*Review* **The Neuroprotective Role of Polydatin: Neuropharmacological Mechanisms, Molecular Targets, Therapeutic Potentials, and Clinical Perspective**

**Sajad Fakhri 1, Mohammad Mehdi Gravandi 2, Sadaf Abdian 2, Esra Küpeli Akkol 3, Mohammad Hosein Farzaei 1,\* and Eduardo Sobarzo-Sánchez 4,5,\***


**Abstract:** Neurodegenerative diseases (NDDs) are one of the leading causes of death and disability in humans. From a mechanistic perspective, the complexity of pathophysiological mechanisms contributes to NDDs. Therefore, there is an urgency to provide novel multi-target agents towards the simultaneous modulation of dysregulated pathways against NDDs. Besides, their lack of effectiveness and associated side effects have contributed to the lack of conventional therapies as suitable therapeutic agents. Prevailing reports have introduced plant secondary metabolites as promising multi-target agents in combating NDDs. Polydatin is a natural phenolic compound, employing potential mechanisms in fighting NDDs. It is considered an auspicious phytochemical in modulating neuroinflammatory/apoptotic/autophagy/oxidative stress signaling mediators such as nuclear factor-κB (NF-κB), NF-E2–related factor 2 (Nrf2)/antioxidant response elements (ARE), matrix metalloproteinase (MMPs), interleukins (ILs), phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt), and the extracellular regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). Accordingly, polydatin potentially counteracts Alzheimer's disease, cognition/memory dysfunction, Parkinson's disease, brain/spinal cord injuries, ischemic stroke, and miscellaneous neuronal dysfunctionalities. The present study provides all of the neuroprotective mechanisms of polydatin in various NDDs. Additionally, the novel delivery systems of polydatin are provided regarding increasing its safety, solubility, bioavailability, and efficacy, as well as developing a long-lasting therapeutic concentration of polydatin in the central nervous system, possessing fewer side effects.

**Keywords:** polydatin; neurodegeneration; neuroprotection; therapeutic targets; pharmacology; novel delivery system

#### **1. Introduction**

Neurodegenerative diseases (NDDs) are amongst the most common factors of disability and death in humans, which refer to the gradual, symmetrical, and specific decreases in sensory, motor, and mental nerve activity resulting in the death of neurons [1,2]. Nerve death accounts for various signs of neurological dysregulations, both chronic and acute, consisting of Parkinson's disease (PD), Alzheimer's disease (AD), central nervous system (Brain/Spinal Cord) injuries, and stroke [3]. Additionally, autism, neuropathic pain, aging, and depression are other NDDs that result from nerve cell death [4,5]. From a mechanistic

**Citation:** Fakhri, S.; Gravandi, M.M.; Abdian, S.; Akkol, E.K.; Farzaei, M.H.; Sobarzo-Sánchez, E. The Neuroprotective Role of Polydatin: Neuropharmacological Mechanisms, Molecular Targets, Therapeutic Potentials, and Clinical Perspective. *Molecules* **2021**, *26*, 5985. https:// doi.org/10.3390/molecules26195985

Academic Editors: Paula Silva and Norbert Latruffe

Received: 6 September 2021 Accepted: 30 September 2021 Published: 2 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

point of view, various factors cause neurological problems, such as oxidative stress [6], inflammation [7], and apoptosis [5,8]. The aforementioned pathological pathways play a harmful role in neuronal cell death mechanisms. The microglia activity, inflammatory cytokines, reactive oxygen species (ROS), and related mitochondrial disruption of oxidative pathways have shown negative results on the process of nerve regeneration that eventually leads to cell death [9,10].

Despite advances in clinical healthcare, neuroprotective agents are still clinically challenged in nerve destruction and NDDs. Thus, there is an emerging need to develop new multi-target therapies that further help to attenuate dysregulated signaling pathways in NDDs [11–13]. Several natural compounds isolated from edible and medicinal plants that exhibit anti-inflammatory properties have been investigated for potential application as pharmaceutical candidates [14]. Natural products are rich sources of polyphenolic compounds, consisting of stilbenoids, which are a big group of resveratrol substances such as monomers, dimers, and oligomers. Stilbenoids are naturally occurring compounds in a variety of plant families, such as Vitaceae, Gnetaceae, Cyperaceae, and Rocarpaceae. Consequently, the wine grape, *Vitis vinifera* L., is considered the primary nutritional source of these compounds [15].

Polydatin is a stilbenoid that passively penetrates cells. It also launches into the cells through an active mechanism by a glucose carrier. The glucose moiety of polydatin causes a higher resistance rate to enzymatic oxidation than resveratrol and has much better water solubility [16,17]. Polydatin has been shown to suppress oxidative stress, inflammation, and apoptosis as major pathways for nerve cell regeneration. The biological activity of polydatin and certain derivatives entails preventing or interfering with several neurodegenerative mechanisms [18].

In a previous study, the protective mechanisms of polydatin were evidenced in cerebral ischemia [19]. Recently, dementia-related disorders are also targeted by polydatin [20]. Besides, the general pharmacology and pharmacokinetic properties of polydatin were developed by Du et al. [21]. As of yet, no review article has discussed the entire set of neuroprotective mechanisms of polydatin. This review focuses on the pharmacological targets, molecular mechanisms, therapeutic potentials, and clinical perspectives of polydatin in NDDs. The pharmacological mechanisms of action of polydatin in the treatment or prevention of NDDs are provided.

#### **2. Polydatin: Chemical Structure, Sources, and Pharmacokinetic Properties**

Several studies concerning the chemical characterization of stilbenoids have been motivated by their numerous promising biological functions, especially those of polydatin. Polydatin (3,4- ,5-trihydroxystilbene-3-β-D-glucoside) is a natural resveratrol glucoside known as resveratrol-3-β-mono-D-glucoside, an active product from the *Polygonum cuspidatum* Sieb. et Zucc roots (Figure 1). However, it is also found in grapes, red wines, hop cones, peanuts, cocoa/chocolate products, and several other meals [21].

Two isomeric types (*cis* and *trans*) of polydatin are found in nature. *Cis*-polydatin is often detected in lower levels. Moreover, they are less biologically active than the *trans* forms [22]. The most common sources of polydatin are grape juice and red/white wines. *Cis*-polydatin is the predominant isoform in carbonated wines and rosé, while the *trans* isomer is abundant in berries, peanuts, grapes, and pistachios [23]. The major sources of polydatin isomers are the rhizomes and roots of *Fallopia japonica* (Houtt.) Ronse Decraene (Polygonaceae), which have long been used in traditional Chinese and Japanese Medicine as an anticancer, diuretic, analgesic, anti-pyretic, and expectorant agent in the management of atherosclerosis [24]. However, this product is present in various other genera such as *Rumex, Picea, Rosa, Quercus,* and *Malus*. Polydatin has received similar consideration to resveratrol because glucoside concentrations are usually higher than aglycone ones in red wine and other grape products. The exact ratio of glycosylated forms to aglycones in wine relies on various aspects such as the fermentation method and ecological conditions in the vineyards [25].

**Figure 1.** Polydatin, a glycosylated form of resveratrol.

Pharmacokinetic studies are often required for the effective and safe clinical use of drugs. The absorption, distribution, and metabolism of polydatin are connected to its bioactivity. Polydatin might have higher bioavailability and a better antioxidant function compared to resveratrol. In addition, intestinal absorption of polydatin is higher than resveratrol made by glucose groups [26]. Polydatin enters the cell through an active glucose carrier mechanism and passive diffusion, while resveratrol just passively penetrates cell membranes [27]. The active transport of polydatin mainly passes through a sodiumdependent glucose transporter 1 (SGLT1), chiefly present in the intestines and stomach [16]. Since the cell content of polydatin is not very low, it indicates an active transfer of polydatin by SGLT1 [21,27].

Polydatin employs two possible pathways to be deglycosylated from *trans*-resveratrol. The primary pathway is cleavage by cytosolic-β-glucosidase following the SGLT1 mediated by passing through the brush-border membrane. The second mechanism, which happens on the luminal side of the epithelium, is deglycosylation by the membrane-bound enzyme lactase-phlorizin hydrolase. This mechanism is followed by passive diffusion of the released aglycone and additional glucuronoconjugation [17]. Although resveratrol is more accumulated and leaves more residue in cells than polydatin, the half-life of polydatin is approximately four hours with a higher level of resveratrol Cmax at the same dose [27]. However, more analytical methods need to be investigated for the determination of *trans*-stilbene glycoside during pharmacokinetics studies [28].

Accordingly, polydatin as a glycosylated resveratrol could be a potential therapeutic agent with fewer pharmacokinetic limitations in comparison to resveratrol.

#### **3. Polydatin against NDDs**

Polydatin has demonstrated several biological/pharmacological effects, such as antiinflammatory [29], anti-apoptotic [30], and antioxidant [31], against NDDs [32]. To combat oxidative stress, polydatin increased antioxidant capacity through associated antioxidant mediators, nuclear factor erythroid 2-related factor 2 (Nrf2) and sirtuin 1 (Sirt1), and antioxidant response elements (AREs) [18]. Polydatin suppresses oxidative stress through phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt)-interconnected mediators [33]. It also blocks oxidative stress and reduces microglial apoptosis through the Nrf2/heme oxygenase (HO-1) pathway [34]. From the inflammatory point of view, by suppressing nuclear factor kappa B (NF-κB), polydatin can stop intercellular adhesion molecule-1 (ICAM-1) protein/mRNA production. Polydatin has also been shown to reduce pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) by down-regulating toll-like receptor-2 (TLR-2) and the

NF-κB p65 pathway [35]. As mitochondria are the major source of ROS in cells, when the intracellular mitochondria are damaged, electron transfer is abnormal, and ROS production is increased, which ultimately accelerates the onset of apoptosis [36]. Several studies have shown the beneficial influence of polydatin on mitochondria from a new perspective. Polydatin has been considered to suppress mitochondria-related cytochrome c release, moreover suppressing caspase-9 and caspase-3 [37]. Polydatin has been thought to decrease ROS release and improve mitochondrial activity by modulating the Sirt3/superoxide dismutase 2 (SOD2) pathway. SOD2 is a mitochondrial antioxidant enzyme whose activity is mediated by Sirt3 [38].

Overall, by modulating several mediators in inflammatory/apoptotic/autophagy/ oxidative stress pathways, polydatin could be a hopeful candidate in combating NDDs.

#### *3.1. Polydatin against AD, and Cognition/Memory Dysfunction*

As the most common form of NDDs, AD is characterized by a gradual decline in memory and mental impairment in all aspects of a person- s ability to perform daily activities, with unknown causes [39]. Studies have shown that the accumulation of old extracellular plaques, mainly consisting of the amyloid beta peptide (Aβ) and intracellular fiber nodules composed of hyperphosphorylated proteins, plays an essential role in the neuropathology of AD [40–42]. Besides, several inflammatory, apoptotic, and oxidative pathways are behind the pathogenesis of AD. Due to numerous pathophysiological mechanisms for AD, effective treatment has not yet been developed. Natural products have shown beneficial therapeutic effects on AD [43]. Amongst natural entities, oral administration of polydatin could dramatically reduce the production of malondialdehyde (MDA) and increase the activity of the antioxidants SOD and catalase (CAT) to protect learning and memory impairments in vivo. In addition, it lessened the damage caused by an oxygen-glucose deficiency in cultured neurons [44]. Tong et al. investigated the protective effect of polydatin in cancer patients undergoing chemotherapy, most of whom had cognitive impairments due to the use of chemotherapy drugs. In their study, polydatin, at a daily dose of 50 mg/kg, reduced doxorubicin-induced cognitive impairment and restored the hippocampal structure of the hippocampus. In addition, polydatin reduced doxorubicin-induced stress by regulating Nrf2, activating the NF-κB pathway, and reducing apoptosis [45,46]. In another study, polydatin was reported to defend against learning and memory failure in neonatal rats with hypoxic-ischemic brain injury (HIBI) caused by unilateral carotid artery ligation. In addition, polydatin decreased memory deficiency and increased the expression of the hippocampal brain-derived neurotrophic factor (BDNF) in rats with HIBI [47]. Moreover, in a study on rat's cognitive function exposed to chronic ethanol, polydatin increased cell survival while decreasing the expression level of cyclin-dependent kinase 5 (cdk5), and reversed functional defects in ethanol-treated mice evaluated by the Morris water test [48]. In another recent study, polydatin has shown protective roles against dementia-related disorders by attenuating several dysregulated pathways, including suppressing neuroapoptosis, oxidative stress, N-methyl D-aspartate receptor subtype 2B (NR2B), senile plaques, neurofibrillary tangles, and cholinergic dysfunctions [20]. Polydatin-mediated in vitro inhibition of Aβ25–35 polymerization and associated fibrils/oligomers was also reported by Rivière et al. [49,50]. As another anti-AD mechanism of polydatin, an in vitro increase in α3 and α7 nicotinic acetylcholine receptors (nAChRs) could help combat NDDs [51]. During an in vivo study, the modulation of NR2B by polydatin in rats' prefrontal cortex reduced learning and memory impairments [52].

Therefore, polydatin could be a helpful candidate in preventing AD and cognitive/memory impairment in various cases. Such an effect is exerted through the modulation of several dysregulated mechanisms, including neurological deficit scores, oxidative stress (e.g., Nrf2, SOD, CAT), inflammation (e.g., NF-κB), as well as Aβ, BDNF, and nAChRs.

