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J. Agric. Food Chem. 2007, 55, 4258−4263
A Commercial Extract of Fruits and Vegetables, Oxxynea, Acts
as a Powerful Antiatherosclerotic Supplement in an Animal
Model by Reducing Cholesterolemia, Oxidative Stress, and
NADPH Oxidase Expression
THIBAULT SUTRA,†,‡ KELLY DEÄCORDEÄ,† JEÄROME RISS,† CONSTANT DALLAS,§ JEAN-PAUL CRISTOL,‡ AND JEAN-MAX ROUANET*,† EA 3762 Nutrition & Aliments, Place E. Bataillon, Universite´ Montpellier 2, 34095 Montpellier, France, EA 2993 Nutrition Humaine & Athe´roge´ne`se, Universite´ Montpellier 1, CHU La Peyronie, 34925 Montpellier, France, and Nutraceutic Business Consulting (NB Consulting), 24 rue Rene´ Caillie, 34500 Be´ziers, France The effects of fruit and vegetable extract (Oxxynea) on plasma cholesterol, early atherosclerosis,cardiac production of superoxide anion, and NAD(P)H oxidase expression were studied in an animalmodel of atherosclerosis. Thirty six hamsters were divided into two groups of 18 and fed an atherogenicdiet for 12 weeks. They received by gavage either water or Oxxynea in water at a human doseequivalent of 10 fruits and vegetables per day. Oxxynea lowered plasma cholesterol and non-HDLcholesterol, but not HDL-cholesterol, and increased plasma antioxidant capacity. It also stronglyreduced the area of aortic fatty streak deposition by 77%, cardiac production of superoxide anion by45%, and p22phox subunit of NAD(P)H oxidase expression by 59%. These findings support the viewthat chronic consumption of antioxidants supplied by fruits and vegetables has potential beneficialeffects with respect to the development of atherosclerosis. The underlying mechanism is related mainlyto inhibiting pro-oxidant factors and improving the serum lipid profile.
KEYWORDS: Atherosclerosis; hamsters; fruits and vegetables; antioxidant compounds; NADPH oxidase
INTRODUCTION
stress observed in atherosclerosis awaits clarification. Recent Mortality from cardiovascular disease is the leading cause findings have suggested that the major source of ROS in the of death in the industrialized world. Diet is believed to play a vascular wall, and also in vascular smooth muscle cells, is the major role in the development of this disease, and much research NAD(P)H oxidase system. This is a membrane-associated is being focused on identifying ways to prevent it through enzyme, composed of five subunits, catalyzing the one-electron changes in dietary habits. Oxidation of low-density lipoproteins reduction of oxygen, using NADH or NADPH as the electron (LDL) is traditionally accepted as initiating processes leading donor. NAD(P)H oxidase generates significant amounts of to the development of atherosclerosis. The earliest events in superoxide radicals, and an association between enzymatic the development of the pathology are endothelial dysfunction activity and clinical risk factors in atherosclerosis has been and oxidative stress in the vascular cell wall, activation of shown (3). Moreover, expression of membrane subunits of inflammatory cells, and migration of vascular smooth muscle NAD(P)H oxidase, such as p22phox, Nox 1, and Nox 4, is cells to the intima with the modification of the extracellular modulated in atherosclerotic arteries (4) and in vascular injury matrix, leading to the artery remodeling (1). Development of (5) by various cytokines like interferon (IFN)-γ and transforming atherosclerosis is thought to be closely dependent upon increased growth factor (TGF)- 1. Azumi et al. (6) found that the severity oxidative stress, that is, an imbalance between reactive oxygen of atherosclerotic lesion correlated with p22phox overexpression species (ROS) generation (chiefly superoxide anions, hydrogen in coronary arteries. Excessive generation of superoxide anion peroxide, hydroxyl radicals) and natural cell antioxidant capacity by phagocyte NADPH oxidase is responsible for LDL oxidation, in favor of the former (2). ROS can regulate many signaling which is the key factor in the initation and progression of pathways, such as infiltration of monocytes in intima and atherosclerosis (1, 7). The contribution of NADPH oxidase to vascular smooth muscle cell proliferation. The cause of oxidative the pathogenesis of atherosclerosis overshoots LDL oxidationprocess. NAD(P)H oxidase induces the expression of adhesion * Author to whom correspondence should be addressed [telephone/fax 33 (0)4 67 14 35 21; e-mail [email protected]].
molecules in endothelial cells for recruitment of monocyte- derived macrophages (8), leading to an amplification system (9) and vascular smooth muscle cells proliferation (10).
