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© 2006 American Society for Nutrition J. Nutr. 136:2213-2219, August 2006

Nutrition and Disease

The Olive Constituent Oleuropein Exhibits Anti-Ischemic, Antioxidative, and Hypolipidemic Effects in Anesthetized Rabbits1,2

Ioanna Andreadou3,4,*, Efstathios K. Iliodromitis3, Emmanuel Mikros4, Maria Constantinou4, Apostolos Agalias5, Prokopios Magiatis5, Alexios Leandros Skaltsounis5, Elli Kamber6, Anna Tsantili-Kakoulidou4 and Dimitrios Th Kremastinos3

3 Second University Department of Cardiology, Medical School, Attikon General Hospital, University of Athens, Athens 12462, Greece; 4 Department of Pharmaceutical Chemistry, 5 Department of Pharmacognosy, School of Pharmacy, University of Athens, Panepistimioupolis, Zografou 15571, Greece; and 6 Department of Experimental Physiology, Medical School, University of Athens, Athens, Greece

* To whom correspondence should be addressed. E-mail: jandread{at}pharm.uoa.gr.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Thgoal of this study was to evaluate the efficacy of the antioxidant olive constituent, oleuropein, on infarct size, oxidative damage, and the metabolic profile in rabbits subjected to ischemia. Oleuropein, 10 or 20 mg/(kg·d), was administered to 8 groups that consumed a normal or hypercholesterolemic diet for 6 wk or only the higher dose for 3 wk. Circulating levels of malondialdehyde, protein carbonyl, nitrite+nitrate, cholesterol, triglycerides, SOD activity, and the metabolic profile were measured using 1H NMR spectra. In rabbits that consumed the normal diet, the infarct size (percentage of infarct to risk areas) was reduced by the administration of 10 mg oleuropein/(kg·d) (16.1 ± 2.9%) or 20 mg oleuropein/(kg·d) for 3 wk (21.7 ± 2.2%) or for 6 wk (24.3 ± 1.3%) compared with the control group (48.05 ± 2.0%, P < 0.05). Only the higher dose of 20 mg/(kg·d) reduced the infarct size in hypercholesterolemic rabbits (34.7 ± 4.4% for 6 wk and 34.8 ± 6.1% for 3 wk) compared with the cholesterol-fed control group (52.8 ± 2.4%, P < 0.05). Oleuropein decreased the plasma lipid peroxidation product and protein carbonyl concentrations compared with the control groups, in which these factors increased relative to baseline due to ischemia and reperfusion. Furthermore, in rabbits administered oleuropein, RBC superoxide dismutase activity did not change during ischemia and reperfusion. This activity was significantly higher than in both control groups in which it was reduced by ischemia and reperfusion compared with baseline. Treatment for 6 wk with both doses of oleuropein reduced total cholesterol and triglyceride concentrations. 1H NMR spectra revealed a different profile of glycolysis metabolites in the oleuropein-treated groups compared with the controls. Oleuropein, for 3 or 6 wk, reduced the infarct size, conferred strong antioxidant protection and reduced the circulating lipids. This is the first experimental study in vivo that suggests the possibility of using an olive constituent in the treatment of ischemia.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Epidemiological data support a lower incidence of cardiovascular diseases in the Mediterranean area compared with the rest of Europe (1,2). These findings were attributed to the traditional Mediterranean diet (3), with its characteristically high consumption of olive oil and olives. The olive products constitute a rich source of polyphenols such as oleuropein and its derivatives, including hydroxytyrosol, which scavenge free radicals and inhibit the chemical oxidation of LDL (4–6). Diets rich in olive oil reduce tissue oxidative stress and enhance the glutathione antioxidant defense system in atherosclerotic rabbits (7), whereas diets rich in polyphenols are currently recommended for the prevention of atherosclerosis (8,9).

