Direct liquid chromatographic analysis of resveratrol derivatives and flavanonols in wines with absorbance and fluorescence detection

Analytica Chimica Acta 458 (2002) 103–110

Direct liquid chromatographic analysis of resveratrol derivatives and flavanonols in wines with absorbance and fluorescence detection Xavier Vitrac, Jean-Pierre Monti, Joseph Vercauteren, Gérard Deffieux, Jean-Michel Mérillon∗ Groupe d’Etude des Substances, Naturelles à Intérêt Thérapeutique, EA 491, Faculté des Sciences Pharmaceutiques, Université de Bordeaux, 2,3 ter, Place de la Victoire, 33076 Bordeaux Cedex, France Received 20 June 2001; received in revised form 31 August 2001; accepted 30 October 2001

Abstract Wine contains a large number of polyphenols including stilbenes, flavanols and anthocyanins. Of these, stilbenes have been reported to have potential chemopreventive activities. We describe the simultaneous determination of six resveratrol derivatives in wines by liquid chromatography (LC) with fluorescence detection. The levels of pallidol, the symmetrical dimer of resveratrol, are reported for the first time. Quantifications were carried out at optimal wavelengths for each compound during separation. A total of 19 red and 30 white commercial wines from South-Western France were analysed, and the highest stilbene concentrations were found in red wines. In addition, the levels of catechins and two flavanonols recently isolated in red wine are reported. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Stilbenes; Pallidol; Astilbin; Dihydromyricetin-3-O-rhamnoside; Wine; Fluorescence; Liquid chromatography

1. Introduction Stilbenes are biologically active secondary metabolites found in numerous families of plants, and their major dietary sources are grapes, wine and peanuts. Among stilbene monomers, trans-resveratrol has been the most widely studied grapevine phytoalexin, both for its role in grapevine–pathogen interactions and on human health. It may be responsible, in part, for the decreased mortality by cardiovascular diseases observed in moderate and regular wine-consuming ∗ Corresponding author. Tel.: +33-5-57-57-18-22; fax: +33-5-56-91-79-88. E-mail address: [email protected] (J.-M. M´erillon).

populations [1]. According to in vitro and animal studies, it has anticancer properties, inhibiting cellular events associated with tumour initiation, promotion and progression. Moreover, it inhibits cyclooxygenase-2 activity which reduces the synthesis of prostaglandins, thus, slowing tumour cell growth [2]. However, only a few studies have set out to determine the levels of the other stilbenes found in wines, with the exception of the cis-isomer of resveratrol, and their 3-O-␤-glucosides, the so-called piceids. Trans-astringin (3,5,3 ,4 -tetrahydroxystilbene-3-O-␤glucoside) has recently been determined in Portuguese and French wines [3], and some resveratrol dimers have also been characterised such as pallidol monoand di-glucosides [4], the resveratrol dimer ε-viniferin [5], parthenocissin A and pallidol [6].

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 4 9 8 - 2

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Since Siemann and Creasy [7] described the presence of trans-resveratrol in wine, several methods to determine its concentration have been described. They generally require multi-step sample pre-treatments such as solid-phase extraction or pre-concentration prior to separation [8,9]. Gas chromatography (GC) has been used by several authors [10–12] but it requires derivatisation to enhance the volatility of resveratrol. More recently, a capillary electrophoresis technique has also been proposed [13,14]. Although liquid chromatography (LC) techniques are the most commonly used procedures, the difficulty in interpreting chromatograms makes it necessary to optimise detection. To this end, fluorescence detection has been used by several authors and good sensitivity and specificity have been obtained in most cases [15–18]. The aim of the present study was to monitor several stilbene derivatives and catechins in wine by LC with direct injection and detection by fluorescence. In addition, flavanonols recently isolated in red wine were determined with UV absorbance detection since these compounds are not fluorescent. This theme is of particular relevance since it is important to monitor polyphenols in food and beverages to evaluate their antioxidant potential. Indeed, numerous stilbenes have properties similar to those of trans-resveratrol in inhibiting platelet aggregation [19,20] and the oxidation of low density lipoproteins (LDLs) [21,22].

