Grapevine as a Rich Source of Polyphenolic Compounds

Grapes are rich in primary and secondary metabolites. Among the secondary metabolites, polyphenolic compounds are the most abundant in grape berries. Besides their important impacts on grape and wine quality, this class of compounds has beneficial effects on human health. Due to their antioxidant activity, polyphenols and phenolic acids can act as anti-inflammatory and anticancerogenic agents, and can modulate the immune system. In grape berries, polyphenols and phenolic acids can be located in the pericarp and seeds, but distribution differs considerably among these tissues. Although some classes of polyphenols and phenolic acids are under strict genetic control, the final content is highly influenced by environmental factors, such as climate, soil, vineyard, and management. This review aims to present the main classes of polyphenolic compounds and phenolic acids in different berry tissues and grape varieties and special emphasis on their beneficial effect on human health.


Introduction
In 2017 the worldwide grape production was 73 million tons and wine consumption was 244 hectolitres, which represents the grapevine as one of the main horticultural crops in the world [1]. The grapevine is part of the large genus Vitis which consists of 70 species divided into two subgenera, Muscadinia and Euvitis. Based on the geographical origin, Euvitis group can be divided into European (V. vinifera L.), East Asian (V. amurensis Rupr.), and North American species (V. labrusca L., V. riparia Michx., V. berlandieri Planch., etc.). One more group that can be considered as a specific group of grape cultivars includes interspecific hybrids, made by crossing V. vinifera cultivars with cultivars belonging to other Vitis species. In recent years there has been a growing interest in studying these cultivars [2][3][4][5] because of their quite different polyphenolic content than V. vinifera cultivars. Furthermore, they possess a level of resistance to some fungal diseases and pests.
Vitis vinifera has a long domestication history, and during the long cultivation period, thousands of grape varieties were developed, many of which are still in production [6]. Grapes are rich in primary and secondary metabolites which affect the quality. There are three distinct tissues in a grape berry (skin, pulp, seeds) which contain different groups of compounds, such as organic acids, sugars, volatile compounds, polyphenols, and phenolic acids. Polyphenolic compounds, together with phenolic acids, are a large group of plant secondary metabolites that can be divided based on structure. Polyphenols are defined as substances that possess multiple aromatic rings with one or more hydroxyl Even though there is a very large number of studies on the topic, determining the composition and contents of different phenols is still a great challenge. Depending on the purpose of the research, different instrumental techniques for phenolic analysis are used. When it is necessary to estimate the total phenolic content, or the total contents of anthocyanins, flavan-3-ols, or flavonoids, different spectrophotometric methods can be applied. Various chromatographic techniques are used to determine the exact composition and content of phenolics in grape extracts, and nuclear magnetic resonance (NMR) and mass spectrometry (MS) are used to determine the structures of individual phenols [20].
Numerous spectrophotometric methods based on different principles have been developed to determine the content of phenolic compounds in different plant matrices and are used to determine the different functional groups present in phenol molecules. The simplest method for the rapid estimation of total phenolics content (TPC) in grape skin extracts is the measurement of absorbance at a wavelength of 280 nm (with a prior dilution of the sample). This method is based on the characteristic absorption of benzene phenol rings at 280 nm. The advantages of this method are the speed of analysis and good reproducibility. Some compounds, such as hydroxycinnamic acids, do not have absorption maxima at the specified wavelength. In addition to phenols, grape skin extracts may contain other benzene ring-containing compounds, which may lead to interference during analysis. Methods based on the determination of absorbances at wavelengths of 320, 360, and 520 nm, respectively, can be used to estimate the total contents of hydroxycinnamic acids, flavonols, and anthocyanins. The most commonly used method for estimating TPC is the Folin-Ciocalteu (FC) method based on the reducing properties of phenols. During the analysis procedure, phosphomolybdate is reduced in the presence of phenol under alkaline conditions to a blue-colored complex having a characteristic absorption maximum in the wavelength range from 725 to 765 nm. This method is not specific because the FC reagent also forms complexes with other compounds, such as proteins and other reducing agents (e.g., ascorbic acid), which may be contained in grape skin extracts. The results are expressed in equivalents of gallic acid (GAE) [20]. The total flavonoid content can be determined by a method based on flavonoid complexation reactions with aluminum ions, where complexes with an absorption maximum at 510 nm are formed in the presence of sodium nitrite and sodium hydroxide [61]. The content of total flavan-3-ol can be determined by the spectrophotometric method with vanillin as a reagent. The reaction between flavan-3-ol and vanillin takes place under acidic conditions to form adducts with an absorption maximum at 500 nm. The second method for determining the flavan-3-ol content is based on their reaction with 4-(dimethylamino)-cinnamaldehyde to form adducts with an absorption maximum at the wavelength of 640 nm [20,61]. There are several spectrophotometric methods for estimating the total anthocyanin content. One of the methods is based on the use of different pH values, using the property of anthocyanins that their wavelengths of absorption maxima change depending on the pH. By reducing the pH of the extract to values between 0.5 and 0.8, anthocyanins are converted to red-colored flavyl forms. Another way to determine the total anthocyanin content is to apply the method by bleaching with sodium bisulfite. The addition of this reagent to a sample of an extract containing anthocyanins leads to the formation of a colorless adduct of anthocyanin-sulfonic acid [20,39,40].
Electroanalytical techniques are groups of analytical techniques based on measurements of resistance, current, and potential, and all of them have in common that they take place in an electrochemical cell which is usually composed of two electrodes immersed in the corresponding electrolyte. These techniques can be divided into voltammetric, potentiometric, conductometric, coulorimetric, etc. Different voltammetric methods are the most commonly used among them, for phenol analysis. A common feature of all voltammetric techniques is that they involve applying a potential to the electrode and monitoring the current flowing through the electrochemical cell, so all voltammetric techniques can be described as functions of potential, current, and time. These techniques are considered active because the applied potential causes oxidation or reduction of electroactive species on the electrode's surface, leading to changes in their concentration. Voltammetric techniques are a reliable tool for the rapid and inexpensive determination of phenolic compounds in various matrices. Methods have been developed that are precise and sensitive enough to be applied to samples containing very low analyte concentrations. Currently, these techniques are slowly replacing spectrophotometric methods for determining TPC. The great advantage of voltammetric techniques lies in the fact that it is not necessary to process the samples before the measurement, which reduces the duration of the analysis but also avoids errors associated with a large number of steps during sample preparation. Today, linear, cyclic, and differential pulse voltammetry techniques are most often used in practice. A glass or graphite electrode is used as the working electrode, while a silver electrode is used as the reference electrode [62][63][64].
High-performance liquid chromatography (HPLC) is most commonly used method to determine the precise composition and phenol content of grape extracts [65][66][67][68][69]. The efficiency of the HPLC method is influenced by the following factors: the composition of the stationary and mobile phases, the elution conditions, and the method of analyte detection. Reverse phase systems with octadecyl silicon dioxide (C18) are the most applied stationary phase to determine individual phenols in grape extracts [39,65,67]. In addition to this stationary phase, monolithic columns made of porous material in one piece can also be used. The use of such columns allows the faster flow of the mobile phase, which can be up to 5 mL/min, thereby shortening the analysis time and balancing the chromatographic system [69]. Today on the market there are special columns with a stationary phase made of nanoparticles and a solid core of silica gel coated with a porous layer of polymer, e.g., Core Shell columns. Using these columns can improve selectivity and resolution at the same time, thereby achieving a very low detection limit and high reproducibility [70]. Gradient elution using two solvent systems is the most commonly used elution method in phenol analysis. One of the solvents is polar, consisting of an aqueous solution of acetic, phosphoric, or formic acid, and the other solvent consists of acidified methanol or acetonitrile. The mobile phase flow rate ranges from 0.2 to 2.5 mL min −1 and the volume of the injected sample from 2 to 20 µL [52,[65][66][67]. Phenolic compounds are good chromophores, and depending on the group to which they belong, have absorption maxima in a wide range of wavelengths from 280 to 530 nm, so a diode array detector (DAD) is most often used for their detection. Flavan-3-ols, stilbenes, and some hydroxycinnamic and hydroxybenzoic acids are also fluorophores that have characteristic excitation and emission spectra and can be determined using a fluorescence detector (FLD) which represents a very selective method of analysis. The HPLC-MS system is used to identify individual components in grape skin extracts, using mild ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). A positive ESI is used to detect anthocyanins, whereas a negative ionization method is used to detect other groups of phenols. Each phenol has characteristic molecular ions and fragments based on which it is possible to determine its structure [71][72][73].

Phenolic Compounds in Grapevines
Grapes and leaves are a rich source of phenolic compounds, and in the grape berry, they can be found in the skin, pulp, and seed. These different tissues of the grape berry have different contents and compositions of phenolic compounds. The skin of a grape berry contains tannins and pigments, the pulp contains juice but no pigments, and seeds contain tannins. Biosynthesis of all phenolic compounds goes through the phenylpropanoid pathway from the amino acid phenylalanine, and two classes of compounds can be produced, flavonoids and stilbenes [11,74]. Phenolic compounds, as secondary metabolites, are frequently accumulated as glycosides. Thus, nonflavanoids accumulate in the vacuoles of mesocarp cells while flavonoids accumulate in the dermal cells of the skin tissue [75]. Many factors influence the biosynthesis of phenolic compounds, among which the most important is the genotype (cultivar). Other factors are related to environmental conditions in which the cultivar is grown, especially light, temperature, soil, and water availability. Additionally, the different management practices, such as irrigation, fertilization, yield management, and canopy management, can also have considerable influences on grape phenolic composition [76]. One more factor that has a considerable influence on determining the content of phenols in grapes is the analysis procedure, especially the extraction method used. Over the years many publications related to the analysis of grape and wine phenolic compounds have been published. Nonetheless, there is still no available standardized procedure for sample preparation, extraction, and analysis [15]. Furthermore, the content of phenolic compounds is expressed in different ways, which hinders the comparison of results.

