*2.5. Toxicity*

EVOO consumption—of course—is safe and the only drawback of excessive use is heightened caloric intake. In light of the use of olive biophenols as nutraceuticals or functional foods ingredients, international bodies require proof of absence of toxicity. HT has been tested in a variety of models and a NOAEL of 500 mg/kg/d has been proposed [36,37]. The recent Novel Food (NF) status granted to HT outlines that "Taking into account that the anticipated daily intake of the NF would be in the range of or even less than the exposure of HT from the consumption of olive oils and olives, which has not been associated with adverse effects, and considering the similar kinetics of HT in rats and humans, [ . . . ..] the Margin of Exposure for the NF at the intended uses and use levels is sufficient for the target population. The EFSA Panel concludes that the novel food, HT, is safe under the proposed uses and use levels" [38]. Finally, HT is generally recognized as safe (GRAS) in the USA and, in summary, there is no clear evidence of toxicity even at high doses.

In any event, caution should be exerted when using any kind of supplements/functional foods in the absence of clear health benefits and as a replacement for a healthful and balanced diet.

#### **3. Molecular Insights into Mechanisms of Action**

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important inflammatory effectors contributing to the elimination of invading pathogens and supporting tissue repair, accelerating the resolution of inflammation. However, ROS/RNS can trigger the generation of inflammatory initiators (e.g., inflammatory cytokines) and damage macromolecules such as lipids, proteins, and nucleic acids. This damage eventually leads to cell death and tissue deterioration [39], which stimulates the development of several diseases, including those of a neurodegenerative nature [40], atherosclerosis [41], metabolic syndrome (MS) [42], type 2 diabetes (T2DM) [43], liver diseases [44], and cancer [45].

Numerous studies performed with animal and cell models sugges<sup>t</sup> that biophenol intake may be beneficial for the prevention and adjuvant treatment of such diseases [46]. In particular, olive oil and its phenolic compounds exert beneficial health effects that encompass anti-inflammatory and antioxidant (direct or indirect) mechanisms, as reflected in many reviews [47–51]. We will briefly review recent evidence arising from studies carried out in the most recent decade (especially in the last lustrum), pointing to the protective effects of olive oil and its phenolic compounds in the context of neurodegenerative disease, CVD, liver disease, cancer, and rheumatic disease.

#### **4. Cardiovascular Disease, Metabolic Syndrome, Type 2 Diabetes**

A possible link between inflammation, endothelial dysfunction, and CVD is increased oxidative stress (now called redox code [52]) [53]. Inflammation participates in atherosclerosis from its inception and development to its ultimate endpoint, thrombotic complications. Oxidative stress has been identified as critical in most of the key steps in the pathophysiology of atherosclerosis [54]. Endothelial dysfunction involves deviations in the regulation of vascular tone and vascular smooth muscle growth, monocyte adhesion, platelet function, and fibrinolytic activity, which are critical in the development and progression of atherosclerosis and its complications. Reduction of nitric oxide (NO) availability is a main alteration responsible for endothelial dysfunction [55]. Regular consumption of high-fat and high-carbohydrate diets promote increased oxidative stress and inflammation that can result in a host of inter-related metabolic abnormalities and endothelial dysfunction [56,57].

