Anthocyanin Profile and Antioxidant Activity from 24 Grape Varieties Cultivated in Two Portuguese Wine Regions

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ANTHOCYANIN PROFILE AND ANTIOXIDANT ACTIVITY FROM 24 GRAPE VARIETIES CULTIVATED IN TWO PORTUGUESE WINE REGIONS

Elisa CoSTA 1, Fernanda CoSME 2, António M. JorDÃo 1* and Arlete MEnDES-FAIA 2 1: Polytechnic Institute of Viseu (Centre for the Study of Education, Technologies and Health), Agrarian Higher School, Estrada de nelas, Quinta da Alagoa, ranhados, 3500-606 Viseu, Portugal 2: Institute for Biotechnology and Bioengineering – Centre of Genomics and Biotechnology (IBB-CGB-UTAD), University of Trás-os-Montes and Alto Douro, School of Life Science and Environment, 5001-801 Vila real, Portugal

Abstract Résumé Aims : The purpose of this work was to evaluate the general Objectifs : Le but de ce travail était d’évaluer la phenolic composition and the anthocyanin profile of composition phénolique générale et le profil d’anthocyanes 24 grape varieties from two Portuguese wine regions as well de 24 cépages de deux régions viticoles portugaises ainsi as their antioxidant activity in the different grape berry que leur activité antioxydante dans les différentes fractions fractions (skins, pulps and seeds). de baie de raisin (pellicules, pulpes et pépins). Methods and results : Individual anthocyanin composition Méthodes et résultats : Les anthocyanes obtenues à partir of grape skin extracts was analyzed by HPLC, whereas total d’extraits de pellicules de raisin ont été analysées par HPLC antioxidant activity was evaluated by two methods: DPPH tandis que l’activité antioxydante totale a été évaluée par and ABTS. In general, a high variability was found among deux méthodes : DPPH et ABTS. En général, une grande the different autochthonous and non-autochthonous grape variabilité a été observée parmi les différentes variétés de varieties in relation to the polyphenolic compounds raisins autochtones et non autochtones en ce qui concerne analyzed, especially the individual anthocyanins. The les composés polyphénoliques analysés comme les individual anthocyanins in grape skin extracts were mainly anthocyanes. Dans les extraits de pellicules de raisin, les malvidin (1.40-7.09 mg/g of skin), in particular malvidin-3- anthocyanes étaient principalement la malvidine (1.40- glucoside (0.62-6.09 mg/g of skin). The highest antioxidant 7.09 mg/g de pellicule), et en particulier la malvidine-3- activity was consistently detected in the seed extracts; glucoside (0.62-6.09 mg/g de pellicule). Dans tous les however, it was not possible to establish a clear difference cépages étudiés, l’activité antioxydante la plus élevée a été among the grape varieties analyzed. mesurée dans les pépins. Par contre, il n’a pas été possible d’établir une différence claire entre les cépages. Conclusion : High variability in polyphenolic content, individual anthocyanin composition and antioxidant activity Conclusion : Une grande variabilité a été trouvée parmi les was found among the diverse autochthonous and non- divers cépages autochtones et non-autochtones étudiés par autochthonous grape varieties studied. Seeds showed the rapport à la teneur en polyphénols, ainsi que la composition highest antioxidant activity, followed by skin and pulp, en anthocyanes et l’activité antioxydante. Les pépins étaient irrespective of the grape variety. la fraction de la baie de raisin à l’activité antioxydante la plus élevée suivie par les peaux et les pulpes, quel que soit Significance and impact of the study : Most vineyards in le cépage. Portugal grow Portuguese cultivars of Vitis Vinifera L. and other cultivars grown worldwide. The phenolic compounds Signification et impact de l’étude : Au Portugal, la plupart and antioxidant activity of these grape cultivars have never des vignobles possèdent des variétés portugaises de Vitis been characterized under the environmental conditions of vinifera L. et d’autres espèces largement cultivées dans le the Douro and Dão regions. The variability in phenolic monde. L’évaluation et la comparaison des composés content among the grape varieties studied confirms the phénoliques et de l’activité antioxydante de ces cépages hypothesis that genetic factors have an important role in the n’ont pas été exploitées, à savoir sous les conditions biosynthesis of these compounds and, consequently, in the environnementales des régions du Douro et du Dão . La antioxidant activity of grapes. variabilité observée dans le contenu phénolique des cépages étudiés confirme l’hypothèse que des facteurs génétiques Key words : antioxidant activity, individual anthocyanins, jouent un rôle important dans la biosynthèse de ces red grapes, pulp, seed, skin composés et, par conséquent, sur l’activité antioxydante du raisin. Mots clés : activité antioxydante, anthocyanes, cépage rouge, pulpe, pépin, pellicule

manuscript received 4th January 2013 - revised manuscript received 8th August 2013

