**1. Introduction**

The grea<sup>t</sup> progress of medical research has highly contributed to decreased mortality due to severe pathologies. But, on the other hand, a longer life expectancy has been associated with a greater incidence of illness and disability.

Non-communicable diseases (NCDs) are a group of long-lasting and slowly progressive chronic disorders [1]. The World Health Organization (WHO) recently reported that NCDs are the leading causes of death and disability for the general population, regardless of age, region, or gender [2]. NCDs have been deeply studied and some common key features have been identified; these include the intracellular presence of oxidative stress due to abnormal production of reactive oxidative species (ROS), inadequate antioxidant defense, and dysregulation of the autophagy pathway, responsible for the maintenance of cellular proteostasis [3]. Also inflammation is implicated in NCDs [4], since its level in an organism is closely related to cellular redox and an autophagic state [5,6].

Moreover, the health care costs associated with NCDs highlight the importance of finding new therapies for these pathological conditions, and it has been shown that healthy and equilibrated dietary patterns are useful in the prevention of NCDs [7].

The consumption of extra virgin olive oil (EVOO) is common in the Mediterranean Diet, which is largely known to have several health benefits and to increase longevity, as reported by the United Nations Educational Scientific and Cultural Organization (UNESCO) in 2010 [8,9]. As recently reported in the III International Conference on Virgin Olive Oil and Health Consensus Report, EVOO intake is also associated with reduced risk of most ageing-related diseases including cardiovascular and neurodegenerative diseases (CVD and NDD), and some types of cancer [10]. Initially, the beneficial properties of EVOO were attributed to functional components such as monounsaturated and polyunsaturated fatty acids (MUFAs and PUFAs), like oleic acid (55 to 83% of total fatty acid (FA)), the essential FA, linoleic acid (3 to 21% of total FA), and linolenic acid (0 to 1.5% of total FA). However, recent epidemiological and experimental studies also show that minor bioactive compounds, including phenolic alcohols, such as hydroxytyrosol (HT, 3,4-dihydroxyphenylethanol, 3,4-DHPEA) and tyrosol (*p*-hydroxyphenylethanol, *p*-HPEA), secoiridoid derivatives, phenolic acids, lignans, and flavonoids contribute to the beneficial e ffects of EVOO [11–13]. A high biophenol content confers a high stability to EVOOs, preventing EVOO autoxidation and contributing to a long shelf-life.

Oleuropein (Ole) is the major phenolic compound in the olive tree, *Olea europaea* L., and is particularly abundant in unprocessed olive fruit and leaves, with concentrations up to 140 mg g<sup>−</sup><sup>1</sup> on a dry matter basis in young olives [14], and 60–90 mg g<sup>−</sup><sup>1</sup> of dry matter in the leaves [15]. In *Olea europaea*, Ole, demethyloleuropein, ligstroside, and oleoside 11-methyl ester are abundant secoiridoids [16] whereas verbascoside [17] is the main hydroxycinnamic derivative of olives [18]

Ole belongs to the secoiridoids, which are abundant in *Oleaceas*, *Gentianales Cornale*s, as well as many other plants. Iridoids and secoiridoids are compounds that are usually glycosidically bound, and are produced from the secondary metabolism of terpenes, as precursors of various indole alkaloids. The secoiridoids in *Oleaceae* are usually derived from the oleoside type of glucosides (oleosides), which are characterized by an exocyclic 8,9-olefinic functionality, a combination of elenolic acid and a glucosidic residue [16]. Ole is an ester of elenolic acid and HT, and has a oleosidic skeleton that is common to the secoiridoid glucosides of *Oleaceae* (Figure 1)

