Hydroxytyrosol and Cytoprotection: A Projection for Clinical Interventions

Hydroxytyrosol (HT) ((3,4-Dihydroxyphenyl)ethanol) is a polyphenol mainly present in extra virgin olive oil (EVOO) but also in red wine. It has a potent antioxidant effect related to hydrogen donation, and the ability to improve radical stability. The phenolic content of olive oil varies between 100 and 600 mg/kg, due to multiple factors (place of cultivation, climate, variety of the olive and level of ripening at the time of harvest), with HT and its derivatives providing half of that content. When consumed, EVOO’s phenolic compounds are hydrolyzed in the stomach and intestine, increasing levels of free HT which is then absorbed in the small intestine, forming phase II metabolites. It has been demonstrated that HT consumption is safe even at high doses, and that is not genotoxic or mutagenic in vitro. The beneficial effects of HT have been studied in humans, as well as cellular and animal models, mostly in relation to consumption of EVOO. Many properties, besides its antioxidant capacity, have been attributed to this polyphenol. The aim of this review was to assess the main properties of HT for human health with emphasis on those related to the possible prevention and/or treatment of non-communicable diseases.


Introduction
According to the Global Health Observatory (GHO) of the World Health Organization (WHO), non-communicable diseases (NCDs), also known as chronic diseases, are the main cause of mortality worldwide. These illnesses include diabetes, hypertension, metabolic syndrome, non-alcoholic fatty liver disease, heart diseases, stroke, cancer, and chronic respiratory diseases, and it is estimated that 38 million of the 56 million global deaths in 2012 were due to NCDs [1,2]. NCDs are mostly characterized by mitochondrial alterations, impaired metabolism, oxidative stress, and inflammation [2], molecular and cellular alterations that are key issues to be dealt with for prevention and treatment [3]. In this context, hydroxytyrosol (HT) (3,4-Dihydroxyphenyl)ethanol; Figure 1) is a polyphenol mainly present in extra virgin olive oil (EVOO) [4] that has a potent antioxidant activity [5][6][7]. Moreover, HT provides other beneficial benefits that may be of importance for human health [8], namely anti-inflammatory [9] and anticancer [10] effects, enhancement of endothelial and vascular function [11], anti-steatotic [7,12] properties, and improvement of endoplasmic reticulum stress [13], autophagy [14] and mitochondrial function [15]. Interestingly, HT is the only polyphenol recognized by the European Food Safety Authority as a protector of low density lipoproteins (LDL) from oxidative damage [16], considering the finding that HT reduces the expression of adhesion molecules in endothelial cells, preventing the oxidation of LDL [17]. In order to obtain this effect, 5 mg of HT and its derivatives should be consumed in the form of extra virgin olive oil (EVOO) per day, which can be easily achieved in a healthy diet [16,18]. Moreover, HT exhibits a preventive effect on the metabolic syndrome in obese subjects, by decreasing fat deposition in liver and muscle, enhancing antioxidant capacity with both direct and indirect effects, and improving mitochondrial function. Furthermore, HT has a significant effect in attenuating glucose and lipid metabolic disorders in mice [19]. In view of these considerations, the aim of this review was to assess the main properties of HT for human health with emphasis on those related to the possible prevention and/or treatment of NCDs.

Material and Methods
The review included several searches that considered the metabolic and beneficial effects of HT in in vivo and in vitro models, using the PubMed database from the National Library of Medicine-National Institutes of Health. A particular emphasis was placed in the participation of HT as an antioxidant, anti-inflammatory, anti-cancerogenic, and anti-steatotic molecule, affording endothelial and vascular function, endoplasmic reticulum stress, autophagy, and mitochondrial function improvements.