The main constituent of the leaves and unprocessed olive drupes of Olea europaea is oleuropein, and the majority of the polyphenols found in olive oil or table olives are products of its hydrolysis. Oleuropein has high antioxidant activity in vitro, comparable to a hydrosoluble analog of tocopherol (10). Oleuropein scavenges superoxide anions and hydroxyl radicals, and inhibits the respiratory burst of neutrophils and hypochlorous acid–derived radicals (5,11). The oxygen-derived free radicals contribute to the pathogenesis of atherosclerosis by oxidizing LDL and generating several species that react with oxygen in the vascular wall (12). Diets rich in saturated fatty acids lead to increased oxidative stress in different tissues of rabbits. In contrast, dietary supplements containing olive oil reduce lipid peroxidation and favor tissue antioxidant defensive mechanisms mediated by the glutathione system (13). Furthermore, olive oil reduces hyperlipidemia and decreases the atherogenic index (7).

Studies (14–16) showed that oleuropein is rapidly absorbed after oral administration, with maximum plasma concentration occurring 2 h after administration. Hydroxytyrosol was its most important metabolite. Both compounds are rapidly distributed and excreted in urine mainly as glucoronides or in very low concentrations as free forms.

In light of the above considerations and of increasing interest in the Mediterranean diet, we investigated the effect of oleuropein, in normal and hypercholesterolemic rabbits subjected to ischemia and reperfusion, on the following: 1) reduction in myocardial infarct size; 2) oxidative stress during ischemia-reperfusion; 3) circulating cholesterol and triglyceride levels; and 4) metabolic profile using 1H-NMR-based metabonomics.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Oleuropein isolation. The oleuropein used in this study was isolated from Olea europaea leaves. Air-dried and pulverized leaves (5 kg) were extracted by mechanical stirring for 12 h with acetone (2 x 25 L). The extract was evaporated completely and washed with a mixture of CH2Cl2:methanol: 98/2 (3 L). The insoluble material (360 g) was separated, dried and subjected to medium pressure LC with Si gel 60 Merck (15–40 µm), using the CH2Cl2-methanol gradient as the eluent to extract pure oleuropein (180 g). The purity of oleuropein was 95% using analytical RP-HPLC with a THERMO Finnigan SPECTRA system (column: LiChrosorb RP-18, 250 x 4.0 mm, 5µm, elution solvent: H2O:acetonitrile gradient, flow:1 mL/min, UV-detection: 254 nm).The purified oleuropein was entirely in the glucoside form and was free of any aglycone forms. The chemical structure of oleuropein is shown in Fig. 1.


Figure 1
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  		Figure 1  Chemical structure of oleuropein.

 
    Dosage protocol. The dose used in the in vivo experiments was based on the average consumption of olive drupes and olive oil (17) in the Mediterranean area. Oleuropein is the major polyphenol of olive tree leaves, unprocessed drupes, and bitter table olives. The majority of polyphenolic metabolites found in olives or in olive oil (hydroxytyrosol, oleuropein aglycone, demethyloleuropein, elenolic acid) originate from the hydrolysis of oleuropein (18). Therefore, we expressed total polyphenol consumption from olive drupes or olive oil as the molecular equivalent of oleuropein, calculated to be ~100 mg/d.

For the extrapolation of the dosage from humans to rabbits, we used the metabolic body size or food intake rather than body weight as a criterion (19). The estimated quantity of oleuropein expressed per unit of human diet was 0.2 mg/g of dry food. Considering an energy density of ~16.4 kJ/g of dry food, the dose was calculated to be 0.012 mg/kJ. For rabbits, this consumption corresponded to a dose of 10 mg/(kg body weight·d). A higher dose of 20 mg/(kg·d) was also given to test for a dose response.

    Rabbits and diets. New Zealand white male rabbits (n = 52) weighing between 2.5 and 3.3 kg completed the study. All rabbits received proper care in compliance with the Principles of Laboratory Animal Care, published by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the Academy of Sciences and published by the National Institutes of Health (20).

