Biophenols are a family of over 8000 polyphenolic structures (those presently described) found in almost all plant families mainly as secondary metabolites, including several hundred isolated from edible plants [36,40]. These molecules include non-flavonoids or flavonoids; the latter are further classified as flavonols, flavononols, flavones, anthocyanins, procyanidins, phenolic acids, stilbenes and tannins on the basis of the number of hydroxyls in the molecule and the type and the position of other substituents [41]. The plant sources of plant polyphenols are, among others, bark, leaves, fruits, spices, berries, vegetables, roots, nuts and seeds, herbs, and whole grain products, from which they are transferred in processed foods of plant origin, including EVOO, red wine, green tea, coffee and turmeric. These compounds are characterized by a broad spectrum of biological activities and exert positive effects in a large number of human diseases, including cancer, CVDs, T2DM and neurodegenerative conditions, with molecular mechanisms often related to their antioxidant activity. In the case of EVOO, its healthy properties have been associated with its peculiar chemical composition [42]. EVOO contains both major components (triglycerides and other fatty acid derivatives where mainly monounsaturated fatty acids, in particular oleic acid, are present) and minor components (over 230 different chemicals including aliphatic and triterpenic alcohols, phytosterols, hydrocarbons, tocopherols, and polyphenols) [43]. In the past, the health effects of EVOO were attributed mainly to the presence of oleic acid; however, more recently, attention has been focused on phenolics, a class of bioactive compounds including phenolic acids, phenolic alcohols, flavonoids, secoiridoids and lignans [44].

In particular, olive tree polyphenols include flavonols, lignans and glycosides. Olive glycosides are iridoids, geraniol-derived monoterpenes, whose chemical structure results from a cyclopentane ring fused to a six-member heterocycle with an oxygen atom. In particular, the bicyclic H-5/H-9β, β-cis-fused cyclopentanepyran ring system is the most common structural feature and the basic skeletal ring of iridoids. Cleavage of the cyclopentane ring of iridoids produces seco-iridoids, while cleavage of the pyran ring produces iridoid derivatives [45]. Iridoids and secoiridoids, mainly in the glycated form, are found in many medicinal plants belonging to the subclass Asteridae that includes several plant families, particularly Oleaceae.

The polyphenols produced by the olive tree are found in the lipid fraction and in the water fraction (dispersed as minute droplets) of olive oil mainly in the glucose-free form (aglycones), resulting from deglycosylation by plant glycosidases during olive squeezing. The most abundant secoiridoid in olive oil is 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA), whose glucose-bound form is commonly known as oleuropein; the latter is the main cause of the bitter taste of olive leaves and drupes. Other secoiridoids include oleuropein derivatives, both in the glucose-bound form or as aglycones, such as the dialdehydic form of decarboxymethyl elenolic acid bound to either HT (3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, 3,4-DHPEA-EDA, also known as oleacein) or to tyrosol (p-hydroxyphenylethanol-elenolic acid dialdehyde, p-HPEA-EDA, also known as oleocanthal, or ligstroside aglycone [46,47] (Figure 1). Oleocanthal produces the burning sensation in the back of the throat that accompanies the consumption of freshly squeezed EVOO. Olive oil also has a rich composition in simple phenols; these include tyrosol (p-hydroxyphenylethanol, p-HPEA) and hydroxytyrosol (3,4-dihydroxyphenylethanol, 3,4-DHPEA, DOPET), two phenolic alcohols mostly derived from their secoiridoid precursors. Olive polyphenols also include verbascoside, the caffeoylrhamnosylglucoside of HT, 1-acetoxypinoresinol and pinoresinol (two lignans).

Olive polyphenols are considered to be responsible for some of the recognized pharmacological properties of the olive tree (anti-atherogenic, antihepatotoxic, hypoglycemic, anti-inflammatory, antitumoral, antiviral, analgesic, purgative and immunomodulatory activities) [28,48,49], together with the protection against aging-associated neurodegeneration [29]. For these reasons, the EVOO quality depends not only on the content in free fatty acids resulting from triacylglycerol breakdown (acidity), but also on its content in polyphenols, the molecules responsible for its taste and for many of its healthy properties. Several factors affect the content of polyphenols in olive oil; these include olive cultivar, environmental cues (altitude, meteorological factors and irrigation), cultivation practices, and ripening stage of the fruits [50], together with extraction techniques, systems to separate oil from olive pastes. The conditions of storage (temperature, time) are also of importance, affecting the rate of oxidation/photooxidation reactions and the deposition of suspended water particles rich in polyphenols [51]. Under optimal conditions, the content of polyphenols in EVOO can exceed 60 mg/100 g.

