Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: Focus on atherosclerosis Mariana Appel Hort a,⇑ , Marcos Raniel Straliotto a , Mariana Silveira Duz b , Paula Moro Netto b , Christiane Borges Souza b , Tamirys Schulz b , Heros Horst c , Moacir Geraldo Pizzolatti c , Andreza Fabro de Bem a , Rosa Maria Ribeiro-do-Valle b a Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Brazil b Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Brazil c Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Brazil article info Article history: Received 10 April 2012 Accepted 25 July 2012 Available online 3 August 2012 Keywords: Croton celtidifolius Proanthocyanidins Atherosclerosis Oxidative stress Endothelial cells abstract Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several cor- onary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu 2+ -induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxida- tion and decreasing in the oxidation rate (V max ), moreover, the protein moieties from LDL were protected against Cu 2+ -induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr À/À ), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxi- dative stress in endothelial cells and improve endothelial function in mice. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Atherosclerosis is a systematic, multifactorial and progressive disease characterized by the accumulation of lipids and fibrous ele- ments in the large arteries (Ross, 1999). The formation of oxidized low-density lipoprotein (oxLDL) and oxidative stress are thought to play a critical role in early atherogenesis, eliciting endothelial dys- function and macrophage activation. OxLDL stimulates the macro- phage to accumulate cholesterol and to form foam cells, which compose fatty streak, the hallmark in early atherosclerotic lesions, followed by the development of fibrous and atheromatous plaques (Stocker and Keaney, 2004). After formation of the fatty streak, the nascent atheroma typically evolves into a more complex lesion, which eventually leads to clinical manifestations (Brown and Gold- stein, 1983; Ross, 1999). Natural antioxidants and lipid-lowering interventions such as food supplements, spices, and herbs find extensive application in the prevention of atherosclerosis because of their ability to prevent in vitro LDL oxidation (Chang et al., 2006) and plaque formation (Ho et al., 2010). Thus, proanthocyanidins have proved to be potent antioxidants in different in vitro models, and in some in vivo inter- vention studies. However, their potential beneficial effect on car- diovascular health is not merely due to this property but includes the different mechanisms implicated in cardiovascular conditions or problems, i.e., hypertension, inflammation, cellular proliferation, thrombogenesis, hyperglycemia, and hypercholester- olemia (de Pascual-Teresa et al., 2010). Many reports have demonstrated the important effects of pro- anthocyanidins on the cardiovascular system, including the antiox- idant activity in several lipid systems, particularly in LDL oxidation (da Silva Porto et al., 2003); ability to increase the antioxidant 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.07.050 Abbreviations: Å NO, nitric oxide; Ach, acetylcholine; CD, conjugated dienes; DCFH-DA, 2 0 7 0 -dichlorodihydrofluorescein diacetate; GSH, reduced glutathione; GSSG, oxidized glutathione; HC, hypercholesterolemic chow; HDL, high density lipoprotein; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; LDL, low density lipoprotein; LDLr À/À , LDL receptor knockout mice; MECK, micellar electrokinetic chromatography; O 2 ÅÀ , superoxide anion; ONOO À , peroxynitrite; oxLDL, oxidized low density lipoprotein; Phe, phenyleph- rine; PRF, proanthocyanidin-rich fraction; RNS, Reactive Nitrogen Species; ROS, reactive oxygen species; TC, total cholesterol; TNF-a, tumor necrosis factoralpha; Trp, tryptophan; VCAM-1, vascular cell adhesion protein-1. ⇑ Corresponding author. Address: Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88040-900 Florianóp- olis, SC, Brazil. Tel.: +55 48 37215047; fax: +55 48 37219672. E-mail address: marianaappel@gmail.com (M.A. Hort). Food and Chemical Toxicology 50 (2012) 3769–3775 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox