Journal of Food and Nutritional DisordersISSN: 2324-9323

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Research Article, J Food Nutr Disor Vol: 3 Issue: 4

Olive Cake Reduce Lipid Peroxidation Associated with Antioxidant Defense in Red Blood Cell and Heart, in Rats Fed a Cholesterol-Enriched Diet

Sherazede Bouderbala*, Khalid Naman Al-Hiti Mohammed, Adila Ougouag, Jihane Benmansour, Nadia Mahdad and Malika Bouchenak
Département de Biologie, Faculté des Sciences de la Nature et de la Vie, Université d'Oran, Algérie
Corresponding author : Sherazede Bouderbala
Laboratoire de Nutrition Clinique et Métabolique, Département de Biologie, Faculté des Sciences de la Nature et de la Vie, Université d'Oran, BP 1524 El M’Naouer, 31000 Oran, Algérie
E-mail: sherazede.bouderbala@yahoo.fr
Received: March 17, 2014 Accepted: August 04, 2014 Published: August 07, 2014
Citation: Bouderbala S, Naman Al-Hiti MK, Ougouag A, Benmansour J, Mahdad N, et al. (2014) Olive Cake Reduce Lipid Peroxidation Associated with Antioxidant Defense in Red Blood Cell and Heart, in Rats Fed a Cholesterol-Enriched Diet. J Food Nutr Disor 3:4. doi:10.4172/2324-9323.1000151

Abstract

Olive Cake Reduce Lipid Peroxidation Associated with Antioxidant Defense in Red Blood Cell and Heart, in Rats Fed a Cholesterol-Enriched Diet

Background: In Mediterranean areas, the olive oil industry produces substantial amounts of by-products, with one of the most important being the olive cake (OC). OC is the solid residue obtained after oil extraction. We hypothesized that administration of OC would prevented hypercholesterolemia and oxidative stress in rats fed a cholesterol-rich diets. Methods: Male Wistar rats (n=24) weighing 45 ± 5 g were divided into four groups fed diet with 1% cholesterol (HC) and supplemented or not with OC at 2.5%, 5% and 7.5% (OC2.5- HC, OC5-HC and OC7.5-HC or HC, respectively) for 28 days. Results: Compared with the HC group, serum total cholesterol values were respectively lower in the OC2.5-HC, OC5-HC and OC7.5-HC groups. Red Blood cells (RBC) thiobarbituric acid reactive substances (TBARS) were 1.3-fold lower in the OC7.5- HC group. In heart, TBARS concentration was significantly lower. In RBC, superoxide dismutase (SOD) activity was 1.13- fold increased in the OC5-HC group. Glutathione peroxidase (GSH-Px) activity was higher in all the OC groups. Glutathione reductase (GSSH-Red) activity was 2.3-fold higher in the OC5- HC and OC7.5-HC. Catalase activity was 1.3-fold increased in the OC2.5-HC and OC5-HC groups. Liver SOD and GSH-Px activities were significantly higher in all groups consumed the OC. Catalase activity was 1.5- fold increased in the OC7.5-HC group. In heart, SOD activity was significantly higher in all OC groups. Conclusion: It appears that olive cake attenuates hypercholesterolemia induced by cholesterol-enriched diet. In addition, olive cake may reduce lipid peroxidation associated with antioxidant defense in RBC and heart, compared to other tissues, in particular liver.

Keywords: Hypercholesterolemic rat; Olive cake; Serum cholesterol; TBARS; Antioxidant enzymes

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Keywords
Hypercholesterolemic rat; Olive cake; Serum cholesterol; TBARS; Antioxidant enzymes

Abbreviations
BW: Body Weight; CVD: Cardiovascular Diseases; GSH-Px: Glutathione Peroxidase; GSSH-Red: Glutathione Reductase; HC: Hypercholesterolemic Rat; OC: Olive Cake; RBC: Red Blood Cells; TBARS: Thiobarbituric Acid Reactive Substances; SOD: Superoxide Dismutase; TC: Total Cholesterol; TG: Triacyglycerols; VLDL: Very Low Density Lipoprotein

