0.1% HCl in 95% ethanol and 4.55 mL of 2% HCl. The solution was mixed and after 15 min the absorbances at 280, 340, 360 and 520 nm were read. Standard used were gallic acid in 10% ethanol for total phenolics, caffeic acid in 10% ethanol for tartatic esters, quercetin in 95% ethanol for flavonols, and malvidin-3-glucoside in 10% ethanol for anthocyanins. The total flavanol content was estimated using the p-dimethylaminocinnamaldehyde (DMACA) method (Arnous et al., 2001). Wine appropriately diluted in methanol (0.4 mL), were mixed with 2 mL of DMACA solution (0.1% in 1 N HCl in methanol). The mixture was vortexed and allowed to react at room temperature for 10 min. The absorbance at 640 nm was then read against a blank prepared similarly without DMACA. Catechin was used as a standard. All samples were analyzed by HPLC-DAD for individual phenolic compounds. Samples were filtered using syringe filter (PTFE 0.45, Altech) prior to the injection. Waters 600E system with a 996-photodiode array detector and a 600E pump was used. Chromatograms were treated using the Millenium 32 program. The column used was a C18 reversed phase Spherisorb (4.0 250 mm2) with 5-mm packing. The mobile phases were, A: water/glacial acetic acid (98:2), B: methanol/water/glacial acetic acid (60:38:2) and C: methanol/ glacial acetic acid (98:2). The gradient used was 0–30 min 100% A at 0.20 mL/min, 30–40 min 58.3% A–41.7% B at 0.60 mL/min. 40–120 min 41.7% A–58.3% B at 0.20 mL/min, 120–155 min 25% A–75% B at 0.30 mL/min, 155–165 min 100% C at 0.60 mL/min, 165–180 min 100% C 0.90 mL/min. The purity of each classified peak was confirmed by the low peak angle and the purity threshold values and the single spectrum that corresponded to each peak. Peak identification and classification was carried out as described previously (Roussou et al., 2004). Some peaks were identified on the basis of the retention time and the UV–Vis spectra of several standards used. All peaks were classified using absorbance characteristics of the phenolic classes derived from the literature (Robards et al., 1999; Lee and Widmer, 1996) and from our observations using several standards. The absorbance wave lengths of phenolic classes were as follows: benzoic acids at 250–280 nm; hydroxycinnamic acids at 305–330 nm, and several of them also at 290–300 nm; anthocyanins at 450–560 nm and at 240–280 nm, and some of them at 315–325 nm; flavanols at 270–280 nm and around 230 nm; flavonols at 350–380 nm and at 250–270 nm, and some of them around 300 nm; flavones and isoflavones at 300–350 nm and at 245–270 nm; flavanones at 270–295 nm, and some of them at 300–320 nm. Peaks exhibited maximum absorbance at 280–305 nm expressed as unclassified 280 nm. Peaks exhibited maximum absorbance at around 230 nm and also absorbed at around 280 nm expressed as unclassified 230 nm. Subsequently, all peaks were classified into nine groups. As main phenolic peaks were taken those exhibiting high area at 280, 255, 320, 360 or 520 nm.
2.4. Antioxidant activities Antioxidant activity of samples was determined using the methods of 1,10 -diphenyl-2-picryl-hydrazyl radical (DPPHd) and bleaching of b-carotene. The ability of samples to scavenge the DPPHd was evaluated as described previously (Larrauri et al., 1998). In test tubes, 0.1 mL of sample (wine or extract in 10% ethanol) or 0.1 mL of 10% ethanol (control) was added to 3.9 mL of DPPHd solution (6 105 mol/L in methanol), and the mixture was mixed well. The absorbance at 515 nm was measured at various time intervals. A blank was prepared for each sample using methanol instead of the DPPHd solution.
