Hydroxytyrosol (HOTYR) is a simple phenolic compound naturally occurring in olive and olive oil, the main source of fat in Mediterranean diet. Nowadays, it is very popular the benefits of this diet for the maintenance of human health and wellbeing. Anyway, the concept of Mediterranean diet may be misunderstanding, being necessary its clarification. It was originally defined as the dietary pattern found in some of the olive-growing regions in the Mediterranean Basin of the 1960s. Thus, a current simple accepted definition is closely related to the diet characteristics as source of separate types of fats. In this way, Mediterranean diet highlights by its high proportion in plant foods where the main source of fat is olive oil, resulting in a higher proportion of mono unsaturated/polyunsaturated fats relative to saturated ones (1).

The healthy benefits of the Mediterranean diet are popularly known and are supported by numerous studies, as the well-known “The Seven Countries Study,” which highlighted the cardiovascular disease related mortality in Mediterranean region compared with other countries with distinct dietary habits in the 1960s (2). This study can be considered as pioneer and opened a high number of researches on those foods with critical influence in the benefits of the Mediterranean diet. Among them, the PREDIMED trial (“Prevención con dieta Mediterránea”) is a large (7447 subjects), parallel-group, multicenter, randomized trial for a follow-up period of 4.8 years, whose participants were assigned to three dietary intervention: Med-diet supplemented with virgin olive oil, Med-diet supplemented with nuts, and low-fat diet. The results showed an important reduction of three of every thousand persons per year in the risk of major cardiovascular events among high-risk persons.

Among the separate foods taking part of the Mediterranean diet, olive oil is considered a crucial element, being one of the most traded and consumed product in the world directly related to the healthy attribution of this popular diet (1). In November 2004, the Food and Drug Administration (FDA) allowed the health benefits of olive oil due to monounsaturated fat in reducing the risk of coronary heart disease and recommended a daily intake of two tablespoon (23 g) (3).

The responsible parameters of the healthy attributions of olive oil and table olives remain poorly understood. Olive oil is mainly composed of two groups of compounds: saponifiable lipids (98.5–99% of the total composition) and an unsaponifiable fraction. The main triglyceride fatty acid is the oleic acid with 55–83% of the total fatty acid content. It also includes moderate amounts of palmitic and linoleic acids and a low percentage of stearic and linolenic acids. On the other hand, the unsaponifiable fraction contains a large variety of compounds responsible for stability and organoleptic characteristics of olive oil. This fraction consists of primarily of hydrocarbons (squalene, carotene, lutein), terpene compounds, sterols, phenolic compounds, and aliphatic alcohols among others. Phenolic compounds from olive oil include four different groups: simple phenols, i.e., tyrosol (TYR), hydroxytyrosol, polyphenols (flavonoids), secoiridoids (oleuropein), and lignans (4).

Although the major components of olive oil are its monounsaturated fatty acids, mainly oleic acid, we cannot attribute the beneficial effects of the Mediterranean diet only to these substances because other oils, such as rape-seed or canola oil are also rich in monounsaturated fatty acids and may produce the same benefit. For this reason, attention has focused on the phenolic compounds, which have showed a strong anti-oxidant capacity, of virgin olive oil that distinguish themselves from other types of fat. The amount of phenolic compounds present in olive oil greatly varies depending on cultivar, degree of maturation, climate, and manufacturing process (5). Thus, extra virgin olive oil, obtained from the cold pressing or centrifugation of the olives, is the one with higher content of total phenolic compounds. An early study showed a total phenolic content of 232 mg/kg in virgin olive oil versus 62 mg/kg values of refined olive oil (up to 80% less usually lost during the refining process) (6).

In this sense, olives, olive oil source, have higher amounts of phenolic compounds that extra virgin olive oil (oil with higher anti-oxidant capacity). Depending on the variety of olive and the type of processing, the phenolic content varies significantly. As an example, total phenolic content estimated in black olives was 16.40 g/kg dry weight, representing hydroxytyrosol 5.78 g/kg. Tyrosol, phloretic acid, dihydrocaffeic acid, acteoside, luteolin, and apigenin were also found. The green olives content was much lower with 4.48 g/kg of hydroxytyrosol and only traces of other phenolics (6, 7).

Hydroxytyrosol, 2-(3,4-dihydroxyphenyl)-ethanol (HOTYR), is a phenolic compound present in the fruit and leaf of the olive (Olea europaea L.), which belongs to the family Oleaceae, comprising species distributed throughout the temperate regions of the world, and essentially localized in the Mediterranean basin. Hydroxytyrosol is one of the main components of virgin olive oil, olive mill waste water (OMWW), and olive leaf extract (OLE), which was early identified as the strongest in vitro anti-oxidant potential among all the olive oil polyphenols (8). As mentioned above, the content of this compound is largely dependent on the oil quality. Amounts range from 14 mg/kg in extra virgin olive oil, up to <2 mg/kg (7). Other estimations increased the estimation to total phenolic content of olive oil in ranges from 487.5 to 1950 mg/l, and HOTYR specifically from 20 to 84 mg/l (9). Hydroxytyrosol in oil is found free, in acetate form or as a part of more complex compounds like oleacein, oleuropein, and verbascoside (10) (Figure 1). Oleuropein is responsible for the bitter taste of olives and decreases as the fruit ripens turning into unglycosylated form, oleuropein aglycone by enzymatic hydrolysis, and finally converted into hydroxytyrosol, being this one an indicator of maturation of the olives (11, 12) (Figure 2).