General Characteristics of Hydroxytyrosol
Olive oil can be separated into a larger fraction and a smaller portion. The larger fraction (98-99% of total oil weight) corresponds to the saponifiable portion that is mainly composed of triglycerides (TGs), oleic acid being their most important component [4]. The minor fraction includes two types of components, namely, the unsaponifiable fraction and the soluble part, the latter containing the phenolic compounds that exhibit antioxidant activity, such as acids and alcohols, with HT belonging to the last group [4]. The olive oil phenols can be divided in three categories, simple phenols (HT and tyrosol), secoiridoids (SEC; oleuropein), and lignans [20]. HT can be present as a simple phenol or esterified with elenoic acid, forming oleuropein [4]. Both HT and tyrosol are formed from the hydrolysis of the SEC aglycones of oleuropein and ligstroside [20]. Hydrolysis of oleuropein takes place during the storage of olive oil and results in the formation of HT, tyrosol and ethanol. HT and oleuropein are catecholes [20], and it has been shown that the antioxidant effect of EVOO phenolic compounds is due to the presence of the catechol group [21]. The antioxidant properties of HT are related to hydrogen-atom donation and the ability to improve radical stability by forming an intramolecular hydrogen bond between the free hydrogens of their hydroxyl groups and their phenoxyl radicals [22]. The beneficial effects of HT have been demonstrated in human, cellular and animal models, mainly consumed as EVOO [17,23] The HT from EVOO has also shown to increase insulin sensitivity and the secretory capacity of β-pancreatic cells, thus reducing the risk of developing metabolic syndrome. The phenolic content of olive oil depends on the place of cultivation, climate, variety of the olive, and its level of ripening at the time of harvest, reaching levels of 100-600 mg/kg, and approximately 50% of that phenolic content corresponds to HT and its derivatives [24,25].

Material and Methods
The review included several searches that considered the metabolic and beneficial effects of HT in in vivo and in vitro models, using the PubMed database from the National Library of Medicine-National Institutes of Health. A particular emphasis was placed in the participation of HT as an anti-oxidant, anti-inflammatory, anti-cancerogenic, and anti-steatotic molecule, affording endothelial and vascular function, endoplasmic reticulum stress, autophagy, and mitochondrial function improvements.