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For each sample concentration tested, the reaction kinetics was plotted. Moreover, the EC50 values of each wine, i.e. the concentration of total phenolics (mg/L) required to lower the initial DPPHd concentration by 50%, was also calculated (linear equation). The exact DPPHd concentration was calculated from a calibration curve with the equation C ¼ 9.5 A, where C the concentration of DPPHd (mg/L) and A the absorbance at 515 nm. EC50 value of quercetin was also determined (linear equation). The ability of samples to inhibit the coupled oxidation of b-carotene and linoleic acid was evaluated as described previously (Gazzani et al., 1998). In test tubes, 0.1 mL of sample (wine or extract in 10% ethanol) or 0.1 mL of 10% ethanol (control) was added to 3.0 mL of b-carotene and linoleic acid emulsion. The mixture was mixed well and the tubes were put in a water bath at 50 1C. The absorbance at 470 nm was measured after 0, 20, 40, 60, 80, 100 and 120 min of incubation. The b-carotene and linoleic acid emulsion was prepared by adding 22.2 mL linoleic acid and 185 mL Tween 40 in 1 mL of b-carotene solution (200 mg/L) in chloroform. Chloroform was evaporated to dryness by rotary evaporation at 25 1C and 80 mbar. Then, 50 mL aerated distilled water were added and the mixture was shaken. Each sample was read against an emulsion prepared as described but without b-carotene (blank). For each sample concentration tested, the reaction kinetics was plotted. Moreover, the EC50 value of each sample, i.e. the concentration of total phenolics (mg/L) required to lower the initial absorbance at 470 nm by 50%, was calculated (logarithmic equation). EC50 value of quercetin was also determined (logarithmic equation). 2.5. Statistical analysis Each experiment was repeated three times and the results reported are the means of the three trials. The one way analysis of variance (ANOVA), using the Duncan test at a level of significance Po0.05 was used for the statistical analysis (SPSS 11.5).
3. Results and discussion
Robola exhibited higher tartaric esters than the others followed by Debina. Similar total phenolics of red wines (1000–4000 mg/L) and of white wines (some hundreds mg/L), and similar values for the determined phenolic classes have also been reported by others (Sanchez-Moreno et al., 2000; De Beer et al., 2003; Minussi et al., 2003; Fernadez-Pachon et al., 2004). All red and white wines were analyzed by HPLC for their phenolic composition. Red wines contained various phenolics (benzoic acids, cinnamic acids, flavanols, flavonols, flavanones, flavones, anthocyanins, unclassifieds at 230 nm, and unclassified at 280 nm). On the other hand, white wines contained mainly benzoic and cinnamic acids. These phenolic classes for red and white wines have also been reported by others (Landrault et al., 2001; Waterhouse, 2002). The main phenolic peaks in chromatograms of each wine are presented in Table 2. As it can be seen some wines, such as Debina, Robola, Limnio and Syrah, exhibited one very high peak of benzoic or cinnamic acid each. All wines scavenged DPPHd, and their scavenging activities decreased by dilution. Red wines were more active than whites. Red wines diluted 10 or more times exhibited comparable activity to undiluted white ones. This may be attributed to their higher phenolic content. Moreover, most red wines exhibited lower EC50 values at 5 min (initial scavenging capacity) and at 40 min (total scavenging capacity) than most white wines indicating the high potency of red wine phenolics (Table 3). However, Limnio red Table 2 Peaks of phenolic compounds with high area in the chromatograms of wines Wine
Major peaks of phenolics and their area
Agiorgitiko
Cinnamic acid, 77.0a; flavanol, 57.1; cinnamic acid, 42.8; gallic acid, 31.3; unclassified 230 nm, 6.7; anthocyanin, 3.4; anthocyanin, 1.0; anthocyanin, 0.3 Cinnamic acid, 152.9; cinnamic acid, 40.9; cinnamic acid, 38.1; gallic acid, 28.0; flavanol, 20.4; unclassified 230 nm, 5.9; anthocyanin, 0.5; anthocyanin, 0.2; anthocyanin, 0.1 Cinnamic acid, 71.8; flavanol, 24.8; cinnamic acid, 21.7; gallic acid, 18.9; cinnamic acid, 15.3; cinnamic acid, 11.0; unclassified 230 nm, 7.6; anthocyanin, 5.9; flavonol, 3.2; anthocyanin, 1.2 Benzoic acid, 652.4; benzoic acid, 132.2; cinnamic acid, 55.6 Cinnamic acid, 28.2; cinnamic acid, 9.4; cinnamic acid, 8.4; feroulic acid, 7.3; benzoic acid, 6.9; flavonol, 5.0 Cinnamic acid, 126.2; flavone, 19.9; cinnamic acid, 14.3; caffeic acid, 8.6; cinnamic acid, 7.2; benzoic acid, 5.3; benzoic acid, 3.9 Cinnamic acid, 28.1; benzoic acid, 8.8; caffeic acid, 7.2; benzoic acid, 4.7 Cinnamic acid, 12.8; tyrosol, 5.3; caffeic acid, 4.5; benzoic acid, 3.4; p-coumaric acid, 2.8; benzoic acid, 2.7; benzoic acid, 2.1; flavone, 1.8
Limnio
Syrah
Debina Athiri
3.1. Antioxidant activities and phenolic composition of various wines Total phenolics, tartaric esters, flavonols, anthocyanins, and flavanols of various red and white wines of the Greek vineyard are presented in Table 1. As expected, red wines contained much higher phenolics than whites. Among red wines, Syrah exhibited the higher total phenolics, tartaric esters, flavonols, anthocyanins, and flavanols. All white wines exhibited similar total phenolics, while they did not contain anthocyanins and flavanols. However,
Robola
Sauvignon blanc Traminer
a
106, is the area of the peak.
Table 1 Total phenolics (Folin and A280 nm), tartaric esters, flavonols, anthocyanins and flavanols of red and white wines of the Greek vineyard Wine
Total phenolics (mg/L gallic acid)
A280 nm (mg/L gallic acid)
A320 nm (mg/L caffeic acid)
A360 nm (mg/L quercetin)
A520 nm (mg/L malvidin3-glucoside)
Flavanols (mg/L catechin)
Agiorgitiko Limnio Syrah Debina Athiri Robola Sauvingnon blanc Traminer
2082b738 2704c7145 3184d7169 261a710 213a79 258a710 272a719 277a76
1138c725 1450d734 1700e733 314b79 206a76 219a717 212a77 208a79
208d719 279e713 308f716 62b75 40a75 96c76 48ab76 57ab77
144b710 174c712 219d713 35a74 24a73 52a76 28a74 28a75
335c721 224b714 523d714 0a70 0a70 0a70 0a70 0a70
230.8b77.5 338.5c721.5 527.1d77.5 0.6a70.1 0.7a70.1 0.3a70.0 0.7a70.1 1.4a70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at Po0.05. Standard deviation.
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wine exhibited EC50 values at 5 min comparable to those of white wines. On the contrary, Sauvignon blanc white wine exhibited EC50 values at 5 min and at 40 min comparable to those of red wines. Quercetin appeared to be more active (lower EC50) than red wines. Quercetin activity was dose dependent in the range 0–5.0 mg/L. DPPHd scavenging by wines and also a correlation between antiradical efficiency and total phenolic content or flavanols has been reported by others (Frankel et al., 1995; Soleas et al., 1997; Sanchez-Moreno et al., 1999; De Beer et al., 2003; Katalinic et al., 2004; Fernandez-Pachon et al., 2006; Kovacevic Ganic et al., 2006). Red wines inhibited the coupled oxidation of b-carotene and linoleic acid, and their activities decreased by dilution. The EC50 values of red wines (Table 4) indicate that their phenolics were more active or equivalent to quercetin. Quercetin activity was
Table 3 EC50 values (mg/L) of red and white wines of the Greek vineyard and quercetin for the scavenging of DPPHd Wine
EC50 at 5 min
EC50 at 40 min
Agiorgitiko Limnio Syrah Debina Athiri Robola Sauvingnon blanc Traminer Quercetin
6.7b70.3
3.1b70.0 4.0cd70.1 3.5bc70.1 6.1e70.4 5.6e70.8 4.4d70.3 4.2d70.1 5.8e70.5 1.6a70.2
10.4d70.1 6.1b70.6 12.2e70.5 12.7e70.5 11.5e70.7 7.8c70.8 10.7d70.6 2.1a70.3
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
Table 4 EC50 values (mg/L) of red wines of the Greek vineyard and quercetin for the inhibition of coupled oxidation of b-carotene and linoleic acid Sample
EC50 at 20 min
EC50 at 120 min
Agiorgitiko Limnio Syrah Quercetin
5.8 e17b73.8 e17 1.6 e27a71.0 e27 4.6 e27a72.3 e27 1.7 e17b70.9 e17
0.15b70.05 0.25b70.06 0.01a70.01 0.26b70.05
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
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dose dependent in the range 0–6.5 mg/L. White wines exhibited only limited activity. Twenty-fold diluted white wines exhibited some activity, while less diluted white wines were inactive or prooxidants. Inhibition of b-carotene bleaching by wines and also a correlation between antioxidant activity and anthocyanins and catechins has been reported by others (Katalinic et al., 2004). Other researchers have reported that in b-carotene assay anthocyanins and flavonols are very active followed by flavanols, while phenolic acids are less active (Fukumoto and Mazza, 2000). 