RED WINE: A FUNCTIONAL BEVERAGE?

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1 In: Wine ISBN: Editor: Youssef El Rayess 2014 Nova Science Publishers, Inc. Chapter 5 RED WINE: A FUNCTIONAL BEVERAGE? Giovanna Giovinazzo 1 *, Ilaria Ingrosso 1, Mariana Tristezza 1, Francesco Grieco 1 and Youssef El Rayess 2,3,4 1 National Research Council. - Institute of Sciences of Food Production (ISPA), Unit of Lecce, via Prov. Lecce-Monteroni, Lecce, Italy 2 Faculty of Agricultural and Food Sciences, Holy Spirit University of Kaslik, Jounieh, Lebanon 3 Université de Toulouse, INPT, UPS, Laboratoire de Génie Chimique, 4 Allée Emile Monso, Toulouse, France. 4 Centre de Viticulture et dřœnologie de Midi-Pyr n es, Avenue de lřagrobiopôle, Castanet-Tolosan, France ABSTRACT Phenolic compounds present in grape berries are extracted from the skin, seeds and pulp during the winemaking process. These substances have a potentially positive effect on human health, thus giving to red wine Ŗbioactive propertiesŗ that can contribute to decrease the incidence of a number of human pathologies. However, basic and clinical sciences are showing that the reality is much more complex than this and that several issues, notably daily intake, bioavailability, or in vivo antioxidant activity are yet to be resolved. Viticulture and winemaking practices, which vary in different countries, determine the concentration of phenolic compounds in wine. In particular poor knowledge is available on the effects of different yeast strains on the final concentration of polyphenols in red wine. In this chapter, we summarize the recent findings concerning the effects of wine polyphenols on human health and propose future directions for research to increase the amount of these healthy compounds in wine. Keywords: Wine, polyphenols, antioxidant, biological effects, cardiovascular disease, bioavailability * Corresponding author: National Research Council. - Institute of Sciences of Food Production (ISPA), Unit of Lecce, via Prov. Lecce-Monteroni, Lecce, Italy. giovanna.giovinazzo@ispa.cnr.it.

2 132 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. INTRODUCTION Grapevine (Vitis spp) is the most cultivated fruit crop in the world, with an area dedicated to viticulture of 7.8 million hectares million tons of berries corresponding to 252 million hectoliters of wine were been produced during the 2012 vintage ( The berries are harvested primarily for winemaking (68%) but also to provide fresh table grapes (30%), raisins (2%) and other minor products. Grape metabolism elaborates a vast array of secondary compounds, which confer selective advantages to the plant against environmental stresses. Among such products are the polyphenols, a large family of secondary metabolites, which are involved in plant responses to biotic and abiotic stresses. The most present polyphenols in plants are the flavonoids, the cinnamic and benzoic acids and the stilbenes. They derive from the phenylpropanoid metabolism, but flavonoids are ubiquitous in plants, whereas stilbenes are specific to certain plant families. Other polyphenols, as the phytoalexins, are antimicrobial compounds synthesized in response to pathogen or herbivore attack. However, other roles have been described for stress-induced polyphenols, including the defense signaling of responses and protection against ultraviolet (UV) light damage (Chong et al., 2009; Jandet et al., 2002) Phenolic compounds are secondary metabolites that are synthesized by grape (Chong et al., 2009) during normal development in response to stress conditions. Although structurally diverse, phenolics are classified into two groups: the flavonoids and the non-flavonoids. The flavonoid family includes the flavonols, such as myricetin, quercetin and kaempferol, which exist both as aglycons and sugar conjugates; the flavan-3-ols (such as (+)-catechin and (Ŕ)- epicatechin) and the anthocyanins, such as malvidin-3-glucoside. The non-flavonoids encompass hydroxybenzoic acids such as gallic acid; hydroxycinnamic acids, including p- coumaric, caffeic and caftaric acids and the stilbenes, such as trans-resveratrol and cisresveratrol (Jandet et al., 2002). The synthesis of stilbenes in grape berry tissues is activated in response to fungal attack, to berry injury and also to ultraviolet irradiation (Jandet et al., 2002). The anthocyanins and the proanthocyanidins are among the most important compounds for the quality of the red wine, since they are responsible for major characteristics of this beverage, i.e., color, bitterness, astringency and chemical stability towards oxidation. Red wine represents a concentrated source of anthocyanidins, catechins, and proanthocyanidins and other phenolics (Mattivi et al., 2002). The anthocyanins, the resveratrol and the flavonols are especially contained in the berry skins. During winemaking, only a fraction of the grape flavonoids is selectively transferred to the wine and a final yield strongly depending on the management of the contact of the liquid must, containing berry skin and seeds, with the solid parts of the grape bunches and on the grape variety. The data concerning the extractable phenolics of the grape cannot be simply generalized to predict the wine composition, since a high variability in the extraction yield from grape to wine is introduced by the technological factors governing the winemaking process (such as temperature, duration and intensity of the liquid-solid contact, final ethanol concentration). Moreover, many other factors (chemical microbiological and physical) have been

