Antioxidant Potential of White Grape Pomaces: Phenolic Composition and Antioxidant Capacity Measured by Spectrophotometric and Cyclic Voltammetry Methods

  Antioxidant potential of white grape pomaces: Phenolic composition and antioxidant capacity measured by spectrophotometric and cyclic voltammetry methods M. Jos´e Jara-Palacios, Dolores Hernanz, M. Luisa Escudero-Gilete, Francisco J. Heredia PII: S0963-9969(14)00599-7 DOI: doi: 10.1016/j.foodres.2014.09.009 Reference: FRIN 5483 To appear in: Food Research International Received date: 21 July 2014 Accepted date: 8 September 2014 Please cite this article as: Jos´e Jara-Palacios, M., Hernanz, D., Luisa Escudero-Gilete, M. & Heredia, F.J., Antioxidant potential of white grape pomaces: Phenolic composition and antioxidant capacity measured by spectrophotometric and cyclic voltammetry methods, Food Research International (2014), doi: 10.1016/j.foodres.2014.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 Antioxidant potential of white grape pomaces: phenolic composition and antioxidant capacity measured by spectrophotometric and cyclic voltammetry methods M. José Jara-Palacios1, Dolores Hernanz2, M. Luisa Escudero-Gilete1, Francisco J. Heredia1 1Food Colour& Quality Laboraty, Dept. Nutrition & Food Science, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. 2Department of Analytical Chemistry, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. * Corresponding author: Francisco J. Heredia Food Colour& Quality Laboratory, Dept. Nutrition & Food Science, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. Tel.: +34 954556495 Fax: +34 954556110 e-mail: here[email protected] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 Abstract Antioxidant potential of white grape pomaces from nine different varieties has been evaluated and compared to Zalema variety. The detailed phenolic composition was measured by RRLC/MS, the total phenolic content (TPC) by Folin-Ciocalteu method, and the antioxidant activity by ABTS assay and cyclic voltammetry (CV). Grape pomaces exhibited a different quantitative phenolic profile and different antioxidant activities, with significant differences (p<0.05). Parellada, Zalema, Sauvignon blanc and Moscatel showed the highest values of TPC and ABTS. The total flavanols, flavonols and phenolic acids contents were significantly correlated to the electrochemical parameter anodic peak current (Ip,a). Finally, a stepwise linear discriminant analysis (SLDA) was carried out, and Zalema variety was differentiated from other varieties based to the total flavonols content, mainly quercetin-3-O- glucoside. Keywords Antioxidant potential, grape pomace, phenolic compounds, cyclic voltammetry (CV), rapid resolution liquid chromatography (RRLC). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 1. Introduction Grapes are one of the major fruit crops and about 80% of the harvest is used by the winemaking industry. Winemaking is a seasonal activity, which leads to the generation of large quantities of wastes during a short period every year (grape harvesting), especially in high production regions. Accumulation of these wastes is a serious environmental problem and its removal is necessary. Traditionally, winemaking byproducts have been sent to distilleries for obtaining ethanol, or to be used as fertilizers or biomass but these activities are usually carried out by external companies representing economic costs for the wine industry. Therefore, alternative solutions for the exploitation and valorization of those byproducts are very interesting because it would involve economic, social and environmental advantages (Devesa-Rey et al., 2011; Pedroza, Carmona, Pardo, Salinas, & Zalacain, 2012; Lavelli, Sri Harsha, Torri, & Zeppa, 2014). Byproducts from winemaking, such as grape pomace, have received much attention because they contain large amounts of phenolic compounds, which have antioxidant properties