**Figure 1.** Chemical structure of oleuropein.

Ole present in green olives, during the oil mechanical extraction process, is hydrolysed by the activity of endogenous β-glucosidases to form oleuropein aglycone (OleA), responsible for the bitter and pungen<sup>t</sup> taste of EVOO. OleA together with other derivative secoiridoid species, such as the dialdehydic derivative of decarboxymethyl elenolic acid bound to either HT (3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, 3,4-DHPEA-EDA, oleacein), or to tyrosol (*p*-hydroxyphenylethanol-elenolic acid dialdehyde, *p*-HPEA-EDA, oleocanthal), and ligstroside aglycone (*p*-HPEAEA), represents the minor polar compounds that determine the antioxidant capacity of EVOO. In recent years, oleocanthal and oleacein have attracted interest from the scientific community [19] due to their inflammatory effects. The first study was due to its similar structure to ibuprofen [20], the second for its ability to stimulate the expression of CD163, an anti-inflammatory gene [21]. The secoiridoid most extensively studied is OleA, whose content is dependent on the oil production process, as previously reported [22].

The relevance of bioactive components in EVOO has been strengthened by the European Food Safety Authority (EFSA), that in 2011, released a health claim [23,24] on the efficacy of oil phenols (5 mg/day per 20 g of EVOO, HT and OleA) in protecting low-densitiy lipoprotein (LDL) from oxidation, the initial event of atherosclerotic plaque formation. This is of interest because it is unique as a health claim that associates a specific dosage of a natural bioactive component of food with cardiovascular risk (LDL cholesterol oxidation) reduction [25].

Leaves of olive tree are a hystorical Mediterranean herbal drug, used as a traditional remedy for health promotion and a therapy for chronic conditions. The differences in Ole content depend on the cultivar, production area, and leaf tissue conditions (fresh, frozen, dried, or lyophilized). Commercial *Olea* leaf extracts, standardized in Ole content, were used to obtain food supplements with specific biological and biomedical properties [26].

Various methods have been developed for the qualitative and quantitative analysis of the occurrence of phenolic and secoiridoid compounds, from the simplest techniques, such as TLC [27], to the more sophisticated ones, such as reversed phase HPLC [22,28,29], GC-MS, FAMS, or TMS [30]. In the fruits, phenyl acids, flavonoids, and secoiridoids have been reported, the phenolic compounds representing 1–3% (*w*/*v*) [31]. In the leaves, 19% (*w*/*w*) is Ole and 1.8% (*w*/*w*) is flavonoids, of which 0.8% is luteolin 7-glucoside [15].

Recently, both oleuropein isoforms, glycosidic form and aglycones, have attracted scientific attention by virtue of their health benefits, such as antioxidant, anti-inflammatory, cardio- and neuro-protective, and anti-cancer effects. These pharmacological activities are mainly due to their putative radical scavenging features, due to the ortho-diphenolic group. Mechanistic studies indicate that these compounds are also able to act at different sites, interfering with protein function and gene expression, or modifying cellular pathways relevant to the NCDs pathological processes [25,32], suggesting that the actions of oleuropein in various disorders may result from shared molecular mechanisms. As reported above, dysregulated autophagy is a common feature of NCDs. This dysregulation seems to be due to increased oxidative stress, so, although these mechanisms are generally viewed as cell autonomous, recent evidence suggests an occurrence of an interplay between autophagy and oxidative stress that influences the inflammatory state of tissues, linked with NCD development [3] (Figure 2). The aim of this review is to collect and discuss the data available in the literature concerning the effect of oleuropein isoforms, Ole and OleA, in NCDs by their putative antioxidant and anti-inflammatory activities, but also through their other peculiar actions as autophagy inducers and amyloid fibril growth inhibitors. The last part of this review is dedicated to the anti-cancer effect of oleuropein and its ability to sensitize and potentiate the action of current therapies.