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INTRODUCTION been conflicting evidence regarding the contribution of anthocyanins to red grape and wine antioxidant Grapes are the chief dietary sources of anthocyanins properties. (rechkemmer and Pool-Zobel, 1996). Anthocyanins are mainly localized in berry skin (Jordão et al ., Dão and Douro are two important wine-producing 1998a) , where they are found at concentrations of 200 regions in northeastern Portugal. The Dão region is to 5,000 mg/Kg of fresh grapes (ribéreau-Gayon , sheltered on nearly all sides by high granite mountains 1982) . Anthocyanin pigments are of noticeable and is situated in the transition zone between maritime importance in grapes and wines because of their dual and continental climate, with abundant rainfall in the role: first, they constitute an essential part of the winter months and long warm dry summers leading up sensory attributes, as their concentration, different to harvest . The Douro region, which is situated around forms and derivatives directly affect the color of the the Douro river basin, is sheltered from Atlantic final wine and second, they are considered to have winds by several mountains and has a continental diverse biological properties and therefore are climate , with hot and dry summers and cold winters. regarded as secondary metabolites with potential Although Portuguese Vitis vinifera L. autochthonous nutritional value. Grape anthocyanins are the 3- O- cultivars are the most frequently used cultivars for the monoglucosides of delphinidin, cyanidin, petunidin, production of high quality red wines in both regions, peonidin, and malvidin. Glucosylated derivatives of other varieties from other countries (especially from these anthocyanins, esterified at the C 6 position of France) have been introduced in the past few years. glucose with acetyl or coumaroyl groups, have also been detected, yet generally at low concentrations. A few studies have been performed on the anthocyanin Malvidin is the predominant monomeric anthocyanin profile of Portuguese grape varieties. However, these in grapes and is the reddest of all anthocyanins, studies were restricted to a small number of varieties providing the characteristic color of young red wines. from southern (Dallas and Laureano, 1994a; Jordão et al ., 1998a) and northern Portugal (Dallas and Anthocyanin concentration and profile is influenced Laureano, 1994a; Dopico-García et al ., 2008; Mateus by several factors such as varietal diversity et al ., 2002) and did not address the antioxidant (Kallithraka et al ., 2005), soil type (Yokotsuka et al ., activity of the different grape berry fractions. 1999), environmental conditions (Jackson and Lombard, 1993; Mateus et al ., 2002 ), vineyard Thus, the purpose of this work was to evaluate the management (Jordão et al ., 1998b), and grape anthocyanin profile in grape skin as well the maturity (Jordão et al ., 1998a). Additionally, some antioxidant activity in different grape berry fractions grape varieties can have a high anthocyanin (skins, pulps and seeds) of 24 grape varieties from concentration but a low extractability index (romero- both Portuguese wine regions. The results obtained in Cascales et al ., 2005). this work could be useful for selecting winemaking techniques in order to improve anthocyanin extraction According to several studies, flavan-3-ols, flavonols and hence increase the antioxidant capacity of the and anthocyanins are the most important compounds wines produced in these two regions. Finally, we that contribute to red grape and wine antioxidant should note that this is the first report of the proprieties (Beecher, 2003; Simonetti et al ., 1997). anthocyanin content and antioxidant activity of grape For rivero-Pérez et al . (2008), the free anthocyanin cultivars from the Dão and Douro wine regions. fraction is mainly responsible for the total antioxidant capacity and scavenger activity in red wines. MATERIALS AND METHODS However, other authors have reported that there is no 1. Samples correlation between antioxidant activity and spectral anthocyanin content in grapes and finished wines In 2010, 24 grape varieties ( Vitis vinifera L.) were (Arnous et al ., 2002; Kallithraka et al ., 2005). For harvested at technological maturity in good sanitary example, considering some individual anthocyanins conditions from 6-year-old grapevines in two (cyanidin-3-glucoside, peonidin-3-glucoside and experimental vineyards of northeastern Portugal (one malvidin-3-glucoside) from Italian wines, Di Majo et in the Douro region and the other in the Dão region) . al . (2008) reported low correlations between these Grape berry samples (samples of 200 berries in individual compounds and their antioxidant capacity duplicate) were picked randomly from 20 different (ranging from 0.29 to 0.54). In addition, Jordão et al . vine plants in all possible conditions (e.g., different (2012) reported differences in the correlation values height and exposure to sunlight). All grape samples between individual anthocyanins and antioxidant were kept frozen at -20 ºC until processing. The grape activity during the maceration process. Thus, there has varieties studied are listed in Table 1.

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Table 1. Grape variety and origin 3. Sample preparation for antioxidant activity and of the grape samples studied. individual anthocyanin composition analysis

Autochthonous/ Skins, pulps and seeds were removed manually from Grape variety Non-autochthonous Origin the berries, washed separately several times with ! grape variety distilled water, and dried on blotting paper. Before the Camarate! AG! Dão! extraction process, seeds were crushed. The extraction Gewürztraminer*! NAG! Dão! process followed the procedure described by Sun et Monvedro! AG! Dão! al . (1996): 50 mL of 80 % methanol followed by Moreto Boal! AG! Dão! 50 mL of 75 % acetone, each for 3 h with agitation. Negro Mole! AG! Dão! After clarification of the suspension by centrifugation Negro Mouro! AG! Dão! Alfrocheiro! AG! Douro! (10 min at 3500 rpm), all the extracts were combined. Alvarilhão! AG! Douro! Aliquots of each extract were filtered (Whatman Aramon! NAG! Douro! 0.45 µm) and frozen at -20 ºC until processing for Bastardo! AG! Douro! antioxidant activity (skin, pulp and seed extracts) and Cabernet Franc! NAG! Douro! individual anthocyanin composition (skin extracts) Carignan Noir! NAG! Douro! analysis. Extractions were done in duplicate for each Cornifesto! AG! Douro! Gamay! NAG! Douro! grape berry fraction.

Grenache! NAG! Douro!

Jean! AG! Douro! 4. Antioxidant activity determination

Malvasia Preta! AG! Douro!

