The effect of olive leaf supplementation on the constituents of blood and oxidative stability of red blood cells

journal of functional foods 9 (2014) 271–279

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The effect of olive leaf supplementation on the constituents of blood and oxidative stability of red blood cells Fátima Paiva-Martins a,b,*, Susana Barbosa a,b, Marco Silva b, Divanildo Monteiro c, Victor Pinheiro c, José Luis Mourão c, João Fernandes d,e, Susana Rocha e,f, Luís Belo e,f, Alice Santos-Silvae,f a

Centro de Investigação em Química (CIQ), Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, Porto 4169-007, Portugal b Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, Porto 4169-007, Portugal c Centro de Ciência Animal e Veterinária and Departamento de Zootecnia, Universidade de Trás-os-Montes e Alto Douro, Apartado 1013, Vila Real, Portugal d IBILI – Instituto de Imagem Biomédica e Ciências da Vida, Faculdade de Medicina da Universidade de Coimbra, Azinhaga Santa Comba, Celas, Coimbra 3000-548, Portugal e Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, Porto, Portugal f Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre, 823, Porto 4150-180, Portugal

A R T I C L E

I N F O

A B S T R A C T

Article history:

Olive leaf (OL) supplements are marketed as promoting health and supporting the body in

Received 18 February 2014

preventing free radical damage. This study examined the effect of different concentrations

Received in revised form 17 April

of OL supplement on the haematological and lipid profile and on the oxidative stability of

2014

red blood cells (RBCs). A cohort of healthy pigs was used as a model in a single-centre, ran-

Accepted 23 April 2014

domized, prospective pilot comparison. Twenty four pigs were assigned to three experi-

Available online

mental diets: a control group fed the conventional diet and two groups fed the conventional diet supplemented at 50 and at 100 g/kg with OL, during 8 weeks. Blood was collected for

Keywords:

haematological, biochemical, and haemostatic studies. OL supplementation resulted in a

Olea europaea

significant decrease in plasmatic triacylglycerols (TAGs) concentration, aligned with a lower

Polyphenols

body mass and fat storage but no significant reductions were found for low-density lipo-

Erythrocytes

protein cholesterol (LDLc) and oxLDL levels. The use of the highest dose resulted in signifi-

Olive leaf

cant RBC membrane destabilization.

Oleuropein

© 2014 Elsevier Ltd. All rights reserved.

Pigs

* Corresponding author. Tel.: +351 22 6082956/856; fax: +351 22 6082959. E-mail address: [email protected] (F. Paiva-Martins). Abbreviations: aNDFom, neutral detergent fibre; OL0, conventional diet; OL5, conventional diet supplemented with dried olive leaf powder at 50 g/kg; OL10, conventional diet supplemented with dried olive leaf powder at 100 g/kg; TAGs, triacylglycerols; TC, total cholesterol; HDLc, high density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; oxLDL, oxidized low density lipoprotein; %MBH, percentage of erythrocyte membrane bound haemoglobin; PT, prothrombin time; aPTT, activated partial thromboplastin time; INR, international normalized ratio http://dx.doi.org/10.1016/j.jff.2014.04.027 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

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1.

