Cardiovascular Research 63 (2004) 593 602 www.elsevier.

com/locate/cardiores

Review

Vascular effects of wine polyphenols

` , Enrica Bosisio Mario DellAgli *, Alessandra Busciala
Faculty of Pharmacy, Department of Pharmacological Sciences, Via Balzaretti, 9-20133 Milan, Italy Received 30 January 2004; received in revised form 4 March 2004; accepted 23 March 2004 Available online 6 May 2004 Time for primary review 20 days

Abstract Moderate consumption of red wine has been putatively associated with lowering the risk of developing coronary heart disease. This beneficial effect is mainly attributed to the occurrence of polyphenol compounds such as anthocyanosides (ACs), catechins, proanthocyanidins (PAs), stilbenes and other phenolics in red wine. This review focuses on the vascular effects of red wine polyphenols (RWPs), with emphasis on anthocyanosides and proanthocyanidins. From in vitro studies, the effect of red wine polyphenols on the vascular tone is thought to be due to short- and long-term mechanisms. NO-mediated vasorelaxation represents the short-term response to wine polyphenols, which exert the effect by increasing the influx of extracellular Ca2 +, and the mobilization of intracellular Ca2 + in endothelial cells. Polyphenolic compounds may also have long-term properties, as they increase endothelial NO synthase expression acting on the promoter activity. In addition, they decrease the expression of adhesion molecules and growth factors, involved in migration and proliferation of vascular smooth muscle cells. Moreover, they inhibit platelet aggregation. However, a paucity of data as regards the bioavailability and metabolism of these compounds in human studies is a limiting factor to proving their efficacy in vivo. D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Red wine polyphenols; Atherosclerosis; Endothelial function; Signal transduction; Vasodilation

1. Introduction For many numbers of years, considerable attention has been directed towards human behavioural habits that could either be considered risk factors or even protective elements for developing chronic pathologies. In particular, much effort has been devoted to elucidate the role of diet in preventing cardiovascular diseases. A so-called Mediterranean diet is thought to prevent cardiovascular diseases [1], as a consequence of its high content of antioxidants, which are crucial in ameliorating oxidative events implicated in many diseases. Similarly, moderate consumption of red wine has been associated with a lowering of the risk of coronary heart disease [2 4]. Red wine represents a rich source of polyphenols such as anthocyanosides (ACs), catechins, proanthocyanidins (PAs), stilbenes and other phenolics. Anthocyanosides (ACs) are flavonoids widely distributed in fruits and vegetables. They provide colour to red wines and to skins of red and black grapes [5]. Proanthocyanidins are another class of plant phenol metabolites widely occurring in fruits
* Corresponding author. Tel.: +39-02-50318345; fax: +39-02-50318391. E-mail address: mario.dellagli@unimi.it (M. DellAgli).

including grapes [6]. ACs and PAs are among the most important compounds in determining the quality of the red wine, because they greatly influence colour, bitterness, astringency, and chemical stability toward oxidation [7]. In addition to antioxidant/antiradical activity, red wine polyphenols (RWPs) were shown to possess many biological properties including the inhibition of platelet aggregation, vasorelaxing activity, modulation of lipid metabolism, and inhibition of low-density lipoprotein oxidation [2,3,8 11]. Thus, the health benefits of moderate consumption of red wine are founded on a multiplicity of actions. The biological properties of red wine stilbenes (trans-resveratrol) have been extensively reviewed [12 14]. Because ACs and PAs exert cardioprotective effects which are not only due to antioxidant/ antiradical activity, the present review will consider the effects of ACs and PAs at vascular level with special emphasis on the vasorelaxing and the antiaggregating properties. 2. Proanthocyanidins and anthocyanosides in red grapes (Vitis vinifera) and wines PAs (or so-called condensed tannins) are high-molecular weight polymers, made of flavan-3-ol units (Fig. 1) linked

0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cardiores.2004.03.019

594

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

Fig. 1. Basic structure of proanthocyanidins: R1, R2 = H, propelargonidins; R1 = H, R2 = OH, procyanidins; R1, R2 = OH, prodelphinidins.

together by carbon carbon bonds. Oxidative condensation occurs between carbon C4 of the heterocycle and C6 or C8 of the adjacent units. The flavan-3-ol unit may have a 3V ,4V dihydroxy substitution on ring B (catechin and epicatechin) and 3V ,4V ,5V trihydroxy substitution (gallocatechin and epigallocatechin). The term procyanidins indicates the polymers of catechin and epicatechin, the term prodelphinidins indicates the polymers made by gallocatechin and epigallocatechin. Dimers, trimers (among which procyanidin C1 is the most abundant), tetramers and oligomers up to 8 units are present in grape (V. vinifera L.) seeds, stems, and skins [6]. The procyanidin dimers B1 B4, characterized by the 4 ! 8 linkage, are the most common in grapes (Fig. 2). The corresponding 4 ! 6 linked isomers B5 B8, may also occur (Fig. 3). Until recently, complete characterization of PAs in grapes and wines has been impaired because of the difficulty of analysing high-molecular weight compounds. However, newly developed methods based on HPLC coupled with mass spectrometry has allowed the characteriza-

Fig. 3. Structure of procyanidins B5 B8.

tion of complex mixture of grape seed extracts and in beverages [8,15 17]. ACs are water-soluble plant pigments, localized in the skin of the berries of red grape cultivars. The term ACs (or anthocyanins) refers to the glycosides of anthocyanidins (free aglycons). ACs may be acylated on the sugar moiety with aromatic and aliphatic acids. The 3-O-h-glucosides of cyanidin, peonidin, delphinidin, petunidin and malvidin are the most abundant in V. vinifera and red wines (Fig. 4).

Fig. 2. Structure of procyanidins B1 B4.