In vitro, EVOO phenolic-rich extracts counteract oxidative stress. They decrease ROS production and levels of malondialdehyde (MDA) [58], downregulate inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression, reduce MAPK (JNK, p38) phosphorylation and nuclear factor κB (NF-κB) translocation [59,60], and reduce VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity [61]. They also decrease the expression of selective NADPH oxidase subunits. In rat hearts, diet supplementation with oil or oil products containing EVOO-polar biophenols attenuated a hypercholesterolemia-induced increase in MDA and TNFα [62], and HT administration improved doxorubicin-enhanced cardiac disturbances, probably by affecting the mitochondrial electron transport chain [63]. Regarding human studies, in healthy subjects, supplementation with olive oil, either low or high in phenolics (18 vs. 286 mg CAE/kg, respectively), improved the proteomic coronary artery disease (CAD) score compared with baseline [64]. Positive effects were also seen in another study with healthy subjects, in this case, in a dose-dependent manner, since consumption of an olive oil with high phenolic content (366 mg/kg) decreased systolic blood pressure as compared to low content (2.7 mg/kg) and to pre-intervention values, and it downregulated the expression of genes related to the renin–angiotensin–aldosterone system in peripheral blood mononuclear cells (PBMCs) [65]. Additionally, in an acute intake study, healthy participants ingested functional virgin olive oils (FVOOs) differing in phenolic content (250, 500, and 750 ppm) and in a sustained intake study, hypercholesterolemic participants ingested a control VOO (80 ppm) or FVOO (500 ppm) [66]. Acute and sustained intake of VOO and FVOO resulted in changes associated with diminished atherosclerotic activity as shown by decreased PON1 protein and increased PON1-associated specific activities [67]. Furthermore, mechanistic studies revealed that the intake of isolated phenolic compounds modulated mitogen-activated protein kinases and peroxisome proliferator-activated receptors regulating PON synthesis [66]. With hypercholesterolemic subjects, using 3-week supplementation with VOO (either enriched or not in its own phenolic compounds), 15 HDL-associated differently expressed proteins were found, mainly involved in pathways of LXR/RXR activation, acute phase response, and atherosclerosis [68]. Recently, it was reported that the ingestion of an olive pomace-enriched biscuit (olive pomace being a waste product of olive oil production containing biophenols and fibers (~17 mg/100 g of HT and its

derivatives)) by hypercholesterolemic subjects led to increased levels of homovanillic acid and 3,4-dihydroxyphenylacetic acid (possibly involved in reducing oxidative LDL cholesterol) as compared to an isoenergetic control. No statistically significant changes were found in either ox-LDL or urinary isoprostane [69]. In this context, the intake of a virgin oil enriched in phenolic compounds (500 mg/kg) led to an increase in HDL antioxidant compounds in hypercholesterolemic volunteers while increasing the levels of fecal HT and dihydroxyphenylacetic acids [70], as compared with pre-intervention values and a lower-phenolic VOO (80 mg/kg) [71]. Of note, in MS patients the consumption of a high-phenol (398 ppm) VOO-based breakfast, as compared to low (70 ppm) or intermediate (149 ppm) phenol content, limited the increase of postprandial lipopolysaccharide (LPS) plasma levels, and reduced TLR4 and SOCS3 proteins, the activation of NF-κB, and postprandial gene expression of IL6, IL1B, and CXCL1 in PBMCs [60].

With regard to studies where olive oil phenolic compounds were administrated alone, several cardioprotective properties have been reported [72–78]. In murine models with induced injury or toxicity, treatment with OLE or its aglycone resulted in recurrent features, such as reduction of pro-inflammatory cytokines production (TNF-α and IL-1β), NF-κB expression and translocation, iNOS expression, adhesion molecules, and apoptosis markers, among others [73–75]. OLE aglycon has also been reported to interfere with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity [79]. Regarding human studies, in patients suffering from ulcerative colitis, OLE-treated colonic samples showed an amelioration of LPS-induced inflammatory damage, accompanied by decreased expression of COX-2 and IL-17 compared to samples exposed to LPS alone [80].