The composition of the normal diet (A717, produced by the Greek Factory "Farma Gefyra") per 100 g was: protein 17.5 g, fat 2.8 g, carbohydrate 15 g, ash 6.5 g, moisture 12 g, calcium 1 g, phosphorus 0.7 g, NaCl 0.5g, all-trans retinol 3.6 mg, cholecalciferol 0.05 mg, all-rac-a-tocopherol 30 mg. The hypercholesterolemic diet was prepared by adding 2 g of cholesterol (product USP23, Dolder) mixed with 6% corn-oil to 1 kg normal diet and tap water.

    Experimental protocol. Oleuropein was dissolved in 5% dextrose solution and was administered orally via a small feeding tube. The rabbits were divided randomly into 8 groups and were subjected to a 30-min period of regional ischemia of the heart followed by a 3-h reperfusion with the following interventions before ischemia was introduced: 4 groups consumed the normal diet. The control group (n = 8) was administered 5% dextrose for 6 wk. Groups O-6-10,7 (n = 8) and O-6-20 (n = 7) were administered 10 and 20 mg oleuropein /(kg·d) for 6 wk and group O-3-20 (n = 7) was untreated for 3 wk, and was administered 20 mg oleuropein /(kg·d) for the last 3 wk. The other 4 groups were treated as described above but they consumed the hypercholesterolemic (CH)7 diet. Thus, the hypercholesterolemic groups were CH (n = 7), CH-O-6-10 (n = 5), CH-O-6-20 (n = 5), and CH-O-3-20 (n = 5).

Before surgical intervention, blood samples were drawn from food-deprived rabbits and centrifuged for 10 min at 2500 x g for measurement of baseline cholesterol and triglyceride concentrations. Blood samples from all groups were collected at baseline, at min 20 of sustained ischemia, and 1 and 20 min of reperfusion for measurement of plasma malondialdehyde (MDA), protein carbonyl (PC), and nitrite + nitrate concentrations, RBC superoxide dismutase (SOD) activity, and plasma metabolic profiles as depicted by the 1H NMR spectra.

In a second series of experiments, 5 additional rabbits were used to obtain blood samples before and after surgical intervention (chest opening) but before the induction of sustained ischemia for plasma MDA and PC assays.

    Surgical preparation. Under general anesthesia and mechanical ventilation, the rabbits were subjected to regional ischemia of the heart as previously described (21).

    Risk area and infarct size. At the end of the experiment, hearts were removed and mounted on a perfusion apparatus and perfused for 2 min in a retrograde manner via the aorta with normal saline (20 mL/min, 50 mm Hg, and room temperature). With the aid of fluorescent particles (10–20 µm, suspended in saline) and 1% triphenyl tetrazolium chloride solution, the borders between the infracted (I), the risk (R), and the normal areas were clarified as previously described (18). I and R area volumes were expressed as cm3 and the percentage of infarct to risk area (I:R) was calculated (21,22).

    Plasma malondialdehyde concentration. The MDA concentration was determined spectrophotometrically at 586 nm and expressed as µmol/L (Oxford Biomedical Research Colorimetric Assay for lipid peroxidation) (23,24).

    Plasma protein carbonyl concentration. A modification of the technique of Levine et al., (25), based on spectrophotometric measurement of 2,4-dinitrophenylhydrazine (DNPH) derivatives of PCs, was used to quantify protein carbonyl content. Briefly, 100 µL of plasma was incubated either with 500 µL DNPH or 2 mol/L HCl (blank) for 1 h at room temperature. The samples were then reprecipated with 600 µL 20% trichloroacetic acid, incubated for 5 min on ice, and subsequently extracted with ethanol:ethyl acetate (1:1, v:v), 3 times at 11,000 x g for 10 min at 4°C. The pellets were carefully drained and dissolved in 6 mol/L-guanidine solution in H2O. The difference between the spectra of the DNPH-treated sample and the HCl control was determined at 360 nm, and the results are expressed as nmol PC/mg protein, using a molar extinction coefficient of 22,000 mol–1 L cm–1 (25). Protein concentration was determined using the BioRad Bradford assay.