Introduction
Cardiovascular diseases (CVD), particularly coronary heart disease, have become a growing problem, especially in developing countries. Dyslipidaemia may be defined as any change of lipids or lipoproteins, quantitatively or qualitatively, in the blood that signifies an increased risk of disease [1]. Hypercholesterolemia is widely known as a dominant risk factor for the development of cardiovascular diseases [2].
It has been reported that oxidative stress induced by reactive oxygen species, plays an important role in the etiology of several diseases including atherosclerosis and coronary heart disease [3]. Hyperlipidemia has also been found to induce oxidative stress in various organs such as the liver, heart, and kidney [4]. To lower high blood cholesterol, a number of lifestyle changes are recommended including smoking cessation, limiting alcohol consumption, increased physical activity and diet control [5].
In Mediterranean areas, the olive oil industry produces substantial amounts of by-products, with one of the most important being the olive cake (OC) [6]. Olive Cake is the solid residue obtained after olive oil extraction. It is one of the most abundant agro-industrial byproducts in the Mediterranean area constituting a source of environmental problems resulting from its accumulation and disposal [7]. Olive mill waste which results from the olive oil extraction, is potentially a rich source of a diverse range of biophenols with a wide array of biological activities [8]. Among biophenol substances, hydroxytyrosol (3,4-dihydroxyphenylethanol) stands out as a compound of high added-value, due to its interesting antioxidant activity and its potentially beneficial human health properties [9]. Hydroxytyrosol has various biological activities, such as downregulation of the immunological responses [10], insulino-stimulatory action and preventing liver, kidney, and pancreas from oxidative damage induced by hyperglycemia [8], anti-inflammatory and hypocholesterolemic effects [11].
The hypothesis that the antioxidant defense status in serum and tissues in rats fed a cholesterol-rich diet and supplemented with olive cake may be related to OC was tested following 2 objectives. The first was to study the effect of different doses of OC on the lipid peroxidation in Red Blood Cells (RBC), serum and tissues. The second was to measure the antioxidant enzymes activity in RBC and different tissues in rats.

Materials and Methods
Preparation of olive cake (OC)
Olive cakecontainingthe skinand the core is collectedafter olive oil extraction. was dried at 60°C until obtaininga constant weight and then
Animals and diets
Male Wistar rats (n=24) (Iffa Credo, l'Arbresle, Lyon, France) weighing 45 ± 5 g were fed a diet containing 20% casein (C) combined with 5% sunflower oil for 4 days. After this adaptation period, rats were then divided into 4 groups consuming for 28 days the same diet containing 1% cholesterol and 0.5% cholic acid and supplemented or not with olive cake (2.5, 5 and 7.5%) (OC2,5-HC, OC5-HC and OC7.5- HC) or HC, respectively (Table 1).
Table 1: Diet composition (g/kg diet)
Diets and tap water were freely available. Animals were kept in wire bottom cages at a temperature of 24°C and relative humidity of 60%, and lights were automatically turned on from 7:00 AM to 7:00 PM. The General Guidelines on the Use of Living Animals in Scientific Investigations [12] were followed, and the protocol and use of rats were approved by our Institutional Committee on Animal Care and Use.
To evaluate the digestibility of lipid in hypercholesterolemic diet, six rats from HC and OC-HC groups were placed individually into metabolism cages. Food intake was measured daily. Faeces were collected from d22 to d28 of the experiment. Total lipids were extracted according to the method of Delsal [13]. The fecal cholesterol content was determined by enzymatic colorimetric method (kit Caymen).
Blood and tissues samples
At day 28, rats were food deprived for 12 hours, anesthetized with sodium pentobarbital (60 mg/kg BW), and euthanized with overdose. Blood was collected from abdominal aorta into dried tubes and centrifuged at 4°C,1000g for 15 min. Serum was taken, and separated red blood cells (RBC) was then washed 3-times by resuspending in 0.9% NaCl solution and repeating the centrifugation. The washed cells were lysed in an equal volume of water and mixed thoroughly. Liver, adipose tissue, gastrocnemius muscle, heart, and kidney were also excised in ice-cold saline, blotted on filter paper, and weighed.
Lipids parameters
Total cholesterol (TC) and triacyglycerols (TG) concentrations were determined by enzymatic colorimetric methods (kit Caymen)
Lipid peroxidation
As a marker of the lipid peroxidation, thiobarbituric acid reactive substances (TBARS) concentrations of serum were measured according to the method of Quintanilha et al. [14] and those of tissues by the method of Ohkawa et al. [15], as previously described in Bouderbala et al. [16].
Serum and tissues carbonyls determinations
Carbonyls contents were determined according to the method of Levine et al. [17] using the 2,4-dinitrophenylhydrazine (DNPH) reagent. The carbonyl content was determined by taking the spectra of the representative samples at 250–300 nm. Each sample was read against the control sample (treated with 2.5 mol/lHCl). The carbonyl content was calculated using an absorption coefficient (ε) of 22,000moll-1 cm-1.
Antioxidant enzyme measurements
Superoxide dismutase (EC. 1.15.1.1) is a metalloenzyme that catalyzes the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide. Superoxide dismutase assay uses a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine (kit, Cayman).
Glutathione peroxidase (EC. 1.11.1.9) catalyzes the reduction of hydroperoxides, including hydrogen peroxide, by reduced glutathione and functions to protect the cell from oxidative damage (kit, Cayman).
Glutathion reductase (GR, EC 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to GSH. The oxidation of NADPH to NADPis accompanied by a decrease in absorbance at 340 nm and is directly proportional to the GR activity in the sample (kit, Cayman).
Catalase (EC. 1.11.1.6) catalyses the decomposition of hydrogen peroxide to water and oxygen. Catalase activity was assayed in tissues by measuring the rate of hydrogen peroxide (H2O2) decomposition according to the method described by Aebi et al. [18].
Protein concentrations were measured using bovine serum albumin (EC 232.936. 2) as a standard (Sigma) according to the method of Lowry et al. [19].
Statistical analyses
Results were expressed as means ± SEM for 6 rats per group. Statistical evaluation of the data was carried out by the parametric Student t test. The calculations were performed using STATISTICA 6.0 (for Windows, StatSoft Inc software, Tulsa, OK). The limit of statistical significance was set at P<0.05 between the different groups consumed or not olive cake.