Phenolic Acids
Phenolic acids are divided into two main groups: benzoic acids, with seven carbon atoms, and cinnamic acids, containing nine carbon atoms. These compounds exist predominantly as hydroxybenzoic and hydroxycinnamic acids that may occur in either the free or the conjugated form [19].
Hydroxybenzoic acids are necessary for the synthesis of other compounds involved in the growth and development of a grape berry. In a grape berry, acids that can be found are gallic, vanillic, gentisic, protocatechuic, syringic, and p-hydroxybenzoic ( Figure 1). Gallic acid is considered the most abundant benzoic acid and an important phenolic compound because it is a precursor of all hydrolyzable tannins and is part of condensed tannins [21]. Other hydroxybenzoic acids are components of lignins and higher content can be found in grape seeds. Table 1 presents the different grapevine cultivars and their contents of hydroxybenzoic acids in grapes and leaves. In most studies presented, gallic acid was analyzed, both in red and white grape cultivars. Even though the contents of vanillic and syringic acids are not analyzed in many studies, we can see that in most of the investigated cultivars their content is higher than the content of gallic acid. Hydroxycinnamic acids are present in hypodermal cells, along with tannins and anthocyanins, and mesocarp and placental cells of the pulp. These compounds are the major class of phenolic acids in grape berries and white wine. In white wines, they can cause color browning under oxidation but also they are precursors of volatile phenolic compounds [19,89]. The most common acids are caffeic, p-coumaric, ferulic, and sinapic ( Figure 1). These acids can be found in all grape tissues but predominantly in the vacuoles of pericarp cells. They differ by the type and number of substituents on the benzene ring. Furthermore, they can be found as cis or trans isomers, although grapes contain more trans isomers. These isomers can be converted enzymatically or through the action of light [19]. Hydroxycinnamic acids can occur in the free form or form of esters created with tartaric acid, sugars, or flavonoids. The list of hydroxybenzoic acid in Table 2 was chosen based on the literature data. Data about other acids, such as sinapic acid and 5-hydroxyferulic acid, are extremely rare.    In the research of Liang et al. [82], more than 340 genotypes of V. vinifera were analyzed. Among other phenolic compounds, phenolic acids were analyzed. The authors observed that the contents of total hydroxybenzoic and hydroxycinnamic acids were higher in wine grapes than in table grapes. Additionally, the contents of these acids tended to increase as berry color became darker, although it was not statistically significant. In the group of hydroxybenzoic acids, the content of vanillic acid was generally higher than that of gallic acid and accounted for 70% of total hydroxybenzoic acids. In the group of hydroxycinnamic acids, caftaric acid was the most abundant and accounted for 74% of total hydroxycinnamic acids. The second most important was courtaric acid, which accounted for 22% of total hydroxycinnamic acids. The contents of hydroxycinnamic acids of some grapevine cultivars are presented in Table 2.   Results are expressed as follows: a mg/g DM; b mg/kg frozen sample; c mg/kg skin; d mg/kg DW e µg/g DM; f mg/kg; g mg/100 g db; h mg/kg FW skin; i µg/g fresh sample; j mg/; k mg/kg berry, dry basis; l mg/100 g DM; m mg/100 g FM; n µg/100 g FW grape; o mg/kg dry sample.

Stilbenes
Stilbenes are a class of polyphenolic compounds that act as phytoalexins, protecting the berry from abiotic and biotic stress. In recent years there has been increased interest in this class of compounds, especially resveratrol, thanks to its beneficial effect on human health. They comprise two aromatic rings linked by an ethane bridge. The major content of stilbenes is located in grape skins but can also be found in grape seeds, stems, and leaves [102]. Stilbenes are formed through the phenylpropanoid and acetate-malonate pathway. The key enzyme is stilbene synthase (StSy) which catalyzes the formation of trans-resveratrol via condensation of 4-coumaroyl-CoA and malonyl-CoA [103]. The simplest stilbene is trans-resveratrol, while cis-resveratrol is a less stable isomer. Their 3-O-glucosides are known as transand cis-piceid, respectively. The more complex compounds formed by oligomerization and polymerization are known as viniferins. Viniferins are produced by oxidation of the basic stilbene by 4-hydroxystilben peroxidases. In grapes are found α-viniferin (a cyclic dehydrotrimer of resveratrol), β-viniferin (a cyclic dehydrotetramer of resveratrol), γ-viniferin (a more polymerized oligomer of resveratrol), δ-viniferin (an isomer of the resveratrol dehydrodimer), and ε-viniferin (a cyclic dehydrodimer of resveratrol) ( Figure 2) [11]. Table 3 presents the contents of transand cis-resveratrol, and transand cis-piceid in the skins of different grapevine cultivars. In grape skins of Gamay and Chardonnay, in addition to trans-resveratrol, and transand cis-piceid, these cultivars contain 8.5 and 2.2 µg/g FW skin of ε-viniferin [104]. In grape cultivars Raboso Piave and Primitivo, considerable amounts of Eand Z-astringin and picetannol were found-in Raboso Piave, 106, 101.7, and 41.8 µg/kg grape; and in Primitivo, 884.2, 121.4, and 281.5 µg/kg grape [105].     The important compounds in the defense of grape berries against Botrytis cinerea are ε-viniferin, resveratrol, and piceid [106,107]. Furthermore, infection by Plasmopara viticola and UV radiation can induce the synthesis of δ-vinferin and ε-viniferin. Furthermore, the content of stilbene compounds depends on the ripening stage, and generally an increasing trend has been observed from vérasion to ripening stage [104]. Infection with a fungal disease, such as Botrytis, can induce the production of resveratrol. In research by Jeandet et al. [106] they observed that the distribution of stilbenes in an infected grape bunch strongly depends on the distribution of infected berries. The resveratrol content was predominantly present in grape berries close to the necrotic area and lesions caused by Botrytis. In non-infected berries, the content of stilbene compounds was very low. This localized response of phytoalexin production can help arrest the spread of Botrytis lesions.   Results are expressed as follows: a mg/kg FW grape berry; b mg/kg berry dry basis; c mg/kg FW skins; d mg/100 g db; e mg/kg; f mg/L; g µg/g DW; h mg/g DW; i mg/kg frozen sample; j mg/100 g DM; k µg/kg grape; l mg/100 g FM; m mg/kg DM; n µg/100 g FW.

Flavonoids
A great number of polyphenolic compounds in grapes are flavonoids. The general structure of flavonoids is two phenyl rings (A and B) and a heterocyclic ring (C) (Figure 3). Flavonoids are grouped into several classes that differ in the oxidation state of the C-ring, and they include anthocyanins, flavonols, and flavan-3-ols. They are synthesized from the combination of shikimic and polyketide pathways [110]. Flavonoids can occur either in free or in conjugated form, often esterified to one or two sugar molecules with at least one hydroxyl group [19,79]. Grape flavonoids are mainly localized in both the peripheral layers of a berry's pericarp and in some layers of the seed coat. The inner thick-walled layers of hypodermis are where the major class of flavonoids is located, represented by anthocyanins, proanthocyanidins (tannins), and simple flavan-3-ols and flavonols [111].

Flavonoids
A great number of polyphenolic compounds in grapes are flavonoids. The general structure of flavonoids is two phenyl rings (A and B) and a heterocyclic ring (C) (Figure 3). Flavonoids are grouped into several classes that differ in the oxidation state of the C-ring, and they include anthocyanins, flavonols, and flavan-3-ols. They are synthesized from the combination of shikimic and polyketide pathways [110]. Flavonoids can occur either in free or in conjugated form, often esterified to one or two sugar molecules with at least one hydroxyl group [19,79]. Grape flavonoids are mainly localized in both the peripheral layers of a berry's pericarp and in some layers of the seed coat. The inner thick-walled layers of hypodermis are where the major class of flavonoids is located, represented by anthocyanins, proanthocyanidins (tannins), and simple flavan-3-ols and flavonols [111].

