Olive oil and health effects : from epidemiological studies to the molecular mechanisms of phenolic fraction

Olive oil is a key component of the Mediterranean diet which is recognized to contribute to its health benefits. Recent prospective studies point towards a protective effect from an olive oil-rich diet in relation to the incidence of cardiovascular diseases and an improvement of cardiometabolic markers such as blood pressure, glycaemia and dyslipidemia, notably by reducing LDL cholesterol and LDL oxidation. The role of minor phenolic fraction was evidenced in intervention trials where lipid profiles showed greater improvement in participants receiving olive oil with higher phenolic content. The phenolic fraction of olive oil is composed of simple phenols (hydroxytyrosol), phenolic secoiridoids (oleuropein aglycone), lignans (pinoresinol), flavonoids and hydroxyisochromans. All these compounds have diverse biological activities that are described in the present review, supporting the protective effects of olive oil against degenerative diseases found in large cohorts monitored in Southern European countries.


Introduction
Olive oil is a key component of the Mediterranean diet and recognized to contribute to its health benefits, especially in preventing cardiovascular diseases (CVD) (Lopez-Miranda et al., 2010).Recent trials on olive oil supplementation point towards a protective effect against cardiometabolic risk factors.
Correspondence: marie-josephe.amiot-carlin@univ-amu.fr The dietary benefits of olive oil were initially linked to its high oleic acid content, a monounsaturated fatty acid (18:1 n-9), which ranges from 55% to 83% of total fatty acids.This has been the basis of the authorized health claim for olive oil labelling by the food and drug administration in 2004.However, experimental-based evidence has accumulated on the health benefits of minor bioactive components of olive oil such as polyphenols which have specific structures and are found in high quantities in virgin and extra-virgin olive oil.Among these structures, hydroxytyrosol was recently reported to protect the blood lipids against oxidative damage as reported by the European food safety authority (EFSA) panel on dietetic products, nutrition and allergies.The Panel considered that in order for olive oil to bear the "heart-health" claim, 5 mg of hydroxytyrosol and its derivatives (e.g.oleuropein complex and tyrosol) in olive oil should be consumed daily (EFSA, 2011;Martin-Pelaez et al., 2013).
The present review compiles the latest results from epidemiological studies and dietary intervention in large cohorts.The mechanisms by which the minor phenolic fraction of olive oil could protect against degenerative chronic diseases are described using the modulation of disease biomarkers and gene expression assessed in human trials.

Prospective studies, meta-analysis and dietary intervention studies with olive oil
The analysis of epidemiological studies on different outcomes, cardiovascular disease, cancer, cognitive decline and weight gain are reported in Table 1.
-Cardiovascular diseases: all prospective studies pointed towards a beneficial effect on health from an olive oilrich diet on the incidence of cardiovascular diseases.The biggest studies were carried out in Southern European countries.In a Spanish population, the association between olive oil intake and all-cause as well as cause-specific mortality was studied.The greatest reduction in risk was observed for CVD mortality.This amounted to 44% for the highest olive oil quartile of consumers in comparison with nonconsumers.A gradual increased intake of olive oil was associated with a decreased risk of CVD mortality (each 10 g per 2000 kcal increase was linked to a 13% decrease in risk) (Buckland et al., 2012).In two recent prospective studies, the level of olive oil consumption was compared with the incidence of coronary heart disease (CHD).In a large cohort of Italian women (EPI-COR STUDY), a significant reduction in CHD risk (-44%) was associated with the highest quartile of olive oil consumption compared with the lowest one (> 31, 2 g/d versus ≤15 g/d) (Bendinelli et al., 2011).In a Spanish cohort taken from the European Prospective Investigation into Cancer study, a more modest reduction of CHD (-22%) was observed in the upper quartile consumers > 28.9 g/d (Buckland et al., 2012).There was a 7% reduction in CHD risk for each 10g/d per 2000 kcal.In the Three-City Study conducted in France, participants who used olive oil intensively had a 41% lower risk of stroke than those who never used it (Samieri et al., 2011).All these prospective studies confirmed the cardiovascular protective effect of olive oil previously reported in case-control studies (Bertuzzi et al., 2002;Fernandez-Jarne et al., 2002;Kontogianni et al., 2007).-Diabetes: there is no epidemiological study specifically regarding olive oil consumption and diabetes.However, olive oil-rich diets do appear effective in the prevention of diabetes.A lower incidence of diabetes was associated to a higher adherence to a Mediterranean diet in healthy subjects (Martinez-Gonzalez et al., 2008) as well as in patients with recent myocardial infarction (Mozaffarian et al., 2007).-Obesity: no incidence on weight gain was found in the SUN prospective cohort study (Bes-Rastrollo et al., 2006).-Cancer: a recent systematic review and meta-analysis of observational studies revealed that, overall, olive oil consumption was associated with lower odds of developing cancer (Psaltopoulou et al., 2011).The greatest reduction in risk was found in breast cancer and digestive system cancers.-Cognitive decline: Berr et al. (2009) et al., 2007;Salas-Salvado et al., 2008).All these findings in large cohort studies are consistent with the protective role of olive oil consumption on health.The role of minor phenolic fraction was evidenced in the EUROLIVE controlled study (Covas et al., 2006) where the improvement of the lipid profile is linked to increasing phenolic content As concerns blood pressure, the Greek European Prospective Investigation into Cancer and Nutrition (EPIC) study showed that olive oil intake was inversely associated with both systolic and diastolic blood pressure (Psaltopoulou et al., 2004).A moderate consumption of olive oil was suggested as an effective recommendation to reduce systolic blood pressure in healthy men who do not typically consume a Mediterranean diet (Bondia-Pons, et al., 2007).