journal of functional foods 9 (2014) 271–279

Introduction

Interest in the Mediterranean diet (MD) has grown worldwide due to its linkage with greater longevity and reduction in cardiovascular diseases, cancer, and age-related cognitive decline. Despite the high complexity of its nutrient composition, olive oil emerges as the principal source of lipids in MD, providing a higher percentage of energy and several bioactive compounds, namely biophenols. Due to their potential antioxidant activity, much research has focused on the biophenols present in olive oil. These compounds appear to show a protective mechanism by delaying or preventing the development of heart and vascular diseases and some cancers, as seen in Mediterranean populations (Cicerale, Conlan, Sinclair, & Keast, 2009; Pérez-Jiménez, Ruano, Perez-Martinez, Lopez-Segura, & Lopez-Miranda, 2007). As an extension of the interest in natural products and the apparent health benefits of the Mediterranean diet, olive leaf extracts have been studied (Eidi, Eidi, & Darzi, 2009; Elamin et al., 2013; Flemmig, Kuchta, Arnhold, & Rauwald, 2011; Kendall et al., 2009; Odiatou, Skaltsounis, & Constantinou, 2013; Park, Jung, Yang, & Kim, 2013; Perrinjaquet-Moccetti et al., 2008; Saberi, Kazemisaleh, & Bolurian, 2008; Wang et al., 2008). Olive leaves are agricultural residues from beating olive trees for fruit removal and represent about 10% of the total weight of fruit arriving to mills. Moreover, the production of olive leaf from pruning has been estimated to be 25 kg per olive tree (Delgado Pertiñez, Chesson, Provan, Garrido, & Gómez-Cabrera, 1998). Considered as an industrial byproduct, olive leaf contains up to 1–3% polyphenols (Paiva-Martins, Correia, Félix, Ferreira, & Gordon, 2007). These polyphenols have the same structure as those found in olives and olive oil, but they are present at different relative concentrations (Paiva-Martins et al., 2007; Zbakh & El Abbassi, 2012). The quantity and quality of the different polyphenols in olive leaf extracts depend on the olive cultivar and post-harvest treatment of leaves (Paiva-Martins et al., 2007; Paiva-Martins & Gordon, 2001). Because of their low chemical and microbiological stability (Paiva-Martins, Barbosa, Pinheiro, Mourão, & Outor-Monteiro, 2009), leaves are usually solvent extracted or dried before being used in infusions. This last treatment destroys several polyphenols but increases the concentration in other phenolic compounds. Indeed, fresh olive leaf is normally rich in polyphenolic compounds, such as the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEAEDA) and an isomer of oleuropein aglycone (3,4-DHPEA-EA), but dry olive leaf contains oleuropein as a major component, even in a higher concentration than before the treatment (Paiva-Martins & Gordon, 2001). Olive leaves have been used for the treatment of wounds, fever, diabetes, gout, atherosclerosis, and hypertension since ancient times and are believed to have a strong antioxidant activity (Gordon, Paiva-Martins, & Almeida, 2001). In vitro studies have shown that olive leaves present hypoglycaemic, hypotensive, anti-human immunodeficiency viral, and antitumour qualities (Cicerale et al., 2009; Elamin et al., 2013; Flemmig et al., 2011; Odiatou et al., 2013; Park et al., 2013; Pérez-Jiménez et al., 2007; Saberi et al., 2008). Commercial olive leaf extracts

are available in powdered capsule forms or in liquid tonics. Olive leaf extract supplements are marketed as promoters of heart health, supporting the body in preventing free radical damage. However, scientific research supporting these claims is lacking. Although there has been a moderate amount of scientific work published about the olive oil biophenol hydroxytyrosol, there is limited literature examining the contribution of olive leaf, or its major biophenol, oleuropein, to health outcomes. These studies are mostly limited to in vitro/ ex vivo (Elamin et al., 2013; Flemmig et al., 2011; Odiatou et al., 2013) and animal (Eidi et al., 2009; Park et al., 2013; Wang et al., 2008) studies. Limited human trials (Kendall et al., 2009; Perrinjaquet-Moccetti et al., 2008; Susalit et al., 2011) have been conducted to assess the health effects of supplemental olive leaf, oleuropein, or of other olive leaf phenols, but conflicting results have been obtained. Encouraging results were obtained with leaf extract in the reduction of hypertension in borderline hypertensive patients (Perrinjaquet-Moccetti et al., 2008; Saberi et al., 2008). However, no effects of multiple dosages of olive leaf supplements on urinary biomarkers of oxidative stress in healthy humans were observed by Kendall et al. (2009). One of the possible reasons for the lack of correlations could have been caused by confounders and compliance to the experimental protocol. In a previous study (Paiva-Martins et al., 2009), the effect of olive leaf supplementation on the growth performance and stability of pork meat showed interesting results. On one hand, leaf supplementation decreased the growth performance of pigs while, on the other hand, it increased the tocopherol content of meat and its stability. These results, concerning human nutrition, may encourage the use of olive leaf in the control of body weight. Moreover, the intramuscular α-tocopherol increase with enriched diets, may suggest an improvement in antioxidant defences that might contribute to preventing oxidative stress associated diseases. Swines have a digestive tract similar to humans and their diet, age, weight, body mass index and environmental stress can be strictly controlled during all the protocol. These animals have become an important resource in biomedical research as they have proven to be excellent models for studying cardiovascular disease (Dyson, Alloosh, Vuchetich, Mokelke, & Sturek, 2006; Turk, Henderson, Vanvickle, Watkins, & Laughlin, 2005), obesity (Dyson et al., 2006), diabetes (Bellinger, Merricks, & Nichols, 2006), hypertension (Dyson et al., 2006), lipoprotein metabolism (Ginsberg & Taskinen, 1997) and intestinal function (Domeneghini, Di Giancamillo, Arrighi, & Bosi, 2006). Moreover, the similarity of pig and human organs has led to a large effort to use them as a source of organs for xenotransplantation (Cooper, Ezzelarab, Hara, & Ayare, 2008). Therefore, this study examined the effect of different concentrations of OL supplement on the haematological and lipid profile and on the oxidative stability of red blood cells (RBCs) of a cohort of young, healthy pigs used as a model, in a singlecentre, randomized, prospective pilot comparison. RBCs are a good model for studying antioxidant related effects since these cells are particularly susceptible to endogenous and exogenous oxidative damage because of their specific role as oxygen carriers and their poor biosynthetic capacity and limited repair mechanisms. Moreover, these cells, the most abundant cells in blood, are closely related to vascular tonus regulation and