Rufete! AG! Douro! The total antioxidant activity of each grape berry

Sousão! AG! Douro! fraction was determined from the extracts according

Tinta Amarela! AG! Douro! to two previously described methods: ABTS and

Tinta Barca! AG! Douro! DPPH. The ABTS method is based on the Tinta Barroca! AG! Douro! discoloration that occurs when the radical cation Tinta Miúda! AG! Douro! .+ ABTS is reduced to ABTS (2,2’-azinobis-3- Tinto Cão! AG! Douro! ethylbenzothiazoline-6-sulfonic acid) (re et al ., AG - autochthonous grape variety; nAG - non- 1999). The radical was generated by reaction of a autochthonous grape variety; * white grape variety with colored skin 7 mM solution of ABTS in water with 2.45 mM potassium persulphate (1:1). The assay was made with The evolutions of the climatic characteristics (rainfall, 980 μL of ABTS .+ solution and 20 μL of sample temperature and relative humidity) for the 2010 (diluted 1:50 in water). The reaction was incubated for vintage in the two regions considered are summarized 15 min in darkness at room temperature, and then in Figure 1. In general, it was clear that during the absorbance at 734 nm was measured. three months (June, July and August) prior to sampling (at the end of September) the average The procedure using the DPPH method is described temperature and average relative humidity was higher by Brand -Williams et al . (1995). Briefly, 0.1 mL of in the Dão region than in the Douro region. In different sample concentrations was added to 3.9 mL addition, average rainfall for the months of July, of 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanolic August and September was lower in the Dão region. solution (25 mg/L). The DPPH solution was prepared daily and protected from the light. The reaction was 2. General physico-chemical and phenolic carried out for 30 min at 20 ºC in closed Eppendorf parameters tubes under shaking, and then absorbance at 515 nm was measured. Methanol was used as a blank Estimated alcohol degree, pH, titratable acidity and reference. The antioxidant activity results were tartaric acid were measured directly from the must expressed as Trolox equivalents (TEAC mM), using obtained after grape pressing, using the analytical the relevant calibration curve. All measurements were methods recommended by the oIV (2006). Total phenolic compounds were determined by measuring performed in duplicate. absorbance at 280 nm (ribéreau-Gayon et al ., 1982). 5. Chromatographic analysis of individual non-flavonoids and flavonoids were determined using anthocyanins the method described by Kramling and Singleton (1969). Color intensity (A 420 + A 520 + A 620 ) and hue The analysis of individual anthocyanins from skin (A 420 /A 520 ) were estimated using the method described extracts was performed on a HPLC Dionex Ultimate by the oIV (2006). All analyses were done in 3000 Chromatographic System (Sunnyvale, duplicate. California, USA) equipped with a quaternary pump

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Figure 1. Evolution of climatic characteristics (temperature, relative humidity and rainfall) for the 2010 vintage in the two regions considered.

Mod el LPG-3400 A, an auto sampler Model ACC- treatment means were carried out using Statistica 7 3000, a thermostated column compartment (adjusted software (StatSoft, Tulsa, USA). Scheffle’s test was to 25 ºC) and a multiple Wavelength Detector MWD- applied to the data as comparison test to determine 300. The column (250 x 4.6 mm, particle size 5 μm) when samples are significantly different after AnoVA ® was a C 18 Acclaim 120 (Dionex, Sunnyvale, (p < 0.05). Principal component analysis (PCA) was California, USA) protected by a guard column of the used to analyze the data and study the relations same material. The solvents were (A) 40 % formic between the grape varieties and their anthocyanin acid, (B) pure acetonitrile and (C) bidistilled water. profile or antioxidant activity. The anthocyanin composition was analyzed by HPLC using the method described by Dallas and Laureano RESULTS AND DISCUSSION (1994b). Thus, initial conditions were 25 % A, 10 % 1. General physico-chemical composition B and 65 % C, followed by a linear gradient from 10 to 30 % B and 65 to 45 % C for 40 min, with a flow The physico-chemical parameters of the grape rate of 0.7 mL/min. The injection volume was 40 µL. varieties at technological maturity are summarized in Detection was made at 520 nm using Chromeleon Table 2. The estimated alcohol degree ranged from 6.8 software (Sunnyvale, California, USA). Individual 9.53 % (v/v) ( Grenache ) to 14.84 % (v/v) ( Malvasia anthocyanins were quantified by means of calibration Preta ) with an average of 12.3 % (v/v). Titratable curve obtained with standard solutions of malvidin-3- acidity, expressed as equivalent of tartaric acid, varied glucoside chloride (>95 % purity) from Extrasynthese from 3.9 ( Moreto Boal ) to 13.5 g/L ( Jean and Tinta (Genay, France). The chromatographic peaks of Miúda ) with an average of 7.3 g/L. The high titratable anthocyanins were identified according to reference acidity and low pH values found in the majority of the data previously described by Dallas and Laureano grape varieties are probably a consequence of two (1994b). All analyses were done in duplicate. main factors: their natural acidity and the ripening process (associated to low degradation rates of organic 6. Statistical analysis acids). In addition, these results are in agreement with The data are presented as mean ± standard deviation. the tartaric acid content of all grape varieties. To determine whether there is a statistically significant 2. General phenolic composition and anthocyanin difference between the data obtained for antioxidant profile activity and for the diverse phenolic compounds quantified in the different grape varieties, an analysis It is well known that the genetic potential for of variance (AnoVA, one-way) and comparison of polyphenol biosynthesis and the degree of ripeness of

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Table 2. General physico-chemical composition of grape varieties at technological maturity.

Grape berry Must volume Estimated alcohol degree pH Titratable acidity Tartaric acid Grape variety weight (g)a (mL)a (%, v:v) (g/L tartaric acid) (g/L)