**Figure 2.** Effect of oleuropein on interplay between oxidative stress, autophaghy and inflammation in non-communicable diseases. AMPK, 5' adenosine monophosphate-activated protein kinase; Beclin-1 autophagy-specific marker; COX, Cyclooxygenase; CRP, C Reactive Protein; Hcy, homocysteine; ICAM-1, Intercellular Adhesion Molecule 1; IL-1β, interleukin-1β; IL-6, interleukin-6; iNOS, inducible form of nitric oxide synthase; LC3 autophagy-specific marker; MMP-9, metalloproteinases-9; mTOR, mammalian target of rapamycin; NF-kB, Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells; oxLDL, oxidized low-density lipoprotein; p62 autophagy-specific marker; PARP1, Poly (ADP-ribose) polymerase; ROS, Reactive Oxygen Species; SIRT-1, NAD-dependent deacetylase sirtuin-1; TFEB, Transcription factor EB; TNF-<sup>α</sup>, tumour necrosis factor-α; VCAM-1, Vascular Cell Adhesion Molecule 1.

## **2. Oleuropein As an Antioxidant**

The strong antioxidant properties of oleuropein is well known, and is shared with other phenols present in olive leaves and olive oil. In its chemical structure oleuropein contains an ortho-diphenolic group able to scavenge ROS through hydrogen donation, and to stabilize oxygen radicals with an intramolecular hydrogen bond. In particular, a *o*-diOH substitution confers a high antioxidant property, whereas single hydroxyl substitutions, e.g., tyrosol, provide none [33]. Ole in vitro can inhibit, in a dose-dependent manner, copper sulphate-induced oxidation of LDLs, assessed through a decrease in thiobarbituric acid-reacting substances and lipid peroxide by-product content [34,35]. In vivo, rabbits fed with an Ole-rich diet showed a higher serum antioxidant levels able to counteract LDL oxidation, and a reduction of total, free, and esterified cholesterol levels compared to animals receiving a standard diet [36]. Moreover, in humans, Visioli et al. [37] proved that Ole supplementation in healthy volunteers decreased, in a dose-dependent manner, the urinary excretion of 8-iso-PGF2<sup>α</sup>, suggesting a lower lipid peroxidation. A scavenging effect of Ole, similar to ascorbic acid and α-tocopherol, was shown in vitro against hypochlorous acid, a potent oxidant species produced in vivo by neutrophil myeloperoxidase at the site of inflammation [33], as well as against nitric oxide, as reported by De la Puerta et al. [38]. The first experimental evidence of the direct antioxidant cardioprotective effect of Ole against the post-ischemic oxidative burst after coronary occlusion, was reported by Manna et al. [39]. Using isolated rat hearts pretreated with Ole, subjected to global ischemia and then reperfused, they observed a decrease of creatine kinase and a reduced glutathione release in the perfusate, as well as a decrease of oxidized glutathione and in the lipid peroxidation level. Ole was also able to exert, in an indirect way, its antioxidant action by stimulating the expression of intracellular antioxidant enzymes *via* the activaction of Nuclear factor erythroid 2-related factor 2 (NrF2) transcription [40], as well as by increasing the level of non enzymatic antioxidants such as glutathione, α-tocoferol, β-carotene, and ascorbic acid [41–43].

#### **3. Oleuropein As an Anti-Inflammatory and CVD Protective Agent**

Inflammation is a crucial and defensive response induced by tissue damage or infection, and represents the "common soil" of multi-factorial diseases, playing a crucial role in promoting many disabling illnesses, such as atherosclerosis, diabetes mellitus, metabolic syndrome, cancer, chronic kidney diseases, and neurodegenerative diseases [44]. It can be divided into two types, acute and chronic. Chronic inflammation is correlated with the production of ROS that may cause oxidative damage and the depletion of antioxidants [45]. Macrophages, one of the main factors in the inflammatory response, produce ROS, but also pro-inflammatory cytokines and chemokines, including IL-1, IL-6, TNF-a, and IFN-γ. IL-6 seems to be the central mediator of the inflammatory response and an index of increased frailty [46–49]. Therefore, besides the release of several inflammatory cytokines or mediators, damaged tissues also release monocyte chemoattractant proteins (MCP-1), cyclooxygenase (COX), inducible form of nitric oxide synthase (iNOS), metalloproteinases (MMP), and adhesion molecules. In addition, nuclear factor Kappa β (NF-kβ) occupies a key upstream position in a complex signal transduction pathway, controlling the production of countless pro-inflammatory mediators [50].