3.2. Antioxidant activities and phenolic composition of Xinomavrored and Roditis-white must and wine Total phenolics, tartaric esters, flavonols and anthocyanins of Xinomavro-red and Roditis-white must, young and aged wines are presented in Table 5. Xinomavro young wine exhibited much higher phenolics than must, which may be attributed to phenolic extraction during maceration, and aged wine higher than young wine, which may be due to migration from oak barrel (Waterhouse, 2002). However, aged wine exhibited lower anthocyanin content than young wine, as determined by the absorbance at 520 nm. Similar results have also been reported by others and have attributed to the transformation of monomeric anthocyanins to more stable oligomeric forms which absorb at wavelength lower than 520 nm (Gomez-Plaza et al., 2000). Roditis must exhibited higher total phenolics and also tartaric esters and flavonols than young and aged wines. This may be due to precipitation of some phenolics during winemaking. The amount of anthocyanins of Roditis must may be attributed to the light pink color of Roditis grape. Young and aged wines exhibited similar phenolic levels. HPLC chromatograms of Xinomavro-red and Roditis-white must, young and aged wines are presented in Figs. 1 and 2, respectively. In chromatogram of Xinomavro wine, as well as in chromatograms of all the other red wines analysed, an increase of baseline at 50–85 min was observed. Similar behavior during red wine analysis was observed by others, and had been attributed to polymeric compounds exhibiting the same size and polarity (Waterhouse et al., 1999). Phenolic levels and composition of Xinomavro and Roditis wines were similar to those reported for red and white wine, respectively (Sanchez-Moreno et al., 2000; Landrault et al., 2001; De Beer et al., 2003; Minussi et al., 2003; Fernadez-Pachon et al., 2004). Xinomavro and Roditis musts exhibited scavenging activity of DPPHd, which decreased by dilution. Similarly, antiradical activity of commercial grape juices has been reported by others (Davalos et al., 2005). However, activity of each must was much lower than young and aged wines. Moreover, Xinomavro and Roditis musts
Table 5 Total phenolics (Folin and A280 nm), tartaric esters, flavonols and anthocyanins of Xinomavro-red and Roditis-white must, young and aged wines Total phenolics, Folin (mg/L gallic acid)
Total phenolics (A280 nm, mg/L gallic acid)
Tartaric esters (A320 nm, mg/L caffeic acid)
Flavonols (A360 nm, mg/ L quercetin)
Anthocyanins (A520 nm, mg/L malvidin-3-glucoside)
Xinomavro Must Young wine Aged wine
1790a759 3476b771 3802c736
832a720 1652b725 1916c716
201a713 263b72 315c77
155a79 193b76 225c76
306a716 361b75 325a78
Roditis Must Young wine Aged wine
371b713 291a75 304a78
264b713 210a76 248b74
83b74 73a71 75a71
79b73 39a72 40a72
23b73 1a70 3a71
Sample
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at Po0.05. Standard deviation.
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11
0 200.
Must, 280 nm 17
AU
0 150.
16 18
0 100.
15
23
19 20 21
0 050. 1
4
5
6
7
0 000. 0 015.
14 12 13
2223
40 39
24 25 26 27
28
8 9 10
29
30
31
33 34
32
36
35
Must, 520 nm
AU
0 010. 0 005. 37 38
0 000. 2223 24 25
1 000. 14
0 800.
21
AU
9
20
0 600. 0 400.
1
15 11
0 200.