Anthocyanins
Compounds that act as natural colorants present in the skin of red grape varieties are anthocyanins. These compounds are co-located with tannins in the thick-walled hypodermal cells of the skin [74] and are a glycosidic form of anthocyanidins. Grapes contain five out of 17 anthocyanidins, and these are delphinidin, peonidin, cyanidin, petunidin, and malvidin, which is usually the predominant anthocyanin in most red grapes [112]. In addition, pelargonidin was identified in berry skins of Cabernet Sauvignon and Pinot noir [113]. The anthocyanin structure has the typical C6-C3-C6 flavonoid skeleton, which contains one heterocyclic benzopyran ring (C ring), one fused aromatic ring (A ring), and one phenyl constituent (B ring) ( Figure 4) [114]. In most V. vinifera grape berries, the glycosylation of anthocyanidins takes place exclusively at the 3-position by the activity of 3-O-glucosyltransferase, while in non-V. vinifera and their hybrids both 3-and 5positions are glycosylated [115]. In 2015 evidence was provided confirming and explaining the existence of anthocyanidin-3-O-diglucoside in Cabernet Sauvignon grape berries. Until 2015 it was accepted that V. vinifera can contain only anthocyanidin-3-O-monoglucosides due to double mutation in the anthocyanin 5-O-glucosyltransferase gene which disrupts the enzymatic activity of the encoded protein [116]. However, the evidence showed otherwise, revealing a recombinant protein (designated as Vv5GT3) that can glycosylate the anthocyanidins on the 3-O and 5-O-positions [117]. The distribution of anthocyanins in different branches of a grape cluster is highly variable depending on many environmental and physiological factors. Anthocyanidins are synthesized in the

Anthocyanins
Compounds that act as natural colorants present in the skin of red grape varieties are anthocyanins. These compounds are co-located with tannins in the thick-walled hypodermal cells of the skin [74] and are a glycosidic form of anthocyanidins. Grapes contain five out of 17 anthocyanidins, and these are delphinidin, peonidin, cyanidin, petunidin, and malvidin, which is usually the predominant anthocyanin in most red grapes [112]. In addition, pelargonidin was identified in berry skins of Cabernet Sauvignon and Pinot noir [113]. The anthocyanin structure has the typical C6-C3-C6 flavonoid skeleton, which contains one heterocyclic benzopyran ring (C ring), one fused aromatic ring (A ring), and one phenyl constituent (B ring) ( Figure 4) [114]. In most V. vinifera grape berries, the glycosylation of anthocyanidins takes place exclusively at the 3-position by the activity of 3-O-glucosyltransferase, while in non-V. vinifera and their hybrids both 3-and 5-positions are glycosylated [115]. In 2015 evidence was provided confirming and explaining the existence of anthocyanidin-3-O-diglucoside in Cabernet Sauvignon grape berries. Until 2015 it was accepted that V. vinifera can contain only anthocyanidin-3-O-monoglucosides due to double mutation in the anthocyanin 5-O-glucosyltransferase gene which disrupts the enzymatic activity of the encoded protein [116]. However, the evidence showed otherwise, revealing a recombinant protein (designated as Vv5GT3) that can glycosylate the anthocyanidins on the 3-O and 5-O-positions [117].     The distribution of anthocyanins in different branches of a grape cluster is highly variable depending on many environmental and physiological factors. Anthocyanidins are synthesized in the cytoplasm but accumulate in vacuoles. During ripening, which affects anthocyanin biosynthesis, a two-stage accumulation is shown. In the first stage, there is a rapid increase of anthocyanins at the early stages of development, closely correlated with sugar accumulation, followed by the second stage characterized by slower accumulation [118]. Furthermore, the total amount of anthocyanins and the relative abundances of single anthocyanins are extremely variable among grapevine cultivars and are under strict genetic control [119]. The composition of anthocyanins for each variety is relatively stable, while absolute concentrations are strongly affected by both environmental and agronomical factors [120]. These can be seen in Table 4, where the distribution of anthocyanins is not identical in the case of same the same grape cultivar grown in different locations and growing seasons. The most abundant anthocyanin for almost all presented cultivars is malvidin-3-O-glucoside.
Besides anthocyanidin-3-O-monoglucosides, there are also 3-O-acetylglucosides, 3-O-coumaroylglucosides, and 3-O-caffeoylglucosides present in different cultivars of grapevine. Their share is-aside from genotype-largely modified by the environmenatal conditions with dominant influences from light intensity and temperature. Anthocyanins are extracted from grape skin during vinification process in red wine production. The red wine hue and intensity differ among cultivars based on the total content of anthocyanins and shares of specific anthocyanin compouds. For same reason, by products of red grapes after red wine production are relatively poor sources of anthocyanins in comparison to fresh grape skins. There is an exeption in red grape byproducts from rosé wine production, in which the majority of anthocyanins are not extracted from grape skins. They can serve as a rich sourse of anthocyanins due to the fact that more than 15% of total red grapes produced are used for rosé wine production.
After the transportation of anthocyanins to the vacuole, they adopt their distinct color, and this compartmentation decreases the feedback inhibition of cytosolic biosynthetic enzymes. What produces the red, violet, and blue coloration comes down to the presence and number of hydroxyl groups, methylations, and sugar moieties. Anthocyanins are red when conditions are acidic, colorless at pH 4.0, and purple above pH 4.5. Under alkaline conditions, blue color can be produced [75]. Table 4. Contents of individual anthocyanins in the grapes of different red grapevine cultivars.

Flavonols
Flavonols constitute a group of flavonoids that vary in color from white to yellow and are closely related in structure to flavones. The main representatives are kaempferol, quercetin, and myricetin. Common as well are simple derivatives such as isorhamnetin [128]. Moreover, the presence of both laricitrin and syringentin was also reported [65]. Flavonols have a double bond between positions 2 and 3 and a ketone group in position 4 of the C ring ( Figure 5). Different sugars can be bound to position 3 of the flavonoid skeleton, producing glucosides, glucuronides, galactosides, and diglycosides [21]. Flavonol's biosynthesis is closely related to that of anthocyanin and is under genetic control. Thus, like anthocyanins, they can be used in the taxonomical classification of both red and white varieties [120,129]. In the biosynthesis of flavonols, activity of flavonol synthase (FLS) activates enzymes that convert dihydriflavonols to flavonols. Between flowering and vérasion, the activity of FLS is commensurate with the pattern of flavonol accumulation in the berry. There are two distinct periods of flavonol synthesis in grape berries. The first period is around flowering and the second during the ripening of the developing berries [130]. Furthermore, it was shown that light and low temperatures have a synergistic effect on the expression of genes within the flavonoid biosynthesis pathway [131].
Molecules 2020, 25, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules After the transportation of anthocyanins to the vacuole, they adopt their distinct color, and this compartmentation decreases the feedback inhibition of cytosolic biosynthetic enzymes. What produces the red, violet, and blue coloration comes down to the presence and number of hydroxyl groups, methylations, and sugar moieties. Anthocyanins are red when conditions are acidic, colorless at pH 4.0, and purple above pH 4.5. Under alkaline conditions, blue color can be produced [75].

Flavonols
Flavonols constitute a group of flavonoids that vary in color from white to yellow and are closely related in structure to flavones. The main representatives are kaempferol, quercetin, and myricetin. Common as well are simple derivatives such as isorhamnetin [128]. Moreover, the presence of both laricitrin and syringentin was also reported [65]. Flavonols have a double bond between positions 2 and 3 and a ketone group in position 4 of the C ring ( Figure 5). Different sugars can be bound to position 3 of the flavonoid skeleton, producing glucosides, glucuronides, galactosides, and diglycosides [21]. Flavonol's biosynthesis is closely related to that of anthocyanin and is under genetic control. Thus, like anthocyanins, they can be used in the taxonomical classification of both red and white varieties [120,129]. In the biosynthesis of flavonols, activity of flavonol synthase (FLS) activates enzymes that convert dihydriflavonols to flavonols. Between flowering and vérasion, the activity of FLS is commensurate with the pattern of flavonol accumulation in the berry. There are two distinct periods of flavonol synthesis in grape berries. The first period is around flowering and the second during the ripening of the developing berries [130]. Furthermore, it was shown that light and low temperatures have a synergistic effect on the expression of genes within the flavonoid biosynthesis pathway [131]. Flavonols are considered to act as UV-and photo-protectors because they absorb strongly at both UV-A and UV-B wavelengths. Thus, they can be mainly found in the outer epidermis of the skin [10]. Flavonols have also an important role in the stability of red wine color because they act as copigments with anthocyanins [132]. Table 5 represents the flavonol profiles of different grapevine cultivars. Castillo-Muñoz et al. [133] analyzed the flavonol profiles of 21 white grape cultivars. It was shown that the skins of white grape cultivars contain flavonols as 3-O-glycoside derivatives of only kaempferol, quercetin, and isorhamnetin. Quercetin-type flavonols dominated the flavonol profile of white grapes (60.8-90.7%), followed by kaempferol-type (8.8-38.3%) and isorhamnetin-type flavonols. Although isorhamnetintype flavonols occurred as minor compounds, some cultivars (Gewürztraminer, Verdejo, Riesling) were characterized by relatively high and significantly different proportions of this compound. By analyzing 91 grape cultivars, among which 64 were red, Mattivi et al. [63] determined a pattern of the flavonols. The main compound in red grape cultivars was quercetin (12.34-87.76%), followed by myricetin (2.35-81.61%), kaempferol (0-17.52%), laricitrin (0-13.99%), isorhamnetin (0.42-11.88%), and syringetin (0-9.88%). Flavonols are considered to act as UV-and photo-protectors because they absorb strongly at both UV-A and UV-B wavelengths. Thus, they can be mainly found in the outer epidermis of the skin [10]. Flavonols have also an important role in the stability of red wine color because they act as co-pigments with anthocyanins [132]. Table 5 represents the flavonol profiles of different grapevine cultivars. Castillo-Muñoz et al. [133] analyzed the flavonol profiles of 21 white grape cultivars. It was shown that the skins of white grape cultivars contain flavonols as 3-O-glycoside derivatives of only kaempferol, quercetin, and isorhamnetin. Quercetin-type flavonols dominated the flavonol profile of white grapes (60.8-90.7%), followed by kaempferol-type (8.8-38.3%) and isorhamnetin-type flavonols. Although isorhamnetin-type flavonols occurred as minor compounds, some cultivars (Gewürztraminer, Verdejo, Riesling) were characterized by relatively high and significantly different proportions of this compound. By analyzing 91 grape cultivars, among which 64 were red, Mattivi et al. [63] determined a pattern of the flavonols. The main compound in red grape cultivars was quercetin (12.34-87.76%), followed by myricetin (2.35-81.61%), kaempferol (0-17.52%), laricitrin (0-13.99%), isorhamnetin (0.42-11.88%), and syringetin (0-9.88%).   Results are expressed as follows: a mg/kg FW skin; b µg/g DW; c mg/kg FW grape berry; d mg/kg DW; e mg/kg; f mg/kg skins; g mg/L; h mg/g DM; i mg/kg berry, dry basis; j mg/100 g FW; 1 Glucoside; 2 Glucuronide; 3 Rutinoside; 4 Galactoside.