Results
Body weight (BW), lipid digestibility and serum TC and TG contents
At day 28, BW and food intake were decreased, respectively, by 65%, 62% and 79% in the OC2.5-HC, OC5-HC and OC2.5-HC groups compared with the HC group.
Fecal lipid content was 1.6- and 2.7-fold lower in the OC2.5-HC and OC5-HC groups than in the HC group. Fecal cholesterol value was respectively, 2.9-, 2.3- and 2.3-fold higher in the OC2.5-HC, OC5-HC and OC7.5-HC groups compared with the HC group.
Serum TC concentration was 1.5-, 1.7- and 2.1-fold lower in the OC2.5-HC, OC5-HC and OC7.5-HC groups compared with the HC group. Serum TG values were respectively, 4.3- and 3.6-fold decreased in the OC5-HC and OC7.5-HC groups compared with the HC group (Table 2).
Table 2: Body weight, lipid digestibility and serum total cholesterol (TC) and triglycerides (TG) contents.
Serum and tissues lipid peroxidation
Compared with the HC group, TBARS were 1.3-fold lower in RBC of the OC7.5-HC group. Serum TBARS values were 1.4-fold lower in the OC5-HC and OC7.5-HC groups. Moreover, in the liver, there was no significant difference in all the groups. In adipose tissue and muscle, TBARS concentrations were 1.5-, 1.7- and 5-, 4.2-fold lower in the OC5-HC and OC7.5-HC groups. In heart, TBARS concentration was 1.8-, 2.7- and 3.5-fold lower in the OC2.5-HC, OC5-HC and OC7.5- HC groups. In contrast, kidney, TBARS contents were 3.5-, 3.2- and 3.8-fold higher in the OC2.5-HC, OC5-HC and OC7.5-HC groups (Table 3).
Table 3: Thiobarbituric acid reactive substances (TBARS) contents in Red Blood Cell, serum and in tissues
Serum and tissues carbonyls
Serum carbonyls values represented 86% in the OC2.5-HC and 80% in the OC5-HC groups of the value noted in the HC group. In liver, adipose tissue and muscle, the values represented, respectively 91%, 75% and 65% in the OC2.5-HC, OC5-HC and OC7.5-HC groups, 85% and 84% in the OC2.5-HC and OC5-HC groups, and 78% in the OC5- HC group (Table 4).
Table 4: Carbonyls contents in serum and in tissues
Antioxidant enzymes activity in RBC and in different tissues
When compared with the HC group, the RBC superoxide dismutase (SOD) activity was 1.13-fold increased in the OC5-HC. Glutathione peroxidase (GSH-Px) activity was 1.5-fold, 2- and 1.5- fold higher in the OC2.5-HC, OC5-HC and OC7.5-HC groups. Glutathione reductase (GSSH-Red) activity was 2.3-fold higher in the OC5-HC and OC7.5- HC. Catalase activity was 1.3-fold increased in the OC2.5-HC and OC5- HC groups.
The liver SOD activity was respectively, 3.2-, 2.7- and 2.6-fold in the OC2.5-HC, OC5-HC and OC7.5-HC groups. GSH-Px activity was significantly increased in all the olive cake groups (P<0.05). Catalase activity was 1.5- fold higher in the OC7.5-HC group.
In heart, SOD activity was 1.5-fold higher in all groups consumed the OC and in adipose, it was 1.2- and 1.3-fold higher in the OC2.5-HC and OC7.5-HC groups. The Kidney GSH-Px and GSSH-Red activities were significantly increased in the OC7.5-HC group (P<0.05), whereas that of catalase was 1.2-fold lower in the OC2.5-HC group (Table 5).
Table 5: Antioxidant enzymes activity in Red Blood Cell and tissues