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

595

3. Red wine polyphenols as protective agents against atherogenesis Dysfunction of the vascular endothelial cells, and proliferation and migration of smooth muscle cells are among the factors contributing to atherosclerosis. Vascular endothelium synthesises and releases nitric oxide (NO), which in turns, promotes vasorelaxation (endothelium-dependent), reduces platelet aggregation, and limits the flux of atherogenic plasma proteins into the artery wall (Fig. 5). The vasorelaxant effect of NO occurs through the activation of guanyl cyclase leading to the accumulation of cGMP [22]. The interaction between wine polyphenols and cell metabolism in the vascular tissue has been extensively investigated. These studies aimed to support for the beneficial effects of wine consumption in preventing cardiovascular diseases. In several animal models, the administration of red wine, grape juice, dealcoholized red wine and PAs extract from grape seeds attenuated the development of atherosclerotic lesions [23 26]. In a pig model, the consumption of RWPs induced a partial reduction of thickness of intimal proliferation even if not statistically significant [27]. Several processes of vascular reactivity seem to be influenced by wine ingredients. Red wine supplementation modulated haemostatic function and prevented thrombosis in experimental animals [10,28,29]. Wine polyphenols were shown to modulate blood pressure, promote vasodilatation, inhibit smooth muscle cell migration and proliferation, and inhibit platelet aggregation. 3.1. Red wine polyphenols and vasorelaxation Red wines and grapes exhibit endothelium-dependent relaxation of blood vessels via enhanced generation and/or increased biological activity of NO, leading to the elevation of cGMP levels [30,31,35,36]. In vivo RWPs were shown to reduce blood pressure in normo and hypertensive rats [33,34,37]. The administration of purple grape juice improved the endothelium dependent, flow-mediated vasodilation in coronary artery disease patients with impaired endothelial function [38]. The amplitude of vasorelaxation changed as a function of the variability of wine constituents according to grape varieties, area of cultivation, and vinification methods. Consequently, the vasodilatatory effect does not apply to all wines and the degree of vasorelaxation is correlated to the content and type of phenols. Endothelium-dependent relaxation was greatest for red wines produced en barrique, a procedure leading to high concentration of phenolic compounds [32]. A correlation between the phenolic content with vasodilatatory effect was later confirmed by Burns et al. [21]. Sixteen red wines were selected to provide a range of origins, grape varieties, and vinification methods. The ability of the wines to act both as vasodilators ex vivo, and as antioxidants in vitro was strongly correlated with the phenolic content of wines. In addition, while the antioxidant

Fig. 4. Structure of the ACs of red wines.

The six-hydroxyl of the glucose may be acylated with acetyl, coumaroyl and caffeoyl groups. During storage and aging of red wines, ACs react with PAs to produce more complex pigments which may partially precipitate [8]. The AC content of red wines may be estimated using a pH reau-Gayon and Stoneshift method adapted from Ribe street [18] where, at pH < 1, ACs are easily detected in their red flavylium form. ACs are then quantified spectrophotometrically as malvidin-3-glucoside equivalents, using the extinction coefficient e = 28,000. Alternatively, ACs are separated from the other polyphenols and directly quantified by their maximal absorbance in the visible range (536 542 nm) [7]. Very recently, a capillary zone electrophoresis method for the quantitative determination of ACs in wine has been validated as alternative to HPLC [19]. Because of their unstability, free aglycons of ACs are never found in grape or wine, except in trace quantities. The levels of ACs and PAs are highly variable due to differences in fruit source (cultivars of V. vinifera) and wine processing [20]. According to this author, a representative content of ACs is 400 mg/l in a red wine less than 6 months of age, not having fermented in oak barrels, and 90 mg/l in a red wine aged for about 2 years or fermented in oak barrels. White wines contain only trace amounts. Red wine contains greater amounts of PAs (750 and 1000 mg/l, young and aged respectively) vs. white wines (20 25 mg/l). The content of extractable PAs (1600 4300 mg/kg of grape) and ACs (300 1900 mg/kg of grape) in different cultivars grown in Italy was reported by Mattivi et al. [7]. Burns et al. [21] reported the total phenol content in 16 red wines selected to provide a range of origins, grape varieties and vinification methods. Total AC levels, measured using a colorimetric assay ranged from 101 to 325 AM (expressed as malvidin 3glucoside equivalents).

596

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

Fig. 5. Protective effects of red wine polyphenols on the arterial wall.

activity was associated with different classes of phenols (gallic acid, resveratrol and catechins), vasodilatation activity was correlated only with the total content of ACs [21]. Investigations devoted to characterize the RWPs responsible for the endothelium-dependent relaxation activity [35,39 42] agree that monomeric catechins and simple phenols (benzoic acid, gallic acid and hydroxycinnamic acids) are devoid of effect. On the contrary, AC enriched fractions and oligomeric PAs (dimers, trimers and tetramers) were the active compounds. Threshold for relaxation by PAs oligomers was between 0.5 and 4 Ag/ml [35,42]. Much higher concentration (>0.1 mg/l) were required for ACs [41]. The endothelium-dependent relaxation activity was lost when higher molecular weight polymers were assayed [41]. RWPs enhanced NO synthesis and cGMP accumulation only in the presence of functional endothelium. In denuded aortic rings, RWPs concentration 103-fold higher was necessary to induce relaxation [43,45]. Besides NO, red wine affected the formation of other mediators of vascular tone, such as endothelium-derived hyperpolarizing factor [43] and prostacyclin [44]. In addition, the synthesis of a potent vasoconstrictor such as endothelin-1 is reduced by red wine in bovine aortic endothelial cells. The suppression occurred at transcriptional level. The decreased synthesis of endothelin-1 was associated with the inhibition of tyrosine kinase family of phosphorylating enzymes. The effect was correlated with the polyphenol content of red wine [45]. The mechanisms underlining NO-dependent vasorelaxation caused by RWPs were investigated [36,46,47]. All studies support for an increase in intracellular Ca2 + as the critical step for the activation of NO-synthase. RWPs increased cytosolic free Ca2 + by enhancing extracellular Ca2 + entry and by increasing Ca2 + mobilization from

