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

Authors

Ron H. Brown, University of Reading
Irene Murray-Harvey, University of ReadingFollow
Wayne E. Zeller, USDA-ARSFollow
Laurie Reinhardt, USDA-ARSFollow
Elisabetta Stringano, University of Reading
An Gea, University of Reading
Christopher Drake, University of Reading
Honorata M. Ropiak, University of Reading
Christos Fryganas, University of Reading
Aina Ramsay, University of Reading
Emily E. Hardcastle, USDA-ARS

Date of this Version

2017

Citation

J. Agric. Food Chem. 2017, 65, pp. 8072−8082, DOI: 10.1021/acs.jafc.7b03489.

Comments

U.S. government work.

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.