The protective actions of HT, tyrosol (Tyr), and other phenolic compounds present in olive oil against oxidative damage and inflammatory response have been recurrently demonstrated in vitro and in vivo [81]. Recently, in the context of inflammatory response in immune blood cells, pure HT, Tyr, and homovanillic alcohol (HVA) at physiologically relevant concentrations (0.25–1 μM) were able to inhibit oxysterol-induced production of proinflammatory cytokines (IL-1β, MIF, and RANTES), ROS production, and redox-based MAPK phosphorylation (JNK, p38) [82]. In addition, both HT and metabolites (1, 2, 5, and 10 μM) provided protection against endothelial dysfunction in human aortic endothelial cells (HAECs) co-incubated with TNF-α by significantly reducing the secretion of E-selectin, P-selectin, ICAM-1, and VCAM-1, and HT metabolites further reduced levels of monocyte chemoattractant protein 1 (MCP-1) [83]. In TNF-α-treated human umbilical vein endothelial cells (hECs), Tyr and its chemically synthesized metabolites Tyr-glucuronate and Tyr-sulfate (particularly the latter) prevented the phosphorylation of NF-κB signaling proteins. Both metabolites also prevented the over-expression of adhesion molecules and the adhesion of human monocytes to hECs [84]. In addition, Tyr and Tyr-sulfate counteracted TNF-α-induced oxidative stress in these cells and ameliorated edema in mice models of acute and chronic inflammation in a dose-dependent manner. In terms of other phenolic compounds found in VOOs, 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and in particular 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA), were shown to significantly protect red blood cells from oxidative damage [85]. In a recent study, MDA levels increased in human endothelial (HECV) cells exposed to a mixture of oleate/palmitate to mimic the condition of atherosclerosis. Treatment with isolated phenolic compounds, apigenin, caffeic acid, coumaric acid, Tyr, and OLE (extracted from olive pomace) significantly decreased MDA levels in these cells. In addition, in these steatotic HECV cells, NO release and NF-κB p65 levels increased significantly with respect to the control. This was counteracted by exposure to phenolic compounds extracted from olive pomace (PEOP) [86]. Regarding recent studies in animal models, in a DSS-induced acute colitis mouse model, hydroxytyrosyl acetate supplementation ameliorated the inflammatory response by modulating cytokine production, along with a reduction in COX-2 and iNOS protein expression, likely through MAPK (p38, JNK) and NF-kB signaling pathways [87]. In a study aiming to assess how HT supplementation differentially affects the adipose and liver tissue proteome, oxidative stress-related proteins were modulated by HT supplementation in both tissues, including a consistent repression of peroxiredoxin 1, which may be indicative of a better antioxidant status [12]. In Wistar rats, both HT- and in particular secoiridoid-supplemented diets (5 mg/kg/day) modulated the aorta and heart proteome compared to the standard diet, downregulating proteins related to proliferation and migration of endothelial cells and occlusion of blood vessels in the former and proteins related to heart failure in the latter [88]. In another study in rats, a high-carbohydrate high-fat diet (MS-inducing diet) + HT (20 mg/kg/day) was effective towards the mobilization of lipids as compared to only an MS-inducing diet, with branched fatty acid esters of hydroxy oleic acids lipids being regulated in the HT-supplemented group, denoting the alleviation of MS [89]. With regard to research in humans, clinical trial-derived evidence where a diet supplemented with phenol-rich olive oils or phenolic extracts is administered is increasing (Table 1, Figure 1). The PREDIMED trial has provided clear proof about the beneficial consequences of a long-term phenol-rich olive oil-supplemented diet in comparison to a low-fat control diet, which are not restricted to cardioprotection [90]. These benefits include improvements in several parameters associated to oxidation, inflammation, hypertension, metabolic syndrome, and diabetes, among others, which translate into lower risk of CVD and total mortality, for instance. Other, recent, short-term (duration of weeks to a few months) and acute studies also support the positive consequences attributed to the consumption of olive oil phenolic compounds (Table 1). Fewer studies in healthy [91] and hyperlipidemic subjects [92] have reported an absence of effect in surrogate markers of CVD, including lipid profile, inflammation, and oxidation, after supplementation with olive oil biophenols.

**Figure 1.** Clinical trials-derived evidence regarding biophenol-rich olive oils' benefits and mechanisms.

It should be underscored that the oxidative stress hypothesis is still debated following the null results of antioxidant trials. Therefore, the true contribution of antioxidant actions (unlikely to be direct due to the low bioavailability of biophenols) to cardioprevention is ye<sup>t</sup> to be fully elucidated.