and benefits on human health (Jayaprakasha, Selvi, & Sakariah, 2003; Rockenbach et al., 2011). Seeds, skins and stems of grape pomace exhibit a different qualitative and quantitative phenolic profile and different antioxidant activities ( a, & García Romero, 2006; Jara-Palacios et al., 2014a). White grape must is not usually fermented with the solid parts of the grape and then higher proportions of phenolic compounds remain in the grape pomace from white grapes than from red grapes. On the other hand, the phenolic composition of grape pomace depends on the variety of grape and, is influenced by agroclimatic factors. There are reports emphasizing the influence of the grape variety, agricultural practices, ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 agroclimatic factors and type of soil on the chemical composition of the grapes (Rodríguez Montealegre et al., 2006; Ruberto et al., 2007). Different techniques have been used for the separation, identification and quantification of phenolic compounds, being the high performance liquid chromatography (HPLC) the most commonly used (Fontana, Antoniolli, & Bottini, 2013). However, the advantages of the rapid resolution liquid chromatography (RRLC), which shows high resolution and sensitivity, and short retention times, are increasing the use of this technique. Antioxidant activity has been widely measured by several in vitro methods such as spectrophotometric methods (ABTS, FRAP, DPPH and ORAC assays) (Floegel, Kim, Chung, Koo, & Chun, 2011) and electroanalytical methods, such as cyclic voltammetry (CV). CV has been successfully applied to the total antioxidant capacity measurement in plant extracts, wines, and juices (Chevion, Chevion, Chock, & Beecher, 1999; Kilmartin, Zou, & Waterhouse, 2002; Kilmartin, & Hsu, 2003; Sousa, da Rocha, Cardoso, Silva, Zanoni, 2004; Makhotkina, & Kilmartin, 2012) and it has become an alternative to traditional spectrophotometric techniques (Sánchez Arribas, Martínez- Fernández, & Chicharro, 2012; Tufan, Baki, Güçlü, Ozyürek, & Apak, 2014). Spain is a geographical area with the typical climatological conditions of warm climate. Many white grape varieties grow in different areas and are used to the wine production of wines with differences in sensory characteristics and phenolic composition, for example, Spanish autochthonous white varieties such as Zalema, Verdejo, Airén, Moscatel, Montepila, Pedro Ximénez, Baladí, Parellada, and originating in other regions such as Sauvignon blanc. Zalema is a white grape variety grown exclusively in southwestern Spain. Previous studies about the phenolic composition and antioxidant activity of winemaking byproducts from Zalema have reported the possibility of applications based on reusing ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 these byproducts (Jara-Palacios et al., 2013; Jara-Palacios et al., 2014a; Jara-Palacios et al., 2014b). In this sense, the aim of this work was to evaluate the differences, in the antioxidant potential, between nine white grape pomaces and to compare them to Zalema variety. 2. Materials and methods 2.1. Standards and Reagents Formic acid, acetic acid, HPLC-grade acetonitrile, methanol, and Folin Ciocalteu reagent were obtained from Panreac (Barcelona, Spain). ABTS (2,2-azino-bis-(3- ethylbenzothiazolne-6-sulfonic acid) diammonium salt) and Trolox (6-hydroxy-2,5,7,8- tetramethyl-chroman-2-carboxylic acid) were purchased from Fluka (Madrid, Spain). Gallic acid, (+)-catechin, (-)-epicatechin, quercetin, kaempferol, ferulic acid, caffeic acid, p-coumaric acid, sodium carbonate, sodium acetate, potassium persulphate and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (Madrid, Spain). Quercetin-3-O-glucoside and kaempferol-3-O-glucoside were obtained from Extrasynthese (Lyon, France). 