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have an important function as mobile free radical scavengers and, therefore, any functional abnormality in these cells will have major health consequences.

2.

Materials and methods

2.1.

Olive leaves procurement

Olive leaves were obtained at a local olive oil press (Cooperativa dos Olivicultores de Murça, Murça, Portugal) during the olive oil production period (November–December). In order to prevent the rejection of leaves in the diet, leaves were given to the animals as a powder (hammer mill, particle size < 1 mm) mixed with conventional diet. The chemical stability of olive leaf preparation introduced into the diet should not be compromised during the manipulation. Therefore, leaves were previously dehydrated at 37 °C for 2 days (Paiva-Martins et al., 2009).

2.2.

Treated olive leaves Chemical composition (g/kg dry matter) Dry matter (as feed basis) Organic matter Crude protein Crude fat Neutral detergent fibre Starch Phytochemicals (g/kg) Total polyphenols Simple phenols Hydroxytyrosol Flavonoids Secoiridoids Oleuropein α-Tocopherol

925.0 918.0 112.0 41.0 520.0 22.0 25.1 0.33 0.21 2.17 22.6 22.1 0.37

Animals and diets

Thirty crossbreed male pigs (Large White × Landrace × Pietrain) were housed individually in galvanized metal crates and were fed on a conventional commercial diet for growing pigs until they were 12 weeks old (67.4 ± 4.7 kg). At this age, the pigs were randomly assigned to three experimental diets (10 pigs/diet): a control group fed a conventional diet (OL0) and two groups fed with conventional diets supplemented with dried olive leaf powder at 5% (OL5) and 10% (OL10). Water was available ad libitum via a low-pressure drinking nipple. The ingredients and chemical composition of experimental diets are shown in Table 1.

2.3.

Table 2 – Chemical composition and major classes of phytochemicals in pre-treated olive leaves.

Food analysis

Feed samples were analyzed for dry matter (DM) by drying at 65 °C for 48 h, for organic matter (OM) according to AOAC, and for neutral-detergent fibre (aNDF) according to the procedures described by Van Soest, Robertson, and Lewis (1991), with a 100% neutral detergent solution and the use of amylase and sulphite (for the reduction of starch and protein).

2.3.1. Isolation and quantification of polyphenols from leaves and feed The leaves or the leaf supplemented feed were macerated in 250 mL of ethanol for 5 days in the dark at room temperature. The extract was separated by filtration and the solvent was evaporated under vacuum. The residue was taken up in 50 mL of acetone/water (1:1, v/v) and an internal standard (syringic acid) was added. The aqueous mixture was successively extracted with n-hexane and ethyl acetate. Each organic solution was washed with water, the solvent was evaporated, and the extracts were dissolved in ethanol in volumetric flasks (25 mL). The compositions of these extracts were determined by a Knauer K-1001 HPLC system (Knauer, Berlin, Germany), on a 250 mm × 4.6 mm i.d. reversed-phase C18 column (Merck, Lisbon, Portugal); detection was performed using a Knauer K-2800 diode array detector (Knauer), with a gradient previously described (Paiva-Martins et al., 2007). The extraction for each sample was performed in duplicate. Chemical composition of olive leaf is shown in Table 2.