General Characteristics of Hydroxytyrosol
Olive oil can be separated into a larger fraction and a smaller portion. The larger fraction (98-99% of total oil weight) corresponds to the saponifiable portion that is mainly composed of triglycerides (TGs), oleic acid being their most important component [4]. The minor fraction includes two types of components, namely, the unsaponifiable fraction and the soluble part, the latter containing the phenolic compounds that exhibit antioxidant activity, such as acids and alcohols, with HT belonging to the last group [4]. The olive oil phenols can be divided in three categories, simple phenols (HT and tyrosol), secoiridoids (SEC; oleuropein), and lignans [20]. HT can be present as a simple phenol or esterified with elenoic acid, forming oleuropein [4]. Both HT and tyrosol are formed from the hydrolysis of the SEC aglycones of oleuropein and ligstroside [20]. Hydrolysis of oleuropein takes place during the storage of olive oil and results in the formation of HT, tyrosol and ethanol. HT and oleuropein are catecholes [20], and it has been shown that the antioxidant effect of EVOO phenolic compounds is due to the presence of the catechol group [21]. The antioxidant properties of HT are related to hydrogen-atom donation and the ability to improve radical stability by forming an intramolecular hydrogen bond between the free hydrogens of their hydroxyl groups and their phenoxyl radicals [22]. The beneficial effects of HT have been demonstrated in human, cellular and animal models, mainly consumed as EVOO [17,23] The HT from EVOO has also shown to increase insulin sensitivity and the secretory capacity of β-pancreatic cells, thus reducing the risk of developing metabolic syndrome. The phenolic content of olive oil depends on the place of cultivation, climate, variety of the olive, and its level of ripening at the time of harvest, reaching levels of 100-600 mg/kg, and approximately 50% of that phenolic content corresponds to HT and its derivatives [24,25]. HT is also present in wine, and it is considered a secondary metabolite of tyrosine formed by yeasts during alcoholic fermentation, having an approximate concentration up to 5 mg/L [26]. In humans, there is an endogenous formation of HT from dopamine, which is favored in the presence of ethanol [24]. De la Torre et al., 2006 compared short-term and postprandial effects of moderate doses of EVOO (1.7 mg HT) and wine (0.35 mg HT), and found that the urinary recovery of HT was greater from wine intake than from EVOO consumption, despite having a lower HT content, which is explained by the endogenous formation from dopamine favored by the alcohol content of the wine [27]. EVOO's phenolic compounds are rapidly hydrolyzed against alkaline conditions in the stomach and intestine, resulting in the increase of free HT to be absorbed in the small intestine where it forms phase II metabolites [11]. The in vitro colon fermentation of HT showed production of phenolic acids (phenylacetic and phenylpropionic acids) and degradation of HT-acetate and oleuropein by fecal culture medium, thus stabilizing HT and tirosol levels [28]. Furthermore, a moderate intake of a phenol-rich olive oil in males significantly raised the concentration in human feces of free HT [28]. These data suggest a role for colon microbiota in HT biotransformation, leading to an enhanced biodisponibility of the polyphenol [28], and that the cardioprotective effects of EVOO observed in hypercholesterolemic subjects could be contributed by the increased populations of bifidobacteria and in phenolic microbial metabolites with antioxidant properties [29]. HT and its metabolites accumulate in a dose-dependent manner in the liver, kidney and brain [30]. Auñon-Calles et al. [31] performed a toxicological evaluation of HT in rats, assessing doses of 5, 50 and 500 mg/kg/d for 13 weeks, which did not induce toxicologically relevant changes at any dose, proposing a no observed adverse effect level (NOAEL) of 500 mg/kg/d. Thus, HT consumption is safe even at high doses, without having genotoxic or mutagenic actions in vitro [32]. HT is mostly absorbed at the intestinal level, and is eliminated through urine, reaching a peak at 0-4 h after ingestion [33]. Miró-Casas et al. [34] investigated the absorption of tyrosol and HT after consumption of either a moderate dose (50 mL once) or sustained doses (25 mL/d for a week) of EVOO in humans. After both dosages, the urinary HT levels increased similarly with an estimated half-life of approximately 8 h, suggesting that urinary levels of HT are not a reliable biomarker of EVOO intake [34].

Antioxidant Effect
The antioxidant properties of HT are associated with its high degree of absorption and bioavailability, which is essential for its pharmacokinetic properties [35], and with its catecholic structure that allows (1) free radical scavenger and radical chain breakdown actions; and (2) a metal chelation feature reducing free-radical generation with production of very stable derivatives [36]. The assessment of the effects of a long-term diet (8 weeks) supplemented with HT (0.03 gm %) in C57BL/6 mice showed lower levels of plasma cholesterol and circulating leptin, in addition to a decreased glutathione disulfide (GSSG)/reduced glutathione (GSH) ratio in adipocytes [37]. Moreover, the HT-supplemented diet modulated gene expression of pathways related to oxidative stress (OS) in adipose tissue, particularly those of GSH and related enzymes [37]. The study of the effect of HT in rats with breast cancer and doxorubicin-induced cardiotoxicity revealed improvement in the drug-induced cardiac alterations by reducing mitochondrial damage and OS, suggesting that HT could enhance the electron transport chain [38]. Under OS conditions, the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) is activated, a transcriptional regulator that controls the expression of phase II enzymes and of genes involved in oxidative defense. Mechanistically, Nrf2 is inactive in the cytoplasm due to the formation of a complex with its inhibitor Keap-1 [39]. Following the release of Keap-1 from complex induced by OS or electrophilic agents, Nrf2 is translocated to the nucleus, where it binds to promoters containing antioxidant response elements (AREs), resulting in the transactivation of the respective genes for phase II and antioxidant enzymes [39]. The effect of HT on pigment epithelial cells of the retina submitted to OS induced by acrolein indicated that HT was able to increase the translocation of Nrf2 to the nucleus, which resulted in increased protein expression and activity of γ-glutamyl cysteine ligase (GCL), NAD(P)H quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), GSH reductase (GR), GSH peroxidase (GPx) and catalase (CAT) [40]. These results demonstrate that HT confers additional indirect antioxidant protection in addition to its already known direct antioxidant properties [40]. In addition, porcine pulmonary artery endothelial cells exposed to HT in the presence of H 2 O 2 exhibited prevention of the intracellular increase of reactive oxygen species (ROS) levels through increased CAT mRNA and enhanced expression and activity of the transcription factor forkhead transcription factor 3a (FOXO3a), both at cytosolic and nuclear levels [41]. Activation of FOXO3a directly increased the expression of antioxidant enzymes, through the phosphorylation of AMP-activated protein kinase (AMPK) that translocates FOXO3a to the nucleus. Therefore, these findings demonstrate that HT positively regulates the antioxidant defense system in the cells studied [41] (Table 1).

