Epicatechin equivalent

Results obtained with colorimetric methods are highly empirical. Estimations of total PAs are often expressed as catechin or epicatechin equivalents, which make the data difficult to interpret and compare across different samples. Qualitative data such as subunit structures, interflavan bond types, and proportions of oligomers with different degrees of polymerization - is not available with any of these non-chromatographic methods. 

The total phenolic content of apples of different varieties has been reported to be in the range 110-600 mg epicatechin equivalents/100 g FW [30], which is much higher than the level of 48 mg GAE/100 g FW [13]. 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

Polymeric fractions were obtained from wines, seed and skin extracts by fractionation on a Toyopearl HW-40 column as described by Souquet et al (4). Two aliquots of the fractions containing polymeric material were t en to dryness under vacuum. The first one was used to determine proanthocyanidin composition by thiolysis followed with HPLC analysis (17). The other one was dissolved in MeOH acidified with 2% HCl and used to estimate the concentration of total polymeric polyphenols and polymeric pigments by measuring the absorbance, respectively at 280 nm and 530 nm. Absorbance data were converted to equivalent epicatechin and equivalent malvidin-3-glucoside, respectively, using the extinction coefficients determined for each compounds under similar conditions. 

All HPLC injections were performed in duplicate. (+)- Catechin and (-)-epicatechin, 280 nm, and quercetin, 365 nm (Aldrich, Milwaukee, WI), caffeic acid, 316 nm(Sigma, St. Louis, MO), gallic acid (MCB Manufacturing Chemists, Cincinnati, OH) and malvidin-3-glucoside, 520 nm (Pfaltz Bauer, Waterbury,CT) were used as external standards at the indicated wavelengths. Caftaric acid was purified in our laboratory by a previously described method (31). The quercetin glycoside is expressed in quercetin equivalents, and all anthocyanins in malvidin-3-glucoside equivalents. 

As the chromatographic process in CCC is based on the partition of a solute between the mobile and stationary phases, the value is the most important parameter in CCC. A Ad value of around 1.0 is most desirable in CCC, wherein a solute with Ad =1.0 elutes with its retention volume equivalent to the total column capacity. In the above two-phase solvent systems, the Ad values of monomers (catechin and/or epicatechin) were greater than 1.0, and those of the ACTs are always smaller than 1.0, suggesting that monomers are more hydrophobic than their oligomers present in ACTs. Among these four solvent systems, we selected a simple binary system of methyl acetate/water for the separation of procyanidin oligomers from ACTs by CCC. 

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