Oxxynea as a Powerful Antiatherosclerotic Supplement J. Agric. Food Chem., Vol. 55, No. 10, 2007 The importance of antioxidants in human health has become oxygen radical generator, and antioxidant (20 µL, either trolox [1-8 increasingly clear, and some epidemiological studies showed µM, final concentration] or Oxxynea [at different concentrations]). The the potential health benefits of dietary antioxidants (11). Fruits reaction was performed at 37 °C, and fluorescence was recorded every and vegetables consumption is inversely related to cancer and minute for 80 min. ORAC values was expressed as trolox equivalentsby using the standard curve calculated for each experiment. The final coronary heart disease mortality, and this appears not to be due ORAC value for Oxxynea was 6100 µmol of trolox equiv/g.
exclusively to high levels of vitamins and fibers (12, 13). Several Animals. Male golden Syrian hamsters (Janvier, Le Genest-St-Isle,
studies have shown that flavonoids also contribute to the overall France) weighing 85-95 g were randomly divided into groups with antioxidant capacity of fruits and vegetables and also to the approximately equal mean group body weights. The animals were beneficial effects, a view supported by recent research demon- housed in plastic cages in a temperature-controlled room (23 ( 1 °C) strating that dietary flavonoids protect against vascular diseases subjected to a 12 h light:dark cycle (lights on at 0700 h) with free and reduce the risk of myocardial infaction (13). There is access to both food and water. Hamsters were handled according to growing interest in flavonoids and phenolic compounds because the guidelines of the Committee on Animal Care at the University of they are potent antioxidants and inhibit low-density lipoprotein Montpellier and NIH guidelines (27).
(LDL) oxidation in vitro (14), properties that are associated with Diets and Feeding Procedures. Two experiments were carried out
their ability to scavenge free radicals and chelate metals. An concomitantly. Experiment 1 was used to determine at what pointhamsters on the atherogenic diet exhibited oxidative stress and increased consumption of phenolics has been correlated with a hypercholesterolemia. In such a way, four groups of six hamsters each reduced risk of cardiovascular diseases and certain types of received either a standard or an atherogenic diet for 15, 30, 45, and 84 cancers (15, 16). Moreover, polyphenols have been shown to days. The standard diet consisted of 200 g/kg casein and 3 g/kg directly interact with NAD(P)H oxidase, inhibiting most of the L-methionine, 447 g/kg corn starch, 175 g/kg sucrose, 50 g/kg cellulose, ROS production in the vessel wall (17). Cumulatively or 80 g/kg vegetable oil (corn oil/sunflower oil, 1/1), mineral mix (35 synergistically, these dietary antioxidants provide bioactive g/kg), and vitamin mix (10 mg/kg). The atherogenic diet has been mechanisms to reduce oxidative stress.