#### *3.2. Polydatin against PD*

PD is an aging-associated condition and the second-most significant reason for NDDs [53]. PD is known for midbrain dopaminergic neuronal loss and the accumulation of α-synucleins called Lewy bodies. Furthermore, damages to non-dopaminergic pathways cause non-motor and motor malfunctions [54]. Owing to their poor effectiveness and adverse side effects, traditional therapies for PD are challenging to implement, and the development of novel innovative and safe agents is now needed. Oxidative stress and neuroinflammation play a significant role in PD pathogenesis [55]. Therefore, preventing the dysregulated mediators of these pathways has a considerable role in prohibiting the dissemination of PD. From a pathophysiological perspective, the degradation of substantia nigra dopaminergic neurons is caused by the hereditary sensitivity and response to harmful environmental stimuli [56]. Bai et al. reported that polydatin could play a critical role in combating PD. Besides, polydatin meaningfully decreased apoptosis and mitochondrial dysfunction during rotenone/Parkin deficiency induced in a human dopaminergic neuronal cell line, SH-SY5Y. In their study, polydatin suppressed the rotenone-induced cell death, mitochondrial membrane potential (MMP), Sirt 1, DJ1, and ROS production. Their study found that when autophagy-related gene 5 (Atg5) is biologically inhibited, the beneficial effects of polydatin are partly inhibited, implying Atg5-mediated neuroprotection [57]. Parkin knockdown-induced oxidative stress, mitochondrial malfunction autophagy deficiency, and mitochondrial fusion expansion were all alleviated by polydatin [58]. Polydatin therapy may also reverse abnormalities in mitochondrial morphology and motor malfunction in a Drosophila model of PD caused by Parkin insufficiency [57].

In the pathogenicity of PD, neuroinflammation hyperactivates microglia and results in the destruction of dopaminergic neurons. As a result, reducing microglial activity could help in the management of PD [59]. Polydatin crosses the blood–brain barrier to protect motor deterioration of substantia nigra and preserves dopaminergic neurons and motor function by suppressing pro-inflammatory mediators and microglia [60,61]. Huang et al. indicated that polydatin caused an increase in Nrf2, p-Akt, and p-glycogen synthase kinase-3β (GSK-3β) Ser9, activated microglial BV-2 cells, and suppressed NF-κB and proinflammatory mediators in the substantia nigra of PD rat-induced by lipopolysaccharide (LPS). Polydatin also inhibited dopaminergic neurodegeneration caused by microglial activation through modulating the Akt/GSK-3β/Nrf2/NF-κB signaling pathway [62]. It is worth noting the discrepancies on the anti/pro-inflammatory cytokines following microglia activation. It reveals the complexity of the brain microglial regulation, including the critical M1 (inflammatory microglia) and M2 (anti-inflammatory microglia). Microglia activations, especially the M1 type, have been considered a critical orchestrator in triggering inflammatory responses during NDDs. However, the production/release of inflammatory cytokines has been highlighted as a common feature associated with the microglial response, which is closely related to imbalanced protein homeostasis in NDDs [63]. So, modulating microglia activation could be a promising strategy for polydatin in combating NDDs.

The disturbance of glycolysis and the decrease in ATP production are other factors involved in the dysfunction of dopaminergic neurons and developing PD [64]. Zhang et al. showed that polydatin might improve glycolysis, glucose metabolism, ATP production, and motor dysfunction in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced early dopaminergic neuronal degeneration. In their study, polydatin prevented the loss of dopaminergic neurons in the striatum and substantia nigra, thereby suppressing neural apoptosis (Bax and cleaved caspase-3) and improving motor function in mice [65]. Suppressing complex I of the electron transport chain and heightened oxidative stress are among the first triggers in the pathogenesis of PD [66]. In an in vitro study, reducing lipid peroxidation, inhibiting apoptosis, and activating the mitogen-activated protein kinase (MAPK) are introduced as the primary neuroprotective mechanisms of polydatin on dopaminergic neurons [67]. A study by Ahmed et al. showed that polydatin (3 mg/kg, intraperitoneally) possessed a neuroprotective effect in attenuating the degeneration of dopaminergic neurons in nigro-striatal regions of the brain. They also indicated

that polydatin improved neuromotor behavior in a rat model of rotenone-induced PD. Thus, the protective effect of polydatin against striatal degeneration is presented in their report [68]. In a similar report, polydatin meaningfully prevented the rotenone-induced dysregulations of MDA, manganese SOD, glutathione, and thioredoxin in the striatum. Besides, polydatin inhibited the rotenone-induced neurodegeneration of dopaminergic neurons in the substantia nigra [61].

Polydatin, as a balancer, may thus be a treatment strategy in PD by reducing oxidative stress, as well as controlling autophagic mechanisms and mitochondrial fusion.

#### *3.3. Polydatin against Central Nervous System (Brain/Spinal Cord) Injuries*

Traumatic brain injury (TBI) is the leading cause of corporality, and permanent dysfunction has become a global public health problem [69]. People with severe TBI sometimes necessitate lengthy therapy. Treatments are missing due to the complexity and obscurity of the pathophysiological pathways in TBI [70]. TBI induced mitochondrial neuronal damage, as evidenced by an increase in ROS mitochondria and a reduction in MMP, causing the previous mitochondrial transition pore to open [69]. Polydatin has shown various pharmacological benefits, including antioxidation, anti-inflammation, anti-apoptosis, and brain-associated injuries [71,72].

Sprague–Dawley rats receiving 30 mg/kg polydatin intraperitoneally after TBI decreased in ROS and blocked TBI-induced MDA expression while increasing SOD levels in damaged cortices. In their study, polydatin prevented MMP collapse and the previous mitochondrial transition pore from opening TBI and reduced the endoplasmic reticulum stress response following TBI [69]. Consistently, polydatin significantly lowered endoplasmic reticulum stress-related unfolded protein activation, containing blocked p-extracellular regulated kinase (ERK) phosphorylation, declined spliced XBP-1, and cleaved activating transcription factor 6 (ATF6) production, as well as increasing the expression of glucoseregulated proteins (GRP78). Besides, polydatin regulated the p38MAPK signaling pathway and the mitochondrial apoptotic pathway (e.g., caspase-3/9) and improved neurological scores and the length of survival in TBI rats [69]. In another report, polydatin protected against SCI by suppressing oxidative stress and apoptosis passing through Nrf2/HO-1 signaling in vitro and in vivo [34]. Polydatin also increased neuronal viability and protected against oxygen-glucose deprivation/re-oxygenation-induced mitochondrial injury and apoptosis in a dose-dependent manner. Besides, polydatin modulated the activity of neuronal mitochondria, including MMP, intracellular calcium levels, the opening of the mitochondrial permeability transition pore (mPTP), ROS generation, and adenosine triphosphate levels. From a mechanistic perspective, polydatin suppressed Keap1 and upregulated Nrf2/HO-1 and NAD(P)H Quinone Dehydrogenase 1 (NQO-1) in oxygenglucose deprivation/re-oxygenation-treated spinal cord motor neurons. Additionally, polydatin reversed the mitochondrial and neuronal damage induced by spinal cord ischemia/reperfusion in a mouse model, partially suppressed by the Nrf2 inhibitor. This represents that the neuroprotective effects of polydatin pass through the Nrf2/ARE pathway [73]. The engagement of Nrf2 on neuronal differentiation in both in vivo and in vitro studies are also provided by Zhan et al. [74]. The involvement of Nrf2/ARE in the protective effects of polydatin is also presented in other reports [75]. In this line, the inhibitory effect of polydatin on ferroptosis was shown both in vitro and in TBI mice. Those responses were applied by preventing the accumulation of free Fe2+, increasing MDA, and decreasing glutathione peroxidase (GPx) [76].

The most common causes of traumatic spinal cord injury (SCI) are motor/car collisions, abuse, and falls [77]. Not unexpectedly, epidemiological trials discovered that SCI mainly existed in young males and resulted in lifelong cognitive defects that significantly reduce their life quality [78]. SCI is characterized by various symptoms, including limb paralysis, a loss of feeling in the lower extremities, and uracratia or uroschesis. A growing body of research suggests the aggregation of inflammatory cytokines across the compromised spinal cord and is amongst the main risk aspects for SCI pathological symptoms [10,11].

Findings indicated that several pro-inflammatory cytokines, including the macrophage migration inhibitory factor (MIF), interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α, are intensified steadily after compression-induced SCI [9]. To modulate these mechanisms, polydatin was injected into adult male Sprague–Dawley rats in a single intraperitoneal dose. In this line, polydatin significantly reduced spinal cord edema and morphological changes in vivo. It also decreased nitric oxide (NO) in spinal cord tissues of SCI rats, which was consistent with the pattern of inducible nitric oxide synthase (iNOS) production. Accordingly, LPS increased protein and mRNA levels of iNOS in BV2 cells, and polydatin reversed these changes [78]. Consequently, polydatin decreased the LPS-induced rise in NO and response to inflammatory microglia. Polydatin also significantly reduced IL-6, IL-1, and TNF-α after a single injection and inhibited the development of inflammatory cytokines in spinal cord tissues following SCI. Besides, polydatin blocked LPS-induced NF-κB activation in BV2 microglia and inhibited the activity of NLRP3 inflammasomes [78]. This stilbene attenuated TBI-induced acute lung injury by suppressing the S100B-mediated formation of neutrophil extracellular traps [79]. Polydatin also meaningfully decreased MDA while increasing SOD, GPx, CAT, and the level of total antioxidant capacity in the brain and liver. Besides, polydatin reduced inflammatory mediators of serum, such as IL-6, IL-1β, and TNF-α. It also modulated the D-galactose-induced caspase-3 and Bcl-2/Bax ratio elevation in the liver and brain [30].

Altogether, the critical role of polydatin in the modulation of Nrf2/ARE, ERK/MAPK, and interconnected apoptotic/inflammatory pathways could pave the road in the modulation of brain/SCI injuries.

#### *3.4. Polydatin against Stroke: As a Coupled Complication to NDDs*

Stroke is one of the most severe cerebrovascular disorders, affecting patients' quality of life [80]. Further pieces of evidence and mechanisms of polydatin protect against cerebral ischemia. Two different shreds of evidence have been mentioned, namely the inhibition of the neurological deficit score and limiting the brain infarction volume in rats with middle cerebral artery occlusion after being treated with polydatin. Several mechanisms have been provided for these two effects of polydatin [81].

Ischemic stroke increases neuroinflammation and ROS. Shah et al. investigated the neuroprotective activity of polydatin against ischemic brain damage in a rat model of chronic middle cerebral artery occlusion (MCAO). Their results indicated that polydatin minimized infarction volume and mitigated neurobehavioral defects by limiting the activation of p38MAPK and c-Jun N-terminal kinase, thereby suppressing neuroinflammation and ROS. They also demonstrated that polydatin upregulated the endogenous antioxidants Nrf2, HO-1, and the thioredoxin pathway, and reduced inflammation and ROS in cortical tissue [82]. As previously mentioned, inflammation and oxidative stress are two major factors in cerebral ischemic pathogenesis. In this line, NF-κB activation plays a critical role in inflammation. Besides, low levels of glioma-associated oncogene Patched-1 (Ptch1), homolog1 (Gli1), and SOD1 will lead to oxidative stress. Ji et al. demonstrated that polydatin could protect the brain of rats with permanent MCAO. Such effects were exerted by modulating inflammation via lowering NF-κB and the attenuation of oxidative stress through increasing Ptch1, Gli1, SOD1 expression, as well as ameliorating blood–brain barrier permeability [83]. Besides, the neuroprotective effects of polydatin on neurological function and the Nrf2 pathway of rats with cerebral hemorrhage were identified. Their study showed that polydatin enhanced neurological function and decreased oxidative stress in rats by controlling the Nrf2/ARE pathway and downstream gene production [84]. Mitochondrial dysfunction and apoptosis are involved in the process of ischemic stroke. In the study of Gao et al., the neuroprotective effect of polydatin was evaluated. Their results demonstrated the anti-apoptotic effect of polydatin and improved mitochondrial dysfunction due to ischemic/reperfusion injury in a rat MCAO model. Increasing Bcl-2 and decreasing cytochrome c, Bax, and caspases-3/9 are centrally associated protective mechanisms [37].

Considering the role of cell adhesion molecules (CAMs) in developing ischemia/ reperfusion-induced cerebrovascular diseases in a rat MCAO model, Cheng et al. found that polydatin can reduce the volume of brain infarction by decreasing the levels of CAMs in comparison to the control group, as well as the involvement of E-selectin, L-selectin, integrins, ICAM-1, and vascular cell adhesion molecule-1 (VCAM-1) [85]. Metastasisassociated lung adenocarcinoma transcript 1 (MALAT1) is a non-coding RNA that has a role in protecting the blood–brain barrier after an ischemic event. In the study of Ruan et al., it has been demonstrated that polydatin could upregulate the expression of MALAT1. Polydatin initiated a MALAT1/CREB/PGC-1α/PPARγ cascade that eventually led to protecting cerebrovascular endothelium and blood–brain barrier integrity from ischemia [81]. Moreover, Chen et al. discovered that high doses of polydatin could reduce edema, inflammation, and apoptosis after an ischemic event in the brain tissue of rat models with MCAO by regulating the expression of p53 and Notch1. The scores for the neurological function and behavioral scores were also improved in such models [86]. During an in vitro study, the protective effects of polydatin have also been shown in influencing the regulation of neuroglobin (Ngb) promotor activity and mRNA expression [87]. Polydatin might also regulate gene expression of Ngb through the attenuation of CREB, HIF-1α, p56, and early growth response protein 1 (Egr1). Besides, a polydatin-associated reduction in NO was also related to Ngb up-regulation [88,89]. From another point of view, polydatin meaningfully inhibited cerebral edema in cerebral hemorrhage rats by suppressing excitatory amino acids [90].

Beyond the stroke, polydatin has shown several other neuroprotective effects. For instance, in the study of Guan et al., polydatin potentially showed anxiolytic effects and suppressed neuroinflammation in a chronic pain mouse model by reducing proinflammatory cytokines, including TNF-α and IL-1β in the amygdala [91].

Different mechanisms are employed by polydatin to combat stroke and anxiety, including Nrf2/HO-1/ARE, Bax/caspases, Egr1/Ngb, CREB, and PGC-1. Additionally, antioxidant activity, an improvement in mitochondrial health, free-radical scavenging, anti-apoptotic/anti-inflammatory activities, up-regulation of BDNF/Shh/Ngb pathway, and down-regulation of CAMs are other protective mechanisms of polydatin [19,92].

