4.1.2. Flavonols

The flavonols exhibit a double bond between the carbons C2 and C3 and the C3 is hydroxylated. In this position, the aglycones can be linked to different sugars (often glucose and rhamnose) [147]. They are found in big amounts in the grape skin because their biosynthesis is stimulated by the sun light, from which they protect the plant. Quercetin, one of the major flavonoids in grapes, reduces inflammatory signaling pathway by inhibiting inflammatory receptors in mice with obesity-induced skeletal muscle atrophy [148] and attenuates adipogenesis and fibrosis in a human muscle-derived mesenchymal progenitors cells model [149]. In human myotubes from a healthy donor, at a physiological dose, quercetin modestly increases the insulin signaling pathway and glycogen storage which could participate in improving insulin sensitivity [150]. Their signaling pathway expresses an anti-inflammatory activity through the activation of Nrf2/Antioxidant Responsive Element (ARE) pathways with a subsequent upregulated synthesis of antioxidant endogenous enzymes [92]. Furthermore, it was described that quercetin reduces inflammation in the adipocytes and macrophages by reducing the expressions of genes encoding for TNF-α, IL-6, IL-1β, Cyclooxygenase-2 (COX-2), Inducible Nitric Oxide Synthase (iNOS) and also by keeping away from the activation of NFkB [93]. Myricetin, another of the flavonoids most present in red grapes, has been shown to have anti-diabetic effects in numerous studies [94]. On C2C12 myotubes, it was demonstrated that myricetin increases glucose uptake thanks to the activation of AMPK and Akt signaling pathways, thus decreasing insulin resistance [151]. Kaempferol, as well, is known to be an anti-inflammatory compound. The different mechanisms were summarized by Alam et al. in 2020 and they include: decreased release of IL-6, IL-1β, 18, and TNF-α, activation of the Nrf2 pathway and synthesis of target enzymes, and inhibition of TLR4 [95].

#### 4.1.3. Anthocyanes

The anthocyanes (or anthocyanins) are, with the chlorophyll and the carotenoids, the most important vegetal pigments [152]. In grapes, anthocyanins are located in the skin and exhibit a strong antioxidant power, to protect the plant from the damage caused by UV radiation [153]. The base structure is the 3,5,7,4'-tetrahydroxyflavylium cation or flavylium cation. The OH group in position 3 is always glycosylated and the one in position 5 is very frequently. There are many studies that show the beneficial antioxidant and anti-inflammatory effects of anthocyanin supplementation on obesity state meticulously reported by Sivamaruthi et al. [97]. To cite one, high fat diet obese mice supplemented with 250 mg/kg/d grape pomace extract, rich in anthocyanin, decreased the levels of plasma C-reactive protein after 12 weeks, thus exerting an anti-inflammatory activity [154]. Many studies have already described their disparate biological activities. Among them, anthocyanins have been shown to exhibit an inhibitory effect on the enzymes COX-1 and COX-2, thus reducing systematic and cardiovascular inflammation [96]. Moreover, glucose and lipid metabolism are also improved following anthocyanin supplementation. For example, cyanidin-3-*O*-glucoside supplementation increases the expression of Peroxisome Proliferator-Activated Receptors (PPARs), thus reducing dyslipidemia and increasing sensitivity to insulin in mice after eight weeks of supplementation by increasing lipid oxidation [98] and plays a role to retrieve IR in diabetes by re-establishing insulin secretion and decreasing IL-1β and IL-6 concentrations [99].

#### 4.1.4. Flavones

These molecules, vegetal yellow pigments, have the fundamental skeleton of the flavone (also known as 2-phenylchromone), with the presence of a double bond between C2 and C3 and no hydroxyl group in C3. It is therefore an oxidized form of flavanones. Like other flavonoids, they mostly appear in the form of water-soluble glycosides. Luteolin and apigenin showed in lipopolysaccharide (LPS)-activated macrophages a strong anti-inflammatory effect thanks to the decreased production of NO and prostaglandin E2 [100]. It was also described as a beneficial effect of apigenin in mitigating obesityinduced atrophy in mice. After the treatment, the size of muscle fibers was enhanced, and mitochondria were increased in number and volume. The effect has been scribed to the counteracting of oxidative species and improving the activity of antioxidant enzymes such as SOD and GPx [101].