2.2. Samples The pomaces from white grapes varieties grown in “ - ” D f Origin (Cordoba, south-eastern Spain), with the typical climatological conditions of warm climate regions, were pp by “I Andaluz de Investigación y Formación Agraria, Pesquera, Alimentaria y de la Producción E (IFA A)” experimental vineyard (Cabra, Spain), when the grapes were at technological ripeness (12°-13° Baumé). Nine varieties, Zalema (Z), Pedro Ximénez (PX), Moscatel (MG), Baladí (B), Parellada (P), Sauvignon blanc (SB), Montepila (M), Airén (A) and Verdejo (V) were evaluated. All varieties were grown in the same warm climate vineyard in ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 order to evaluate the differences only due to the grape variety and not influenced by different climates. 2.3. Sample preparation and extraction A sample of 100 g of clusters (including grapes and stems) was manually pressed to mix the skins, the seeds and the stems. The obtained must was discarded, and the resulting solid sample (grape pomace) was weighed and freeze-dried. The dry pomaces were extracted with 75% methanol according to the methodology described by Jara-Palacios et al. (2014a). The extractions were carried out in triplicate and the obtained extracts were used for analysis. 2.4. Total phenolic content Total phenolic content (TPC) was determined using the Folin-Ciocalteu assay (Singlenton, & Rossi, 1965) with some modifications (Jara-Palacios et al., 2014a). Gallic acid was employed as a calibration standard and results were expressed as gallic acid equivalents (mg GAE/ 100 g of dry pomace (DP)). 2.5. Individual phenolic compounds The individual phenolic compounds were determined by RRLC/MS following the method described by (Jara-Palacios et al., 2014a). Phenolic compounds were identified by their retention time, UV-vis spectra and mass spectra, as well as by comparison with our data library and standards when available. The corresponding calibration curves were made up of ten standards: catechin, epicatechin, gallic acid, caffeic acid, ferulic acid, p-coumaric acid, quercetin-3-O-rutinoside, quercetin-3-O-glucoside and kaempferol-3-O-glucoside. Procyanidins were quantified with the calibration curve of catechin. Caftaric, fertaric and coutaric acids were quantified using the calibration curves of caffeic, ferulic and coumaric acids, respectively. Quercetin and isorhamnetin ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 derivatives were quantified as quercetin-3-O-glucoside and kaempferol derivates as kaempferol-3-O-glucoside. Each extract was injected three times (n=9) to quantify each compound, and the results were expressed as mg phenolic compound/100 g of DP. Total flavanols, total flavonols and total phenolic acids were also estimated by summing the content of each individual phenolic compound identified by RRLC. 2.6. ABTS/persulphate assay The ABTS•+ radical was produced by the oxidation of 7 mM ABTS with potassium persulphate (2.45 mM) in water (Re et al., 1999). The mixture was allowed to stand in k p f 16 b f ABTS•+ w diluted with PBS at pH 7.4 to give an absorbance of 0.7±0.02 at 734 nm. The extracts (50 µL) of pomace were mixed with 2 mL of the ABTS•+ diluted solution, vortexed for 10 s, and the absorbance measured at 734 nm after 4 min of reaction at 30 ºC. Different dilutions of each extract were assayed and the results were obtained by interpolating the absorbance on a calibration curve obtained with Trolox (30-1000 M). Three independent experiments were performed in triplicate for each of the assayed extracts and the results were expressed as Trolox-equivalent antioxidant capacity (TEAC; millimoles of Trolox with the same antioxidant capacity as 100 g DP). 2.7. Electrochemical assays A potentiostat/galvanostat (AUTOLAB model PGSTAT 302 N) controlled by a General Purpose Electrochemical System (GPES) software (Metrohm Autolab B.V., Utrecht, The Netherlands), was used for all electrochemical measurements. 