2. Experimental 2.1. Reagents All solvents were of liquid chromatographic grade (Scharlaud), except H2 O which was distilled and filtered through a Millipore membrane (0.22 ␮m). Solutions were degassed before use. Chemically pure standards of stilbenes (trans- and cis-piceid, trans-astringin, pallidol) were purified from grape cell suspension cultures and unambiguously characterised by spectrometric methods [23,24]. Trans-resveratrol was from Sigma, and cis-resveratrol was obtained by enzymatic hydrolysis of cis-piceid with a ␤-glucosidase (EC 3.2.1.21, Sigma) as follows: 10 mg of enzyme was mixed with 10 mg of compound in 10 ml of H2 O adjusted to pH 6 with NaOH (0.1 M). The mixture was incubated at 25 ◦ C overnight (15 h)

and extracted twice with EtOAc. Cis-resveratrol was purified by semi-preparative LC. Astilbin and dihydromyricetin-3-O-rhamnoside were isolated from red wine by a combination of chromatographic techniques as previously described [6]. Stock solutions (10 mg l−1 ) were prepared in 50% aqueous MeOH and kept at 4 ◦ C in the dark. Working solutions were prepared by dilution with the mobile phase. 2.2. Apparatus The analyses were carried out in a Bischoff liquid chromatograph equipped with two pumps (model 2250), an automated gradient controller (Normasoft software) and an automated injector (Alcott, model 708). Detection was carried out either with a programmable fluorescence detector (Groton, model FD-500) or with a UV–VIS detector (Kontron, model 430) at room temperature. 2.3. Chromatographic conditions The chromatographic analysis was carried out with a Prontosil C18 4.0 mm × 250 mm column (4 ␮m particle size, Bischoff) with a guard cartridge of the same material. For determination of stilbenes, we used H2 O/trifluoro acetic acid (TFA) (99/1, v/v) as solvent A, and acetonitrile/solvent A (80/20, v/v) as solvent B, at a flow rate of 1.0 ml min−1 with the following gradient: 15% B (0–10 min), 15–18% B (10–13 min), 18% B (13–15 min), 18–23% B (15–17 min), 23–25% B (17–21 min), 25% B (21–24 min), 25–32% B (24–28 min), 32% B (28–30 min), 32–40% B (30–33 min), 40% B (33– 38 min), 40–70% B (38–44 min), 70–80% B (44–47 min), 80–90% B (47–52 min), 90–100% B (52–53 min) and 100% B (53–58 min). This was followed by a 10 min equilibrium period with initial conditions prior to injection of the next sample. Wine samples were filtered (0.45 ␮m, Millipore) and 20 ␮l was directly injected. Detection was carried out by monitoring the emission signals of the programmable fluorescence detector (Groton, model FD-500) set at optimal wavelengths for each compound during the chromatographic separation. Excitation and emission scans for each pure compound were done in chromatography solvents with a scan rate of 30 nm min−1 from 200 to 450 nm. The fluorescence detection

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parameters were a 90 Hz lamp frequency, a 200 nA range and a 10% scale autozero. Standard deviations calculated for six analyses of the same sample were <5% for all the compounds studied. Determination of astilbin and dihydromyricetin-3O-rhamnoside was done with the same LC solvents and flow rate as those used for stilbenes, with the gradient as follows: 18% B (0–10 min), 18–23% B (10–17 min), 23–24.5% B (17–21 min), 24.5–31.5% B (21–27 min), 31.5–40% B (27–30 min), 40–80% B (30–32 min), 80–100% B (32–35 min) and 100% B (35–40 min). This was followed by a 10 min equilibrium period with initial conditions prior to injection of the next sample. Wine samples were filtered and 100 ␮l was directly injected. Chromatograms were monitored at 290 nm using the UV detector. 2.4. Wine samples A group of 49 commercially available wines from AOC Bergerac (France) were analysed; 19 were blended red wines, 18 were dry white wines, and 12 were sweet white wines. All the samples were analysed during the year 2001.