3 Red Wine: A Functional Beverage? 133 demonstrated to further modify the phenolics structure and concentration during the fermentation, fining and storage of wine. The various polyphenol families present in wine (Paixao et al., 2008; Arnous, et al., 2001) are important for a number of technological properties of wine such as clarity, hue, and palatal taste (Paixao et al., 2008). In particular, tannins confer astringency and structure to the beverage by the formation of complexes with the proteins of saliva. The knowledge about their qualitative and quantitative profile in grapes is very important to predict wine aging attitude and can help to solve problems related to color stability, especially in the case of red wines that are destined to long aging periods (Mattivi et al., 2002). The wine aging also changes the phenolic composition, as these compounds can suffer diverse transformations, like oxidation processes, condensation and polymerization reactions and extraction from wood, usually associated to the changes in color and colloidal stability (Saucier et al., 1997), flavor, bitterness and astringency (Sánchez-Iglesias et al., 2009). The polyphenolic fingerprint can be useful for the classification of wines, since it can give us information about the variety, the geographic and winery origin and even the applied winemaking technology (Spranger et al., 2004) There is emerging evidence that a functional diet can help in modulating the immune system responses to the inflammation processes through a variety of mechanisms based on the absorption and utilization by the human metabolism of specific compounds. Polyphenols are the principal compounds related to the wine consumption benefits due to their antioxidant and free radical scavenging properties. These physiological effects are especially associated to flavonoids and stilbenes (Paixao, et al., 2008), namely quercetin, (+)- catechin, gallic acid and trans-resveratrol (Soleas et al., 2001). Flavonoids have shown the inhibition of lower density lipoprotein (LDL) oxidation by macrophages and cupric ions, as increasing evidences shows that oxidized LDL and very LDL (VLDL) may be involved in the pathogenesis of atherosclerosis (Carluccio et al., 2007). Flavanols, such as (Ŕ)-epicatechin have many beneficial health effects such as anti-tumorgenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties and has attracted interest because their concentrations in red wine are higher than other flavonoids (Tarola and Giannetti, 2007). The stilbene trans-resveratrol has gained great attention and a number of scientific papers have appeared related to the moderate consumption of red wine the ability to inhibit platelet aggregation and LDL oxidation and its beneficial effects in health. Since trans-resveratrol is postulated to be involved in the health benefits associated with a moderate consumption of red wine, it is one of the most extensively studied natural products. Indeed, hundreds of studies have reported the beneficial effects of trans-resveratrol on neurological (Anekonda 2006) and cardiovascular systems (Lippi et al., 2010a-2010b). Soleas and coworkers had related that trans-resveratrol may be the most effective anticancer polyphenol present in red wine (Soleas et al. 2001). One of the most striking biological activities of trans-resveratrol is its anticancer activity that prevents carcinogenesis in the three stages of tumor development (Cai et al., 2011). Further data provide interesting insights into the effect of this compound on the life span of different organisms, suggesting that trans-resveratrol might be regarded as a potential antiaging agent in treating age-related human diseases (Pearson et al., 2008). In addition, the effects described in mice subjected to a high-calorie diet (Kundu et al., 2006) open up new approaches for treating not only age-related diseases but also obesity-related disorders (Subbaramaiah et al., 1998).

4 134 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. In this chapter, we attempt to summarize the recent findings concerning the effects of wine polyphenols on human health and propose future directions for research to increase the amount of these healthy compounds in wine. TECHNOLOGICAL DETERMINANTS OF ANTIOXIDANT CONTENTS AND ACTIVITY IN GRAPES AND WINES The grape cultivar plays an essential role in establishing the absolute amount of the flavonoids, thus determining the final content of polyphenols in red wines. Indeed, it has been proposed that the knowledge of the qualitative and quantitative analysis of polyphenolos in the berry allows the prediction of the final polyphenol content in the derived wine (Mattivi et al., 2002; Vinci et al., 2008). Mattivi et al. (2002) have studied the Ŗphenolic potentialŗ of twenty-five red grape cultivars (Vitis vinifera L.), including 21 Italian and 4 international red grape varieties, by analyzing the amount and the localization of the extractable flavonoids in the grape. The obtained results showed that the grape cultivar has an essential part in determining the absolute amount of the flavonoids, and the distribution between the berry skin and seeds of the flavan-3-ols. However, the terroir also detains a strong influence on the concentration of these compounds in the individual wines. Significant evidence about the influence of the terroir on the amount of polyphenols in red wines has been supplied by Kumšta et al. (2012), who analyzed grape growth under the same geographical region during four vintages. The Authors have studied the relationship between the properties of 43 wines of the cv. Riesling from six wine-growing sub-regions and their specific ecosystem, showing that the phenolic composition of grapes and wines is likely to be related to the terroir. A clearcut association was detected in wines between wine-growing area and trans-resveratrol concentration, thus allowing the authors to demonstrate the capacity to distinguish different wine terroir by using the trans-resveratrol concentrations in the derived wine. Because of the large influence of phenolic compounds on red wine quality, many winemaking techniques have been developed to influence the extraction of these compounds during vinification and most have been directed at enhancing extraction. Different factors limit the extraction of different classes of phenolic compounds. The impact of winemaking processes on phenolic extraction is well-known and several winemaking steps and practices are known to influence the phenolic composition of red wines. Higher fermentation temperatures have been reported to increase phenolic extraction. A number of studies examined the effect of temperature on extraction of winemaking practices on phenolic compounds (Gao et al., 1997; Girard et al., 1997; 2001; Atanacković et al., 2012), showing that total phenolics content was greater at higher fermentation temperatures. In particular, Atanacković et al. (2012) have studied the influence of thermo-vinification on the resveratrol content, total phenolic content and antioxidant potential of red wines obtained from the cultivars Merlot, Cabernet Sauvignon, Pinot Noir and Prokupac. The obtained results indicated that thermo-vinificated wine samples resulted after thermo-vinification contained higher amount of phenolic compounds. Several investigations have analyzed the effects of the maceration time, SO 2, pectolytic enzymes and fining agents addition on the final content of polyphenols in the produced wines.

