BACKGROUND AND PURPOSE: Antioxidants may protect against atherosclerosis and thus prevent cerebrovascular disease. We studied the association between dietary antioxidants and subtypes of stroke. METHODS: The study cohort consisted of 26 593 male smokers, aged 50 to 69 years, without a history of stroke. They were participants of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study in Finland. The men completed a validated dietary questionnaire at baseline. Incident cases were identified through national registers. RESULTS: During a 6.1-year follow-up, 736 cerebral infarctions, 83 subarachnoid hemorrhages, and 95 intracerebral hemorrhages occurred. Neither dietary flavonols and flavones nor vitamin E were associated with risk for stroke. The dietary intake of beta-carotene was inversely associated with the risk for cerebral infarction (relative risk [RR] of highest versus lowest quartile 0.74, 95% CI 0.60 to 0. 91), lutein plus zeaxanthin with risk for subarachnoid hemorrhage (RR 0.47, 95% CI 0.24 to 0.93), and lycopene with risks of cerebral infarction (RR 0.74, 95% CI 0.59 to 0.92) and intracerebral hemorrhage (RR 0.45, 95% CI 0.24 to 0.86). Vitamin C intake was inversely associated with the risk for intracerebral hemorrhage (RR 0.39, 95% CI 0.21 to 0.74). After simultaneous modeling of the antioxidants, a significant association remained only between beta-carotene intake and risk for cerebral infarction (RR 0.77, 95% CI 0.61 to 0.99). CONCLUSIONS: Dietary intake of beta-carotene was inversely associated with the risk for cerebral infarction. No association was detected between other dietary antioxidants and risk for stroke.

OBJECTIVES: To investigate the relationship between gastric cancer and the intake of specific carotenoids (alpha-carotene, beta-carotene, lutein, and lycopene) and flavonoids (quercetin, kaempferol, myricetin, and luteolin) using new data on their concentration in foods. METHODS: Case-control study carried out in Spain that included 354 cases of gastric cancer and 354 controls, matched by age, gender, area of residence and hospital. Usual food intake was assessed using a dietary history questionnaire. RESULTS: In a multivariate model adjusted for several dietary factors, no association was found between intake of any of the studied carotenoids and the risk of gastric cancer. The adjusted OR of gastric cancer for the highest quartile of total flavonoid intake versus the lowest quartile was 0.44 (95 percent confidence interval [CI] = 0.25-0.78; P for trend = 0.003). Kaempferol intake was found to be protective (OR = 0.48; CI = 0.26-0.88; P for trend = 0.04) comparing the highest versus the lowest quartile of intake. A trend toward lower risk of stomach cancer with higher intake of quercetin was also found. CONCLUSIONS: The results of this study support the hypothesis that the well-established protective effect of fruit and vegetables against gastric cancer could, in part, be due to the presence of flavonoids.

Antioxidant properties and cytoprotective activity of flavonoids (rutin, dihydroquercetin, quercetin, epigallocatechin gallate (EGCG), epicatechin gallate (ECG)) were studied. All these compounds inhibited both NADPH- and CCl4-dependent microsomal lipid peroxidation, and the catechins were the most effective antioxidants. The I(50) values calculated for these compounds by regression analysis were close to the I(50) value of the standard synthetic antioxidant ionol (2,6-di-tert-butyl-4-methylphenol). The antiradical activity of flavonoids to O2-* was studied in a model photochemical system. Rate constants of the second order reaction obtained by competitive kinetics suggested flavonoids to be more effective scavengers of oxygen anion-radicals than ascorbic acid. By competitive replacement all flavonoids studied were shown to be chelating agents capable of producing stable complexes with transition metal ions (Fe2+, Fe3+, Cu2+). The flavonoids protected macrophages from asbestos-induced damage, and the protective effect increased in the following series: rutin < dihydroquercetin < quercetin < ECG < EGCG. The cytoprotective effect of flavonoids was in strong positive correlation with their antiradical activity to O2-*.

Biochem Biophys Res Commun. 2002 Jul 5;295(1):9-13.
The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids.
Rezk BM, Haenen GR, van der Vijgh WJ, Bast A.
Department of Pharmacology and Toxicology, Faculty of Medicine, Universiteit Maastricht P.O. Box 616, 6200 MD Maastricht, The Netherlands.

Phloretin is a dihydrochalcone flavonoid that displays a potent antioxidant activity in peroxynitrite scavenging and the inhibition of lipid peroxidation. Comparison with structurally related compounds revealed that the antioxidant pharmacophore of phloretin is 2,6-dihydroxyacetophenone. The potent activity of 2,6-dihydroxyacetophenone is due to stabilisation of its radical via tautomerisation. The antioxidant pharmacophore in the dihydrochalcone phloretin, i.e., the 2,6-dihydroxyacetophenone group, is different from the antioxidant pharmacophores previously reported in flavonoids.