intracellular stores. The authors suggested that Ca2 + signalling pathways leading to NO production could involve the activation of multiple cellular targets (G-proteins, phospholipase C, tyrosine kinase), depending on the composition of the polyphenol mixture. In addition to increased NO synthase activity, RWPs may prolong the half-life and increase the bioavailability of NO, by reducing its degradation mediated by reactive oxygen species [27]. 3.2. Modulation of gene expression by red wine polyphenols All events previously described are responsible for a short-term response mediated by RWPs. Recent studies pointed out that polyphenolic compounds from several sources may also have long-term properties, as they are able to modulate gene expression in different cancer cell lines and in macrophages [48 50]. RWPs significantly increased NO synthase expression, acting on the promoter activity [51]. Thus, polyphenols not only activate the enzyme, but also increase its levels, which might lead to a long-lasting effect. Adhesion of leukocytes, monocytes and T-lymphocytes to the vascular endothelium is among the earliest events in inflammatory response and atherogenesis. Successively, accumulation of leukocytes in the arterial intima can make atherosclerotic plaque worsening. Adhesion process is facilitated by the action of endothelial-leucocyte adhesion molecules, which include intercellular adhesion molecule 1 (ICAM-1), E-selectin and vascular cell adhesion molecule 1 (VCAM-1). The expression of these molecules can be transcriptionally regulated by inflammatory cytokines such as interleukin-1 alpha and tumor necrosis factor alpha (TNF-a). It was reported that at 5 Ag/ml, PAs extracted from grape seeds downregulated the TNF-a induced expression of VCAM-1

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

597

in primary human umbilical endothelial cells, leading to a reduced adherence with leukocytes and T-cells [52,53]. Similar results were reported in one human study [54]. In systemic sclerosis patients, plasma levels of VCAM-1, ICAM-1 and P-selectin were found to be higher than in normal volunteers. The administration of 100 mg/day of PAs derived from grape seeds for 1 month attenuated the increased expression of these adhesion molecules. Another study used ACs from blackberry administered to carrageenan-treated rats, a model of acute lung inflammation. ACs reduced the upregulation of ICAM-1 in lung tissue [55]. The AC mixture used in this study differs from that found in wine, since cyanidin-3-O-glucoside was predominant (80% of the total ACs); nevertheless, it might be expected that a similar behaviour by ACs occurs in red wines. 3.3. Red wine polyphenols and vascular smooth muscle cells Vascular smooth muscle cells contribute to the pathogenesis of atherosclerotic lesions, in as much as their proliferation and migration are critical events for progressive intimal thickening and development of arterial wall sclerosis. At the site of the lesion formation, the most potent mitogenic and chemotactic agent for vascular smooth muscle cells is platelet-derived growth factor (PDGF) released by platelets, endothelial cells and vascular smooth muscle cells themselves. PDGF exerts its biological effects via activation of two subtypes of transmembrane receptor tyrosine kinases, termed a and h PDGF receptor. Ligand binding to the h receptor promotes the activation of signalling enzymes which are important for cell migration and proliferation. Activation of phosphatidylinositol 3V -kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways as response to PDGF is implicated in vascular smooth muscle cells motility [56 58]. Then, the effect of RWPs on the proliferation and the migration of cultured vascular smooth muscle cells has also been investigated [59,60]. RWPs (1 100 Ag/ml) inhibited rat aortic smooth muscle cells proliferation and DNA synthesis. Polyphenol fractions of different molecular weights, c 200 400 for monomeric components (ACs, catechins and flavonoids) and 1600 2000 for oligomeric PAs, showed similar antiproliferative effects. Two distinct mechanisms have been postulated. One involves the downregulation of cyclin A gene expression, through the decreased expression of transcription factors ATF-1 and CREB (cAMP-responsive element). The second is associated with the downregulation of PI3K activity, which is known to interfere with cell-cycle regulation via the upregulation of p27kip1 acting as inhibitor of cyclin-dependent kinase [61,62]. In addition to proliferation, RWPs (1 100 Ag/ml) inhibited vascular smooth muscle cell migration through the specific inhibition of PI3K and p38MAPK, but not through other MAPKs and ERK 1/2 (extracellular signal-regulated

protein kinase 1 and 2). The attenuation of the signals leading to vascular smooth muscle cell proliferation and migration could also be the consequence of the inhibition of PDGF h receptor by RWPs [63]. Vascular endothelial growth factor (VEGF) released from vascular smooth muscle cells is a powerful endothelial mitogen and stimulates the expression of adhesion molecules and monocyte chemotactic protein-1 [64]. Red wine administered to cholesterol-fed rabbits inhibited the expression of monocyte chemotactic protein-1, but this effect was found also in animals receiving white wine [65]. The exposure of vascular smooth muscle cells to RWPs ( z 3 Ag/ml) markedly reduced the overexpression of VEGF induced by PDGF and other growth factors such as athrombin and transforming growth factor-h1 [66]. This effect was due to a reduced phosphorylation of p38MAPK, in agreement with previous findings [60]. Because the phosphorylation of p38MAPK and VEGF expression in vascular smooth muscle cells were stimulated in the presence of reactive oxygen species, the authors concluded that the inhibition of VEGF expression by RWPs involves their antioxidant properties. 3.4. Red wine polyphenols and platelet aggregation Platelets contribute to the rate of development of atherosclerosis and coronary artery diseases through several mechanisms [67]. While it is widely demonstrated the antiaggregating effect of alcohol [68], flavonoids [69 72] and resveratrol [12], the literature survey provided only fragmentary and contradictory information about the contribution of ACs and PAs to the inhibition of platelet aggregation. Demrow et al. [10] compared the effect of red and white wines (ml/kg), and grape juice (ml/kg) on platelet activity and thrombus formation in an in vivo dog model. Red wine and grape juice were effective as antiplatelet and antithrombotic compounds when administered intragastrically. Administration of white wine did not produce significant effects. Because the amount of ethanol necessary to produce the antiplatelet and antithrombotic activity is greatly reduced when red wine rather than pure ethanol was given, it was concluded that red wine contained platelet inhibitors in addition to ethanol. Rein et al. [73] reported the effects of dealcoholized red wine on platelet function in vitro and in vivo in healthy humans. In nonstimulated platelets, dealcoholized red wine added to whole blood induced platelet activation while suppressed the activation in response to epinephrine. A partial confirmation of these findings derives from the study by Russo et al. [74], who found in vitro a significant antiaggregating activity associated with a fraction of dealcoholized red wine containing procyanidins, ACs and catechins. No activity was associated to the other constituents: phenolic acids, flavonols, and polymeric PAs. However, dealcoholized red wine showed no effects when given orally to healthy subjects [73,75]. Other studies on

598

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

humans failed to show differences in platelet aggregation after red and white wine intake [76,77]. Further studies are needed to sort out these discrepancies.