5 Red Wine: A Functional Beverage? 135 The influence of different maceration stage on phenolic composition of Aglianico, Montepulciano, Nero di Troia and Sangiovese wines has been recently studied by Gambacorta (2011). The authors compared three different maceration technologies, i.e., the traditional (5 days of maceration at 25 C with three daily punching-down), the prolonged maceration (10 days) and the cryomaceration (24 h at 5 C), concluding that the phenols extraction from grapes was found to be mostly dependent on the grape variety rather than on the applied maceration technology. Gambuti et al. (2007) have investigated the effect of the above parameters during vinification of grapes of the red the red cv. Aglianico. The authors concluded that the combination of the addition of SO 2 and pectolytic enzymes during the pre-fermentative phases enhanced the potential health protective effect of red wine, since these practices enhance the transfer of bioactive polyphenols from grape berries to the wine. A similar investigation has been recently carried out on the Vranec red grape cultivar (Ivanova et al., 2012; Kostadinović et al., 2012). The effects of maceration time and SO 2 and yeast on polyphenolic during vinification and wine aging were compared and the obtained results suggested that all parameters affected the content of total phenolics, anthocyanins, flavonoids and flavan-3-ols. Moreover, during aging, wines produced after 6 and 10 days of maceration showed no changes in their phenolic content, whereas a slight reduction of total phenolics was detected in wines obtained after 3 days of grape maceration. The clarification stage is performed during the vinification process in order to obtain limpid and bright wines by the eliminating substances in suspense as well as the wine-protein instability. However the introduction into the wine of different fining agents affects the quality of the final product. In fact, fining agents such as PVPP, gelatin, egg albumin and casein have demonstrated that they can decrease the concentration of a large number of phenolic compounds and can alter the color in treated (Castellari et al., 1998; Maury et al., 2001; Castillo-Sanchez et al., 2008). An interesting comparison on the effects of nine different winemaking procedures (traditional, delestage, saignée, delayed punching-down, addition of grape-seed tannins, addition of ellagic-skin-seed tannins addition, must-wine heating, cryomaceration, prolonged maceration) on the phenolic content and antioxidant activity of musts and wines derived from the economically relevant red grape cultivar ŖPrimitivoŗ has been documented by Baiano et al. (2009). A number of evidence was revealed by the authors: i) tannins addition raised the phenolic content of musts and wines in a greater amount when compared to the other technologies; ii) wine aging, independently on the winemaking procedure, determined an enhancement of the antioxidant activity: iii) wine obtained by traditional technology, saignée and tannins addition were denoted by the highest antioxidant activities; iv) wines produced through traditional winemaking had the highest content in anthocyanins. There has also been an increasing interest in understanding the potential of wine yeast and their derivate to influence the phenolic profile of red wines. Yeast lees has been demonstrated to adsorb anthocyanins (Morata et al. 2003) and to reduce the polyphenolic content (Mazauric and Salmon, 2005; Lesko et al., 2011) of wines. Polyphenols are adsorbed by the macromolecules released during yeast autolysis in a similar manner to protein-fining agents. A number of studies on the effect of different yeast strains used for vinification of Pinot noir (Girard et al., 2001; Lorenzini 2001), Cabernet franc and Merlot (Mazza et al., 1999),

6 136 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. Vranec wines (Ivanova et al., 2012) and Sangiovese (Zini et al., 2002) showed that the influence of the yeast starter on the phenolic content of produced wines was not significant. However, several other investigations produced positive evidences about the potential correlation between yeast starter cultures and total antioxidant capacity of wines. A study on the phenolic profiles of wines produced from Pinot noir cultivar indicated that the dissimilarity of their polyphenolic profile was due to the different yeast strain utilized (Dumont et al., 1993). Cuinier (1997) compared results obtained from Gamay grape must fermentations carried out using eighteen different yeast strains. The obtained wines showed differences up to 100 mg/l in anthocyanins and 10 absorbance units in total phenolics content. Caridi et al. (2004) described a correlation between the yeast strain employed in the fermentative process and the polyphenolic composition of wine, highlighting that strain properties are able to alter chromatic properties, phenolic profile and antioxidant activity of the final product. Similar results were obtained in a recent study using Vranec and Merlot wines form Macedonia, where the increase of trans-resveratrol concentration and antioxidant activity in the produced wines was influenced by the yeast strain used during fermentation (Kostadinović et al., 2012). Brandolini et al. (2007) carried out a large scale analysis testing the effect of 19 strains of Saccharomyces cerevisiae, isolated from different grapes and wines, on the total polyphenol content and the antioxidant capacity of obtained wines. The authors demonstrated that the tested parameters were significantly influenced by the yeast strain, and contributing positively to the final quality of the wine and to its functional properties. POLYPHENOLS BIOLOGICAL PROPERTIES The epidemiological data from France (French Paradox) strongly suggests a protective effect of red wine despite a high-fat diet. The conclusive studies showed that the dealcoholized red wine had cardiovascular protective effects in short-term studies on humans (Karatzi et al., 2005; Papamichael et al., 2006). Both flavonoids and non-flavonoid phenolic compounds have been implicated in the protective effects of wine on the cardiovascular system. These compounds were also described as potent antioxidants as they reduce lowdensity lipoprotein (LDL) cholesterol oxidation, modulate cell signaling pathways, reduce platelet aggregation, inhibit the growth of some tumor types, and exhibit anti-inflammatory, antibacterial, antifungal, antiviral, neuroprotective, anti-proliferative and anti-angiogenic activities. However, the beneficial effects of moderate wine consumption may be attributed to the overall mix of all of its components and not to a specific action of one. Oxidative stress results in direct or indirect reactive oxygen species ROS-mediated damage of nucleic acids, proteins, and lipids, and has been implicated in carcinogenesis, neurodegenerative disease, atherosclerosis, diabetes, and aging. As antioxidants, polyphenols may protect cell constituents against oxidative damage and, therefore, limit the risk of various degenerative diseases associated to oxidative stress. The antioxidant effect of polyphenols limits the damage by acting directly on ROS. Numerous studies in vitro as well as in animals and humans demonstrate beneficial effects of grape polyphenols on traditional cardiovascular risk factors as: hypertension, hypercholesterolemia, diabetes and smoking leading to atherosclerosis (Auger et al., 2005;