    Erythrocyte superoxide dismutase activity. SOD activity was determined by its inhibitory action on the xanthine-xanthine oxidase generated superoxide anions which, in the presence of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, react to form a red formazan dye (26) (RANSOD SOD SD125) as described previously (24).

    Plasma nitrate + nitrite concentrations. The concentrations of nitrite + nitrate (NOx) in blood plasma were measured using the Griess reaction as previously described (27) (Cayman's Nitrate/Nitrite Colorimetric Assay Kit 780001), with results were expressed as µmol/L.

    Plasma total cholesterol and triglycerides concentrations. Plasma cholesterol and triglyceride concentrations were measured spectrophotometrically in the hypercholesterolemic groups using commercial kits (DiaSys Diagnostic Systems GmbH, Cholesterol FS 10130021, and Triglycerides FS 10571 021).

    Sample preparation and 1H NMR spectroscopy. Deuterated water (40 µL) for a magnetic field lock was added to 500 µL of plasma samples, and CPMG 1H NMR spectra were acquired on a Bruker DRX-400 Avance spectrometer as described previously (28).

    Data reduction of the NMR spectra and principal component analysis (PCA). Spectra ({delta} 0.5–8.5 ppm) were segmented into 181 chemical shift regions of 0.04 ppm width using the software package AMIX (Analysis of Mixtures, version 2.7, Bruker). The integral was calculated for each of the spectral regions. All integrals were normalized to the total intensity of each spectrum. Regions containing free nonendogenous EDTA and EDTA metal complex resonances were removed ({delta} 2.98–3.38, {delta} 3.54–3.70) together with the HDO region ({delta}4.6–5.26). All integrals were collected in Excel (Microsoft, Excel XP) and then exported into the SIMCA-P (Version 8.0, Umetrics) software for PCA (29,30).

Before PCA, all NMR data variables were mean-centered and pareto-scaled. PCA was used to reduce the dimensionality of the data sets (29,30). The plot of the samples as a function of the first 2 principal components results in a 2-dimensional map (scores plot) providing the maximum information in the data set concerning any patterns, groupings, or outliers, whereas the loadings plot highlights the influence of different input variables (metabolite NMR signals) in the final classification (29,31).

    Data analysis and statistics. Data are presented in the text as means ± SEM. Myocardial infarct size (%I:R), and plasma NOx and lipid concentrations were evaluated by 1-way ANOVA with Bonferroni correction. A calculated P-value < 0.05 was considered significant. Groups at each time were compared by 1-way ANOVA and pairwise multiple comparisons were performed using the Bonferroni test. One-factor RM-ANOVA was used for the comparison of each variable during the observation period (baseline vs. min 20 ischemia, min 1 and min 20 reperfusion, respectively) for each group separately. Pairwise multiple comparisons were performed using the method of Tukey critical difference. The absolute changes from baseline after 20 min of ischemia, and 1 or 20 min of reperfusion for MDA and SOD were tested by analysis of covariance (ANCOVA) using baseline measurements as covariates. A paired t test was used for the comparison of MDA and PC concentration before and after the opening of the chest. All tests were 2-sided with 95% significance level. Statistical analysis was done using the statistical package SPSS v. 10.00 (Statistical Package for the Social Sciences).


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Infarct size. Administration of oleuropein at a dose of 10 or 20 mg/(kg·d) for 6 wk and 20 mg/(kg·d) for 3 wk reduced the infarct size compared with the control and CH groups (P = 0.001, Table 1). The infarct size in hypercholesterolemic rabbits was less than that in the CH group in the groups treated with the 20 mg/(kg·d) dose for 6 or 3 wk, (Table 1, P < 0.05). The CH-O-6-10 group had an infarct size of 42.2 ± 2.9%, which did not differ from control and CH groups but was larger than in the O-6–10 group (P = 0.001).