previously described (22) and consisted of 200 g/kg casein and 3 g/kg With the exception of recent research by Adams et al. (18) L-methionine, 393 g/kg corn starch, 154 g/kg sucrose, 50 g/kg cellulose, with a transgenic mice model, few studies have investigated 150 g/kg lard, 5 g/kg cholesterol, mineral mix (35 g/kg), and vitamin the effect of plant material on atherosclerosis and oxidative stress mix (10 mg/kg). Vitamin and mineral mixes were formulated accordingto AIN-93 guidelines (28) and supplied by Scientific Animal Food & in rodents (19, 20); moreover, these studies were only focused Engineering (SAFE, Augy, France). The atherogenic diet did not contain on the effect of vegetables. Golden Syrian hamsters fed a fat- rich diet develop dyslipidemia and atherosclerotic plaques, In experiment 2, two groups of 18 hamsters were fed the atherogenic similar in many respects to human atheroma (21-23). Hamsters diet for 84 days. The hamsters of each group were fed daily by gavage were selected for this study because of their responsiveness to either tap water (group 1; control) or a solution of Oxxynea in water plasma cholesterol lowering and anti-atherogenic interventions (group 2; experimental). The volume of the solutions force-fed was (24). Moreover, hamster has a plasma lipoprotein distribution adjusted daily to the weight of the hamsters and was established by similar to that of humans and LDL as the major plasma extrapolating 500 mL/d water drinking for a 70 kg human. This cholesterol carrier. To induce an oxidative stress, their high represents a volume of 7.14 mL/(kg of body weight‚d). Based upon cholesterol and high fat diet was rendered deficient in vitamin the ORAC value of fruits and vegetables starting material and issuingOxxynea, and according to a recommended consumption of 10 servings C and E and in selenium. This study was designed to trigger of fruits and vegetables/d for a human, that is, ∼800 g/d, hamsters the arterial wall response to such a stress (fatty streak formation from the experimental group received 21.4 mg of Oxxynea/(kg body and aortic atherosclerosis emergence) and then to look at the modulation of this effect by a commercial fruit and vegetable Analytical Procedures. At the end of each experimental period,
extract, Oxxynea. In addition, for the first time, modulation of hamsters were deprived of food for 18 h and were anesthetized with oxidative stress parameters including cardiac production of an IP injection of pentobarbital (60 mg/mL at a dosage of 60 mg/kg superoxide anions and NAD(P)H oxidase expression was body weight). In experiment 1, only plasma cholesterol, cardiac superoxide anion production, and NADPH oxidase expression weremeasured as described below. In experiment 2, blood was drawn bycardiac puncture with heparin moistened syringes, and plasma was MATERIALS AND METHODS
prepared by centrifugation at 2000g for 10 min at 4 °C, then stored at Fruits and Vegetable Extract. According to the manufacturer (NB
-80 °C prior to analysis. Plasma concentrations of total cholesterol Consulting, Be´ziers, France), the powdered Oxxynea extract was (TC) and HDL cholesterol (HDL-C) were measured by an enzymatic obtained from 22 varieties of antioxidant-rich fruits and vegetables technique (Konelab, Thermo Electron Corp., Vantaa, Finland). Plasma including apple, asparagus, bilberry, apricot, black currant, broccoli, nonHDL-C was calculated from the difference between TC and HDL- carrot, cherry, cucumber, garlic, grapefruit, green cabbage, olive, onion, orange, papaya, pineapple, red and white grapes, strawberry, tea, tomato, The antioxidant capacity of plasma was measured as trolox and wheat germ. Oxxynea contains high level of catechins, that is, sum equivalent, that is, a quantitative value for general antioxidant levels of procyanidin dimers B1, B2, B3, and B4 (1.14 g/100 g) and in biological samples (29, 30), which was assayed in plasma with a monomeric catechins (catechin, 0.55 g/100 g; epicatechin, 3.08 g/100 quantitative colorimetric technique according to the kit supplier’s g; epichatechin-3-O-gallate, 4.10 g/100 g; epigallocatechin, 4.17 g/100 instructions (Kit NX2332; Randox, Mauguio, France). The assay is g; epigallocatechin-3-O-gallate, 21.33 g/100 g). Other phenolic com- based on the incubation of a peroxidase and H2O2 with 2,2′-azino-di- pounds such as gallic acid and anthocyanins were detected in lower (3-ethylbenzthiazoline sulfonate) (ABTS) to produce the radical cation amounts (0.15 and 0.6 g/100 g, respectively). The extract also contained ABTS°+. This has a relatively stable blue-green color, which is low levels of lycopene (28 mg/100 g) and vitamin C (4.92 mg/100 g).
measured at 600 nm. Antioxidants (albumin, uric acid, ascorbic acid, Oxygen Radical Absorbance Capacity (ORAC) Value. The
R-tocopherol, glutathione, beta-carotene, etc.) in the sample suppressed ORAC-fluorescein assay was based on the method of Ou et al. (25) ABTS°+ color production to a degree proportional to their concentration.
