Effects of Olive Oil Phenolic Compounds on Inflammation in the Prevention and Treatment of Coronary Artery Disease

Coronary artery disease (CAD) is responsible for more than 7 million deaths worldwide. In the early stages of the development of atherosclerotic plaques, cardiovascular risk factors stimulate vascular endothelial cells, initiating an inflammatory process, fundamental in the pathogenesis of CAD. The inclusion of potentially cardioprotective foods, such as olive oil, to the diet, may aid in the control of these risk factors, and in the reduction of cytokines and inflammatory markers. The present review aims to address the interaction between phenolic compounds present in olive oil, and inflammation, in the prevention and treatment of CAD. In vitro and in vivo studies suggest that phenolic compounds, such as hydroxytyrosol, tyrosol, and their secoiridoid derivatives, may reduce the expression of adhesion molecules and consequent migration of immune cells, modify the signaling cascade and the transcription network (blocking the signal and expression of the nuclear factor kappa B), inhibit the action of enzymes responsible for the production of eicosanoids, and consequently, decrease circulating levels of inflammatory markers. Daily consumption of olive oil seems to modulate cytokines and inflammatory markers related to CAD in individuals at risk for cardiovascular diseases. However, clinical studies that have evaluated the effects of olive oil and its phenolic compounds on individuals with CAD are still scarce.


Introduction
Cardiovascular diseases account for 17.7 million deaths every year, and are the leading cause of death worldwide. Of these deaths, it is estimated that 7.4 million are caused by coronary artery disease (CAD) [1]. More than 90% of the events related to CAD-acute myocardial infarction and death-occur in individuals with at least one of the risk factors for CAD [2,3]. Therefore, primary and secondary prevention strategies aim to reduce traditional risk factors (diabetes mellitus, hypertension, dyslipidemia, obesity) and lifestyle-related disorders, such as inadequate diet, smoking, physical inactivity, and abusive use of alcohol [3,4]. In the early stages of development of atherosclerotic plaques, risk factors stimulate vascular endothelial cells to express chemotactic and adhesion molecules, initiating the inflammatory process, fundamental in the pathogenesis of CAD [5][6][7].
In the context of a dietary pattern considered to be healthy, the inclusion of potentially cardioprotective foods, including sources of unsaturated fats and phenolic compounds, such as olive oil, may modulate the concentration of pro-inflammatory cytokines and markers of inflammation [8,9], and assist in the control of modifiable risk factors, such as diabetes mellitus [8], hypertension [10], concentrations of polyunsaturated fatty acids (linoleic fatty acid: 3.5-21%) and saturated fatty acids (palmitic fatty acid: 7.5-20%, stearic fatty acid: 0.5-5%) [16].

Phenolic Compounds of Olive Oil
Phenolic compounds are secondary plant metabolites synthesized during normal development or in stressful situations [74]. In virgin olive oils, the synthesis of these compounds occurs when the olive fruits are crushed during the industrial process to obtain the olive oil. Thus, the presence of phenolic compounds is directly related to glycosides initially present in the fruit tissue, and the activity of hydrolytic and oxidative enzymes [75]. In terms of chemical structure, they have at least one hydroxyl attached to an aromatic ring [74].
The phenolic compounds are mainly responsible for the organoleptic characteristics (aroma and flavor) [82,83] and oxidative stability of the olive oil [20,84]. Several factors influence their concentration: plant variety [77,84,85], environmental factors [86,87], olive storage and maturation conditions [88,89], oil extraction conditions [90,91] and commercial storage of the final product [92]. The mean phenolic content in EVOO is 551.4 mg/kg [93,94] (ranging from 50-800 mg/kg) [95]; in fine oil, it is 206.7 mg/kg [94], and ROO has the lowest indices, 198-62.0 mg/kg [17,94]. The phenolic content of EVOO has a wide variation. Montano et al., evaluated eight varieties of plants grown at extreme altitudes, and found that Cornicabra presented the highest concentrations (mean of 632.6 mg/kg) while Arbequina had the lowest values (200.2 mg/kg) [84]. Baiano et al., evaluated the effect of planting location on the content of phenolic compounds and found significant variations, from 195.2 to 32.3 mg/kg for the same type of crop [96].