Camarate 241 ± 10a 128 ± 5c 11.00 ± 0.02e 2.93 ± 0.04ef 5.1 ± 0.1abc 3.19 ± 0.23abc Gewürztraminer 444 ± 1l 177 ± 1f 12.76 ± 0.01m 2.41± 0.00a 8.8 ± 0.2fgh 6.99 ± 0.02ijkl Monvedro 273 ± 8bcd 130 ± 4cd 12.35 ± 0.01k 3.11± 0.00hij 4.7 ± 0.3ab 2.56 ± 0.02a Moreto Boal 325 ± 2fg 152 ± 1e 13.59 ± 0.02p 3.17 ± 0.00ijk 3.9 ± 0.3a 3.35 ± 0.02bc Negro Mole 318 ± 8efg 159 ± 4e 11.47 ± 0.01f 2.75 ± 0.00cd 6.2 ± 0.1cd 5.97 ± 0.08gh Negro Mouro 319 ± 10efg 152 ± 5e 12.15 ± 0.02j 3.22 ± 0.00jk 4.1 ± 0.1a 3.38 ± 0.01bc Alfrocheiro 260 ± 8ab 131± 6cd 11.96 ± 0.02h 2.91 ± 0.00ef 6.0 ± 0.4bcd 3.19 ± 0.23abc Alvarilhão 297 ± 9def 144 ± 5de 13.45 ± 0.02o 2.83 ± 0.00de 8.0 ± 0.1ef 6.95 ± 0.06ijkl Aramon 727 ± 6o 322 ± 3k 10.59 ± 0.02c 2.72 ± 0.01bc 9.5 ± 0.3gh 7.70 ± 0.12k Bastardo 408 ± 2k 181 ± 1f 12.08 ± 0.02i 3.49 ± 0.00m 4.1 ± 0.2a 2.49 ± 0.68a Cabernet Franc 268 ± 4abc 66 ± 0a 13.73 ± 0.01q 2.94 ± 0.01ef 6.0 ± 0.4bcd 5.56 ± 0.04fg Carignan Noir 450 ± 3l 179 ± 1f 12.49 ± 0.02l 3.14 ± 0.01hij 8.5 ± 0.3fg 6.01 ± 0.11gh Cornifesto 303 ± 6ef 108 ± 2b 14.01 ± 0.02r 3.26 ± 0.00k 6.0 ± 0.5bcd 3.35 ± 0.02bc Gamay 487 ± 5m 228 ± 2h 11.67 ± 0.01g 3.09 ± 0.09hi 8.0 ± 0.1ef 6.38 ± 0.01hij Grenache 378 ± 6ij 203 ± 4g 9.53 ± 0.01a 3.22 ± 0.07jk 10.0 ± 0.4h 6.22 ± 0.00ghi Jean 387 ± 3jk 179 ± 1f 13.45 ± 0.02o 2.92 ± 0.01ef 13.5 ± 0.5i 7.04 ± 0.05jkl Malvasia Preta 295 ± 8cde 129 ± 4cd 14.84 ±0.02s 3.38 ± 0.01l 8.5 ± 0.4fg 3.88 ± 0.17cd Rufete 484 ± 8m 269 ± 5j 12.08 ± 0.01i 3.05 ± 0.01gh 5.5 ± 0.3bcd 2.87 ± 0.02ab Sousão 336 ± 7gh 178 ± 4f 9.66 ± 0.02b 2.62 ± 0.01b 8.5 ± 0.1fg 7.24 ± 0.02kl Tinta Amarela 530 ± 9n 270 ± 9j 10.93 ± 0.01d 2.91 ± 0.02ef 6.3 ± 0.1cd 3.50 ± 0.16bc Tinta Barca 459 ± 9lm 230 ± 5hi 11.67 ± 0.02g 2.88 ± 0.01ef 9.0 ± 0.2fgh 6.53 ± 0.03ijk Tinta Barroca 527 ± 7n 246 ± 3i 12.76 ± 0.01m 3.15 ± 0.01hij 5.5 ± 0.4bc 4.57 ± 0.37de Tinta Miúda 358 ± 7hi 197 ± 4g 13.45 ± 0.02o 2.88 ± 0.00ef 13.5 ± 0.3i 7.25 ± 0.24kl Tinto Cão 272 ± 6bcd 111 ± 2b 13.04 ± 0.01n 2.94 ± 0.00fg 6.8 ± 0.5de 4.96 ± 0.16ef

a Grape berry weight and must volume of 200 berries; data are the average of two replicates ± standard deviation; different letters above means indicate statistically significant differences between grape varieties ( p < 0 .05).

individual grape varieties may affect the polyphenolic aging processes, but also on the potential content in grape berries at harvest. In addition, the concentration of anthocyanins in grape skin as well as concentration of phenolic compounds is highly the easiness of anthocyanin extraction from skin into influenced by viticulture and environmental factors must (ortega-regules et al ., 2006). such as sunlight, temperature, altitude, soil type, and water and nutritional status (Jackson and Lombard, The individual anthocyanin composition of the skin 1993; Yokotsuka et al ., 1999). As expected, due to the extracts of the grape varieties at technological reasons mentioned above, differences in phenolic maturity is presented in Table 4. Malvidin-3-glucoside composition were observed among grape varieties was the major individual anthocyanin (concentrations (Table 3). ranging from 0.62 to 6.09 mg/g of skin) in all varieties except Alvarilhão and Rufete , where the major The total phenolic compounds, expressed as individual anthocyanins were peonidin-3-glucoside equivalent of gallic acid, ranged from 989 to (1.04 mg/g of skin) and malvidin-3- p-coumaroyl 3033 mg/L with an average of 1608.5 mg/L. The glucoside (1.48 mg/g of skin), respectively. Malvidin- highest concentration of total phenols (ranging from 3- p-coumaroyl glucoside was the second main 2119 to 3033 mg/L) was detected in Tinta Barca , Tinta individual anthocyanin for the majority of the Barroca , Sousão and Tinta Miúda , while Camarate , varieties and the values ranged from 0.12 to 3.44 mg/g Moreto Boal , Cornifesto and Grenache showed the of skin. These results are in agreement with previous lowest values (ranging from 989 to 1088 mg/L). findings in other grape varieties (Dallas and Laureano, 1994a; Jordão et al ., 1998a; Kallithraka et al ., 2005; The interest of winemakers for grape polyphenolic ortega-regules et al ., 2006). According to ribéreau- content is increasing, as it offers tools to influence the Gayon et al . (2006), malvidin derivative forms are color, bitterness, astringency, ‘mouth-feel’ and ‘age- stable molecules and their presence give stability to ability’ of wines. Therefore, the color of red wines and the wine during the winemaking process because of its evolution depend not only on the winemaking and their relative resistance to oxidation. Mateus et al .