that was subsequently modified by Davalos et al. (26). Briefly, the Aortic Tissue Processing. Following blood collection and liver
reaction was performed in 75 mM phosphate buffer (pH 7.4), and the removal, the intact aorta from 12 hamsters was first perfused with final assay mixture (200 µL) contained fluorescein (120 µL, 70 nM phosphate buffered saline containing 1 mmol/L CaCl2 and 15 mmol/L final concentration) as oxidizable substrate, 2,2′-azobis(2-amidinopro- glucose for 5 min, then with 0.1 mmol/L sodium cacodylate buffer pH pane) dihydrochloride (AAPH, 60 µL, 12 mM final concentration) as 7.4 containing 2.5 mmol/L CaCl2, 2.5% paraformaldehyde, and 1.5% J. Agric. Food Chem., Vol. 55, No. 10, 2007 glutaraldehyde for the fixation of the vasculature. The aorta wascarefully dissected between sigmoid valves and 3-4 cm after the aorticarch and thoroughly cleaned of loose adventitial tissue; the aortic archwas cut free, open longitudinally along the outside of the arch, pincorked, immersed in fresh fixative solution, and stored at 4 °C untilstaining. The aortic arches were then first rinsed for 48 h in 0.1 mol/Lsodium cacodylate buffer pH 7.4 containing 30 mmol/L CaCl2 and 250mmol/L sucrose. The arches were then rinsed in distilled water, stainedfor 40 s in Harris hematoxylin, rinsed in distilled water, and then quicklyin 70% isopropyl alcohol; finally, they were stained in Oil red O for30 min according to Nunnari et al. (31), rinsed in 70% isopropyl alcohol,and back to distilled water. Each aortic arch was then directly displayedon a glass slide, endothelium side up, covered with Aquamountmounting medium and cover slips, and observed en face by lightmicroscopy. All segments were photographed using a video digitizer.
A computerized image analysis system (ImageJ, Scion Corp., Frederick,MD) attached to a compound light microscope was used to measurethe total Oil Red O stained area of each aortic arch. The area coveredby foam cells (aortic fatty streak lesion area or AFSA) was expressedas a percentage of the total area surveyed.
Determination of Superoxide Anion Production. Superoxide anion
production was evaluated in hamsters that were not used for AFSAmeasurement (n ) 6 per group). Briefly, the left ventricle (150 mg)(41) was placed in Krebs buffer containing 250 µM of lucigenin, andthe intensity of luminescence was recorded on a luminometer (Perkin-Elmer Wallac, Victor, Turku, Finland). Results were expressed as count/mg of protein.
Immunoblotting. Proteins were extracted as previously described
(32) from the frozen left ventricles of six hamsters previously used formeasurement of superoxide anion production. Samples were homog-enized using an ultra turrax T25 basic (Irka-Werke) in an ice coldextraction buffer containing 120 mM NaCl, 25 mM KCl, 2 mM CaCl2,15 mM Tris-Cl pH 7.5, 0.5% Triton X-100, 1 mM PMSF, 0.1 mMDTT, 10 M leupeptin, and 1 M pepstatin. Protein concentrations insample were determined by Bio-Rad Dc protein assay using BSA as astandard. Proteins (50 µg) were separated with 12% SDS-PAGE andthen transferred to a nitrocellulose membrane (45 min, 100 V).
Membranes were incubated for 2 h with primary antibody againstp22phox (1/200, Santa Cruz Biotechnology, Santa Cruz, CA) in blockingbuffer. After six washes (6 °C, 5 min) in TBS/Tween under gentle Figure 1. Time course experiment of plasma cholesterol concentration
agitation, blots were incubated for 45 min with horseradish peroxidase- (A) and superoxide anion production (B) in hamsters fed a standard diet
labeled antibody (1/5000). After further washes, blots were treated with (white bars) and in hamsters fed an atherogenic diet (black bars) during enhanced chemiluminescence detection reagents (ECL, Amersham), and experiment 1. Values are expressed as mean ± SEM of triplicate wells areas (mm2) were measured using the BIO-Profil 1D software (Fisher (n ) 6). For each dietary treatment, bars with different index letters differ Statistical Analyses. Data are shown as the means ( SEM. Data
were subjected to logarithmic transformation where necessary to achieve tively, such differences being only significant at 84 days (Figure
homogeneity of variances. Statistical analysis of the data was carried 1b). In addition, cardiac superoxide levels increased by 20.5%
out using the Stat View IV software (Abacus Concepts, Berkeley, CA) (NS) from day 15 to day 45 and were highest at 84 days (Figure
by one-way ANOVA followed by Fisher’s protected least significant 1b) in hamsters fed atherogenic diet. In agreement with the
difference test. Differences were considered significant at P < 0.05.