1 ml of the extracts was diluted with 0.1 M sodium acetate-acetic acid buffer at pH 3.6 (Rebelo, Rego, Ferreira, & Oliveira, 2013). The diluted sample was transferred into a glass water-jacketed electrochemical cell (EG&G, Princeton, NJ) connected to a ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 circulator that held the sample temperature at 25.0 ± 0.5 °C. Prior to the measurements, the electrolyte solutions were de-aerated with an inert gas (N2) for 10 min. All measurements were carried out at room temperature using a conventional threeelectrode system consisting of a glassy carbon working electrode, platinum auxiliary electrode and Ag/AgCl reference electrode. The cyclic voltammograms scans were made from 0.0 to 1.0 V at a scanning rate of 5 mV/s. The electrochemical parameters extracted from the cyclic voltammetry curves were the anodic current area (Q), the anodic peak current (Ip,a), and the anodic peak potential (Ep,a) of the main peaks in the cyclic voltammograms. QI represents the integrated area of the cyclic voltammogram for scans taken from 0.12 to 0.32 V, QII from 0.35 to 0.55 V, and QIII from 0.65 to 0.85 V. All of the cyclic voltammograms were recorded in duplicate. 2.8. Statistical analysis One way analysis of variance (ANOVA) was applied to determine whether significant differences (p<0.05) exist among the different grape pomace vatieties. In addition, simple and multiple correlations between the contents of phenolic compounds and the antioxidant activity, measured by ABTS method and CV, were studied. In all cases, statistically significant level was considered at p<0.05. Pattern recognition techniques (PR), like stepwise linear discriminant analysis (SLDA), were applied on experimental standardized data in order to classify different grape pomace varieties. These statistical analyses of the data were performed using the Statsoft Statistica V 8.0 software (StatSoft, 2007). 3. Results and discussion 3.1. Phenolic composition ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 15 irreversible oxidation of the -OH group at position 3 on the C-ring (Cosio, Buratti, Mannino, & Beneditti, 2006; Janeiro & Brett, 2004). Makhotkina et al., (2012) reported that flavonols that are derivatives of quercetin are able to produce the peak II at approximately 0.5 V. The third voltammetric peak could be due to phenolic acids such as p-coumaric acid, or flavanols, such as catechin (Kilmartin et al., 2001; Rebelo et al., 2013). Zalema and Moscatel extracts had the highest values of IIIIp,a (1.95 µA) while Airén and Parellada extracts showed the lowest values (IIIIp,a = 1.36 and 1.35 µA, respectively). Finally, a non-linear correlation between cyclic voltammetric parameters and results of antioxidant activity by ABTS assay or TPC by Folin-Ciocalteu method was obtained. This is in accordance to other authors and it could indicate that the CV technique not provides only phenolic information (Rebelo et al., 2013). 3.4. Stepwise linear discriminant analysis To ascertain whether it was possible to discriminate between different varieties of grape pomaces as a function of TPC, ABTS, IIp,a, IIIp,a, IIIIp,a values and total flavanols, flavonols and phenolic acids contents (independent variables), a SLDA was carried out. All variables were found statistically significant (p<0.05). Two classification functions were obtained, which yielded a good separation (100% correct classification) among samples (Figure 4). The discriminant function 1 was mainly related to total flavonols content, ABTS and TPC values (with negative sign), and total flavanols content and IIIIp,a values (positive sign), whereas the discriminant function 2 was mainly linked to IIp,a and IIIp,a (positive sign), and total flavanols (negative sign). As can be seen in Figure 4, the discriminant function 1 mostly discriminates between Zalema and Moscatel varieties, and the discriminant function 2 between Parellada and Sauvgnon blanc, and Zalema and Moscatel varieties. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 16 4. Conclusions Grape pomaces of nine varieties exhibited different quantitative phenolic profile and different antioxidant activities measured by the ABTS assay and CV, showing statistically significant differences (p<0.05) among varieties. In all varieties, flavanols were the main phenolic group, being catechin and procyanidin B1 the most abundant compounds. Moscatel and Parellada varieties showed highest levels of flavanols, and phenolic acids levels were highest in Pedro Ximénez and Baladí varieties. Zalema variety showed the highest concentration in flavonols, showing significant differences with the other varieties. The antioxidant activity measured by the ABTS assay showed a good correlation to the TPC and particularly to the flavanols and flavonols contents, however a linear correlation was not found between CV results and ABTS or TPC data. The electrochemical parameters IIp,a, IIIp,a and IIIIp,a were significantly different among varieties, being IIp,a mainly ascribed to flavonols and phenolic acids, IIIp,a to flavanols, and IIIIp,a to the three mentioned phenolic groups. Eight variables allowed classify correctly 100% of the grape pomace samples. Total flavanols content was the variable which had the most influence in the discrimination of Zalema variety, mainly due to quercetin-3-O-glucoside concentration; therefore, as reported by some authors, flavonolic profile could be used to differentiate the white grape pomace of this variety. Results suggest that the electrochemical response of phenolic compounds in grape pomace extracts could be used as a measurement of the antioxidant potential. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 17 Acknowledgements The authors acknowledge the collaboration of “ j D.O. H v ” and IFAPA (Córdoba) by suppling samples, and the assistance of the technical staff of Biology Service (SGI, Universidad de Sevilla). M.J. Jara-Palacios holds a predoctoral Research Grant (FPU) from the Spanish Ministry of Education. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 18 References Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A., & Haroutounian, S. (2010). Bioactive non-coloured polyphenols content of grapes, wines and vinification by products: evaluation of the antioxidant activities of their extracts. Food Research International, 43, 805-813. Andrade, P.B., Mendes, G., Falco, V., Valentao, P., & Seabra, R.M. (2001). Preliminary study of flavonols in port wine grape varieties. Food Chemistry, 73, 397-399. Castillo-Muñoz, N., Gómez-Alonso, S., García-Romero, E., & Hermosín-Gutiérrez, I. (2010). Flavonol profiles of Vitis vinifera white grape cultivars. Journal of Food Composition and Analysis, 23, 699-705. Chevion, S., Chevion, M., Chock, P. B., & Beecher, G. R. (1999). The antioxidant capacity of edible plants: extraction protocol and direct evaluation by cyclic voltammetry. Journal of Medicinal Food, 2, 1-11. Cosio, M. S., Buratti, S., Mannino, S., & Beneditti, S. (2006). Use of an electrochemicalmethod to evaluate the antioxidant activity of herb extracts from the Labiatae family. Food Chemistry, 97, 725-731. Devesa-Rey, R., Vecino, X., Varela-Alende, J.L., Barral, M.T., Cruz, J.M., & Moldes, A.B. (2011). Valorization of winery waste vs. the costs of not recycling. Waste Manage, 31, 2327-2335. Floegel, A., Kim, D.O., Chung, S.J., Koo, S.I., & Chun O.K. (2011). Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods. Journal of Food Composition and Analysis, 24, 1043-104. Fontana, A.R., Antoniolli, A., & Bottini. R.J. (2013). Grape Pomace as a Sustainable Source of Bioactive Compounds: Extraction, Characterization, and Biotechnological Applications of Phenolics. Journal of Agricultural and Food Chemistry, 61, 8987-9003. Gordillo, B., Cejudo-Bastante, M.J., Rodríguez-Pulido, F.J., Jara-Palacios, M.J., Ramírez-Pérez, P., González-Miret, M. L., & Heredia, F. J. (2014). Impact of Adding White Pomace to Red Grapes on the Phenolic Composition and Color Stability of Syrah Wines from a Warm Climate. Journal of Agricultural and Food Chemistry, 62, 2663- 2671. Guendez, R., Kallithraka, S., Makris, D. P., & Kefalas, P. (2005). Determination of low molecular weight polyphenolics constituents in grape (Vitis vinifera sp.) seed extracts: Correlation with antiradical activity. Food Chemistry, 89, 1-9. Janeiro, P., & Brett, A. M. O. (2004). Catechin electrochemical oxidation mechanisms. Analytica Chimica Acta, 518, 109-115. Jara-Palacios, M. . -Manzano, S., Escudero-Gilete, M. . H D. D . - s, A.M., Heredia, F.J., & Santos-Buelga, C. (2013). Study ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 19 of Zalema grape pomace: phenolic composition and biological effects in Caenorhabditis elegans. Journal of Agricultural and Food Chemistry, 61, 5114-5121. Jara-Palacios, M. J., Hernanz, D., González-Manzano, S., Santos-Buelga, C., Escudero- Gilete, M. L., & Heredia, F. J. (2014a). Detailed phenolic composition of white grape by-products by RRLC/MS and measurement of the antioxidant activity. Talanta, 125, 51-57. - . . B. -Miret, M.L., Hernanz, D., Escudero-Gilete, M.L., & Heredia, F.J. (2014b). Comparative Study of the Enological Potential of Different Winemaking Byproducts: Implications in the Antioxidant Activity and Color Expression of Red Wine Anthocyanins in a Model Solution. Journal of Agricultural and Food Chemistry, 62, 6975-6983. Jayaprakasha, G.K., Selvi, T., Sakariah, K.K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Research International, 36, 117- 122. Kilmartin, P. A., Zou, H., & Waterhouse, A. L. (2001). A cyclic voltammetry method suitable for characterizing antioxidant properties of wine and wine phenolics. Journal of Agricultural and Food Chemistry, 49, 1957-1965. Kilmartin, P. A., Zou, H., & Waterhouse, A. L. (2002). Correlation of wine phenolic composition versus cyclic voltammetry response. American Journal of Enology and Viticulture, 53, 294-302. Kilmartin, P.A., & Hsu, C.F. (2003). Characterisation of polyphenols in green, oolong, and black teas, and in coffee, using cyclic voltammetry. Food Chemistry, 82, 501-512. Lavelli, V., Sri Harsha, P.S., Torri, L., & Zeppa, G. (2014). Use of winemaking byproducts as an ingredient for tomato puree: the effect of particle size on product quality. Food Chemistry, 152, 162-168. Makhotkina, O., & Kilmartin, P.A. (2012). The phenolic composition of Sauvignon blanc juice profiled by cyclic voltammetry. Electrochimica Acta, 83, 188-195. Pedroza, M. A., Carmona, M., Pardo, F., Salinas, M. R., & Zalacain, A. (2012). Waste grape skins thermal dehydration: potential release of colour, phenolic and aroma compounds into wine. CyTA-Journal of Food, 10, 225-234. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231-1237. Rebelo, M.J., Rego, R., Ferreira, M., Oliveira, M.C. (2013). Comparative study of the antioxidant capacity and polyphenol content of Douro wines by chemical and electrochemical methods. Food Chemistry, 141, 566-573. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 20 Rockenbach, I.I., Gonzaga, L.V., Rizelio, V.M., Goncalves, A., Genovese, M.I. & Fett, R. (2011). Phenolic compounds and antioxidant activity of seed and skin extracts of red grape (Vitis vinifera and Vitis labrusca) pomace from Brazilian winemaking. Food Research International, 44, 897-901. Rodríguez Montealegre, R., Romero Peces, R., n Vozmediano, J.L., a, J., & García Romero, E. (2006). Phenolic compounds in skins and seeds of ten grape Vitis vinifera varieties grown in a warm climate. Journal of Food Composition and Analysis, 19, 687-693. Ruberto, G., Renda, A., Daquino, C., Amico,V., Spatafora, C., Tringali, C., & De Tommasi, N. (2007). Polyphenol constituents and antioxidant activity of grape pomace extracts from five Sicilian red grape cultivars. Food Chemistry, 100, 203-210. Sánchez