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Wine phenolics: contribution to dietary intake and bioavailability
Food Research International 33 (2000) 461±467
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Wine phenolics: contribution to dietary intake and bioavailability Pierre-Louis Teissedre *, Nicolas Landrault Centre de Formation et de Recherche en Oenologie, Universite de Montpellier I, Faculte de Pharmacie, Av. Charles Flahault, 34060 Montpellier cedex 2, France Received 10 September 1999; accepted 3 January 2000
Abstract Current research suggests that wine contains substances that may reduce the mortality rate from coronary heart diseases. The oxidation of low-density liporoteins (LDL) is thought to be a key step in the development of atherosclerosis. In previous research, catechin oligomers, procyanidin dimers and trimers were extracted, isolated and puri®ed from grapes seeds and tested for their inhibition on LDL oxidation, along and with other monomeric wine phenolics.We quantify the level of phenolics, major catechins [(+)-catechin, (ÿ)-epicatechin, procyanidin dimers B1, B2, B3, B4], phenolics acids, caftaric acid and malvidine-3-glucoside by HPLC with UV detection for 60 French varietal commercial wines taken from the South of France in order to appreciate the daily dietary intake of these compounds for French population. Based on a still signi®cant French red wine consumption of 180 ml/day, the current daily intake of phenolics can be estimated at 400.2 mg/day/resident and catechins (monomers, dimers B1, B2, B3, B4) at 83.2 mg/day/resident, including 40% of monomers for the French population. Red wine, and particularly Egiodola, and CabernetSauvignon varieties, contributes to a very signi®cant proportion of phenolics dietary intake and, in particular catechins. These results provide a basis for epidemiological evaluation of phenolics and catechins intake by wine consumption in the French population. Few data exist on absorption and metabolism of phenolics from wine. Further studies are necessary to determine the behaviour of this class of compounds in vivo after wine absorption. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phenolics; Catechins; Levels; Dietary intake; Absorption
1. Introduction Epidemiological studies conducted during the last 20 years have shown that coronary heart diseases are less prevalent in countries consuming a regular and moderate consumption of wine (Friedman, & Kimball, 1986; Klatsky, & Armstrong, 1993; St-Leger, Cochrane & Moore, 1979; WHO., 1989). In one of the best known studies, Renaud and De Lorgeril (1992) suggest an explanation of the phenomenon particularly favourable to the French population with regard to cardiovascular disease, known as the ``French paradox'', which was ®rst described in 1987 by Richard. The results of the Monica program (1989), a worldwide CAD surveillance system organised by the World Health Organization (WHO), con®rm that mortality levels provoked by coronary artery disease are much lower in France than in other industrialised countries, even though the * Corresponding author. Tel.: +33-4-6754-4520; fax: +33-4-67544526. E-mail address: [email protected] (P.-L. Teissedre).
consumption of saturated fats in France is much the same and blood cholesterol levels are generally higher. Furthermore, other factors associated with risk of coronary artery disease, such as arterial blood pressure, body weight and smoking, are no lower in France than in the other countries. This is the French paradox. Renaud and De Lorgeril (1992) used WHO and OECD data to study which food parameters could be correlated with cardiovascular disease (CVD) mortality levels. Among the numerous dierent foods, only the consumption of dairy fats (between 1980 and 1985) showed positive correlation with CVD mortality (1987 levels), but this correlation was highly signi®cant (r=0.73, P<0.001). However, although the consumption of dairy fats is the same in France as in the UK, Australia and Germany, mortality from coronary artery disease is lower in France. Using multifactor analysis, Renaud and De Lorgeril revealed that in 17 winedrinking countries, the only dietary factor correlating signi®cantly with coronary artery disease mortality, apart from dairy fats, was wine (r=0.87, P <0.001). Moreover, wine consumption had a negative coecient,
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indicating a protective eect. It was thus realised that the French case was not paradoxical when wine consumption was taken into account. In this study, the protection aorded by wine consumption was also detected in Switzerland and other industrialised countries. From this, attention turned to the non-alcoholic fractions of wine. Wine, particularly red wine, is an important source of polyphenols which are capable of inhibiting the processes behind coronary artery disease. This hypothesis is supported by the results of recent epidemiological studies concerning foodstu polyphenols, particularly ¯avonoids. A correlation was also noticed between increasing levels of ¯avonoid ingestion from fruit and vegetables and reduction in coronary artery disease. The studies carried out by Hertog, Feskens, Hollman, Katan and Kromhout (1993), Knekt, Jarvinen, Reunanen and Maatela (1996) and Rimm, Katan, Ascherio, Stampfer and Willett. (1996) reveal the bene®ts of a diet rich in ¯avonoids. Polyunsaturated fatty acids occur as a major part of the low-density lipoproteins in blood and oxidation of these lipids components in low-density lipoproteins (LDL) may play a role in atherosclerosis (Steinberg, Parthasary, Carew, Khoo & Witzum, 1989). With a continued high level of oxidized lipids, the blood vessels damage reaction process continues and can lead to generation of foam cells and plaque, the symptoms of atherosclerosis. In addition, in vitro studies show that oxidation of human LDL can be inhibited by physiological levels of vitamins C and E (Jialal, Vega, & Grundy, 1990), and certain ¯avonoids (De Whalley, Rankin, Hoult, Jessup & Leake, 1990). Thus, the hypothesis was advanced that potent antioxidative phenolics in wine can prevent oxidation of LDL and explain the French paradox. Thus, the inhibition of human LDL was demonstrated by the addition of the mixture of polyphenols from wine (Frankel, Kanner, German & Kinsella, 1993). Red wine diluted 1000-fold inhibited the in vitro oxidation of human LDL signi®cantly more than a-tocopherol. Other eects of red wines include inhibition of chronic in¯ammation and thrombotic tendencies (Kinsella, Frankel, German & Kanner, 1993). (+)-Catechin and (ÿ)-epicatechin are the basic units of the catechins group. The procyanidins are formed from the association of several of these monomeric units: 2±5 units for catechin oligomers (e.g. Fig. 1), over 5 units for catechin polymers. The procyanidins dier in the position and con®guration of their monomeric linkages. The structures of procyanidin dimers B1, B2, B3 and B4 are the best known. These molecules possess a structure that confers them with an antioxidant property which can inhibit the processes leading in the long term to atherosclerosis and arterial thrombosis.