4. Bioavailability of red wine polyphenols Two-thirds of the total intake of polyphenols in the human diet has been estimated to be accounted by flavonoids, the most abundant being flavan-3-ols (catechins and PAs), ACs and their oxidation products [78]. Information about the absorption, distribution, metabolism and excretion of individual flavonoids in humans is scarce. Furthermore, most studies have been designed using flavonoids in the aglycon form rather than their glycosides, which predominate in plants. Because the term flavonoid is applied in general to a multitude of compounds, in this section we will focus on absorption and metabolism of ACs and PAs (dimers, trimers, oligomers and long chain polymers). The question whether ACs are absorbed as free aglycon or in their glycoside form was approached for the first time by Paganga and Rice-Evans [79] who demonstrated the presence of ACs in human plasma, but without quantification. Later, several other studies confirmed that ACs are absorbed in the glycosilated form after oral consumption [80 85]. All studies performed in animals and humans agree that the bioavailability of ACs is limited [86 90]. In humans, plasma levels of total ACs are low, the order of magnitude being around 0.1 AM or less [82,83,91 93] and most of the ACs are excreted in urines during the first 4 h [83]. Between 1.5% and 5.1% of the ingested ACs were recovered in the urine of healthy subjects, within 12 h after consumption of 300 ml of wine containing around 218 mg of anthocyanosides. The AC levels in the urine reached a peak within 6 h from consumption [87]. More recent studies indicated that the amount of ACs excreted in urine was in much lower range, 0.04 0.1% of the dose [84,85]. After the ingestion of 400 ml of Lemberger wine (containing 270 mg of ACs) by healthy volunteers, red wine ACs were found in human plasma (Cmax: 42 F 8 Ag/l) and urine (cumulative excretion: 0.20 0.23% of the oral dose) as unchanged glucosides [93,94]. Other peaks detected in the urine samples showed similar absorption spectra as the ACs. These additional AC-like compounds may be metabolites of ACs, but the nature of the compounds was not elucidated [85,93]. Neither the aglycon nor glucoronide and/or sulphate derivatives were found in plasma and urines from subjects taking wine or other sources of ACs [80,83,88,91], but in some studies plasma and urines were not enzymatically treated. Recently, Wu et al. [95] reported methylated and glucoronidated AC metabolites in elderly women after ingestion of eldberry extract. Then the issue whether ACs undergo metabolism pre/post absorption requires further investigations. As ACs are bulky and polar molecules, it is expected that they are absorbed at gastro-

intestinal level through a carrier-mediated system. Indeed, Passamonti et al. [90,96] reported that ACs are competitive inhibitors of bilitranslocase (Ki ranging from 1.4 to 22 AM), a carrier protein expressed on gastric mucosa [97] and in vivo experiments proved the involvement of the stomach in the absorption of grape anthocyanosides in rats. The structure activity relationship revealed that monoglucosyl and diglucosyl ACs were better substrates than the corresponding aglycon [96]. The latter observation represents a further confirmation that glycosides are the preferred form for absorption. Furthermore, the ability of ACs to penetrate the gastric mucosa could be the explanation for the rapid appearance ( c 5 20 min) of ACs in systemic plasma [83,88,89,91]. At the same time, the contribution of the upper part of the intestine in the process of absorption cannot be excluded. Other questions that remain open concern the influence of alcohol, tartaric acid, a major organic acid in wine, and PAs to the efficiency of absorption. Results from the study by Bub et al. [88] tend to exclude the effect of alcohol on AC bioavailability, but the low number of subjects (6) is a limit to draw clearcut conclusions. On the other hand, tartaric acid increased the AUC of (+)-catechin metabolites in plasma of rats treated orally with a dose of 100 mg/kg of RWPs, suggesting that it can enhance the absorption and the bioavailability of polyphenols in red wine [98]. This observation needs to be confirmed in human studies. In comparison with ACs, the bioavailability of PAs has received much less attention, maybe partially due to the complexity of structures, which makes their analysis difficult, and for the lack of commercially available pure standards. Few data are available in animals and in humans. Early studies were performed with radiolabelled PA extracts which did not allow discrimination between monomers, oligomers and/or metabolites. Feeding rats and mice with a radiolabelled mixture of grape PAs [largely dimers, plus (+)-catechin and ( )-epicatechin] permitted the identification in urines and faeces of some ill-defined PAs and of hippuric acid, ethylcatechol and various phenolic acids. The radioactivity was found in all the tissues of the animals within few hours after the ingestion [99,100]. Other data obtained from studies in chickens and sheep indicated that the radioactivity associated with PA polymers was completely recovered in the bowel and the faeces of the treated animals, thus showing a lack of absorption of these large molecules [101,102]. Procyanidin polymers were also shown to be degraded in vitro into low-molecular weight phenolic acids by a human colonic microflora grown anaerobically [103,104]. Dimers and trimers were uptaken at similar extent into Caco-2 cell layer, a well-established model for human absorption, while polymers adhered partially to the cell surface [105]. The fate of procyanidin dimers has been studied recently: procyanidin B2 was found in plasma of subjects receiving 0.375 g cocoa/kg body weight, as beverage. The compound appeared in plasma as early as 0.5 h (16 nM). The maximal