Hydroxytyrosol, with a molecular weight of 154.16 is a white powder with melting point around 55°C and is fairly soluble in water and polar organic solvents such as low-molecular weight alcohols. Like other polyphenols, it is easily oxidized in aqueous solutions unless adequate precautions, acid pH, and absence of oxidants, are taken. As it was noted at the beginning, HOTYR is considered by far the main simple phenolic compound of table olives (13) although the concentration depends on olive variety (11), maturation, and the processing method (7). Besides, the concentration of phenolic compounds is different when comparing table olives and raw fruit due to manufacturing process. For example, in the preparation process of the Californian-type black olives, levels of HOTYR and caffeic acid decreases in the darkening process (14). Therefore, the beneficial properties for human health found in olive oil could also be supposed and extended in table olives. Moreover, HOTYR was identified in olive pits as shown by a 2001 patent explaining the synthesis of this compound by a process of quick autohydrolysis (15). Another natural source of HOTYR is red wine (16), and although the concentrations found are lower than those found in olive oil, unsettling bioavailability studies reveal the emergence of higher quantities of HOTYR concentrations than those administered, in urinary recoveries after administration of red wine. An interaction between ethanol and dopamine leading to the formation of hydroxytyrosol was suggested as a possible explanation to this fact (17). Therefore, all these data reveal the relevance of HOTYR consumption by olive products, which are regularly consumed in the Mediterranean diet and ensure a daily contribution to the human health.

Reactive oxygen species (ROS) are critically involved in the endothelial dysfunction that contributes to atherosclerosis development. Oxidative stress-induced endothelial dysfunction probably represents one of the first stages in the development of atherosclerotic lesions (31, 32). Accordingly, the atherosclerotic vessel wall contains increased levels of ROS, which affect several redox–sensitive pathways in vascular cells, resulting in a markedly altered cellular composition of the tissue. Migration and proliferation of vascular smooth muscle cells in the area is induced, as well as expression of adhesion molecules and chemotactic factors by the endothelium (33). Consequently, increased arterial adhesiveness at predisposed sites provides an excellent environment for local infiltration of circulating immune cells, resulting in chronic inflammation (34). Thus, direct reduction of levels of ROS and/or stimulation of anti-oxidant defenses at these levels could avoid atherosclerosis development.

It has been observed in animal models that levels of several anti-oxidant enzymes decline during atherogenesis (35). Incubation of porcine pulmonary artery endothelial cells (VECs) with HOTYR in the presence of H2O2 prevents the increase in intracellular ROS levels (36). HOTYR increases catalase mRNA, protein, and activity of both cytosolic and nuclear protein levels of forkhead transcription factor 3a (FOXO3a), which directly increases the expression of anti-oxidant enzymes, as well as the phosphorylation of AMP-activated protein kinase (AMPK) that translocates FOXO3a to the nucleus. Thus, these findings demonstrate that HOTYR positively regulates the anti-oxidant defense system in VECs and provides a molecular basis for the prevention of cardiovascular diseases by HOTYR.

Another study analyzed the effects of HOTYR on anti-oxidant status and the progression of aortic lesions in hyperlipemic rabbits. After 1-month of controlled diet 50 and 42% decrease in total cholesterol and triacylglycerols, respectively, and a 2.3-fold increase in HDL-cholesterol were observed. The HOTYR-supplemented groups also improved their anti-oxidant status and reduced the size of atherosclerotic lesions measured as intimal layer areas of the aortic arch when compared with control animals (37).

In 2003, Carluccio et al. (38) found that at nutritionally relevant concentrations, oleuropein and hydroxytyrosol, among all the tested molecules present in olive oil and red wine, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein in human umbilical vein endothelial cells incubated for 30 min with the phytochemicals, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. The relevance of nuclear factor-NF-κB and activator protein-1 confirmed by electrophoretic mobility shift assays, and possibly GATA binding sites in mediating VCAM-1 transcriptional inhibition, was observed in this case.

In 1995, an experimental study conducted by Visioli showed in vitro the inhibitory effect of hydroxytyrosol and two derivative compounds on LDL oxidation (39). Different indices of lipid oxidation such as vitamin E content, formation of thiobarbituric acid-reacting substances (TBARS), lipid peroxidases, levels of polyunsaturated fatty acids, protein modification, and conjugated diene formation were used. Copper sulfate and horseradish peroxidase/hydrogen peroxidase were used as LDL oxidation systems. Hydroxytyrosol and two derivatives at 10⁻⁵ M almost completely inhibited conjugated diene formation induced by 5 μM copper sulfate as oxidant in LDL incubation samples. The mechanism of action proposed for this phenolic compounds were the scavenging free radicals and/or acting as chelating agents and thus depressing the superoxide-driven reactions and breaking the propagation chain of peroxides (40).

