The effect of consuming red wine, or its major polyphenol constituents catechin or quercetin, on the development of atherosclerotic lesions, in relation to the susceptibility of plasma LDL to oxidation and to aggregation, was studied in atherosclerotic apolipoprotein E deficient (E degree) mice. Forty E degree mice at the age of 4 weeks were divided into four groups, 10 mice in each group, and were supplemented for up to 6 weeks in their drinking water with placebo (1.1% alcohol); catechin or quercetin (50 micrograms/d per mouse), or red wine (0.5 mL/d per mouse). Consumption of catechin, quercetin, or red wine had no effect on plasma LDL or HDL cholesterol levels. The atherosclerotic lesion area was smaller in the treated mice by 39%, 46%, and 48%, respectively, in comparison with E degree mice that were treated with placebo. In accordance with these findings, cellular uptake of LDL derived after catechin, quercetin, or red wine consumption was found to be reduced by 31%, 40%, and 52%, respectively. These results were associated with reduced susceptibility to oxidation (induced by different modes such as copper ions, free radical generator, or macrophages) of LDL isolated after red wine or quercetin and, to a lesser extent after catechin consumption, in comparison with LDL isolated from the placebo group. Similar results were obtained when LDL was preincubated in vitro with red wine or with the polyphenols prior to its oxidation. Even in the basal oxidative state (not induced oxidation), LDL isolated from E degree mice that consumed catechin, quercetin, or red wine for 2 weeks was found to be less oxidized in comparison with LDL isolated from E degree mice that received placebo, as evidenced by 39%, 48%, and 49% reduced content of LDL-associated lipid peroxides, respectively. This effect could be related to enhanced serum paraoxonase activity in the polyphenol-treated mice. LDL oxidation was previously shown to lead to its aggregation. The present study demonstrated that the susceptibility of LDL to aggregation was reduced in comparison with placebo-treated mice, by 63%, 48%, or 50% by catechin, quercetin, and red wine consumption, respectively, and this effect could be shown also in vitro. The inhibition of LDL oxidation by polyphenols could be related, at least in part, to a direct effect of the polyphenols on the LDL, since both quercetin and catechin were found to bind to the LDL particle via the formation of an ether bond. We thus conclude that dietary consumption by E degree mice of red wine or its polyphenolic flavonoids quercetin and, to a lesser extent, catechin leads to attenuation in the development of the atherosclerotic lesion, and this effect is associated with reduced susceptibility of their LDL to oxidation and aggregation.

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Polyphenolic flavonoids are powerful antioxidants. In the present study we investigated the antioxidative activity against low-density-lipoprotein (LDL) oxidation of a not yet studied subclass of polyphenols, the isoflavans, which are present in licorice alcoholic extract. The study was performed in humans as well as in atherosclerotic apolipoprotein E-deficient mice (E zero), because their LDL is highly susceptible to oxidation. LDL oxidation was induced by incubating it with copper ions as well as with the aqueous or lipid-soluble free radical generators 2,2'-azobis'2-amidino propane hydrochloride (AAPH) and 2,2'-azobis 2,4-dimethylvaleronitrile (AMVN), respectively. The extent of LDL oxidation was determined by measuring the formation of conjugated dienes, thiobarbituric acid reactive-substances (TBARS), and lipid peroxides. By all methods in human studies, licorice ethanolic extract as well as a pure material, which was identified by gas chromatography-mass spectroscopy as the isoflavan glabridin, were shown to inhibit LDL oxidation by a mechanism involving scavenging of free radicals. In an ex vivo study, LDL isolated from the plasma of 10 normolipidemic subjects who were orally supplemented for 2 wk with 100 mg licorice/d was more resistant to oxidation than was LDL isolated before licorice supplementation. Dietary supplementation of each E zero mouse with licorice (200 micrograms/d) or pure glabridin (20 micrograms/d) for 6 wk resulted in a substantial reduction in the susceptibility of their LDL to oxidation along with a reduction in the atherosclerotic lesion area. These results could be related to the absorption and binding of glabridin to the LDL particle and subsequent protection of the LDL from oxidation by multiple modes as shown in humans and in E zero mice.

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 g of glabridin/mg of cell protein after 2 h of incubation, and this process was time-and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by ϳ70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E 0 ) mice. MPMs harvested from glabridin-treated E 0 mice (20 g/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size.We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E 0 mice, induced by glabridin.The LDL 1 oxidation hypothesis of atherosclerosis is supported by evidence for the accumulation of oxidized LDL in the atherosclerotic lesion, by increased LDL oxidizability in patients with increased risk for atherosclerosis, and by the antiatherogenicity of several potent antioxidants against LDL oxidation (1-4). Extensive investigation is being made to identify natural food products that can offer antioxidant defense against LDL oxidation. Flavonoids are polyphenolic compounds naturally present in fruits and vegetables and are an integral part of the human diet (5). Consumption of flavonoids in the diet was shown to be inversely associated with morbidity and mortality from coronary heart disease (6, 7). Flavonoids are powerful antioxidants and their antioxidative capacity is related to their chemical structure (8,9). We have recently shown that dietary supplementation of healthy human volunteers with flavonoid-rich nutrients such as olive oil, red wine, or licorice root, resulted in a reduction in LDL oxidizability (10 -12). F...