The entire set of neuropharmacological characteristics of polydatin against AD, PD, TBI/SCI, and stroke are presented in Table 1. Overall, by employing several mechanisms and the modulation of various dysregulated pathways, polydatin could be a promising neuroprotective phytochemical against PD, AD, TBI/SCI, and stroke (Figure 2).


**Table 1.** Neuropharmacological mechanisms of polydatin against different NDDs.


**Table 1.** *Cont.*

AD: Alzheimer's disease, Akt: Protein kinase B, Atg5: Autophagy Related 5, ATP: Adenosine triphosphate, Aβ: Amyloid beta, Bcl-2: B-cell lymphoma 2, BDNF: Brain-derived neurotrophic factor, BMSCs: Bone marrow mesenchymal stem cell, CAT: Catalase, Cdk5: Cyclin dependent kinase 5, DPPH: 2,2-diphenyl-1-picrylhydrazyl, Egr1: Early growth response 1, ERK: Extracellular-signal-regulated kinase, GRP78: Glucose-regulated protein, GPx: Glutathione peroxidase, GSH: Glutathione, GSK-3β: Glycogen synthase kinase-3β, GSSG: Glutathione disulfide, HEK-293T: Human embryonic kidney cells, HIBI: Hypoxic-ischemic brain injury, HIF-1α: Hypoxia-inducible factor 1-alpha, HO-1: Heme oxygenase-1, ICAM-1: Intercellular adhesion molecule-1, IL: Interleukin, iNOS: Inducible nitric oxide synthase, LPO: Lipid peroxidation, LPS: Lipopolysaccharides, MALAT1: Metastasis associated lung adenocarcinoma transcript 1, MAPK: Mitogenactivated protein kinase, MCAO: Middle cerebral artery occlusion, MDA: Malondialdehyde, MMP: Matrix metalloproteinase, MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells, Ngb: Neuroglobin, NO: Nitric oxide, Nrf2: Nuclear factor E2-related factor 2, OGD: Oxygen-glucose deprivation, PD: Parkinson's disease, PTCH1: Protein patched homolog 1, ROS: Reactive oxygen species, SCI: Spinal cord injury, SMNs: spinal motor neurons, SOD: Superoxide dismutase, TBI: Traumatic brain injury, TNF-α: Tumor necrosis factor α.

**Figure 2.** Polydatin employs several mediators to combat PD, AD, TBI/SCI, and stroke.

#### **4. Polydatin Novel Delivery Systems: Nanoformulations, and Targeted Therapy**

Nanomedicine is the medicinal use of nanotechnology that employs biocompatible, low-toxicity nanomaterials and nanoparticles to control drug pharmacokinetics, administration rate, and bioavailability [96]. In addition, polydatin may guard against brain injury, kidney problems, heart failure, and improve glucose and lipid metabolism [97,98]. However, therapeutic activities of polydatin are constrained due to weak water solubility, the chemical imbalance in aqueous alkaline medium, and substantial first-pass metabolism. To address these limitations, recyclable nanostructures have sparked wide attention because of their potential in drug delivery and successful removal from the body [11]. In this way, chitosan-loaded nanoparticles administrated daily by gastric intubation for about one month improved the effect of polydatin in male Wistar albino rats [99].

In diabetes mellitus (DM), polydatin was used because of its various therapeutic mechanisms consisting of controlling free-radical production and mitochondrial activity, as well as regulation of inflammation and oxidative stress [97,98]. The anti-hyperglycemic and antioxidant effects of polydatin resulted in a substantial reduction in hemoglobin A1C in treated diabetic rats, and treatment resulted in a significant increase in hepatic glycogen levels, which may be secondary to improved insulin levels and intervention [98].

Apart from its low water solubility, the reduced effectiveness and safety risk of polydatin must be addressed before being used in clinical trials. In this way, microenvironmentsensitive nanoparticles have shown considerable promise in increasing the bioavailability of lipophilic substances [100]. The depletion of liver fibrosis in mice given a polydatinloaded micelle (PD-MC) was verified by measuring hydroxyproline and fibrotic parameters, including collagen type 1 (Col1), tissue inhibitor of metalloproteinases 1 (TIMP-1), transforming growth factor-beta (TGF-β), and PD-MC, which not only inhibited hepatocyte apoptotic cell death but also showed anti-inflammatory properties. The anti-inflammatory activity of PD-MC was linked to its ability to suppress the ROS and TLR4/NF-B p65 signaling pathway. The mice treated with PD-MC had significantly less hepatic oxidative stress due to the lower levels of 4-Hydroxynonenal (4-HNE) [101].

Polydatin has a clear impact on the cardiac system, acting as an anticoagulant, antiinflammatory, anti-atherosclerotic, anti-hypercholesterolemic, and anti-ischemic agent. It reduces platelet accumulation, increases microcirculation, strengthens the endothelium and nervous system, and relieves coughing and asthma, which can be found to manage shock [21]. However, the limited oral bioavailability (half-life 8–14 min) and low solubility (the highest solubility is estimated to be 30 g/mL in water at 25 ◦C) of polydatin has restricted its administration [21,102]. Accordingly, liposomes have shown increased solubilization and stabilization while also providing good drug concentrations for water-soluble and lipid-soluble medicines. The polydatin-loaded liposomes (10 mg/kg) system was balanced in Sprague–Dawley rats. The long-lasting characteristics of the polydatin-loaded liposomal system can improve the absorbance of polydatin in the digestive system, but there are no organ histopathologic modifications after treatment with the polydatin-loaded liposome [102].

In cancer, the traditional treatment options, such as surgery, chemotherapy, radioactivity, immunotherapy, and hormonal treatments, are inadequate for controlling cancer progression [103]. In this way, polydatin possesses various properties such as anti-proliferative, antioxidant, anti-inflammation, and immunomodulatory. For improving the anticancer effectiveness of polydatin and other novel therapies, the production of nanoparticles has received much attention [104]. So, oral administration of polydatin-loaded poly (lactic-coglycolic acid) [PLGA] nanoparticles (polydatin-PLGA-NPs) in Syrian hamsters resulted in lower amounts of lipid peroxidative byproducts. Polydatin-PLGA-NP therapy decreased tumor histological symptoms from extreme to mild and blocked the development of squamous cell carcinoma. Besides, the administration of polydatin-PLGA-NPs led to a substantial reduction in tumor volume and occurrence. Polydatin-PLGA-NPs significantly increased enzymatic antioxidant rates such as SOD, CAT, and GPx, while decreasing the rate of cytochrome (Cyt) p450, Cyt b5, glutathione S-transferase, gammaglutamil transferase, and glutathione reductase activities, which are among the metabolizing enzymes of phases I and II. Polydatin-PLGA-NPs treatment caused apoptosis via sheared caspase-3 overexpression and the prevention of dimethyl benzyl anthracene-induced mutant p53 and cyclin-D1 production in a dose-dependent manner [105]. As another disorder, irritable bowel syndrome is currently thought to result from dysfunction in the brain—gut axis, including both central and peripheral pathways concerned, and in particular, involving cannabinoid receptors and affecting the activity of most cells. To modulate these dysregulated mechanisms, the effect of a co-micronized form of palmitoylethanolamide/polydatin was examined in 157 patients with irritable bowel syndrome [106].

Altogether, in addition to its high effectiveness and the more appropriate pharmacokinetic characteristics of polydatin, using novel delivery systems for this secondary metabolite could increase the associated efficacy and reduce some of the remaining limitations of phytochemicals, by increasing solubility/bioavailability and decreasing safety risks. Figure 3 shows the novel delivery systems of polydatin.

**Figure 3.** Novel delivery systems of polydatin: Reduction in the pharmacokinetic limitations.

#### **5. Conclusions**

Polydatin is a multi-target stilbenoid secondary metabolite extracted from herbal sources. As polydatin is a glycosylated form of resveratrol, several biological activities and health benefits are connected to the administration of polydatin, including cardioprotective, hepatoprotective, and neuroprotective factors. Prevailing studies focus on the neuroprotective potential of polydatin by employing several mechanisms, including Nrf2/Keap1/ARE, PI3K/Akt, ERK/MAPK, TLR/NF-κB/TNF-α/ILs, and Bax/Bcl-2/caspases (Figure 4). In this line, polydatin critically modulates inflammatory, apoptotic, and oxidative mediators towards combating AD, PD, stroke, CNS injuries, and miscellaneous neuroprotective responses. On the other hand, the pharmacokinetic drawbacks of polydatin, including their poor bioavailability, low solubility/selectivity, low plasma concentration, rapid metabolism, and chemical degradation, limit the associated therapeutic uses. It reveals the importance of novel drug delivery systems to reduce the restrictions in modulating tumor cell senescence. It is also worth noting that providing a novel delivery system could potentially help the polydatin to pass through the blood–brain barrier and develop a long-lasting therapeutic concentration of drugs in the CNS, while possessing fewer side effects [107–109].

In the present study, the pharmacological targets, molecular mechanisms, and therapeutic potentials of polydatin are highlighted through the attenuation of inflammatory/apoptotic/oxidative pathways to tackle multiple dysregulated pathways in NDDs. The need to provide novel delivery systems of polydatin, including nanoformulations, and targeted therapy is also considered. Further pre-clinical studies are needed to elucidate the precise neuroprotective mechanisms of polydatin followed by well-controlled clinical trials.

**Figure 4.** Neuroprotective mechanisms of polydatin.

**Author Contributions:** Conceptualization, S.F., M.H.F. and E.S.-S.; drafting of the manuscript, S.F., M.M.G. and S.A.; software, S.F., reviewing and editing of the paper: S.F., E.K.A., M.H.F. and E.S.-S.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** E.S.-S. thanks Proyecto Interno I+D+I UCEN (CIP2020036) for financial support.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **Abbreviations**



#### **References**


### *Review* **Effects of Wine Components in Inflammatory Bowel Diseases**

**Josip Vrdoljak 1,2, Marko Kumric 1, Tina Ticinovic Kurir 1,2, Ivan Males 3, Dinko Martinovic 1, Marino Vilovic <sup>1</sup> and Josko Bozic 1,\***


**Abstract:** With the rising prevalence of Inflammatory bowel disease (IBD) worldwide, and the rising cost of treatment with novel biological drugs, there is an increasing interest in various diets and natural foods as a potential way to control/modulate IBD. As recent data indicates that diet can modify the metabolic responses essential for the resolution of inflammation, and as wine compounds have been shown to provide substantial anti-inflammatory effect, in this review we aimed to discuss the current evidence concerning the impact of biological compounds present in wine on IBD. A number of preclinical studies brought forth strong evidence on the mechanisms by which molecules in wine, such as resveratrol or piceatannol, provide their anti-inflammatory, anti-oxidative, antitumor, and microbiota-modulation effects. However, concerning the effects of alcohol, it is still unclear how the amount of ethanol ingested within the framework of moderate wine consumption (1–2 glasses a day) affects patients with IBD, as human studies regarding the effects of wine on patients with IBD are scarce. Nevertheless, available evidence justifies the conductance of large-scale RCT trials on human subjects that will finally elucidate whether wine can offer real benefits to the IBD population.

**Keywords:** wine; inflammatory bowel disease; resveratrol; polyphenols; Crohn's disease; ulcerative colitis; diet; inflammation

#### **1. Introduction**

Inflammatory bowel disease (IBD) consists of a spectrum of chronic, non-communicable, multifactorial diseases of the gastrointestinal tract. Two main types of IBD are Crohn´s disease (CD) and ulcerative colitis (CD) [1]. The global prevalence of IBD is rising, increasing the load on the working population and the healthcare system [2–4]. There are a lot of unknowns in the etiology and pathophysiology of IBD. It is considered that IBD occurs in a complex interplay of susceptible genes, an ill-fitted diet, changed intestinal microbiota, and a pathologic immune response to dietary elements and intestinal microbes [5–7]. The current treatment modalities for IBD consist of anti-inflammatory drugs (salicylates etc.), immunosuppressants (azathioprine, corticosteroids, etc.), biological medications (anti-TNF-α, anti-integrin, cytokine-targeted therapy), and surgical treatment [8–10]. Besides these therapeutic modalities, there are also dietary therapies such as Exclusive Enteral Nutrition [11,12]. In addition, there is an increasing interest in various diets and natural foods as a potential way to control/modulate IBD [13]. The usual suspects being the Paleolithic diet, low-FODMAP diet, gluten-free diet, specific carbohydrate-based diet, and the Mediterranean diet (MedDiet) [12–14]. Interestingly, recent data indicates that diet can modify the metabolic responses essential for the optimal healing of injury-induced inflammation, as nutrients can act as signaling agents [14]. Lately, the MedDiet, primarily through the usage of red wine and olive oil, is coming to the forefront of prevention and

**Citation:** Vrdoljak, J.; Kumric, M.; Ticinovic Kurir, T.; Males, I.; Martinovic, D.; Vilovic, M.; Bozic, J. Effects of Wine Components in Inflammatory Bowel Diseases. *Molecules* **2021**, *26*, 5891. https:// doi.org/10.3390/molecules26195891

Academic Editor: Paula Silva

Received: 7 September 2021 Accepted: 24 September 2021 Published: 28 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

management of chronic diseases, including cardiovascular disease (CVD), diabetes mellitus type 2, cancer, and IBD [15–17].

The Mediterranean diet is a dietary pattern commonly found in the olive oil treegrowing parts of the Mediterranean basin [15,18]. Red wine and olive oil have been a central part of the Mediterranean diet since the days of the ancient Greeks and Romans. Even since those days, people have talked about the health benefits of olive oil and wine, but only now, in modern times, can we dissect these foods and look at the molecules that give them the desired effects on health.