In 2006, the PREDIMED trial first showed the anti-inflammatory e ffect of the Mediterranean Diet (MD) supplemented with EVOO for three months (compared with a low fat diet), through a remarkable decrease of serum C-Reactive Protein (CRP), IL-6, endothelial and monocytary adhesion molecules (ICAM-1 and VCAM-1), and chemokines, in a group of 722 partecipants [51]. Many other sub-studies of the PREDIMED trial have confirmed the anti-inflammatory properties of MD with EVOO by studying the changes in biomarkers associated with atherogenesis, such as peripheral blood mononuclear cell expression of cell surface inflammatory mediators (adhesion molecule and pro-inflammatory ligand CD40 expression on T lymphocytes and monocytes) or other molecules associated with systemic inflammation, such as those that induce the expression of adhesion molecules and activating NF- kB (TNFR60), or playing a role in T cell proliferation (TNFR80) [52–54]. Di Daniele et al. [55] demonstrated that the Italian MD in nephropatic patients could be a useful tool in the treatment of cardiovascular comorbidity related to renal dysfunction, causing a significant decrease in serum homocysteine (Hcy), dependant on a methylenetetrahydrofolate reductase genotype. In fact, Hcy, during the autoxidation process, induces impairment of the endothelium by producing ROS, with consequent involvement in atherosclerosis [56,57]. All these studies support that adherence to the MD with EVOO can modify inflammation, regardless of shared genetic and environmental factors.

In this context, many scientists have focused their attention on the possibility of using individual EVOO polyphenolic compounds like oleuropein, as a promising alternative anti-inflammatory agent, due to its capability to inhibit the synthesis of pro-inflammatory cytokines [58,59] and lipoxygenase activity [4], or to modulate inflammatory parameters [60].

Starting from in vitro results, Miles et al. [61] observed that Ole was the most powerful inhibitor of the production of IL-1β, compared with other phenols from EVOO, from human whole blood cells stimulated by lipopolysaccharide (LPS). Ryu S. et al. [62] verified that Ole was able to modulate the phenotype of LPS-activated murine macrophages, RAW 264.7, through the downregulation of key markers in inflammation pathways such as iNOS, COX-2, NFKB, and JNK, and of the two pro-inflammatory interleukins, IL-6 and IL-1β. The same authors also showed the e fficacy of Ole in the reduction of LPS-induced NO in a zebrafish embryo model.

OleA is able to modulate the tumor microenvironment, at least in part, through its anti-inflammatory properties, as reported by Margheri et al. [63]. Using "senescence-associatedsecretory-phenotype" (SASP) fibroblasts, that show features of cancer-associated fibroblasts, they found that treatment with OleA decreased both the levels of SASP pro-angiogenic factors in the fibroblasts, and the release of the same in cell media, particularly IL-8. Interestingly, when endothelial progenitor cells and resident mature microvascular endothelial cells were exposed to these latter cell media, vasculogenesis and angiogenesis was inhibited by a decrease of MMPs and by the urokinase-type plasminogen activator, suggesting a mechanistic interpretation of the anti-angiogenic activities for cancer prevention by OleA.

The anti-inflammatory e ffect of Ole is better appreciated by studies using in vivo animal models. Giner et al. [64] demonstrated that Ole was able to ameliorate the symptoms of dextran sulfate sodium(DSS)-induced colitis in mice through the reduction of COX-2, iNOS, and MMP-9, and the suppression of p38 MAPK phosphorylation, which may be due to the up-regulation of annexin A1 [33]. Impellizzeri et al. [59] found that OleA could attenuate TNFα and IL-1β production in a mouse model of carrageenan-induced pleurisy. The same pathways of TNFα and IL-1β were also a ffected by Ole in a rat model of post-traumatic stress disorder [65], and in rats with spinal cord trauma [66]. Ole also showed its beneficial e ffect in an ovariectomy/inflammation experimental model of bone loss in rats, modulating parameters of inflammation, such us fibrinogen and spleen weight [67].