The levels of phenolic acids, flavonoids and hydroxy-isocromans are relatively low in virgin olive oil. Phenolic acids were the first compounds identified in the olive oil; at least 14 have already been described and are generally present in amounts of less than 1 mg/kg [99][100][101]. Luteolin and apigenin are the two flavonoids found in the highest concentration in olive oil; however, this is lower than that of other phenolic compounds [102]. Bianco et al., identified two hydroxy-isocromans [1-phenyl-6,7-dihydroxy-isochroman and 1-(3 -methoxy-4 -hydroxy)-6,7-dihydroxy-isochroman] in commercial virgins olive oils formed from the HT reaction with benzaldehyde and vanillin, respectively [103]. The average concentrations of the phenolic compounds found in the different types of olive oil are presented in Table 1. After ingestion, the phenolic compounds are metabolized through two phases: hydrolysis (phase 1) that occurs in the stomach and small intestine (where most are promptly absorbed); and conjugation (phase 2), in the small intestine and mainly in the liver-this process basically involves methylation, sulfation, and glucuronidation [104][105][106][107][108]. Oleuropein, specifically, does not follow the same route, as it is degraded by the colon microbiota to HT, which can then be absorbed [106].
In humans, the absorption of phenolic compounds of virgin oils, especially HT and tyrosol, is dose-dependent, and these compounds are excreted in the urine in the conjugated form [109]. Vissers et al., studied healthy subjects (one group submitted to ileostomy and another with intact colon) and estimated that the absorption rate would be at least 55-66% of the dose of ingested olive oil [110]. The absorption of HT also depends on the food matrix: higher percentages of urinary excretion were observed after ingestion of this phenolic compound as a natural component of virgin olive oils (42% of ingested HT), compared to ROO (23%) or to yogurt (5.8%) [111].

Phenolic Compounds of Olive Oil and Inflammation: In Vitro and Animal Model Studies
Evidence has shown that regular consumption of foods rich in phenolic compounds may decrease the risk for the development of chronic diseases [112,113], mainly due to their ability to modulate low-grade inflammation [114]. The mechanisms by which these compounds may exert an anti-inflammatory effect, specifically on cardiovascular diseases, involves: (1) antioxidant activity; (2) modification of the signaling cascade and transcription network (blocking the signaling and expression of nuclear factor kappa B); (3) decrease of the adhesion of immune cells (T lymphocytes and monocytes) to the endothelium; and (4) improvement of endothelial dysfunction [114]. Due to the complex chemical composition of the oil, particularly the EVOO, we tried to elucidate which phenols would be more involved in these mechanisms.
At nutrient-relevant concentrations (<10 µM), close to those found in human fluids following EVOO intake [118], HT inhibited the production of ON and PGE2, but had no effect on the expression of inducible nitric oxide synthase, TNF-α or IL -1β, in granulocytes and monocytes [119]. In peripheral blood mononuclear cells, HT culture reduced MMP-9 concentrations, and inhibited PGE2 production and COX-2 expression, without affecting COX-1 [120]. In endothelial cell culture, HT and oleuropein phenols reduced the inflammatory process in angiogenesis through the inhibition of COX-2 and MMP-9 [121], suggesting that the mechanism of action of HT on the inflammatory process is similar to that of nonsteroidal anti-inflammatory drugs (NSAIDs) [122,123]  HT significantly reduced COX-2 expression (mRNA and protein level) and release of PGE2, the latter being dose-dependent. HT also increased the production of TNF-α by monocytes. COX-2 mRNA was also inhibited by secoiridoid derivatives [123]. Tyrosol and hydroxyl-isocroman compounds also have an effect on AA. In macrophage culture (RAW 264.7) stimulated by phorbol-12-myristate-13-acetate esters, tyrosol (≥100 µM) inhibited the release of AA and synthesis of metabolites (PGE2 and LTB4) induced by exogenous oxygen-reactive species. This further reduced the release of NO induced by phorbol-12-myristate-13-acetate stimulus [124]. 1-Phenyl-6,7-dihydroxy-isochroman significantly inhibited the production of TXA2 and PGE2, and of TNF-α in LPS-primed human monocytes; this action was mediated by the suppression of NF-κB activation, leading to a decrease in COX-2 synthesis [125].