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(2002) found that malvidin-3-glucoside and its important group, ranging from 0.23 ( Bastardo ) to 2.87 acylated esters were the major anthocyanin (Tinto Cão ) mg/g of skin, except in four varieties monoglucosides in Touriga Nacional and Touriga (Camarate , Gewürztraminer , Monvedro and Jean ) Francesa at harvest date. where the acetyl glucoside group was the second main group. A high concentration of p-coumaroyl- In general, cyanidin derivatives (cyanidin-3-acetyl anthocyanin derivatives, mainly from malvidin and glucoside and petunidin-3-acetyl glucoside) were the petunidin, has been associated with warm climates less abundant individual anthocyanins (ranging from (Downey et al ., 2006). However, according to 0.01 to 0.05 and 0.01 to 0.39 mg/g of skin, Férnandez-Lopez et al . (1998) it is commonly respectively), which is in line with the results accepted that the anthocyanin composition of each published by several authors (Dimitrovska et al ., cultivar is closely linked to its genetic inheritance and, 2011; ortega-Meder et al ., 1994; roggero et al ., from a qualitative point of view, is quite independent 1986). In addition, these two anthocyanins were not of seasonal conditions or production area. detected in a great number of the grape varieties studied. According to Di Stefano and Flamini (2008), The acetyl glucoside group, which participates in an cyanidin is the precursor of peonidin-3-glucoside by intra-molecular copigmentation process leading to an the action of the UFGT (which transform cyanidin increase in wine color intensity (ortega-regules et into cyanidin-3-glucoside) and MT enzymes (which al ., 2006), was the minor group, ranging from 0.01 transform cyanidin-3-glucoside into peonidin-3- (Grenache ) to 2.03 ( Gewürztraminer ) mg/g of skin. glucoside). Although the anthocyanin profile may be complex and quite different for each variety studied, in general The glucoside group was the main anthocyanin Carignan Noir , Monvedro and Negro Mole showed chemical group in all grape varieties, ranging from the highest individual anthocyanin concentration 1.40 ( Rufete ) to 7.09 ( Carignan Noir ) mg/g of skin while Bastardo , Aramon , Grenache and Alvarilhão (sum of all five 3-glucoside anthocyanins). The showed the lowest concentration. It is important to coumaroyl glucoside group was the second most note that grape anthocyanin concentration is largely

Table 3. General phenolic composition of grape varieties at technological maturity.

Total phenolic Flavonoid Non-flavonoid Color intensity Color hue Grape variety compounds compounds compounds (abs.u.)a (abs.u.)a (mg/L gallic acid) (mg/L gallic acid) (mg/L gallic acid) Camarate 989 ± 13a 946 ± 11abc 43 ± 2a 13.57 ± 0.83h 6.31 ± 0.35cde Gewürztraminer 1124 ± 17ab 1031 ± 16bcd 93 ± 1abc 9.34 ± 0.24cde 7.96 ± 0.25ghi Monvedro 1240 ± 14abc 1118 ± 13bcde 122 ± 1bcd 7.62 ± 0.16b 7.46 ± 0.15efgh Moreto Boal 1032 ± 39a 961 ± 9abc 71 ± 3ab 5.21 ± 0.13a 11.33 ± 0.26j Negro Mole 1375 ± 17abcd 1278 ± 16defg 97 ± 1abc 10.15 ± 0.38ef 5.54 ± 0.08abc Negro Mouro 1593 ± 26def 1408 ± 23efgh 185 ± 3def 12.17 ± 0.11gh 6.46 ± 0.12cdef Alfrocheiro 1492 ± 13bcd 1262 ± 11defg 229 ± 2efgh 9.94 ± 0.20def 7.79 ± 0.11fghi Alvarilhão 1820 ± 39fgh 1582 ± 34ghi 238 ± 5efgh 9.17 ± 0.16cde 7.28 ± 0.22defgh Aramon 1590 ± 13def 1341 ± 11fgh 248 ± 2fghi 8.56 ± 0.14bcd 8.74 ± 0.10i Bastardo 2024 ± 37gh 1662 ± 30hi 362 ± 7k 9.93 ± 0.23def 8.59 ± 0.25hi Cabernet Franc 1900 ± 17fgh 1535 ± 14fghi 365 ± 6k 13.26 ± 0.15h 8.26 ± 0.07hi Carignan Noir 1476 ± 22bcd 1268 ± 19defg 208 ± 5efg 10.28 ± 0.87ef 8.35 ± 0.51hi Cornifesto 1088 ± 18ab 874 ± 13a 214 ± 3efg 8.25 ± 0.04bc 6.82 ± 0.10cdefg Gamay 1529 ± 18cde 1309 ± 12efgh 220 ± 2efgh 12.86 ± 0.43h 8.08 ± 0.48ghi Grenache 1038 ± 52a 907 ± 9ab 131 ± 7bcd 5.41 ± 0.60a 7.43 ± 0.35efgh Jean 1530 ± 24cde 1334 ± 10efgh 196 ± 5defg 10.99 ± 0.30fg 5.94 ± 0.29bcd Malvasia Preta 1352 ± 28abcd 1083 ± 23bcde 269 ± 6ghij 10.05 ± 0.14ef 6.18 ± 0.27bcde Rufete 1297 ± 33abc 1133 ± 28bcde 164 ± 4cde 11.16 ± 0.33fg 5.86 ± 0.00bc Sousão 2130 ± 74h 1898 ± 12i 232 ± 8efgh 18.68 ± 0.21jk 5.78 ± 0.22bc Tinta Amarela 1490 ± 28bcd 1197 ± 23cdef 294 ± 6hijk 16.22 ± 0.37i 4.86 ± 0.03ab Tinta Barca 3033 ± 19i 2700 ± 16j 333 ± 13jk 19.15 ± 0.12k 5.99 ± 0.02bcd Tinta Barroca 2783 ± 13i 2418 ± 18j 365 ± 15k 17.68 ± 0.10f 6.14 ± 0.16bcde Tinta Miúda 2119 ± 37h 1857 ± 32i 263 ± 5ghij 21.68 ± 0.16l 4.19 ± 0.12a Tinto Cão 1562 ± 33def 1254 ± 26defg 308 ± 11ijk 13.35 ± 0.42h 5.78 ± 0.18bc

a abs.u. – absorbance units referred to a 1 mm path length cell used; data are the average of two replicates ± standard deviation; different letters above means indicate statistically significant differences between grape varieties ( p < 0 .05).