Recently, a study by Larussa T. et al. [68] demonstrated that the administration of OleA on colonic biopsies taken from ulcerative colitis patients led to a decrease of COX-2 and IL-17 levels, considerably reducing the inflammation of the colonic tissue. This evidence encourages the use of oleuropein as an inflammation-modulator.

#### *Lipid-Regulating, Anti-Hypertensive and Antidiabetic E*ff*ects of Oleuropein*

CVD is a group of disorders a ffecting heart and/or blood vessels. CVD, including coronary heart disease (CHD), cerebrovascular disease, and peripheral arterial disease, are characterized by fatty deposits in the inner walls of the blood vessels supplying the heart and brain, that may cause an arrest of blood flow to these organs. The cardiovascular protective e ffect of oleuropein is supported by many in vivo animal studies and human clinical trials that showed, in addition to its antioxidant and anti-inflammatory properties, its lipid-lowering activity, anti-hypertensive, and hypoglycemic action [69–71].

Lockyer et al. [72] conducted a randomized, controlled trial on pre-hypertensive volunteers, who after an intake of Ole-enriched olive leaf extract for six weeks (136 mg Ole; 6 mg HT), showed significantly lower blood pressure (BP), plasma total cholesterol, LDL cholesterol, and triglycerids relative to the control, with a 5.76% reduction in coronary heart disease risk. Another trial on patients with stage-1 hypertension, showed that a daily dose of 2 × 500 mg of olive leaf extract (with 16–24% Ole) for four weeks, lowered systolic and diastolic BP with an e ffect comparable to that exerted by an e ffective dose (12.5–25 mg twice daily) of Captopril (the standard therapy for stage-1 hypertension), and reduced total plasma LDL and triglyceride levels. The authors concluded that the dual e ffect of olive leaf extract in lowering BP, probably due to angiotensin converting enzyme inhibition and calcium channel blocking activities, and improving lipid profiles, is advantageous for reducing the risk for CVD [73].

Insulin resistance is a systemic disorder, in which there is a reduced action of insulin despite an "hyperinsulinaemia" condition, that a ffects many organs, in particular the liver and adipose tissue, and leads to development of two NCDs, type 2 diabetes mellitus (T2DM) and metabolic syndrome, well known cardiovascular risk factors. Recent research has described the beneficial properties of OleA and Ole-enriched olive leaf extracts against T2DM, and other metabolic syndrome associated conditions. In particular, OleA prevents amylin aggregation into amyloid fibrils, whose pancreatic presence is considered one of the causes of the su fferance and functional impairment of insulin-secreting cells in T2DM (see 5: Oleuropein as anti–amiloid mean) [74] Therefore, many studies conducted in animal and cell models have reported that Ole has the property of decreasing blood glucose and cholesterol levels, and improving oral glucose tolerance and insulin sensitivity [41,75,76]. These findings were confirmed

by human clinical trial results showing that treatment with Ole improved glucose homeostasis, reduced glycated hemoglobin and fasting insulin levels, suggesting a significant anti-diabetic e ffect [77–79]. Interestingly, in the context of these latter metabolic disorders, both characterized by insulin-resistance, de BocK et al. [78] showed a recovery of insulin sensitivity and pancreatic β-cell secretion capacity, in a group of overweight middle-aged men that received capsules of oleuropein-leaf extracts for 12 weeks, corroborating previous findings on the hypoglycemic e ffect of oleuropein [41,71,80].

Another disease highly associated with insulin resistance and the metabolic syndrome is non-alcoholic fatty liver disease (NAFLD), that a ffects about 25% of the world population, and'the following non-alcoholic steatohepatitis (NASH). Research on cell and animal models have reported that oleuropein may counteract these conditions through di fferent actions, including (i) an anti-lipidemic activity [81], (ii) protection and prevention of liver damage [82–84], and (iii) by interfering with signaling pathways involved in lipogenesis and in the onset of fatty liver disease [69]. Unfortunately, today these findings are not adequately supported by human studies, and remains unproven.