Discussion
In the present study, we have demonstrated that olive cake has hypocholesterolemic, hypotriglyceridemic effects and significant antioxidant defense status in RBC and tissues, in young rats fed a cholesterol-rich diet. Food consumption in the hypercholesterolemic rats enhanced with the increased in olive cake intake.
Generally, a high-cholesterol diet could increase the body weight [20]. In this investigation, after 28d, body weight was significantly lower in the OC2.5-HC and OC5-HC, compared with the HC group; this was probably due to low food consumed by these animals. In the OC7.5-HC group, BW was similar to that of HC group, suggested that olive cake at this dose have been accepted by rat. On the other hand, this study showed that food consumption in the hypercholesterolemic rats increased with the increase in the olive cake doses,this can be explained by organoleptic factors such as flavor or odor.
Hypercholesterolemia is a major risk factor for cardiovascular disease. Research on natural substances that affect cholesterol metabolism for the prevention of hypercholesterolemic has therapeutic importance. In particular, the investigation of dietary components that can be added to foods to lower or regulate cholesterol levels is of special interest [21].
Several studies have shown the effect of dietary cholesterol on blood cholesterol levels. Feeding of atherogenic diet (0.5% cholic acid and 1% cholesterol) for 3 weeks resulted in an increase of total cholesterol [22]. These data confirmed and extended those reported by other finding diets enriched by different amounts of cholesterol, ranging between 0.5% and 4% [23], for different periods of times, supplied or not with cholic acid. Our results indicated that in the hypercholesterolemic rat, the consumption of olive cake regardless dose administered (2.5%, 5% or 7.5%) decreases serum cholesterol values. It could be suggested that low cholesterol levels inducted by olive cake ingestion may be explained by the action on the two key enzymes of cholesterol metabolism: by decreasing the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase activity, enzyme responsible for the regulation of cholesterol biosynthesis or by increasing the cholesterol 7α- hydroxylase activity, enzyme responsible in the conversion of cholesterol into bile acids. In the OC2.5-HC, OC5-HC and OC7.5-HC groups compared with the HC group, considerable excretion of fecal cholesterol content was noted, probably due to decreased in cholesterol intestinal absorption. Moreover, low TG values were noted in the OC5-HC and OC7.5-HC groups than in the HC group. The triglycerides lowering effect noted in these groups may be due to an increased catabolism of very low density lipoprotein (VLDL) fraction by an increased in the activity of lipoprotein lipase post-heparin, enzyme localized in the endothelium of the blood capillaries of the many peripherals tissues which hydrolysis VLDL-TG releasing fatty acids which are either received by the adipose tissue for storage, or captured by muscle to be oxided, leading to the formation of particles having lost 2/3 of their TG contentremnants" that are themselves captured via specific receptors in the liver.
Products collected after extraction of olive oil contain pectin [24], and several studies have shown that pectin contents in vegetables and fruits reduce cholesterol and increase lipoprotein lipase activity in adipose tissue which may be responsible for decreased serum TG [25].
Free radical-induced lipid peroxidation or oxidative stress has been shown to participate in the pathogenesis of several diseases [26]. Hypercholesterolemia induces not only atherosclerosis, but also produces a lot of free radicals in blood and tissues [27]. Red blood cells are susceptible to oxidation by oxygen radical because they are very rich in Fe++ containing molecules, primarily hemoglobin [28]. Thiobarbituric acid reactive substances (TBARS) level is a good indicator of lipid peroxidation and our results indicated that these substances were decreased only in RBC of the OC7.5-HC reflecting, the enhancement of antioxidant enzymes activity. This increase might be due to enzyme activation by olive cake supplementation.