2.3.2. Isolation and quantification of tocopherol from leaves and feed Table 1 – Chemical composition and nutritional value of the experimental diets. Chemical composition (% dry matter)

OL0

OL5

OL10

Dry matter (% feed) Organic matter Crude protein Crude fat aNDFoma Cellulose Total polyphenols (g/kg feed) Oleuropein (g/kg feed) Hydroxytyrosol (g/kg feed) Tocopherol (mg/kg feed)

89.2 94.3 19.1 5.6 14.8 4.6 0.0 0.0 0.0 8.2

89.0 94.4 18.4 5.3 16.1 5.0 1.26 1.10 0.01 26.6

89.5 94.2 18.1 4.9 17.6 5.3 2.51 2.21 0.02 45.1

a

aNDFom, neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash.

Leaves or leaf supplemented feed were homogenized at 10,000 g for 3 min using a homogenizer (AM-3) in 100 mL of methanol/ isopropanol (8:2, v/v) for 2 min. The extract was separated by filtration. After repeating this procedure twice, the solvent was evaporated under vacuum. The residue was dissolved in 10 mL of methanol/isopropanol/hexane (1:3:1, v/v/v). The composition of this solution was determined by HPLC. The HPLC system was composed of a Merck-Hitachi chromatograph with a 250 mm × 4.6 mm Waters Spherisorb ODS2 5 µm column (Supelco Inc., Bellefonte, PA, USA) coupled to a Merck Hitachi L-4200 UV-Vis detector and with a Merck Hitachi L-6200 Intelligent Pump (Mannheim, Germany). Detection was performed at 292 nm at room temperature. The flow rate was 1 mL/min; the mobile phase used was 2% acetic acid (pH 3.1) in water (A), methanol (B), and isopropanol (C) for a total running time of 70 min, according to the method previously described (Paiva-Martins et al., 2009). Twenty microlitres of each

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solution were injected into the column twice. Extraction for each sample was performed in duplicate.

2.4.

Blood analysis

Venous blood was collected to tubes with and without anticoagulant (ethylenediaminetetraacetic acid, EDTA, for haematological studies and citrate for haemostatic studies) in the morning after an overnight fast of at least 12 h. After preparation of erythrocytes membranes, aliquots were prepared and stored at −80 °C. To study the lipid profile, serum and plasma were separated and kept frozen at −80 °C until assays were performed.

2.4.1.

Haematological and haemostatic indices

Haematological parameters and platelet indices were determined in whole blood samples using a VetScanHM2 automated haematology analyser (Abaxis Veterinary Diagnostics, Union City, NJ, USA). The following parameters were determined: platelet and RBC counts, haematocrit, haemoglobin (Hb) concentration, mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), red cell distribution width (RDW), and total and differential leucocyte counts. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured in citrated plasma samples using automated STA Compact Coagulation Analyzer (Diagnostic Stago, Roche, Basilea, Switzerland) and commercially available reagents. The PT was determined by adding 0.2 mL rabbit thromboplastin reagent (Simplastin, Organon Tekina Corp., Durham, NC, USA) to 0.1 mL plasma, while aPTT was measured by adding 0.1 mL actin-activated cephaloplastin reagent (Organon Teknika Corp., Dublin, Ireland) to 0.1 mL of the plasma, and mixed with 0.1 mL CaCl2 solution. Plasma samples for determination of both PT and aPTT were incubated at 37 °C for 3 min before adding respective reagents. As control for the PT and aPTT assays citrated human reference plasma was used (Baxter Diagnostics Inc., Deerfield, IL, USA). The PT index as well as the international normalized ratio (INR) was calculated.

2.4.2.