This review will discuss the current evidence concerning the impact of biological compounds found in wine on IBD. We will describe the composition of wine and the impacts of wine as a whole or its isolated compounds on IBD pathophysiology by reviewing in vitro, animal, and human studies, whilst respecting that a limited amount of these compounds are found in wine.

#### *1.1. Wine Composition*

Wine, one of the oldest alcoholic beverages, is created during a process of grape must fermentation. The main constituents of wine are water, ethanol (usually between 9–15%), carbohydrates, organic acids (malic acid, citric acid, tartaric acid, etc.), as well as polyphenols, and volatile compounds [19,20]. The technological process of winemaking and the different types of grapes used offer a plethora of different wines with varying levels of alcohol and polyphenols [19,21]. Specifically, the concentration of phenolic compounds within grapes is dependent on grape variety, growing season, soil type, maturity of wine, as well as environmental and climatic conditions [22]. The phenolic composition (both concentration and composition) changes mainly in the first steps of vinification and continues during storage. In line with this, it is important to address that phenolic composition of the final wine differs from the composition of the corresponding grapes as a consequence of production of new derivatives, such as tyrosol, flavenes, and free phenolic acids [23]. The total content of phenolic compounds in grapes is affected by several factors: cultivar, the geographic origin, year of production, soil chemistry, degree of maturation, as well as solar radiation and temperature [23]. For all these reasons, estimation of polyphenol content in wine is rather challenging, and consequently, it represents a major obstacle in creation of "standardized moderate consumption" of wine.

Polyphenols are considered the main bioactive components in wine that positively affect health (prevention and management of non-communicable diseases) [19,24]. Because all grape parts are used during the winemaking process of red wine, the polyphenolic content is much higher in red wine when compared to white wine (1–5 g/L vs. 0.2–0.5 g/L) [18]. Polyphenols consist of a wide variety of chemical compounds that are generally classified into two main branches: flavonoids, and non-flavonoids [25]. Flavonoids are represented by flavonols (quercetin and myricetin), chalcones, flavononols, flavanols (catechin and epicatechin), flavones, anthocyanidins and isoflavonoids. Non-flavonoids are represented by phenolic acids, stilbenes (resveratrol), coumarins, lignans, and tannins [25,26]. Most of these compounds have demonstrated some or all of these desired effects: antioxidant, anti-inflammatory, anti-cancer, and anti-microbial (Figure 1) [27,28].

**Figure 1.** Multiple molecular targets of wine polyphenols contributing to its anti-inflammatory and anti-oxidant effects, changes in intestinal permeability and gut microbiota. Abbreviations: PAMPs: Pathogen-associated molecular patterns; ZO-1: Zonula occludens-1; TLR: Toll-like receptor; Nrf2: nuclear factor erythroid-derived 2; MyD88: Myeloid differentiation primary response 88; iRAKS: Interleukin-1 receptor associated kinase; TRAF6: Tumor necrosis factor receptor (TNFR) associated factor 6; TAK1: transforming growth factor-β-activated kinase 1; NF-κB: nuclear factor kappa-light-chainenhancer of activated B cells; IκB: inhibitor of nuclear factor kappa B.

#### *1.2. Wine Composition*

Since IBD is a chronic, non-communicable, inflammatory disease, a rising number of research papers are trying to ascertain the potential positive effects of wine or its components on IBD. Many of the phenolic compounds in wine have low bioavailability, and hence, reach low concentrations in the bloodstream, while their high content present in the gut can produce a more significant effect on enterocytes and the bacterial flora [29,30].

#### 1.2.1. The Evidence In Vitro

The pathophysiology of IBD consists of an aberrant immune response of the gut, with an increased expression of pro-inflammatory cytokines and an increased creation of reactive oxygen species (ROS). This cascade's main factors are COX-2, iNOS, IL-8, TNF-α, and NF-κB. Furthermore, a critical mechanistic determinant of IBD is a dysfunctional intestinal barrier, as seen through altered expression and subcellular distribution of tight junction (TJ) proteins [31]. Nunes et al. have demonstrated how a polyphenolic extract from Portuguese red wine decreased the paracellular permeability in cytokine-stimulated HT-29 colon epithelial cells. The red wine extract induced a significant increase in the mRNA of the barrier-forming TJ proteins occludin, claudin-5, and *zonula occludens* (ZO)-1 compared to control cells. It also led to less formation of claudin-2 mRNA, which is a channel-forming protein usually induced by pro-inflammatory conditions [31]. One other paper that focused on the impact of wine-digested fluids on gut microbiota has shown an increase in *Akkermansia*, *Selenomonadaceae*, and *Megasphaera* genus levels, as well as a positive change in short-chain fatty acid (SCFA) levels which could lead to decreased paracellular permeability [32].

On the other hand, a study by Asai et al. exhibited how a low and acute dose of ethanol leads to apoptotic cell death in confluent Caco-2 cells and, therefore, impairs intestinal barrier function [33]. Interestingly, these positive impacts of wine polyphenols on intestinal permeability in vitro are in contrast to in vivo findings by Swanson et al., where moderate wine consumption led to increased intestinal permeability [34].

Considering intestinal permeability, in vitro evidence on the impact of red wine extract and wine digested fluids suggests a protective effect on the cellular barrier [31,32]. This is in stark contrast to the in vitro evidence using an acute dose of ethanol and the in vivo evidence provided by Swanson et al. [33,34]. Hence, it is probable that the polyphenolic content per se has a positive effect on intestinal permeability, while the alcoholic content potentially negates that effect.

Moreover, another study with Portuguese red-wine extract enriched in anthocyanins exhibited an anti-inflammatory effect in HT-29 colon epithelial cells stimulated with proinflammatory factors (TNF-α, IFN-γ, and IL-1). It was shown how the wine extract decreases COX-2 activity, the synthesis of iNOS and IL-8, as well as decreases the degradation of inhibitor of NF-κB [35]. Nevertheless, the polyphenol-enriched red wine extract contains a higher concentration of polyphenols than the concentration ingested in the usually recommended one glass of wine a day.

Considering the role of oxidative stress in IBD pathophysiology, many researchers have investigated the potential benefits of antioxidants. A study by Deiana et al. has shown how extracts from three different Sardinian grape varieties applied to Caco-2 cell monolayers counteracted the oxidative activity in a tert-butyl hydroperoxide (TBH) induced oxidative damage model [36]. Furthermore, Tannin procyanidin B2 has also exhibited protective activity against oxidative stress in the human colonic Caco-2 cell model by up-regulating glutathione S-transferase P1 (GSTP1) via s ERK and p38 MAPK activation and Nrf2 translocation [37]. A study on the potential anti-oxidative effect of resveratrol on porcine intestinal-epithelial cell line (IPEC-J2) treated with deoxynivalenol (DON), has shown a reduction in ROS levels via Nrf2 signaling pathway activation [38]. Moreover, numerous studies have exhibited the anti-inflammatory effects of resveratrol in intestinal cells. In one study on Caco-2 cells exposed to bacterial lipopolysaccharide, resveratrol reduced the rate of degradation of an endogenous NF-κB inhibitor (IκB), and therefore led to a reduction of NF-κB activity with a decrease in COX-2 expression [39]. Another beneficial effect of resveratrol is on alleviating mitochondrial dysfunction [40]. When resveratrol was applied in extremely high concentrations it prevented indomethacininduced mitochondrial dysfunction in Caco-2 cells [40,41]. In another study on Caco-2 cells stimulated with LPS, where researchers studied the effects of polyphenols from red wine, cocoa and green tea they found how a dietary dose moderately modulates intestinal inflammation, but does not increase HDL production [42].

A number of in vitro experiments using batch culture fermentation have shown beneficial effects of wine components on fecal microbiota. The common impact seen is a growth enhancement of *Bifidobacterium* spp. and *Lactobacillus* spp., with growth inhibition of the *Clostridium* group [43–47]. These experiments show how wine and its components have a prebiotic effect on the "good" gut bacteria while also showing anti-microbial results on those bacteria that could lead to intestinal pathology.

Overall, the above-mentioned in vitro experiments provide evidence that wine and wine polyphenols have the following benefits: (i) they decrease the activity of NF-κB and therefore decrease the production of pro-inflammatory cytokines, (ii) activate Nrf2 signalling pathway and therefore reduce ROS levels, (iii) polyphenols (quercetin) bind to the ubiquinone site of complex I protecting it from inhibitors like indomethacin and decreasing mitochondrial dysfunction, (iv) wine polyphenols support the growth of healthy microbiota and inhibit the growth of pathologic microbiota (Table 1).


**Table 1.** In vitro studies on wine polyphenols and intestinal inflammation.

#### 1.2.2. The Evidence on Animal Models

As reviewed by Nunes et al., the benefits of resveratrol, a key active molecule present in red wine, have also been confirmed in animal models of IBD and intestinal cancer [38]. For example, Martin et al. have shown that, in an early colonic inflammation model caused by trinitrobenzenesulphonic acid (TNBS) instillation in rats, resveratrol (5–10 mg/kg/day) administration has significantly decreased the index of neutrophil infiltration and levels of proinflammatory cytokine IL-1β, whilst reducing the degree of colonic injury [48]. Furthermore, the same researches later showed how resveratrol extended its benefits to

a rat model of chronic gut inflammation caused by TNBS. Resveratrol treatment led to decreased neutrophil infiltration, reduced TNF-α levels, reduced COX-2 and the NF-κB p65 protein expression, and also led to a significant increase of TNBS-induced apoptosis in colonic cells [49]. Moreover, another study on the DSS-induced colitis mouse model, but with a diet enriched with resveratrol, showed attenuation of colitis signs and symptoms. The mice that ate a resveratrol enriched diet (at 20 mg/kg of diet) maintained their body weight and had less diarrhoea and rectal bleeding. All the mice in the treatment group survived compared to the 40% mortality rate in the non-resveratrol group. The same study also showed a significant reduction in proinflammatory cytokines, TNF-alpha and IL-1beta, and an increase of the anti-inflammatory cytokine IL-10 [50].

Moreover, Larrosa and associates demonstrated on rats with DSS-induced colitis how low doses of resveratrol, similar to the dosage contained in a hypothetical daily diet of a person weighing 70 kg, lead to a reduction in mucosal levels of inflammatory markers [51]. Similar to the previously mentioned studies, prostaglandin E (PGE)-2, COX-2, and PGE synthase-1 were affected. In addition, this study showed an increase in *Bifidobacterium* and *Lactobacillus* spp. with a reduction in *E. coli* growth [51]. This is in line with other studies that have shown that resveratrol enhances the growth of *Lactococcus lactis*, whilst inhibiting the growth of *Enterococcus faecalis* [52]. Importantly, the relation between resveratrol and gut microbiota is a two-way street [53]. On one hand, resveratrol modulates gut microbiota, yet on the other, resveratrol can be transformed by gut microbiota into various bioactive metabolites.

In a DSS-induced colitis mouse model, Li et al. have exhibited that mice fed with muscadine grape phytochemicals (MGP) or muscadine wine phytochemicals (MWP) for 14 days had decreased levels of proinflammatory cytokines (IL-6, TNF-α), reduced myeloperoxidase activity, while also preventing weight loss and preserving colonic length [54]. A study researching the effects of grape seed extract (GSE) in a rat model of DSS-induced ulcerative colitis yielded promising results. Male Sprague-Dawley rats were fed daily (days 0–10) with GSE (400 mg/kg), and compared with no-GSE controls, GSE-fed rats had significantly decreased ileal villus height (14%; *p* < 0.01) and mucosal thickness (13%; *p* < 0.01), approaching the values of healthy controls. GSE also reduced the qualitative histological severity score (*p* < 0.05) in the proximal colon, but there was no significant effect in the distal colon [55]. In a study investigating the effects of proanthocyanidins from grape seed (GSPE) in a TNBS-induced recurrent ulcerative colitis rat model, Wang et al. demonstrated how GSPE treatment led to a recovery of pathologic changes in the colon, reduced the colonic weight/length ratio, and improved the macroscopic and microscopic damage scores. Furthermore, iNOS and myeloperoxidase activities were significantly reduced in the GSPE group, while the superoxide dismutase and gluthatione peroxidase activities were significantly increased [56].

These data indicate that proanthocyandins and wine/grape phytochemicals have similar and probably synergistic effects on the colonic mucosa. The main characteristics exhibited were modulation of the inflammatory response, inhibition of inflammatory cell infiltration, a reduction in ROS-damage, and a promotion of colonic tissue repair and regeneration [53–56].

Correspondingly, and as reviewed previously, a number of animal studies have exhibited the benefits of resveratrol in decreasing the risk of colon cancer in animal models with chronic intestinal inflammation [40]. In one study on a DSS colitis mouse model, they found how resveratrol inhibited the formation of polyps, and also reduced cell damage and subsequent proliferation of epithelial cells in the intestinal mucosa [57]. By these effects, resveratrol inhibited the tumour initiation process, and we can argue that by these mechanisms it can potentially lead to a decrease in colon cancer incidence in chronic intestinal inflammation, such as IBD-. Moreover, in a study by Cui et al., the authors demonstrated that resveratrol reduces tumour incidence and tumour multiplicity. In mice treated with azoxymethane (AOM) + DSS, tumour incidence was 80%, while the mice treated with AOM + DSS + resveratrol (300 ppm) had a 20% tumour incidence. Moreover, AOM + DSS-treated mice had 2.4 +/−0.7 tumours per animal, while the resveratrol treated group had 0.2 +/− 0.13 tumours per animal [58]. In another study, rats were fed with processed meat, and red wine and pomegranate extracts were added to their diet. The rats that were fed polyphenols had significantly less precancerous lesions, with a full suppression of faecal excretion of nitrosyl iron, therefore suggesting that nitrozation could be a promoter of carcinogenesis [59].