The normal daily dietary intake of plant polyphenols is in the 0.1–1.0 g range; however, the bioavailability of these molecules, including the olive ones, in humans is poor due to reduced intestinal absorption and fast biotransformation that favors their urinary excretion. In addition, in the case of the brain, the circulating polyphenols must also cross the blood–brain barrier before reaching the parenchyma. With few exceptions, polyphenol aglycones can be partially absorbed in the small intestine by passive diffusion [52] much better than their glycated counterparts [53], although important amounts proceed to the large intestine to be eliminated [54]. A review of many studies on polyphenol bioavailability reported a 0 to 4.0 µmol/L plasma concentration of total metabolites produced from the oral administration of 50 mg aglycone equivalents of a polyphenol [55]. After intestinal absorption and passage to the lymph, most polyphenols undergo phase I and phase II metabolism, with substantial biotransformation and production of methylated, sulphated, hydroxylated, thiol-conjugated and glucuronide derivatives and degradation products [56]. These modifications alter the chemical properties of plant polyphenols, favor their excretion and, possibly, provide them new biological activities [57]. The importance of the colonic microflora for polyphenol bioavailability, due to its ability to metabolize and chemically modify polyphenols, has been reported recently [55]. Anyway, recent studies indicate that plant, including olive, polyphenols are absorbed in discrete amounts from the intestine and rapidly distributed through the blood flow to the whole organism, including the brain, both in rats [58,59] and in humans [60,61]. Plant polyphenols do interact with, and cross, synthetic and cell membranes. The interaction of oleuropein aglycone with synthetic phospholipid membranes favored by the presence of anionic lipids has been reported in a very recent study [62]. Another study reported that several polyphenols (the olive ones were not included) protected the mitochondria against membrane permeabilization by amyloid oligomers, suggesting some interference with oligomers’ interaction with the membrane [63]. Finally, oleuropein aglycone (OLE) was the main polyphenol found in breast cancer cells treated with an olive leaf extract in a recent metabolite-profiling study, suggesting its ability to cross the plasma membrane of these cells [64].

Due to the rising interest in natural phenols as possible new drugs, strategies to improve their bioavailability are under study, with encapsulation being probably the most actively investigated, in some cases with encouraging results [65,66]. Most of these molecular tools have not been tested in clinical trials, yet this strategy appears promising to improve the efficacy of natural phenols as drugs while reducing the amount of the administered dose. Actually, accurate studies on the effective dose of olive polyphenols to be administered daily to humans to obtain significant protection are still lacking; at any rate, the amount of OLE and other plant polyphenols taken daily in foods appears not adequate to ensure a dose suitable to produce short-term acute effects. However, clinical and experimental evidence indicates that a continuous consumption of moderate amounts of these molecules can be effective in the long term; this can also result in the accumulation in body tissues of these lipophilic molecules, leading to a low-intensity continuous stimulus of cell defenses against amyloid deposition, protein and metabolism dyshomeostasis, oxidative stress and other alterations underlying age-associated pathologies. These effects, although not proven experimentally, could, at least in part, explain the healthy properties of the MD. Nevertheless, the intake of moderate amounts of olive, and other plant, polyphenols provided by a typical MD supports the usefulness of the integration of polyphenol-enriched olive leaf extracts and other polyphenol-enriched nutraceuticals that can intensify, in the short term, the beneficial effects of these molecules.

