Isolation and characterization of endothelium-dependent vasorelaxing compounds from grape seeds

Previous work has shown that red wines, grape juices, and other grape produ cts cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolat ion and characterization of some of the compounds responsible for EDR activ ity. Concord grape seeds were extracted with methanol and the compounds wer e separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (pr imarily phenolic acids, catechins, and proanthocyanidins) were further sepa rated semipreparatively by reversed-phase HPLC, and peaks were collected an d bioassayed for EDR activity using the rat aorta preparation. EDR-active c ompounds were subsequently characterized by HPLC retention times and electr ospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR ac tivity were proanthocyanidin trimers, tetramers, pentamers, and polymers an d their gallates, as well as a dimer gallate (EC50 values in the range of 0 .6-2.5 mug catechin equivalents/mL). These compounds should be useful for i n vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.