#### 4.1.5. Isoflavones

Isoflavones, commonly known as phytoestrogens, are isomers of flavones in which the phenyl group (ring B) is linked to the C3 and not to the C2 of ring C. They are synthetized following a microbial attack or in stress conditions, thus acting as phytoalexins [155]. It described their anti-inflammatory activity and their effects on the mitigation of T2D. Daidzein acts as an inhibitor of the enzymes α-glucosidase and α-amylase, thus decreasing post-prandial glycemia [102]. Additionally, daidzein acts on different targets involved in the insulin response (AMPK, Glycerol Kinase (GK), Glucose 6-Phosphatase (G6Pase), Phosphoenolpyruvate Carboxykinase (PEPCK), PPARγ, Glucose Transporter 4 (GLUT4), Insulin Receptor Substrate 1 (IRS1), IRS2, etc.) and in the anti-inflammatory response (PPARγ, TNFα, NFkβ, IL-6, Chemokine ligand 2 (Ccl2), Chemokine (C-X-C motif) ligand 2 (Cxcl2), etc.) [103]. In obese patients, genistein (50 mg/day for 2 months) ameliorates IR associated with an increase in skeletal muscle AMPK activation, thus increasing fatty acid oxidation and insulin sensitivity [104].

#### 4.1.6. Flavanones

All the molecules of this class have a structure based on the progenitor flavone, with a double bond between C2 and C3. We can find flavanones in grapes and some representative molecules such as naringenin and hesperetin which exhibit important anti-inflammatory, antioxidant, and antidiabetic action. In fact, naringenin is able to ameliorate hyperglycemia and to improve the secretion of insulin. Moreover, it has an action on the inflammatory status by decreasing cytokines like TNFα and IL-6 and increasing the activity of SOD [105]. Naringenin and hesperetin are aglycones, but they are often found in their glycosylated form called naringin (naringenin + neohexperidose) and hesperidin (hesperetin + rutinose). It was demonstrated that hesperidin has an antidiabetic action, exerted by upregulating IRS, Akt, and GLUT4 in muscle cells, which is higher than the aglycone hesperetin [106].

#### *4.2. Non-Flavonoids*

#### 4.2.1. Stilbenes

Stilbenes are known as phytoalexins, protective compounds secreted by the plant following contact with a pathogen or an abiotic stress [156]. Stilbenes are diaryl ethers, ethenes substituted with a phenyl group on both carbon atoms of the double bond. Thus, there are two possible geometric isomers of stilbene, *trans* and *cis* [157]. Grape berries are an excellent source of trans-resveratrol, the most notable compound among stilbenes, with a concentration of 10–100 higher than in other berries [158]. Trans-resveratrol is the molecule belonging to this class that has been most investigated for its biological properties [159] due to the activation of Sirtuin 1 (SIRT1). Lagouge et al., in a groundbreaking paper, showed that resveratrol activates the deacetylase SIRT1 and the coactivator Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1α (PGC-1α), thus increasing mitochondrial activity, its decrease is a cause of aging and metabolic diseases, and miming caloric restriction and exercise [107]. Then, resveratrol can be used successfully

against different pathologies [160]. Another beneficial effect on metabolism is given by the fact that resveratrol improves insulin sensitivity by activating Akt, and AMPK pathways and inhibiting NFkB favoring insulin signaling, lipid oxidation, and decreasing inflammation in rodents [107,108]. Besides its beneficial metabolic effect, resveratrol has been efficient in animal models or cell cultures for preventing muscle atrophy due to dexamethasone [128], mechanical unloading [129], cancer cachexia [161], and sarcopenia in obese rodents [109,110]. Interestingly, resveratrol has been shown to decrease oxidative stress and inflammation associated with aging without a reversal of muscle atrophy [162,163].

