Stilbenes: Quantitative extraction from grape skins, contribution of grape solids to wine and variation during wine maturation

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Abstract

With the objective of studying the relationship between stilbenic composition in grape skins and that in corresponding red wine, we have firstly optimized a method for quantitative extraction and purification of stilbenes from grape skins, used for posterior HPLC analysis. Stilbene levels in grape skins of three Vitis vinifera varieties (Castelão, Syrah and Tinta Roriz) and in their corresponding wines made by traditional winemaking technologies were analyzed. Stilbene composition in grape skin varied considerably, depending on the grapevine variety. The contents of trans-resveratrol in grape skins of the three varieties were not significantly different. However, their contents of trans-piceid on dry skin were very significantly different (67.24, 10.43 and 11.57 mg kg−1, respectively). Moreover, cis-piceid, which was absent in Syrah and Tinta Roriz grape skins, was detected in Castelão grape skin with an important amount (58.9 mg kg−1 on dry skin). The high concentrations of stilbenes were found in stem tissue (cv. Castelão): 145.52 mg kg−1 of trans-resveratrol, 61.43 mg kg−1 of trans-piceid and 143.85 mg kg−1 of cis-piceid (on dry stem), while only trans-resveratrol was quantified in the grape seeds with a low concentration (6.8 mg kg−1 on dry seed). Simulated maceration of each solid part of grapes using model wine solution indicated that both stem and seed have little contribution of stilbenes to wines while skins are the major source of these compounds in wine. On the other hand, cis-piceid was not detected in the corresponding wines made by these grapevine varieties, but its isomer trans-piceid was detected in an important concentration, suggesting that cis-piceid, once extracted, isomerized to its trans-form. The evolution of these compounds during the elaboration of wines (from the end of alcoholic fermentation until second racking, i.e., just before bottling) showed that in general, trans-resveratrol concentration decreases gradually, trans-piceid kept constant, while cis-resveratrol increases slightly. The important finding of this work is that there was a significant correlation between the concentration of total piceid in grape skins and that in the respective wine. On the contrary, no such relationship was found for resveratrol.

Introduction

Stilbenes (1,2-diarylethenes) belong to non-flavonoid class of phenolic compounds. Their occurrence in plant tissues is associated to the resistance of plant against fungal diseases such as Botrytis cinerea, although they can also occur to abiotic stress, such as UV irradiation. Generally, stilbenes are considered as phytoalexins, and their formation in grape leaves has been correlated with disease resistance [1], [2]. Phytoalexins are a group of plant chemicals of low molecular mass which are inhibitory to microorganisms and their accumulation in plants is initiated by interaction of the plant with microorganisms. Stilbenes are present in both edible and non-edible plant tissues. Grape and red wine are among the major dietary sources of stilbenes. During the last two decades, interest in human-health benefit of moderate consumption of red wines has increased dramatically. It is generally accepted that polyphenols in wines are responsible for this beneficial effect, and stilbenes are one of the major groups of such compounds.

Stilbenes can be also found to be presented in oligomeric and polymeric forms, so-called viniferins [3]. ɛ-Viniferin (dimeric resveratrol), α-viniferin (trimeric resveratrol), as well as stilbenoids like ampelopsin A (dimeric resveratrol) and hopeaphenol (dimeric ampelopsin A) have been identified [4]. However, the major stilbene is its monomeric form—resveratrol (3,5,4′-trihydroxystilbene). This is why the majority of published works over many years have focused on this compound [5], [6], [7], [8], [9], [10].

Wine stilbenes include essentially both cis- and trans-form of resveratrol and piceid [11]. The most important factors affecting the content of these compounds in wine are their concentrations in grape, the winemaking technology and their changes during wine aging process.

In grape cluster, stilbenes are considered to be located essentially in skins [12] and mainly in glucosylated form [13], [14]. These compounds were also reported to be present in grape seeds [15] and grape stems [16]. Resveratrol, the major stilbene of grapes, may present in two isomer forms, but only its trans-isomer has been identified in Vitis vinifera grapes [12], [17], [18]. The concentration of trans-resveratrol in the skins of ripening grapes varied considerably, depending on the variety [19]. On the other hand, resveratrol concentration in grape skin was found to be negatively correlated with the developmental stage of berries [12].