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602

599

concentration was reached by 2 h (41 nM). The single monomers and their metabolites were also detected indicating cleavage of the dimer in the digestive tract and biotransformation [106]. The cleavage of procyanidin dimers B2 and B5 to the corresponding monomers at gastrointestinal level has been confirmed by other studies [107 109]. Monomers are then O-methylated and conjugated as glucoronides as described for other flavonoids [110]. In contrast, Donovan et al. [111] found that procyanidin B3, which is also present in wine, was not bioavailable: no trace of the compound was detected in plasma and urine after treating rats with a grape seed extract or procyanidin B3 alone. Nor procyanidins were cleaved to the bioavailable monomers. The reason for these discrepancies might reside in the experimental protocols and in the use of animal models. Rats were fasted before receiving procyanidin B2 [109]; in the study by Holt et al. [106], subjects consumed a flavanolrich cocoa; in the study by Donovan et al. [111], rats were fed a standard semipurified diet supplemented with procyanidin B3 or the grape seed extract. No studies thus far have addressed the issue of evaluating absorption and metabolism of PAs after wine drinking. It is then of utmost priority to eliminate this deficiency.

dosing and in conditions simulating the regular consumption of this beverage.

Acknowledgements This review is dedicated to the memory of Professor Giovanni Galli. We gratefully acknowledge Dr. Germana Galli for her help during the preparation of this manuscript.

References
[1] Ulbright TLV, Southgate DAT. Coronary heart disease: seven dietary factors. Lancet 1991;338:985 92. [2] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 1993;341(8843):454 7. [3] Maxwell S, Cruickshank A, Thorpe G. Red wine antioxidant activity in serum. Lancet 1994;344(8916):193 4. [4] Aviram M, Fuhram B. Wine flavonoids protect against LDL oxidation and atherosclerosis. Ann N Y Acad Sci 2002;957:146 61. [5] Francis FJ. Food colorants: anthocyanins. Crit Rev Food Sci Nutr 1989;28:273 314. [6] Bombardelli E, Morazzoni P. Vitis vinifera L. Fitoterapia 1995;66(4): 291 317. [7] Mattivi F, Zulian C, Nicolini G, Valenti N. Wine, biodiversity, technology and antioxidants. Ann N Y Acad Sci 2002;957:37 56. [8] Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-like compounds-nature, occurrence, dietary intake and effects on nutrition and health. J Sci Food Agric 2000;80:1094 117. [9] Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 1998;56:317 33. [10] Demrow HS, Slane PR, Folts JD. Coronary artery disease/platelets: administration of wine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries. Circulation 1995;91:1182 8. [11] Tedesco II, Russo M, Russo P, et al. Antioxidant effect of red wine polyphenols on red blood cells. J Nutr Biochem 2000;11(2): 114 9. [12] Fremont L. Biological effects of resveratrol. Life Sci 2000;66: 663 73. [13] Bhat KPL, Kosmeder JW, Pezzuto JM. Biological effects of resveratrol. Antioxid Redox Signal 2001;3(6):1041 64. [14] Kimura Y. Pharmacological studies on resveratrol. Methods Find Exp Clin Pharmacol 2003;25(4):297 310. [15] Vivas N, Bourgeois G, Vitry C, Glories Y, de Freitas V. Determination of the composition of commercial tannin extracts by liquid secondary ion mass spectrometry (LSIMS). J Sci Food Agric 1996;72:309 17. [16] De Freitas VAP, Glories Y, Bourgeois G, Vitry C. Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry. Phytochemistry 1998;49: 1435 41. [17] Gabetta B, Fuzzati N, Griffini A, et al. Characterization of proanthocyanidins from grape seeds. Fitoterapia 2000;71:162 75. reau-Gayon P, Stonestreet E. Le dosage des anthocyanes dans [18] Ribe les vins rouges. Bull Soc Chim Fr 1965;9:2649 52. ` en-Lo ` pez R, Fernandez-Zurbano P, Tena MT. Development and [19] Sa validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine. J Chromatogr, A 2003;990:247 58. [20] Waterhouse AL. Wine phenolics. Ann N Y Acad Sci 2002;957: 21 36. [21] Burns J, Gardner PT, ONeil J, et al. Relationship among antioxidant

5. Conclusions The interest in compounds present in red wine was stimulated by the claim that the regular consumption of this beverage could help in preventing cardiovascular diseases. In comparison with white wine, which does not share the same putative beneficial effects as red wine, the latter contains ACs and PAs in greater amounts. Then most of the studies reported in this review aimed to ascertain the contribution of these two classes of polyphenolic compounds to the overall effect of red wine. As shown by the large number of publications, ACs and PAs possess in vitro relevant biological activities and interact with many targets of the cell signalling pathways. However, the physiological significance of these findings depends on the assumption that ACs and PAs are bioavailable in the same chemical form as that used for the in vitro studies and can attain plasma levels as high as those used for in vitro studies. As reported in the previous paragraph, both assumptions are questionable. Even if most of the data were not obtained after wine drinking, few studies available about the plasma levels of ACs and PAs report values as 100 nM or lower, while concentrations used in vitro exceeded these values. In addition, no data are available to assess the distribution of ACs and PAs at the site of action in the unmetabolized form, as it would be required to mimic the situation of in vitro experiments. Among PAs, it is apparent that only dimers appear in plasma as the native compounds, but this observation needs to be confirmed in humans drinking wine. It is then imperative to acquire knowledge on the fate of red wine polyphenols after single