Arribas, A., Martínez-Fernández, M., & Chicharro, M. (2012). The role of electroanalytical techniques in analysis of polyphenols in wine. Trends in Analytical Chemistry, 34, 78-96. Singlenton, V.L., Rossi, J.A. (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American Journal of Enology and Viticulture, 16, 144-158. Sousa, W.R., da Rocha, C., Cardoso, C.L., Silva, D.H.S., Zanoni, M.V.B. (2004) Determination of the relative contribution of phenolic antioxidants in orange juice by voltammetric methods. Journal of Food Composition and Analysis, 17, 619-633. StatSoft Inc. STATISTICA (data analysis software system), v 8.0; Tulsa, OK, 2007. Tufan, A.N., Baki, S., Güçlü, K., Ozyürek, M., & Apak, R.A. (2014). Novel Differential Pulse Voltammetric (DPV) Method for Measuring the Antioxidant Capacity of Polyphenols-Reducing Cupric Neocuproine Complex. Journal of Agricultural and Food Chemistry, 62, 7111-7117. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 21 Figure captions Figure 1. Relative proportion of the three phenolic groups in the grape pomaces from the nine grape varieties. Figure 2. Representative cyclic voltammogram of a grape pomace extract of the Zalema grape variety. Figure 3. Cyclic voltammograms of grape pomace extracts from the nine varieties. Figure 4. Scatterplot of the grape pomaces samples in the plane defined by the canonical function when phenolic composition and antioxidant activity are considered for discrimination. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 22 Abbreviations Dry pomace (DP) Zalema (Z) Pedro Ximénez (PX) Moscatel (MG) Baladí (B) Parellada (P) Sauvignon blanc (SB) Montepila (M) Airén (A) Verdejo (V) Total phenolic content (TPC) Cyclic voltammetry (CV) Rapid resolution liquid chromatography (RRLC) Mass spectrometry (MS) Anodic current area (Q) Anodic peak current (Ip,a) Anodic peak potential (Ep,a) One way analysis of variance (ANOVA) Stepwise linear discriminant analysis (SLDA) Pattern recognition techniques (PR) Trolox-equivalent antioxidant capacity (TEAC) Trolox-equivalent (TE) Oxygen radical absorbance capacity (ORAC) 1,1-diphenyl-2-picrylhydrazyl (DPPH) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 23 2 2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Ferric reducing antioxidant power (FRAP) Phosphate buffered saline (PBS) High performance liquid chromatography (HPLC) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 24 Figure 1 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Z PX MG B P SB M A V Relative proportion variety Phenolic acids Flavonols Flavanols ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 31 Table 3. Electrochemical parameters of three anodic peaks extracted from the cyclic voltammetry curves of the grape pomaces. Peak I Peak II Peak III QI EIp,a IIp,a QII EIIp,a IIIp,a QIII EIIIpa IIIIp,a Z 0.26a 0.22 1.34a 0.30a,d 0.42 1.97a 0.28a,b 0.76 1.95a PX 0.27a 0.22 1.50b 0.34b 0.42 2.20b,c 0.25a,b 0.77 1.45b,c MG 0.26a 0.22 1.43c,f 0.35b,c 0.42 2.28b 0.33b 0.76 1.95a B 0.27a 0.22 1.52b 0.33b,d 0.43 2.12c 0.25a,b 0.77 1.58b,d P 0.28a 0.22 1.56d,g 0.34b 0.42 2.23b,c 0.24a 0.77 1.35c SB 0.28a 0.22 1.53b,d 0.37c 0.43 2.41d 0.27a,b 0.76 1.54b M 0.28a 0.22 1.57e,g 0.33b 0.43 2.13c 0.24a 0.77 1.45b,c A 0.27a 0.22 1.52b 0.34b 0.42 2.24b 0.24a,b 0.77 1.36c V 0.26a 0.22 1.41f 0.31d 0.42 1.95a 0.29a,b 0.76 1.73c Each value represents mean (n=6) ± SD. Different letters in the same column indicate significant differences by ANOVA test (p<0.05). Ep,a is expressed as V and Ip,a as µA. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 32 Highlights 1. Antioxidant potential of white grape pomaces from nine varieties has been evaluated 2. Cyclic voltammetry was used to measure the antioxidant activity 3. A RRLC method has been used to determine the phenolic composition of grape pomaces 4. Samples exhibited different phenolic profiles and antioxidant activities 5. Zalema variety was well differentiated from other varieties