3 5 2 4 6 7 8
0 000.
10 1213
26
Young wine, 280 nm
28 29 30 27 31 36 37 32 35 3334
19 16 18 17
38
39 40
43 44 45 47 49 50
48
Young wine, 520 nm
0.020
AU
42
51 52
46
0 010.
53 41
0 000. 17
0 800.
21 19
AU
0 600.
22
23 24 25
20
27
26
18
0 400. 31 32
11 57
0 200.
3 12 4 6 8
0 000.
8
15 10 12 13 14 16
28
Aged wine, 280 nm
29 30 33 34 35 37
36 38 39 40
41
4243 44
46
47
Aged wine, 520 nm
0. 008
50 49
51
AU
0. 006 0.004
45
48
0. 002 0. 000 0
20
40
60
80
100
120
140
160
180
Minutes Fig. 1. HPLC chromatograms of Xinomavro-red must, young and aged wines at 280 and 520 nm. Must: 1–4, unclassifieds 230 nm; 5, 6 benzoic acids; 7, gallic acid; 8, flavanol; 9, cinnamic acid; 10, unclassified 280 nm; 11, cinnamic acid; 12, unclassified 280 nm; 13, cinnamic acid; 14, flavanone; 15, cinnamic acid; 16, flavanol; 17, flavanone; 18, 19, flavanols; 20, flavanone; 21, benzoic acid; 22, 23, cinnamic acids; 24, unclassified 280 nm; 25–27, flavanols; 28–30, unclassifieds 280 nm; 31, 32, flavonols; 33, rutin; 34, flavonol; 35, anthocyanin; 36, unclassified 280 nm; 37, 38, anthocyanins; 39, benzoic acid; 40, flavanol. Young wine: 1, 2, unclassifieds 230 nm; 3, 4, cinnamic acids; 5–7, benzoic acids; 8, unclassified 280 nm; 9, gallic acid; 10, benzoic acid; 11, cinnamic acid; 12, unclassified 280 nm; 13, benzoic acid; 14, 15, cinnamic acids; 16, flavanone; 17, cinnamic acid; 18, flavanone; 19, flavanol; 20, 21, flavanones; 22–29, flavanols; 30, unclassified 280 nm; 31, cinnamic acid; 32, flavanol; 33, cinnamic acid; 34, benzoic acid; 35, and 36, flavanols; 37, 38, unclassifieds 280 nm; 39, 40, benzoic acids; 41, anthocyanin; 42, flavonol; 43, cinnamic acid; 44, flavonol; 45, rutin; 46, anthocyanin; 47, flavonol; 48, anthocyanin; 49, 50, flavonols; 51–53, anthocyanins. Aged wine: 1–3, unclassifieds 230 nm; 4, cinnamic acid; 5, benzoic acid; 6, cinnamic acid; 7–9, benzoic acids; 10, flavone; 11, gallic acid; 12, unclassified 280 nm; 13, benzoic acid; 14–16, flavanols; 17, cinnamic acid; 18–22, flavanols; 23, flavone; 24–32, flavanols; 33, 34, unclassifieds 280 nm; 35, flavanone; 36, 37, flavanols; 38, syringic acid; 39, flavone; 40, cinnamic acid; 41, p-coumaric acid; 42, cinnamic acid; 43, 44, unclassifieds 280 nm; 45, anthocyanin; 46, unclassified 280 nm; 47, rutin; 48, 49, anthocyanins; 50, benzoic acid; and 51, anthocyanin.