Flavonoids are naturally occurring plant compounds with antioxidant properties. Their consumption has been associated with the protective effects of certain diets against some of the complications of atherosclerosis. Low-density lipoprotein (LDL) oxidative modification is currently thought to be a significant event in the atherogenic process. Most of the experiments concerning the inhibition of LDL oxidation used isolated LDL. We used diluted human whole plasma to study the influence of flavonoids on lipid peroxidation (LPO) promoted by copper, and their interaction with uric acid, one of the most important plasma antioxidants. Lipid peroxidation was evaluated by the formation of thiobarbituric acid reactive substances (TBARS) and of free malondialdehyde (MDA). The comparative capability of the assayed flavonoids on copper (II) reduction was tested using the neocuproine colorimetric test. In our assay system, urate disappears and free MDA and TBARS formation increase during the incubation of plasma with copper. Most of the tested flavonoids inhibited copper-induced LPO. The inhibition of LPO by flavonoids correlated positively with their capability to reduce copper (II). The urate consumption during the incubation of plasma with copper was inhibited by myricetin, quercetin and kaempferol. The inhibition of urate degradation by flavonoids correlated positively with the inhibition of LPO. Urate inhibited the copper-induced LPO in a concentration-dependent mode. Luteolin, rutin, catechin and quercetin had an antioxidant synergy with urate. Our results show that some flavonoids could protect endogenous urate from oxidative degradation, and demonstrate an antioxidant synergy between urate and some of the flavonoids.

Intake of dietary flavonols and flavones was inversely associated with risk for cardiovascular disease in several epidemiologic studies. This may have been due to effects on hemostasis because flavonoids have been reported to inhibit platelet aggregation in vitro. We indeed found that 2500 micromol/L of the flavonol quercetin and the flavone apigenin significantly inhibited collagen- and ADP-induced aggregation in platelet-rich plasma and washed platelets by approximately 80-97%. However, lower concentrations, such as might occur in vivo, had no effect. To test this in vivo we fed 18 healthy volunteers 220 g onions/d providing 114 mg quercetin/d, 5 g dried parsley/d providing 84 mg apigenin/d, or a placebo for 7 d each in a randomized crossover experiment with each treatment period lasting 2 wk. Onion consumption raised mean plasma quercetin concentrations to 1.5 micromol/L; plasma apigenin could not be measured. No significant effects of onions or parsley were found on platelet aggregation, thromboxane B2 production, factor VII, or other hemostatic variables. We conclude that the antiaggregatory effects of flavonoids seen in vitro are due to concentrations that cannot be attained in vivo. Effects of dietary flavonols and flavones on cardiovascular risk are possibly not mediated by hemostatic variables.

Endogenous antioxidants such as the lipid-soluble vitamin E protect the cell membranes from oxidative damage. Glutathione seems to be able to regenerate alpha-tocopherol via a so-called free radical reductase. The transient protection by reduced glutathione (GSH) against lipid peroxidation in control liver microsomes is not observed in microsomes deficient in alpha-tocopherol. Introduction of antioxidant flavonoids, such as 7-monohydroxyethylrutoside, fisetin or naringenin, into the deficient microsomes restored the GSH-dependent protection, suggesting that flavonoids can take over the role of alpha-tocopherol as a chain-breaking antioxidant in liver microsomal membranes.

OBJECTIVE: We examined plasma levels of carotenoids, tocopherols, and total antioxidant activity in women before and after dietary intervention to reduce fat and/or energy intakes. Dietary fat and energy may affect intake and bioavailability of carotenoids and tocopherols, and these micronutrient levels in turn can contribute to the antioxidant capacity of plasma. METHODS: Women were randomized onto one of four diets for 12 wk: non-intervention, low fat (15% of energy from fat with maintenance of energy intake), low energy (25% energy reduction with maintenance of percentage of energy from fat), and combined low fat and low energy. Fasting plasma was available for analysis from a subset (n = 41) of women enrolled in the study. RESULTS: Levels of carotenoids and tocopherols did not change significantly over 12 wk on any diet arm, despite a modest but statistically significant increase in fruit and vegetable intake in the women following the low-fat diet (from 3.3 to 5.2 servings/d excluding potatoes). Levels of Trolox-equivalent antioxidant capacity (TEAC), total cholesterol, and two major plasma antioxidants (urate and bilirubin) also did not change significantly. Of the individual micronutrients measured, lycopene and lutein/zeaxanthin correlated most strongly with TEAC values, and the correlation with lycopene was statistically significant before intervention. CONCLUSION: The decreases in dietary fat and energy intakes in this study were quite large, but this did not appear to have detrimental effects on plasma micronutrient levels, nor did it appreciably affect plasma antioxidants. Because lycopene levels were significantly associated with plasma TEAC before intervention, interventions that increase levels of lycopene might be more likely to increase the antioxidant capacity of plasma.

