Strawberry Extract Caused Endothelium-Dependent Relaxation through the Activation of PI3 Kinase/Akt INDIKA EDIRISINGHE, †,‡ BRITT BURTON-FREEMAN, ‡,§ PETER VARELIS, ‡ AND TISSA KAPPAGODA* ,† Departments of Internal Medicine and Nutrition, University of California, Davis, One Shields Avenue, Davis, California 95616, and National Center for Food Safety & Technology, Illinois Institute of Technology, Moffet Campus, Summit-Argo, Illinois 60501 Polyphenolic compounds are vasodilators and help to lower the risk of cardiovascular diseases. We hypothesized that a freeze-dried strawberry powder that is rich in polyphenolic compounds would cause an endothelium-dependent relaxation (EDR) through the activation of phosphatidylinositol-3 (PI3)-kinase/protein kinase B (Akt) in rabbit aorta. The powder was prepared by freeze drying a homogenate of ripe California strawberry fruits. An aqueous extract of strawberry powder was applied to rabbit aortic rings suspended in organ baths containing Krebs-Henseleit buffer maintained at 37 °C. In aortic rings precontacted with norepinephrine, the extract produced a dose-dependent relaxation. The maximum relaxations elicited by the extract (73.1 ( 1.0%) were similar to those elicited by acetylcholine (68.2 ( 1.5%) (n ) 14 for each). The relaxation by strawberry extract was abolished by removal of the endothelium and by prior incubation with N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME), confirming the essential role of endothelial nitric oxide synthase (eNOS). The responses to the strawberry were also abolished by incubation with wortmannin and LY294002, which are inhibitors of PI3 kinase. Using immunoblotting, we also demonstrated that the strawberry extract induced the phosphorylation of both Akt and eNOS in human umbilical vein endothelial cells (HUVECs) via PI3 kinase/Akt pathway. Taken together, our novel findings suggest that the EDR induced by the strawberry extract was mediated by activation of the PI3 kinase/Akt signaling pathway, resulting in phosphorylation of eNOS. KEYWORDS: Bioactive phenols; PI3 kinase/Akt; eNOS; ROS; EDR; strawberry; anthocyanins INTRODUCTION Epidemiological studies have consistently demonstrated an association between the consumption of diets rich in fruits and vegetables and a lower risk for developing chronic diseases including cancer and atherosclerosis (1-3). It has been sug- gested that many of the health benefits from consuming these plant foods may come from bioactive compounds that are present in plants (4). These compounds vary widely in chemical structure and function and are grouped according to their chemical class. Phenolic compounds, including their subcat- egory, flavonoids, are present in all plants and have been studied extensively (5). They have been shown to induce endothelium- dependent relaxation (EDR) in a variety of in vitro experiments (6, 7). We have demonstrated recently that the EDR induced by a grape seed extract (GSE) is mediated by the activation of the phosphatidylinositol-3 (PI3) kinase/protein kinase B (Akt) signaling pathway (7). Strawberries have been shown to be a rich source of phenolic compounds (8, 9). The phenolic compounds re- sponsible for the red color in strawberry flesh, the antho- cyanins, have been widely investigated and identified as glucosides of pelargonidin and cyanidin, with pelargonidin- 3-glucoside as the major compound (10-12). Glucosides and glucuronides of quercetin and kaempferol have been identified as the main flavonols. Numerous studies have shown that strawberries have antioxidant effects that are mainly ascribed to their high content of phenolic compounds (8, 13, 14). Because the activation of the PI3 kinase/Akt signaling pathway (7) is redox sensitive, we hypothesized that an extract of freeze- dried strawberry powder that is rich in phenolic compounds would cause an EDR through activation of PI3 kinase/Akt, potentially due to the antioxidant action of the phenolic compounds. These studies were undertaken on both rabbit aortic rings and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS Study Design and Procedures. This study was approved by the Animal Use and Care Administrative Advisory Committee, University of California (Davis, CA). Unless otherwise stated, all chemicals were * To whom correspondence should be addressed. Tel: 916-734-8407. Fax: 530-734-6474. E-mail: ctkappagoda@ucdavis.edu. † Department of Internal Medicine, University of California, Davis. ‡ Illinois Institute of Technology. § Department of Nutrition, University of California, Davis. J. Agric. Food Chem. 2008, 56, 9383–9390 9383 10.1021/jf801864t CCC: $40.75  2008 American Chemical Society Published on Web 09/25/2008