Lipid analyses

The lipid profile included the evaluation of triacylglycerols (TAGs), total cholesterol (TC), high density lipoprotein cholesterol (HDLc), low density lipoprotein cholesterol (LDLc), and oxidized low density lipoprotein (oxLDL). Cholesterol and TAGs were measured by enzymatic-colorimetric assays using commercially available kits (Randox Laboratories, Antrim, UK); HDLc and LDLc were measured by using direct methods (Randox Laboratories). The concentration of oxLDL in plasma was measured by an ELISA procedure using the murine monoclonal antibody, mAb-4E6, as capture antibody bound to microtitration wells, and a peroxidase conjugated anti-apolipoprotein B antibody recognizing oxLDL bound to the solid phase (Mercodia AB, Uppsala, Sweden).

2.5.

H2O2-induced haemolysis

2.5.1.

Preparation of RBC suspensions

Blood samples were processed within 2 hours of collection. Samples were centrifuged at 400 g for 10 min; plasma and buffy

coat were carefully removed and discarded. RBCs were washed three times with phosphate buffered saline solution (PBS; 125 mM NaCl and 10 mM sodium phosphate buffer, pH 7.4) at 4 °C and, finally, resuspended in PBS, to obtain RBC suspensions at 10% (for erythrocyte membrane studies) or 2% (for haemolysis studies) haematocrit.

2.5.2.

Haemolysis assays

The assays were performed using H2O2 solutions at a final concentration of 7.5 mM, and RBC suspensions of 2% haematocrit. The incubations were carried out at 37 °C for 4 h under gentle shaking. The cells were pre-incubated at 37 °C for 15 min and then H2O2 was added. Four independent assays (n = 4) were performed for each tested antioxidant system, and all tests and controls were in duplicate. Haemolysis was determined spectrophotometrically, according to Ko, Hsiao, and Kuo (1997). In all sets of experiments, a negative control (RBCs in PBS) was used. After 4 hours of incubation, an aliquot of the RBC suspension was taken out, diluted with 20 volumes of saline and centrifuged (1180 g, for 10 min). The absorption (A) of the supernatant was read at 540 nm. To yield the absorption (B) of a complete haemolysis, an aliquot of the RBC suspension was treated with 20 volumes of ice cold distilled water and, after centrifugation, the absorption was measured at the same wavelength. The percentage of haemolysis was then calculated: (A/B) × 100.

2.5.3.

Erythrocyte membrane changes

The changes in membrane bound haemoglobin (MBH) and membrane proteins were evaluated (Fig. 1). RBC suspensions at a higher haematocrit of 10% were prepared in order to obtain sufficient RBC membranes to perform these studies. RBCs were washed in saline solution and immediately lysed, by hypotonic lyses according to Dodge, Mitchell, and Hanahan (1963). The obtained membranes were washed in Dodge buffer, with phenylmethylsulphonyl fluoride added in the first two washes as a protease inhibitor, with a final concentration of 0.1 mM. The total protein concentration of the RBC membrane suspensions was determined by the Bradford’s method (Bradford, 1976). MBH was measured spectrophotometrically after protein dissociation of membrane components with Triton X-100 (5% in Dodge buffer) at 415 nm; the absorbance at this wavelength was corrected by subtracting the absorbance of the background at 700 nm; this value and the membrane protein concentration were then used to calculate the % MBH. To evaluate RBC membrane protein profile, the samples were treated with a solubilization buffer (0.125 M Tris HCl pH 6.8, 4% sodium dodecyl sulphate (SDS), 20% glycerol, 10% 2-mercaptoethanol), heat denatured and submitted to electrophoresis. The electrophoresis was carried out on a discontinuous system of polyacrylamide in the presence of sodium dodecyl sulphate (SDS-PAGE), using a 5–15% linear polyacrylamide gradient gel (8 μg of protein/lane) and a 3.5–17% exponential polyacrylamide gradient gel (6 μg of protein/lane), according to Laemmli and Fairbanks methods, respectively (Laemmli, 1970; Fairbanks, Steck, & Wallach, 1971). The proteins were stained with Coomassie brilliant blue, and finally the gel was scanned

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was regarded as significant. Comparisons of means were carried out with one-way ANOVA and the post hoc Tukey test was used to identify significant differences between the groups.

3.