It is widely recognized that oxidative and nitrosative stress as well as inflammation are the major abnormalities underlying neurodegeneration and that antioxidant molecules, such as olive oil polyphenols, restore neuronal function through the amelioration of the redox status. Some beneficial effects of the MD have been associated with the consumption of EVOO polyphenols; these include antioxidant, hypoglycemic, antimicrobial, antiviral, antitumor, cardioprotective, neuroprotective, antiaging and anti-inflammatory activities [67,68]. It has been reported that EVOO polyphenols are protective against cognitive impairment associated with aging and neurodegenerative diseases due to their ability to protect DNA against oxidative stress, to inhibit mitochondrial dysfunction and to attenuate lipid peroxidation by scavenging free radicals, thus sustaining endogenous antioxidant stability [69,70]. They are also able to inhibit amyloid β (Aβ) and τ protein aggregation and toxicity, the main causes of the neurodegenerative cascade in AD [39,71,72]. EVOO polyphenols participate in the redox balance of the cell as antioxidants and as mild pro-oxidants, with ensuing upregulation of the antioxidant defenses of the cell. Accordingly, they can be considered as hormetic factors. For instance, in the presence of peroxidases, HT can undergo a redox cycling that generates superoxide [70], and tyrosol also increases C. elegans lifespan by activating the heat shock response [71]. It was reported that HT reduces brain mitochondrial oxidative stress and neuroinflammation in AD-prone transgenic mice by the induction of Nrf2-dependent gene expression [72]. The eight-week administration of oleuropein (60 mg/kg/day) improved mitochondrial function and reduced oxidative stress by activating the Nrf2 pathway in SHR rats [73]. Furthermore, tyrosol (240 mg/kg) was found to be protective against LPS-induced acute lung injury through the inhibition of NF-κB and the activation of AP-1 and of the Nrf-2 pathway [74]. EVOO polyphenols also enhance Nrf-2 activation at the hepatic level and the ensuing release of antioxidant enzymes [75]. Nrf2 is considered the principal regulator of redox homeostasis and its activation inhibits pro-inflammatory mediators such as cytokines, COX-2 and iNOS [76]. EVOO polyphenols limit inflammation by reducing the expression/activity of the transcription factors NF-κB and AP-1 [77] thanks to their free radical scavenging and radical chain breaking capacity and to the reduced formation of ROS and RNS. Moreover, HT inhibits the development of the inflammatory cascade following LPS and carrageenan injection through downregulation of the levels of pro-inflammatory cytokines (TNF-α and IL-1β), COX2, iNOS, NO, PGE2 and NF-kB and reducing DNA damage [78,79,80]. It was reported that the co-injection of OLE (450 µM) with Aβ42 (50 µM) into the nucleus basalis magnocellularis (NBM) of adult rats interfered with Aβ aggregation and significantly counteracted Aβ toxicity against choline acetyltransferase-positive neurons of the NBM and reduced astrocyte and microglia activation [81]. Another study reported that OLE protects transgenic C. elegans strains, constitutively expressing Aβ3-42, by reducing Aβ plaque load and motor deficits [82]. Interestingly, significant anti aggregation and neuroprotective effects of a diet supplemented with OLE, HT or a mix of polyphenols from olive mill wastewater were reported in the TgCRND8 mouse model of Aβ deposition. In these transgenic mice, a significant improvement in cognitive functions and a significant reduction in Aβ plaque number, size, and compactness were found in 3- and 6-month-old mice (at the early and intermediate stage of Aβ deposition, respectively) fed for 8 weeks with the OLE-supplemented diet [83,84,85]. A significant improvement in synaptic function and a significant reduction in the number, size and compactness of both Aβ42 and its 3-42 pyroglutamylated derivative (pE3-Aβ) deposits occurred even when the treatment was started at 10 months, when these mice display increased brain deposits of Aβ and, in particular, of pE3-Aβ in the cortex and hippocampal areas. These data indicate that oral diet supplementation with OLE not only results in the prevention of amyloid deposition but also in the disaggregation of preformed plaques and in a reduction in pE3-Aβ generation [85]. The effect of OLE against Aβ peptide aggregation was dose-dependent and could be reproduced by diet supplementation with a mix of polyphenols from olive mill wastewater or by HT administered at the same dose as that of pure OLE [84,86]. Interestingly, the treatment with OLE (50 mg/kg of diet for 8 weeks) astonishingly activated neuronal autophagy even in TgCRND8 mice at an advanced stage of pathology. In these animals, histone 3 acetylation on lysine 9 (H3K9) and histone 4 acetylation on lysine 5 (H4K5) were increased in the cortex and the hippocampus; such an increase matched both a decrease in HDAC2 expression and a significant improvement in synaptic function [85].