7 Red Wine: A Functional Beverage? 137 Bernatova et al., 2002; Peng et al., 2005; Pinent el al., 2004). Wine polyphenols can reduce the risk of heart disease by their antioxidant and anti-inflammatory activities, by decreasing the platelet aggregation and by improving the endothelial function and increasing fibrinolysis. It is well-known that the LDL oxidation is the key mechanism in atherosclerosis. Red wine intake exhibit positive effect on biomarkers of atherosclerosis including a decrease of LDL/HDL ratio, oxidized LDL levels, lipoprotein, clotting factors and fibrinogen levels (Avellone et al., 2006; Guilford and Pezzuto, 2011). Polyphenols may exert also antithrombotic effect by inhibition of patelet aggregation. A dysfunction of endothelial cells may alter the balance between the synthesis and interaction of proteins that promote clot formation and fibrinolytic proteins that facilitate fibrinolysis. Short-term ingestion of red wine improved enthothelial function in patients with coronary artery disease (whelan et al., 2004). This is could be explained by the promotion of endothelium nitric oxide production which have a vasorelaxation effect. This last effect can reduce also the development of hypertension. In recent years, a large number of studies have been devoted to illustrate the potential cancer chemopreventive activities of red wine polyphenols (Soleas et al., 1997; Yang et al., 2001; Femia et al., 2005). These latter have been shown to block carcinogenesis and to inhibit the growth of tumor in whole animals or in cell culture. Different mechanisms have been suggested to explain the anti-carcinogenic effects and are reviewed by Scalbert et al. (2005) and Rodriguo et al. (2011), Guilford and Pezzuto (2011). Nowadays, literature shows that moderate wine consumption and due to the presence of polyphenols may decrease the risk of several cancers: colon, ovarian, prostate, lung and basal cell carcinoma. The moderate wine consumption was associated with a decreased risk of developing esophageal adenocarcinoma (Kubo et al., 2009) and also lung cancer (Chao, 2007). Hudson et al. (2007) reported that grape polyphenols induced prostate tumor cell lines apoptosis. Oak et al. (2005) showed that red wine polyphenols inhibit angiogenesis by reducing the proliferation and migration of endothelial and vascular smooth muscle cells. Eng et al. (2002) found that red wine polyphenols have an inhibitory activity against aromatase (a cytochrome P450) involved in breast tumor growth. Wine polyphenols were shown to exhibit anti-inflammatory effect. This effect appears to be associated with the inhibition of nitric oxide production. A critical step in both inflammation and atherosclerosis is the adhesion of circulating monocytes to vascular endothelial cells, which involves vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (Hidalgo et al., 2012). Studies showed that polyphenols contribute to the attenuation of inflammatory diseases by decreasing ICAM-1 and VCAM-1 levels (Kwon et al., 2005; Lotito and Frei, 2006). The ani-inflammation effect of polyphenols has been mainly ascribed to immunomodulation and antioxidative action (Chacona et al., 2009). In fact, polyphenols modulate cytokine expression as basic pathway to anti-inflammation. Also, wine polyphenols can exhibit anti-inflammatory activities by their capacities to scavenge nitric oxide and to decrease tne nitric oxide synthase activity. Epidemiological and animal studies have shown that wine polyphenols can reduce the risk of developing of neurological disorders such as dementia, Parkinsonřs and Alzheimerřs diseases (Basli et al. 2012). Oxidative stress resulting in ROS generation is responsible of many forms of cellular deterioration leading to various chronic pathologies like neurodegenerative disorder. The antioxidant effect of polyphenols protects cell constituents against oxidative alteration and thus limits the risk of developing degenerative disorders. Also, wine polyphenols may regulate the nitric oxide activity at the level of endothelial nitric