Fig 1. Antitoxidants wine ¯avonoids.
The ¯avonoids are the most lipophilic of the natural antioxidants, but less so than a-tocopherol. The atocopherol seems to be located in the lipid membrane within the phospholipid bilayer while the ¯avonoids are probably mainly located at the polar surface of the bilayer. The aqueous, i.e. transported in the plasma, free radicals would therefore be captured more easily by the ¯avonoids than by the less accessible a-tocopherol. Thus, the ¯avonoids could be concentrated near to the membranous surface of the low-density lipoprotein (LDL) particles, ready to capture the oxygenated aqueous free radicals. They would in this way prevent the consumption of lipophilic a-tocopherol and thus delay oxidation of the lipids contained in the LDL. Moreover, if, as research suggests, the initiation phase and the propagation phase of lipid peroxidation take place, respectively, at the surface and the interior of the membranes, then the ¯avonoids could well hinder the correct course of the reaction by limiting the initiation phase. Catechins and procyanidins have been shown in vitro to be powerful inhibitors of LDL oxidation, more so than a-tocopherol (Teissedre, Frankel, Waterhouse, Peleg & German, 1996), and of platelet aggregation (Ruf, Berger & Renaud, 1995). Moreover, it has recently been shown that the consumption of wine by humans leads to an increase in the antioxidant capacity of plasma (Furhmann, Lavy & Aviram, 1995). Other studies have been carried out on the antioxidant activity Ð through inhibition of copper-catalysed oxidation of human LDL Ð of a selection of Californian wines, made from Cabernet-Sauvignon, Merlot, Zinfandel, Petite Syrah, Pinot-noir, Sauvignon
P.-L. Teissedre, N. Landrault / Food Research International 33 (2000) 461±467
blanc and Chardonnay. The relative inhibition of LDL oxidation (calculated with respect to the total phenol concentration of each sample) varied from 46 to 100% for red wines and from 3 to 6% for white wines (Frankel, Waterhouse & Teissedre, 1995). The antioxidant activity of wines made with long maceration times exclusively from RhoÃne Valley Syrah and Grenache varieties was 60% relative inhibition of LDL oxidation (Teissedre, Waterhouse & Frankel, 1995). Comparison of these results with the dierent Californian red wines showed that the Rhodanian wines made from Syrah and Grenache gave values of relative inhibition of human LDL oxidation identical to those of Cabernet-sauvignon but less than those of Petite Syrah (80%) or Merlot (74%). In contrast, the Syrah wines produced with short extractions gave a low mean value of relative inhibition (16%) which is nevertheless 4 times higher than that obtained for Californian white wines (4.4%). It was deduced from this that each phenolic compound of wine could play a role in the protection against LDL oxidation. All the properties and studies reported support the present hypotheses for explaining the reduced risk of mortality from coronary artery disease in moderate and regular consumers of wine (particularly red wine). In the present study we evaluated total phenols content, the major catechins [(+)-catechin, (ÿ)-epicatechin, dimers B1, B2, B3, B4], some phenolic acids (cafeic acid, para-hydroxycoumaric acid, protocatechuic acid), a cinnamate, caftaric acid, and a major anthocyanine, mavidine-3-glucoside, for 60 commercial varietal wines taken from the same area of France, to provide concentrations data of phenolics for varietal Languedoc wines and to appreciate the daily dietary intake of phenolics for the French population.
