Reagents and standards

Methanol and acetonitrile were obtained from Scharlab® S.L. (Sentmenat, Spain), trifluoroacetic acid (TFA) from Sigma-Aldrich® and deuterated solvents from Euriso-Top. Water was purified using an Elga water purification system (Bucks, UK) with a resistivity of no less than 18 MΩ/cm.

Wine samples

Seven Tunisian red wines from the Mornag appellation (Clipea, Distinto, Mornag, Selian, Sidi Brahim, Sidi Rais and Sidi Zahia) were examined for their stilbene content by using UPLC-MS and HPLC-DAD methods. The wines were purchased from local stores and kept in the dark until analyzed.

Stilbene pre-purification

Red wine is a complex mixture of molecules. Among these, stilbenoids are in very low concentration and are often masked by other compounds, which sometimes makes it difficult to determine and quantify the stilbenes by techniques such as HPLC-DAD. Thus, in order to remove compounds such as sugars, anthocyanins or proanthocyanidins for example, we had to carry out a pre-purification step. The different wines were evaporated and purified on XAD16 and DOWEX columns (figure 1). These methods for the pre-purification of polyphenols have been developed and adapted from techniques already used in our laboratory and previously described concerning DOWEX columns (Decendit and Mérillon, 1996 ; Waffo-Téguo et al., 1996) or XAD16 columns (Pawlus et al., 2013). Each 750-mL bottle of wine was concentrated down to 650 mL in vacuo at 30 °C and diluted with 100 mL of water. The concentrated wine was poured over 500 g of Amberlite XAD-16 resin (Sigma-Aldrich), in an open column (5.1 cm id × 35 cm), and rinsed with 5 L of water to remove sugars, small organic acids and other non-polyphenolic compounds. The polyphenols were then eluted with 3 L of methanol and 1 L of acetone. The solvents were removed in vacuo and the samples were lyophilized. The concentrated XAD16 residue was then chromatographed over a cation-exchange resin column (DOWEX, Sigma-Aldrich), rinsed with distilled water (4 L), and eluted with 75% (v/v) aqueous methanol (2 L) to yield a solid powder after lyophilization. In order to evaluate the accuracy of the purification method, we proceeded to verify the chromatographic steps with mini-columns (0.8 mm id × 5 cm) and two pure stilbenoid standards ((E)-resveratrol and (E)-piceid); the proportions between the amount of deposited stilbenes, the amount of resins, and the volumes of water and eluents for each step were conserved. For example, 10 µg of each stilbene were dissolved in 2 mL water and submitted to these two chromatographic steps in these conditions. The various steps were followed by HPLC as described below. We were not able to detect stilbene in the rinsing waters of the columns, which means that there is apparently no loss of stilbenoids at this level. Elution with 75% methanol at the last step with the DOWEX column allowed the recover 96.9 ± 0.9% and 92.9 ± 1.6% of piceid and resveratrol, respectively. These experiments were confirmed a second time. It should also be noted that, when the amount of stilbenes deposited on the columns was 10 times greater, we observed a loss of resveratrol in the rinsing waters during the step of the XAD16 column (approximately one third). Under these conditions, the limit of solubility of resveratrol in water is exceeded and we have a precipitate, which cannot be retained by the XAD-16 resin. These stilbene concentrations here (equivalent to 100 mg/L) are also much too high compared to those found in wine.

Analytical HPLC-DAD

Analyses were performed by analytical HPLC-DAD using a HPLC system model 1100 (Agilent technologies) coupled with a diode array detector (DAD). 20 µL of each sample was injected for each chromatographic analysis. Polyphenolic extracts were separated on a Bischoff® (Stuttgart, Germany) Prontosil® reverse-phase C₁₈ column (5 µm packing, 4 mm id × 250 mm) protected with a guard column of the same material. The solvent system used was ultrapure water acidified with 0.1% TFA (solvent A), and acetonitrile acidified with 0.1% TFA (solvent B). The elution program at 1 mL/min was 10% B (0-5 min), 10-60% B (5-35 min), 60% B (35-40 min) followed by a 5 min wash with 100% B and a 5 min reequilibration step. The chromatograms were monitored at 290 and 306 nm and the spectra (200-600 nm) continuously recorded.

