Preparative isolation of apple flavan-3-ols monomers and oligomers using pH-zone-refining centrifugal partition chromatography combined with reversed-phase liquid chromatography.

Abstract

Flavan-3-ols (catechin monomers and procyanidins) are the main class of polyphenols in apples and are found in high concentrations in cider apple varieties. They are known to be involved in bitterness and astringency in apple-based beverages, and also contribute to polyphenol nutritional intake.Therefore, highly purified flavan-3-ol fractions isolated from raw materials are needed to study their various properties. For this purpose, a gentle strategy combining pH-zone-refining centrifugal partition chromatography (pH-ZRCPC) and preparative reversed-phase liquid chromatography (Prep-RPLC) was developed to recover one hundred milligrams of a high purity apple flavan-3-ol fraction. First, pH-ZRCPC fractionation in descending mode was optimized to remove hydroxycinnamic acid derivatives using a biphasic mixture composed of ethyl acetate/n-butanol/water (3/2/5, v/v). Trifluoroacetic acid and sodium hydroxide were used as retainer and eluter, in the upper and lower phases, respectively. Secondly, Prep-RPLC separation was carried out in isocratic mode at 20% ACN to remove dihydrochalcones. Finally, from one gram of a crude polyphenol extract, four hundred and nine milligrams of a highly purified fraction of flavan-3-ols with an average degree of polymerization close to 3.1 was obtained with 73% recovery.