2. Materials and methods 2.1. Materials (+)-Catechin, (ÿ)-epicatechin, gallic acid, cafeic acid, protocatechuic acid, para-hydroxycoumaric acid were obtained from Aldrich, malvidine-3-glucoside from ExtrasyntheÂse. Caftaric acid was provided by Ursa Vorshek. Procyanidins dimers B1, B2, B3, B4, were obtained from grape seeds as detailed below. 2.2. Wine samples We analysed 60 samples of dierent French varietal wines: 50 red (Merlot, Cabernet-Sauvignon, Grenache, Syrah, Egiodola); 10 white (Chardonnay) in commercial bottles from Domaine Virginie (BeÂziers, France). The wine samples analysed were from all viticultural areas of
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Languedoc and of the same vintage year, 1998. All wines analysed are frequently consumed in France. 2.3. Total phenols content Total phenols were analyzed by the Folin±Ciocalteu method (Singleton & Rossi, 1965), calibrating against gallic acid standards, and expressing the results as GAE. 2.4. Extraction and isolation of crude procyanidins Grape seeds (vitis vinifera), 150 g, were extracted with methanol as described by Bourzeix, Weyland and Heredia (1986) and by Weinges and Piretti (1971). The extract (3 ml, 300 mg) was chromatographed on Fractogel TSK HW-40(s) (25±40 mm) (450 25 mm id) with methanol as eluant, using an ISCO (Lincoln, NE, USA) model UA-5 absorbance monitor set at 280 nm, a peristaltic Miniplus2 pump (Gilson Inc, Middletin, WI, USA) and an ISCO 328 fraction collector. Ten fractions containing procyanidins were collected. 2.5. Isolation of puri®ed procyanidins Semi-preparative HPLC was performed with a Waters 510 pump (Waters, Miford, MA, USA) a U6K injector, and a Hewlett-Packard (Palo Alto, CA, USA) model 1050 UV±Vis detector set at 280 nm. The column was a Water RCM Novapak C18 25100 mm, 4 mm particle size. Elution was carried out by a linear gradient of 0± 500 ml/l methanol with the solvent described below at 2 ml/min. 2.6. TLC analysis Silica plates (DC Alufolien-Kieselgel 60, 0.2 mm thick, Merk, EM Separation technology, Gibbstown, NJ, USA), were developed with toluene/acetone/formic acid (3:3:1 v/v/v) as described by Lea, Bridle, Timberlake and Singleton. (1979). The plates were visualised by spraying with a solution of vanillin (100 g/l) in concentrated HCL. 2.7. HPLC analysis A Hewlett-Packard model 1090 with three low-pressure pumps and a diode array UV detector coupled to a Hewlett-Packard Chem station was used for solvent delivery system and detection. A Hewlett-Packard column Nucleosil 100 C18, 2504 mm, 5 mm particle size was used for the stationary phase with a ¯ow of 0.7 ml/ min. The solvents used for separation were: solvent A: 50 mM dihydrogen ammonium phosphate adjusted to pH 2.6 with orthophosphoric acid; solvent B: 20% A with 80% acetonitrile; and solvent C: 0.2 M orthophosphoric acid adjusted with ammonia to pH 1.5, and solvent
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gradient conditions as described by Lamuela-Raventos and Waterhouse. (1994). Temperature was thermostated at 25 C. 3. Results and discussion Analytical studies on phenolics and ¯avonoids in wine revealed that this beverage contains high levels and that the dierent wine-making techniques are important factors (Bourzeix et al., 1986). However, the results cited here concern wines made from California (Waterhouse & Teissedre, 1997) or from micro-vini®cation and not intended for consumption. The aim of this study was to analyse the phenolics and catechins content of varietal wines from the south French region made from diverse varieties and using varied vini®cation techniques. The results obtained were used to evaluate the phenolics and catechins intake from regular, moderate wine consumption. Red wines had generally much higher levels of phenolics than white wines. This dierence is the result of dierent wine-making techniques. The catechins in grapes, present mainly in the pips, diuse into the must during the course of maceration. The maceration phase constitutes a fundamental dierence in the vini®cation of red and white wine. It is essential for the production of red wine and is deliberately avoided for the production of white, where rapid pressing separates the liquid phase from the solid matter. The concentrations of total phenol as determined by the Folin±Ciocalteu method varied from 1847 to 2600 mg/l GAE, for the red wines and are of 245 mg/l GAE for the Chardonnay white wines (Table 1). The most interesting varieties for high levels of total phenol are Cabernet-Sauvignon, Egiodola and Syrah.
Thus, the red wines studied had high overall levels of catechins (sum of monomers and procyanidin dimers analysed) Ð 462.6 mg/l Ð but there were considerable dierences between the red wines (Table 1). Levels of catechins in the white wines were lower: 6.5 mg/l. All the compounds studied were present in each of the 50 red wines analysed. For the catechin monomers, the mean concentration [sum of (+)-catechin and (ÿ)-epicatechin] was 202.6 mg/l. The quantity of procyanidin dimers (sum of B1, B2, B3, B4) was 260 mg/l and that of malvidine-3-glucoside was 164.4 mg/l. The white wines (n=10) showed very low concentrations of monomers (5 mg/l) and absence of malvidine-3-glucoside. The highest concentrations of catechins in red varietal wines were found in Egiodola (732 mg/l), Cabernet-Sauvignon and Grenache (close to 500 mg/l). Egiodola was found richest in malvidine-3-glucoside (382 mg/l), up to 4 times more than the other red varieties. The mean contents of each phenolic are given in Table 1. 3.1. Estimation of phenolics and catechins intake in moderate wine consumers in France Over the last few years, the consumption of wine in France has fallen considerably. In 1986, the mean consumption was 305 ml/person/day (Darret, Couzy, Antoine, Magliola & Mareschi, 1986). This level fell sharply to 180 ml/person/day in 1995 (Boulet, Laporte, Aigrin & Lalanne, 1995). An estimation of the intake of phenolics and catechins was calculated from these latest consumption ®gures and our own analytical results on the catechin (monomers, dimers,) content of 60 wines. Although the levels of these compounds vary considerably from one varietal wine to another, the regular consumption of the dierent products available to
Table 1 Average levels of phenolics in Languedoc varieties wines Varieties a
Phenolics
Merlot
Cabernet-Sauvignon
Grenache
Syrah
Egiodola
Chardonnay
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol contentb
195.3 192.5 409.2 317.7 10.13 7636.5 8760.9 5026.5 4727.6 2512.2 4513.5 9122.3 2000514
5312.1 3010.1 41.2 102.6 20.18 12027.6 12040.8 9915.1 5320.3 4411.5 5323.2 9738.9 2600774
227.1 4911.2 20.6 377.8 10.09 11044.5 10542.8 776.8 5623.4 8018.2 7030.1 13217.8 1847332
237.2 124.1 41 203.1 20.14 5120.2 5022.6 388.7 9014.5 254.9 203.9 12020.5 2266294
5323.6 184.7 60.9 4012.1 50.43 12935.8 16541.8 14041.2 8334.5 9137.8 11347.9 382123 2400240
20.3 71.4 10.4 2215.1 41.1 42.3 1O.9 0.50.1 0.30.67 0.20.14 0.50.45 NFc 24563
a b c
Values expressed in mg/lS.D. Expressed in mg Gallic acid equivalent/lS.D. NF, not found.