3. Results and discussion 3.1. Optimisation of the LC procedure In the last 10 years, several methods to determine trans-resveratrol and its derivatives (mainly cis-resveratrol, trans- and cis-piceid) in wine have been reported. They include GC, GC–mass spectrometry (MS) methods and more recently, capillary electrophoresis. However, LC has been the most frequently used technique due to its widespread availability. Many of the published LC methods require liquid–liquid extraction or solid phase extraction (SPE) with C-18 or SAX cartridges before the LC analysis, which may result in modifications of the phenolic composition and significant losses of compounds. We resolved this problem by direct injection, a procedure which has already been successfully used [5,15,17,25,26]. The complexity of wine samples makes it necessary to optimise the gradient in order to separate in the same run both typical and unusual stilbenes whose content

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is of interest. The chromatographic solvent system reported by other workers for the analysis of stilbenes in wines was first used, i.e. a solvent mixture of H2 O/acetic acid/acetonitrile [26]. However, it did not produce sufficiently separated peaks. The resolution was improved by replacing acetic acid by TFA, which led to the satisfactory separation of the six stilbenes in the same run. Since (+)-catechin and (−)-epicatechin are highly fluorescent, we attempted to achieve good separation from stilbenes using a gradient elution technique. Several gradients were tried which gave different profiles. The optimal gradient was started at a low concentration of solvent B (15%), which allowed the separation of catechins from the void time. Fig. 1 shows the chromatograms corresponding to a solution of standards and a directly injected sample of red wine. As previously reported, stilbenes are easily detected by fluorimetry [15]. Moreover, their fluorimetric detection is more sensitive and specific than UV absorbance detection. The main stilbenes detected in wine in this study (Fig. 2) were transand cis-resveratrol and their glucosides (piceids), together with trans-astringin and the recently isolated symmetrical dimer of resveratrol, pallidol [6]. The fluorescence properties of each compound were then investigated in order to determine the optimal excitation and emission wavelengths to use during the chromatographic separation. They were automatically modified according to the retention time of each compound and the ease with which they could be precisely changed within the elution profile of the sample. Unlike Adrian et al. [5] who used fixed excitation and emission wavelengths, we did not find the same characteristics for all stilbenes (Table 1), a result certainly due to our different solvent system. Considering the different spectral characteristics of the compounds studied, we thought that by obtaining a calibration graph for each stilbene at optimal wavelengths, this would improve sensitivity. We, therefore, injected standard solutions in the range 0.1–10 mg l−1 and obtained graphs by plotting concentration against peak area, each point being the mean value from three independent area measurements. Table 1 shows the regression coefficients, the detection and quantification limits, and the linearity intervals obtained for each compound. The quantification and detection limits were calculated as the concentrations giving signals 10 times and 3 times as high

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Fig. 1. Chromatographic profiles using fluorescence detection for a standard mixture of polyphenols (A) and a directly injected sample of red wine (B): 1, (+)-catechin; 2, (−)-epicatechin; 3, trans-astringin; 4, trans-piceid; 5, cis-piceid; 6, trans-resveratrol; 7, pallidol; 8, cis-resveratrol. Chromatographic conditions are described in Section 2 and the wavelengths changes are shown at the top of the figure.

as the standard deviation (S.D.) of the blank value, respectively. 3.2. Stilbenes concentrations in wines Once the chromatographic conditions for the separation and detection were optimised, the procedure was used to determine stilbenes in wines from South-Western France (AOC Bergerac). Table 2 show

the concentrations of the main resveratrol derivatives determined by the LC method. Determination of trans- and cis-resveratrol levels in red wines showed mean values of 2.3 and 0.1 mg l−1 , respectively. Moreover, white wines showed total values <1 mg l−1 for both isomers. This result corroborates other studies which show that red wines from various countries and regions have a low mean concentration of trans- and cis-resveratrol, <5 mg l−1 [26,27].

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Fig. 2. Structures of the compounds studied.

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Table 1 Parameters for the calibration of polyphenolic compounds using fluorescence detectiona Compound

Wavelengths (nm) (λEx /λEm )

Linear range (mg l−1 )

LOQ (mg l−1 )

LOD (mg l−1 )

Intercept

Slope

(+)-Catechin (−)-Epicatechin Trans-astringin Trans-piceid Cis-piceid Trans-resveratrol Pallidol Cis-resveratrol

280/310 280/310 290/390 290/390 260/400 300/390 260/400 260/400

0.5–10 0.5–10 0.5–10 0.1–10 1.0–10 0.5–10 0.5–10 1.0–10

0.07 0.05 0.06 0.05 0.1 0.03 0.03 0.07

0.02 0.01 0.01 0.01 0.03 0.01 0.02 0.02

−5.50 0.50 0.89 1.89 −0.09 0.60 3.78 −0.10

86.70 75.75 30.36 149.67 23.95 50.78 45.26 22.60

a

LOQ: limit of quantification; LOD: limit of detection.