Therefore, in addition to the reported properties above, the ability of oleuropein to inhibit endothelial activation, monocyte cell adhesion and platelet aggregation within the concentration range expected after the nutritional intake from MD, sugges<sup>t</sup> that oleuropein may also be considered an anti-atherogenic agent, reflecting its CVD protective activity [85–89]

#### **4. Oleuropein As an Autophagy Inducer**

Autophagy is a process by which the cells removes damaged organelles, malformed proteins or amyloid aggregate accumulation through lysosomal degradation. This is a process highly conserved and is required to maintain cellular homeostasis. It starts with the formation of a phagophore (that coincides with membrane isolation) that grows and terminates in auto-phagosome completion, which follows its fusion with lysosomes to form auto-phagolysosomes. Beclin-1 and LC3 are typical markers of autophagy activation, involved in the first steps of phagophore formation, while p62 participates in cargo recognition by lysosomes [90]. The target of rapamycin complex 1 (mTORC1) and the AMP-activated protein kinase (AMPK) are the stress sensors that control autophagy. However, while mTORC1 is an autophagy inhibitor activated by serum, nutrients, growth factors, etc., AMPK is instead an autophagy inducer, activated by low energy conditions and polyphenols.

Dysregulated autophagy is a common feature in NCDs implicated in NDD, metabolic syndrome, diabetes, CVDs, gastrointestinal diseases, and cancer [3]. As a master regulator of protein, lipid and carbohydrate metabolism, altered autophagy may concomitantly promote metabolic disorders and diseases associated with ageing, unhealthy diets, and inflammation. Indeed, knockout of the Atg7 gene in mice, an essential gene for autophagy, shows in vivo typical Parkinson' disease (PD) features like Lewy bodies (LBs) formation, including endogenous synuclein and neuronal loss, as well as hepatomegaly with mutant hepatocytes showing accumulation of ubiquitin-positive aggregates [91,92]. A high fat-diet and genetically obese mice showed a decrease in autophagy flux, linked to elevated inflammatory gene expression [93–95]. Interestingly, autophagy seems to have a role in hypothalamic agouti-related peptide neurons in the regulation of food intake and energy balance, suggesting that the ability to regulate hypothalamic autophagy for modulating energy homeostasis may have implications in the development of new therapeutic options for obesity, and metabolic syndrome conditions [96]. Autophagic flux is also inhibited in pancreatic β-cells exposed to fatty acids, thus suppressing insulin secretion, a crucial factor for promoting T2DM to type-I diabetes conversion [97]. In the context of CDV, several studies show that autophagy might have beneficial or detrimental roles depending on the stage and type of the considered cardiovascular disease [4]. A beneficial function of autophagy has been observed in ischemia-reperfusion, cardiac hypertrophy, and atrial fibrillation. However, the majority of cardiac disorders suggests that autophagy may be a common cellular pathway that can be targeted for therapeutic gain, and the growing number of cardioprotective therapies a ffecting autophagic activity confirms this evidence [3,98,99]. Autophagy is also pivotal for intestinal homeostasis, appropriate intestinal immune responses, and anti-microbial protection, as well as neuronal and microglial

functions [100]. In cancer cells, autophagy may exert either a tumor-promoting or tumor-suppressing effect [3]. Thus, it is still debated whether autophagy induction or inhibition may represent the most promising approach for future cancer treatments. Interestingly, cancer cells may also use autophagy as a resistance mechanism against chemotherapy [101]. In conclusion, autophagy is a key factor in the pathogenesis and regulation of various kinds of diseases, serving as a potential and e ffective target for their intervention. Therefore, the use of substances, such as polyphenols, that modulate autophagy and minimize the collateral e ffect, may be a valid therapeutic approach [102].

Some of the studies that contribute to demonstrating the healthful actions of oleuropein against pathologies involving autophagy dysfunction, acting as an autophagy enhancer through several mechanisms, and its potential use as a nutraceutical agen<sup>t</sup> in several NCDs are summarized below