Flavan-3-ols
One of the principal classes of grape polyphenolic compounds is flavan-3-ols. In grapes, they are present as monomers or polymers called proanthocyanidins (PAs) or condensed tannins. The main monomers found in grapes are (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, and (−)-epicatechin-3-O-gallate ( Figure 6) [73,135]. Proanthocyanidin structures vary depending upon the nature (stereochemistry and hydroxylation pattern) of flavan-3-ol units, the degree of polymerization, and the presence or absence of modifications [136]. PAs are mainly oligomeric and polymeric forms of (+)-catechin and (−)-epicatechin. The monomers are linked through C4-C6 or C4-C8 linkages (B type) with sometimes additional C2-O-C5 or C2-O-C7 bonds (A-type) [21,83]. PAs are the most abundant class of soluble polyphenolics in grape berries and can be found in hypodermal layers of the skin and the soft parenchyma of the seed coat between the cuticle and the hard seed coat. They are accumulated in specific vacuoles (tannin vacuoles) and act as deterrents to herbivores and fungi. Skin tannins have a much larger average size than seed tannins, and they contain epigallocatechin, whereas in seed tannins they are generally absent [74,75]. Flavan-3-ols in the grape berry start to accumulate before vérasion, especially during the early stage of development, and then decrease in concentration during ripening [137].

Flavan-3-ols
One of the principal classes of grape polyphenolic compounds is flavan-3-ols. In grapes, they are present as monomers or polymers called proanthocyanidins (PAs) or condensed tannins. The main monomers found in grapes are (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, and (−)-epicatechin-3-O-gallate ( Figure 6) [73,135]. Proanthocyanidin structures vary depending upon the nature (stereochemistry and hydroxylation pattern) of flavan-3-ol units, the degree of polymerization, and the presence or absence of modifications [136]. PAs are mainly oligomeric and polymeric forms of (+)-catechin and (−)-epicatechin. The monomers are linked through C4-C6 or C4-C8 linkages (B type) with sometimes additional C2-O-C5 or C2-O-C7 bonds (A-type) [21,83]. PAs are the most abundant class of soluble polyphenolics in grape berries and can be found in hypodermal layers of the skin and the soft parenchyma of the seed coat between the cuticle and the hard seed coat. They are accumulated in specific vacuoles (tannin vacuoles) and act as deterrents to herbivores and fungi. Skin tannins have a much larger average size than seed tannins, and they contain epigallocatechin, whereas in seed tannins they are generally absent [74,75]. Flavan-3-ols in the grape berry start to accumulate before vérasion, especially during the early stage of development, and then decrease in concentration during ripening [137].  Table 6 depicts the contents of flavan-3-ols in the skins of different cultivars. In white grapevine cultivars, Semillon and Ugni blanc, the highest concentrations of procyanidins are present in the early stages of development. The major constituent of flavan-3-ols was (+)-catechin, along with procyanidin dimers B1, B4, and B6. During the maturation, the predominant dimers were B2 and B4 [138]. Cabernet Sauvignon is richer in low molecular weight procyanidins than Merlot. Additionally, dimers B2, B4, and B1 were found in both Merlot and Cabernet Sauvignon [139].  Table 6 depicts the contents of flavan-3-ols in the skins of different cultivars. In white grapevine cultivars, Semillon and Ugni blanc, the highest concentrations of procyanidins are present in the early stages of development. The major constituent of flavan-3-ols was (+)-catechin, along with procyanidin dimers B1, B4, and B6. During the maturation, the predominant dimers were B2 and B4 [138]. Cabernet Sauvignon is richer in low molecular weight procyanidins than Merlot. Additionally, dimers B2, B4, and B1 were found in both Merlot and Cabernet Sauvignon [139].    Results are expressed as follows: a mg/kg DW; b mg/kg; c mg/kg FW grape berries; d µg/g DW; e mg/kg berry, dry basis; f mg/g grape; g mg/L; h mg/g DM; i mg/g berry; j mg/100 g db; k mg/kg fresh grape; l mg/kg berry; m mg/g DW skins; n mg/kg frozen sample; o µg/100 g DM; p mg/100 g fresh grape; r µg/100 g FW.
Flavan-3-ols are important in winemaking and affect wine's sensory properties, contributing to astringency and bitterness. The intensity and duration of astringency can be attributed to structural characteristics (chain length, the stereochemistry of subunits, site of the bond between subunits). Bitterness is highest in the monomers and lowest in the trimers, whereas monomers are rated lower in astringency than the dimers or trimers [142,145]. Despite the differences in the content of flavan-3-ols depending on the cultivar, the contents of those compounds in the wine industry by-products are dominantly the results of the differences in grape processing technology of red and white wines. In the case of red wine production, which has longer contact of skins and seeds with juice (typically 7-10 days), their extraction is much higher than in the case of white wines. For the same reason, the resulting skins and seeds have higher contents of flavan-3-ols after white wine production, typically from white cultivars of grapevine.
Astringency and bitterness are especially expressed in the case of young wines, especially in cases when grapes are not fully mature. This is common in cases of late-ripening cultivars and years with lower temperatures. Wines with higher levels of flavan-3-ols, despite their positive health influences, are usually not preferred by consumers, and wines with high contents are subjected to aging in wooden barrels, which results in their sedimentation.

Biosynthesis of Phenolic Compounds
There are many review papers and book chapters which describe the biosynthesis of phenolic compounds in grapes [22]. In brief, the biosynthesis of phenolic compounds can be divided into several interconnected pathways. The first pathway, the phenylpropanoid pathway, includes the conversion of phenylalanine by three successive enzymatic reactions to 4-coumaroyl-CoA. The above-mentioned enzyme reaction is catalyzed by the following enzymes: phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxyliase (C4H), and 4-coumaroyl: CoA ligase (4CL). Phenolic acids are the products of modifications of intermediates of the phenylpropanoid pathway, and 4-coumaroyl-CoA, the end product of this branch, can be converted to an intermediate (tetrahydroxychalcone) for flavonoids' biosynthesis by the action of chalcone synthase (CHS) or by stilbene synthase (STS) to an intermediate for stilbene biosynthesis of resveratrol (Figure 7) [111].

Grape Phenolics and Their Impact on Human Health
In the last few decades, there has been a growing interest in establishing a healthy diet and lifestyle that will maintain overall health and prevent stress-related diseases, such as cardiovascular disease (CVD), cancer, and diabetes [35]. Wine, grapes, and grape products have been consumed since ancient times. Grapes contain various nutrient elements, such as minerals, vitamins, edible fibers, and phytochemicals, among which the most important are polyphenolic compounds [24]. A lot of positive properties are attributed to phenolic compounds, such as antioxidant, antiinflammatory, anticancerogenic, and antibacterial activity.

Antioxidant Activity
Oxidative stress is an imbalance between antioxidant molecule production and the oxidative reactive species. This delicate balance aims to maintain a suitable redox potential. If the redox potential increases, there is a risk of disease occurrence, such as cardiovascular, metabolic, and neurodegenerative diseases. Thus, research in dietary intake that may reduce the incidence of these chronic diseases has become important [146].
Phenolic compounds can act as antioxidants mainly due to their redox potential, which allows them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers [147]. Oxidation of lipoproteins, such as LDL, is an important step in the development of atherosclerosis. Therefore, dietary supplementation with antioxidant preparations containing polyphenols may reduce the risk of atherosclerosis [148]. In a study on healthy and hemodialysis subjects, the effect of concentrated red grape juice was studied. It was shown that grape juice consumption increased the antioxidant capacity of plasma, and reduced the concentration of oxidized LDL [149]. Leong et al. [150] studied

Grape Phenolics and Their Impact on Human Health
In the last few decades, there has been a growing interest in establishing a healthy diet and lifestyle that will maintain overall health and prevent stress-related diseases, such as cardiovascular disease (CVD), cancer, and diabetes [35]. Wine, grapes, and grape products have been consumed since ancient times. Grapes contain various nutrient elements, such as minerals, vitamins, edible fibers, and phytochemicals, among which the most important are polyphenolic compounds [24]. A lot of positive properties are attributed to phenolic compounds, such as antioxidant, anti-inflammatory, anticancerogenic, and antibacterial activity.