#### 4.2.2. Phenolic Acids

Phenolic acids are one of the classes of phenolic compounds found in higher concentrations in the plant world [164]. This class of compounds is divided into hydroxybenzoic acids and hydroxycinnamic acids, in the function of the position of the carboxylic group on the aromatic ring (C1-C6 or C3-C6 structure) [143]. They are often found in conjugated form with tartaric acid or glucose, forming soluble compounds [165]. Phenolic acids are known to have a beneficial impact on human health, acting as oxidative species scavengers but also regulating some key signaling pathways. As an example, phenolic acids exert an adjuvant effect on diabetes by activating the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway, increasing the translocation of GLUT4 in adipose and muscle tissues and thus insulin sensitivity [111]. Gallic acid and *p*-coumaric acid showed to have a potent hypoglycemic and lipid-lowering effect on diabetic rats, exerted by decreasing TNF-α and modulating PPAR-γ in adipose tissue [112] and by modulating muscle AMPK [166]. Caffeic acid phenetyl ester, a derivative of caffeic acid found in grapes, inhibits the enzymes COX and lipoxygenase (LOX), the main enzymes involved in inflammation [113]. To bring some examples of biological activities among the hydroxybenzoic acids, vanillic acid can be successfully employed in obesity because it decreases the adipogenic PPAR and CCAAT-enhancer-binding proteins α (C/EBPα) and increases lipid oxidation through AMPKα [114]. Syringic acid is an antidiabetic agent, which combines an antihyperglycemic effect with the mitigation of diabetic neuropathy. The effect is given by the activation of PGC-1α and Nrf1, with consequent increases in mitochondrial biogenesis, and decreased secretion of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) [115].

It is evident nowadays that introducing dietary grape polyphenols through alimentation is essential for maintaining a good state of health. Several in vitro and in vivo studies and clinical trials demonstrated that their action on the body is exerted by regulating metabolism, weight, and muscle function, mitigating oxidative stress, inflammation, and chronic diseases [122,126,167].

Although their antioxidant action is considered their main mechanism of action, this action alone is not able to explain all the biological effects of grape polyphenols. In fact, it has been proven that they also act through the modulation of receptors [168], transcription factors during myogenesis [169,170], enzymes activities [171,172], and also through epigenetic modulation [173], and non-coding (nc) RNA regulation [174].

### **5. Metabolism of Polyphenols**

Structures and activities of polyphenols could be altered by their interaction with other molecules contained within the food matrix and of course by hepatic and intestinal metabolism. Consequently, human plasma concentrations of polyphenols are not comparable to those concentrations described as necessary to achieve a great biological activity as demonstrated by in vitro studies. Their metabolism and bioavailability vary considerably from molecule to molecule and there is a strong hypothesis that polyphenols' metabolites produced in vivo are also responsible for the biological action of polyphenols [175].

The series of processes involved in the metabolization, and absorption of polyphenols begins in the oral cavity with saliva and then continues in the gastrointestinal tract involving the gut microbiome. After these processes, a part of the polyphenols will be absorbed in their original form, a part will be excreted in the feces and a part will be transformed into new molecules with biological effects. The destiny of polyphenols in the digestive system depends on their original chemical structure: different polyphenols will undergo different transformations.

Saliva is composed mainly of water, salts, enzymes, and proteins (albumin, α-amylase, sulphomucins, sialomucins, glycoproteins, sulfated cystatins, agglutinins, histatins, lysozymes, mucins, immunoglobulins, proline-rich proteins) among which amylase is the most abundant [176]. In the mouth, through mastication polyphenols are mixed with saliva and solubilized. Molecules such as tannins can form a complex and precipitate with the tanninbinding salivary proteins (TBSPs). These complexes remain stable during the transit in the stomach, while they are solubilized in the intestine in presence of bile salts [177]. Lipophilic polyphenols such as resveratrol, curcumin, and quercetin are poorly bioavailable because of their lack of solubility, thus limiting their antioxidant action in the body. Saliva has been described as able to solubilize lipophilic polyphenols, thus increasing their bioavailability and their antioxidant activity [178].

