Biochemical and Molecular Action of Nutrients Walnut Polyphenolics Inhibit In Vitro Human Plasma and LDL Oxidation 1,2 Koren J. Anderson,* Suzanne S. Teuber,* Alayne Gobeille, † Peader Cremin,** Andrew L. Waterhouse † and Francene M. Steinberg ‡3 *Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, School of Medicine, † Department of Viticulture and Enology, and ‡ Department of Nutrition, University of California, Davis, CA; and **Sequoia Sciences, San Diego, CA ABSTRACT Recent epidemiologic studies have associated nut consumption with a reduced incidence of car- diovascular mortality. However, little is known about the contribution of nut polyphenols to antioxidant and cardiovascular protection. In this investigation, polyphenol-rich extracts from English walnuts (Juglans regia) were studied and compared with ellagic acid for their ability to inhibit in vitro plasma and LDL oxidation, as well as their effects on LDL -tocopherol during oxidative stress. In addition, the Trolox equivalent antioxidant activity (TEAC) was determined and liquid chromatography electrospray detection mass spectrometry (LC-ELSD/MS) analyses of the walnut extracts were performed. 2,2'-Azobis'(2-amidino propane) hydrochloride (AAPH)-induced LDL oxidation was significantly inhibited by 87 and 38% with the highest concentration (1.0 mol/L) of ellagic acid and walnut extract, respectively. In addition, copper-mediated LDL oxidation was inhibited by 14 and 84% in the presence of ellagic acid and walnut extract, respectively, with a modest, significant LDL -tocopherol sparing effect observed. Plasma thiobarbituric acid reacting substance (TBARS) formation was significantly inhibited by walnut extracts and ellagic acid in a dose-dependent manner, and the extracts exhibited a TEAC value greater than that of -tocoph- erol. LC-ELSD/MS analysis of the walnut extracts identified ellagic acid monomers, polymeric ellagitannins and other phenolics, principally nonflavonoid compounds. These results demonstrate that walnut polyphenolics are effective inhibitors of in vitro plasma and LDL oxidation. The polyphenolic content of walnuts should be considered when evaluating their antiatherogenic potential. J. Nutr. 131: 2837–2842, 2001. KEY WORDS: ● polyphenol ● ellagic acid ● LDL ● antioxidant ● English walnuts Recent epidemiologic studies have shown that consuming diets rich in plant-derived foods that are high in phenolic compounds, even while consuming high amounts of saturated fatty acids (1), is associated with a reduced incidence of cardiovascular mortality (2–5). Polyphenolics have been shown to possess free radical–scavenging and metal-chelating activity in addition to their reported anticarcinogenic proper- ties (6). These plant-based, nonnutrient phytochemicals may have a protective effect on the susceptibility of LDL to oxi- dative modification and ultimately, on atherosclerosis. The oxidative modification and inflammation hypothesis of atherogenesis is widely accepted and supported by experimen- tal data in hypercholesterolemic animal models and human epidemiologic studies, recently reviewed by Chisolm and Steinberg (7). Atherogenesis is a multifactorial process that includes oxidatively modified LDL, which triggers pathological events through multiple pathways, leading to atherosclerosis (8). Numerous in vitro studies using a variety of oxidation methods and measurements have shown that polyphenolics from red wine (9), green tea (10) and chocolate (11) can inhibit LDL oxidation. In vivo, it is unclear whether diet- derived polyphenolics can indeed influence the atherogenic process, but it is thought that the antioxidant potential of plant-derived foods may be one factor in reducing cardiovas- cular risk (12). A correlation between nut consumption and a reduced incidence of ischemic heart disease has been observed, most notably by the Adventist Health Study, the Iowa Women’s Health Study and the Nurses’ Health Study (13–15). Con- sumption of walnuts has favorable effects on human serum lipid profiles, with a decrease in total and LDL cholesterol as well as triglycerides (16 –19) and an increase in HDL choles- terol and apolipoprotein A1 (20). Tree nuts are an important source of beneficial dietary lipids, and a potentially rich source of phenolic compounds that contribute to antioxidant capac- ity. However, there has been very little investigation into the contribution of nut polyphenols to antioxidant protection and reduction in cardiovascular risk. This study, therefore, sought to determine the antioxidant capacity of walnut polyphenolic compounds in vitro. Walnut polyphenolics are found in the highest concentration in the pellicle, the thin tan-brown “skin” that lines the meat of the nut. Thus, this paper reports on a brief characterization of the polyphenolic-rich extract of English walnuts, its relative antioxidant activity, and demon- 1 Presented in preliminary form at Experimental Biology 2001, March 31–April 4, Orlando, FL [Anderson, K. J., Teuber, S., Steinberg, F. M. (2001) Walnut polyphenolic extracts inhibit in vitro plasma and LDL oxidation. FASEB J. 15: A607 (abs.)]. 2 Supported in part by a grant from the California Walnut Commission. 3 To whom correspondence should be addressed. E-mail: fmsteinberg@ucdavis.edu. 0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences. Manuscript received 18 May 2001. Initial review completed 26 June 2001. 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