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due, among other factors, to the berry size of each of skin) and malvidin-3- p-coumaroyl glucoside grape variety. on the other hand, Gewürztraminer , (ranging from 0.88 to 3.44 mg/g of skin). The Monvedro , Moreto Boal , Negro Mole , Alvarilhão , varieties Moreto Boal , Monvedro , Gewürztraminer Cabernet Franc , Jean and Tinta Amarela presented and Camarate appeared in the positive part of PC2, the widest variety of individual anthocyanins while due to their high content in malvidin-3-acetyl Rufete presented the narrowest variety of individual glucoside (ranging from 1.01 to 1.65 mg/g of skin). anthocyanins quantified. From the description presented above, anthocyanins To better understand the relationship between grape could be considered useful markers to distinguish variety and anthocyanin concentration, a principal grape varieties; however, this characteristic should be component analysis (PCA) was performed used with caution since anthocyanin concentration is (Figure 2A). The first two principal components (PCs) influenced not only by genetic background but also by explained 92.22 % of the total variance and showed agroecological factors, such as maturation (Conde et that grape varieties can be distinguished according to al ., 2007; Jordão et al ., 1998a), ripening stage (Segade their individual anthocyanin concentration. Figure 2A et al ., 2008), climate (Gil and Yuste, 2004), stress shows the corresponding loading plots that established levels (Gatto et al ., 2008) and cultural practices the relative importance of each variable. The first (Jordão et al ., 1998b). principal component (PC1), which explained 81.89 % of the variance, was negatively correlated with the 3. Antioxidant activity from different grape berry variables malvidin-3-glucoside and malvidin-3- p- fractions coumaroyl glucoside. The second PC (PC2, 10.33 % of the variance) was positively correlated with the The data in Table 5 show the antioxidant activity variable peonidin-3-glucoside and negatively quantified in the different grape berry fractions (pulps, correlated with malvidin-3-acetyl glucoside. Five skins and seeds) from the grape varieties studied. The groups could be distinguished. The varieties highest antioxidant activity was found in seeds Camarate , Gewürztraminer , Monvedro , Negro Mole , (ranging from 77.59 to 867.81 and from 75.52 to Negro Mouro , Cabernet Franc , Carignan Noir , 363.47 µmol/g of seed, for ABTS and DPPH method, FigureFiguCreo 22.r. nifesto and Tinta Barroca were rather grouped on respectively), followed by skins (ranging from 1.13 to the negative side of PC1, due to their high content in 292.05 and from 1.78 to 299.99 µmol/g of skin, for malvidin-3-glucoside (ranging from 4.10 to 6.09 mg/g ABTS and DPPH method, respectively) and pulps A B

Figure 2. Principal component analysis score plot (PC1 and PC2) of grape varieties: individual anthocyanins (A) and antioxidant activity (B).

CA, Camarate; GW, Gewürztraminer; Mn, Monvedro; MB, Moreto Boal; nM, negro Mole; nMr, negro Mouro; AF, Alfrocheiro; AV, Alvarilhão; Ar, Aramon; BT, Bastardo; CF, Cabernet Franc; Cn, Carignan noir; Cr, Cornifesto; GY, Gamay; Gr, Grenache; J, Jean; MP, Malvasia Preta; r, rufete; S, Sousão; TA, Tinta Amarela; TB, Tinta Barca; TBr, Tinta Barroca; TM, Tinta Miúda; TC, Tinto Cão. Delp gluc, delphinidin-3-glucoside; Cyan gluc, cyanidin-3-glucoside; Petun gluc, petunidin-3-glucoside; Peon gluc, peonidin-3- glucoside; Malv gluc, malvidin-3-glucoside; Cyan acet-gluc, cyanidin-3 acetyl glucoside; Petun acet-gluc, petunidin-3-acetyl glucoside; Peon acet-gluc, peonidin-3-acetyl glucoside; Malv acet-gluc, malvidin-3-acetyl glucoside; Peon coum-gluc, peonidin-

31 J. Int. Sci. Vigne Vin , 2014, 48 , n°1, 51-62 - 57 - ©Vigne et Vin Publications Internationales (Bordeaux, France) 08-jordao_05b-tomazic 01/04/14 20:26 Page58

Elisa CoSTA et al.

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J. Int. Sci. Vigne Vin , 2014, 48 , n°1, 51-62 ©Vigne et Vin Publications Internationales (Bordeaux, France) - 58 - 08-jordao_05b-tomazic 01/04/1420:26Page59

Table 5. Antioxidant activities of gTraabplee s5k. iAnn, ptiuolxpid aanndt saecetidv ietxietsr aocft sg raasp me eskasinu,r epdu lbpy a tnhde sAeeBdT eSx tarnadc tDs aPsP mHe maseutrheodd sb yin t hger aApBe TvSarieties at technological maturity. and DPPH methods in grape varieties at technological maturity.

Skins Pulps Seeds Grape variety ABTS DPPH ABTS DPPH ABTS DPPH

abc efgh kl j abc Camarate 47.64 ± 0.62 58.14 ± 2.02 2.57 ± 0.00 3.13 ± 0.27 161.13 ± 7.23bcde 129.00 ± 18.91 gh defg ijk ef ij Gewürztraminer 78.78 ± 7.45 45.30 ± 0.00 2.14 ± 0.07 2.02 ± 0.05 276.21 ± 6.03fghi 330.89 ± 1.89 b ghi fghi de ab Monvedro 34.48 ± 0.62 66.71 ± 0.00 1.58 ± 0.02 1.84 ± 0.07 77.59 ± 0.00a 96.91 ± 11.35 h ab n j def Moreto Boal 93.28 ± 1.86 9.63 ± 1.09 3.65 ± 0.02 3.12 ± 0.03 310.31 ± 1.21hi 210.56 ± 9.45 fg hi bcde b cde Negro Mole 71.78 ± 4.96 88.11 ± 2.02 0.95 ± 0.04 1.05 ± 0.09 137.26 ± 4.82bcd 185.15 ± 15.13 cdef defg ijk ghi efg Negro Mouro 57.74 ± 4.96 48.60 ± 3.78 1.95 ± 0.07 2.48 ± 0.01 145.79 ± 14.47bcd 239.97 ± 39.71 abc bc a a def Alfrocheiro 47.20 ± 3.72 15.34 ± 4.04 0.04 ± 0.03 0.18 ± 0.06 105.72 ± 1.21ab 229.27 ± 54.83 a defg a ef ghij Alvarilhão 9.03 ± 3.10 38.79 ± 20.18 0.05 ± 0.03 2.01 ± 0.06 174.77 ± 4.82bcde 300.14 ± 3.78 i k o hi hij Aramon 133.21 ± 4.96 203.27 ± 16.40 4.80 ± 0.22 2.59 ± 0.07 505.52 ± 16.88l 314.84 ± 1.89 a a bcd ef j Bastardo 1.13 ± 0.62 1.78 ± 0.01 0.82 ± 0.07 2.02 ± 0.05 410.05 ± 19.29jk 363.47 ± 11.82 bcdef cde fghi cd fghi Cabernet Franc 52.91 ± 1.86 30.32 ± 1.01 1.71 ± 0.12 1.48 ± 0.02 506.37 ± 10.85l 264.87 ± 4.73 j k nm fgh abc Carignan Noir 192.01 ± 9.93 182.37 ± 1.26 3.42 ± 0.13 2.25 ± 0.01 867.81 ± 18.08n 130.34 ± 9.45 bcdef bcd cdef bc ab Cornifesto 52.91 ± 0.62 23.40 ± 3.47 1.16 ± 0.07 1.25 ± 0.08 306.89 ± 44.61hi 106.27 ± 5.67 bc ghi hij fgh efgh Gamay 42.38 ± 3.10 68.22 ± 11.35 1.92 ± 0.07 2.24 ± 0.02 224.21 ± 19.29efgh 253.57 ± 19.24 b l lm i a

- ij

Grenache 33.16 ± 6.21 299.99 ± 5.04 2.95 ± 0.06 2.71 ± 0.05 75.52 ± 11.35

5 348.67 ± 14.47 9 Jean 63.88 ± 13.65defg 94.08 ± 12.61i 3.30 ± 0.56nm 1.12 ± 0.06bc kl 122.31 ± 1.89abc

- 453.52 ± 20.49 a a ab bc ab Malvasia Preta 1.57 ± 1.24 3.21 ± 1.01 0.55 ± 0.04 1.12 ± 0.06 237.85 ± 16.88efgh 95.57 ± 13.24 bc fghi ghij b cde Rufete 37.55 ± 2.48 63.76 ± 7.57 1.86 ± 0.03 1.09 ± 0.05 539.62 ± 69.92l 189.49 ± 13.24 © efg defg fgh b cdef efg V Sousão 68.27 ± 0.00 43.88 ± 4.04 1.30 ± 0.16 1.09 ± 0.02 189.26 ± 1.21 249.65 ± 5.67 i bcd ab abc a def g Tinta Amarela 45.89 ± 1.86 7.75 ± 0.96 0.63 ± 0.28 0.49 ± 0.00 m 219.92 ± 0.00 n 711.81 ± 14.47 e bcde j a a bcd

abc e Tinta Barca 50.28 ± 0.62 137.33 ± 7.06 0.16 ± 0.06 0.49 ± 0.01 122.77 ± 3.62 161.09 ± 11.35 t

a hi defg bc efg V Tinta Barroca 7.20 ± 1.96 87.39 ± 1.01 1.24 ± 0.13 1.12 ± 0.00 defg 237.30 ± 1.89 i 206.31 ± 13.26 n k cdef kl efg efg

efgh P Tinta Miúda 292.05 ± 3.72 32.24 ± 6.73 2.55 ± 0.06 2.14 ± 0.11 236.99 ± 22.91 239.97 ± 1.89 u i bcd jkl ghi hij b Tinto Cão 123.55 ± 7.45 25.33 ± 4.04 2.40 ± 0.15 2.48 ± 0.01 ghi 318.35 ± 14.18 l 283.88 ± 31.34 i c J a . t I i n o Data are the average of two replicates ± standard deviation; values are expressed as !mol/g of skin, pulp or seed; different letters above means indicate t n . s Data are the average of two replicates ± standard deviation; values are expressed as µmol/g of skin, pulp or seed; different letters above means indicate statistically S

statistically significant differences between grape varieties (p < 0.05). I c n i

. significant differences between grape varieties ( p < 0.05). t e V r i n g a n t i e o

V n a i n l e , s 2

( 0 B 1 o 4 r , d 4 e 8 a , u

n x ° , 1

F , r 5 a 1 n - c 27 6 e 2 ) 08-jordao_05b-tomazic 01/04/14 20:26 Page60

Elisa CoSTA et al.