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the consumer tends toward a mean value which we considered to be close to that calculated for the 60 wines. For this reason, this estimation can only be considered for regular consumption of the same variety wine over a suciently long period of time (which remains to be determined statistically). Daily intake of each phenolic compound by variety wine is indicated in Table 2. The consumption of 180 ml of CabernetSauvignon or Egiodola wine, for which the mean phenolics concentration are, respectively, 2600 and 2400 mg/l, gives a mean daily intake of 468 and 432 mg of total phenolics. This reasoning applied to white wine for regular (daily), moderate (180 ml) consumption gives estimations of phenolics intake of only 44.1 mg for white Chardonnay wines, 10 times less than for the red (Table 3). Daily intake of catechins from varieties wines (Fig. 2) ranged from 0.9 to 59.2 mg/person for monomers (catechin and epicatechin), and from 1.2 to 129.8 mg/ person for total catechins (including monomers and dimers). The best results were obtained with Egiodola, Cabernet-Sauvignon, and Grenache varieties. Amounts of total catechins daily intake of Egiodola was twice than for Merlot or Syrah, and almost 110 times more than for Chardonnay. Cabernet-Sauvignon and Grenache gave interesting daily intake results for total catechins close to 90 mg/person. 3.2. Absorption of phenolics and catechins in the plasma after wine consumption. Although epidemiological studies note a correlation between the reduction of risk of coronary artery disease and the regular, moderate consumption of wine, no causal relationship has been demonstrated. The ¯avonoids in wine constitute a considerable LDL antioxidant and platelet anti-aggregant potential, but it
465
is dicult to know whether this potential is slightly, partially or totally realised during metabolism. It would appear necessary, therefore, to supply irrefutable biochemical proof of the envisaged protective mechanisms. Although compounds such as (+)-catechin, (ÿ)-epicatechin, procyanidin dimers B1, B2, B3, B4 and trimers C1, C2 inhibit LDL oxidation in vitro (Teissedre et al., 1996), no account can be taken of the large variability in the composition of blood plasma in order to verify whether these compounds or their metabolites can be suciently active in vivo to aord protection, even though epidemiological studies suggest that they do. Current research concerns absorption of these compounds in human plasma after wine consumption. The intestinal ¯ora (which varies in function of the
Table 3 Daily intake of phenolics from white or red wines Average intake in mg/day/persona Phenolics
White wine
Red wine
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol
0.36 1.26 0.18 3.96 0.72 0.72 0.18 0.09 0.054 0.036 0.09 NFb 44.1
6.12 4.61 2.02 4.97 0.40 17.50 18.97 14.54 11.84 9.54 10.84 29.59 400.2
a b
Based on a daily wine consumption of 180 ml. NF, not found.
Table 2 Daily intake of phenolics from varieties of wines consumption for the French population Daily intake in mg/day/persona Phenolics
Merlot
Cabernet-Sauvignon
Grenache
Syrah
Egiodola
Chardonnay
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol
3.42 3.42 7.2 5.58 0.18 13.68 15.66 9 8.46 4.5 8.1 16.38 360
9.54 5.4 0.72 1.8 0.36 21.6 21.6 17.82 9.54 7.92 9.54 17.46 468
3.96 8.82 0.36 6.66 0.18 19.8 18.9 13.86 10.08 14.4 12.6 23.76 333
4.14 2.16 0.72 3.6 0.36 9.18 9 6.84 16.2 4.5 3.6 21.6 408
9.54 3.24 1.08 7.2 0.9 23.22 29.7 25.2 14.94 16.38 20.34 68.76 432
0.36 1.26 0.18 3.96 0.72 0.72 0.18 0.09 0.054 0.036 0.09 NFb 44.1
a b
Based on a daily wine consumption of 180 ml. NF, not found.
466
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Fig 2. Daily intake of catechins from consumption of varieties of wines for the French population.
environment of the population groups) is likely to metabolise some of these compounds. The ®rst step consists of identifying the intact molecules and/or their metabolites in the plasma. This analysis is very delicate to carry out since these compounds have dierent stabilities depending on the physical and biological conditions. A method developed recently associates a simple, rapid phase of plasma sample preparation with a highly sensitive detection method: ¯uorescence (Carando, Teissedre & Cabanis, 1998). This method has enabled us to identify (+)-catechin in plasma and to quantify it for several persons having consumed wine with their meal. A very large variability in plasma (+)-catechin concentrations (260±810 ng/ml) was found. At present, this method has only been applied to (+)catechin, but it would be of interest in the future to apply it to the analysis of other phenolics compounds. It would also be interesting to determine whether the plasma (+)-catechin comes directly from the wine (+)catechin or whether it is also likely to arise from procyanidin metabolism. 4. Conclusion Phenolics intake is 10 times higher from red wines (400.2 mg/person/day) than for white wines (44.1 mg/ person/day) consumption. Catechins intake can be highest with Egiodola (129.8 mg/day/person), CabernetSauvignon and Grenache varieties. Catechins monomers can represent 40% of total catechins. It would, of course, be important in the future to investigate other compounds, such as anthocyanines, in order to re®ne this estimation. Furthermore, it would be desirable to extend this estimation to stilbenes components of wine.
However, it will also be very important in the future to obtain data on the bioavailability of these compounds in the plasma. It has been established that (+)-catechin is present in the plasma after a meal during which wine had been consumed, but these data must now be extended to other phenolics and correlated to plasma antioxidant activity to give proof of the pharmacological action of phenolics molecules from wine in vivo. Acknowledgements This work was supported by Domaine Virginie (BeÂziers, France) We are grateful to F. Gasc for glass expertise.