Minor compounds of olive oil and phenolic fraction: characterisation and bioavailability
Minor compounds of olive oil are classified into two categories; unsaponifiable (non-polar) and the soluble (polar) fractions, including phenolic compounds (Cicerale et al., 2009).The phenolic fraction of olive oil (olive oil phenols, or OOP's) varies both in quantity and quality.Owen et al. (2000) reported an average concentration of phenolic compounds of 230 mg/kg in extra virgin olive oil.The phenolic quantity varied from 50 D512, page 2 of 8 M.J. Amiot: OCL 2014, 21(5) D512

Table 1.
Epidemiological studies on olive oil and health outcomes.to 800 mg/ (Perona et al., 2006) while the phenolic composition depends on various factors, including the variety of olive, the environment (soil and climate), farming practices, the maturity of olive fruits, the manufacturing process and storage conditions (Amarowicz et al., 2009).OOP's can be classified into five main structures.These are simple phenols, phenolic secoiridoids, lignans, and flavonoids and hydroxyisochromans (Fig. 1).Oleuropein aglycone, found in ripening olives and olive oil, is a phenolic secoiridoid liberated from the glucoside form, oleuropein, upon the action of a ß-glucosidase.In olive oil, oleuropein is degraded into elenolic acid, the secoiridoid moiety, and hydroxytyrosol, the phenolic moiety.Oleuropein aglycone and hydroxytyrosol are characterized by the orthodiphenolic structures, but in olive oil, there are also corresponding mono-phenolic structures of ligstroside aglycone and tyrosol.Tyrosol, hydroxytyrosol, and their corresponding secoiridoid derivatives constitute around 90% of the total phenolic content of virgin olive oil (de la Torre-Carbot et al., 2006).Hydroxyisochromans are formed by reaction between hydroxytyrosol and aromatic aldehydes (vanillin and benzaldehyde) under very mild conditions (Bendini et al., 2007).OOP's were reported to be highly bioavailable compared to other plant polyphenols.OOP absorption efficiency was evaluated about 55-66 mol% in humans (Vissers et al., 2004).Tyrosol and hydroxytyrosol are absorbed by humans in a dosedependent manner in relation to the phenolic content of the olive oil administered (Visioli et al., 2000).Tyrosol and hydroxytyrosol are extensively metabolized and found in urine as conjugates, glucuronide and monosulphate as well as methylated derived simple phenols, homovanillic alcohol and homovanillic acid (Corona et al., 2009).These metabolites have been identified in LDL (de la Torre-Carbot et al., 2007).

