Oxygen Radical Scavenging Capacity of Phenolic and Non-Phenolic Compounds in Red and White Wines
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Cent. Eur. J. Biol. • 7(1) • 2012 • 146-158 DOI: 10.2478/s11535-011-0095-8 Central European Journal of Biology Oxygen radical scavenging capacity of phenolic and non-phenolic compounds in red and white wines Research Article Roberto Lo Scalzo1,*, Massimo Morassut2, Paolo Rapisarda3 1National Council for Agricultural Research (C.R.A.), Research Unit of Food Technology (CRA-IAA), 20133 Milano, Italy 2National Council for Agricultural Research (C.R.A.), Research Unit for Enology of Central Italy (CRA-ENC), 00049 Velletri, Italy 3National Council for Agricultural Research (C.R.A.), Research Center of Citriculture and Mediterranean Crops (CRA-ACM), 95024 Acireale, Italy Received 12 May 2011; Accepted 19 October 2011 Abstract: The aim of the present study was the evaluation of the antioxidant content in phenolic and non-phenolic extracts of ten wine samples, trying to elucidate the potential role of unusual antioxidant compounds. Samples of wines processed from red and white grapes (Vitis vinifera L.), deprived of the volatile fraction at low temperature and buffered at physiological pH, were fractionated by C18 into two fractions: FR1 and FR2. Non-phenolics, such as tartaric, malic, lactic, and succinic acids; glucose; fructose; and glycerin were mainly found in FR1, while polyphenols were present exclusively in FR2. Peroxyl radical quenching was assayed by the ORAC method, while superoxide and hydroxyl radical scavenging activity were assayed by electron paramagnetic resonance. In the ORAC and superoxide assays, most of the activity was found in FR2, while in hydroxyl radical assay, the activity was found in FR1. Model solutions were used to attribute a role to the single compounds in the evaluation of wine’s ROS scavenging capacity: the ORAC and superoxide anion scavenging effects were mainly attributed to the polyphenols, averaging 94.8%, with some contribution from glycerin, particularly in white wines. Unexpectedly, the main chemical responsible for hydroxyl radical scavenging activity was glycerin (56.1%), with the polyphenols scavenging at 18.1%. Keywords: Wine • Phenolic compounds • Antioxidant • C18 fractionation • Peroxyl radical • Superoxide anion • Hydroxyl radical © Versita Sp. z o.o. 1. Introduction Wine is known for its healthy properties, if appropriately assumed. These properties are strictly Wine represents an application of a traditional related to polyphenols, known as antioxidants, able to biotechnology known for thousands of years. It is now a scavenge free radicals produced by oxygen catabolism very important worldwide aspect of food culture, having or fatty acid peroxidation [2,3]. spread from the Mediterranean basin countries to other Many studies have been made to identify the active emerging producers, such as the United States, Chile, compounds of wine antioxidant action, both on whole Australia and South Africa. extracts and separately on its polyphenol classes, such The chemical composition of the grapes as anthocyanins, flavones, stilbenes, cinnamates and is subjected to deep changes during the phenolic acids [4-11]. biotechnological processing into wine [1]. The wine The common opinion is that the free radical scavenging composition profile is obtained by the progressive capacity of grape and wine extracts is related to its alcoholic extraction of grape components, usually polyphenol content, mainly located in the grape skin [12-16]. with maceration for red-skinned and not for white- It has been found that phenols show different skinned grape varieties. rankings of reactivity with different antioxidant assays * E-mail: [email protected] 146 R. Lo Scalzo et al. [17-19]. In addition, some studies on the antioxidant 2. Experimental Procedures potential of grape extracts found that the use of different tests gave different responses, not directly correlated 2.1 Rationale of the experiment with the polyphenolic content of wine. For example, the Different red and white wines made from ten grape activity of grape components against hydroxyl radicals, varieties (Vitis vinifera L.) were evaporated to subtract one of the most harmful free radicals generated as an the volatile and ethanolic fraction and re-solubilized oxygen byproduct, showed that the activity of grape in phosphate buffer to give the unfractionated (UN) extracts was poorly related to polyphenol content. samples. An aliquot of UN was fractionated on C18, A previous work by Lo Scalzo et al. [20] demonstrated obtaining two fractions: FR1, the directly eluted fraction that the comparison of two different antioxidant assays, deprived of polyphenols, containing the non-phenolic such as DPPH and hydroxyl radical scavenging, resulted hydrophilic compounds, mainly