8 138 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. oxide synthase (enos) protein expression in endothelial cells (Wallerath et al., 2003). A preventive approach to reduce tissue injury associated with the risk of cerebral ischemia is constituted by enos up-regulation by wine polyphenols. Type 2 diabetes is characterized by insulin resistance, hyperglycemia, overproduction of glucose by the liver and defection of β-cells in the pancreas. Long term effects of diabetic patients include an increased risk for cardiovascular disease, blindness, nerve and kidney damage, and limb amputations. Several studies have reported the anti-diabetic effect of grape and wine polyphenols (Marfella et al., 2006; Kar el al., 2009; Zumino, 2009). Wine polyphenols may affect glycemia through different mechanisms including: the inhibition of glucose adsorption in the gut or its uptake by peripheral tissues; the inhibition of β- glucosidase, α-amylase and sucrose in rats; the inhibition of gluconeogenesis, adregenic stimulation of glucose intake or the stimulation of insulin by pancreatic β-cells; the modulation of SIRT1 gene improving whole-body glucose homeostasis and insulin sensitivity in rats. Also, the antioxidant and the anti-inflammatory properties of wine polyphenols may be responsible for the positive response in type 2 diabetes. Phenolic acids: This group of phenols is defined non-coloured phenols abundant in white grape berries and responsible for the bitter and astringent properties of wine (Tian et al., 2009). In wine, there are two groups of phenolic acids; hydroxybenzoic acids and hydroxycinnamic acids (Cabrita et al., 2008). Hydroxybenzoic acids, including gallic acid, protocatechuic acid, gentisic acid, p-hydroxybenzoic acid, vanillic acid and syringic acid, derive from benzoic acid. Chlorogenic acid, as the main constituent of the hydroxycinnamic acid derivative group, increased with harvest time delay, and the same occurred with sinapic acid, while the converse was the true of caffeic acid and ferulic acid, which were also esterified with tartaric acid as the known compounds caftaric acid and fertaric acid, respectively (Gil-Muñoz et al., 1999; Cabrita et al., 2008). Hydroxycinnamic acids have gained an increasing interest in health because they are known to be potent antioxidants. These compounds have been described as chain-breaking antioxidants acting through radical scavenging activity that is related to their hydrogen or electron donating capacity and to the ability to delocalize/stabilize the resulting phenoxyl radical within their structure. The free radical scavenger ability of antioxidants can be predicted from standard one-electron potentials. Phenolic acids represent important fraction of wine phenolics, but their biological effects have been scarcely investigated. The interrelationship between antioxidative capacity and vasodilatory activity, two potentially beneficial biological effects, of phenolic acids from wine were examined. Antioxidative and vasodilatory effects of phenolic acids relate to the number of hydroxyl groups in the phenyl ring, degree of compactness and branching of molecules, and three-dimensional distributions of atomic polarisability of the tested molecules (Teixeira et al., 2013). Caffeic acid has been shown to have neuroprotective effects against injury induced by 5-S-cysteinyl-dopamine, against Aβ-induced neurotoxicity and by inhibiting peroxynitrite-induced neuronal injury (Sul et al., 2009; Vauzour et al., 2010). Ferulic acid has been cited as an anti-diabetic effect by lowering blood glucose and by increasing plasma insulin (Jung et al., 2007) Anthocyanins: Anthocyanins are plant pigments belonging to a subset of flavonoids with a particularly high antioxidant capacity and concomitantly strong health-promoting effects

9 Red Wine: A Functional Beverage? 139 (Bártíková et al., 2013; Yoo et al., 2010). Anthocyanins have been credited with capacity to modulate cognitive and motor function, to enhance memory, and to have a role in preventing age-related declines in neural function (Youdim et al., 2002). As part of the human diet, they offer protection against cancer, inhibiting the initiation and progression stages of tumor development (Martin et al., 2013). They also reduce inflammatory inducers of tumor initiation, suppress angiogenesis, and minimize cancerinduced DNA damage in animal disease models. Anthocyanins also protect against cardiovascular diseases and age-related degenerative diseases associated with metabolic syndrome (Aggarwal and Shishodia, 2004; Renaud and de Lorgeril, 1992). DellřAgli et al. (2005) have shown that wine anthocyanins inhibit the activity of phosphodiesterase-5 reducing the risk of cardiovascular disease by vasorelaxation. Grape juice and wine anthocyanins in synergy with other flavonoids have been cited as responsible for antiplatelet activity in human and dog systems (Shanmuganayam et al., 2002). Anthocyanins have also neuroprotective benefits by reducing age-associated oxidative stress and by improving cognitive brain function (Tarozzi et al., 2007). Flavonols: Dietary flavonols inhibit LDL oxidation and so reduce the primary risk factor for atherosclerosis and related diseases. Long-term dietary administration of flavonols (such as quercetin) offers cardio-protection and improves the levels of CVD risk factors in animals (Vina et al., 2007), and the animal studies are supported by human epidemiological studies, which show inverse correlations between the occurrence of CVD, certain cancers, and agerelated degenerative diseases and the consumption of flavonol rich diets (Mink et al., 2007; Seeram et al., 2004; Soobrattee et al., 2006). Flavonols have been linked to protective effects against several specific cancers, including leukemia and pancreatic, breast, cervical, prostate, uterine, and urinary tract cancers. Subjects with regular flavonol intake have a 10Ŕ60% lower incidence of these types of cancer compared with subjects with low flavonol intake. This protective activity results from both the action of flavonols as stimulators of antioxidant defenses and their direct inhibitory effects on cellular proliferation. Quercetin consumption has been reported to be inversely associated with breast cancer incidence (Fink et al., 2007). Resveratrol: Resveratrol is a stilbene phytoalexin produced by specific plant species in response to biotic and abiotic challenges. It is thought to be one of the principal agents in the health-promoting effects of red wine (Baur and Sinclair, 2006). Resveratrol is a stilbene phenolic compound, it being in the same family of molecules that includes the cis or trans isomers of glucosides (piceid) and polymers (viniferins). Resveratrol and piceid are present mainly in grape and grape products, but also in lower amounts in peanuts, pistachios, some berries, and chocolate. Results of clinical studies show that the most important source of resveratrol and piceid are wines (98.4%), grape and grape juices (1.6%), whereas peanuts, pistachios, and berries contribute to less than 0.01% (Zamora-Ros et al., 2008). Wine is the major source of resveratrol and piceid to the diet, ranging from 95 and 98.7% for transresveratrol and trans-piceid to 99.9% and 99.7% for cis-resveratrol, cis-piceid, respectively. Other foods items such as grapes contribute by amount of 3.8% of total trans-resveratrol, whereas other contributors such as pistachios or berries provide less than 1% of the dietary total amount of trans-resveratrol and trans-piceid.

