Crown procyanidin: a new procyanidin sub-family with unusual cyclic skeleton in wine

Abstract

Condensed tannins (also called proanthocyanidins) are a widely distributed throughout in plants kingdom and are one of the most important classes of secondary metabolites, in addition, they are part of the human diet. In wine, they are extracted during the winemaking process from grape skins and seeds. These compounds play an important role in red wine organoleptic characteristics such as color, bitterness and astringency. Condensed tannins in red wine are oligomers and polymers of flavan-3-ols unit such as catechin, epicatechin, epigallocatechin and epicatechin-3-O-gallate. The monomeric units can be linked among them with direct interflavanoid linkage or mediated by aldehydes. During our investigation on red wine condensed tannins, three surprisingly polar tannins oligomers (one tetramer and two pentamers) were detected by HPLC-UV-MS (Tof) and their concentrations remained stable during wine aging. The objective of this study was (1) to develop a purification method for these three oligomers of condensed tannins observed in red wine, (2) to determine their structures by high resolution mass spectrometry, chemical depolymerization strategy, as well as NMR, (3) to quantify them in various red wine and to estimate their organoleptic properties. The new procyanidin tetramer and the two new procyanidin pentamers have been purified by a “three steps-two gels” strategy with the first step of C-18 Solid Phase Extraction, the second step of TSK-40S Gel Filtration Chromatography and the last step of C-18 HPLC semi-preparative. Their fragmentation pattern obtained by MS/MS analysis using a high resolution mass spectrometry revealed that these three compounds belong to the procyanidin family. Moreover the inter-flavanoid linkages, sub-units information as well as overall configuration of the tetramer were established by 1D and 2D NMR. The structure of the tetramer have been determined to be a symmetric procyanidin with four sub-units of (−)-epicatechin link together by B-type interflavanoid linkage in the following sequence of Unit 1-(4-8)-Unit 2-(4-6)-Unit 3-(4-8)-Unit 4 (4-6)-Unit 1 with the first unit linked with the last unit via the forth interflavanoid linkage C6-C4 to form the macrocyclic structure. Since such carbon skeleton has never been reported before for procyanidins in wine, neither in plants kingdom, we decide to name this new group of procyanidins “crown procyanidins”. This new procyanidin sub-family has been quantified in three different red wines (merlot, cabernet sauvignon and shiraz) with various vintages (from 1991 to 2011). The concentration of the crown procyanidin remains stable in wine during ageing and their concentration shows higher stability than linear B-type procyanidins which their content decrease during aging. Moreover, a strong correlation between these new procyanidin and the red wine astringency level has been observed.