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Wine phenolics: contribution to dietary intake and bioavailability Pierre-Louis Teissedre *, Nicolas Landrault Centre de Formation et de Recherche en Oenologie, Universite de Montpellier I, Faculte de Pharmacie, Av. Charles Flahault, 34060 Montpellier cedex 2, France Received 10 September 1999; accepted 3 January 2000
Abstract Current research suggests that wine contains substances that may reduce the mortality rate from coronary heart diseases. The oxidation of low-density liporoteins (LDL) is thought to be a key step in the development of atherosclerosis. In previous research, catechin oligomers, procyanidin dimers and trimers were extracted, isolated and puri®ed from grapes seeds and tested for their inhibition on LDL oxidation, along and with other monomeric wine phenolics.We quantify the level of phenolics, major catechins [(+)-catechin, (ÿ)-epicatechin, procyanidin dimers B1, B2, B3, B4], phenolics acids, caftaric acid and malvidine-3-glucoside by HPLC with UV detection for 60 French varietal commercial wines taken from the South of France in order to appreciate the daily dietary intake of these compounds for French population. Based on a still signi®cant French red wine consumption of 180 ml/day, the current daily intake of phenolics can be estimated at 400.2 mg/day/resident and catechins (monomers, dimers B1, B2, B3, B4) at 83.2 mg/day/resident, including 40% of monomers for the French population. Red wine, and particularly Egiodola, and CabernetSauvignon varieties, contributes to a very signi®cant proportion of phenolics dietary intake and, in particular catechins. These results provide a basis for epidemiological evaluation of phenolics and catechins intake by wine consumption in the French population. Few data exist on absorption and metabolism of phenolics from wine. Further studies are necessary to determine the behaviour of this class of compounds in vivo after wine absorption. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phenolics; Catechins; Levels; Dietary intake; Absorption
1. Introduction Epidemiological studies conducted during the last 20 years have shown that coronary heart diseases are less prevalent in countries consuming a regular and moderate consumption of wine (Friedman, & Kimball, 1986; Klatsky, & Armstrong, 1993; St-Leger, Cochrane & Moore, 1979; WHO., 1989). In one of the best known studies, Renaud and De Lorgeril (1992) suggest an explanation of the phenomenon particularly favourable to the French population with regard to cardiovascular disease, known as the ``French paradox'', which was ®rst described in 1987 by Richard. The results of the Monica program (1989), a worldwide CAD surveillance system organised by the World Health Organization (WHO), con®rm that mortality levels provoked by coronary artery disease are much lower in France than in other industrialised countries, even though the * Corresponding author. Tel.: +33-4-6754-4520; fax: +33-4-67544526. E-mail address: [email protected] (P.-L. Teissedre).
consumption of saturated fats in France is much the same and blood cholesterol levels are generally higher. Furthermore, other factors associated with risk of coronary artery disease, such as arterial blood pressure, body weight and smoking, are no lower in France than in the other countries. This is the French paradox. Renaud and De Lorgeril (1992) used WHO and OECD data to study which food parameters could be correlated with cardiovascular disease (CVD) mortality levels. Among the numerous dierent foods, only the consumption of dairy fats (between 1980 and 1985) showed positive correlation with CVD mortality (1987 levels), but this correlation was highly signi®cant (r=0.73, P<0.001). However, although the consumption of dairy fats is the same in France as in the UK, Australia and Germany, mortality from coronary artery disease is lower in France. Using multifactor analysis, Renaud and De Lorgeril revealed that in 17 winedrinking countries, the only dietary factor correlating signi®cantly with coronary artery disease mortality, apart from dairy fats, was wine (r=0.87, P <0.001). Moreover, wine consumption had a negative coecient,
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indicating a protective eect. It was thus realised that the French case was not paradoxical when wine consumption was taken into account. In this study, the protection aorded by wine consumption was also detected in Switzerland and other industrialised countries. From this, attention turned to the non-alcoholic fractions of wine. Wine, particularly red wine, is an important source of polyphenols which are capable of inhibiting the processes behind coronary artery disease. This hypothesis is supported by the results of recent epidemiological studies concerning foodstu polyphenols, particularly ¯avonoids. A correlation was also noticed between increasing levels of ¯avonoid ingestion from fruit and vegetables and reduction in coronary artery disease. The studies carried out by Hertog, Feskens, Hollman, Katan and Kromhout (1993), Knekt, Jarvinen, Reunanen and Maatela (1996) and Rimm, Katan, Ascherio, Stampfer and Willett. (1996) reveal the bene®ts of a diet rich in ¯avonoids. Polyunsaturated fatty acids occur as a major part of the low-density lipoproteins in blood and oxidation of these lipids components in low-density lipoproteins (LDL) may play a role in atherosclerosis (Steinberg, Parthasary, Carew, Khoo & Witzum, 1989). With a continued high level of oxidized lipids, the blood vessels damage reaction process continues and can lead to generation of foam cells and plaque, the symptoms of atherosclerosis. In addition, in vitro studies show that oxidation of human LDL can be inhibited by physiological levels of vitamins C and E (Jialal, Vega, & Grundy, 1990), and certain ¯avonoids (De Whalley, Rankin, Hoult, Jessup & Leake, 1990). Thus, the hypothesis was advanced that potent antioxidative phenolics in wine can prevent oxidation of LDL and explain the French paradox. Thus, the inhibition of human LDL was demonstrated by the addition of the mixture of polyphenols from wine (Frankel, Kanner, German & Kinsella, 1993). Red wine diluted 1000-fold inhibited the in vitro oxidation of human LDL signi®cantly more than a-tocopherol. Other eects of red wines include inhibition of chronic in¯ammation and thrombotic tendencies (Kinsella, Frankel, German & Kanner, 1993). (+)-Catechin and (ÿ)-epicatechin are the basic units of the catechins group. The procyanidins are formed from the association of several of these monomeric units: 2±5 units for catechin oligomers (e.g. Fig. 1), over 5 units for catechin polymers. The procyanidins dier in the position and con®guration of their monomeric linkages. The structures of procyanidin dimers B1, B2, B3 and B4 are the best known. These molecules possess a structure that confers them with an antioxidant property which can inhibit the processes leading in the long term to atherosclerosis and arterial thrombosis.