Molecular mechanisms of specific olive oil compounds
The main mechanisms by which OOP's could act are reported in Figure 2.
Lipid metabolism: high levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) are recognized risk markers for atherosclerosis.This is considered to be the primary cause of cardiovascular diseases (CVD) while high density lipoprotein cholesterol (HDL-C) is assumed to be protective.As previously mentioned, lipid profiles were improved in participants of the crossover human study EU-ROLIVE, which enrolled 200 European participants receiving olive oil for three weeks (Covas et al., 2006).Participants were randomly assigned to three groups of olive oil differing in their phenolic content (low, medium and high).HDL-C linearly increased with the phenolic content, whereas TC/HDL-C ratio linearly decreased.LDL-C/HDL-C ratio and triglycerides decreased in those consuming medium and high phenolic olive oils.Smaller human trials confirmed that phenol rich olive oil improves lipid profiles in terms of circulating HDL-C, decreasing LDL-C (Damasceno et al., 2011) and larger but fewer triacylglycerol-rich lipoproteins (TRL) (Cabello-Moruno et al., 2007).
Oxidative stress: there is a recognised link between oxidative stress produced by reactive oxygen species (ROS) and degenerative diseases such as atherosclerosis, certain cancers and neurodegenerative diseases.OOP's were reported to have antioxidant effects on lipid and DNA oxidation that were greater than those of vitamin E (Fito et al., 2000;Masella et al., 2004;Owen et al., 2000).LDL oxidation (oxLDL) is considered to be a major risk factor for the development of atherosclerosis and CVD.In the EUROLIVE study, oxidized LDL levels decreased linearly with increasing phenolic content (Covas et al., 2006).The increased resistance of LDL to oxidation following a phenolic rich olive oil supplementation was confirmed in different short term studies, although three studies compiled by Covas (2007) and Cicerale et al. (2010) showed no effect.The binding capacity of phenolic compounds to LDL could explain the increased resistance of LDL to oxidation (Fito et al., 2007).Besides LDL oxidation, other oxidative markers also showed improvements.Thus, phenol-rich virgin olive oil administered to human subjects reduced F2-isoprostanes (Visioli et al., 2000), improved the balance between reduced and oxidized gluthatione GSH and GSSG (Covas et al., 2006) or increased glutathione peroxidase activity GSH-Px (Weinbrenner et al., 2004).
Inflammation: elevated concentrations of inflammation markers in serum are associated with increased cardiovascular risk.Plasma thromboxane B2 (TXB2) and leukotriene B4 (LTB4) are considered as proinflammatory agents.In the inflammatory pathway, the inhibition of cyclooxygenases (COX) results in the reduction of arachidonate into eicosanoids, prostaglandins and thromboxane.OOP's were shown to have anti-inflammatory properties (Bogani et al., 2007).Oleocanthal and an ester of tyrosol, was found to inhibit COX-1 and COX-2, mimicking the effect of ibuprofen (Beauchamp et al., 2005;Smith et al., 2005).Moreover, inflammatory markers like C-reactive protein (CRP) and Interleukin-6 (IL-6) have also been shown to be predictors for CVD.The PREDIMED study showed that CRP and IL-6, as well ICAM-1, and VCAM-1 were reduced in participants following the Mediterranean diet supplemented with olive oil compared with the low-fat diet group (Estruch et al., 2006).In a placebocontrolled crossover randomized trial on 28 stable coronary heart disease patients, IL-6 and CRP levels significantly decreased after virgin olive oil intervention (Fito et al., 2008).
Platelet function: blood platelets were shown to play a role in the development of atherosclerosis.The development of CHD has been linked to high plasma levels of coagulation and fibrinolytic factors such as plasminogen activator inhibitor-1 (PAI-1) and factor VII (FVII).Virgin olive oil with a high phenolic compound content (400 mg/kg) has been demonstrated to inhibit PAI-1 and FVII (Delgado-Lista et al., 2008).Hydroxyisochromans were shown to inhibit platelet aggregation (Togna et al., 2003) and cAMP-phosphodiesterase inhibition was shown as one mechanism by which platelet aggregation was inhibited in presence of OOP's (Dell'Agli et al., 2008).
Endothelium dysfunction: in different human trials, the consumption of meals with phenolic rich olive oil was shown to improve endothelium function in the postprandial period (Vogel et al., 2000;Karatzi et al., 2008;Fuentes et al., 2008;Ruano et al., 2007).Experimental studies carried out in different animal models of atherosclerosis, hypertension, hypercholesterolemia supported the link between endothelial dysfunction and oxidative stress.The different actions of polyphenols on endothelial and smooth muscle cells through nitric oxide (NO) stimulation have been reviewed (Andriantsitohaina et al., 2012).OOP's were reported to contribute to increased NO levels and prevent the powerful oxidant peroxynitrite forming (Perona et al., 2006).Moreover, OOP's were found to decrease homocysteine, which has been linked to increased adhesiveness of the endothelium (Manna et al., 2009).