Fig. 1 – SDS polyacrylamide-gel electrophoresis (exponential gradient gel) of pig red blood cell membrane proteins and correspondence to human red blood cell membrane proteins. The gel was stained with Coomassie blue. See Table 5 for band correspondence.

Results and discussion

All pigs were healthy throughout the experimental period. The significant effects (P < 0.05) of these treatments on growth performances (Table 3) was previously reported (Paiva-Martins et al., 2009). Leaf addition to the diet decreased both the final weight and the daily feed intake. Considering that oleuropein, the main polyphenol found in leaves (Table 2), is a bitter glycoside (Boskou, Blekas, & Tsimidou, 2005), there was probably a decrease in the palatability of diets that induced a lower feed intake. Nevertheless, a worse feed to weight gain ratio for pigs having lower final weight was also observed. In fact oleuropein has inhibitory effects towards digestive and metabolic enzymes, namely, trypsin, glycerol dehydrogenase, glycerol-phosphate dehydrogenase, glycerokinase, and lipase (Polzonetti, Egidi, Vita, Vincenzetti, & Natalini, 2004). Therefore, the high content of fibre and oleuropein in olive leaves (Table 2) may contribute significantly to the lower digestibility of diets. When pigs are reared in extensive systems, their performance is a crucial factor. However, concerning human nutrition these results may encourage the use of olive leaf in the control of body weight. Moreover, the intramuscular α-tocopherol increased with enriched diets, suggesting an improvement in the antioxidant defenses of the animals that may contribute to the prevention of oxidative stress associated diseases.

3.1. Effect of leaf supplementation on the blood cells and haemostasis (Darkroom CN UV/wl, BioCaptMW version 99, Vilber Lourmat, France) for identification and evaluation of proteins by densitometry.

2.6.

Statistical analysis

For the statistical analysis we used the IBM SPSS 20.0 statistical package (IBM Corp., Armonk, NY, USA). For comparisons of continuous variables between cases and controls, univariate ANOVAs were performed. A P value of less than 0.05

In the present study, we did not observe significant differences either in erythrocyte concentration or in its morphology; however, a trend towards lower values of RBCs, Hb concentration, and haematocrit were observed (Table 4). No differences were observed for WBCs, for the percentage of the different types of leukocytes, and for the (Fig. 2) the number of platelets (Table 4). Moreover, no differences were found for the haemostatic studies, suggesting no interference in the blood coagulation process.

Table 3 – Effects of dietary treatment on growth performance, measurements and tocopherol content. Group

Initial weight/kg Final weight/kg Daily gain (DG)/kg Daily feed (DF)/kg Feed:gain Backfat thickness (P2) (mm) Backfat α-tocopherol (mg/kg) Intramuscular α-tocopherol (mg/kg)

OL0

OL5

OL10

SE

P

68.8 130.5a 1.10a 2.90a 2.64a 13.4a 0.11a 0.26a

67.1 113.9b 0.84b 2.44b 2.95b 9.8b 2.81b 2.05b

67.5 111.7b 0.79b 2.44b 3.11b 9.6b 4.41c 3.30c

1.6 3.7 0.05 0.12 0.09 1.1 0.70 0.43

0.751 0.003 <0.001 0.019 0.003 0.047 <0.001 <0.001

Mean values in the same row with different superscript letters are significantly different (P < 0.05). Adapted from Paiva-Martins et al. (2009).

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Table 4 – Haematological and haemostatic indices. Red blood cells (n × 10 /L) Haemoglobin (g/dL) Haematocrit (%) Mean cell volume (fL) Mean cell haemoglobin (pg) Mean cell haemoglobin concentration (g/dL) Red cell distribution width (%) White blood cells (n × 109/L) Neutrophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%) Platelets (n × l09/L) Activated partial thromboplastin time (s) Prothrombin time (s) Prothrombin index (%) INR mean 12

OL0

OL5

OL10

SE

P

8.4 13.9 44.8 53.9 16.8 31.1 21.5 22.0 21.7 2.0 0.56 69.0 6.7 440.6 12.0 11.1 98.9 1.02

8.0 13.6 43.3 54.4 17.2 31.5 21.7 21.9 24.0 2.6 0.25 67.1 6.0 391.4 13.0 11.5 96.3 1.07