In vitro results on AA have also been observed in vivo [126,127]. In healthy subjects, consumption of a meal (150 g of tomatoes) with EVOO (607 mg/kg phenolic content, 300 mg/kg of HT derivatives) reduced the inflammatory markers TXB2 and LTB4 after 2 and 6 h [127].
Oleocanthal is another phenol with anti-inflammatory effects similar to those of NSAIDs. Oleocanthal was able to induce dose-dependent inhibition of COX-1 and COX-2 inflammatory enzymes in vitro, and had higher potential at equimolar concentrations when compared with ibuprofen; 25 mM of oleocanthal inhibited the COX enzyme activity by 41-57% while 25 mM ibuprofen inhibited it by 13-18% [128,129].
In addition to inhibiting the expression of endothelial adhesion molecules (VCAM-1) [115], oleuropein may reduce the inflammatory response by inhibiting TLR and the signaling of mitogen-activated protein kinases in a zebrafish model [130]. Oleuropein administration inhibited proliferation of vascular smooth muscle cells in vitro [131].
Male Sprague Dawly rats were allocated into five groups: (1) the sham group previously treated with vehicle; (2) the acute myocardial infarction group previously treated with vehicle (1 mL of distilled water/day); (3) three acute myocardial infarction groups that received different concentrations of oleuropein (10, 20, and 30 mg/kg) for 7 days before acute myocardial infarction. The groups receiving previous treatment with oleuropein (20 and 30 mg/kg) had lower IL-1β and TNF-α values, when compared to the group with acute myocardial infarction that received only the vehicle [132].
Hyperhomocysteinemia has been associated with a high risk of cardiovascular disease because it increases vascular endothelial adhesiveness [134]. Phenolic compounds, such as tyrosol and p-coumaric acid, may decrease homocysteine-induced cell adhesion and ICAM-1 expression; however, they do not reduce the expression of ICAM-1 induced by TNF-α, demonstrating the potential selective effect of these compounds [135].
Recent studies have suggested the importance of the concomitant presence of non-alcoholic fatty liver disease and systemic inflammation (elevated CRP) in the development of atherosclerosis [136]. The effects of HT were investigated using a high-fat diet in an animal model of insulin resistance and non-alcoholic fatty liver disease. The rats were divided into three groups: (1) control diet (10.5% of lipids); (2) high fat diet (58% lipids); (3) high-fat diet + HT (10 mg/kg/day). After 6 weeks, HT attenuated, significantly, the metabolic impairment induced by the high-fat diet. It had also reduced hepatic inflammation and nitrosative−oxidative stress through decreased protein nitrosylation, lipid peroxidation, and production of oxygen-reactive species [137].

Studies on Olive Oil Phenolic Compounds and Inflammation in Individuals at Risk for CAD
Studies on primary prevention have demonstrated the association between consumption of EVOO, naturally rich in phenolic compounds, and reduced risk of major cardiovascular events in patients at high risk for developing cardiovascular diseases [15,138]. Such an effect may be mediated by the control of modifiable risk factors and potential anti-inflammatory mechanisms of olive oil phenols [9].
Cardiovascular risk factors, such as hypertension, dyslipidemia, diabetes mellitus, and smoking, cause endothelial dysfunction, contributing to the onset of the inflammatory process in atherosclerosis [5]. Obesity and metabolic syndrome are characterized by a chronic and low-grade inflammatory state, increasing the contribution of inflammation to the genesis and evolution of CAD [139,140]. Thus, nutritional strategies and interventions that minimize the inflammatory process in individuals at high cardiovascular risk would help in the primary prevention of CAD.