cardiac superoxide production at 84 days of atherogenic diet,the measure of cardiac NADPH oxidase expression by western blot showed that cholesterol diet triggered a significant expres- Evolution of Cholesterol Concentration, Superoxide Pro-
sion of p22phox (Figure 3) by 146% (p ) 0.001).
duction, and NADPH Oxidase Expression during Early
Oxxynea Improves Blood Lipid Profile. Nutritional param-
Development of Atherosclerosis (Experiment 1). Plasma
eters are shown in Table 1. No significant difference appeared
cholesterol significantly increased in the hamsters fed the in food intake and final body weight between the two groups.
atherogenic diet just from the first 15 days as compared to the Plasma lipids are summarized in Table 2. Oxxynea significantly
controls animals. No alteration in cholesterol was observed in reduced plasma total cholesterol by 11.7% (p < 0.0001) and the control hamsters fed the standard diet, whereas in hamsters non-HDL cholesterol by 14% (p ) 0.0066), but not HDL- fed the atherogenic diet, plasma cholesterol level significantly cholesterol, as compared to the control group. Consequently, increased from day 30 (7.76 ( 0.46 mmol/L) to day 45 (10.18 the atherogenic index calculated as total cholesterol/HDL- ( 1.47 mmol/L) and leveled to 84 days (Figure 1a).
cholesterol was lowered by 8.3% (p ) 0.0139) in hamsters The time course of cardiac superoxide production was also established. Whereas the cardiac superoxide level was constant Oxxynea Improves Antioxidant Status and Decreased
during 84 days in the control hamsters, we noted an increase by Preventing NADPH Oxidase Expression. In experi-
of superoxide production in atherogenic hamsters compared at ment 2, Oxxynea significantly increased by 10% the plasma 15, 30, 45, and 84 days by 18.5% (not significant, NS), 21.5% antioxidant capacity induced by the atherogenic diet (p ) (NS), 21.8% (p ) 0.0548), and 94.1% (p < 0.0001), respec- 0.0244) (Table 2). As shown in Figures 2 and 3, superoxide
Oxxynea as a Powerful Antiatherosclerotic Supplement J. Agric. Food Chem., Vol. 55, No. 10, 2007 Table 2. Effects of Ingestion of a Fruit and Vegetable Extract
(Oxxynea) on Plasma Lipid Concentrations and on Plasma Antioxidant
Capacity (PAC) in Hamsters Fed an Atherogenic Dieta (Experiment 2)
a Values are means ± SEM, n ) 18. Data were analyzed by one-way ANOVA followed by the least significant difference test. For each dietary treatment, meansin a column with different letters differ, P < 0.05. b TC : total cholesterol. c HDLC: high-density lipoprotein cholesterol. d Total cholesterol/HDL-cholesterol.
Figure 2. Cardiac superoxide anion production in hamsters fed a standard
or an atherogenic diet with (OXX) or without (ATH) Oxxynea. Values are
expressed as mean ± SEM of triplicate wells (n ) 6). For each dietary
treatment, bars with different index letters differ (P < 0.05).
Figure 4. Effects of ingestion of water (ATH) or a fruit and vegetable
extract Oxxynea (OXX), on aortic fatty streak area in hamsters fed an
atherogenic diet for 84 days (experiment 2). Values are expressed as
mean ± SEM (n ) 12). Bars with different index letters differ (P < 0.05).