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  		TABLE 1 Infarct size in the hearts of rabbits fed normal or hypercholesterolemic diets and administered 2 doses of oleuropein for 3 or 6 wk after 30 min of ischemia and 180 min of reperfusion1

 
The infarct zone differed between the control group and groups treated with oleuropein in normal rabbits, as well as in hypercholesterolemic rabbits that were administered 20 mg/kg of oleuropein. In contrast, there were no effects of 3 h of reperfusion in the risk areas exposed to sustained ischemia in any of the groups (Table 1).

The hemodynamic variables (blood pressure and heart rate) at baseline, at min 30 of ischemia, and at min 180 of reperfusion were not affected by oleuropein.

    Plasma MDA. The MDA concentration was significantly greater at min 20 of sustained ischemia and at min 1 and 20 of reperfusion compared with baseline in the control and CH groups (P < 0.05, Table 2). In contrast, in normal and hypercholesterolemic rabbits treated with oleuropein, plasma MDA concentrations did not change from baseline. At min 1 and 20 of reperfusion, all groups treated with oleuropein had lower MDA concentrations than the control and CH groups (P < 0.05, Table 2).


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  		TABLE 2 Plasma MDA concentrations at baseline, min 20 of ischemia, and min 1 and min 20 of reperfusion in anesthetized rabbits fed normal or hypercholesterolemic diets and administered 2 doses of oleuropein for 3 or 6 wk.1

 
Our primary endpoint was to determine what changes occur during the 20 min of reperfusion, not to study the interaction among factors such as diet, dose administered, and treatment duration. The statistical ANCOVA (Supplemental Table 1) showed that at min 20 of ischemia, all groups treated with oleuropein had significantly lower plasma MDA concentrations that the control group. In addition, the CH-O-6-10 and CH-O-3-20 groups had a lower MDA concentration than the CH group (P < 0.05). At min 1 and 20 of reperfusion, all groups treated with oleuropein had a concentration lower than that of either the control or CH groups (P < 0.005).

In the rabbits studied separately, the plasma MDA concentration did not differ before (1.38 ± 0.3 µmol/L) and after (1.52 ± 0.4 µmol/L) the opening of the chest.

    Plasma protein carbonyls. The PC concentration was higher in the control and CH groups after 20 min of reperfusion compared with all of the treated groups (Table 3). In the control and CH groups, the PC levels were higher at min 20 of reperfusion compared with baseline and min 20 of ischemia (P < 0.05). In the oleuropein-treated groups, the plasma PC levels did not change. The PC levels were 5.0 ± 0.8 and 4.8 ± 0.5 nmol/mg protein, respectively, before and after the opening of the chest.


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  		TABLE 3 Protein carbonyl content at baseline, at min 20 of ischemia, and at min 1 and min 20 of reperfusion in anesthetized rabbits fed normal or hypercholesterolemic diets and administered 2 doses of oleuropein for 3 or 6 wk1

 
    Erythrocyte SOD. In the control and CH groups, SOD activity was significantly lower at min 20 of ischemia and at min 1 and 20 of reperfusion compared with baseline. In all rabbits administered oleuropein, SOD activity did not change relative to baseline (Table 4). At min 1 and 20 of reperfusion, SOD activity in erythrocytes was higher in all groups treated with oleuropein than in the corresponding control groups (P < 0.05). The ANCOVA (Supplemental Table 2) showed that at min 20 of ischemia, groups O-6-20 and CH-O-3-20 had a significantly higher activity than the control and CH groups. At 1 min of reperfusion, all groups treated with oleuropein had significantly higher SOD activity compared with the control and CH groups (P < 0.05). Among the treated groups, CH-O-6-20 had lower SOD activity than the CH-O-3-20 and O-6-20 groups (P < 0.05). At min 20 of reperfusion, all treated rabbits had greater activity than the control and CH groups (P < 0.005). The CH-O-6-20 group had lower RBC SOD activity than the O-6-20 group (P < 0.005) at min 20 of reperfusion.


