Isolation of oligomeric proanthocyanidins from flavonoid-producing cell cultures

Abstract

The extraction, fractionation, and chromatographic separation of a series of proanthocyanidin monomers and oligomers were facilitated using a flavonoid-rich cell culture of Vaccinium pahalae Skottsberg as the donor tissue. The cell cultures, after exposure to light, readily accumulated anthocyanin pigments and other flavonoids in relatively large amounts, with minimal concurrent production of pectins, enzymes, and complex sugars produced in field-grown Vaccinium berries. The absence of these interfering compounds greatly simplified the isolation and purification of proanthocyanidins and other phenolic compounds from cell cultures, primarily using vacuum chromatography. Subsequently, the structures and molecular weights of several individual compounds and the general composition of unresolved fractions were established with 1H- and 13C-NMR and MS. The initial extract of V. pahalae cell cultures was readily fractionated on silica gel to yield a series of fractions containing proanthocyanidin B-2, a series of increasingly polar proanthocyanidin oligomers ranging from dimers to heptamers largely based on (−)-epicatechin structures (some with A-type linkages), a mixture of E- and Z-p-coumaric acid, the corresponding 4-O-glucoside, and other compounds containing E- and Z-p-coumaric acid moieties. Cell culture extracts demonstrated broad antioxidant capacity and significant ability to inhibit tumor promotion in vitro, as indicated in an ornithine decarboxylase assay.