Concerning trans- and cis-piceid, their levels exceeded those of the free isomers and reached maximal concentrations of 26 and 24 mg l−1 , respectively, in red wines; cis-piceid is typically found at lower concentrations than trans-piceid in wines. The maximal value for cis-piceid in white wines was found to be 0.9 mg l−1 , that of the trans-isomer being 2.9 mg l−1 . Trans-astringin is sometimes found at higher concentrations than piceids in wines, some wines having concentrations >30 mg l−1 . However, there were wide variations between the wine samples and trans-astringin was not detected in all of them. In this study, levels of pallidol are reported in red wine for the first time. Concentrations ranged from 0.5 to 4.8 mg l−1 for red wines, with a mean value of 2.5 mg l−1 . It could not be detected in dry or sweet white wines. It has been shown that pallidol and some other resveratrol dimers are fungal metabolites of resveratrol [28]. Thus, the occurrence of this compound in wine is certainly due to the oxidation

of resveratrol by fungus in infected berries used for vinification. Red wines contain larger amounts of stilbenes than white wines, regardless of the oenological technology applied. The extent of maceration with skins and seeds during fermentation is the main factor determining the concentration of stilbenes in wines. They generally require long maceration on the skins to be extracted efficiently [29,30]. As already reported [31], we found that total stilbene levels reached mean concentrations up to 20 mg l−1 often with a predominance of the glucoside isomers, depending on multiple factors such as grape cultivar, fungal pressure and climate. Moreover, ␤-glucosidase could hydrolyse glycosidically-bound derivatives and release the corresponding resveratrol isomer [29]. Furthermore, it has been demonstrated that the addition of a recombinant yeast strain expressing the BgiN gene encoding a ␤-glucosidase during vinification increases the amounts of total resveratrol derivatives, especially the non-glycosylated

Table 2 Stilbene levels in red and white wine samplesa Compound

Red wines Range

Trans-astringin Trans-piceid Cis-piceid Trans-resveratrol Cis-resveratrol Pallidol a

(mg l−1 )

N.D.–38.1 0.1–26.0 N.D.–24.1 0.9–3.8 N.D.–0.9 0.5–4.8

White wines Average 6.3 6.2 3.8 2.3 0.1 2.5

± ± ± ± ± ±

1.8 0.5 0.5 0.4 0.2 0.2

Range (mg l−1 )

Average

N.D.–8.5 N.D.–2.9 N.D.–0.9 N.D.–0.2 N.D.–0.1 N.D.

1.4 ± 0.5 ± 0.3 ± 0.06 ± 0.03 ± N.D.

N.D.: not detected. Red wines, n = 19; white wines, n = 30. Values are given in mg l−1 ± S.D.

0.2 0.8 0.2 0.05 0.03

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Table 3 Flavonoid levels in red and white wine samplesa Compound

Red wines Range

(+)-Catechin (−)-Epicatechin Astilbin Dihydromyricetin-3-O-rhamnoside a

(mg l−1 )

1.4–85.1 N.D.–106.6 6.4–15.6 20.9–69.1

White wines Average 46.4 32.9 10.0 44.7

± ± ± ±

10.3 6.1 1.2 14.8

Range (mg l−1 )

Average

2.3–28.7 0.2–71.7 0.6–4.4 1.8–6.0

28.3 33.8 2.2 3.0

± ± ± ±

8.0 10.0 3.3 1.3

N.D.: not detected. Red wines, n = 19; white wines, n = 30. Values are given in mg l−1 ± S.D.

forms [32]. These findings are interesting since such oenological practices may enhance the quantity of resveratrol available in the human diet. In addition, hydrolysis of resveratrol glucosides by a ␤-glucosidase can occur in human small intestine and liver, as reported by Day et al. for flavonoid and isoflavonoid glucosides [36]. Wine stilbenes appear to have properties that may contribute to the reduction of the incidence of coronary heart disease observed in moderate wine drinkers. The data on the levels of all the resveratrol derivatives in wine reported in this study suggest that the amount available to induce physiological effects may be greater than previously thought. All our samples showed significant stilbene levels and the compounds are now recognised as potent antioxidants. For example, trans- and cis-resveratrol, trans- and cis-piceid, and trans-astringin are known to be significantly active molecules against LDL oxidation in vitro [22]. 3.3. Flavonoids levels in wines The progressive gradient run developed in this study allowed the analysis of (+)-catechin and (−)-epicatechin. Concentrations of catechin have a mean value of 46 mg l−1 in red wines and 28 mg l−1 in white wines (Table 3). These levels are lower than those found by other authors [33,34]. Epicatechin has lower values (33 mg l−1 ) than catechin in red wines and surprisingly higher values (34 mg l−1 ) in white wines. The unexpected high epicatechin levels encountered in white wines may be due to a possible coelution with other wine components also showing fluorescence, although the LC procedure has been carefully optimised.