**Table 1.** Randomized clinical trials-based evidence on the effects and mechanisms after the consumption (acute or sustained) of phenol-rich olive oil and olive oil



OO, olive oil; TP, total phenols; HT, hydroxytyrosol; OLE, oleuropein; CVD, cardiovascular disease; TAS, total antioxidant status; TAC, total antioxidant capacity; SOD, superoxide dismutase; MDA, malondialdehyde; HVA, homovanillyl alcohol; HDL, high density lipoproteins; ox-LDL, oxidized low density lipoproteins; TG, triglycerides; TC, total cholesterol; PBMC, peripheral blood mononuclear cell; CAT, catalase; JUN, Jun proto-oncogene, AP-1 transcription factor subunit; PTGS2, prostaglandin-endoperoxide synthase 2; EGR1, early growth response protein 1; IL, interleukin; MCP1, monocyte chemoattractant protein 1; CD40L, CD40 ligand, ADRB2, adrenoceptor Beta 2; OLR1, oxidized low-density lipoprotein receptor 1, GH-PX, glutathione peroxidase; DPP4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide 1; Apo B-48, apolipoprotein B-48; ACE, angiotensin-converting enzyme; NR1H2, nuclear receptor subfamily 1 group H member 2; EVOO, extra virgin olive oil; T2DM: type 2 diabetes.

## **5. Neurodegenerative Diseases**

Neurodegenerative disorders are age-dependent disorders which are becoming increasingly prevalent, in part because human longevity keeps increasing [113]. These disorders are defined by a multifactorial nature and have common neuropathological hallmarks such as abnormal protein dynamics with defective protein degradation and aggregation, oxidative stress and free radical formation, impaired bioenergetics and mitochondrial dysfunction, and neuroinflammatory and apoptotic processes [114]. Examples of neurodegenerative diseases include AD, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, among many others.

Either included in EVOOs or in the form of extracts, administration of phenolic-rich compounds has been demonstrated to exert neuroprotective effects in several in vitro and in vivo studies, as recently reviewed [115]. Olive oil or olive oil extracts containing a mix of phenolic compounds have been demonstrated to counteract age-related dysfunctions in several neuropathology-induced models. The neuroprotective effects seen include the improvement in cognitive behavior and motor coordination, accompanied by a reduction of total Aβ (due to enhanced Aβ clearance pathways and reduced brain production), and tau brain levels, a rise in the activity of detoxifying enzymes, and reduced lipid peroxidation [28,116]. Moreover, in ischemia–reperfusion models, administration of phenolic-rich olive oil reduced infarct volume, brain edema, blood–brain barrier permeability, and improved neurologic deficit scores, as well as brain ceramide levels [117,118]. Furthermore, an olive oil extract (45.5% biophenols, 4.2% HT, 2.2% Tyr, and 9.2% OLE) modulated inflammatory response in LPS-activated astrocytes and serum of multiple sclerosis patients by diminishing MMP-9 and MMP-2 levels and activity [119]. Finally, in amyotrophic lateral sclerosis (ALS) models, in vivo exposure to EVOO phenols resulted in higher survival and better motor performance, with improved muscle status and autophagy markers, and diminished endoplasmic reticulum (ER) stress [120], while in vitro it protected motoneurons from LPS-induced lethality, and inhibited IL-1β and NO release [121].

Concerning studies where pure phenolic-compounds were tested, OC, OLE, HT, and Tyr have been the subject of most research. OC, a naturally occurring phenolic secoiridoid of EVOO, has been attributed several neuroprotective activities. It interacts with relevant actors in different disease-related pathways (ex. inflammation, cancer, neurodegenerative diseases), such as heat-shock proteins (for example by inhibiting Hsp90) [122], and tau-441; this induces stable conformational modifications of the protein secondary structure and also interferes with tau aggregation [123]. This phenolic compound is capable of altering the oligomerization state of Alzheimer's-associated Aβ oligomers while protecting neurons from their synaptopathological effects [124]. Both in vitro and in vivo, OC was reported to enhance Aβ clearance from the brain via up-regulation of P-glycoprotein and LDL lipoprotein receptor-related protein-1 (major Aβ transport proteins) at the blood–brain barrier [125]. More recently, OC was reported to prevent oligomer (Aβo)-induced synaptic protein SNAP-25 and PSD-95 down-regulation in neurons, and to attenuate Aβo-induced inflammation, glutamine transporter (GLT1), and glucose transporter (GLUT1) down-regulation in astrocytes [126]. In addition, it reduced the Aβo-induced increase of interleukin-6 and glial fibrillary acidic protein (GFAP). As a cautionary note, OC is a high-molecular weight molecule for which bioavailability needs to be ascertained. In addition, the fact that OC crosses the blood–brain barrier remains unproven.