Antioxidant Activity
Oxidative stress is an imbalance between antioxidant molecule production and the oxidative reactive species. This delicate balance aims to maintain a suitable redox potential. If the redox potential increases, there is a risk of disease occurrence, such as cardiovascular, metabolic, and neurodegenerative diseases. Thus, research in dietary intake that may reduce the incidence of these chronic diseases has become important [146].
Phenolic compounds can act as antioxidants mainly due to their redox potential, which allows them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers [147]. Oxidation of lipoproteins, such as LDL, is an important step in the development of atherosclerosis. Therefore, dietary supplementation with antioxidant preparations containing polyphenols may reduce the risk of atherosclerosis [148]. In a study on healthy and hemodialysis subjects, the effect of concentrated red grape juice was studied. It was shown that grape juice consumption increased the antioxidant capacity of plasma, and reduced the concentration of oxidized LDL [149]. Leong et al. [150] studied the effect of Pinot noir juices on the protection of human intestinal Caco-2 cells from H 2 O 2 -induced stress. It was observed that the cell viability of Caco-2 under oxidative stress is positively correlated with the content malvidin-3-O-glucoside and total phenolics. Similar research was conducted by Wang et al. [151], wherein grape phenolic extract (GPE), rich in hydroxybenzoic acids, flavonols, and hydroxycinnamic acids, was used to observe the antioxidant protection of Caco-2 cells. The results showed that treatment with GPE was able to considerably reduce oxidative and protein damage, and reduce pro-oxidant-cytotoxicity. A study by Lingua et al. [152] on Caco-2 cells evaluated the effect of gastrointestinal (GI) digestion on antioxidant activity (AC) of white grapes, comparing them to those of their white wines. It was shown that the winemaking process modified the phenolic profile of grapes and on average only 12% of the TP (total polyphenols) content in grapes was present in wines. Furthermore, digestion reduced the phenolic content, with only 31% and 67% of native polyphenols from grapes and wines being potentially bioaccessible. It was also shown that cellular AC of non-digested and digested food was the same at the same polyphenol concentration. This indicates that changes in phenolic profile did not modify the bioactivity.
A high-fat-diet (HFD) in mice can induce leaky gut syndrome combined with low-grade inflammation, which can increase reactive oxygen species (ROS) in the intestines and may contribute to dysbiosis and metabolic syndrome (MetS). A study on mice fed a HFD were administered GPE and β-carotene [153]. The results showed that GPE, rich in proanthocyanidins (PCA), decreased HFD-induced ROS. This reduction of ROS may benefit anaerobic and microaerophilic gut bacteria, such as Akkermansia muciniphila. This implies that the bloom of beneficial gut bacteria associated with improved metabolic status may be linked to the protective antioxidant activity of polyphenolic compounds. The influences of grape skin polyphenols may differ depending on their concentrations and intracellular ROS. In research on human intestinal cells (HT-29), in basal and stressed conditions, polyphenols showed pro-oxidant and anti-oxidant effects. In basal conditions, the pro-oxidant effect, corresponding to high ROS and low reduced glutathione (GSH) content, was due to the polyphenolic oxidation in cell culture with the production of hydrogen peroxide. On the other hand, in stressed conditions grape skin polyphenols showed anti-oxidant effects up to 1.3 × 10 −6 µg/g and restored the stress-related GSH reduction [154].
Grape pomace, containing skins, seeds, and stems, is considered a waste in the winemaking process, and is usually discarded, despite its high content of bioactive compounds, including polyphenols. However, with the growing interest in sustainable winemaking, there is an awareness of using this waste and its bioactive compounds for different purposes, such as food coloring agents, nutritional additives, or as agents in pharmaceutical formulations. Cristea et al. [155] proposed using the grape marc extract for the development of a new food dye with antioxidant properties of natural origin. Grape pomace (GP) obtained from Nero d'Avola grape, rich in polyunsaturated fatty acids and polyphenols, presents strong antiradical and antiproliferative activity. The incubation of human hepatoma cell lines Hep-G2 with polyphenols showed a considerable effect on cell inhibition. The antioxidant activity was shown on fibroblasts HS-68, exposed to inducers oxidative stress. The incubation of cells for 24 h with polyphenols considerably inhibited cytotoxicity of the pro-oxidants [156]. Besides the grape pomace, the leaves can also be used as a source of many polyphenolic compounds. Maia et al. [157] showed that Pinot noir leaves are rich in phenolic compounds with antioxidant activity, such as caffeic acid, kaempferol, quercetin. All that should be very interesting for the pharmaceutical and food industries.

Anti-Inflammatory Activity
Inflammation is a protective response of a tissue against cell injury, irritation, or pathogen invasion, and several environmental stress factors may cause inflammation [158]. The mechanism of proanthocyanidin's (PAs) anti-inflammatory activity was investigated. Li et al. [159] evaluated grape seed proanthocyanidin's anti-inflammatory activity in vitro and in vivo on rats and mice. It was observed that PAs had an inhibitory effect on paw edema in rats and ear swelling in mice. The author proposed that the major anti-inflammatory mechanism of PAs is related to its suppressive effect on the formation of inflammatory cytokines. Similar research was conducted on diet-induced obesity rats. The results showed that the daily consumption of PAs prevents inflammation in the adipose tissue, muscle, and liver, which might improve obesity-induced insulin resistance in these tissues [112]. Stilbenes are another class of polyphenols that exhibit anti-inflammatory activity, and plants containing stilbenoids have been used extensively in folk medicine [160]. Piceatannol is a stilbene compound that can be found in relatively low concentrations in wine but has strong antioxidant and anti-inflammatory activity. This was shown in a study where piceatannol inhibited mast cell-mediated allergic inflammatory reactions and their possible mechanisms, such as histamine release and MAPK pro-inflammatory cytokines. Resveratrol, the most known stilbene compound, also showed anti-inflammatory activity. It can have a protective effect in acute colitis, an inflammatory disorder, by reducing PGD2 production and the overexpression of COX-2. Additionally, it caused a considerable increase in TNBS-induced apoptosis [161]. Furthermore, resveratrol can suppress apoptosis and inflammatory signaling through its actions on the nuclear factor kappaB (NF-kB) pathway in human chondrocytes. These results suggest that resveratrol should be considered for the prophylactic treatment of osteoarthritis in human and companion animals [162].

Cardiovascular Protection
CVD is a major health problem worldwide affecting considerable proportions of the populations of developed countries. CVD is associated with high cholesterol levels in the blood-in particular, low-density lipoproteins (LDL). It is widely accepted that an excessive dietary intake of saturated fats and an unhealthy diet increases cholesterol and LDL levels in the blood [15,110,163] Thus, the research by Renaud et al. [12] that the French population suffered a relatively low incidence of coronary heart disease (CHD) despite the high intake of saturated fat, has sparked the interest of many researchers. The so-called "French paradox" was attributed to moderate wine consumption and its polyphenolic compounds. In a study on 69 male and female subjects, the effects of red wine and red grape extracts were studied. It was observed that moderate red wine consumption increased HDL-C by 11-16% and decreased fibrinogen by 8-15% compared with drinking water. Additionally, the red wine group had a mean weight loss of 0.11 kg. This suggests that moderate red wine consumption is associated with beneficial changes in blood lipids and fibrinogen that may reduce the CV risk factors [164]. Furthermore, alcohol, red wine, and polyphenolic grape extracts may inhibit platelet adhesion to fibrinogen which could attribute to the cardioprotective effects of prolonged and moderate alcohol or red wine consumption [165]. Consumption of capsules with polyphenolic extracts, containing 800 mg of polyphenolic compounds, lowers systolic blood pressure by 3 mmHg and diastolic blood pressure by 3 mmHg. Catechins and procyanidins are likely the class of flavonoids contributing to this blood pressure-lowering effect [166]. Chaves et al. [167] showed that acute consumption of grape extracts (GPE), equivalent to 1.25 cups of fresh grapes, considerably improved brachial artery flow-mediated dilation (FMD) within 3 h of consumption. Further GPE consumption twice a day for 3 weeks further improved FMD, and total antioxidant capacity in plasma was slightly increased. Additionally, the GPE consumption concomitant with high-fat meal prevented high fat-induced vascular endothelial dysfunction. A study conducted on rats during 14 months studied the long term impact of phenolic compounds (PC) on age-associated cardiac remodeling [168]. The three-month-old rats were daily treated till they were middle-aged with different doses of PC. It was observed that long-term daily consumption of PC preserves cardiac morphology and performance with less hypertrophy, inflammation, fibrosis, and cardiomyocyte apoptosis. These results showed that daily consumption of PC could have a positive effect on heart protection during aging. Poti et al. [169] performed a meta-analysis on data extracted from randomized controlled clinical trials involving subjects taking polyphenol-based supplements. The authors included 34 studies and performed a meta-analysis of the main parameters evaluated in the selected studies. The analyzed data showed high heterogeneity because of the differences in the treatment, in terms of formulation, dose, source, and identity of the evaluated polyphenol. Despite that, the overall analysis revealed a considerable effect of polyphenols in positively modulating the cardiovascular parameters considered. The polyphenols positively affected blood pressure, lowering systolic and diastolic pressure. The treatments lowered plasma levels of LDL-C while increasing HDL-C levels and FMD percentage. No considerable effects were detected for high-sensitivity C-reactive protein (hs-CRP). The authors concluded that even though these effects are statistically considerable, the detected differences are of modest size and their clinical benefits need further research.

Neuroprotective Activity
Phenols possess multiple neuroprotective effects that include protecting neurons from damage caused by neurotoxins, possessing the ability to prevent neuronal inflammation, and improving memory, learning, and cognitive functions. These effects can be achieved through two mechanisms. Phenols interact with important signaling pathways in the brain, causing inhibition of neurotoxininduced apoptosis and promoting neuronal survival and differentiation. This group of compounds selectively acts on many protein kinases and the mode of signal transduction that regulate certain transcription factors responsible for the expression of certain genes. Polyphenols have a positive effect on the vascular system and thus affect the cerebrovascular circulation, which ultimately results in angiogenesis, neurogenesis, and changes in the morphology of neurons, thereby improving the ability to remember and learn. The formation of accumulations of amyloid fibrils is a common feature of many neurological diseases, such as Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and prion diseases. The formation of amyloid fibrils is due to aggregation and deposition of misstructured proteins resulting from errors in axon signaling, inhibition of proteasomal activity, errors in DNA transcription, and increased levels of oxidative stress, which ultimately results in neuronal dysfunction. Phenols inhibit the formation of amyloid fibrils through special aromatic interactions, thereby preventing the formation of structured fibrils with a cytotoxic effect [170].
Youssef et al. [171] investigated the effect of grape seed and skin extract (GSSE) in a model of Parkison's disease (PD). They discovered that GSSE has a neuroprotective effect on midbrain dopaminergic neurons by reducing apoptosis, the level of ROS, and inflammation. The GSSE protectd against neural loss and improved motor function in a model of PD.
The grape polyphenolic compounds can delay the initiation and/or hamper the progression of cognitive decline and AD. They can neutralize accumulated neurotoxins and inhibit apoptosis of neural cells caused by oxidative stressors and pro-inflammatory factors [172].