Chemoprevention: while the antioxidant properties of OOP's against DNA damage and their anti-inflammatory actions are well characterized, OOP's may also exert anticarcinogenic effects through their ability to act on the metabolism of carcinogens (Hashim et al., 2005).The various anticarcinogenic mechanisms of phenolic compounds have been reviewed by Yang et al. (2001).Furthermore, olive oil is rich in lignans (Owen et al., 2000;Bendini, 2007) and it is assumed that foods containing high amounts of lignans appeared to protect against breast cancer via their anti-estrogenic effects (Saarinen et al., 2007).In addition, in cell experiments, oleuropein and hydroxytyrosol have been shown to induce cell death of MCF-7 human breast cancer cells (Han et al., 2009).Recent findings have provided clearer evidence that hydroxytyrosol is a potent inhibitor of the vascular endothelial growth factor (VEGF-2) signalling pathway that regulates tumour angiogenesis (Lamy et al., 2014).
Other actions: as reducing agents, OOP's are supposed to spare vitamin E. As vitamin E (tocopherols) has been shown to exert antioxidant activities, OOP's could act indirectly in preventing CVD.
Nutrigenomic effects: the beneficial effects for atherosclerotic processes could be explained by the variation in gene expression following a diet rich in olive oil, resulting in improving risk biomarkers as reported in Figure 2. Two human trials demonstrated that the minor phenolic fraction of olive oil plays a role in the downregulation of pro-atherogenic genes in microarray analyses (Konstantinidou et al., 2010;Camargo, 2010).In a randomised trial, in vivo gene expression changes were assessed in the mononuclear cells of healthy volunteers after an intake of high or low-phenolic olive oil as part of the Mediterranean diet (Konstantinidou et al., 2010).Significant changes in inflammation and oxidative stress-related gene expression were found between the Mediterranean diet pattern and the control group.Moreover, a significant linear decreasing trend was observed in connection with the phenolic content of olive oil in inflammation-related gene expression (interferon gamma, Rho-GTPase-activating protein 15) and oxidative stress (adrenergic beta 2 receptor).In another study involving patients with metabolic syndrome, the intake of a breakfast enriched in high phenolic content virgin olive oil decreased the inflammation-related gene expression of NF-κB and COX-2 (Camargo et al., 2010).Moreover, another diet intervention with 737 participants showed that a Mediterranean diet rich in virgin olive oil may reverse the effects of the IL6 (-174G/C) gene variant on 3-year body weight change (Razquin et al., 2010).In a study context of oxidative stress and chemoprevention, changes in gene expression were evaluated using genome-wide mRNA-Sequencing in various cell lines exposed to hydroxytyrosol (Rafehi et al., 2012).The results indicated that hydroxytyrosol promotes the cell's own protection against oxidative stress by a significant upregulation of enzymes involved in antioxidant defence systems.The greatest upregulation was found for heme oxygenase-1.The growth of breast tumours could be stimulated by estradiol leading to the regulation of gene expression and signal transduction pathways inducing cell proliferation.It was shown that hydroxytyrosol could act as a chemopreventive agent in breast cancer cell proliferation via the inhibition of estrogendependent rapid signals involved in uncontrolled tumour cell growth (Sirianni et al., 2010).

Conclusions
The added value of virgin olive oil in protecting against cardiovascular disease found in large prospective studies has been supported by numerous dietary intervention trials.These provided evidence that phenolic compounds display a broad spectrum of bioactive properties, including lipid-improving, antioxidant, anti-inflammatory and vasodilatory effects.Numerous studies have been focused on hydroxytyrosol as compiled in Hu's review (Hu et al., 2014).Olive oil is also rich in lignans which are considered to be promising molecules in decreasing the risk of cardiovascular disease, despite the fact that their mechanisms have not yet been clarified (Peterson et al., 2010).Moreover, the role of olive oil polyphenols in human microbiotia remains to be explained, taking into account recent findings in this field (Cardona et al., 2013).Since it was recently shown that a long-term intervention of a virgin olive oil-rich diet resulted in a better cognitive function compared to a control diet (Martinez-Lapiscina et al., 2013), further studies are needed to specify the role of the minor phenolic fraction of olive oil in cognition and neurodegenerative diseases.