Anti-Cancerogenic Effect
The antioxidant, antiproliferative, pro-apoptotic, and anti-inflammatory properties attributed to HT suggest an anti-carcinogenic potential of the polyphenol [8], which are linked to the prevention of some types of cancer through different mechanisms. In the study of Warleta et al. [47], HT contribute to the preventive activity against breast cancer attributed to EVOO, through the reduction of OS and the protection of DNA oxidation in normal cells of the breast at physiological concentrations. Furthermore, the antitumor effect of HT could be exerted through down-regulation of epidermal growth factor receptor (EGFR) expression, as shown in human colon tumor cells in which HT induced ubiquitination and degradation of the receptor, effects that are associated with a decrease in cell proliferation, thus complying with its anti-cancerous effect [48]. Besides, Notarnicola et al. [49] studied the effect of HT on human colorectal cancer cells and found that HT had antiproliferative and pro-apoptotic effects. The inhibitory effects of cell growth are mediated through inhibition of fatty acid synthase (FAS), which has been found to be directly related to the aggressiveness of the tumor [49]. Moreover, HT has shown to inhibit the cell proliferation and induce apoptotic cell death in breast cancer cells, having a cytotoxic effect on MCF-7 cells in a dose dependent manner [50] (Table 1).

Effects on Endothelial and Vascular Function
In addition to the properties already discussed, HT has a beneficial effect on endothelial and vascular function [51,52]. Evaluation of endothelial function in a double-blind, randomized controlled clinical trial was performed in 13 patients with pre-hypertension and grade 1 hypertension who were given either functional olive oil (961 mg/kg phenolic compounds) or regular olive oil (289 mg/kg phenolic compounds) [51]. Data shown revealed that after 5 h of the intake, the functional olive oil showed significant differences with respect to the regular olive oil, namely, increased reactive hyperemia and plasma C max of HT sulfate (metabolite), and decreased oxidized LDL, TG and glucose levels, with greater benefits for endothelial function than olive oil with a lower phenolic content [51]. Furthermore, a randomized controlled double-blind crossover trial in 18 apparently healthy volunteers who consumed one capsule of olive leaf extract (51 mg oleuropein, 10 mg HT) or control capsule in a single ingestion, revealed that extract consumption induced significant diminution in arterial stiffness, digital pulse volume, and IL-8 levels compared to control, with increased excretion of metabolites derived from oleuropein and HT [52]. Hence, phenolic compounds of olive leaf extract positively modulated vascular function and inflammation [50]. Additionally, HT and its metabolites significantly decreased secretion of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in human aortic endothelial cells (HAEC) at physiological concentrations (1, 2, 5 and 10 µM) co-incubated with TNF-α for 18 and 24 h [11], concomitantly with reduction in markers of endothelial dysfunction that may contribute to the prevention of atherosclerosis [11]. Finally, the effect of HT in both free and complex forms (SEC) on the aortic and cardiac proteome of female Wistar rats, revealed a higher free HT content after supplementation with both forms of HT, together with the detection of the phase-II metabolite HT-3-O-sulfate, which appeared only after the diet with HT [53]. The proteome of both heart and aorta changed after supplementation with EVOO's phenolic compounds, the difference being greater when supplementation was with SEC, which may be due to the higher HT content in the tissue after the SEC diet, with a negative regulation of proteins involved in blood vessel occlusion, endothelial cell proliferation, pro-inflammatory processes, and cell death [53] (Table 1).