hydroxy acids, glycerin in a strong difference between grape skin and flesh and residual simple sugars; and FR2, the retained extracts. The scavenging activity of DPPH was higher fraction eluted with MeOH, with practically all the in grape skin than flesh, as was expected in accordance polyphenols present in the original wine sample. with their significantly different phenol content. The UN, FR1 and FR2 samples were analyzed and activity against hydroxyl radicals did not give the same compared for their content in hydroxy acids such as result: the hydroxyl radical scavenging was about the tartaric, malic, lactic and succinic; for glycerin, glucose same between the grape skin and flesh extracts. The and fructose; and for total polyphenols index (Table 1). experiment was repeated on table grape samples [21], Three free radical scavenging tests against the by comparing three antioxidant assays: DPPH, Fremy’s main Reactive Oxygen Species (ROS) on the different salt, and hydroxyl radical scavenging on grape skin and fractions of red and white wines were also performed. flesh extracts. The results were confirmed as previously The scavenging of peroxyl radicals was evaluated with described, with the indices of DPPH and Fremy’s salt the fluorescein quenching by a peroxyl radical generated scavenging yielding higher results in skin than flesh, by thermal degradation of an azo-bis-compound, with no difference for hydroxyl radical scavenging. well known as the ORAC test (Oxygen Radical Furthermore, the grape extracts were fractionated by Absorbance Capacity). Superoxide anion (SASC) and solid phase extraction on C18, and the result was that hydroxyl radical scavenging (HRSC) were measured the activity against DPPH and Fremy’s salt was found by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin- in the polyphenol-enriched fraction, while most of the trap reaction and electronic paramagnetic resonance activity against hydroxyl radical was in the fractions detection, respectively. poor in polyphenols. These results were confirmed unexpectedly in vivo, where experimental rats were 2.2 Winemaking and sampling divided into three groups, two separately fed with grape Wine samples were obtained from the experimental wine skin or flesh, and the third used as a control, fed without cellar of the CRA-ENC, Research Unit for the Enology grape supplementation. The interesting result was that of Central Italy, Velletri (Rome). 10 wine samples were the two groups separately fed with grape skin and flesh obtained from the winemaking of single certified grape were equally cardioprotected with respect to the control varieties (red: 1 Abbuoto, 2 Bombino nero, 3 Marzemino, group [22]. 4 Montepulciano, 5 Petit verdot, 6 Refosco dal peduncolo At this point, the identification of the compounds rosso, 7 Teroldego; white: 8 Bombino bianco, 9 Greco, responsible for the antioxidant activity in wine grapes 10 Malvasia del Lazio), harvested in the experimental has become the most relevant question, starting with vineyard of the CRA-ENC during the 2007 season, and the most abundant grape compounds, such as simple processed according to established protocols. Red grapes sugars and organic acids. Previous experiments (about 100 kg) were destemmed and crushed. The must [23,24] demonstrated that •OH could be scavenged by was treated with 6 g/hL of potassium metabisulfite and simple sugars, with disaccharides more active than inoculated with 20 g/hL of Saccharomyces cerevisiae r.f. monosaccharides [25], and also that organic acids can uvarum strain S6u (LALLEMAND Inc 151 Skyway Avenue act as antioxidants [26,27]. Rexdale, Ontario M9W 4Z5). Fermentation temperature Many types of grape are destined to become wine was 25°C, the maceration time of skins was 10 days. and the logical continuation of these studies is to shift White grapes (about 100 kg) were destemmed, crushed, previous considerations of grapes to wine, so the aim and 10 g/hL of potassium metabisulfite was added. After of the present study was to investigate the role of clarification, musts were racked and inoculated with polyphenols in the antioxidant capacities of different 25 g/hL of of Saccharomyces cerevisiae r.f. uvarum, wine samples before and after fractionation. strain S6u. Fermentation took place in 100-L stainless 147 148 tartaric acid (mg/100 ml) malic acid (mg/100 ml) lactic acid (mg/100 ml) succinic acid (mg/100 ml) Samples UN FR1 FR2 UN FR1 FR2 UN FR1 FR2 UN FR1 FR2 1 Abbuoto 198.9 a 205.6 a 0.2 b 66.1 a 63.9 a 0.0 b 41.9 a 42.3 a 0.0 b 28.3 a 26.1 a 0.0 b 2 Bomb nero 258.9 a 257.7 a 2.1 b 148.0 a 145.0 a 0.0 b 13.2 a 12.5 a 0.0 b 20.2 a 17.7 a 0.0 b 3 Marzemino 187.6 a 190.4 a 0.8 b 22.5 a 18.2 a 0.0 b 179.8 a 184.9 a 0.0 b 16.1 a 16.6 a 0.0 b 4 Montepulciano 84.5 a 81.7 a 0.3 b 24.8 a 21.6 a 0.0 b 207.5 a 207.1 a 0.0 b 22.2 a 21.7 a 0.0 b 5 Pet verdot