Additionally, Dolara et al. have showed how red wine polyphenols can influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles in rats [60]. The rats were treated with Azoxymethane (AOM) and 1,2-dimethylhydrazine dihydrochloride (DMH) for colon cancer/adenoma induction, and statistically significant reduction in adenoma number was seen in DMH group, while a significant reduction in total tumor number (cancer + adenoma) was seen in AOM group. What's more, the main bacterial strains in the polyphenol treated group were *Bacteroides*, *Lactobacillus* and *Bifidobacterium* spp., whereas in the control group the predominant strains were *Bacteroides*, *Clostridium* and *Propionibacterium* spp. [60]. The authors used wine polyphenols which contained 4.4% anthocyanins, 0.8% flavonols, 2.0% phenolic acids, 1.4% catechin, 1.0% epicatechin and 28.0% proanthocyanidin units, consisting of 18.0% epigallocatechin, 13.2% catechin, 65.0% epicatechin and 3.8% epicatechin gallate [59]. In addition, in a study by Femia et al., the authors researched the effects of red wine polyphenols on AOMinduced colon carcinogenesis in rats [61]. The results showed that rats treated with total polyphenolic extracts from red wine (WE) had significantly less colorectal adenomas, while there was no noticeable difference in rats treated with high molecular weight polyphenols (HMWP) or low molecular weight polyphenols (LMWP), respectively, suggesting that a synergistic effect of polyphenols is needed to exert a beneficial outcome [61].

Interestingly, a recent study investigated piceatannol, an analogue of resveratrol found in grapes and wine as well, different just by an additional hydroxyl group located at the 3- -carbon and metabolically more stable than resveratrol [61–63]. They showed how piceatannol significantly inhibited VEGF-mediated signalling and cell proliferation in VEGF-treated colon cancer cells (HT-29), as well as suppressed VEGF-mediated angiogenesis in zebrafish embryos [64]. Piceatannol is abundant in wine, with an average concentration of 13.1 ng/mL in French red wine (3× that of resveratrol) [65]. Furthermore, as it was shown that piceatannol has a higher oral bioavailability than resveratrol, added that it is also more metabolically stable than resveratrol, whilst having similar anti-inflammatory, anti-cancer, and cardioprotective properties, piceatannol positioned itself as a molecule on which additional future research should be focused [61,64,65].

Overall, the presented studies on animal models confirm the findings from in vitro studies (Table 2). These studies show how bioactive compounds found in wine (resveratrol, piceatannol, proanthocyanidins, total phenolic extracts) exert an anti-inflammatory, antioxidative, and anti-tumor effect, as well as positive effect on intestinal flora [39,50,51,58,59,61].




#### **Table 2.** *Cont.*

#### 1.2.3. The Evidence on Humans

While the evidence of the beneficial effects of wine and/or wine polyphenols on intestinal inflammation are plentiful in in vitro and animal models, the human in vivo studies are still lacking large randomized control trials (RCTs) and meta-analyses. Nevertheless, the current evidence is also promising and mandates further research on the matter.

An RCT study investigating the effects of resveratrol on patients with ulcerative colitis, in which the patients were given 500 mg resveratrol or placebo capsule for 6 weeks, showed that resveratrol supplementation led to a significant decrease in plasma levels of TNF-a, hs-CRP, as well as decrease in activity of NF-kB in PBMCs [66]. Moreover, the score of inflammatory bowel disease questionnaire-9 (IBDQ-9) increased, while the clinical colitis activity index score has significantly decreased in the treatment group [66]. In a small sample study by Sabzevary-Ghahfarokhi et al., it was demonstrated on patients with UC that resveratrol can reverse the inflammatory effects of TNF-α by reducing IL-1β and increasing IL-11 production, thereby providing protective effects on UC patients [67]. Additionally, a study by Gonzalez et al. analyzed intestinal immune markers in healthy volunteers before and after red wine consumption. They demonstrated that in a subgroup of participants with a high basal cytokine level, red wine ingestion led to a significant reduction in pro-inflammatory markers (TNF-α, IL-6, and IFN-γ) that usually promote initial inflammation [68]. However, poor water solubility and low bioavailability of resveratrol limit its clinical applications [69]. Hence, Intagliata et al. recently reported multiple modalities that could reverse these issues using different delivery systems such as liposomes, polymeric and lipid nanoparticles, but also by chemical modifications thus improving its physicochemical properties [70].

Furthermore, the aforementioned study by Swanson et al. investigated the effects of moderate red wine consumption on intestinal permeability and stool calprotectin, which are associated with recurrent IBD disease activity [34]. Interestingly, the study had mixed results, as 1–3 glasses of daily red wine consumption led to decreased stool calprotectin levels in inactive IBD patients, while it also led to increased intestinal permeability measured by urinary lactulose/mannitol excretion (small bowl permeability) and urinary sucralose secretion (large bowl permeability). Nevertheless, the study had several notable limitations: small sample size (21 subjects), short follow-up duration (1 week), and a lack of assessment

of mucosal activity. We can argue that these effects result from harmful effects of alcohol on those areas of the gut that are sensitive because of previous inflammatory damage and match the location of the disease. Given all the evidence from in vitro and animal studies regarding the anti-inflammatory effects from compounds found in wine, we hypothesize how these biologically active compounds are causing the decrease in stool calprotectin levels. In a previous study, the same author found how patients with inactive IBD drink alcohol in quantities similar to the general population, and how 75% of IBD patients reported a worsening of GI symptoms after drinking alcohol [71]. Another prospective cohort study also indicated how alcohol consumption increases the risk of exacerbation in patients with UC [72].

Moreover, in a crossover study, Hey et al. investigated the effects of five different alcoholic drinks on patients with CD [73]. Twenty patients with CD in remission and twelve healthy controls were randomly given red wine, white wine, Smirnoff Ice, Elephant Beer and pure ethanol. No differences in alcohol absorption were found between the groups, but CD patients reported a higher abdominal pain symptom score after ingesting Smirnoff Ice and Elephant beer. Authors argue how high sugar content present in these drinks leads to more intestinal fermentation that could present itself with symptoms of abdominal pain and bloating [73].

In our previous observational study investigating MedDiet adherence in patients with IBD, only 4.5% of patients in the UC group and 8% of patients in the CD group reported daily red wine intake in the MedDiet framework. Notably, when the participants were asked about the suspected foods that aggravate IBD-related symptoms, 50% in the UC group and 36% in the CD group reported alcohol as a suspect [74]. Interestingly, in a Swedish prospective cohort study that showed how the MedDiet lowers the risk of lateonset Crohn's disease, moderate alcohol intake increased with the MedDiet adherence score. In the highest MedDiet score bracket [6–8], 61% of participants moderately consumed alcohol, while the number of moderate alcohol consumers in the lowest bracket [0–2] was only 14% [75]. While some of the IBD patients associate wine intake with symptom aggravation, it seems that moderate wine consumption in the framework of the MedDiet could lead to IBD prevention [74,75].

#### **2. Precautions and Future Directions**

In general, alcohol was shown to increase gut permeability by causing transepithelial and paracellular permeability [76]. Chronic alcohol ingestion also leads to gut dysbiosis (less *Lactobacillus* and *Bifidobacterium* spp.), bacterial overgrowth, and a disruption of intestinal immune response [76–78]. The research shows that chronic and uncontrolled ethanol ingestion disrupts intestinal homeostasis and increases intestinal and, later on, systemic inflammation [76,79]. The other well-established deleterious effects of wine on liver function, metabolism, brain, and alcohol addiction, must not be neglected as well [80]. On the other hand, as we have discussed in this review, other studies show how biologically active compounds found in alcoholic beverages such as wine (polyphenols, tannins, organic acids) have a completely different effect on intestinal homeostasis, and exert antiinflammatory, anti-oxidative, and positive microbiota effects, making wine capable of assisting in disease control and affecting disease monitoring [29,40]. Nevertheless, most of the alcohol-mediated effects seem to aggravate intestinal inflammation and consequently impact disease onset, recurrence, and control of symptoms. Furthermore, British Society of Gastroenterology consensus guidelines address the importance of alcohol reduction because alcohol further reduces bone mineral density, which is already substantially struck by corticosteroids [81]. Finally, alcohol use interferes with the metabolism of most IBD medications, leading to either increase in side effect occurrence rate or loss of drug's effect [82]. Specifically, mesalamine, azathioprine, methotrexate, and biologic medications can all be affected by concomitant alcohol intake via a variety of mechanisms. Nevertheless, a large number of authors advocates moderate wine consumption based on inferences drawn from large-scale populational studies. Although questioned by certain authors, the

J-shaped curve explaining the relationship between alcohol use and total mortality has been well established [83,84]. Namely, the lowest mortality risk was observed at 6 g/day of alcohol (half of a drink/day), but with lower mortality with up to 4 drinks/day in men and 2 drinks/day in women when compared with no alcohol consumption, even after adjustment for a myriad of confounding variables. Furthermore, as presented by Xi et al., light alcohol consumption appears to be protective against cancer mortality, unlike heavy alcohol use which is associated with increased cancer risk [85–87]. Unlike the cardiovascular effects of wine, which have been extensively studied, the role of wine in IBD, or any other gastrointestinal pathology for that matter, has been poorly elucidated. Although certain inferences from studies exploiting the effects of wine on vascular function can be drawn to IBD because of the overlapping mechanisms, such as anti-inflammatory properties and protection from oxidative stress, the evidence on the effect of wine and alcohol on IBD course is still inconclusive.

Hence, as detrimental effects seem to prevail, at least for now, future research should focus on finding the optimal dose of red wine for these patients. We are casting about for dosage (if it exists) in which the beneficial effects of polyphenols, tannins, and organic acids will outweigh the detrimental effects of ethanol. It should also be noted that even if we found optimal dosage, adherence to the exact dosage of wine will be very challenging, markedly owing to the addictive nature of alcohol consumption. In summary, before we have firm evidence from more extensive prospective studies, caution should be advised in recommending red wine consumption to patients with IBD.

#### **3. Conclusions**

This review summarized the current evidence concerning the effects of wine compounds on IBD. A number of in vitro and animal model studies provide strong evidence on the mechanisms by which molecules found in wine, such as resveratrol or piceatannol provide their anti-inflammatory, anti-oxidative, anti-tumor, and microbiota-modulation effects. However, concerning the effects of alcohol, it is still unclear how the amount of ethanol ingested within the framework of moderate wine consumption (1–2 glasses a day) affects patients with IBD, as human studies on the effects of wine and its molecules on IBD/intestinal inflammation are scarce. In addition, it is doubtful whether the above-noted effects can be obtained by drinking wine exclusively, as this beverage contains only scarce amount of these compounds. Since more and more patients are turning to dietary options, such as the Mediterranean diet, as a means to control their diseases, there is an increasing need for high-quality, evidence-based information. Therefore, we have a strong foundation for translation into clinical studies and human research. With the rising prevalence of IBD worldwide and the rising cost of treatment with novel biological drugs, wine polyphenols could serve as a cheaper therapeutic modality accessible to more patients. We conclude that the evidence provided can serve as a basis for large-scale RCT trials on human subjects that will finally elucidate whether wine can offer real benefits to the IBD population.

**Author Contributions:** Conceptualization, J.V., T.T.K. and I.M.; writing—original draft preparation, M.K., D.M., M.V. and J.V.; writing—review and editing, I.M., T.T.K. and J.B.; visualization, J.V.; supervision, D.M., M.V. and J.B.; project administration, M.K. and J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The paper has been proofread by language professional Dalibora Behmen, M.A. The figure was kindly provided by Zrinka Miocic M. Arch.

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

#### **Abbreviations**


#### **References**


## *Review* **Wine, Polyphenols, and Mediterranean Diets. What Else Is There to Say?**

**Celestino Santos-Buelga \*, Susana González-Manzano and Ana M. González-Paramás**

Grupo de Investigación en Polifenoles (GIP-USAL), Universidad de Salamanca, E-37007 Salamanca, Spain; susanagm@usal.es (S.G.-M.); paramas@usal.es (A.M.G.-P.)

**\*** Correspondence: csb@usal.es; Tel.: +34-923-294-500

**Abstract:** A considerable amount of literature has been published claiming the cardiovascular benefits of moderate (red) wine drinking, which has been considered a distinguishing trait of the Mediterranean diet. Indeed, red wine contains relevant amounts of polyphenols, for which evidence of their biological activity and positive health effects are abundant; however, it is also well-known that alcohol, even at a low level of intake, may have severe consequences for health. Among others, it is directly related to a number of non-communicable diseases, like liver cirrhosis or diverse types of cancer. The IARC classifies alcohol as a Group 1 carcinogen, causally associated with the development of cancers of the upper digestive tract and liver, and, with sufficient evidence, can be positively associated with colorectum and female breast cancer. In these circumstances, it is tricky, if not irresponsible, to spread any message on the benefits of moderate wine drinking, about which no actual consensus exists. It should be further considered that other hallmarks of the Mediterranean diet are the richness in virgin olive oil, fruits, grains, and vegetables, which are also good sources of polyphenols and other phytochemicals, and lack the risks of wine. All of these aspects are reviewed in this article.

**Keywords:** olive oil; resveratrol; alcohol; phytochemicals; tyrosol

#### **1. Introduction**

In November 2010, following a transnational nomination submitted by Spain, Greece, Italy, and Morocco, the UNESCO decided to inscribe the Mediterranean Diet as an Intangible Cultural Heritage of Humanity (https://ich.unesco.org/en/Decisions/5.COM/6.41, accessed on 1 September 2021), further enlarged in December 2013 with the incorporation of three other countries: Croatia, Cyprus, and Portugal (https://ich.unesco.org/en/ Decisions/8.COM/8.10, accessed on 1 September 2021). In its decision, the UNESCO recognized the Mediterranean diet (MedDiet) as "a set of skills, knowledge, practices and traditions ranging from the landscape to the table, including the crops, harvesting, fishing, conservation, processing, preparation and, particularly, consumption of food ( ... ) characterized by a nutritional model that has remained constant over time and space, ( ... ) always respecting beliefs of each community."