10 140 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. Resveratrol, possess numerous important bioactivities, including anti-inflammatory, antioxidant, anti-aggregatory functions, and modulation of lipoprotein metabolism (Gerard and Mullin, 2011; DřIntrono et al., 2009; Frei, 2004). It has also been shown to detain chemopreventive properties against certain forms of cancer, and cardiovascular disorders and to have positive effects on longevity (Kundu and, Surh, 2008; Vergara et al., 2012). Aside from cardiovascular disease, resveratrol has been reported to potentially benefit a number of conditions, including cancer (Kaminski et al., 2011). Anticancer activity of this compound is mainly due to induction of apoptosis via several pathways, as well as alteration of gene expressions, all leading to a decrease in tumor initiation, promotion, and progression. (Udenigwe et al., 2008) Resveratrol blocks the growth of lymphoma cells and also increases their rate of cell death (Bruno et al., 2003). Resveratrol sensitizes chemotherapy-resistant lymphoma cells to treatment with paclitaxel-based chemotherapy. (Jazirehi et al., 2004) Resveratrol also reduces the production of growth factors, such as vascular endothelial growth factor and interleukin- (Baur et al., 2006), and may reduce the ability of lymphoma cells to spread to other organs (Dulak et al., 2005) Resveratrol has been studied for its effect on cell cycle signaling for colon cancer. (Fonar et al., 2011; Nguyen et al., 2009) Finally, was demonstrated that in vitro administration of resveratrol favorably altered gene expression in the androgen axis and in cell cycle regulators, providing potential anticancer benefit for prostate cancer (Jones et al., 2005). Moreover, trans-resveratrol appears to protect against diabetes (Sharma et al., 2007) and neurodegenerative disorders (Anekonda, 2006), due to induction of Sirtuin-1 genes (Wood et al., 2004). Trans-resveratrol might also contribute to increasing the lifespan of metazoans and mice by miming the effect of caloric restriction and thus decreasing agerelated signs (Park et al., 2012; Pearson et al., 2008). Experimental studies have shown that resveratrol exhibits both an anti-inflammatory and cardio-protective potential by inhibiting the expression of inflammatory mediators and the monocyte adhesion to vascular endothelial cells (Carluccio et al., 2007; Csiszar et al., 2006). Although resveratrol exhibits potent anticancer activities against transformed cells, its effectiveness is limited by its poor bioavailability and as a dietary phytonutrients it is most effective against tumors with which it comes in direct contact, such as skin cancers and tumors of the gastrointestinal tract. Furthermore inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein de-acetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein d-eacetylation. (Pietrocola et al., 2012) Flavanols: Flavanols are present either as monomers, (-)-epicatechin, (+) catechin and gallocatechin gallate and as oligomers and polymers also called condensed tannins or proanthocyanidins. Catechin and proanthocyanidins have proved to be potent antioxidants in different in vitro models, and in some in vivo intervention studies. However their potential beneficial effect on cardiovascular health is not merely due to this property but includes the different mechanisms implicated on cardiovascular conditions or problems, i.e., hypertension, inflammation, cellular proliferation, trombogenesis, hyperglycemia and hypercholesterolemia. Catechin and proanthocyanidins have been shown to have free radical scavenging and antioxidant activities. It seems that the proanthocyanidin dimer have the most antioxidant