600

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602 activity, vasodilation capacity and phenolic content of red wines. J Agric Food Chem 2000;48:220 30. Li H, Fo rstemann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000;190:244 54. Vinson JA, Teufel K, Wu N. Wine Red wine, dealcoholized red wine, and especially grape juice, inhibit atherosclerosis in a hamster model. Atherosclerosis 2001;156:67 72. Klurfeld DM, Kritchevsky DM. Differential effects of alcoholic beverages on experimental atherosclerosis in rabbits. Exp Mol Pathol 1981;34:62 71. da Luz PL, Serano JCV, Chacra AP, et al. The effect of red wine on experimental atherosclerosis: lipid-independent protection. Exp Mol Pathol 1999;65:150 9. Yamakoshi J, Kataoka S, Koga T, Ariga T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 1999;142: 139 49. de aetano G, Cerletti C. Wine and cardiovascular disease. Nutr Metab Cardiovasc Dis 2001;11:47 50. Wollny T, Aiello L, Di Tommaso D, et al. Modulation of haemostatic function and prevention of experimental thrombosis by red wine in rats: a role for increased nitric oxide production. Br J Pharmacol 1999;127(3):747 55. De Curtis A, Porto AL, Vischetti M, Amore C, Donati MB, Iacoviello L. Effects of alcohol-free red wine on experimental thrombosis in hyperlipidemic rats. Thromb Haemost 2001;86:P1617. Fitzpatrick DF, Hirschfield SL, Coffey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol: Heart Circ Physiol 1993;265(34):H774 8. Fitzpatrick DF, Hirschfield SL, Ricci T, Jantzen P, Coffey RG. Endothelium-dependent vasorelaxation caused by various plant extracts. J Cardiovasc Pharmacol 1995;26(1):90 5. Flesch M, Schwarz A, Bohm M. Effects of red and white wine on endothelium-dependent vasorelaxation of rat aorta and human coronary arteries. Am J Physiol: Heart Circ Physiol 1998;275(44): H1183 90. Mizutani K, Ikeda K, Kawai Y, Yamori Y. Extract of wine phenolics improves aortic biomechanical properties in stroke-prone spontaneously hypertensive rats (SHRSP). J Nutr Sci Vitaminol (Tokyo) 1999;45(1):95 106. Diebolt M, Bucher B, Andriantsitohaina R. Wine polyphenols decrease blood pressure, improve NO vasodilatation, and induce gene expression. Hypertension 2001;38:159 65. Fitzpatrick DF, Fleming RC, Bing B, Maggi DA, OMalley R. Isolation and characterization of endothelium-dependent vasorelaxing compounds from grape seeds. J Agric Food Chem 2000;48(12): 6384 90. o ` ova O, Andriantsitohaina R. Red wine polypheZenebe W, Pecha nols induce vasorelaxation by increased nitric oxide bioactivity. Physiol Res 2003;52:425 32. tova I, Pecha o ` ova O, Baba l P, Kysela S, Stvrtina S, AndriantBerna sitohaina R. Wine polyphenols improve cardiovascular remodelling and vascular function in NO-deficient hypertension. Am J Physiol: Heart Circ Physiol 2002;282:H942 8. Stein JH, Keevil JG, Wiebe DA, Aeschlimann S, Folts JD. Purple grape juice improves endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients with coronary artery disease. Circulation 1999;100(10):1050 5. Andriambeloson E, Kleschyov AL, Muller B, Beretz A, Stoclet JC, Andriantsitohaina R. Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta. Br J Pharmacol 1997;120:1053 8. Freslon JL, Mendes A, Desgranges C, Vercauteren J. Procyanidin oligomers from grape seed induce NO-dependent relaxation of aortic rings via P2Y1 receptor activation. Br J Pharmacol 1997;122:200 [Abstract]. Andriambeloson E, Magnier C, Haan-Archipoff G, et al. Lobstein Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta. J Nutr 1998;128:2324 33. Fitzpatrick DF, Bing B, Maggi DA, Fleming RC, OMalley R. Vasodilating procyanidins derived from grape seeds. Ann N Y Acad Sci 2002;957:78 89. Ndiaye M, Chataigneau T, Andriantsitohaina JCS, Schini-Kerth VB. Red wine polyphenols cause endothelium-dependent EDHF-mediated relaxations in porcine coronary arteries via a redox-sensitive mechanism. Biochem Biophys Res Commun 2003;310:371 7. Derek D, Pearson DA, German JB. Endothelial cell basal PGI2 release is stimulated by wine in vitro: one mechanism that may mediate the vasoprotective effects of wine. J Nutr Biochem 1997;8:647 51. Corder R, Douthwaite JA, Lees DM, et al. Khan Endothelin-1 synthesis reduced by red wine. Nature 2001;414(6866):863 4. Andriambeloson E, Stoclet JC, Andriantsitohaina R. Mechanism of endothelial nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta. J Cardiovasc Pharmacol 1999;33: 248 54. Martin S, Andriambeloson E, Takeda K, Andriantsitohaina R. Red wine polyphenols increase calcium in bovine aortic endothelial cells: a basis to elucidate signalling pathways leading to nitric oxide production. Br J Pharmacol 2002;135:1579 87. Lin YL, Lin JK. ( )-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharideinduced activity of transcription factor nuclear factor-nB. Mol Pharmacol 1997;52:465 72. Okabe S, Fujimoto N, Sueoka N, Suganuma M, Fujiki H. Modulation of gene expression by ( )-epigallocatechin gallate in PC-9 cells using a cDNA expression array. Biol Pharm Bull 2001;24:883 6. Bellosta S, DellAgli M, Canavesi M, et al. Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae). Cell Mol Life Sci 2003;60:1440 8. Leikert JF, Rathel TR, Wohlfart P, Cheynier V, Vollmar AM, Dirsch VM. Red wine polyphenols enhance endothelial nitric oxide synthase expression and subsequent nitric oxide release from endothelial cells. Circulation 2002;106:1614 7. Sen CK, Bagchi D. Regulation of inducible adhesion molecule expression in human endothelial cells by grape seed proanthocyanidin extract. Mol Cell Biochem 2001;216(1 2):1 7. Bagchi D, Bagchi M, Sthos SJ, Ray SD, Sen CK, Preuss HG. Cellular protection with proanthocyanidins derived from grape seeds. Ann N Y Acad Sci 2002;957:260 70. Kalin R, Righi A, Del Rosso A, et al. Activin, a grape seed-derived proanthocyanidin extract, reduces plasma levels of oxidative stress and adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in systemic sclerosis. Free Radic Res 2002;36(8):819 25. Rossi A, Serraino I, Dugo P, et al. Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radic Res 2003;37(8):891 900. Knall C, Worthen GS, Johnson GL. Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc Natl Acad Sci U S A 1997;94(7):3052 7. Imai Y, Clemmons DR. Roles of phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways in stimulation of vascular smooth muscle cell migration and deoxyribonucleic acid synthesis by insulin-like growth factor-I. Endocrinology 1999;140(9): 4228 35. Hedges JC, Dechert MA, Yamboliev IA, et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 1999;274:24211 9. Iijima K, Yoshizumi M, Ouchi Y, et al. Red wine polyphenols inhibit proliferation of vascular smooth muscle cells and downregulate expression of cyclin A gene. Circulation 2000;101:805 11. Iijima K, Yoshizumi M, Hashimoto M, et al. Red wine polyphenols