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  		TABLE 4 SOD activity during ischemia reperfusion at baseline, min 20 of ischemia, min 1 and min 20 of reperfusion in anesthetized rabbits fed normal or hypercholesterolemic diets and administered 2 doses of oleuropein for 3 or 6 wk1

 
    Plasma NOx. Hypercholesterolemia markedly increased the production of NO2– + NO3– at baseline compared with the normal myocardium (Table 5). Treatment with 20 mg oleuropein/(kg·d) did not produce a further increase in NOx in hypercholesterolemic rabbits. The concentration of NOx at baseline as well as at min 1 of reperfusion was greater in the CH-O-6-10 group compared with the O-6-10 group (P < 0.001). Moreover, a reduction in NOX concentration occurred at min 1 of reperfusion in the CH-O-6-20 and CH-O-3-20 groups compared with the baseline value (P < 0.05).


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  		TABLE 5 Plasma nitrite/nitrate concentrations at the baseline and at min 1 of reperfusion in anesthetized rabbits fed normal or hypercholesterolemic diets and administered 2 doses of oleuropein for 3 or 6 wk1

 
    Plasma total cholesterol and triglycerides. In the hypercholesterolemic rabbits, treatment for 6 wk with either a 10 or 20 mg/(kg·d) dose of oleuropein reduced the total cholesterol concentration by 28.7 and 33.1%, respectively, and the triglyceride concentration by 41.1 and 61.4%, respectively (P < 0.05, Table 6). Plasma lipids were not affected when oleuropein was administered for only 3 wk. Plasma cholesterol and triglyceride concentrations in rabbits fed the normal diet were 2.1 ± 0.2 and 0.38 ± 0.04 mmol/L respectively.


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  		TABLE 6 Plasma total cholesterol and triglyceride concentrations in rabbits fed a hypercholesterolemic diet and administered 2 doses of oleuropein for 3 or 6 wk and in the control group.1

 
    Metabolic profiles based on 1H NMR spectra. In the 1H NMR spectra of plasma samples, the signals for many low-molecular-weight metabolites were detected. There were minor differences in amino acids, e.g., Phe, Tyr, Leu, Ile, Gly, Gln, Glu, Lys, creatine + creatinine. Among the integral regions for alanine, pyruvate, lactate, acetate, ß-hydroxybutyrate, and glucose metabolites related to glycolysis, there were differences in the acetate and glucose signals among the groups. However, the various time points during ischemia/reperfusion injury did not differ in any of the groups.

    PCA. The NMR spectra were subjected to PCA using the metabonomic approach (27,29) to investigate variations in the metabolic profile after treatment with oleuropein. PCA analysis was performed either with respect to the dose of oleuropein and/or cholesterol or during the timed events of ischemia/reperfusion injury within the same group. The scores plots (PC1/PC2) showed a classification for the samples of the different oleuropein and cholesterol doses at each specific time point. The most satisfactory classification was achieved at min 20 of reperfusion. The samples corresponding to the O-6-10 and O-6-20 groups were well separated from the control group, whereas samples from the hypercholesterolemic rabbits with or without oleuropein treatment formed their own cluster (Fig. 2). Within a group, no classification with respect to the different time points was observed.


Figure 2
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  		Figure 2  PCA scores plot showing the mapping position of 1H NMR plasma spectra of samples obtained from rabbits fed a normal or hypercholesterolemic diet and administered 2 doses of oleuropein for 6 wk after min 20 of reperfusion. The plot illustrates that the separation between groups is due to differences in the metabolic profile. A separation between O-6-10, O-6-20 and the control groups occurred. The CH, CH-O-6-10, and CH-O-6-20 groups are clustered together, separate from the groups fed the normal diet.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The present study shows that the natural olive product oleuropein reduces the infarct size in normal rabbits and, at higher doses, in hypercholesterolemic rabbits in vivo. Furthermore, oleuropein protects against oxidative damage during ischemia-reperfusion, reduces the PC content and enhances SOD activity. Treatment with the low or high dose of oleuropein reduces total cholesterol and triglyceride levels, alters the metabolic profile of normal rabbits and provides cardioprotection even before the onset of ischemia. To our knowledge, this is the first in vivo study that tests the effectiveness of oleuropein in reducing myocardial injury.