11 Red Wine: A Functional Beverage? 141 effect. These effects confer to the flavanols a cardioprotection action by limiting the oxidative stress factors. In breast cancer cell lines, epicatechin inhibits metastatic cell, hepatocyte growth factor signaling and cell motility; causes cell arrest in S phase; modulates NO signaling; induces killer caspases; and inhibits NF-κB signaling (Butt and Sultan 2009). Grape seed proanthocyanidins exert their anti-cancer effects through the inhibition of the constitutive expression of various NF-κB responsive genes/ proteins such as cyclooxygenase-2, inducible nitric oxide synthase, proliferating cell nuclear antigen and MMP-9 in human epidermoid carcinoma A431 cells. Furthermore, treatment of human epidermal keratinocytes with grape seed proanthocyanidins inhibits UV-induced oxidative stress-mediated activation of ERK1/2, JNK and p38 of MAPK family (Nandakumar et al., 2008). Also, grape seeds proanthocyanidins have anit-inflammatory effects by modulating the adipokine and cytokine gene expression. Wine catechins have strong antimicrobial activity against Porphyromonas gingivalis and Prevotella intermedia. POLYPHENOL MECHANISMS OF ACTION AGAINST CHRONIC DISEASES Reactive oxygen and nitrogen species are considered major determinants of many degenerative pathologies, including CVD, certain cancers, neurodegenerative diseases, chronic inflammation and tissue aging (Closa and Folch-Puy, 2004; Valko et al., 2006). Essentially, these molecules can act by damaging biological structures and molecules, but can also influence cell signaling either indirectly (by changing the redox cellular status) or directly (by participating in intracellular signaling) (Virgili and Marino, 2008). Although the antioxidant properties of phytonutrients have been a major focus of research, there is an emerging view that they may act not only by scavenging reactive oxygen and nitrogen species or suppressing their production but also by enhancing the endogenous antioxidant capacity of cells/tissues (e.g., glutathione synthesis) or by influencing signaling pathways through interaction with proteins, enzymes, and nuclear receptors (Virgili and Marino, 2008). Red wine contains varying levels of resveratrol, flavonols, anthocyanins, and catechins, which are all effective antioxidants. However, the cardio-protection afforded by these polyphenols is likely the result of multiple biological activities independent of their antioxidant activities, including improvement of endothelial function, reduction in LDL uptake, oxidation and aggregation, reduction of blood pressure and inhibition of platelet aggregation (Bose et al., 2008), Interestingly, dietary polyphenols have been suggested to prevent LDL oxidation by direct binding to specific sites in LDLs (Wang and Goodman, 1999). The chemopreventive activity of phytonutrients has been suggested to be associated with the ability to block the progression of latent tumors through different mechanisms (Beliveau and Gingras 2007). Polyphenols can modulate the action of these enzymes that detoxify carcinogens or proteins that export them from cells (Conney, 2003), such as the plateletderived growth factor receptor (for example, several anthocyanin) (Martin et al., 2011). In particular, the resveratrol promotes apoptosis and cell death of tumor cells (Jung et al., 2006)

12 142 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. vascularizes the microtumors by blocking the metalloproteases and reduces inflammation by inhibiting cyclooxygenase 2 (DřIntrono et al., 2009; Aggarwal and Shishodia, 2004). Polyphenols have been shown to modulate energy metabolism and consequently reduce insulin resistance, type 2 diabetes and metabolic syndrome (Hanhineva et al., 2011). AMPactivated protein kinase (AMPK) is a conserved sensor of cellular energy (Khan et al., 2005). Low energy stores determined by caloric restriction increase the AMP/ATP ratio and activate AMPK activity, which in turn activate sirtuin 1 (SIRT1), a class III histone deacetylase (HDAC), and influence glucose/lipid metabolism and age-related diseases (Wood et al., 2004). Therefore, activation of AMPK has been proposed as a strategy for treating metabolic syndrome and delaying aging (Curtis et al., 2005). Recent studies have demonstrated that many dietary polyphenols can act as mimics of caloric restriction and activate AMPK, thus suppressing hepatic gluconeogenesis and inducing hepatic fatty-acid β-oxidation. Wine polyphenols can stimulate the glucose transporters in muscle and adipose tissues, with an overall reduction of the glucose level in the blood and the lipid content of the liver as well as an improvement in insulin sensitivity (Hanhineva et al., 2011). Resveratrol, quercetin and anthocyanins also extend life span in different species (Baur and Sinclair, 2006) and, in particular, resveratrol directly inhibits camp-degrading phosphodiesterases, leading to an increase in camp and subsequent activation of AMPK (Park et al., 2012). POLYPHENOL BIOAVAILABILITY AND DELIVERY The biological effects of grape polyphenols in human and experimental models demonstrate antioxidant properties closely associated with the maintenance of endothelial function, increase in antioxidant capacity, protection against LDL oxidation and neuroprotective effects. Recent patents regarding grape polyphenols show a tendency to return to natural products with a minimum use of severe extraction processes and organic solvents. Moreover, the recent patents regarding human health show more pharmaceutical use of grape juice and other polyphenol-rich products. The application of such products in clinical trials as a substitute or co-adjuvant with drugs may be useful in future research. (Gollücke and Ribeiro, 2012) Several investigations have proposed that untargeted metabolomics serve as a powerful approach for providing new clues about metabolism, bioavailability, and excretion of wine polyphenols-intake biomarkers. To our knowledge, there are a limited number of studies applying metabolomics to wine intake. In this context, Grun et al. (2008) have identified ten metabolites that were significantly increased after consumption for four weeks, of 800 mg of red per week, wine and juice of grape polyphenols. Indeed, metabolomic approaches represent an interesting tool for unraveling the complex relations between the consumption of polyphenols and human health. Bioavailability is a challenging subject to study, because the various biotic factors that may affect it. In particular, little information are available on the effect of the food matrix on the bioavailability of polyphenols, which may affect their absorption (Ortega et al., 2009; Perez-Jimenez et al., 2009). One of the main factors influencing polyphenols bioavailability