Studies have shown the anti-inflammatory effects of virgin olive oils supplementation at different stages of development of atherosclerosis. In individuals with endothelial dysfunction, Widmer et al. evaluated the effects of VOO (340 mg/kg total polyphenols) and VOO enriched with epigallocatechin 3-gallate (EGCG), a catechin naturally found in green tea (VOO + EGCG: 300 mg/kg total polyphenols + 280 mg/Kg EGCG) on inflammatory mediators. The main difference in phenolic composition between the two oils was the secoiridoid content (VOO 61% vs. VOO + EGCG 48%), whereas the lignan content was similar (VOO 33% vs. VOO + EGCG 37%). The authors did not find differences between the VOO and VOO + EGCG groups, but concluded that the supplementation of olive oil to the usual diet of the participants for 4 months had a positive effect on the reduction of cell adhesion molecules (sICAM-1), platelets, monocytes, and lymphocytes involved in the inflammatory process [141].
Two randomized crossover clinical trials evaluated the anti-inflammatory effects of VOO in patients with mild dyslipidemia (no drug treatment). In the VOLOS study (Virgin Olive Oil Study), participants underwent two interventions for 7 weeks each, 40 mL/day of EVOO containing 166 mg/L of HT (free and esterified in oleuropein), and ROO with only 2 mg/L. The predominant fatty acids in the two samples were oleic (70.9% EVOO vs. 72.7% ROO), palmitic (11.5% vs. 10.7%), and linoleic (8.5% vs. 7.6%). The results demonstrated a 20% reduction in serum TXB2 concentration only in the EVOO group containing HT [127]. The second trial compared MeDiet supplemented with three different dietary sources of fats (35-50 g VOO, 40-65 g nuts, and 50-75 g almonds), accounting for 40% of the dietary lipid content (20% of the total energy value). Interventions were performed for 4 weeks, and although VOO contained the highest total polyphenol content (343 mg/kg vs. 13 mg/kg of walnuts and 11 mg/kg of almonds), there was no significant difference in serum levels of CRP and adhesion molecules (sVCAM-1 and sICAM-1) after follow-up [144].
A high-fat meal, in addition to promoting postprandial hypertriglyceridemia, stimulates the intestinal absorption of endotoxins such as LPS. This endotoxin is able to bind TLR4, which in turn triggers various signaling pathways, including NF-κB, leading to transcription of genes related to the inflammatory response [146,147]. With the objective of investigating the mechanisms by which the VOO polyphenols reduce the postprandial inflammatory response, Camargo et al., administered a VOO-rich meal with different concentrations of phenolic compounds (high 398 mg/kg, intermediate 149 mg/kg, and low 70 mg/kg) to subjects with metabolic syndrome. After 4 h, high concentrations of VOO inhibited NF-κB and decreased the expression of IL-1β (vs. intermediate), and IL-6 (vs. low/intermediate). The VOO-rich meal with low phenol concentration promoted increased serum levels of IL-6, as well as the protein NF-κB subunit p65, TLR4, and postprandial LPS. These results suggest that the ingestion of phenolic VOO reduces postprandial inflammation mainly by decreasing the activation of NF-κB, secondary to the reduction of LPS absorption [148].
In addition to the postprandial anti-inflammatory effect of VOO, the ingestion of a EVOO-rich meal (72% of the caloric intake) (1125 mg/kg total polyphenols and 350 mg/kg tocopherols) resulted in lower elevations of adhesion molecules (ICAM-1 and VCAM-1) in hypertriglyceridemic men, and in healthy subjects after 8 h, and compared to a high ROO breakfast [150]. Table 2 shows the main randomized clinical trials that evaluated the effect of different concentrations of olive oil phenolic compounds on inflammation markers in patients with cardiovascular risk.

Anti-Inflammatory Effects of Olive Oil Phenolic Compounds in Patients with CAD
The anti-inflammatory effects of VOO phenolic compounds have been extensively investigated in vitro, in animal models and in clinical trials involving subjects at risk for cardiovascular diseases.