exhibited higher EC50 values at 5 min (initial scavenging activity), and also at 60 min (total scavenging activity) than young and aged wines (Table 6). Consequently, the lower antiradical potency of must may be attributed to its lower phenolic content or to the lower potency of must phenolics. Both young wines exhibited
lower EC50 values at 5 min than the respective aged wine, indicating the potency of their phenolics. Xinomavro-red must exhibited significant antioxidant activity in the b-carotene assay, which decreased by dilution. Xinomavro must diluted 10 times exhibited total antioxidant activity
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2
0.120
619
Must
3 25 24
0.080
26
23
AU
4 5 8
0.040
12 11
6 7 9
1
0.000 0.600
13
10
29
27
16 19 18 2122 15 17 20
28 31 33 34 32 35
14
37
43 44
39
42
38
45
40 41
30
Young wine
4 1 6
0.400
30
AU
23 11
22
5 9
7
0.200
12 8
2
0.000
3
10
17
32
28
35
24 21
18
14
37 33
19
16 13
26 25 27
20
15
29
31
43 36
38 39
34
40 41
42
AU
47 46 48
4
6
0.400 5
19
7 10
1
8 9 3
22 21
11 18
13
0.000
44
Aged wine
2
0.800
45
15 14
16
27
23
20
17
24
26 25
33 34 36 29 32 28 35 37 31 38
12
20
46
39 40
48 45 47
42 43 44
41
30
40
60
80
100
120
140
160
180
Minutes Fig. 2. HPLC chromatograms of Roditis-white must, young and aged wines at 280 nm. Must: 1–3, unclassifieds 230 nm; 4–6, cinnamic acids; 7, 8, benzoic acids; 9, cinnamic acid; 10, benzoic acid; 11, flavonol; 12, flavanol; 13, cinnamic acid; 14, flavone; 15, cinnamic acid; 16, flavanol; 17, benzoic acid; 18, cinnamic acid; 19, benzoic acid; 20–22, cinnamic acids; 23, benzoic acid; 24, unclassified 280 nm; 25, 26, benzoic acids; 27, 28, cinnamic acids; 29, benzoic acid; 30, unclassified 230 nm; 31, 32, unclassifieds 280 nm; 33–35, benzoic acids; 36, cinnamic acid; 37, flavanol; 38, benzoic acid; 39, unclassified 280 nm; 40, rutin; 41, flavonol; 42, benzoic acid; 43–45, flavanols. Young wine: 1–3, unclassifieds 230 nm; 4, 5, flavanones; 6, flavone; 7, 8, benzoic acids; 9, cinnamic acid; 10–12, benzoic acids; 13, unclassified 280 nm; 14, flavanone; 15, 16, benzoic acids; 17–21, cinnamic acids; 22, flavanone; 23, cinnamic acid; 24, flavone; 25, flavanol; 26, benzoic acid; 27, cinnamic acid; 28, benzoic acid; 29, unclassified 230 nm; 30, caffeic acid; 31, cinnamic acid; 32, 33, benzoic acids; 34, flavone; 35, unclassified 280 nm; 36, benzoic acid; 37, p-coumaric acid; 38, cinnamic acid; 39, flavanol; 40, benzoic acid; 41, unclassified 280 nm; 42, benzoic acid; 43–45, cinnamic acids; 46, benzoic acid; 47, unclassified 280 nm; 48, flavanol. Aged wine: 1–3, unclassifieds 230 nm; 4–5, cinnamic acids; 6–12, benzoic acids; 13, unclassified 280 nm; 14, benzoic acid; 15, unclassified 280 nm; 16–18, cinnamic acids; 19, benzoic acid; 20, flavanone; 21, flavone; 22–24, flavanones; 25, benzoic acid; 26, caffeic acid; 27, cinnamic acid; 28, flavanone; 29, cinnamic acid; 30, unclassified 280 nm; 31–34, benzoic acids; 35, flavanone; 36, cinnamic acid; 37, 38, cinnamic acids; 39, unclassified 280 nm; 40, benzoic acid; 41, 42, unclassifieds 280 nm; 43–45, cinnamic acids; 46, benzoic acid; and 47, 48, flavanols.