Consistent associations of this Mediterranean dietary pattern with cardiovascular benefits were first reported in the 1960- s from the earlier results of the Seven Countries Study (https://www.sevencountriesstudy.com/, accessed on 1 September 2021), describing significantly lower mortality rates and incidences of cardiovascular diseases in the Italian, Greek, and Croatian cohorts than in the rest of the included (non-Mediterranean) countries [1]. Further confirmation of these outcomes was obtained from the HALE project (Healthy Ageing—a Longitudinal study in Europe), analysing data on lifestyle, dietary, and biological determinants of healthy ageing from individuals of 13 European countries, collected from the Seven Countries, as well as the FINE (Finland, Italy, Netherlands Elderly study) and SENECA (Survey in Europe on Nutrition in the Elderly—a Concerted Action) prospections. It was found that adherence to a MedDiet, together with a healthful lifestyle

**Citation:** Santos-Buelga, C.; González-Manzano, S.; González-Paramás, A.M. Wine, Polyphenols, and Mediterranean Diets. What Else Is There to Say? *Molecules* **2021**, *26*, 5537. https:// doi.org/10.3390/molecules26185537

Academic Editors: Norbert Latruffe, Paula Silva and Francesco Cacciola

Received: 25 June 2021 Accepted: 9 September 2021 Published: 12 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(i.e., being physically active, non-smoking for more than 15 years, and moderate alcohol intake) was associated with a more than 50% lower rate of all-causes and cause-specific mortality, including coronary heart disease (CHD), cardiovascular disease (CVD), and cancer [2].

Similar observations were made from many other epidemiological and intervention studies. A comprehensive analysis of the results from observational studies and randomised clinical trials—comprising a total population of over 12,800,000 individuals—was made by Dinu et al. [3], concluding that there was robust evidence to suggest that greater adherence to a Mediterranean diet style is associated with a reduced risk of overall mortality, CVD, overall cancer incidence, neurodegenerative diseases, and type-2 diabetes. In a previous screening across intervention trials, Serra-Majem et al. [4] also concluded that there was good evidence to suggest that a MedDiet improves the lipid profile, endothelial function, and blood pressure, despite the fact that the authors also highlighted that there were discrepancies on how the different studies defined and formulated the Mediterranean diet.

Indeed, the Mediterranean diet does not constitute a close and unique nutritional model, but it is rather a compendium of diverse dietary habits traditionally followed by countries around the Mediterranean basin. In spite of their heterogeneity, some common patterns are observed across these countries, namely a high consumption of plant products such as fruits, vegetables, legumes, and nuts, as well as cereals (bread, pasta, rice, and whole grains); a moderate intake of dairy products, fish, poultry, and eggs as main protein sources, with small amounts of red and processed meat; the use of olive oil as a main fat source and water as a beverage of choice. Additionally, the diet is characterized by infusions and optional moderate amounts of wine taken with meals, and a preference for seasonal, fresh, and locally low-processed products. This was summarized in the Pyramid of the Mediterranean diet proposed by the "Fundación Dieta Mediterránea" (https://dietamediterranea.com/en/fundacion, accessed on 1 September 2021) (Figure 1).

**Figure 1.** Pyramid of the Mediterranean diet (https://dietamediterranea.com/en/fundacion, accessed on 1 September 2021).

Relevant nutrients and bioactive compounds contributed by the main food items in the Mediterranean diet are summarized in Table 1.


**Table 1.** Main bioactive compounds provided by representative foodstuffs of the Mediterranean diets.

The dietary patterns of the MedDiet have been suggested to likely overlap with those for optimal prevention of both cardiovascular diseases and cancer. Thus, it is considered that, whatever the particular food choices, Mediterranean diets provide adequate intakes of total fat and long-chain polyunsaturated fatty acids, dietary fiber, antioxidant vitamins, carotenoids, and polyphenols, as well as a balanced n-6/n-3 ratio of essential fatty acids and low amounts of saturated fatty acids (SFA) [5–7]. All of these characteristics are related to beneficial effects on endothelial and cardiovascular function. Furthermore, monounsaturated fatty acids (MUFA) present in olive oil (i.e., oleic acid) are acknowledged to improve the blood lipid profiles [8]. Actually, a common and consistent feature of the MedDiet seems to be the existence of a high MUFA/SFA ratio (estimated to be around 2.0 on average) [5], which has been significantly associated with low CVD mortality and overall mortality [9]. Complex carbohydrates and dietary fiber contributed by whole grain products, legumes, and vegetables have also been related with gut health and protection against different cancers, especially colorectal cancer [10,11]. There are also glucosinolates and other organosulphur compounds present in cruciferous vegetables and allium condiments, which have acknowledged anti-inflammatory properties [12]. Another feature of the MedDiet which is usually associated with its health-promoting properties is the supply of significant amounts of different classes of antioxidant polyphenols, which are even higher than those of other dietary antioxidants, such as vitamin C, vitamin E, or carotenoids [13]. Regular intake of these compounds has been related to beneficial effects on the lipids profile, blood pressure, glucose metabolism, adiposity, or inflammatory processes, and are also associated with a reduction in the incidence of several chronic diseases, like cardiovascular diseases, type-2 diabetes, metabolic syndrome, neurodegenerative disorders, or different cancers [14–16].

The purpose of the present review is to discuss the role of wine and wine polyphenols in the health benefits of the Mediterranean diet. A particular mention is made to resveratrol, owing to the special attention that has been paid to its possible contribution to the beneficial effects of moderate wine intake, as associated with the MetDiet. Reference is also made to olive oil as a distinguishing food in the MetDiet with claimed health benefits, which have been proposed to rely, at least in part, on its characteristic polyphenols, which are different to those present in wine.

#### **2. Polyphenols as Key Components of Mediterranean Diets**

Plant phenolic compounds, commonly referred to as polyphenols, are widespread in the diet, and are nowadays considered, at least in part, responsible for the health protective effects of fruit and vegetable-rich diets. They can be classified in two major classes: flavonoids and non-flavonoids, including phenolic acids (i.e., hydroxybenzoic and hydroxycinnamic acids and their derivatives), stilbenes, and lignans (Figure 2), as well as phenolic alcohols and their secoiridoid derivatives.

**Figure 2.** Core structures of the main classes of plant phenolic compounds.

The dietary intake of polyphenols largely varies among individuals, and is estimated to range from a few hundred mg/day to more than 1800 mg/day depending on the region and target population, as well as the methodology used for the assessment [17]. Hydroxycinnamic acid esters, namely caffeoylquinic acids and flavan-3-ols oligo/polymers (i.e., proanthocyanidins), are usually reported as the most important groups of consumed polyphenols, followed by anthocyanins and flavonols [17]. In general, the contribution of phenolic acid derivatives and flavonoids tends to be equilibrated, although there are differences across countries and population groups as a function of their dietary habits. For example, there are higher proportions of flavonoids in Mediterranean regions, while phenolic acids would predominate in non-Mediterranean countries [18–21]. The main food sources for individual polyphenols tend to be similar among individuals, with coffee, tea, and fruits as major items, and vegetables and red wine in a second range [17]. It is suggested that moderate red wine drinkers consume polyphenols at levels well above the population average [6].

Despite the fact that the antioxidant capacity of polyphenols is well-substantiated in vitro and has been recurrently associated in the literature to their health effects, the little bioavailability and large biotransformation of most polyphenols in the organism raise doubts that this activity can have a primary role on their in vivo effects [17]. Although this possibility might not be discarded for particular compounds or situations, nowadays, other alternatives are considered to contribute to the in vivo effects of polyphenols. For example, they could act as modulators of gene expression and intracellular signaling cascades involved in cell function and protection [22,23]. There is also increasing evidence about the crucial role of the interactions between polyphenols and gut microbiota as a mainstay to explain the health benefits of their consumption. A vast majority of the consumed polyphenols reach the large intestine unaltered, where they can be catabolized by the colonic microflora to a variety of metabolites [24,25]. Some of these metabolites can be biologically active, and be responsible for the activity associated with their parent polyphenols. Among others, this would be the case of tyrosols, produced from oleuropein and related phenolics from olive oil, with putative effects against some types of cancer [26]; urolithins, involved in the lipid-lowering effects and improvement in the cardiovascular risk biomarkers of ellagitannins [27]; or estrogenic *S*-equol, enterodiol, and enterolactone, derived from soy isoflavones [28] and lignans [29], respectively. The role of other metabolites, such as phenolic acids and aldehydes resulting from the bacterial breakdown of flavonoids, is still uncertain, although they might be expected to contribute to a part of their effects, both at the local and systemic level. Additionally, unabsorbed polyphenols and phenolic metabolites can also have an impact on the composition of the gut microbiota, acting as prebiotic-like compounds. For instance, they have been suggested to be able to decrease the Firmicutes/Bacteroidetes ratio [30,31], linked to obesity trends in humans [32], and increase the abundance of beneficial *Bifidobacteria* and *Lactobacilli* spp. [30,33–35], while producing a reduction in the levels of Bacteroides, Streptococci, Enterobacteriacae, or Clostridia [36,37]. In the end, several mechanisms might be involved in the biological effects of polyphenols and contribute to their health benefits. This is a current active field of research that is rapidly progressing, so that advances are expected in coming years [38].

Most of the available information on the biological activity and effects of the phenolic compounds has been obtained from in vitro, ex vivo, and animal studies, whereas data directly obtained in humans are scarce, and restricted, in general, to short-term intervention trials on a reduced number of people. Some attempts have been made in Mediterranean cohorts, whose results support the role of polyphenol-rich foods to the health benefits of the Mediterranean diet [16,19,39,40]. Nevertheless, assessing the precise contribution of dietary polyphenols to those benefits remains complex, owing to the fact that the same food sources are also rich in other bioactives, such as vitamins, minerals, dietary fiber, or other antioxidants, which should also contribute to the health effects [17].

#### **3. Olive Oil**

Olive oil, and especially virgin olive oil (VOO), is one of the products most usually associated with the health properties of the MedDiet. Its regular consumption has been claimed to provide benefits against a number of disease conditions, such as atherosclerosis, diabetes mellitus, obesity, cancer, or neurodegenerative diseases [41]. It is well-known that olive oil is very rich in monounsaturated fatty acids—mainly oleic acid, accounting for up to 80% of its total fatty acids—with acknowledged positive effects on the profiles of plasmatic lipoproteins, triglycerides, and platelet aggregation [42,43], which has been linked to protection against cardiovascular and neurodegenerative diseases [44]. Actually, the EFSA has approved health claims regarding the positive effects of "monounsaturated fatty acids (mainly oleic acid)", "oleic acid", and "extra virgin olive oil" in the maintenance of normal blood LDL-cholesterol concentrations and the maintenance of normal (fasting) blood concentrations of triglycerides, when replacing saturated fatty acids (SFAs) in foods or diets [8].

In addition to its fatty acid profile, VOO also contains a series of biologically active polyphenols, in a concentration that oscillates within a large range from 50 to 1000 mg/kg, depending on the olive cultivar and ripening stage, environmental factors (climate, altitude, agricultural practices), extraction techniques, storage conditions, and time [45]. It has been estimated that they may account for up to around 2% of total olive oil weight, contributing not only to olive oil's health properties, but also to its taste and fatty acid stability against oxidation [46].

VOO possesses a unique phenolic composition mainly consisting of secoiridoid derivatives, the most abundant one being oleuropein, the glucosylated form of 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA). Oleuropein is considered the main compound contributing to the bitterness of olives. Other related secoiridoids are the ligstroside aglycone (p-HPEA-EA) and the dialdehydic form of elenolic acid linked to either hydroxytyrosol (3,4-DHPEA-EDA; oleacein) or tyrosol (p-HPEA-EDA; oleocanthal), both existing as aglycones and glucosyl derivatives. Besides, VOO also contains phenolic alcohols such as tyrosol (p-HPEA) and hydroxytyrosol (3,4-DHPEA), mostly derived from their se-coiridoid precursors. The structures of these polyphenols are depicted in Figure 3. Other phenolic compounds also reported in lower amounts in VOO include lignans (pinoresinol, 1-acetoxypinoresinol, and 1-hydroxypinoresinol), verbascoside (i.e., caffeoylrhamnosyl-glucoside linked to hydroxytyrosol), some phenolic acids (vanillic, gallic, coumaric, caffeic acids) and flavonoids (especially flavonols derived from apigenin, luteolin, or quercetin) [46,47].

**Figure 3.** Representative polyphenols present in virgin olive oils.

Olive oil phenolics have been extensively studied for their potential to counteract the onset and progression of a variety of chronic and aging-related diseases, and is attributed to hypoglycemic, anti-obesity, cardioprotective, neuroprotective, antimicrobial, and anti-cancer properties [47–49]. Several in vitro and in vivo studies have associated the health-promoting effects of olive oil phenolics to their antioxidant and anti-inflammatory potential as related to their ability to modulate a series of molecular pathways. Thus, they have been reported to be able to activate AMPK (AMP-activated protein kinase) with subsequent inhibition of the mTOR signaling pathway [22], which is involved in the regulation of adipose tissue functions, such as adipogenesis, thermogenesis, and lipid metabolism. It also modulates processes like mitochondrial biogenesis and functionality, hypoxia signaling, autophagy, and cell cycle progression [50]. In intervention studies, VOO-rich Mediterranean diets were deemed effective in reducing several inflammatory markers, such as C-reactive protein, TNF-α, interleukin-6 (IL6), endothelial adhesion molecules (VCAM-1, ICAM), or chemokines like MCP-1, which has been related to their polyphenol content [40,51]. A compound that demonstrated strong in vitro anti-inflammatory properties is oleocanthal, with a structure that resembles ibuprofen, which was shown to cause a dose-dependent

inhibition of cyclooxygenase enzymes COX-1 and COX-2 [52]. Similarly, hydroxytyrosol was able to inhibit TNF-α, iNOS, and COX-2 in LPS-challenged human monocytic cell lines [53]. Additionally, in vitro and animal studies have reported that oleuropein and hydroxytyrosol may reduce fat tissue accumulation by downregulating the expression of adipogenesis-related genes like PGC-1α, lipoprotein lipase, acetyl CoA carboxylase-1, and carnitine palmitoyltransferase-1 [54]. Recent reviews can be consulted for further information on olive oil phenolic effects and mechanisms of action [44,47,49,55,56].

