Development of a fractionation method for the detection and identification of oak ellagitannins in red wines
Introduction
During maturation and ageing of wines in oak barrels different substances can be released from the wood to the wine and can take part in several reactions which might produce both qualitative and quantitative changes in the wine affecting its organoleptic properties such as colour and astringency [1], [2], [3]. The ellagitannins (hydrolysable tannins) are among these compounds. In oak heartwood they may represent 10% of the dry weight and are responsible for the high durability of this wood [4].
The most representative structures of ellagic tannins are vescalagin and castalagin, two isomers first isolated and described by Mayer [5]. Lyxose/xylose derivatives (grandinin and roburin E, respectively) and dimeric forms (roburins A, B, C, D) have also been described [6], [7]. These compounds are extracted from wood to wine owing to the hydro-alcoholic nature of the wine. The extraction depends mainly on the pool of potential extractable compounds that are originally present in the barrel oak wood and on the conditions and duration of the winemaking process [8].
The content of the ellagitannins in oak wood is influenced by several factors [8]. On the one hand, the oak species (e.g., Quercus robur L., Quercus petraea (Matt.) Liebl., Q. alba L.), the geographical origin (e.g., American or European oak) [9], [10], the age [11] or the sylvocultural treatment of the tree, on the other, the processing of wood in cooperage that changes its chemical composition. The method to obtain the staves, the method of seasoning (natural or artificial, length, location, etc.) or the degree of toasting (no-toasting, light, medium or heavy toasting) have an important influence on the ellagitannins’ content in oak [12], [13]. As a result of all these treatments the ellagitannins can be oxidized and can form polymers and copolymers with cell-wall components. Thus, their solubility would be reduced [14]. Moreover, the age of the barrels is also important, since it has been reported that the levels of extracted ellagitannins are much lower in old barrels [15].
The ellagitannins possess several hydroxyl functions in ortho positions at the periphery of their molecular structures (Fig. 1). For this reason, in wine, they are involved in oxidation reactions, acting as consumers of oxygen which induce an increased oxidizing power that finally causes a large quantity of peroxides and acetaldehyde to be produced by oxidation [1]. Thus, ellagic tannins may take part in the changes of colour during maturation and ageing of wine, helping to improve colour stability and also protecting it against oxidation [1], [16], [17]. Oak wood ellagitannins can also affect the astringency of the wine, since, as procyanidins, they have the ability to precipitate proteins, in particular the salivary proteins in the oral cavity [1], [2], [18].
During maturation and ageing of wine in oak barrels, ellagitannins are solubilised by wine and diffuse through the wood. Once in the wine the ester bonds of the ellagitannins may be easily hydrolysed giving rise, by rearrangement of the HHDP (hexahydroxydiphenic) residue, to ellagic acid. The ellagitannins might also be oxidised in its HHDP residue. However, in this case, the product of its degradation would not be ellagic acid [15], [19]. Furthermore, due to their high reactivity, ellagitannins are involved in many other processes during the ageing of wine. Consequently, their levels in wine are much lower than those of other wine constituents and, as a result, they are not easily detected in the chromatograms even recorded at their maximum wavelength. Moreover, their structural similarities make their chromatographic separation difficult in reversed phases as well as their identification by means of the spectral features (identical UV–vis spectra, pseudo-molecular ions with the same m/z ratios).
There are few studies whose objective has been the qualitative and quantitative determination of the ellagitannin composition in wines. Some of them only determine the total concentration of these compounds [20] following a procedure usually employed in the quantification of total ellagitannins in wood [14], [21], which is based on acid hydrolysis of the ellagitannins and subsequent quantification of the free ellagic acid released. Nevertheless, when the studies had the objective of the qualitative and quantitative characterisation of the wine samples [22] and, in some cases grapes [23], a fractionation step was always required. In the existing methods the high volume of sample required, the complexity of some of the stages and the presence of targeted compounds in more than a single fraction can be considered as the main drawbacks.
The main objective of this study was to obtain a fractionation method which would allow the isolation of the original oak ellagitannins from other wine constituents in order to make their detection and quantification in wine samples possible. This study also had the objective of obtaining an HPLC method which would separate compounds with the same m/z ratios in order to facilitate their identification and quantification by means of mass spectrometric analyses.
Section snippets
Isolation of the ellagitannins from oak chips
One hundred grams of medium toasted chips obtained from Q. petraea (Matt.) Liebl. wood were macerated overnight in 2.5 L of a hydromethanolic solution (1:1) with constant stirring. Chips were extracted twice with the same protocol and both extracts were gathered and concentrated under reduced pressure in order to remove the methanol. Then the aqueous extract was first cleaned three times with diethyl ether and three times with ethyl acetate in order to remove other extracted oak compounds which
Fractionation method
The HPLC-DAD–MS analysis of the spiked wine before its fractionation revealed the complexity of the sample. Fig. 3 shows its chromatogram (dilution 3/5 in 2.5% acetic acid) recorded at 250 nm. The elevation of the baseline in the earlier part of the chromatogram and the presence of a “hump” from 20 to 55 min were indicative of the co-elution of a large number of compounds making the detection and identification of the ellagitannins difficult. Since the concentration of the ellagitannins in the
Conclusions
A two-step fractionation method consisting of a solid phase extraction in C-18 Sep-Pak® cartridges followed by size exclusion chromatography in a hand-packed Sephadex LH-20 minicolumn has been optimised for the detection and identification of the main oak ellagitannins in red wine samples. Oak ellagitannins were only present in one fraction and separated from other phenolic compounds that might interfere in the HPLC-DAD–MS analyses of the fractions, such as, for example, gallic acid which
Acknowledgements
Thanks are due to the Spanish MICINN and FEDER (Grant Ref. AGL2008-05569-C02-01) for financial support. The authors also thank the Spanish MICINN for an F.P.U. predoctoral scholarship to I. García-Estévez and Mr. G.H. Jenkins for his help with the English version of the ms.
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Structural elucidation of the ellagitannin with a molecular weight of 2038 isolated from strawberry fruit (Fragaria ananassa Duch.) and named fragariin A
2019, Food ChemistryCitation Excerpt :Studies on the biological properties of ETs have proved another source of progress in the field of isolation of this group of polyphenols. Currently, the most widespread techniques for obtaining purified preparations are low-pressure chromatography and adsorbent bed resins such as Amberlite XAD-16 (Jurgoński et al., 2017; Juśkiewicz et al., 2015; Seeram et al., 2005), and XAD-1600N (Sójka et al., 2013), and gel resins, such as Sephadex LH-20 (García-Estévez, Escribano-Bailón, Rivas-Gonzalo, & Alcalde-Eon, 2010; Howard, Prior, Liyanage, & Lay, 2012; Navarro et al., 2017). In the second step, preparative reverse-phase HPLC is commonly used (Gasperotti, Masuero, Vrhovsek, Guell, & Mattivi, 2010; Seeram et al., 2005; Sójka et al., 2016).