After the mechanical and chemical transformations in the mouth, polyphenols are transported to the gastrointestinal tract. Absorption can occur by passive transport or, much more frequently because of lipophilia, by carrier-mediated active transport. In rats, phenolic acids [179] and not glycosylated flavonoids [180] can be absorbed at the stomach level. Chen et al. described that among the total polyphenolic compounds only 5–10% can be directly absorbed in the small intestine while the rest must undergo transformations by enzymes in subsequent sections of the gastrointestinal tract before they can be absorbed [179]. For instance, glycosylated flavonoids, such as quercetin, are poorly absorbed due to their hydrophilic character. They must be deglycosylated by β-glucosidases of the small intestine and then absorbed as aglycones [181]. The human digestive tube is populated by a copious microbial population, counting more than 100 trillion different microorganisms, whose name is gut microbiota. Gut microbiota has a big impact on the polyphenols' absorption and bioavailability, because of their own enzymatic capacities. First, the *O*-glycosides are hydrolyzed to aglycones, which will further undergo reactions of glucuronidation or sulfonation [182]. For example, trans-piceid, which is chemically an *O*-glycoside of resveratrol, is hydrolyzed to free resveratrol by the gut microbiota [183]. Subsequently, both molecules are largely sulfonated or glucuronidated but also hydroxylated, to produce different derivatives such as dihydroresveratrol, dihydropiceid, and many others [184]. Moreover, the gut microbiota is able to perform catabolic reactions, like degradation of aromatic rings via carbon-carbon cleavage, decarboxylation, hydrogenation, ihydroxylation, demethylation, thus forming derivatives with simpler structures [182]. Cyanidin, taken as a model of anthocyanidin, is metabolized by the gut microbiota starting by the opening of the pyranic ring followed by a second carbon-carbon cleavage, giving protocatechuic acid and 2-(2,4,6-trihydroxyphenyl) acetic acid as final products [185]. Hydrolysable tannins are complex phenolic compounds, metabolized by the gut microbiota. The first enzymes involved are hydrolases (tannin acyl hydrolase) which release gallic acid (gallotannins) or ellagic acid (ellagitannins). Gallic acid is further transformed by decarboxylation and hydroxylation, while ellagic acid only transforms by dihydroxylation [186].

Ferulic acid is mostly found in its esterified form. Its methyl ester, for example, is readily demethylated to ferulic acid then its double bond is saturated by hydrogenation, the methoxy group on the carbon 3 is demethylated and the carbon 4 is dehydroxylated to obtain *3*-phenylpropionic acid [187]. The polyphenols' metabolites thus formed will be partly absorbed into the systemic circulation and partly excreted as waste to terminate their biological activity. Apart from the metabolization carried out by the gut microbiota, they can enter the enterohepatic cycle and undergo phase I and phase II metabolism, eventually going back from the liver to the intestine through the bile [188]. Phase I metabolism is carried out in the liver by the cytochrome P450 (CYP450) superfamily of enzymes, and it involves reactions of oxidation, hydrolysis, and reduction. Phase II metabolism has the aim to conjugate polyphenols to augment their hydrophilicity and help their rapid elimination from the body. Phase II enzymes include UDP-glucuronosyltransferase, responsible

for glucuronidation, N-acetyltransferase, which catalyzes the transfer of acetyl groups from acetyl-CoA to polyphenols, glutathione-S-transferase which leads the polyphenol to conjugation with a reduced glutathione molecule [189]. It was reported for instance, that naringin and naringenin are susceptible to phase I and phase II metabolism. In fact, they are firstly oxidized or demethylated by CYP450 and subsequently glucuronidated, sulfated and methylated. From the metabolism of the only naringin 32 metabolites are derived, some keeping the flavonoid structure and some only a phenolic one [190]. As another example, it is possible to find in the human intestine quercetin-3 -*O*-glucuronide and quercetin-4 -*O*-glucuronide as a result of phase II metabolism on quercetin [191]. Another well-studied molecule is resveratrol, which when it reaches the human gastrointestinal tract goes through reactions of sulfation and glucuronidation, phase II reactions, generating a variety of reported metabolites, such as trans-resveratrol-3-*O*-sulfate, transresveratrol-4 -*O*-sulfate, *trans*-resveratrol-3,4 -disulfate, *trans*-resveratrol-3-*O*-glucuronide and *trans*-resveratrol-4 -*O*-glucuronide [192].