Anticancerogenic Activity
In the past few years interest in the concept and practice of chemoprevention as an approach to control cancer has increased greatly [173]. Polyphenolic compounds have also been shown to prevent the growth of cancer cells. In the study by Hsieh and Wu [174], the combination of epigallocatechin gallate, resveratrol, and γ-tocotrienol was used on breast cancer cells. This combination used at suboptimal doses elicited synergism in suppressing cell proliferation, modulating gene expression, and increasing antioxidant activity, as compared to each of the three phytochemicals added alone. In vitro and in vivo studies showed that resveratrol inhibits the growth of melanoma cells. When resveratrol was administered to mice, it reduced the growth of established melanoma cells and prolonged survival [175]. In a study by Nivelle et al. [176], resveratrol and various oligomeric derivatives were obtained from elicited grapevine cell suspensions. Four stilbenes (resveratrol, ε-viniferin, pallidol, and newly characterized dimer (6)) were recovered and assessed for their biological activity on the cell growth of two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28) and a healthy human dermal fibroblast HDF line. The obtained results showed that resveratrol has the best anti-cancer properties because its efficiency against cancer cell viability is not affected by the presence of fetal bovine serum (FBS). Furthermore, resveratrol showed a tumor-specificity. Oligomers such as ε-viniferin and dimer (6) greatly reduced cancer cell viability, although this activity was considerably decreased in the presence of FBS. Anthocyanins can also have anticancerogenic activity.
Cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, and an anthocyanin-rich grape extract were used to treat colon and breast cancer cell lines. They were used alone or in the presence of a clinically used drug entacapone. Entacapone in combination with anthocyanins had a greater than additive effect on the growth inhibition of the colon cancer cells. Similar results were obtained with treated breast cancer cells, where entacapone enhanced the growth inhibitory activity of the anthocyanin extract. The drug also had antiproliferative effects when used as a single treatment. It was also shown that an important mechanism for growth inhibition is oxidative stress [177]. Cyanidin-3-O-β-glucopyranoside (C3G) was shown to have anti-proliferative and pro-differentiation properties. Treatment with C3G on two different prostate cancer cells displayed reduced cell viability and increased the levels of tumor suppressor [178]. Another study on prostate cancer cell lines was conducted using grape powder extract (GPE) [179]. The GPE treatment inhibited the cell viability and growth of prostate cancer cells only at 100 µg/mL concentrations. However, at low 1.5-15 µg/mL concentrations, GPE considerably reduced the colony formation and wound healing capabilities of prostate cancer cell lines. A natural red wine extract, containing polyphenols, flavonoids, and anthocyanins, was studied on the colon cancer cell line. The red wine extract can activate molecular pathways, involving Nrf2 signaling and the modulation of structural and signaling sphingolipid mediators that cooperate in promoting differentiation and reducing the proliferation of digestive tract cancer cells [180]. In another study, the peel and seed extract were incubated with human epidermoid carcinoma A431 cell lines to evaluate antiproliferative, apoptotic effects, and the morphological apoptotic changes induced by the extracts [181]. The study demonstrated that seed and peel extracts can inhibit the growth of A431 skin cancer cells by inducing cytotoxicity, generating reactive oxygen species followed by loss of mitochondrial membrane potential, and induction of apoptosis. Another study investigated the in vitro effects and putative action mechanisms of grape seed extract (GSE) on human breast cancer cells (MCF-7). The phenolic compounds were able to induce apoptotic cell death in MCF-7 cells at suitable concentrations. At the same time, GSE induced transient but considerable enhancement of GJIC in non-communicating MCF-7 cells and an early and dose-dependent re-localization of the connexin 43 (Cx43) proteins on the plasma membrane. The gap-junction-mediated cell-cell communications (GJIC) is a basal cellular function strictly related to the carcinogenic process and should be considered a target for potential chemotherapeutic compounds [182].