Fig 1. Antitoxidants wine ¯avonoids.
The ¯avonoids are the most lipophilic of the natural antioxidants, but less so than a-tocopherol. The atocopherol seems to be located in the lipid membrane within the phospholipid bilayer while the ¯avonoids are probably mainly located at the polar surface of the bilayer. The aqueous, i.e. transported in the plasma, free radicals would therefore be captured more easily by the ¯avonoids than by the less accessible a-tocopherol. Thus, the ¯avonoids could be concentrated near to the membranous surface of the low-density lipoprotein (LDL) particles, ready to capture the oxygenated aqueous free radicals. They would in this way prevent the consumption of lipophilic a-tocopherol and thus delay oxidation of the lipids contained in the LDL. Moreover, if, as research suggests, the initiation phase and the propagation phase of lipid peroxidation take place, respectively, at the surface and the interior of the membranes, then the ¯avonoids could well hinder the correct course of the reaction by limiting the initiation phase. Catechins and procyanidins have been shown in vitro to be powerful inhibitors of LDL oxidation, more so than a-tocopherol (Teissedre, Frankel, Waterhouse, Peleg & German, 1996), and of platelet aggregation (Ruf, Berger & Renaud, 1995). Moreover, it has recently been shown that the consumption of wine by humans leads to an increase in the antioxidant capacity of plasma (Furhmann, Lavy & Aviram, 1995). Other studies have been carried out on the antioxidant activity Ð through inhibition of copper-catalysed oxidation of human LDL Ð of a selection of Californian wines, made from Cabernet-Sauvignon, Merlot, Zinfandel, Petite Syrah, Pinot-noir, Sauvignon
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blanc and Chardonnay. The relative inhibition of LDL oxidation (calculated with respect to the total phenol concentration of each sample) varied from 46 to 100% for red wines and from 3 to 6% for white wines (Frankel, Waterhouse & Teissedre, 1995). The antioxidant activity of wines made with long maceration times exclusively from RhoÃne Valley Syrah and Grenache varieties was 60% relative inhibition of LDL oxidation (Teissedre, Waterhouse & Frankel, 1995). Comparison of these results with the dierent Californian red wines showed that the Rhodanian wines made from Syrah and Grenache gave values of relative inhibition of human LDL oxidation identical to those of Cabernet-sauvignon but less than those of Petite Syrah (80%) or Merlot (74%). In contrast, the Syrah wines produced with short extractions gave a low mean value of relative inhibition (16%) which is nevertheless 4 times higher than that obtained for Californian white wines (4.4%). It was deduced from this that each phenolic compound of wine could play a role in the protection against LDL oxidation. All the properties and studies reported support the present hypotheses for explaining the reduced risk of mortality from coronary artery disease in moderate and regular consumers of wine (particularly red wine). In the present study we evaluated total phenols content, the major catechins [(+)-catechin, (ÿ)-epicatechin, dimers B1, B2, B3, B4], some phenolic acids (cafeic acid, para-hydroxycoumaric acid, protocatechuic acid), a cinnamate, caftaric acid, and a major anthocyanine, mavidine-3-glucoside, for 60 commercial varietal wines taken from the same area of France, to provide concentrations data of phenolics for varietal Languedoc wines and to appreciate the daily dietary intake of phenolics for the French population.
2. Materials and methods 2.1. Materials (+)-Catechin, (ÿ)-epicatechin, gallic acid, cafeic acid, protocatechuic acid, para-hydroxycoumaric acid were obtained from Aldrich, malvidine-3-glucoside from ExtrasyntheÂse. Caftaric acid was provided by Ursa Vorshek. Procyanidins dimers B1, B2, B3, B4, were obtained from grape seeds as detailed below. 2.2. Wine samples We analysed 60 samples of dierent French varietal wines: 50 red (Merlot, Cabernet-Sauvignon, Grenache, Syrah, Egiodola); 10 white (Chardonnay) in commercial bottles from Domaine Virginie (BeÂziers, France). The wine samples analysed were from all viticultural areas of
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Languedoc and of the same vintage year, 1998. All wines analysed are frequently consumed in France. 2.3. Total phenols content Total phenols were analyzed by the Folin±Ciocalteu method (Singleton & Rossi, 1965), calibrating against gallic acid standards, and expressing the results as GAE. 2.4. Extraction and isolation of crude procyanidins Grape seeds (vitis vinifera), 150 g, were extracted with methanol as described by Bourzeix, Weyland and Heredia (1986) and by Weinges and Piretti (1971). The extract (3 ml, 300 mg) was chromatographed on Fractogel TSK HW-40(s) (25±40 mm) (450 25 mm id) with methanol as eluant, using an ISCO (Lincoln, NE, USA) model UA-5 absorbance monitor set at 280 nm, a peristaltic Miniplus2 pump (Gilson Inc, Middletin, WI, USA) and an ISCO 328 fraction collector. Ten fractions containing procyanidins were collected. 2.5. Isolation of puri®ed procyanidins Semi-preparative HPLC was performed with a Waters 510 pump (Waters, Miford, MA, USA) a U6K injector, and a Hewlett-Packard (Palo Alto, CA, USA) model 1050 UV±Vis detector set at 280 nm. The column was a Water RCM Novapak C18 25100 mm, 4 mm particle size. Elution was carried out by a linear gradient of 0± 500 ml/l methanol with the solvent described below at 2 ml/min. 2.6. TLC analysis Silica plates (DC Alufolien-Kieselgel 60, 0.2 mm thick, Merk, EM Separation technology, Gibbstown, NJ, USA), were developed with toluene/acetone/formic acid (3:3:1 v/v/v) as described by Lea, Bridle, Timberlake and Singleton. (1979). The plates were visualised by spraying with a solution of vanillin (100 g/l) in concentrated HCL. 2.7. HPLC analysis A Hewlett-Packard model 1090 with three low-pressure pumps and a diode array UV detector coupled to a Hewlett-Packard Chem station was used for solvent delivery system and detection. A Hewlett-Packard column Nucleosil 100 C18, 2504 mm, 5 mm particle size was used for the stationary phase with a ¯ow of 0.7 ml/ min. The solvents used for separation were: solvent A: 50 mM dihydrogen ammonium phosphate adjusted to pH 2.6 with orthophosphoric acid; solvent B: 20% A with 80% acetonitrile; and solvent C: 0.2 M orthophosphoric acid adjusted with ammonia to pH 1.5, and solvent
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gradient conditions as described by Lamuela-Raventos and Waterhouse. (1994). Temperature was thermostated at 25 C. 3. Results and discussion Analytical studies on phenolics and ¯avonoids in wine revealed that this beverage contains high levels and that the dierent wine-making techniques are important factors (Bourzeix et al., 1986). However, the results cited here concern wines made from California (Waterhouse & Teissedre, 1997) or from micro-vini®cation and not intended for consumption. The aim of this study was to analyse the phenolics and catechins content of varietal wines from the south French region made from diverse varieties and using varied vini®cation techniques. The results obtained were used to evaluate the phenolics and catechins intake from regular, moderate wine consumption. Red wines had generally much higher levels of phenolics than white wines. This dierence is the result of dierent wine-making techniques. The catechins in grapes, present mainly in the pips, diuse into the must during the course of maceration. The maceration phase constitutes a fundamental dierence in the vini®cation of red and white wine. It is essential for the production of red wine and is deliberately avoided for the production of white, where rapid pressing separates the liquid phase from the solid matter. The concentrations of total phenol as determined by the Folin±Ciocalteu method varied from 1847 to 2600 mg/l GAE, for the red wines and are of 245 mg/l GAE for the Chardonnay white wines (Table 1). The most interesting varieties for high levels of total phenol are Cabernet-Sauvignon, Egiodola and Syrah.
Thus, the red wines studied had high overall levels of catechins (sum of monomers and procyanidin dimers analysed) Ð 462.6 mg/l Ð but there were considerable dierences between the red wines (Table 1). Levels of catechins in the white wines were lower: 6.5 mg/l. All the compounds studied were present in each of the 50 red wines analysed. For the catechin monomers, the mean concentration [sum of (+)-catechin and (ÿ)-epicatechin] was 202.6 mg/l. The quantity of procyanidin dimers (sum of B1, B2, B3, B4) was 260 mg/l and that of malvidine-3-glucoside was 164.4 mg/l. The white wines (n=10) showed very low concentrations of monomers (5 mg/l) and absence of malvidine-3-glucoside. The highest concentrations of catechins in red varietal wines were found in Egiodola (732 mg/l), Cabernet-Sauvignon and Grenache (close to 500 mg/l). Egiodola was found richest in malvidine-3-glucoside (382 mg/l), up to 4 times more than the other red varieties. The mean contents of each phenolic are given in Table 1. 3.1. Estimation of phenolics and catechins intake in moderate wine consumers in France Over the last few years, the consumption of wine in France has fallen considerably. In 1986, the mean consumption was 305 ml/person/day (Darret, Couzy, Antoine, Magliola & Mareschi, 1986). This level fell sharply to 180 ml/person/day in 1995 (Boulet, Laporte, Aigrin & Lalanne, 1995). An estimation of the intake of phenolics and catechins was calculated from these latest consumption ®gures and our own analytical results on the catechin (monomers, dimers,) content of 60 wines. Although the levels of these compounds vary considerably from one varietal wine to another, the regular consumption of the dierent products available to
Table 1 Average levels of phenolics in Languedoc varieties wines Varieties a
Phenolics
Merlot
Cabernet-Sauvignon
Grenache
Syrah
Egiodola
Chardonnay
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol contentb
195.3 192.5 409.2 317.7 10.13 7636.5 8760.9 5026.5 4727.6 2512.2 4513.5 9122.3 2000514
5312.1 3010.1 41.2 102.6 20.18 12027.6 12040.8 9915.1 5320.3 4411.5 5323.2 9738.9 2600774
227.1 4911.2 20.6 377.8 10.09 11044.5 10542.8 776.8 5623.4 8018.2 7030.1 13217.8 1847332
237.2 124.1 41 203.1 20.14 5120.2 5022.6 388.7 9014.5 254.9 203.9 12020.5 2266294
5323.6 184.7 60.9 4012.1 50.43 12935.8 16541.8 14041.2 8334.5 9137.8 11347.9 382123 2400240
20.3 71.4 10.4 2215.1 41.1 42.3 1O.9 0.50.1 0.30.67 0.20.14 0.50.45 NFc 24563
a b c
Values expressed in mg/lS.D. Expressed in mg Gallic acid equivalent/lS.D. NF, not found.