In wines, both cis- and trans-resveratrol were detected, with the latter predominant [20], [21]. The presence of cis-resveratrol in wines was generally considered to be due to the photochemical isomerization of partial trans-form during winemaking process [5], [14]. However, some authors suggested that cis-resveratrol might be present in grapes in combined form which could be liberated by hydrolysis during fermentation/maceration process of winemaking [18], [22]. Since skin tissue is the major source of such compounds to wine, the high level of stilbenes in grape skin has a practical importance in enology. Grape seeds contain low amount of stilbenes [15], but considerable amount of these compounds may be found in grape stems [16], [23], [24]. Furthermore, red wines contained much higher concentration of both trans- and cis-resveratrol than rosé wines, while only trace amounts of such compounds were found in the white wines [24], [25], [26]. In our previous work [24], we have verified that the wines made by traditional winemaking technologies (skin fermentation with stem and skin fermentation without stem) contained higher levels of resveratrol than those made by carbonic maceration technique. Prolonged maceration after alcoholic fermentation did not affect the total resveratrol concentration in finished wines, suggesting that the extraction of resveratrol from grape skins should be completed before or at the end of alcoholic fermentation.

Several authors have studied the transference of stilbenes from grape to red wines during fermentation [15], [27]. The diffusion of stilbenes to wine was very rapid, reaching their maximum levels at 10–12 d of maceration [27]. Pezet and Cuenat [15] observed that the extraction of resveratrol from the skin correlated well with the increase of ethanol concentration in wine up to 6 d of maceration. Although stilbene compounds are known to be presented in all solid parts of grape cluster (skins, seed and stem) [15], [16], [24], little is known about the transfer ratio of these compounds from each solid to wine. In addition, there has been little information about the evolution of stilbenes in wine during storage and ageing process. Since wine stilbenes are originated from the grapes cluster from which it is made and that stilbenes have been considered to be located essentially in skins, it would be expected that there is a correlation between the levels of stilbenes in grape skins and those in its corresponding wine. Thus, the main objective of this work was to establish a relationship between the levels of stilbenes in grape skin and those of the corresponding wine. For this purpose, we have identified and quantified stilbenes in grape skins of three V. vinifera varieties (Castelão, Syrah and Tinta Roriz) based on an optimized extraction method followed by HPLC and monitored the variation of stilbene levels in the corresponding wines during storage (i.e., at the end of fermentation, after 2 months of storage and at the time of bottling). Moreover, the transfer rates of stilbene compounds from each of the solid parts of grape cluster into wine were verified by imitated maceration using model wine solution.

Section snippets

Reagents, standards and calibration

All solvents used were of analytical or HPLC grade. trans-Resveratrol was purchased from Sigma Chemical Company (St. Louis, MO, USA). trans-Piceid and trans-astringin were purchased from POLYPHENOL AS (Bergen, Norway).

trans-Stilbenes can be partially converted to their respective cis-isomers by UV irradiation [20], [21], [28]. Thus, cis-resveratrol, cis-piceid and cis-astringin were obtained by isomerization of their respective trans-standards under the following optimized conditions: for

Identification of stilbenes in grape skins and in red wines

The typical HPLC chromatograms of grape skins and red wines are presented in Fig. 1, Fig. 2, respectively.

Using internal standards, it was verified that the retention times of peaks a–c in Fig. 1 corresponded to that of trans-piceid, cis-piceid and trans-resveratrol, respectively, while the retention times of peaks d–f in Fig. 2 corresponded, respectively, to that of trans-piceid, trans-resveratrol and cis-resveratrol.

On the other hand, the HPLC-DAD confirmed that the UV–vis spectra of peaks a

Conclusion

Both grape stem and skin contain high amount of stilbenes but their transfer rates to wine are very low. Grape skins are major contributes to wine because of its quantitative importance as compared to stem and seed. Thus, both white and red winemaking by-products (pomace) would be interesting to be used as source of stilbenes. Methanol acidified by 0.1% HCl was the best solvent to extract stilbenes from grape skins. Stilbene levels in grape skins depend on the varieties. Only the level of total

Acknowledgements

The authors thank the financial support for this work from the Project PIDDAC 701 and the Fundação para a Ciência e a Tecnologia (Portugal).

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