Antimicrobial Activity
One of the pharmacologically important properties of polyphenols and phenolic acids is their antimicrobial activity, i.e., antibacterial, antifungal, and antiviral activity. Their antibacterial activity has been the most researched. They can act through several mechanisms on numerous Gram-positive and Gram-negative bacteria. The mechanisms of antibacterial action of phenols include inhibition of intracellular enzymes, removal of substrate for bacterial growth, direct action on metabolic pathways such as oxidative phosphorylation or electron transfer, and prevention of metalloprotein synthesis through metal ion complexation. Inhibition of nucleic acid synthesis in certain bacteria is based on the inhibition of enzymes such as DNA gyrase and DNA topoisomerase. Due to their structure, phenols can interact with proteins, lipids, and certain enzymes of bacterial cell membranes, and thus cause changes in membrane functionality in the form of changes in fluidity and permeability, allowing the loss of protons, ions, and macromolecules, but also allowing other molecules to enter the cell, such as antibiotics. Oxidative phosphorylation is an important metabolic process for obtaining energy in the form of ATP molecules. Inhibition of only one of the enzymes of this process, such as NADH-cytochrome c reductase, inhibits the whole process, which inhibits the growth of bacteria. Some phenols can inhibit the human immunodeficiency virus (HIV) and in particular the pandemic strain of HIV-1 by preventing the virus from entering the host cell or preventing its replication by inhibiting key enzymes, such as HIV-1 reverse transcriptase and other DNA polymerases. Numerous phenolic compounds can also inhibit the activity of other viruses: herpes simplex virus, adenoviruses, respiratory syncytial virus, poliovirus, rabies virus, rotavirus, and Sindbis virus. Regarding those viruses, the polyphenols inhibit various DNA or RNA polymerases [183][184][185][186][187][188][189][190].
Bacteria are a large group of single-celled organisms that are microscopic in size and are some of the most widespread organisms in nature. They are found in water, air, and soil. They are an integral part of every food chain in nature. Bacteria can be found in all parts of the world, both in the tropics and in areas that are constantly under snow and ice. Many bacteria live in or on humans. They live in the intestinal flora, genitals, oral and nasal cavities, on the skin, and in other parts of the human body. Today, several thousand different types of bacteria have been described, some of which are very useful for humans, and about a hundred bacterial species can cause disease in humans. Pathogenic bacteria are those that can cause human disease, on their own or through their harmful products. Non-pathogenic bacteria can live in or on humans without causing any harm. Under certain circumstances, non-pathogenic bacteria can become pathogenic by penetrating tissues or organs that are not their natural habitats. Pathogenic bacteria are specific for a certain type of host and a special type of tissue. Some types of bacteria destroy the cells of their host. Many pathogenic bacteria produce toxins that are dangerous for the metabolism of the host cell. Pathogenic bacteria can be transmitted through water, food, air, coughing, sneezing, various secretions, and feces. Foodborne bacteria are causative agents of a great number of diseases and they have a great impact on human health and the economy. Among foodborne bacteria Salmonella spp., pathogenic Escherichia coli, Shigella spp., Yersinia spp., Listeria monocytogensis, Staphylococcus aureus, Clostridium spp., pathogenic Bacillus spp., Vibrio spp., and pathogenic Campylobacter spp., are of great importance [191,192].
The antimicrobial activity of different antibacterial agents can be expressed by the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The MIC is defined as the lowest concentration of an antimicrobial ingredient or agent that is bacteriostatic (prevents the visible growth of bacteria). MICs are used to evaluate the antimicrobial efficacies of various compounds by measuring the effects of decreasing concentrations of the respective antibiotics/antiseptics over a defined period in terms of inhibition of microbial population growth. The MBC is the lowest concentration of an antibacterial agent required to kill a bacterium over a fixed, somewhat extended period, such as 18 h or 24 h, under a specific set of conditions. The MBC is identified by determining the lowest concentration of an antibacterial agent that reduces the viability of the initial bacterial inoculum by a pre-determined amount, such as ≥99.9%. The MBC is complementary to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that greatly inhibits growth, the MBC demonstrates the lowest level of an antimicrobial agent resulting in microbial death. In other words, if a MIC shows inhibition, plating the bacteria onto agar might still result in organism proliferation because the antimicrobial did not cause death. Antibacterial agents are usually regarded as bactericidal if the MBC is no more than four times the MIC [193].
Listeria monocytogenes is a new pathogen in food microbiology. This bacterium is present everywhere in nature. Although it is isolated primarily from raw foods of plant and animal origins, it can also be present in cooked food as a result of subsequent contamination with dirty utensils and dishes (cooking removes it). Listeriosis (a disease caused by the bacterium L. monocytogenes) has very mild flu-like symptoms and very often goes unrecognized. However, infection with this bacterium is extremely dangerous for small children, pregnant women (in whom it causes abortion, fetal death, neonatal sepsis), the elderly, and immunocompromised persons, having relatively high mortality in those cases. The entry of bacteria into the body is oral, through food. Food from which L. monocytogenes is isolated is considered unhealthy. Anastasiadi et al. investigated the effects of grape seed extracts and grape steam extracts obtained from Voidomato seed and Mandilaria stems on the bacterium L. monocytogenes. The antimicrobial activity was expressed as MIC after 48 h of inoculation. The obtained values were 0.26 and 0.34 for grape seed extracts and grape steam extracts, respectively [194].
Bacteria species belong to the Campylobacter genus are the major cause of bacterial food-borne diarrheal diseases. This genus comprises 17 species, 14 of which are associated with human disease. Among them, Campylobacter jejuni and Campylobacter coli causes more than 95% of infections attributed to this genus. Silvan et al. investigated the effects of grape seed powder on the activity of nine C. jejuni strains (LP1, 118, CIII, CN1, CNL1, CNL2, NCTC 11351, NTCT 11168, and ATCC33291) and three C. coli strains (CNL4, LP2, and ATCC 43478). The extract, at the final concentration of 500 mg GAE/L, inhibits the growth of all examined bacteria strains. The inhibition of bacteria growth is in the range from 5.08 to 6.97 log CFU/mL. A resistant clinical isolate, C. jejuni LP1, was selected for the determination of MIC value (20 mg/L). The obtained MBC was 60 mg/L which is ten times lower than the most concentrated tested extract [195].
Staphylococcus aureus belongs to the family Staphylococcaceae. This family contains four genera, and members of these genera are Gram-positive, facultatively anaerobic, nonmotile cocci. Cocci can appear singly or in pairs, tetrads, short chains, or characteristic "grapelike" clusters. S. aureus produces enterotoxins responsible for food poisoning. The antimicrobial activity of muscadinia grape seed and grape skin extracts obtained from 10 cultivars against three S. aureus strains (ATCC 12600-U, ATCC 35548, and ATCC 29247) was determined. The average total phenol contents in grape seed extracts and grape skins extracts were 51.13 mg and 19.85 mg GAE/g, respectively. In the case of grape seed extracts average MIC values for ATCC 12600-U, ATCC 35548, and ATCC 29247 strains were 60.1, 54.8, and 59.6 µg/mL, respectively, while for grape skin extracts MIC values were 80.2, 70.7, and 76.7 µg/mL, respectively. These results indicate the correlation between total phenol content and antimicrobial activity. Higher polyphenolic content causes stronger inhibition of bacterial growth. The above-mentioned S. aureus strains are used for the antimicrobial study of the effectiveness of Carlos skins (CK) and seed (CS) extracts, and Noble skin (NK) and seed (NS) extracts. The phenolic contents of CK, CS, NK, and NS extracts were 39.0, 35.9, 58.8, and 58.2 mg GAE/g D.M., respectively. The MIC values for CS and NS (67 and 88 µg/mL) against ATCC 33548, ATCC 12600-U, and ATCC 29247 strains were, respectively, for NK, 113, 226, and 113 µg/mL, and for CK, 152, 304, and 152 µg/mL. The MIC values for pure compounds such as gallic acid, caffeic acid, catechin, ellagic acid, and quercetin were around 2500 µg/mL. The MIC values were determined for antibiotic compounds ampicillin, nalidixic acid, and streptomycin. Ampicillin had a MIC value lower than 1000 µg/L (5 µg/mL) only against ATCC 12600-U, while streptomycin had MICs of 20 µg/mL against ATCC 33548 and ATCC 12600-U strains. Nalidixic acid as a very strong antibiotic compound was very active against all analyzed strains with a value range from 20 to 156 µg/mL. Based on these results, grape seed extracts are very potent antimicrobial agents against three S. aureus strains-even stringer inhibitors than some antibiotics. The planktonic cell susceptibility to polyphenols and polyphenols inhibiting biofilm formation was determined by studying the ATCC 35548 strain and results are expressed as minimum biofilm inhibitory concentration (MBIC). The obtained MBIC value for inhibiting biofilm formation was within 2-fold lower than MICs, while MIBCs of preformed biofilm were 16-fold higher than MICs. The MICs for CS and NS against planktonic cells were 40 and 51 µg/mL, respectively; those against biofilm formation were 20 and 25 µg/mL, respectively; and those against preformed biofilm were 641 and 800 µg/mL, respectively. These findings indicate that GSE can inhibit bacterial biofilm formation at sub-MIC concentration [196,197].
Methicillin-resistant Staphylococcus aureus (MRSA) can cause nosocomial and community-acquired infections. The above-mentioned bacterium causes many diseases, such as mild skin infections, pneumonia, septicemia, and deep-seated abscesses. The effect of grape seed proanthocyanins extract (GPSE) on 43 MRSA strains was analyzed. All examined MRSA strains showed sensitivity against GPSE but with different levels of inhibition; thus, they can be divided into three groups based on the diameter of the inhibition zone: weakly sensitive (16 strains), moderately sensitive (15 strains), and highly sensitive (11 strains). At the concentration of 3 mg/mL of crude GPSE (the equivalent of 20.7 µg/mL flavonoid content), complete inhibition of all analyzed bacterial strains was observed. The GPSE has bactericidal activity because it disrupts the bacterial cell wall [198].
Escherichia coli is a Gram-negative, non-spore-forming, facultative anaerobe; a rod-shaped bacterium. It may or may not be mobile, but bacterial motility has principal roles in its adherence to surfaces and the potential formation of biofilms and gut colonization. E. coli encompasses a large and diverse group of bacteria. Most strains are harmless. Pathogenic strains can produce toxins. These strains are divided into six groups according to the mechanism of pathogenesis: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC, also known as Shiga toxin-producing E. coli, STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and attaching and effecting E. coli (A/EEC). The E. coli STEC group includes the following strains: O157:H7, 026:H11, O45:H2: O103:H2, O111:H2, O121:H19 and O145: NT. The major virulence factor of STEC strains is Shiga toxin (Stx). The effects of grape seed extract containing a minimum of 95% total flavonoids and 82% procyanidins on the seven above-mentioned STEC strains were studied. The MIC and MBC values were 2 mg/mL for the O26:H11 strain, and these values for all other strains were 4 mg/mL. The GSE content of 4 mg/mL effectively inhibited the growth of all tested strains, whereas 0.25-2 mg/mL GSE delayed bacterial growth when the level of inoculation was 5 × 10 5 CFU/mL. A marked reduction in the swimming motility of all motile tested STEC strains can be achieved with GSE content of 0.125 mg/mL, while 4 mg/mL GSE inhibited the production of Stx in 103:H2, O111:H2, and O157:H7 strains [199,200].
Delgado Adamez et al. are collected grape seeds from Tempranillo grapes before and after vinification, and they got extracts from dry grape seed obtained before winemaikin (GSEJ) and dry grape seed obtained after winemaikin (GSEW), respectively. The antimicrobial activities of the GSEJ and GSEW were tested against three Gram-positive bacteria (910 Listeria innocua, 847 T Brochothrix thermosphatica, 910 Staphylococcus aureus subsp. aureus) and three Gram-negative bacteria (110 T Pseudomonas aeruginosa, 409 Salmonella enterica subsp. enterica, 45 Escherichia coli). In the seed extracts, dilutions of 100 and 50 µL/mL, inhibition for all analyzed bacteria was nearly 100% which means that both extracts were active against all examined bacteria. They were more effective against Gram-positive bacteria than Gram-negative bacteria. The latter bacteria have a lipopolysaccharide layer which is involved in the reduction of the sensitivity of those bacteria against extracts. The MIC was determined after 24 h of inoculation. This value for the GSEJ was 50 µL/mL for S. aureus subsp. aureus and L. innocua and 100 µL/mL for B. thermosphatica and S. enterica subsp. Enterica, while for E. coli and P. aeruginosa the MIC value exceeding 100 µL/mL In a case of GSEW, MICs for S. aureus subsp. aureus and L. innocua were 100 µL/mL, while for other bacteria MIC values were the same as for GSEJ extract [201].
The antimicrobial effect of grape pomace extract from Cabernet Sauvignon (132.2 mg of GAE/g) and Syrah (102.6 mg of GAE/g) is tested against two Gram-negative bacteria (E. coli ATCC 25922 and Salmonella typhi STH-2370) and two Gram-positive bacteria (L. monocytogenes ATCC 15311 and S. aureus ATCC 6538). Grape pomace extract had antibacterial activity against all the analyzed bacteria strains. At the concentration of 500 µg/mL extracts reach over 90% of inhibition for all studied bacteria apart from S. Typhi (70% of inhibition). In general, S. aureus and E. coli were the most sensitive bacteria exceeding 50% of inhibition at 62.5 µg/L concentration of extracts [202].
Jayaparkasha et al. are isolated grape seeds from Bangalore blue grapes and made an extract. The obtained extract is tested for antibacterial activity by pour plate method against Bacillus cereus, Bacillus cougalans, Bacillus subtilis, S. aureus, E. coli, and P. aeruginosa. The MIC values of GSE are determined by the number of colonies developed after incubation. The MIC was 900 ppm for B. cereus, B. subtilis, and B. couagulans, 1000 ppm for S. aureus, 1250 ppm for E. coli, and 1500 ppm for P. aeruginosa. Based on these values it can be concluded that GSE strongly inhibits grow of tested Gram-positive and Gram-negative bacteria [203].
Helicobacter pylori are the Gram-negative bacterium that is the etiological agent of peptic ulcers and gastritis. It is also associated with mucosa-associated lymphoid tissue lymphoma and gastric cancer. Martini [204,205].
Porphyromodonas gingivalis and Fusobacterium nucleatum are associated with periodontitis and other acute periodontal diseases. These bacteria are embedded in the plaque biofilms. The antibacterial activity of grape seed extracts (GSE) had MIC values 4000 and 2000 µg/mL for P. gingivalis and F. nucleatum, respectively determined 24 h after inoculation with bacteria. The determined MBC values for both bacteria were 8000 µg/mL. The GSE at the concentration of 2000 µg/mL had an inhibitory effect on the formation of the biofilm composed of Lactobacillus, Streptococcus, Actinomyces, Fusobacterium, and Poryformonas bacteria. The presented values indicate that GSE could be used in oral hygiene for the prevention of periodontitis [206].
Hepatitis C virus (HCV) is a positive single-stranded RNA virus belonging to the Flaviviridae family. This virus contains 10 individual proteins, of which six are nonstructural (NS) proteins, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Nonstructural proteins are necessary for replication and other cellular processes, thus they are a potential target for antiviral agents. The HCV NS3 protein has protease and helicase activity so it is an indispensable component for the replication of HCV. Vitisin B has no effect on HCV entry in the host cell via the HCVpp system and does not affect the cleavage of polyprotein by the action of the NS3/NS4A protease, but it can negatively regulate HCV assembly/production, independent of its effect on the replication of HCV. The main inhibitory mode of action of vitisin B and ε-viniferin is the reduction of HCV protein production by inhibition of replication of the viral genome. These compounds specifically targeting the activity of NS3 helicase by direct binding and competing with its natural substrate, ATP. The anti-HCV activity of the vitisin B was EC 50 = 6 nM and CC 50 > 10 µM. [209,210].
Herpesviridae is a family of viruses consisting of 8 species each causes a distinct disease: humane cytomegalovirus (HCMV) herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), human herpesvirus 8 (HHV-8), and Epstein-Barr virus (EBV). According to the Baltimore classification, they belong to the enveloped, nuclear replicating double-stranded DNA viruses. These viruses rely on the host machinery for DNA replication and transcription via RNA [211]. The HCMV is a lymphotropic virus that replicates and establishes latency in tissues associated with the lymphatic system. This virus is a causative agent of mononucleosis. Selective inhibition of HCMV replication can be achieved by inhibition of cellular p38 mitogen-activated protein kinase. Entry and/or signal transduction via epidermal growth factor receptor (EGFR) and downstream activation of phosphatidylinositol 3-kinase (PI3-K) and effectors such as p70S6K and Act are the initial events in the replication of HCMV. The major IE gene promoter (MIEP) of HCMV which is necessary for viral genome expression is activated by transcription factors NF-κB and Sp1. Resveratrol inhibited autophosphorylation of the EGFR and PI3-K signaling and the overall result is the inhibition of viral DNA replication. It also fully inhibits the initial phase of NF-κB and Sp1 activation and thereby reducing activities of DNA binding to those proteins. Resveratrol blocks the appearance of immediate-early, early, and late viral proteins of HCMV. Overall, this stilbene act early in the replication cycle of HCMV, no later than 4 h postinfection. Inhibition of the HCMV was IC 50 = 1-2 µM [212]. The HSV-1, HSV-2, and VZV infect nervous tissue (neurotropic viruses). Herpes simplex virus 1 and 2 are causative agents of recurrent facial and genital herpetic lesions. The genome of the HSV virus is very complex and encodes nearly 100 transcripts. The productive replication cycle is composed of different stages: immediate-early (no viral protein synthesis), early (no DNA replication), and late (after viral genome replication). The entry of the virus to the cell requires interaction between viral membrane glycoproteins and cellular receptors, herpesvirus entry mediators (HVEM) which are related to the proteins that interact with the tumor necrosis factor (TNF). Host nuclear transcription factor NF-κB can be activated by different stimuli such as inflammatory cytokines, growth factors, and bacterial or viral infections. This factor is activated in response to HSV infection and at least two immediate-early herpes proteins, ICP4 and ICP27. The above mention factor interacts directly with the genome of the HSV virus via binding to the promoter of an immediate-early gene, ICP0. Resveratrol suppresses activation of NF-κB in cells infected by HSV-1 and HSV-2. This compound also negatively affects the synthesis of mRNA for ICP0 and ICP4 genes (immediate-early genes), and HSV DNA polymerase and single-stranded DNA binding protein (ICP8) (early genes). This led to the considerable inhibition of od DNA synthesis and late gene activation. Resveratrol in the concentration of 219 µM reduces viral titer by 99.9%. Overall, resveratrol inhibits early phases of HSV replication and has a great impact on the later viral cycle phases [213]. Varicella-zoster virus causes two distinct clinical entities: varicella (chickenpox) and herpes zoster (shingles). The genome of VZV contains double-stranded DNA with 125kb. Resveratrol does not affect the attachment of the virus to the host cell. It interferes with the first stage of the replication of VZV by suppression of activation of an immediate-early gene, ie62. IE62 protein is an essential regulatory protein of VZV. This protein structurally and functionally is similar to the ICP4 from HSV and regulates the expression of the viral genome. Resveratrol by reducing the level of mRNA affects the synthesis of IE62. In the concentration of 219 µM inhibit VZV replication with the lower limit of plaque assay of 10 pfu/mL [214].
Influenza A virus belongs to the family of orthomyxoviruses. It contains a segmented genome composed of minus-strand RNA. Like other orthomyxoviruses, the influenza A virus has enveloped virions with spikes. There are two different types of spikes composed of hemagglutinin (HA) protein and neuraminidase (NA) protein. Characteristics of the above-mentioned virus are that it has a viral ribonucleoprotein (vRNP) made of eight segments of (−)RNA associated with nucleocapsid proteins and enzymes, PB1, PA, and PB2 which are components of RNA-dependent RNA RNA polymerase (RdRp). The viral RNA encodes 17 influenzas A proteins, six of which are phosphorylated. During influenza virus infection, respective kinases cascades are activated including p38MAPK and JNK pathways and Raf/MEK/ERK cascade. Palamara et al. [215] in-depth investigated the effect of resveratrol on the replication of the virus in vivo and in vitro. Based on obtained results they concluded that resveratrol blocks nuclear-cytoplasmic translocation of vRNP complexes decreases expression of late viral proteins, and an inhibition of cellular protein kinase C (PKC) activity and its dependent pathways. Resveratrol inhibits the expression of M1 and HA on the post-transcriptional level by blockage of the nucleocytoplasmic translocation of vRNPs. Resveratrol inhibits the activities of PKC, and thus effects MPAK pathways (p38MPAK and JNK). Resveratrol in a concentration of 40 µg/mL completely blocks viral replication. Administration of resveratrol to mouses infected with influenza A virus considerably improves their survival and decreases pulmonary virus titers.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the causative agent of Coronavirus Disease 2019 (COVID- 19), first emerged in 2019 and triggered a global pandemic affecting more than 215 countries. As of October 2020, there were more than 42,000,000 confirmed cases and more than 1,100,000 deaths. At this moment, there are no specific antiviral drugs or vaccines available for the treatment of COVID-19. This virus belongs to the Coronaviridae family of viruses which are enveloped, single-stranded positive-sense RNA viruses. The genome is ≈29.9 kb long, composed of 12 functional open reading frames and coated with nucleocapsid (N) protein, and enclosed by a lipid bilayer consisting of the three membrane proteins: spike (S), membrane (M), and envelope (E). The genome encodes 12 proteins, such as main protease (M Pro ), RdRp, RNA binding N terminal domain (NTD) of nucleocapsid protein (N protein), viral ion channel (E protein), 2 -O-RIBOSEMethyltransferase, and human angiotensin-converting-enzyme 2 receptor (hACE-2) for entry into target cells. The above-mentioned proteins are the key targets for drug development. Some studies confirmed that kaempferol, quercetin, rutin, catechin, epigallocatechin, epigallocatechin gallate, ferulic acid, caffeic acid, δ-viniferin, and delphinidin-3-O-glucoside inhibit ACE-2 receptor and can potentially inhibit the entry of the virus into the target cells. Malvidin-3,5-O-diglucoside, δ-viniferin, delphinidin-3-O-glucoside, kaempferol, malvidin-3-O-glucoside, myricetin, and procyanidins have a high affinity for binding to M Pro , RdRp, and hACE-2 so could inhibit viral replication in host cells [216][217][218][219].