UPLC-MS analysis

Monitoring of the collected Tunisian red wines was achieved by UPLC-MS. The UPLC analyses were carried out using an UPLC system model 1290 Infinity (Agilent Technologies) coupled to an Esquire 3000+ mass spectrometer (Bruker Daltonics GmbH) with electrospray ionization and ion trap analyzer. The column used was a C₁₈ reversed-phase (100 × 2.1 mm, 1.8 µm, Agilent). Water 0.1% formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) were used as mobile phases. 2.1 µL of each sample was injected and analyzed at 25 °C. The elution program at 0.21 mL/min was 10% B (0-2 min), 10-60% B (2-14 min), 60% B (14-16 min) followed by a 2 min wash with 100% B and a 5 min reequilibration step. The detection wavelengths were set at 290 and 306 nm. Electrospray ionization in positive and negative mode was used.

Figure 1 - Flow diagram of primary method used for the different stages of pre-purification of wine stilbenoids.

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Stilbene quantification

Quantification of individual stilbenes was performed using standards/compounds isolated from Cyperaceae and Vitaceae. Quantification was performed by HPLC-DAD analysis on an Agilent® 1100 Series system by using the same HPLC-DAD conditions previously described. Extracts of red wines were dissolved at 1 mg/mL in methanol and 20 µl of these solutions were injected. The identity of each peak was verified in parallel with UPLC-MS for each wine sample. Stilbene concentrations were determined by reporting the measured integration area of each compound. Mean values of each extract were calculated from three replicates. For the calibrations, the same volume of injection was used. Seven solutions including blank were prepared for each pure isolated stilbenoid compound (0, 0.001, 0.005, 0.01, 0.02, 0.05 and 0.1 mg/mL in 50% methanol). Each concentration was injected five times.

Structure elucidation

Figure 2 - UPLC chromatogram recorded at 290 nm showing the stilbenes detected in the Sidi Zahia red wine. Peak numbering as in Table 1.

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In this study, stilbenes were identified by UPLC-MS/MS analysis. This combination provided an accurate method for the characterization of stilbenes (Stecher et al., 2001). To illustrate the analysis of stilbenes in wine, the UPLC chromatogram of the Sidi Zahia red wine is presented in Figure 2. Stilbenes were tentatively identified by examining the mass spectra (MS and MS/MS). The presence of stilbene oligomers was monitored via extracted ion chromatograms in negative ion mode. Typical m/z values for these compounds were used, including [M−H]⁻ ions of m/z 227 or 242 (monomers), 453 (dimer), 677 (trimer), and 905 (tetramer). The corresponding values for these compounds were also evaluated in positive mode. To confirm the identification of the stilbenes, standards were used (Arraki et al., 2013 ; Papastamoulis et al., 2014).

Using this procedure, seven stilbenes (Figure 3) were identified in Tunisian red wines, including three monomers (resveratrol, piceid and piceatannol), one dimer (ε-viniferin), one trimer (α-viniferin) and two tetramers (hopeaphenol and isohopeaphenol). Retention time (T_R) and molecular ions (positive and negative modes) are shown in Table 1.

Figure 3 - Chemical structures of various stilbenes identified in red wines.

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Concerning the stilbene content in the Tunisian red wine, the Sidi Zahia red wine was qualitatively and quantitatively the richest one (Figure 3). The effective quantification of the stilbene content is in progress. Nevertheless, these studies show that among all studied wines, only Sidi Zahia contains the seven stilbenes (Table 2). Isohopeaphenol is present in all wines except Distinto. The Sidi Rais, Mornag, Sidi Brahim and Clipea wines contain four stilbenes and do not contain ε-viniferin, α-viniferin and hopeaphenol.

Total stilbene contents

The average content of the seven stilbenes in the different red wines is shown in Table 3. The total concentrations of identified stilbenes were thus determined at 4.2 mg/L for Sidi Zahia, 2.5 mg/L for Sidi Rais, 3.9 mg/L for Mornag, 0.7 mg/L for Selian, 0.9 mg/L for Distinto, 1.5 mg/L for Sidi Brahim and 1.8 mg/L for Clipea.