However, few studies have tested its effects specifically in CAD patients. Fitó et al. evaluated the effect of daily supplementation of 50 mL/day of VOO and ROO in patients with stable CAD for two periods of 3 weeks. The oils used in the study had similar monounsaturated fatty acid content and differed mainly in phenolic content (161 vs. 14.7 mg/kg total; 0.15 vs. 0 mg of β-carotene; 8.73 vs. 5.99 mg of α-tocopherol; 6.53 vs. 0.62 mg of caffeic acid equivalents; respectively). Serum concentrations of sICAM-1, sVCAM-1, CRP, and IL-6 were evaluated. The effects on proinflammatory cytokines (IL-6) and inflammation markers (CRP) were observed only in the VOO group, with a reduction of 0.166mg/dL (95% CI −0.261-0.071; p = 0.002) and 0.063 mg/dL (95% CI −0.119-0.007, p = 0.024), respectively [18] ( Table 2).
The main anti-inflammatory effects of olive oil phenolic compounds are briefly presented in Figure 1.

Anti-Inflammatory Effects of Olive Oil Phenolic Compounds in Patients with CAD
The anti-inflammatory effects of VOO phenolic compounds have been extensively investigated in vitro, in animal models and in clinical trials involving subjects at risk for cardiovascular diseases.
However, few studies have tested its effects specifically in CAD patients. Fitó et al. evaluated the effect of daily supplementation of 50 mL/day of VOO and ROO in patients with stable CAD for two periods of 3 weeks. The oils used in the study had similar monounsaturated fatty acid content and differed mainly in phenolic content (161 vs. 14.7 mg/kg total; 0.15 vs. 0 mg of β-carotene; 8.73 vs. 5.99 mg of α-tocopherol; 6.53 vs. 0.62 mg of caffeic acid equivalents; respectively). Serum concentrations of sICAM-1, sVCAM-1, CRP, and IL-6 were evaluated. The effects on proinflammatory cytokines (IL-6) and inflammation markers (CRP) were observed only in the VOO group, with a reduction of 0.166mg/dL (95% CI −0.261-0.071; p = 0.002) and 0.063 mg/dL (95% CI −0.119-0.007, p = 0.024), respectively [18] ( Table 2).
The main anti-inflammatory effects of olive oil phenolic compounds are briefly presented in Figure 1.

Conclusions
The consumption of VOOs rich in phenolic compounds seems to favorably modulate inflammation, which contributes to the development and progression of CAD. In vitro and animal model studies have suggested mechanisms of action of these compounds in inflammatory activity at the cardiovascular level, including effects on the arachidonic acid cascade and on signaling pathways and receptors, improvement of vascular function, and reduction of adhesion molecules and chemokines. Moreover, these studies allow us to evaluate the isolated effects of VOO phenolic compounds.
Clinical trials conducted in individuals at risk for the development of cardiovascular diseases show positive effects of daily intake of different amounts of olive oil on inflammatory markers. The main findings of the randomized clinical trials included in this review reinforce the results found in in vitro and animal models. In humans, these effects were observed at the cell level (immune cells) and in inflammatory markers. A limitation for the discussion of the results is the great variation in the phenolic content of different types of VOOs. Furthermore, dietary supplementation with olive oil is associated with changes in dietary patterns as a whole, which may improve the inflammatory profile of patients at risk for CAD. It is also important to consider that dietary patterns, like MeDiet, include other sources of phenolic compounds.
As we have seen, the effects of olive oil and/or its phenolic compounds specifically on individuals with established CAD are still scarce. In this sense, more clinical trials, preferably long-term studies, are necessary to evaluate and confirm the beneficial effects of the phenolic compounds present in the olive oil on the inflammatory process, both in the prevention and treatment of CAD.
Author Contributions: Priscilla Azambuja Lopes de Souza wrote the manuscript, Aline Marcadenti and Vera Lúcia Portal reviewed the manuscript. All authors read and approved the final version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.