More than 15 years after the first study, the European Food Safety Authority (EFSA) published a scientific opinion related to polyphenols in olive, and The EFSA Panel on Dietetic Products, Nutrition, and Allergies regarded about the relationship between the consumption of olive oil polyphenols and protection of LDL particles from oxidative damage (41). The panel considered that 5 mg of hydroxytyrosol and its derivatives in olive oil should be consumed daily to reach this effect at physiological level. This amount of hydroxytyrosol must be contained in a maximum of 20 g of oil to be considered within a balanced diet. Considering that the oxidation of LDL plays an important role in the development of atherosclerosis, this communication brings robust data on the implication of phenolic compounds in coronary heart disease. This scientific opinion was supported by consistent studies showing the relationship between phenolic compounds in olive oil and LDL oxidation. To provide its opinion EFSA took into account several studies demonstrating the involvement of phenolic compounds as protectors of LDL oxidation. Other studies have been reviewed with results in the same direction as those previously recognized by EFSA. Data are shown in Table 1.

In general, olive oil polyphenols may have anti-oxidant protective properties not only by the free-radical scavenging but also for the modulation of gene expression such the CD40 ligation and its downstream products in human beings. It has been observed that intake of high content of phenolic compounds from olive oil (366 mg/kg) reduced the expression of CD40 ligand gene and its downstream products, and was associated with a decrease in oxidized LDL in plasma and an increase in urinary polyphenols (40).

Another process involved in the development of atherosclerotic lessions is platelet aggregation, which is the previous step in thrombus formation (49). HOTYR can be considered anti-thrombotic, since it significantly reduces platelet aggregation. While Dell’Agli et al. (50) found positive results for HOTYR in the reduction of human platelet aggregability but not through the inhibition of cAMP- and cGMP-phosphodiesterases as the targets of the biological effect as the other olive oil polyphenols tested in their study, early works of Petroni et al. (51) and more recently Léger et al. (52) in human blood samples from healthy volunteers and patients with uncomplicated diabetes type 1, respectively, also suggest anti-platelet aggregation activity. In these studies, a reduction in thromboxane B2 levels, a chemically stable and inactive form of thromboxane A₂, which acts as platelet aggregating and vasoconstrictor, was measured. Another comparative study (53) with mildly dyslipidemic patients reached similar results, finding a decrease in the production of thromboxane B2 and an increase in plasma anti-oxidant capacity after consumption of HOTYR contained in extra virgin olive oil (166 mg/l; estimated ingestion 6.64 mg/day) compared with the absence of results in the case of a low intake from a refined oil (2 mg/l; estimated ingestion 0.02 mg/day).

Gonzalez-Correa et al. (54) also found HOTYR and HOTYR-acetate as inhibitor of platelet aggregation in rats after oral administration for 7 days, with similar effectiveness of acetyl salicylic acid. In this case, mechanisms involved in this effect were a decrease in thromboxane synthesis and an increase in vascular nitric oxide (NO) production.

In vitro and in vivo studies (55) taken as a model of inflammatory response in rats plantar edema induced by carrageenan shows hydroxytyrosol as inhibitor of cyclooxygenases 1 and 2, which convert arachidonic acid to prostaglandins, resulting in pain and inflammation, at the same level as widely used drugs such as ibuprofen and celecoxib. In studies with human monocytic cells, THP-1 (56) treated with lipopolysaccharides to induce inflammatory response HOTYR showed the inhibition of pro-inflammatory cytokines (TNF-α) and reduced the expression of cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) more than 60%.

Beneficial effects were also found (57) in patients with stabilized coronary disease. The intake of virgin olive oil, rich in polyphenols, was shown to be more effective than refined olive oil in producing a decrease in interleukin-6 (IL-6) and C-reactive protein (CRP) levels, recognized inflammatory markers in cardiovascular disease (58).

A DSM study from the human nutrition and health department (59) describes hydroxytyrosol as the most powerful anti-inflammatory compound among polyphenols of olive oil, showing effective inhibition in the production of NO and PGE2, decreasing secretion of cytokines (IL-1α, IL-1β, IL-6, IL-12, TNF-α) and chemokines (CXCL10/IP-10, CCL2/MPC-1) and reducing gene expression of iNOS, IL-1α, CXCL10/IP-10, MIP-1β, matrix metaloproteinase-9, and prostaglandin E2 synthase (PGES).

Very recently, Killen et al. (60) have reviewed the HOTYR ability to modulate the transcription factor NF-κB whose activation triggers the expression of over 150 genes, including those encoding cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, and IL-17, chemokines and adhesion molecules, implicated in both physiological and pathophysiological inflammatory responses.

Data from these studies provide a molecular basis for elucidating the effects of HT on inflammatory processes. Concentrations reached after dietary intake of olive oil results in appropriated values of HOTYR for inhibiting the NO and PGE2 production and significant modulation of gene expression. Therefore, exposure to lower levels of HOTYR by regular dietary intake might repeatedly modulate physiological parameters that provide a systemic health benefit.