Figure 3. Expression of the cardiac p22phox subunit of NAD(P)H oxidase
DISCUSSION
in hamsters fed a standard diet (STD) or an atherogenic diet with (OXX)or without (ATH) Oxxynea during 84 days. The densitometric measurement This study reported the protective effect of fruit and vegetable shows arbitrary area units. Values are expressed as mean ± SEM (n ) antioxidants supplementation against diet-induced oxidative 6). For each dietary treatment, bars with different index letters differ (P < stress and atherosclerosis in hypercholesterolemic golden Syrian The golden Syrian hamster is a good nutritional rodent model Table 1. Effects of Ingestion of a Fruit and Vegetable Extract
of atherosclerosis, which could mimic the early stages of human (Oxxynea) on Body Weight and Food Intake of Hamsters Fed an atherosclerosis, that is, fatty streak (33). As previously reported, a high fat diet led to an early increase in total and non-HDLcholesterol after 15 days of diet leading to lipid deposition on aortic arch at 84 days. Interestingly, the atherogenic diet-induced hypercholesterolemia is in parallel accompanied by a tendency of superoxide anion overproduction, which reaches the signifi- cance at 45 days. In agreement with oxidative hypothesis ofatherosclerosis, it could be postulated that NADPH oxidase a Values are means ± SEM, n ) 18. Data were analyzed by one-way ANOVA expression and activity conspire with high non-HDL cholesterol followed by the least significant difference test. For each dietary treatment, means level to induce foam cells and fatty streak.
in a column with different letters differ, P < 0.05.
As reported in other rodent models of atherosclerosis, such as insulin resistance (32) or hypertension (34), oxidative stress anion production (Figure 2) and expression of p22phox (Figure
could be a key event in diet-induced atherosclerosis and cardiac 3) decreased by 45.5% (p < 0.0001) and 59.1% (p ) 0.001),
remodelling. Hypercholesterolemia has been previously involved respectively, in hamsters receiving Oxxynea.
in enhanced ROS production by NADPH oxidase activity (35) Oxxynea Powerfully Inhibits Lipid Deposition in Aortic
in a model of cholesterol-fed mice. It has been further suggested Arch. Average aortic fatty streak accumulation (AFSA),
that ROS overproduction could be linked to an induction of measured as the percentage of Oil Red O staining relative to NADPH oxidase subunit in particular gp91phox in neutrophils the total area surveyed (Figure 4), was not detected in hamsters
from hyperlipidemic guinea pig (36). Our observation of a fed the standard diet (experiment 1). In addition, AFSA was hypercholesterolemic diet-induced NADPH oxidase expression significantly reduced by 77% (p ) 0.001) in the hamsters (+146%) in cardiac tissue extends these observations. Super- receiving Oxxynea as compared to control animals on the oxide anion and further ROS generation by monocyte-derived macrophages could oxidize LDL, being in turn responsible for J. Agric. Food Chem., Vol. 55, No. 10, 2007 amplification loops by stimulation of phagocyte NADPH associated with lipid and plasma antioxidant capacity improve- oxidase. Beyond generation of foam cells and cholesterol ment without any effect on plasma oxidative stress markers such deposition (37), cholesterol-induced ROS generation could as MDA, AOPP, and AGEs (21). Taken together, these results participate in left ventricle remodelling as suggested by the suggest a specific role of polyphenol in vascular tissue mediated enhanced expression of p22phox in the infarcted sites.
Oxxynea, a fruit and vegetable antioxidant extract, prevented All of these results suggest that this extract acted by the progression of early atherosclerosis in aortic arch of mechanisms operating both inside and outside a hypolipemic cholesterol-fed hamsters (<10% foam cell coverage of aorta).
effect, especially an antioxidant effect. Although the constituent- In agreement with the recent report by Adams et al. (18) that a (s) responsible for these effetcs remain(s) unclear, candidates diet rich in green and yellow vegetables inhibits atherosclerosis such as vitamin C, vitamin E, carotenoids, selenium, and in transgenic mice, we have shown that Oxxynea extract polyphenols could act synergistically or additively to prevent prevents fatty streak formation in aortic arch of cholesterol fed atherosclerosis in the hamster model. These promising results hamster. This effect could be in part due to a slight, but obtained in a diet-induced atherosclerosis animal model give significant, decrease in total and non-HDL cholesterol, without rise to further studies in clinical fields.
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