Proanthocyanidins purification

Tanner, G.J. et al., Proanthocyanidin biosynthesis in plants. Purification of legume leucoantho-cyanidin reductase and molecular cloning of its cDNA. J. Biol. Chem., 278, 31647, 2003. 

Lazarus, S.A. et al.. Analysis and purification of proanthocyanidin oligomers. Methods Polyphenol Anal, 267, 2003. 

Proanthocyanidins Extraction, Purification, and Determination of Subunit Composition by HPLC... 

This unit is composed of three separate procedures describing proanthocyanidin extraction from plant tissue (see Basic Protocol 1), purification (see Basic Protocol 2), and subsequent analysis by reversed-phase HPLC (see Basic Protocol 3). These protocols have been developed and used for analysis of grape skins, grape berries, and grape seeds. Without modification, wine, apples, and pears have also been analyzed using these procedures. 

This purification step is designed to remove impurities from the proanthocyanidin extract. It utilizes liquid-liquid extraction to remove lipophilic material and monomeric flavan-3-ols, and also adsorption chromatography to remove more hydrophilic material such as organic acids, sugars, and residual flavan-3-ol monomers. Following the steps in this protocol, purified and powdered proanthocyanidins are obtained. 

Proanthocyanidins following purification are light buff powders, with a varying degree of yellow depending on the level of oxidation that has occurred. 

Proanthocyanidins need to be extracted from food samples and undergo purification steps before HPLC analyses, and various extraction... 

Figure 8.3 Sample extraction and purification steps for proanthocyanidin analysis.

The approach we have found to be most useful for the isolation and purification of Type 1 or 2 proanthocyanidin polymers is extraction from plant tissue with acetone-water mixtures (25, 37), separation of the monomers and lower oligomers from the resulting aqueous solution with ethyl acetate, adsorption on Sephadex LH-20 of the aqueous solution diluted with an equal volume of methanol and washing with the same solvent to eliminate impurities. The polymer is then displaced with acetone-water to yield freeze-dried analytically pure material (25). In the case of procyanidins, the ethyl acetate fraction contains monomers (catechin or epicatechin), dimers, trimers, and some tetramers, whereas the LH-20 fractions contain tetramers, on up to genuinely polymeric species. 

---