7.8 13.5 42.5 54.2 17.3 31.8 22.3 21.9 21.4 3.1 0.28 69.5 5.8 373.8 11.9 10.6 101.7 0.97

0.2 0.2 0.5 0.6 0.3 0.2 0.4 0.9 1.5 0.3 0.08 1.8 0.5 25.8 0.3 0.3 1.3 0.02

0.316 0.321 0.170 0.756 0.524 0.355 0.826 0.969 0.537 0.215 0.128 0.672 0.406 0.424 0.193 0.352 0.352 0.362

3.2. Effect of leaf supplementation on the blood lipid profile The supplementation of olive leaves did not result in significant changes in the cholesterol containing lipoproteins profile (Fig. 3) as compared with the control group (OL0). However, results revealed that olive leaf supplementation resulted in a significant decrease in TAGs. These groups also presented a significant reduction (27%) of backfat storage (Table 3) and a lower body weight, suggesting that leaves may be a cardioprotective agent. According to the increase in the tocopherol content observed in meat (Table 3), a trend towards lower values of oxLDL was also observed in the OL5 and OL10 groups.

3.3.

Effect of leaf supplementation on red blood cells

ConcentraƟon (mg/dL)

RBCs are particularly useful in the evaluation of the antioxidant protection of several compounds. RBCs, the most abun-

dant blood cells, are particularly susceptible to endogenous oxidative damage because of their specific role as oxygen carriers. RBCs are equipped with efficient antioxidant systems that are crucial to detoxifying the cells from reactive oxygen species (ROS) that are overproduced outside or within the erythrocyte. The development of an “oxidative stress” condition by inducing oxidative damages on erythrocyte constituents, namely on haemoglobin, may ultimately lead to haemolysis and to an increase in the cell-free haemoglobin levels in the plasma (Rother, Bell, Hillmen, & Gladwin, 2005). When haemoglobin is denatured, it links to the erythrocyte membrane at the cytoplasmic domain of band 3 protein, inducing its aggregation and the linkage of natural anti-band 3 antibodies and complement fixation on the erythrocyte surface, marking the cell for removal by the macrophages of the reticuloendothelial system. Whenever haemoglobin is released from erythrocytes, it is potentially dangerous because it can be converted into a powerful promoter of oxidative processes in blood (Rother et al., 2005). We found that enriched diet did not induce oxidative damage on haemoglobin, as showed by MBH that presented similar values for control and enriched diets. However, a slight

Fig. 2 – Percentage of haemolysis of RBC suspension (2% Ht) under oxidative stress conditions (7.5 mM H2O2/1 hour/ 37 °C), percentage of erythrocyte membrane bound haemoglobin (MBH) and oxLDL concentration in blood (U/L). *Mean values are significantly different (P < 0.05).

100

OL0

OL5

OL10

80 60 40

* *

20 0 TAGs

TC

HDLc

LDLc

Fig. 3 – Blood lipid profile. *Mean values are significantly different (P < 0.01). TAGs, triacylglycerols; TC, total cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein.

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Table 5 – RBC membrane protein profile. Band (%)

Correspondence to human RBC protein

OL0

OL5

OL10

SE

P

1 2 3 4 5 6 7 S 9 10 11

α-Spectrin β-Spectrin Ankyrin

17.4 14.4 7.2a 9.1 9.8a 4.3 7.2 18.la 8.6 1.6 1.4 0.14 0.42a 0.71a 3.09 0.67a 5.52a

19.4 15.9 7a 9.3 11.6b 4.2 6.9 14.6b 8.3 1.6 1.3 0.12 0.37a 0.58b 3.07 0.60b 5.97b

19.2 17.1 5.8b 7.7 14.4c 4.3 6.4 14c 9.9 1.3 1.3 0.12 0.29b 0.45c 2.38 0.40c 6.72c

0.5 0.6 0.3 0.5 0.8 0.2 0.3 0.7 0.4 0.2 0.1 0.09 0.04 0.07 0.24 0.07 0.57

0.088 0.154 0.042 0.269 0.025 0.884 0.504 0.034 0.151 0.442 0.619 0.433 0.042 0.046 0.136 0.042 0.041