[22] [23]

[42]

[43]

[24]

[44]

[25]

[45] [46]

[26]

[27] [28]

[47]

[48]

[29]

[30]

[49]

[31]

[50]

[32]

[51]

[33]

[52]

[34]

[53]

[35]

[54]

[36]

[55]

[37]

[56]

[38]

[57]

[39]

[58]

[40]

[59]

[41]

[60]

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602 inhibit vascular smooth muscle cell migration through two distinct signalling pathways. Circulation 2002;105:2404 10. Collado M, Medema RH, Garcia-Cao I, et al. Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1. J Biol Chem 2000;275(29):21960 8. Suzuki E, Nagata D, Yoshizumi M, et al. Reentry into the cell cycle of contact -inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J Biol Chem 2000;275(5): 3637 44. Rosenkranz S, Knirel D, Dietrich H, Flesch M, Erdmann E, Bohm M. Inhibition of the PDGF receptor by red wine flavonoids provides a molecular explanation for the French paradox. FASEB J 2002;16(14):1958 60. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77(7):527 43. Feng AN, Chen YL, Chen YT, Ding YZ, Lin SJ. Red wine inhibits monocyte chemotactic protein-1 expression and modestly reduces neointimal hyperplasia after balloon injury in cholesterol-fed rabbits. Circulation 1999;100:2254 9. Oak MH, Chataigneau M, Keravis T, et al. Red wine polyphenolic compounds inhibit vascular endothelial growth factor expression in vascular smooth muscle cells by preventing the activation of the p38 mitogen-activated protein kinase pathway. Arterioscler Thromb Vasc Biol 2003;23:1001 7. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and acute coronary syndromes. N Engl J Med 1992;326:242 50. Renaud SC, Ruf JC. Effects of alcohol on platelet functions. Clin Chim Acta 1996;246:77 89. Landolfi R, Mower R, Steiner M. Modification of platelet function and arachidonic acid metabolism by bioflavonoids. Biochem Pharmacol 1984;33:1525 30. Gryglewski R, Korbut R, Robak J, Swies J. On the mechanism of antithrombotic action of flavonoids. Biochem Pharmacol 1987;36: 317 22. Beretz A, Stierle A, Anton R, Cazenave JP. Role of cAMP in the inhibition of human platelet aggregation by quercetin, a flavonoid that potentiates the effect of prostacyclin. Biochem Pharmacol 1981;31:3597 600. Slane PR, Qureshi AA, Folts JD. Platelet inhibition in stenosed canine arteries by quercetin and rutin, polyphenolic flavonoids found in red wine. Clin Res 1994;42:162 [Abstract]. Rein D, Paglieroni TG, Pearson DA, et al. Cocoa and wine polyphenols modulate platelet activation and function. J Nutr 2000; 130:2120S 6S. Russo P, Tedesco I, Russo M, Russo GL, Venezia A, Cicala C. Effects of de-alcoholated red wine and its phenolic fractions on platelet aggregation. Nutr Metab Cardiovasc Dis 2001;11(1):25 9. Pellegrini N, Pareti FI, Stabile F, Brusamolino A, Simonetti P. Effects of moderate consumption of red wine on platelet aggregation and haemostatic variables in healthy volunteers. Eur J Clin Nutr 1996;50(4):209 13. Lavy A, Fuhrman B, Market A, et al. Effect of dietary supplementation of red or white wine on human blood chemistry, hematology and coagulation: favorable effect of red wine on plasma high-density lipoprotein. Ann Nutr Metab 1994;38(5):287 94. Pace-Asciak CR, Rounova O, Hahn SE, Diamandis EP, Goldberg DM. Wines and grape juices as modulators of platelet aggregation in healthy human subjects. Clin Chim Acta 1996;246(1 2):163 82. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr 2000;130(8S):2073S 85S. Paganga G, Rice-Evans CA. The identification of flavonoids as glycosides in human plasma. FEBS Lett 1997;401:78 82. Cao G, Prior RL. Anthocyanins are detected in human plasma after oral administration of an elderberry extract. Clin Chem 1999;45(4): 574 6.