It is known that abnormal acetylation takes place in memory and learning disorders such as AD, where a significant increase in HDAC2 inhibits gene expression at specific loci, such as those involving autophagy markers [87]. In addition to the induction of an intense and functional autophagic response in the cortex, other relevant biological effects of OLE were uncovered in the TgCRND8 model; these include increased microglia migration to the plaques for phagocytosis, enhanced hippocampal neurogenesis and reduced astrocyte reaction [83,88]. OLE induced autophagy through the increase in cytosolic levels of Ca²⁺ and the subsequent activation of the enzyme complex AMPK by Ca²⁺/Calmodulin Protein Kinase Kinase β (CaMKKβ) and the ensuing increase in phosphorylation of mammalian target of rapamycin (mTOR) with mTOR inhibition [89]. These data support the idea that autophagy activation by OLE and other olive polyphenols proceeds via modulation of the AMPK–mTOR axis, similarly to data reported for other plant polyphenols [90]. TgCRND8 mice fed with a diet supplemented with OLE or HT (50 mg/kg of food) exhibited increased levels of Beclin-1 and LC3 autophagic markers in the soma and dendrites of neurons of the somatosensory/parietal and entorhinal/piriform cerebral cortex, together with improved autophagosome/lysosome fusion [83,86]. Furthermore, the significant accumulation of PAR polymers and the increase in PARP1 expression found in the cortex at the early (3.5 months) and intermediate (6 months) stage of Aβ deposition in the TgCRND8 mice were rescued to control values by OLE supplementation. OLE-induced reduction in PARP1 activation was paralleled by the overexpression of SIRT1, and by a decrease in the pro-inflammatory NF-κB and the pro-apoptotic p53 marker [88].

The ability of EVOO polyphenols to modulate the action of NF-kB was observed both in vitro and in vivo in different tissues. In vivo, HT attenuated apoptosis in rat brain cells by modulating the levels of caspase-3 and NF-kB p65 subunit [91]; in high-fat diet (HFD)-fed C57BL/6 J male mice, daily doses of HT (5.0 mg/kg) attenuated the increment of NF-κB and SREBP 1c, and increased the activity of Nrf2 and PPAR-γ in the liver [92]. In female BALB/c mice, an EVOO-supplemented diet was protective in the management of induced systemic lupus erythematosus disease, likely through the inhibition of the MAPK, JAK/STAT, and NF-κB pathways in splenocytes [93]. One of the most studied upstream constituents of the NF-κB signaling pathway is the activation of the mitogen-activated protein kinases (MAPKs) [94]. In the TgCRND8 mice, an HT-supplemented diet modulated MAPK signaling by activating ERK and downregulating SAPK/JNK expression, a mechanism that may underlie memory improvements in these mice [86]. These data agree with other findings suggesting an involvement of ERK stimulation in memory formation and synaptic plasticity. In the C57BL/mouse model of AD, the administration of HT and its acetylated derivative significantly improved spatial memory deficits induced by the intracerebral injection of Aβ42 plus ibotenic acid. The latter affected the Bcl-2/Bad levels, activated caspase/cytochrome-dependent apoptosis, and downregulated pro-survival genes also involved in memory functions (Sirt-1, CREB, and CTREB target genes), whereas HT administration alleviated these alterations [95]. Taken together, these data suggest that OLE and/or its metabolite, HT, can be effective to combat cellular alterations underlying AD symptoms in the absence of undesirable side effects.

Finally, HT was shown to inhibit the toxicity associated with α-synuclein aggregation in PD [96]; HT and OLE improved spatial working memory and energetic metabolism in the brain of aged mice [97]; and HT decreased oxidative stress in the brain of db/db mice, a widely used human T2DM animal model, by improving mitochondrial function and inducing phase II antioxidative enzymes through the activation of the Nrf2–ARE pathway [98].

To date, less data have been reported for oleocanthal. Recently, in vitro and in vivo studies reported that oleocanthal enhances β-amyloid clearance as a potential neuroprotective mechanism [99,100].