Band 3 Protein 4.1 Protein 4.2 Band 5 Band 6 Band 7 Protein 4.1/Spectrin Protein 4.1/Band 3 Protein 4.2/Band 3 Spectrin/Band 3 Ankyrin/Band 3 Spectrin/Ankyrin

Mean values in the same row with different superscript letters are significantly different (P < 0.05).

increase in haemolysis was found for OL5, and a significantly higher value for OL10, as compared with the control OL0. Indeed, a trend towards lower values of RBCs was observed, further suggesting a haemolytic effect for the more enriched diet OL10. The RBC membrane is a complex structure comprising a lipidic bilayer, transmembrane or integral proteins, and peripheral proteins, including the cytoskeleton proteins. Modifications in RBC membrane protein composition may account for changes in the deformability of the cell, compromising its circulation in the microvasculature and its survival (Gallagher, 2005; Reliene et al., 2002; Rocha et al., 2005). Spectrin is the major protein of the cytoskeleton, and therefore the most responsible for RBC shape, integrity, and deformability. It links the cytoskeleton to the lipid bilayer by vertical protein interactions with the transmembrane proteins, band 3, and glycophorin C (Gallagher, 2005; Rocha et al., 2005). In the vertical protein interaction of spectrin with band 3, ankyrin (also known as band 2.1) and protein 4.2 are also involved. A normal linkage of spectrin with the other proteins of the cytoskeleton assures normal horizontal protein interactions, and its linkage with the transmembrane proteins assures normal vertical interactions. Figure 1 shows the SDS polyacrylamide-gel electrophoresis (exponential gradient gel) of RBC membrane proteins. Human RBC membrane proteins were also submitted to electrophoresis as a control, considering that the protein profile is similar to that presented by animal species (Lenard, 1970). We found that diet enrichment was associated with a trend to higher values of spectrin, a trend to lower values of ankyrin, and a significant increase in band 3 (P = 0.025) and band 5 (P = 0.034) (Table 5). These changes in membrane protein profile lead to a significant disturbance in horizontal and vertical protein interactions, as suggested by the altered ankyrin/ band 3 (P = 0.042), spectrin/ankyrin (P = 0.041) and protein 4.2/ band 3 (P = 0.046) ratios (Table 5). These disturbances were much higher for OL10 group, which may explain the higher haemolysis observed in this group as compared with the control and OL5 groups. Moreover, the reduction observed in RBC count,

haematocrit and Hb concentration is probably related to this lower stability. The increase in the haemolysis could have been due to a higher level of oxidation; however, an increase was not found in MHB %, showing that this increased haemolysis was not due to an increase in the oxidative damage of haemoglobin (Fig. 2). These RBC membrane alterations are in accordance with previous results obtained in vitro, where interactions of olive leaf polyphenols with red blood cell membrane proteins were also observed (Paiva-Martins et al., 2009, 2010, 2013). Interactions of polyphenolic compounds with membranes are usually regarded as a benefit in the sense that these interactions will bring the antioxidant moiety of an antioxidant molecule to the site of action in the vicinity of the membrane. Nevertheless, our results showed that these interactions with red cell membrane proteins resulted in a significant disturbance in horizontal and vertical protein interactions in the OL10 group, with consequent membrane destabilization. The present study corroborates the wellknown duality of the biological activity of phenolic compounds, namely the concentration-dependent balance between their cytoprotective versus cytotoxic capacities. Moreover, other constituents in leaves besides phenols may contribute to the negative effects obtained with leaf enriched diets.

4.

Conclusions

This work revealed that olive leaf supplementation resulted in a significant decrease in plasmatic TAGs, fat storage and a lower body weight, even at the lower concentration used, without compromising animal health, suggesting that leaves may also be used in the control of human body weight and as a cardio-protective agent. The dosage of numerous commercial olive leaf extracts is usually calculated in terms of oleuropein content. Most of these commercial extracts are recommended to be used in a dose between 100 and 1000 mg of

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oleuropein per day. The lower dose used in this study (OL5) would represent an 800 mg dose in humans and, therefore, the normal human doses should not show any deleterious effects. Nevertheless, further studies are needed in order to confirm the beneficial activity and safety of olive leaf powder and extracts supplements for human beings. REFERENCES

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