(ranging from 0.04 to 4.80 and from 0.18 to and non-autochthonous grape varieties and the grape 3.13 µmol/g of pulp, for ABTS and DPPH method, samples from the two wine regions considered. From respectively). In addition, the average values were these results, it is clear that the antioxidant activity 314.17, 68.24 and 1.82 µmol/g, respectively for seeds, values depend on the method used. According to skins and pulps considering the ABTS method and Villaño et al . (2006), this divergence is due to the 211.01, 89.78 and 1.72 µmol/g, respectively for seeds, different reagents of the polyphenols with each skins and pulps considering the DPPH method. one method applied. For Wang et al . (2004), ABTS ˙+ and possible explanation for this distribution in the DPPH radicals have a different stereochemical different grape berry fractions could be the higher structure and a different method of genesis and thus amount of polyphenols such as monomeric flavanols they lend, after the reaction with the antioxidants, a (catechin and epicatechin), dimeric, trimeric and qualitatively different response to the inactivation of polymeric proanthocyanidins, and phenolic acids in their radical. Some authors reported that no single seeds compared to skins (Di Majo et al ., 2008; Yilmaz assay can provide all the information needed to and Toledo, 2004). These findings are in agreement evaluate antioxidant capacity, and multiple assays are with previous studies conducted with other red Vitis therefore required to build up an antioxidant profile of vinifera grape varieties (Poudel et al ., 2008; Xu et al ., particular foodstuffs (rivero-Pérez et al ., 2007). 2010). To highlight the relation between grape varieties and Taking into account the antioxidant activity of the red their antioxidant activity in the different grape berry grape varieties analyzed here, a large variation was fractions (pulps, skins and seeds), a PCA was applied found in the different grape berry fractions (Table 5). (Figure 2B). The PCA of the antioxidant activity For seeds and considering the ABTS method, values obtained by two methods showed that the first Carignan Noir , Tinta Amarela , Rufete , Cabernet two PCs explained 84.47 % of the total variance. The Franc , Aramon , Jean and Bastardo showed the first PC (70.21 % of the variance) was positively highest antioxidant activity values (ranging from correlated with seeds ABTS and the second PC 410.05 to 867.81 µmol/g of seed; p < 0.05) while (14.26 % of the variance) was positively correlated Monvedro and Alfrocheiro showed the lowest with skins DPPH and negatively correlated with seeds antioxidant values (77.59 and 105.72 µmol/g of seed, DPPH. Grape varieties appeared grouped due to their respectively; p < 0.05). However, considering the different antioxidant activity of skins, seeds and pulps. DPPH method, Bastardo , Gewürztraminer , Tinto Cão , The grape varieties Carignan Noir and Tinta Amarela Aramon and Alvarilhão showed the highest antioxidant activity values (ranging from 300.14 to were on the right side of PC1, disclosing a good 363.47 µmol/g of seed; p < 0.05) while Monvedro , correlation with seeds ABTS. However, the grape Grenache and Malvasia Preta showed the lowest varieties Bastardo , Gewürztraminer , Alvarilhão and antioxidant values (ranging from 75.52 to Tinto Cão were on the negative side of PC2, 96.91 µmol/g of seed; p < 0.05). disclosing a good correlation with seeds DPPH. For skins and considering the ABTS method, Tinta A linear regression analysis was performed to Miúda , Carignan Noir , Aramon and Tinto Cão determine the correlation between polyphenol showed the highest antioxidant activity values composition and respective antioxidant activities in (ranging from 123.55 to 292.05 µmol/g of skin; p < the different grape berry fractions. This analysis found 0.05) while Bastardo , Malvasia Preta and Tinta no significant correlation ( p > 0.05) between general Barroca showed the lowest antioxidant values phenolic parameters and antioxidant activity in the (ranging from 1.13 to 7.20 µmol/g of skin, grape berry fractions. on the one hand, correlations respectively; p < 0.05). The results obtained by the between the antioxidant activity and the total DPPH method showed similar distribution of high polyphenolic content of a great number of grape seed (Carignan Noir and Aramon ) and low ( Malvasia and skin extracts from different grape varieties have Preta and Bastardo ) antioxidant activity values. been reported (Monagas et al ., 2005; Xu et al ., 2010). Finally, for pulps, high antioxidant activity values on the other hand, other authors (Bozan et al. , 2008) were found for Aramon , Moreto Boal and Carignan reported no significant correlations between Noir (4.80, 3.65 and 3.42 µmol/g of pulp for ABTS individual flavanols analyzed by HPLC or total method, respectively) while low antioxidant values polyphenols and antioxidant activity in seed extracts were found for Alfrocheiro , Alvarilhão , Tinta Barca from several grape varieties. Thus, there is conflicting and Malvasia Preta (0.04, 0.05, 0.16 and 0.55 µmol/g evidence in the literature about the correlation of pulp for ABTS method, respectively). overall, between polyphenol content and the antioxidant there is no clear difference between the autochthonous activity of grapes.

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CONCLUSIONS Biochemical changes throughout grape berry development and fruit and wine quality. Food 1, 1-22. In this work, the influence of grape variety on the Dallas C. and Laureano o., 1994a. Effect of So 2 on the phenolic content, anthocyanin profile and antioxidant extraction of individual anthocyanins and colored activity of the different grape berry fractions (skins, matter of three Portuguese grape varieties during pulps and seeds) has been analyzed in twenty-four winemaking. Vitis 33 , 41-47. different grape varieties cultivated in two Portuguese Dallas C. and Laureano o., 1994b. Effects of pH, sulphur wine regions ( Douro and Dão ). In general, a high dioxide, alcohol content, temperature and storage time variability was found among the diverse on colour composition of a young Portuguese red autochthonous and non-autochthonous grape varieties table wine. J. Sci. Food Agric. 65 , 477-485. in relation to polyphenolic content, anthocyanin Di Majo D., La Guardia M., Giammanco S., La neve L. profile and antioxidant activity. Seeds were the grape and Giammanco M., 2008. The antioxidant capacity berry fraction with the highest antioxidant activity, of red wine in relationship with its polyphenolic followed by skins and pulps, irrespective of the grape constituents. Food Chem. 111 , 45-49. variety. Di Stefano r. and Flamini r., 2008. High performance liquid chromatography analysis of grape and wine The results obtained for polyphenolic concentration, in polyphenols. In: Hyphenated Techniques in Grape particular individual anthocyanin concentration, and and Wine Chemistry , pp. 33-79. riccardo Flamini antioxidant activity must be considered in wineries, in (ed.), John Wiley & Sons, Ltd. order to apply the most appropriate winemaking Dimitrovska M., Bocevska M., Dimitrovski D. and techniques. Thus, from the data presented in this work, Murkovic M., 2011. Anthocyanin composition of several specific winemaking techniques could be Vranec, Cabernet Sauvignon, Merlot and Pinot noir suggested depending on the grape variety: extended grapes as indicator of their varietal differentiation. maceration could be used to optimise anthocyanin Eur. Food Res. Technol. 232 , 591-600. extraction and therefore improve color in Bastardo and Dopico-García M.S., Fique A., Guerra L., Afonso J.M., Alvarilhão wines, whereas standard winemaking Pereira o., Valentão P., Andrade P.B. and Seabra process could be a good option for Carignan Noir , r.M., 2008. Principal components of phenolics to Tinta Miúda and Tinto Cão. Increasing pumping over characterize red Vinho Verde grapes: anthocyanins or frequency during the maceration process can also non-coloured compounds? 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