Accumulating evidence suggests that dietary antioxidant vitamins are positively associated with lung function. No evidence exists regarding whether dietary carotenoids other than beta-carotene are related to pulmonary function. In 1995--1998 the authors studied the association of forced expiratory volume in 1 second and forced vital capacity as the percentage of the predicted value (FEV(1)% and FVC%, respectively) after adjustment for height, age, gender, and race with the intakes of several carotenoids (alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein/zeaxanthin, and lycopene) in a random sample of 1,616 men and women who were residents of western New York State, aged 35--79 years, and free from respiratory disease. They observed significant associations of lutein/zeaxanthin and vitamins C and E with FEV(1)% and FVC% using multiple linear regression after adjustment for total energy intake, smoking, and other covariates. When they analyzed all of these antioxidant vitamins simultaneously, they observed the strongest association of vitamin E with FEV(1)% and of lutein/zeaxanthin with FVC%. The differences in forced expiratory volume in 1 second and forced vital capacity associated with a decrease of 1 standard deviation of dietary vitamin E or lutein/zeaxanthin were equivalent to the influence of approximately 1--2 years of aging. Their findings support the hypothesis that carotenoids, vitamin C, and vitamin E may play a role in respiratory health and that carotenoids other than beta-carotene may be involved.

The ability of dietary carotenoids such as beta-carotene and lycopene to act as antioxidants in biological systems is dependent upon a number of factors. While the structure of carotenoids, especially the conjugated double bond system, gives rise to many of the fundamental properties of these molecules, it also affects how these molecules are incorporated into biological membranes. This, in turn, alters the way these molecules interact with reactive oxygen species, so that the in vivo behavior may be quite different from that seen in solution. The effectiveness of carotenoids as antioxidants is also dependent upon their interaction with other coantioxidants, especially vitamins E and C. Carotenoids may, however, lose their effectiveness as antioxidants at high concentrations or at high partial pressures of oxygen. It is unlikely that carotenoids actually act as prooxidants in biological systems; rather they exhibit a tendency to lose their effectiveness as antioxidants.

The antioxidant activities of astaxanthin and related carotenoids have been measured by employing a newly developed fluorometric assay. This assay is based on 4,4-difluoro-3,5-bis(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene (BODIPY 665/676) as an indicator; 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) as a peroxyl radical generator; and 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator in an organic and liposomal media. By employing this assay, three categories of carotenoids were examined: namely, the hydrocarbon carotenoids lycopene, alpha-carotene, and beta-carotene; the hydroxy carotenoid lutein; and the alpha-hydroxy-ketocarotenoid astaxanthin. The relative peroxyl radical scavenging activities of Trolox, astaxanthin, alpha-tocopherol, lycopene, beta-carotene, lutein, and alpha-carotene in octane/butyronitrile (9:1, v/v) were determined to be 1.0, 1.0, 1.3, 0.5, 0.4, 0.3, and 0.2, respectively. In dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tri-HCl buffer (pH 7.4 at 40 degrees C), the relative reactivities of astaxanthin, beta-carotene, alpha-tocopherol, and lutein were found to be 1.00, 0.9, 0.6, and 0.6, respectively. When BODIPY 665/676 was replaced by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C(11)) as an indicator, astaxanthin showed the highest antioxidant activity toward peroxyl radicals. The relative reactivities of Trolox, astaxanthin, alpha-tocopherol, alpha-carotene, lutein, beta-carotene, and lycopene were determined to be 1.0, 1.3, 0.9, 0.5, 0.4, 0.2, and 0.4, respectively.

Much effort has been expended in evaluating the relative antioxidant potency of carotenoid pigments in both in vitro and in vivo experiments. It is quite clear that in vitro, carotenoids can inhibit the propagation of radical-initiated lipid peroxidation, and thus fulfill the definition of antioxidants. When it comes to in vivo systems, it has been much more difficult to obtain solid experimental evidence that carotenoids are acting directly as biological antioxidants. In fact, under nonphysiological circumstances, carotenoids may act as prooxidants. These results can be modified by altering the oxidant stress, the cellular or subcellular system, the type of animal, and environmental conditions, such as oxygen tension. Results of this type raise the question as to whether it is still appropriate to group the carotenoids with such antioxidant vitamins as vitamin E and vitamin C. Thus, the biological properties of the carotenoids may be much more related to the products of the interaction of carotenoids with oxidant stress, that is, such breakdown products as apocarotenoids and retinoids.

Surprisingly, neither the precise pharmacological effect nor the toxicological profile is usually established for food components. Carotenoids are no exception in this regard. Only limited insight into the pharmacology and toxicology of carotenoids exists. It is known that the antioxidant action of carotenoids is determined by 1. electron transfer reactions and the stability of the antioxidant free radical, 2. the interplay with other antioxidants and 3. the reaction with active oxygen. Numerous metabolites of carotenoids are formed upon their action as an antioxidant. Most of these metabolites have an unknown biological activity. It is concluded that a severe lack of knowledge hampers adequate suggestions for human supplementation.