In a recent paper, we reported the isolation of astilbin and its 5 -hydroxylated derivative (dihydromyricetin-3-O-rhamnoside) in red wine [6]. Since these flavanonols show no fluorescence in our experimental conditions, we used the LC method with UV absorbance detection in order to determine their levels in wines. Maximal absorption wavelengths for these two compounds were found to be 290 nm. In this case, the chromatographic conditions were very similar to those used for stilbenes, except that the gradient was shorter since the first and last steps were unnecessary. The corresponding peaks were identified by comparing the retention time obtained for the standard mixture, the wine sample and the wine sample spiked with the standards under identical conditions (not shown). Levels of astilbin in red and white wines are shown in Table 2. The highest levels were found in red wines (15.6 mg l−1 ) with a mean value of 10 mg l−1 . In white wines, astilbin levels were five times lower, averaging 2.2 mg l−1 . Its hydroxylated derivative was found at higher levels in all the wines, with mean values of 44.7 and 3.0 mg l−1 in red and white wines, respectively. The levels of these two compounds are therefore, reported in wines for the first time, and it has already been shown that astilbin is present in grape stems [35]. It would now be interesting to study the effect of maceration with stems on its levels in wines. 4. Conclusions In this paper, we describe a one-step chromatographic method suitable for the analysis of the five major stilbenes of wine, together with the recently characterised stilbene pallidol. The use of fluorescence detection is well adapted to the low

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concentrations of stilbenes in wines. In particular, no prior treatment of the wine sample is required. The method can also be useful to identify and quantify other fluorescent compounds in wine, such as catechins. Although stilbene concentrations depend on multiple factors such as climate, geographical origin, fungal pressure and vinification procedures, the variations of their concentrations in relation to each parameter remain poorly understood. Indeed, wines produced in South-Western France are blends of several grape cultivars in one appellation.

Acknowledgements The authors thank Ray Cooke for reading the manuscript. Research was supported by the Conseil Interprofessionnel des Vins de la Région de Bergerac (CIFRE Fellowship no. 552/98 to X. Vitrac and Grant no. 980305002) and the Région Aquitaine (Grant no. 990305014). References [1] S.C. Renaud, R. Guéguen, G. Siest, R. Salamon, Arch. Intern. Med. 159 (1999) 1865. [2] M.J. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W.W. Beecher, H.H.S. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon, J.M. Pezzuto, Science 275 (1997) 218. [3] M.T. Ribeiro de Lima, P. Waffo-Téguo, P.L. Teissedre, A. Pujolas, J. Vercauteren, J. C Cabanis, J.M. Mérillon, J. Agric. Food Chem. 47 (1999) 2666. [4] B. Baderschneider, P. Winterhalter, J. Agric. Food Chem. 48 (2000) 2681. [5] M. Adrian, P. Jeandet, A.C. Breuil, D. Levite, S. Debord, R. Bessis, Am. J. Enol. Vitic. 51 (2000) 37. [6] X. Vitrac, C. Castagnino, P. Waffo-Téguo, J.C. Delaunay, J. Vercauteren, G. Deffieux, J.M. Mérillon, J. Agric. Food Chem., in press. [7] E.H. Siemann, L.L. Creasy, Am. J. Enol. Vitic. 43 (1992) 49. [8] F. Mattivi, Z. Lebensm Unters Forsch 196 (1993) 522. [9] R.M. Lamuela-Raventos, A.L. Waterhouse, J. Agric. Food Chem. 41 (1993) 521. [10] D.M. Goldberg, E. Ng, A. Karumanchiri, E.P. Diamantis, G.J. Soleas, J. Chromatogr. A 708 (1995) 89.

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