Anti-Steatotic Effect
Supplementation with oleuropein, the HT precursor compound, has a protective effect in reversing the negative effects induced by a HFD in mice, regularizing hepatic steatosis, hyperlipidemia, and increased body weight and liver weight [54]. Also, the study of the effect of the different phenolic compounds (HT, tyrosol, and oleuropein) present in EVOO in rat hepatocytes revealed a decrease in de novo synthesis of fatty acids (FAs), cholesterol, and TG [55]. Moreover, in the study by Pirozzi et al. [42] using HT supplementation (10 mg/kg/d) in rats with HFD-induced NAFLD, protective effects against liver damage caused by HFD were observed, including an anti-inflammatory action, improved glucose tolerance, increased insulin sensitivity, decreased OS, and reduced plasma cholesterol, increasing the hepatic levels of PPAR-α (Table 1).

Effects on Endoplasmic Reticulum (ER) Stress and Autophagy
OS and high levels of saturated FAs induce protein misfolding or unfolding constituting an ER stress condition triggering the unfolded protein response (UPR) to recover protein homeostasis and stability [56]. However, if the latter feature is not reached, pathological responses can be attained, including several liver diseases [56]. It has been demonstrated that HT has an effect on regulating cell ER stress and autophagy. In fact, Giordano et al. [13] studied human hepatocarcinoma HepG2 cells with ER stress induced by tunicamycin and treated with HT at concentrations of 1 and 5 µM. At both HT concentrations, two markers of ER stress involved in the UPR, namely, ER CCAAT-enhancer-binding protein homologous protein (CHOP) and binding immunoglobulin protein (BiP), were significantly reduced over control values, as well as BiP's transcription factor activating transcription factor 6α (ATF6α) that was reduced with the higher HT dose [13]. Furthermore, HT at both doses restored levels of B-cell lymphoma 2 (Bcl2), an anti-apoptotic protein, improving ER homeostasis [13]. Besides, the incubation of HepG2 cells with HT for 24 h (1 and 5 µM) decreased mRNA levels of ER CHOP and BiP, thus evidencing the preventive activity of HT against the ER stress in the hepatocyte [57].
Autophagy has been identified as a protective process during the development of osteoarthritis, which is critical for the survival of chondrocytes. Recently, Cetrullo et al. [14] studied the possibility of modulating autophagy in chondrocytes through the treatment with HT, by assessment of DNA damage and cell death in human C-28/12 chondrocytes undergoing primary osteoarthritis when exposed to hydrogen peroxide. HT increased autophagy markers and protected chondrocytes from DNA damage and cell death induced by OS [14]. Furthermore, in rats submitted to severe exercise for an 8-week period, supplementation with 25 mg/kg/day of HT reduced OS and thereby mitochondrial impairment, with reduction of muscular atrophy induced by autophagy and mitochondrial fission [58] (Table 1).

Effects on Mitochondrial Function
HT is known to improve mitochondrial function either by increasing the activity and/or expression of mitochondrial complexes I, II, III, IV, and V [15,38,59,60]. The 3T3-L1 adipocytes subjected to HT in the concentration range of 0.1-10 µM stimulated the expression and activity of peroxisome-proliferator activator receptor γ co-activator 1α (PGC1α), which promotes mitochondrial function by increasing the expression and activity of all complexes involved in the respiratory chain and oxidative phosphorylation [60]. Accordingly, HT was shown to improve the mitochondrial electron transport chain in rats with cardiotoxicity induced by doxorubicin by increasing the levels of complex-II and complex-III that partially restore the respiratory chain activity [38]. This effect was also observed in diabetic mice after a 2-month treatment with HT at doses of 10 and 50 mg/kg, where the expression levels of complexes I, II, and IV and the activity of complex I were increased in the brain, improving mitochondrial function [15]. In agreement with these views, HT also stimulates mitochondrial biogenesis in ARPE-19 human retinal pigment epithelial cells subjected to OS, through enhancement in the expression of PGC1α that leads to an increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 (UCP2) and mitochondrial complexes [40] (Table 1).