OLE aglycone provided neuroprotection to cultured neuronal cells [127], invertebrate simplified models of Alzheimer's disease and inclusion body myositis [128], and murine models of amyloid-ß deposition by interfering with Aß aggregation, counteracting the associated neuroinflammation, inducting autophagy, and improving cognitive performance [129–131]. Moreover, exposure to OLE protected against apoptosis in murine models of spinal cord injury and cerebral I/R injury, along with reduced infarct volume in the latter [132,133]. Reduced oxidative damage in specific brain areas was also found after OLE administration, as well as increased levels of antioxidant enzymes and improved learning and memory retention [134,135]. Recent studies have supported the protective capacities of HT and Tyr through the reduction in inflammatory markers, downregulation of apoptotic proteins, and ameliorated mitochondrial dysfunction [136–139]. In this sense, both pre- and post-treatment with HT prevented Aβ(25–35)-induced astrocytic cell line C6 cytotoxicity, induced Akt activation, and reduced the activation of mTOR, leading to improved insulin sensitivity and restoration of proper insulin-signaling [27].

Recent studies sugges<sup>t</sup> that olive oil phenolic compounds are processed by the body as xenobiotics via the Keap1/Nrf2/ARE signaling axis and exert their protective actions through the induction of these enzymes. Yet, no induction of phase II enzymes was found in PBMCs from healthy humans supplemented with HT, and further studies are needed to confirm this hypothesis [91]. In a very recent study using cell-free model assays, EVOO phenolic extracts (rich in secoiridoids derivatives, lignans, and vanillic acid) acted as multi-target ligands directly inhibiting neurodegenerative disorder-related enzymes BuChE, 5-LOX, hMAO-A and hMAO-B in a dose-dependent manner [140].

In summary, in vitro and in vitro neuroprotective activities attributed to olive oil phenolics include interference with amyloid and tau protein aggregation, and reduction of Aβ deposition, production, and induced inflammation, as well as enhanced Aβ clearance, decreased inflammatory biomarkers, oxidative stress, and apoptosis, lessening of cerebral infarct volume and damage after induced injury, and attenuation of insulin resistance, mitochondrial dysfunction, and ATP depletion. On the other hand, human evidence on the neuroprotective actions of olive oil phenolics coming from clinical trials is scarce (Table 1, Figure 1). Of note, the PREDIMED study reported an improvement in Mini-Mental State Examination (MMSE) and Clock Drawing Test (CDT) results, as well as in immediate verbal memory (associated with total olive oil consumption) following long-term consumption of a phenol-rich olive oil-supplemented diet compared to a low-fat control diet [111].

## **6. Hepatic Dysfunction**

Continued liver damage can lead to chronic liver diseases, such as simple steatosis and steatohepatitis (steatosis with inflammation and hepatocyte injury and death) and fibrosis, among others, which are highly prevalent worldwide [141]. Accumulating evidence indicates that oxidative stress and inflammation are strongly linked and participate in the pathophysiological processes of liver diseases [44].

