Facile Purification of Milligram to Gram Quantities of Condensed Tannins According to Mean Degree of Polymerization and Flavan-3-ol Subunit Composition

Abstract

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. ¹H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or ¹H-¹³C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

Keywords: Sephadex LH-20; Toyopearl HW-50F; batch chromatography; nuclear magnetic resonance; proanthocyanidins; thiolysis.