The ischemic heart possesses endogenous mechanisms that render it more tolerant against a potentially lethal episode of ischemia. Most of the intracellular mediators involved in the mechanism of protection were upregulated by pharmacological means (21,32). Hypercholesterolemia is one of the major causes of cardiovascular morbitity and mortality. It results in endothelial dysfunction (33), which may be improved by reducing circulating cholesterol levels. In our study, we noted a reduction of circulating cholesterol and triglycerides after 6 wk of treatment with 10 or 20 mg oleuropein/(kg·d). This finding is in accord with the results of Coni et al. (34) who found that the addition of 10% (wt:wt) extra virgin olive oil and 7 mg/kg oleuropein to the standard diet reduces plasma levels of total cholesterol and increases the ability of LDL to resist oxidation. Olive biophenols induce a redistribution of various circulating plasma components with an indirect effect on the size and composition of LDL, that is related mainly to the phospholipid/free cholesterol in LDL (34). Moreover, an olive extract rich in oleuropein reduces cholesterol levels in rats, an effect attributed to its ability to slow down the lipid peroxidation process and to enhance antioxidant enzyme activity (35), evidence that is agreement with our findings.

Normal vascular rings exposed to oxidized LDLs rapidly develop endothelial dysfunction, which is restored by L-arginine and NO (36). NO donors ameliorate the severity of myocardial injury after a period of sustained ischemia in hypercholesterolemic rabbits (37). To determine whether a decrease in NO production occurs in hypercholesterolemia, we measured NOx as an index of NO (38–40). ROS reduce NO bioactivity through the formation of peroxynitrite and NOx (41). In our study, we found higher NOx levels in hypercholesterolemic rabbits compared with normal rabbits at baseline. The higher dose of oleuropein was required to prevent NOx production. In fact, there was a significant reduction in plasma NOx in hypercholesterolemic rabbits treated with high doses of oleuropein at min 1 of reperfusion, which may contribute to the reduction of infarct size in this group.

Lipid peroxidation is evaluated by levels of circulating MDA (24). Reperfusion increases tissue MDA in isolated rabbit hearts, whereas the rate of MDA releases into coronary effluents is only modestly increased (42). MDA production increases in both treated groups early in reperfusion, confirming our previous reports concerning an increased formation of lipid peroxidation products at the beginning of reperfusion (23,24). We also found that the antioxidant effect of oleuropein is exerted in normal and hypercholesterolemic rabbits independently of the dose and duration of treatment. Oxidative stress also causes direct damage to proteins or leads to chemical modification of amino acids in proteins. This increases the protein content in carbonyls, which then serve as biomarkers of general oxidative stress (43). In fact, the PC content was significantly elevated in the untreated groups (control and CH group) at min 20 of reperfusion, indicating greater injury from oxidative stress, whereas pretreatment with oleuropein markedly decreased the PC content at this time, further supporting our findings with respect to its protective action against oxidative stress.

ROS are produced at the beginning of reperfusion, whereas relatively low amounts are generated during ischemia. Because we found a significant increase in MDA at min 20 of ischemia in the control and CH groups, we tested the possibility that surgical intervention may have been responsible for the production of oxidative stress. However, the surgical intervention did not affect the levels of either MDA or PC. At this point, it should be emphasized that the lack of oxygen availability at the time of ischemia does not necessarily mean that oxygen free radicals are not generated (44). It was reported that the metabolic disarrangement that occurs during ischemia may cause a predisposition to the formation of free radicals from residual molecular oxygen. The mitochondrial electron-transport system, the activation of neutrophils, the metabolism of arachidonic acid, and the autoxidation of catecholamines are potential sources of free radical production in the myocardium during ischemia (44). These may provide an explanation for the increased MDA levels at min 20 of ischemia. On the other hand, the absence of an increase in the PC level at the same time point may be due to the slower rate of oxidation of proteins and the higher demands for the latter process to occur compared with lipid peroxidation.