601

[61]

[62]

[63]

[64] [65]

[66]

[67]

[68] [69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78] [79] [80]

[81] Tsuda T, Horio F, Osawa T. Absorption and metabolism of cyanidin 3-O-h-D-glucoside in rats. FEBS Lett 1999;449:179 82. [82] Matsumoto H, Inaba H, Kishi M, Tominaga S, Hirayama M, Tsuda T. Orally administered delphinidin-3-rutinoside and cyanidin-3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms. J Agric Food Chem 2001;49(3):1546 51. [83] Cao G, Muccitelli HU, Sanchez-Moreno C, Prior RL. Anthocyanins are absorbed in glycated forms in elderly women: a pharmacokinetic study. Am J Clin Nutr 2001;73:920 6. [84] Nielsen ILF, Dragsted LO, Ravn-Haren G, Freese R, Rasmussen SE. Absorption and excretion of black currant anthocyanins in humans and watanabe heritable hyperlipidemic rabbits. J Agric Food Chem 2003;51:2813 20. [85] McGhie TK, Ainge GD, Barnett LE, Cooney JM, Jensen D. Anthocyanins glycosides from berry fruits are absorbed and excreted unmetabolized by both humans and rats. J Agric Food Chem 2003; 51:4539 48. [86] Morazzoni P, Livio S, Scilingo A, Malandrino S. S. Vaccinium myrtillus anthocyanosides pharmacokinetics in rats. Arzneimittelforschung 1991;41(2):128 31. [87] Lapidot T, Harel S, Granit R, Kanner J. Bioavailability of red wine anthocyanins as detected in human urine. J Agric Food Chem 1998;46:4297 302. [88] Bub A, Watzl B, Heeb D, Rechkemmer G, Briviba K. Malvidin-3glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and grape juice. Eur J Nutr 2001;40:113 20. [89] Milbury PE, Cao G, Prior RI, Blumberg J. Bioavailability of elderberry anthocyanins. Mech Ageing Dev 2002;123(8):997 1006. [90] Passamonti S, Vrhovsek U, Vanzo A, Mattivi F. The stomach as a site for anthocyanins absorption from food. FEBS Lett 2003;544: 210 3. [91] Miyazawa T, Nakagawa K, Kudo M, Muraishi K, Someya K. Direct intestinal absorption of red fruits anthocyanins, cyanidin-3-glucoside and cyanidin-3,5-diglucoside, into rats and humans. J Agric Food Chem 1999;47(3):1083 91. [92] Mazza G, Kay CD, Cottrell T, Holub BJ. Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects. J Agric Food Chem 2002;50:7731 7. [93] Netzel M, Strass G, Bitsch I, Konitz R, Christmann M, Bitsch R. Red wine anthocyanins and their bioavailability in humans. Ernaehrung 2002;26(2):58 63. [94] Frank T, Netzel M, Strass G, Bitsch R, Bitsch I. Bioavailability of anthocyanidin-3-glucosides following consumption of red wine and red grape juice. Can J Physiol Pharmacol 2003;81(5):423 35. [95] Wu X, Cao G, Prior RL. Absorption and metabolism of anthocyanins in elderly women after consumption of elderberry or blueberry. J Nutr 2002;132:1865 71. [96] Passamonti S, Urhovsek U, Mattivi F. The interaction of anthocyanins with bilitranslocase. Biochem Biophys Res Commun 2002;269: 631 6. [97] Battiston L, Macagno A, Passamonti S, Micali F, Sottocasa GL. Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat. FEBS Lett 1999;453(3):351 5. [98] Yamashita S, Sakane T, Harada M, et al. Absorption and metabolism of antioxidative polyphenolic compounds in red wine. Ann N Y Acad Sci 2002;957:325 8. tique [99] Laparra J, Michaud J, Masquelier J. Etude pharmacocine ` res flavanoliques (Pharmacokinetic study of flavonic des oligome r 1977;11:133 42. oligomers). Plant Med Phytothe [100] Harmand MF, Blanquet P. The fate of total flavanolic oligomers (OFT) extracted from Vitis vinifera L., in the rat. Eur J Drug Metab Pharmacokinet 1978;3:15 30. [101] Jimenez-Ramsey LM, Rogler JC, Housley TL, Butler LG, Elkin RG. Absorption and distribution of C-14-labelled condensed tannins and related sorghum phenolics in chickens. J Agric Food Chem 1994;42: 963 7.

602

M. DellAgli et al. / Cardiovascular Research 63 (2004) 593602 C. Decomposition of cocoa procyanidins in the gastric milieu. Biochem Biophys Res Commun 2000;272(1):236 41. Spencer JP, Schroeter H, Shenoy B, Srai SK, Debnam E, Rice-Evans C. Epicatechin is the primary bioavailable form of the procyanidin dimers B2 and B5 after transfer across the small intestine. Biochem Biophys Res Commun 2001;285(3):588 93. Baba S, Osakabe N, Natsume M, Terao J. Absorption and urinary excretion of procyanidin B2 [epicatechin-(4h-8)-epicatechin] in rats. Free Radic Biol Med 2002;33(1):142 8. Spencer JP, Schroeter H, Crossthwaithe AJ, Kuhnle G, Williams RJ, Rice-Evans C. Contrasting influences of glucuronidation and O-methylation of epicatechin on hydrogen peroxide-induced cell death in neurons and fibroblasts. Free Radic Biol Med 2001;31(9): 1139 46. me sy C. Donovan JL, Manach C, Rios L, Morand C, Scalbert A, Re Procyanidins are not bioavailable in rats fed a single meal containing a grapeseed extract or the procyanidin dimer B3. Br J Nutr 2002; 87:299 306.

[102] Terril TH, Waghorn GC, Wooley DJ, McNabb WC, Barry TN. Assay and digestion of 14C-labelled condensed tannins in the gastrointestinal tract of sheep. Br J Nutr 1994;72:467 77. [103] Groenewoud G, Hundt HKL. The microbial metabolism of condensed (+)-catechins by rat-caecal microflora. Xenobiotica 1986; 16:99 107. prez S, Bre zillon C, Rabot S, et al. Polymeric proanthocyanidins [104] De are catabolized by human colonic microflora into low-molecular weight phenolic acids. J Nutr 2000;130(11):2733 8. prez S, Mila I, Scalbert A, Huneau JF, Tome D. Transport of [105] De proanthocyanidin dimer, trimer and polymer across monolayers of human intestinal epithelial Caco-2 cells. Antioxid Redox Signal 2001;3(6):957 67. [106] Holt RR, Lazarus SA, Sullards MC, et al. Procyanidin dimer B2 [epicatechin-(4h-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am J Clin Nutr 2002;76: 798 804. [107] Spencer JP, Chaudry F, Pannala AS, Srai SK, Debnam E, Rice-Evans

[108]

[109]

[110]

[111]