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the consumer tends toward a mean value which we considered to be close to that calculated for the 60 wines. For this reason, this estimation can only be considered for regular consumption of the same variety wine over a suciently long period of time (which remains to be determined statistically). Daily intake of each phenolic compound by variety wine is indicated in Table 2. The consumption of 180 ml of CabernetSauvignon or Egiodola wine, for which the mean phenolics concentration are, respectively, 2600 and 2400 mg/l, gives a mean daily intake of 468 and 432 mg of total phenolics. This reasoning applied to white wine for regular (daily), moderate (180 ml) consumption gives estimations of phenolics intake of only 44.1 mg for white Chardonnay wines, 10 times less than for the red (Table 3). Daily intake of catechins from varieties wines (Fig. 2) ranged from 0.9 to 59.2 mg/person for monomers (catechin and epicatechin), and from 1.2 to 129.8 mg/ person for total catechins (including monomers and dimers). The best results were obtained with Egiodola, Cabernet-Sauvignon, and Grenache varieties. Amounts of total catechins daily intake of Egiodola was twice than for Merlot or Syrah, and almost 110 times more than for Chardonnay. Cabernet-Sauvignon and Grenache gave interesting daily intake results for total catechins close to 90 mg/person. 3.2. Absorption of phenolics and catechins in the plasma after wine consumption. Although epidemiological studies note a correlation between the reduction of risk of coronary artery disease and the regular, moderate consumption of wine, no causal relationship has been demonstrated. The ¯avonoids in wine constitute a considerable LDL antioxidant and platelet anti-aggregant potential, but it
465
is dicult to know whether this potential is slightly, partially or totally realised during metabolism. It would appear necessary, therefore, to supply irrefutable biochemical proof of the envisaged protective mechanisms. Although compounds such as (+)-catechin, (ÿ)-epicatechin, procyanidin dimers B1, B2, B3, B4 and trimers C1, C2 inhibit LDL oxidation in vitro (Teissedre et al., 1996), no account can be taken of the large variability in the composition of blood plasma in order to verify whether these compounds or their metabolites can be suciently active in vivo to aord protection, even though epidemiological studies suggest that they do. Current research concerns absorption of these compounds in human plasma after wine consumption. The intestinal ¯ora (which varies in function of the
Table 3 Daily intake of phenolics from white or red wines Average intake in mg/day/persona Phenolics
White wine
Red wine
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol
0.36 1.26 0.18 3.96 0.72 0.72 0.18 0.09 0.054 0.036 0.09 NFb 44.1
6.12 4.61 2.02 4.97 0.40 17.50 18.97 14.54 11.84 9.54 10.84 29.59 400.2
a b
Based on a daily wine consumption of 180 ml. NF, not found.
Table 2 Daily intake of phenolics from varieties of wines consumption for the French population Daily intake in mg/day/persona Phenolics
Merlot
Cabernet-Sauvignon
Grenache
Syrah
Egiodola
Chardonnay
Gallic acid Caeic acid Para-hydroxycoumaric acid Caftaric acid Protocatechuic acid Catechin Epicatechin Dimer B1 Dimer B2 Dimer B3 Dimer B4 Malvidine-3-glucoside Total phenol
3.42 3.42 7.2 5.58 0.18 13.68 15.66 9 8.46 4.5 8.1 16.38 360
9.54 5.4 0.72 1.8 0.36 21.6 21.6 17.82 9.54 7.92 9.54 17.46 468
3.96 8.82 0.36 6.66 0.18 19.8 18.9 13.86 10.08 14.4 12.6 23.76 333
4.14 2.16 0.72 3.6 0.36 9.18 9 6.84 16.2 4.5 3.6 21.6 408
9.54 3.24 1.08 7.2 0.9 23.22 29.7 25.2 14.94 16.38 20.34 68.76 432
0.36 1.26 0.18 3.96 0.72 0.72 0.18 0.09 0.054 0.036 0.09 NFb 44.1
a b
Based on a daily wine consumption of 180 ml. NF, not found.
466
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Fig 2. Daily intake of catechins from consumption of varieties of wines for the French population.
environment of the population groups) is likely to metabolise some of these compounds. The ®rst step consists of identifying the intact molecules and/or their metabolites in the plasma. This analysis is very delicate to carry out since these compounds have dierent stabilities depending on the physical and biological conditions. A method developed recently associates a simple, rapid phase of plasma sample preparation with a highly sensitive detection method: ¯uorescence (Carando, Teissedre & Cabanis, 1998). This method has enabled us to identify (+)-catechin in plasma and to quantify it for several persons having consumed wine with their meal. A very large variability in plasma (+)-catechin concentrations (260±810 ng/ml) was found. At present, this method has only been applied to (+)catechin, but it would be of interest in the future to apply it to the analysis of other phenolics compounds. It would also be interesting to determine whether the plasma (+)-catechin comes directly from the wine (+)catechin or whether it is also likely to arise from procyanidin metabolism. 4. Conclusion Phenolics intake is 10 times higher from red wines (400.2 mg/person/day) than for white wines (44.1 mg/ person/day) consumption. Catechins intake can be highest with Egiodola (129.8 mg/day/person), CabernetSauvignon and Grenache varieties. Catechins monomers can represent 40% of total catechins. It would, of course, be important in the future to investigate other compounds, such as anthocyanines, in order to re®ne this estimation. Furthermore, it would be desirable to extend this estimation to stilbenes components of wine.
However, it will also be very important in the future to obtain data on the bioavailability of these compounds in the plasma. It has been established that (+)-catechin is present in the plasma after a meal during which wine had been consumed, but these data must now be extended to other phenolics and correlated to plasma antioxidant activity to give proof of the pharmacological action of phenolics molecules from wine in vivo. Acknowledgements This work was supported by Domaine Virginie (BeÂziers, France) We are grateful to F. Gasc for glass expertise.
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