13 Red Wine: A Functional Beverage? 143 can be found in their chemical structure. In foods, most polyphenols exist as polymers or in glycosylated forms and aglycon. In these native forms, polyphenols cannot be absorbed and have to be hydrolyzed by the intestinal enzymes or by the colonic micro flora before absorption. Anthocyanins represent an exception, because the intact glycosides can be absorbed and detected in the circulation (Nurmi et al., 2009). The specific chemical structures of polyphenols, as well as the type of the sugar in the glycoside, determine their rate and extent of intestinal absorption. Following the ingestion of polyphenols, the absorption of part of them occurs in the small intestine, where the glycosides are hydrolyzed. The fraction of polyphenols that is not absorbed in the small intestine reaches the colon, where it undergoes to substantial structural modifications. In fact, the colonic micro flora hydrolyzes glycosides into aglycones and degrades them to simple phenolic acids. This microbial activity is of great importance for the production of specific active metabolites. It is important to underline that there is great inter-individual variability in producing these active metabolites (Lu and Anderson, 1998; Morton et al., 2002). Once absorbed, and prior to the passage into the blood stream, polyphenols undergo other structural modifications due to conjugation processes, that mainly include methylation, sulfation and glucuronidation. These modifications could affect the bioavailability of polyphenols and, consequently, their biological activity. The conjugation mechanisms are highly efficient and free aglycones are generally either absent or present in low concentrations in plasma after consumption of nutritional doses. The therapeutic potential of resveratrol can only be adopted in vivo only if the limitations related to its bioavailability can be overcome. Research programs are currently exploring other means of enhancing resveratrol bioavailability, including: i) co-administration with metabolism inhibitors in order to prolong its presence in vivo, ii) use of resveratrol analogous endowed with a better bioavailability, iii) drug delivery system employing nanotechnology (Johnson et al., 2011; Goldberg et al., 2003). As far as concerns the first approach, some authors have evaluated the possibility of enhancing the pharmacokinetic parameters of resveratrol by partially inhibiting its glucuronidation by means of co-administration with specific inhibitors (Hoshino et al., 2010). The second strategy consists in the evaluation of new naturally-occurring and/or synthetic analogous of resveratrol endowed with the same structural backbone and some chemical modifications resulting in better efficacy (Cai et al., 2011). In this context, the biological activity exerted by resveratrol metabolites is still debated. Therefore, further studies are needed, including the possibility to induce deglycosylation to release resveratrol into the target tissues (Hassan-Khabbar et al., 2008). However, conventional formulations alone are probably inadequate to overcome the physicochemical and pharmacokinetic limitations of resveratrol bioavailability. To solve this problem, a number of novel drug delivery systems have been proposed to protect and stabilize resveratrol and to enhance its bioavailability: nano- and micro-formulations for resveratrol encapsulation that include liposomes, polymeric nanoparticles, solid lipid nanoparticles, lipospheres, cyclodextrins, polymeric microspheres, yeast cell carriers and calcium or zinc pectinate beads (Amri et al., 2012). At the present time, the research in this field is mainly focused on exploring multiparticulate forms from the millimeter to micrometer range and colloidal carriers in the nanometer range in order to improve the oral intake as these offer the possibility of easy chronic administration. For all current formulation attempts, the main issue is to determine whether the drug delivery system

14 144 Giovanna Giovinazzo, Ilaria Ingrosso, Mariana Tristezza et al. is efficient in improving the pharmacokinetic profile of resveratrol and promoting the in vivo therapeutic effects. Perspectives now lie in the development of innovative formulations able to overcome all the limitations governing resveratrol bioavailability as a pre-requisite to securing efficient and sustained delivery in vivo (Amri et al., 2012). CONCLUSION The precise nature of the role played by polyphenols in human health has been largely highlighted in these last years. The grape is one of the principal sources for the human intake of healthy polyphenols and their concentration in the berry is mainly cultivar-dependent. A better knowledge concerning the composition and dynamics of polyphenols profile in red grape will help vine-dresser and wine makers in producing grape-derived products and wines with high content of phenolic antioxidants and considerable antioxidant activity, maintaining optimal organoleptic properties and a significant link with the original terroir. Even though, a better understanding is still request about the several different cellular mechanisms and complex pathways involved in wine polyphenols metabolism, the present findings suggest that the contribute of antioxidant phenols through a reasonable daily drinking of red wines may offer additional protection against in vivo oxidation and other damages of human cell components. ACKNOWLEDGMENT This chapter was supported by a grant from the Regione Puglia Project PS_008 - INNOWINE- "Biotecnologie innovative per il miglioramento della qualità e sicurezza dei vini tipici pugliesi". REFERENCES Aggarwal, B. B. & Shishodia, S. (2004). Suppression of the nuclear factor-κb activation pathway by spice derived phytochemicals, reasoning for seasoning. Ann. NY. Acad. Sci., 1030, 434Ŕ441. Amri, A., Chaumeil, J. C., Sfar, S. & Charrueau, C. (2012). Administration of resveratrol, What formulation solutions to bioavailability limitations? J. Control Rel., 158, 182Ŕ193. Anekonda, T. S. (2006). Resveratrol a boon for treating Alzheimerřs disease? Brain Res. Rev., 52, Arnous, A., Makris, D. P. & Kefalas, P. (2001). Effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. J. Agr. Food Chem., 49, 5736 Ŕ Atanacković, M., Petrović, A., Jović, S., Gojković- Bukarica, L., Bursać, M. & Cvejić, J. (2012). Influence of winemaking techniques on the resveratrol content, total phenolic content and antioxidant potential of red wines. Food Chem., 131, 513Ŕ518.

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