Serum TBARS was decreased in the OC5-HC and OC7.5-HC groups, suggesting that these OC doses were sufficient to attenuate lipid peroxidation in serum. It is widely known that both liver and heart are primary organs at risk from hypercholesterolemia [27]. In spite of similar lipid peroxidation of liver, heart TBARS were decreased with olive cake consumption. In addition, in adipose tissue and muscle, TBARS were diminished, especially with 5 and 7.5% doses. In kidney, TBARS values were increased in all groups fed OC, this organ seemed to be more sensitive than the other selected tissues, and hence, olive cake doses were probably not sufficient to protect this organ. In addition, the no significant difference in TBARS contents and the increased SOD and GSH-Px activities of liver in all the groups fed the OC compared to the HC, suggested that this tissue protected moderately against free radicals attack leading to oxidative damage caused by the high-cholesterol enriched diet. Oxidative stress is one of causative factors that link hyperlipidemia with atherosclerosis pathogenesis [29], and the addition of nutriments rich in polyphenols could improve activities of free radical scavenging enzymes. Moreover, polyphenols may limit oxidative damage, by acting directly on reactive oxygen species, by acceptance of electron by phenolic groups, or by stimulating endogenous defense systems [30]. Olive cake is considered as a rich source of phenolic compounds, with a wide array of biological activities [31]. A wide range of phenolic compounds have been identified in virgin oil, including phenolic alcoholic, secoirdoid derivatives phenolic acids and flavonoids [32]. However, only about 2% of the total phenols found in olive fruits are transferred to the extracted olive oil, while the other 98% are retained in the olive cake. Analysis of these phenolic extracts has demonstrated their high antioxidant activity and suggested their potential use as additives for the food industry [33]. Our resulted showed also that liver catalase and adipose tissue SOD were increased in the OC7.5-HC groups, compared with HC group, suggesting that these tissues may stimulate the activation of this enzyme. It is known that SOD catalyses superoxide anions to hydrogen peroxide, which is broken down by catalase and GSH-Px, and then prevents further generation of free radicals. Several studies showed that HC diet raised cardiac superoxide anions, without significant change of cardiac SOD activity [34]. As shown in Table 3, HC diet feeding markedly raised TBARS levels, whereas it decreased the SOD activity in heart (Table 4), this might be due to a lot production of free radicals in cardiac tissue induced by HC. Olive cake markedly enhanced cardiac SOD activity, catalyzing free radicals the excess produced in this tissue of HC rats.
In conclusion, olive cake is able to decrease the high serum lipid contents in rats fed with hypercholesterolemic diet for four weeks. It also suppressed the high level of lipid peroxidation in the RBC, serum and cardiac tissues. OC significantly enhanced the activities of the antioxidant enzymes in the RBC, liver and heart. Our data indicate that OC supplementation of diet during 28d was powerful to attenuate serum lipids and provides cardiac protection from hypercholesterolemia. Phenolic compounds contained in olive cake might be responsible for both lipid-lowering and antioxidative actions to protect the liver and heart from hypercholesterolemia. However, further investigations are needed to identify the biologically active ingredients in olive cake tested and to determine the molecular mechanisms for the responses observed in hypercholesterolemic rats.

Acknowledgment
This research was supported by the Algerian Ministry of Higher Education and Scientific Research.

Conflict of interest
The authors declared no conflict of interest. All authors critically reviewed the manuscript and approved the final version submitted for publication

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