Isolation and characterization of complex oligomeric proanthocyanidins from grape: applications of CCC and high field multidimensional NMR methods

Grape seed extract (GSE) is well known to have antioxidant, anti-inflammatory and anticarcinogenic polyphenolic components. The active agents from various Vitis species have been clearly shown to be phenolic in nature and typical extracts contain complex mixtures of epicatechin, catechin, oligomeric proanthocyanidins, as well as large polymeric procyanidins, which can all exist in a form esterified with gallic acid. The isolates are mainly procyanidin components consistently present in GSE of diverse origins. Seven of the major components were isolated from a commercial sample of Activin® using a combination of countercurrent chromatography and preparative HPLC. These include a monomer, three dimers, and three trimers. In order to more rapidly characterize these types of compounds, we have utilized high field nuclear magnetic resonance spectroscopy at 700MHz with a TCI cryoprobe for direct observation of both ¹H and ¹³C nuclei. Complete NMR data sets for the isolated compounds (1D, HSQC, HMBC, NOESY, ROESY, COSY, and hybrid methods such as HSQC-TOCSY) were collected to determine the structures and completely assign all ¹H and ¹³C resonances of these. High resolution TOF MS data were also obtained. Bruker AMIX^TM software was utilized to facilitate NMR and MS spectroscopic analysis. This software provides for powerful evaluation of multi-modality data with a variety of spectral analysis (including indirect covariance processing), assignment, spectral prediction, and data base tools. The data for these compounds are incorporated into a natural product spectral database we are currently expanding.