The beneficial effects of phenolic compounds from olives and olive oil (i.e., hydroxytyrosol and oleuropein complex) were recognized by the European Food Safety Authority (EFSA), which authorized health claims in relation to polyphenols in olives and the protection of LDL particles from oxidative damage, the maintenance of normal blood HDL-cholesterol concentrations, the maintenance of normal blood pressure, and "antiinflammatory properties". It also recognized their contribution to upper respiratory tract health, body defences against external agents, and the maintenance of a normally functioning gastrointestinal tract [57].

Within the PREDIMED study (http://www.predimed.es, accessed on 1 September 2021), a large Spanish trial on the primary prevention of chronic diseases through the Mediterranean Diet carried out in subjects at cardiovascular risk followed since 2013, it was estimated that olive oil and olives may provide about approximately 11% of the total polyphenol intake in a typical MedDiet, representing an important differential contribution to the profile of phenolic compounds consumed by Mediterranean populations [19]. Less optimistic calculations have been made by other authors. Thus, Parkinson and Cicerale [56], assuming a mean VOO intake of 30–50 g/day in Mediterranean countries, estimated that the amount of polyphenols ingested from VOO consumption would not exceed 9 mg/day. Whatever the dietary intake, at present there is not enough evidence to confirm that the consumption of olive phenolic compounds isolated by or as components of the VOO can be healthy [58]. Most of the in vivo studies with olive oil polyphenols have been carried out using supraphysiological concentrations that are difficult to extrapolate to a dietary context, while the number and variety of randomized clinical trials (RCT)—providing the highest level of scientific evidence—are very limited and insufficient to confirm their beneficial effects on humans, except for some markers of cardiovascular risk. Actually, the strongest piece of evidence has been obtained for the ability of VOO polyphenols to protect lipoproteins from oxidation and to reduce systolic blood pressure in hypertensive individuals [56]. Extensive RCT in different population groups with distinct disorders and at phenolic levels adjusted to usual VOO consumptions are, therefore, necessary to achieve high quality scientific evidence before nutritional recommendations can be given [56,58].

The health benefits attributed to olive oil could also be supposed for table olives. Nevertheless, the phenolic composition of table olives differs from that of olive oil, as they are influenced not only by the cultivar and harvesting time (green or fully ripened), but also by the processing conditions used for making them edible, which lead to chemical transformations in the polyphenols [59,60]. Thus, under the alkaline conditions used for the debittering of fruits in Spanish-style olives, oleuropein is hydrolyzed to practical disappearance. Moreover, in Greek-style black olives, in which the fruits are collected fully ripened and directly put into brine, an acid hydrolysis of oleuropein occurs, and orthodiphenols are oxidized and polymerized during the darkening step [60]. Tyrosol and hydroxytyrosol and their acetates have been identified as the most representative phenolic compounds in table olives, with concentrations of total polyphenols ranging between 200 mg/kg to 1200 mg/kg, depending on the cultivar and processing method, with oxidized olives containing the lowest levels. A further decrease of phenolic content is produced in pitted olives due to their loss in the washing liquids, which reduce their concentration to almost half that of the nonpitted fruits [60].

Besides polyphenols, olives also contain other bioactive compounds in the unsaponifiable fraction, such as pentacyclic triterpenoids like maslinic acid and oleanolic acid (Figure 4). A range of biological activities have been shown for maslinic acid, mostly from in vitro

studies, such as anti-inflammatory, antiproliferative, antioxidant, and antidiabetic properties. In regard to oleanolic acid, hepatoprotective, antitumor, and antiviral properties have been reported [60–62]. These compounds are not lost during processing, and they are present in table olives and olive-pomace oil, a byproduct from olive oil extraction submitted to a refining process that leads to the complete loss of polyphenols. Table olives may contain more than 1300 mg/kg (dw) of maslinic acid, which is considered its richest food source [63]. Other dietary sources are spinach and eggplant, aromatic herbs, legumes, and to a lesser extent, some fruits like mandarin and pomegranate. Actually, plant-based diets including olives and olive oil, like the MedDiet, could provide a constant supply of maslinic acid, which might partly contribute to their health-enhancing properties [61].

Oleanolic acid Maslinic acid

**Figure 4.** Structures of oleanolic and maslinic acids.

#### **4. Wine in the Context of the Mediterranean Diet**

Wine is considered another distinguishing food of the Mediterranean diet contributing to its health benefits [44]. Nevertheless, it should not be forgotten that in several countries and regions that follow typical Mediterranean dietary patterns, alcohol, and therefore wine, is excluded for religious reasons.

Since the early St. Leger et al. [64] and Framingham studies [65], a lot of epidemiological evidence has accumulated, pointing to the existence of inverse relationships between light to moderate alcohol consumption—especially wine—and incidences and mortality of cardiovascular diseases (see, e.g., [66–69]), as well as of other chronic disorders like type 2 diabetes [70–73] or dementia and cognitive decline in old age [74,75]. The relationship has been described as a U- or J-shaped curve [76], with a minimum situated at a level of consumption around 10 to 30 g of alcohol/day. These studies are not free from debate, as they have been attributed to suffer from methodological limitations, which may have led to misinterpretations or biased conclusions [77–80]. Nevertheless, despite possible bias, many authors agree that when confounding factors are specifically adjusted, epidemiological trials still continue to be remarkably consistent regarding the beneficial effects from low to moderate alcohol/wine intake on CVD morbidity and mortality, as well as diabetes, osteoporosis, and neurological disorders [81–83].

A point of discussion is whether the purported wine benefits are due to ethanol or to other components. It is known that ethanol itself is able to increase HDL-cholesterol, prevent platelet aggregation, and enhance fibrinolysis, which may have positive effects on the cardiovascular system [84]. However, when differentiation among drinks is made, it is generally concluded that wine provides superior health benefits to other alcoholic drinks especially spirits—either regarding protection against CVD [85–88], type 2 diabetes [73], or dementia [89]. This perception has also been supported by the results obtained in human clinical studies [90–94] and observations over Mediterranean cohorts [19,39,95].

The intended superior benefits of wine have been related to its phenolic compounds, which are absent or in very low concentrations in other alcoholic drinks. Wine contains a variable mixture of flavonoid and non-flavonoid compounds, extracted from the grape during winemaking. Phenolic contents in red wine is usually well above 1 g/L—concentrations that are higher than those that can be found in most fruits and vegetables—while in white wine, it does not commonly exceed a few hundred mg/L, due to the fact that it is not

normally submitted to maceration with grape solids during winemaking [96]. The majority phenolic fraction in red wine is constituted by flavonoids (>85%), especially procyanidins (i.e., flavan-3-ol oligo/polymers; condensed tannins). Actually, red wine is one of the richest dietary sources of procyanidins [97], a type of compound recognized to possess a range of biological activities, and that is related with the disease preventive properties of plant-based diets [98,99]. Red wine is also rich in anthocyanins (especially young red wine) and flavonols, with acknowledged biological activities, including antioxidant, anti-inflammatory, antiproliferative, or gene modulating abilities [100], which are also considered to contribute to the health protective effects of fruits and vegetables. Hydroxycinnamic acids and their tartaric esters are the most important phenolic compounds in white wine, while other phenolics, like hydroxybenzoic acids, stilbenes (e.g., resveratrol), lignans, or dihydroflavonols are usually present in low concentrations in either type of wine, usually not exceeding a few mg/L [96].

Polyphenols, and especially flavonoids, have been proposed to be the main vasoactive components in red wine. They have been reported to be able to modulate the plasmatic lipid profile to a healthy shape, reducing triglyceride and LDL-cholesterol circulating levels [90,101,102]. They may also improve both systolic and diastolic blood pressure, stimulate endothelial-dependent vasodilation by enhancing nitric oxide (NO) generation, decrease platelet aggregation, and inhibit the activity of inflammatory enzymes and the production of several types of proinflammatory and oxidant mediators [103–105]. Many recent reports have been published dealing with the putative health effects of polyphenols, either from wine or other plant sources, and their possible mechanisms of action (see, e.g., [17,106–111]), and thus it does not seem necessary to insist herein.

In addition to polyphenols, other bioactive phenolic and non-phenolic components can also be present in wine that might contribute to the putative health effects and that are usually less considered. Thus, during must fermentation, yeasts catabolize aromatic amino acids—such as tyrosine, tryptophan, and phenylalanine—to their respective aromatic alcohols, tyrosol, tryptophol, and phenyl ethanol, which also possess bioactive properties and are also associated with some of the beneficial effects of moderate wine consumption [112]. Tyrosol has been indicated to be the second most abundant nonhydroxycinnamate phenolic in many wines, with concentrations that may reach up to 95 mg/L. Its antioxidant and anti-inflammatory properties were suggested to contribute to the beneficial effects attributed to a moderate consumption of wine [113,114]. Among others, tyrosol was found to be able to inhibit the LPS-induced production of pro-inflammatory cytokines tumor necrosis, like factor alpha (TNF-α), and interleukins IL-1β and IL-6 in human peripheral blood mononuclear cells at nanomolar concentrations, either alone or in synergy with caffeic acid [113,114]. Hydroxytyrosol is also present in wine in levels under 10 mg/L [115–117], but it can also be formed in the human organism from hydroxylation of tyrosol. De la Torre et al. [118] found that the consumption of moderate doses of wine or olive oil by healthy subjects led to a higher increase in urinary concentrations of hydroxytyrosol in the wine group, despite the fact that the amount of hydroxytyrosol administered was fivefold greater in the olive oil group (1.7 mg vs. 0.35 mg). This was explained by the biotransformation of tyrosol to hydroxytyrosol; besides, the alcohol could help to increase the bioavailability of the tyrosol present in the wine. The authors indicated that a single glass of wine was at least equivalent to 25 mL (22 g) of virgin olive oil in its capacity to increase hydroxytyrosol concentrations in the body, leading to similar beneficial effects. The same group found that there was a direct association between wine consumption and the urinary concentrations of tyrosol and hydroxytyrosol determined in individuals at cardiovascular risk included in the PREDIMED study [119], suggesting that the endogenous formation of hydroxytyrosol might explain part of the cardiovascular benefits associated with light-to-moderate wine consumption.

Another bioactive compound that may contribute to the health benefits of wine is melatonin (*n*-acetyl-5-methoxytryptamine). This is a neurohormone secreted from the pineal gland, with well-characterized antioxidant, anti-inflammatory, and immune-modulating

properties. It also contributes to the regulation of the circadian rhythms and has been attributed to tumor inhibitory activities and positive effects on the cardiovascular system, lipid, glucose metabolism, and neuroprotection [120,121]. It is present in grapes and can also be formed in wine from tryptophan metabolism by yeasts [122]. Actually, its content in wine is mostly influenced by the fermentation process, where the yeast strain and the fermentation time are the most influential factors [122]. It has been shown that blood levels of melatonin and total antioxidant capacity in plasma increased after the dietary intake of food containing it [123–125]. Melatonin concentrations ranging from a few μg/L to more than 150 μg/L have been reported in wine [126], which is higher than those found in most fruits and vegetables. Moreover, most fruits and vegetables are usually situated in the low ng/g level, with only a few products, such as mushrooms, coffee beans, or some berries showing contents in the μg/g range [120]. Therefore, wine can be considered a significant source of dietary melatonin, though it is not unlikely that it could be a contributor to the beneficial effects associated with wine consumption [121].

#### **5. What about Resveratrol?**

A phenolic compound that has been frequently associated with the putative beneficial effects of wine is the stilbene resveratrol (3,4- ,5-trihydroxy-trans-stilbene), a phytoalexin that can be found in grape skin and is extracted into wine during winemaking. The average contents of resveratrol in wine does not usually exceed a few mg/L [96]. Since white wine is not usually submitted to maceration with grape solids, it possesses lower resveratrol concentrations than red wine.

Dietary sources of resveratrol are scarce and, in addition to grapes, they include rhubarb, peanuts, or berries, though they are always present in low levels. Actually, grapes and wine are considered the most relevant food sources for humans [127]. Stilbenes are synthetized by plants in response to biotic or abiotic stress, so that exposure to UV radiation can induce the formation of resveratrol in grapes, increasing its concentration by up to tenfold [128]. Post-harvest UV irradiation has been employed as a strategy to increase resveratrol levels so as to "functionalize" grapes [129].

The presence of resveratrol in wine was firstly described in 1992 [130], suggesting that it might be an active component in the lowering effects of serum lipids associated with wine consumption. Since then, a high number of studies have been published reporting a diversity of bioactivities and multiple potential health outcomes for stilbene, including antioxidant, anti-inflammatory, anti-obesity, chemopreventive, glucose-modulating, cardiovascular protective, or calorie restriction mimicking effects [131]. A departure point in resveratrol research could be established in the study by Jang et al. [132], reporting its ability to inhibit the enzymatic activity of both forms of cyclooxygenase (COX1 and COX2), suggesting that it may behave as an anti-inflammatory and anticarcinogenic agent. Further studies showed that it was able to enhance stress resistance and extend lifespan in various model organisms, including *Saccharomyces cerevisiae*, *Caenorhabditis elegans*, *Drosophila melanogaster*, fish, and mice [133–135]. Those effects were related to the activation of Sir2 proteins (sirtuins), a family of NAD+-dependent deacetylases and mono-ADP-ribosyltransferases involved in key regulation processes, such as glucose and insulin production, fat metabolism, the regulation of the p53 tumour suppressor, and cell survival [136].