Table 6 EC50 values (mg/L) of Xinomavro-red and Roditis-white must, young and aged wines for the scavenging of DPPHd
Table 7 EC50 values (mg/L) of Xinomavro-red must, young and aged wines for the inhibition of coupled oxidation of b-carotene and linoleic acid
Sample
EC50 at 5 min
EC50 at 60 min
Sample
EC50 at 20 min
EC50 at 120 min
Xinomavro Must Young wine Aged wine
5.0c70.2 2.4a70.1 3.6b70.3
2.3b70.2 1.9a70.1 1.7a70.2
Must Young wine Aged wine
6.4 e6a71.8 e6 30.4 e6b72.6 e6 3.6 e6a71.8 e6
1.6b70.3 2.5c70.5 0.4a70.2
Roditis Must Young wine Aged wine
9.8b71.8 4.1a70.3 4.9a70.4
3.8a70.2 2.1b70.1 2.5c70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
comparable to young wine diluted 10 times and aged wine diluted 20 times, eventhough it contained much lower total phenolics than those. Moreover, EC50 value of Xinomavro must was lower
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
than that of young wine (Table 7). Xinomavro aged wine appeared to be more active and exhibited lower EC50 value than young wine and must. Increased antioxidant capacity with aging of red wines has been reported by others (Echeverry et al., 2005). Roditis-white must exhibited some activity in the b-carotene assay. However, its activity was much lower than Xinomavro-red must, even when tested at the same total phenolic content. Young and aged Roditis
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wines exhibited only limited activity. They exhibited prooxidant activity or none activity, respectively, at 20 min (initial antioxidant activity). At 120 min (total antioxidant activity), 8 or 4-fold diluted wines exhibited some activity while less diluted not. 3.3. Antioxidant activities and phenolic composition of Xinomavrored and Roditis-white wine extracts Young and aged Xinomavro-red and Roditis-white wines fractionated into three fractions each by extraction. All these phenolic fractions were examined for scavenging DPPHd and inhibiting b-carotene bleaching. HPLC chromatograms revealed that Xinomavro young or aged wine fractions 1 contained mainly anthocyanins and flavanols, fractions 2 flavanols, flavonols and tyrosol, and fractions 3 cinnamic acids, benzoic acids and flavonols. Chromatographic analysis also indicated that Roditis young or aged wine fractions 1 contained mainly unclassified peaks with max. 230 nm and some phenolic acids, fractions 2 phenolic acids and tyrosol, and fractions 3 phenolic acids. All wine extracts exhibited antiradical potency in a dosedependent manner, indicating that many red and white wine phenolics may be active (Table 8). Similarly, scavenging of DPPHd by various wine phenolics has been reported by others (Frankel et al., 1995; Soleas et al., 1997). Young wine fractions exhibited lower EC50 values than the respective of aged wine at 5 min (initial scavenging activity) and also at 60 min (total scavenging activity). This indicates the higher antiradical potency of all classes of young compared to aged red and white wine phenolics. In Xinomavro, no systematic differences were observed among young wine fractions. However, among aged wine fractions, fraction 1 exhibited lower EC50 values at 5 min than the others, indicating the possible potency of anthocyanins and flavanols. Similarly, flavanols exhibited best correlation among wine phenolics for DPPHd scavenging (Arnous et al., 2002; De Beer et al., 2003), while red wine extract containing anthocyanins and flavanols exhibited best DPPHd scavenging among the other extracts (Ghiselli et al., 1998; Burns et al., 2001; Fernadez-Pachon et al., 2004). In Roditis, young and aged wine fractions 3 exhibited lower EC50 values than the others, indicating the possible potency of cinnamic and benzoic acids. Each of the three Xinomavro-red aged wine fractions was sub-fractionated into non-polymeric (monomeric and dimeric) and polymeric polyphenols. All sub-fractions exhibited antiradical Table 8 EC50 values (mg/L) of Xinomavro-red young and aged wine phenolic fractions for the scavenging of DPPHd Fraction
EC50 at 5 min
EC50 at 60 min
Xinomavro 1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
1.8a70.1 2.0a70.1 2.3a70.1 3.9b70.2 5.0c70.2 4.6c70.3
1.4a70.1 1.6b70.1 1.4a70.1 2.1cd70.2 2.5d70.3 1.8bc70.2
Roditis 1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
3.2c70.1 1.9b70.1 1.2a70.1 6.4d70.5 3.2c70.1 3.3c70.4
1.6b70.1 1.4b70.1 0.9a70.1 3.3d70.3 2.0c70.1 2.0c70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
Table 9 EC50 values (mg/L) of Xinomavro-red young and aged wine phenolic fractions for the inhibition of coupled oxidation of b-carotene and linoleic acid Fraction
EC50 at 20 min
EC50 at 120 min
1, young wine 2, young wine 3, young wine 1, aged wine 2, aged wine 3, aged wine
1.5 e3a70.3e3 1.7 e3ab70.2e3 2.2 e3b70.3e3 0.2 e3c70.2e3 0.4 e3c70.1 e3 1.9 e3b70.1 e3
1.1c70.1 1.1c70.1 1.2d70.1 0.6a70.1 0.9b70.1 0.9b70.1
Values are the means of the three trials (n ¼ 3). Means in every column without common superscript differ significantly at
Po0.05.
