Antioxidant and Cellular Activities of Anthocyanins and Their Corresponding Vitisins AsStudies in Platelets, Monocytes, and Human Endothelial Cells MARI ÄA GARCI ÄA-ALONSO, † GERALD RIMBACH, ‡,§ JULIA Ä N C. RIVAS-GONZALO, † AND SONIA DE PASCUAL-TERESA* ,‡,| Unidad de Nutricio ´n y Bromatologı ´a, Facultad de Farmacia, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain, and School of Food Biosciences, Hugh Sinclair Human Nutrition Unit, University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-R (TNF-R) secretion in lipopolysaccharide plus interferon-γ-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-R-induced monocyte chemoattractant protein production in primary human endothelial cells. KEYWORDS: Anthocyanin; vitisin A; antioxidant activity; nitric oxide production; TNF-r secretion; MCP-1 production; platelet aggregation INTRODUCTION Anthocyanins are a group of phenolic compounds responsible for the red-blue color of many fruits and vegetables. They are glycosylated polyhydroxy or polymethoxy derivatives of 2- phenylbenzopyrylium or flavilium salts (1). The main dietary sources of anthocyanins include red-colored fruits and vegetables and red wine. The intake of anthocyanins in humans has been estimated to be 180-215 mg/day (2) in the Unites States, which is considerably higher than the intake of other flavonoids (23 mg/day) including quercetin, kaempferol, myricetin, apigenin, and luteolin (3). Epidemiological studies suggest that a moderate consumption of anthocyanins may be, at least partly, associated with a protection against coronary heart disease (4, 5). During red wine aging, there is a loss of anthocyanins and it appears that other pigments, so-called vitisins A (Figure 1), are formed through the interaction of the original anthocyanins with pyruvic acid (6). The structure of vitisin A is based on the anthocyanidin- 3-glucoside with an additional C 3 O 2 between position C4 and the 5-hydroxyl group of the molecule (7). The chemical interaction between anthocyanins and pyruvic acid has been shown to influence the color of red wine and may also impact the antioxidant and biological properties of anthocyanins. Although there is evidence that anthocyanins provide bene- ficial effects to human health, their cellular mechanism of action remains largely unknown. The purpose of the present study was 2-fold: to analyze the antioxidant activity of the most abundant anthocyanins present in red wine, Dp-3-glu, Pet-3-glu, and Mv- 3-glu, in comparison to their corresponding vitisins A and to study the effect of anthocyanins and vitisins on NO production and TNF-R secretion in macrophages, collagen-induced platelet aggregation, and MCP-1 production in primary HUVEC. We have chosen these three cell types since they play a key role in the pathogenesis of atherosclerosis. MATERIALS AND METHODS Isolation and Purification of Anthocyanins. Anthocyanins (Dp- 3-glu, Pet-3-glu, and Mv-3-glu) were isolated from a methanol-acid red grape skin extract by semipreparative high-performance liquid * To whom correspondence should be addressed. Tel: +34 91 549 23 00. Fax: +34 91 549 36 27. E-mail: soniapt@if.csic.es. † University of Salamanca. ‡ University of Reading. § Current address: Insitute of Human Nutrition and Food Science, Christian Albrechts University, D-24111 Kiel, Germany. | Current address: Department of Plant Food Science and Technology, Instituto del Frio, CSIC, Jose Antonio Novais 10, E-28040 Madrid, Spain. 3378 J. Agric. Food Chem. 2004, 52, 3378-3384 10.1021/jf035360v CCC: $27.50 © 2004 American Chemical Society Published on Web 05/11/2004