Later on, several authors have also explored the effects of resveratrol on obesity, brain function, and visual performance. The results obtained in a number of studies in cell, animal, and human trials revealed that resveratrol and related stilbenes were able to inhibit adipocyte differentiation and proliferation, decrease lipogenesis, and promote lipolysis and fatty acid beta-oxidation [137], pointing out that it may be used as an anti-obesity agent. Regarding brain function, Kennedy et al. [138] found that the oral administration of a single dose of resveratrol (250 or 500 mg) to healthy adults increased cerebral blood flow during task performance in a dose-dependent way without affecting cognitive function. Furthermore, Evans et al. [139] reported that daily consumption of 150 mg of resveratrol for 14 weeks enhanced verbal memory and overall cognitive performance in postmenopausal

women. Another study in postmenopausal women concluded that supplementation with 75 mg of trans-resveratrol twice a day for a year improved overall cognitive performance and cerebrovascular responsiveness to cognitive stimuli, which was also associated with a reduction of fasting blood glucose [140]. By contrast, a nutritional intervention with 200 mg/day of resveratrol failed to show significant improvements in verbal memory after 26 weeks in healthy elderly individuals [141]. Moreover, in a meta-analysis on the results obtained from four randomized clinical trials, Farzaei et al. [142] did not conclude significant effects on memory and cognitive performance assessed by auditory verbal learning test. Similarly, Marx et al. [143] concluded that, despite the fact that resveratrol supplementation might improve cognitive performance, the results obtained among clinical trials are limited and inconsistent. As for visual performance, studies carried out in different retinal cell lines found that resveratrol at micromolar concentrations was able to protect them from damage caused by oxidative stress and hyperglycemia-induced lowgrade inflammation, suggesting that it might contribute to preventing age-related ocular disorders like cataracts, glaucoma, or macular degeneration [144–146]. Additionally, oral administration of resveratrol (5 to 200 mg/kg for 5 days) to mice was seen to prevent endotoxin-induced uveitis by inhibiting oxidative damage, leading authors to propose that supplementation with resveratrol is a possible strategy to treat ocular inflammation [147].

However, despite the range of evidence on the potential benefits of resveratrol obtained in model and preclinical studies, attempts have failed to come to clear and consistent outcomes in cohort and clinical trials [148,149]. It must also be highlighted that the available studies have been performed using relatively high doses of resveratrol, which are unlikely to be provided by the diet when taking into account the scarcity of food sources and the very low concentrations at which stilbenes are present. It does not seem that Mediterranean diets, either with or without wine, can represent further improvements in this sense. Thus, it should not be expected that resveratrol may have a relevant contribution to the beneficial health effects associated with Mediterranean diets or any other type of diet. Supplementation or therapeutical approaches might, therefore, be the way to take advantage of its potential benefits. Nonetheless, much work seems still required in this respect. As recently reviewed by Ren et al. [148], poor pharmacokinetics and low potency—as well as possible toxicity issues, including gastrointestinal disorders, headache, rash, or nephrotoxicity [131,148]—seem the main bottlenecks to overcome for its nutritional or therapeutical application. The development of more potent analogues and/or novel resveratrol formulations to enhance its bioavailability may be promising strategies to take it from bench to people [148].

#### **6. The Social Context**

Polyphenols are not the only reason that has been argued to support the beneficial effects associated with wine consumption, but socioeconomical and contextual factors also matter and could be even more important. Indeed, when interpreting the relationship between wine consumption and health, the underlying lifestyle and dietary patterns have to be considered, as they can be as influential on the health outcome as the type of drink. Mediterranean diets are themselves considered to constitute healthy dietary and lifestyle behaviour, making it difficult to extract the contribution of wine; otherwise, they might counteract the negative impact of alcohol on the organism.

It has been claimed that the MedDiet involves a "Mediterranean way of drinking", that is, a regular, moderate wine intake mainly consumed with meals [6]. When consumed with meals, wine tends to be sipped more slowly as compared to other alcoholic drinks, which may provide metabolic advantages. Among others, the concomitant presence of food in the stomach slows down gastric emptying and subsequent ethanol absorption which favours hepatic metabolism and clearance, lowering the peak of alcohol concentration in the blood [150]. It has been reported that, when consumed within meals, alcohol intake is associated with a lower risk of acute myocardial infarctions [151]. The concurrent presence of food might also reduce the amount of alcohol available to the oral microbiota, which

has the capacity to metabolize ethanol to acetaldehyde, a compound associated with the tumorigenic effects of ethanol in the upper gastrointestinal tract [152]. It has also been observed that when wine is consumed with food, the onset of the plasma uric acid elevation coincides with the period of postprandial oxidative stress produced after a meal, which may contribute to the wine's protective effects [153]. Moreover, the presence of alcohol may improve the bioavailability of polyphenols in the food bolus, which would thus be more easily assimilated [154].

Studies in countries where wine is not the traditional alcoholic drink have also supported that a preference for wine is associated with healthy outcomes and more favourable dietary patterns [155]. Burke et al. [156], in a health screening on middle-aged men in Australia, found that a preference for wine was related to a greater consumption of fruit, vegetables, and bread, as compared to people that preferred beer. In a survey in Finland, Mannisto et al. [157] observed that wine drinkers had significantly higher intakes of antioxidants in their diet, indicating a greater consumption of fruit and vegetables than groups with other drink preferences. A higher intake of fruits, salads, cooked vegetables, fish, and olive oil was also found by Tjonneland et al. [158] in those that preferred wine, as compared with other alcoholic drinks, in a cross-sectional study conducted in Denmark. Similarly, Sluik et al. [159], in a representative sample of people from the Netherlands, found that wine drinkers consumed less energy and more vegetables and fruit juices, while the choice of beer was associated with a higher intake of meat, soft drinks, margarine, and snacks. All those behaviours associated with wine choice result in diets closer to the MedDiet, supporting the idea that it is not only wine, but the associated dietary and lifestyle patterns which contribute to healthier outcomes. Interestingly, this type of association has not been found in studies performed in some Mediterranean countries, as was the case of some Italian [160] or Spanish cohorts [161,162], where no significant correlation between wine consumption and healthier dietary habits was observed in relation to non-drinkers or consumers of other alcoholic beverages.

#### **7. Risks of Wine Consumption**

Despite the fact that moderate consumption may have health benefits, it is also wellknown that alcohol, even at a low level of consumption, has some risks. It is well-known that there is a causal relationship between alcohol intake and the incidence of a variety of pathologies—particularly liver diseases—which, in their more severe form, such as the alcoholic hepatitis, lead to a mortality rate exceeding 50% in three months. Other manifestations, like steatosis of alcoholic cirrhosis, are initially less severe, although in advanced cirrhosis, the median of survival is situated around 1–2 years [163]. In addition, there are also well-established relationships between alcohol intake and incidence of pancreatitis and diverse types of cancer, as well as some infectious diseases and nonintentional injuries.

A comprehensive report on alcohol-attributable deaths was released in 2018 within the frame of the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 [164]. For that, a meta-analysis of relative risks for 23 health outcomes associated with alcohol use was made using 694 data sources of individual and population-level alcohol consumption, along with 592 prospective and retrospective studies on the risk of alcohol use. The study used 195 locations and a time span from 1990 to 2016, including people aged above 15 of both sexes. It was concluded that alcohol use was a leading risk factor for the global disease burden worldwide, causing substantial health loss from many causes and accounting for nearly 10% of global deaths among people aged 15–49 years. The risk of all-cause mortality, and of cancers specifically, raised with increasing levels of consumption, with no level of consumption that can be considered free of risks [164].

The International Agency for Research on Cancer (IARC) classifies alcohol as a Group 1 carcinogen, causally associated with the development of cancers of the upper digestive tract and liver, and has sufficient evidence to be positively associated with colorectum and female breast cancer, without differences among the type of alcoholic drink [165]. The existence of a high association between alcohol intake and the increased risk of different types of cancers was confirmed in many prospective studies. In a meta-analysis carried out on 156 epidemiological studies, Corrao et al. [166] concluded that the risk for all types of cancer significantly increased for ethanol intakes of above 25 g/day. Also, a highly significant association was found for liver cirrhosis and essential hypertension, although, for coronary heart disease and ischemic stroke, a reduction in the risk was observed with a minimum consumption of 20 g alcohol/day [66].

An aspect to notice is that observational studies on alcohol and health usually consider average alcohol consumption, which may hide risky drinking behaviours, such as irregular binge drinking, that always involves higher health risks and mortality rates [167,168]. A pattern of irregular heavy drinking is associated with pathophysiological mechanisms that increase the risk of sudden cardiac death, hypertension, atrial or ventricular fibrillation, and cardiomyopathy, even if the average consumption is comparable to moderate consumption [169]. Heavy drinking during pregnancy is known to produce foetal alcohol syndrome, leading to abnormalities and mental retardation. Nevertheless, there is also evidence that prenatal exposure to light to moderate levels of alcohol could affect foetal development and result in decreased body weight, neurodevelopmental deficits, and longterm effects on the growth of children [170,171]. All in all, there is no level of alcohol that can be considered safe during pregnancy. As a result, its consumption must be avoided by pregnant women, as well as during the period of breastfeeding. Similarly, alcohol must be avoided by younger people: adolescent alcohol use shows clear positive relationships with total mortality and is associated with an increased risk for development of chronic alcohol use disorders in adulthood [172].

It has been suggested that the detrimental effects of ethanol might be partly counterbalanced by the polyphenols contained in wine and other foods that play a part in the MedDiet, like extra-virgin olive oil [173], although this may be more of a perception that is not supported by consistent studies in humans. In a recent position paper on Dietary Guidelines for the Spanish Population [174], the Spanish Society of Community Nutrition (SENC) established that the consumption of alcoholic beverages is not encouraged or recommended in any case. Nevertheless, taking into account the prevalence of the Mediterranean uses and customs in Spain, an optional consumption of wine in limited amounts (no more than 40 g alcohol/day for men and no more than 20 g alcohol/day for women) and with meals is suggested only for adults who so desire and are not subject to contraindication due to a health condition or medication use. The SENC also highlighted that people who do not use alcoholic beverages should not start drinking because of its potential beneficial effects, and that equivalent results can be achieved through an adequate diet without the potential risks of alcohol.

#### **8. Concluding Remarks**

The Mediterranean diet has been associated with beneficial health outcomes in the prevention of chronic degenerative disorders, including cardiovascular diseases, type-2 diabetes, cognitive decline, or cancers. Its benefits were recognized by the UNESCO, which in 2010 inscribed the Mediterranean diet as an Intangible Cultural Heritage of Humanity. A feature of the MedDiet that has been related to its health benefits is that it contributes significant amounts of antioxidants, and especially polyphenols, whose regular intake is related to beneficial effects on the lipids profile, blood pressure, glucose metabolism, adiposity, and inflammatory processes. Virgin olive oil and moderate wine consumption have been indicated as two distinctive hallmarks of the MedDiet, contributing to its health benefits [44]. Indeed, olive oil represents a differential Mediterranean product, with a peculiar phenolic composition based on secoiridoids and derived phenolic alcohols, described to be able to improve the blood lipidic profile, maintain blood pressure, and provide anti-inflammatory properties, as recognized by the EFSA with a health claim.

On the other hand, a moderate consumption of wine, especially red wine, has been proposed to provide some degree of protection against cardiovascular diseases, diabetes

mellitus, or cognitive decline, which has been related to its polyphenol content. The available studies in this respect are, however, limited by their observational nature, and there is a lack of randomized clinical trials that may prove a causal relationship. Furthermore, wine contains alcohol, which even at moderate consumption increases the risks of liver disorders and several types of cancers, among other diseases, Although the Mediterranean habit of drinking wine with meals may delay ethanol absorption and favour its more rapid clearance, at the same time that it may contribute to a decrease in postprandial oxidative stress produced after a meal. Furthermore, although polyphenols present in wine are also found in fruits and vegetables that lack the risks associated with alcohol, the concomitant presence of ethanol in the food bolus might make wine polyphenols more bioavailable. Some authors have, however, highlighted that a high wine and total alcohol intake, particularly by men, can represent a problematic aspect of the Mediterranean diet that may have not been critically evaluated [175]. Indeed, the potential risks of wine consumption, even at moderate doses, may have been overlooked or undervalued by many authors, which inadvertently may have disclosed a confusing message, although not only restricted to the context of the Mediterranean diets. Certainly, it does not seem wise to think of wine or any other alcoholic drink as an element for health promotion, but the risks of alcohol should always be considered in the first place. Releasing any message that might induce people to drink in the hope of gaining health benefits could likely have more harmful than beneficial consequences.

All in all, it is not easy to give a simple answer to the question of whether wine should be considered a key food contributing to the beneficial health outcomes of the MedDiet. Despite the fact that it is excluded from the diet in many Mediterranean areas for religious reasons, we do think that it definitely constitutes a distinguishing feature of many Mediterranean cultures, and plays an undeniable part of their historical legacy. In those regions, wine can be a relevant contributor to polyphenol intake and could be considered a side element in the beneficial health effects of the MedDiet, provided that it is consumed in the 'traditional' way, that is, light to moderate regular consumption with meals. In our opinion, wine has to be regarded as a fruitive food, to be enjoyed responsibly and in moderation, in a convivial environment and in the context of an adequate diet. In this case, it may constitute another element of a healthy lifestyle, provided that there are no reasons that advise against their intake.

**Author Contributions:** Conceptualization, C.S.-B.; methodology, C.S.-B., S.G.-M. and A.M.G.-P.; resources, C.S.-B. and A.M.G.-P.; writing—original draft preparation, C.S.-B.; writing—review and editing, C.S.-B., S.G.-M. and A.M.G.-P.; project administration, C.S.-B. and A.M.G.-P.; funding acquisition, C.S.-B. and A.M.G.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The GIP-USAL is funded by the Spanish Ministerio de Ciencia e Innovación (Project PID2019-106167RB-I00), Consejería de Educación de la Junta de Castilla y León (Project SA093P20), and Strategic Research Programs for Units of Excellence from Junta de Castilla y León (ref CLU-2018-04).

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

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


*Review*