Standard deviation.
potency (data not shown). It has been reported that polymeric phenolics of red wines contribute to 50% of its scavenging capacity (Fernadez-Pachon et al., 2004). All Xinomavro-red young and aged wine fractions inhibited b-carotene bleaching (Table 9), indicating that many wine phenolics may be active. Similarly, inhibition of b-carotene bleaching by various phenolic compounds has been reported by others (Fukumoto and Mazza, 2000). Most aged wine fractions exhibited lower EC50 values than the respective of young wine. This indicates the higher antioxidant potency of aged compared to young wine phenolics. Fractions 1 of young and aged Xinomavro wines exhibited lower EC50 values than the other fractions, indicating the possible high potency of anthocyanins and flavanols. Similarly, anthocyanins and catechins exhibited best correlation among wine phenolics for inhibition of b-carotene bleaching (Katalinic et al., 2004) and anthocyanins and flavanols for inhibition of corn oil emulsion (Sanchez-Moreno et al., 2000). All Xinomavro-red aged wine sub-fractions inhibited b-carotene bleaching (data not shown), indicating the potency of both nonpolymeric and polymeric phenolics. All Roditis-white wine extracts were examined for their antioxidant potency in b-carotene bleaching at various dilutions, i.e. at total phenolic range 0.5–10 mg/L. Among them, extracts 2 and 3 exhibited only limited antioxidant activity, and extracts 1 were inactive. The most active extract, extract 2 of aged wine, exhibited % inhibition up to 75%, while the others up to 50%. These results indicate possible low effectiveness of phenolic acids in b-carotene bleaching, since extracts 2 and 3 were rich in phenolic acids. Present results indicate that red and white must, especially red one, exhibit antioxidant activities in scavenging of DPPHd and in bleaching of b-carotene. Red wines are also active in these two assays. White wines scavenge DPPHd, while they are not active in b-carotene assay. Results indicate that many red and white wine phenolics may be active in scavenging DPPHd and inhibiting b-carotene bleaching. Moreover, they indicate that anthocyanins and flavanols may be among the most active phenolics. The absence of those phenolics (anthocyanins and flavanols) in white wines may be explaining the lower potency (EC50 values) of white wine phenolics versus those of reds. In present study, white wines and white wine extracts were inactive or exhibited limited activity in b-carotene bleaching. However, white wines and extracts exhibited significant activity in inhibiting LDL oxidation (Tselepis et al., 2005). Their differences in these two assays may be attributed to poor distribution of phenolic acids (the main phenolics of white wines and extracts) between the two phases of the emulsion in the case of b-carotene assay. Results show that the order of antioxidant activities of wines and wine extracts change according to the method used, DPPHd scavenging and b-carotene bleaching. This may be attributed to
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the chemistry of these antioxidant capacity assays. DPPHd scavenging was believed to involve hydrogen atom transfer reaction. However, a recent work based on the kinetic analysis of the reaction between phenols and DPPHd shows that the reaction in fact behaves like a single electron transfer reactionbased assay (Foti et al., 2004). On the other hand, the coupled oxidation of linoleic acid and b-carotene, like other linoleic acid oxidation assays, is a hydrogen atom transfer reaction-based assay (Huang et al., 2005). Consequently, the differences observed in the two assays may be attributed to differences in electron and hydrogen transfer abilities of their phenolics. Moreover, in the case of b-carotene assay the distribution of the antioxidants (phenolics) between the two phases of the emulsion can be critical.
4. Conclusions Present results indicate that must of red and white grapes, especially red, is active in scavenging DPPHd and inhibiting bcarotene bleaching. Red wines of the Greek vineyard exhibit higher activities in these two assays than white wines. Results indicate that wine aging may affect their antioxidant activities, while anthocyanins and flavanols may be among the most active phenolics.
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