Modulation of hepatic lipid metabolism, including protective effects against steatosis [142,143], lipid synthesis [144,145], and endoplasmic reticulum stress [146,147], as well as induction of antioxidant/detoxicant enzymes [148], mitochondrial biogenesis, and mitochondrial function [149] by olive oil and its phenolic compounds has been reviewed recently [150,151]. Recent in vivo studies support a dose-dependent hepatic protective role for olive oil and its phenolic compounds. In C57BL/6J male mice, dietary supplementation with an EVOO (859 mg total biophenols) significantly reduced fat accumulation in liver and the plasmatic metabolic alterations caused by a high-fat diet (HFD) compared to EVOOs with lower amounts (116 and 407 mg) and produced a normalization of oxidative stress-related parameters, desaturase activities, and long-chan polyunsaturated fatty acids (LCPUFA) content in tissues [152]. Moreover, in male Sprague–Dawley rats, a biophenol-rich VOO (0.290 mg phenols/kg/day) was able to (as compared to a phenol-free olive oil), significantly reduce liver inflammation and mitochondrial oxidative stress and restore insulin sensitivity, while limiting HFD-induced insulin resistance, inflammation, and hepatic oxidative stress, preventing nonalcoholic fatty liver disease (NAFLD) progression [153]. Furthermore, the replacement of dietary fat with phenolic-rich EVOO (total phenolic compound concentration: 447 ppm) reversed HFD-induced hepatic steatosis in mice. Also, the use of a phenolics-rich EVOO rather than EVOO (104 ppm) improved the plasma lipid profile and adipose tissue cytokine expression in mice with NAFLD [154]. Olive oil, HT and tyrosol (TY) showed protective effects against TCDD-induced hepatotoxicity in male Wistar rats, restoring ALT, AST, ALP, nitrite, and protein carbonyl content as well as NQO1 and HO. In addition, treatment with olive oil and its phenolic compounds resulted in reduced CYP1A1 and apoptosis (reduction and rise in Bax and Bcl-2 levels, respectively) [155]. In a rat model of NAFLD, the most common chronic liver disease in western countries, HT (10 mg/kg/day) significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome

proliferator activated receptor PPARα and its downstream-regulated gene fibroblast growth factor 21, the phosphorylation of acetyl-CoA carboxylase [156]. HT also reduced liver nitrosylation of proteins, reactive oxygen species production, and lipid peroxidation. In male mice C57BL/6J, HT supplementation (5 mg/day, for 12 weeks) significantly reduced fat accumulation in liver and plasma as well as tissue metabolic alterations induced by HFD, in addition to a normalization of Δ-5 and Δ-6 desaturase activities and oxidative stress-related parameters as compared to control animals [157]. In Wistar rats, a phenolic-rich olive fruit extract and an OLE extract showed protective effects against deltamethrin-induced hepato-renal toxicity by reducing lipid peroxidation (MDA), Cox-2, and apoptosis (reduction in p53 and rise in bcl-2), and by augmenting total antioxidant capacity and superoxide dismutase (SOD) and catalase (CAT) activities [158]. Treatment with a mix of PEOP was performed on rat hepatoma (FaO) cells exposed to a mixture of oleate/palmitate to mimic the conditions of NAFLD. Tyr, OLE and PEOP significantly reduced the triglyceride (TG) content with respect to steatotic cells. PEOP also decreased the number and size of lipid droplets in steatotic cells as compared to control. Furthermore, exposure to apigenin, caffeic acid, coumaric acid, OLE, and PEOP significantly decreased MDA level in steatotic FaO cells as compared to the control. Uptake of fatty acids (FAs) into hepatocytes and their oxidation are regulated mainly by PPAR α, while the anabolic esterification and conversion of FAs to TGs is controlled by PPARγ, for which expression has been shown to increase in NAFLD. Incubation with PEOP resulted in a significant decrease and increase in PPAR α and PPARγ expression, respectively, with respect to steatotic cells. With regard to mitochondrial β-oxidation, PEOP led to a further up-regulation of Cpt1 expression with respect to steatotic cells [86]. In male C57BL/6J mice, supplementation with HT attenuated liver metabolic alterations produced by HFD, activating transcription factors PPARα and Nrf2, and deactivating NF-κB [159]. Finally, in a recent study where individual compounds were administered, a 21-day dietary supplementation (5 mg/kg bw/day) with OLE or HT maintained higher levels of α-tocopherol in female Wistar rats' liver compared to a control diet, even though all diets supplied the same daily dose of α-tocopherol [160].

Human evidence on hepatic protective actions of olive oil phenolics coming from clinical trial is scarce and inconclusive (Table 1). Noteworthy, the PREDIMED study reported an improvement in fatty liver index, with potential implications in the delay or slowdown of NAFLD progression [112]. However, other studies where extracts of phenolic compounds from olive oil have been supplemented to healthy and hyperlipidemic subjects have reported an absence of effect on liver function [91,92,97].