Other Effects
In addition to the aforementioned effects, HT may have an effect at the bone level, decreasing bone loss in femur and formation of multinucleated osteoclasts. These actions were ascribed to the antioxidant effect of HT, taking into account that high levels of ROS suppress bone metabolism and increase the formation of osteoclasts, thus preventing bone mass loss [61].
Another interesting effect of HT is related to the management of pain related to inflammation, as shown by Takeda et al. [62] in a double-blind controlled study in which 25 adults with early osteoarthritis of the knee were given one tablet of 50 mg/day of olive extract (10 mg HT) or placebo for 4 weeks. A significant diminution in pain was observed in the group after 2 and 4 weeks of HT treatment, pointing to HT as effective treatment to reduce pain in patients with gonarthrosis [62]. Table 1. Summary of hydroxytyrosol (HT) properties in different study models. GCL: γ-glutamyl cysteine ligase; GR: GSH reductase; HO-1: heme oxygenase-1; NQO1: NAD(P)H quinone oxidoreductase; CAT: catalase; ROS: reactive oxygen species; FOXO3a: forkhead transcription factor 3a; NO: nitric oxide; PGE2: prostaglandin E2; iNOS: inducible NO synthase; PGES: prostaglandin E2 synthase; NAFLD: non-alcoholic fatty liver disease; HFD: high-fat diet; PPAR-α: peroxisome proliferator-activated receptor-α; GSH: glutathione-ethyl ester; CHOP: CCAAT-enhancer-binding protein homologous protein; BiP: binding immunoglobulin protein; hECs: human umbilical vein endothelial cells; IL: interleukin; EGFR: epidermal growth factor receptor; LDL: low density lipoproteins; UPR: unfolded protein response.

Properties
Reference Rats with NAFLD were divided into three groups: Control diet, HFD and HFD + HT (10 mg/kg/d) for 5 weeks.
Increased levels of PPAR-α. Reduced the expression of pro-inflammatory cytokines TNF-α and IL-6. HT was detected in heart tissue mainly in its free form after supplementation with HT or SEC. Supplementation changed heart and aorta proteome. These proteins are related to cardiovascular function, improving endothelial and vascular function. Reestablishment of cognitive alterations provoked by prenatal stress is a further beneficial effect of HT (10 or 50 mg/kg/d), as shown in rats with alterations of learning capacity and memory performance by improving the alterations of the neural proteins such as brain-derived neurotrophic factor (BDNF), growth associated protein 43 (GAP43), synaptophysin, NMDAR1, NMDANR2A, NMDANR2B, while decreasing OS [63].

Conclusions
Data discussed on this review show that HT is a polyphenol which has not only powerful antioxidant activity but also possesses other cytoprotective properties with beneficial health effects. These include anti-inflammatory, anticancer, and anti-steatotic effects, with improvement of endothelial and vascular function, endoplasmic reticulum stress, autophagy, and mitochondrial function. Therefore, HT has a positive outcome on molecular and cellular alterations commonly associated with NCDs, becoming a potential therapeutic agent for the prevention and/or treatment of these chronic diseases. Interestingly, HT has been studied in cellular, animal and human models that demonstrate the safety of the polyphenol; nevertheless, more studies in humans are needed to determine the therapeutic doses. Main beneficial properties of HT and the metabolic pathways involved in these effects are summarized in Figure 1.