Prolonged ischemia and reperfusion reduce the antioxidant enzymes, SOD, as well as catalase and glutathione peroxidase in the ischemic-reperfused myocardium (45). The loss of these key antioxidant enzymes limits the overall antioxidant reserve of the heart, making it susceptible to ischemia/reperfusion injury and ineffective in providing complete protection against the increased activities of the ROS. Reperfusion plays an important role in the activity of erythrocyte SOD in patients suffering from acute myocardial infarction (46). Furthermore, we showed previously that SOD activity is dramatically reduced after ischemia and reperfusion in control rabbits, whereas it is increased in preconditioned animals (24). To further investigate the effect of oleuropein on the depletion of key plasma antioxidants, we determined the SOD activity in erythrocytes at different time points. Total SOD activity was reduced in the whole blood in the control and hypercholesterolemic groups after 20 min of reperfusion. Pretreatment with 2 doses of oleuropein kept the SOD activity stable at different time points of ischemia and reperfusion and therefore left the myocardium in an "antioxidant state."

Our metabonomic analysis revealed favorable variations in the low-molecular-weight metabolite profile of plasma samples treated with oleuropein, which resulted in a clear discrimination from control in the case of normal rabbits. Variations in the metabolic profile of the hypercholesterolemic groups were not further affected by treatment with oleuropein; ths, all of the above groups formed one separate cluster (Fig. 2, loading plots). This classification appears to be due to metabolites related to glycolysis (plot not shown). More specifically, the different classification as controls and treated normal groups with oleuropein is closely related to an increase in the ratio of glycolytic end products (alanine + lactate) to end products of lipid metabolism (acetate), indicating an impairment of aerobic glucose metabolism (47) in the control group. This is in agreement with the observed decrease in sugars and the concomitant increase in lactate in the control group, demonstrating an increase in anaerobic metabolism (48). In contrast, the above differences were not observed in the hypercholesterolemic groups, which were separated from the normal groups due to increased glucose levels resulted from a shift of glycolysis toward increased lipid metabolism (49).

The above results indicate that treatment with oleuropein influences the metabolic profile of normal subjects, restoring the mechanism of aerobic glycolysis and providing cardioprotection even before the onset of ischemia.

Finally, the reduction in infarct size by oleuropein can be achieved by several mechanisms such as the elimination of reperfusion injury (because of lesser oxidative stress), the improvement of endothelial function, either directly or because of hypolipidemia, or by a possible activation of intracellular mediators that render the heart more tolerant toward a subsequent lethal ischemic insult.

In conclusion, the natural constituent in olive, oleuropein, is a potent antioxidant that decreases total cholesterol and triglyceride levels and considerably reduces infarct size in vivo. This is the first experimental study in vivo that encourages the future use of a novel natural agent for the treatment of coronary heart disease. Further studies may show that the administration of smaller doses of oleuropein, even for shorter periods, may confer similar benefits.


   FOOTNOTES
 
1 Supported in part by the General Secretariat for Research and Technology of Greece (program TP27). Back

2 Supplemental Tables 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

7 Abbreviations used: CH, 6 wk cholesterol-enriched diet with 2 g of cholesterol; CH-O-3-20, 6 wk cholesterol-enriched diet with 3 wk 20 mg/(kg·d) oleuropein; CH-O-6-10, 6 wk cholesterol-enriched diet with 10 mg/(kg·d) oleuropein; CH-O-6-20 6 wk cholesterol-enriched diet and 20 mg/(kg·d) oleuropein; DNPH, 2,4-dinitrophenylhydrazine; I:R, infarct to risk area; MDA, malondialdehyde; NOx, nitrite + nitrate, O-6-20, 6 wk 20 mg/(kg·d) oleuropein; O-3-20, 3 wk 20 mg/(kg·d) oleuropein; O-6-10, 6 wk 10 mg/(kg·d) oleuropein; PC, protein carbonyl; PCA, principal component analysis; SOD, superoxide dismutase. Back

Manuscript received 1 February 2006. Initial review completed 24 February 2006. Revision accepted 22 May 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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