Conclusions and Future Perspectives
Grape phenolic compounds are a large and diverse class of compounds found in grapes. Many factors influence the final contents and composition of phenols in grape, among which are genetic factors, environmental ones, and cultural practices. Phenolic compounds have important roles in plants, as they act as phytoalexins, UV-protectants, and decrease the reactions caused by abiotic and biotic stresses. Furthermore, they are important in winemaking, as they influence wine color and pigmentation, moth-feel, bitterness, and astringency. Additionally, the consumption of wine and fresh grapes and its products have shown the positive influence of polyphenolic compounds on human health. Different studies showed antioxidant, anti-inflammatory, neuroprotective, anticancerogenic, and antimicrobial activity of phenols. Thus, it would be beneficial to include fresh grapes and their products, along with moderate wine consumption, in our diet.
In this review, a comprehensive presentation of phenolic compounds in grapes and grape leaves is given. It includes extraction techniques, methods of analysis, composition, and contents of phenols in individual varieties with a special emphasis on their impacts on human health.
Despite the differences in expression in different studies related to the content of phenolic compounds in grapes and grapevine leaves, it is clear that there is huge variability in the contents of numerous phenolic compounds related to specific cultivars, but for the same reason, it is difficult to compare this results. Some efforts should be made in the future to obtain more comparable datasets.
There are numerous extraction techniques for the isolation of phenolic compounds from the grapevine. The choice of the technique is strongly dependent upon available laboratory equipment and the goal of the extraction. The highest throughput of samples can be achieved by the application of SLE and EAE. These techniques are also suitable for the commercial extraction of phenolic compounds because GRAS solvents can be used.
Non-communicable diseases such as cardiovascular disease, diabetes, cancers, and many neurodegenerative diseases are the leading causes of death in developed countries. A large amount of money is spent on the treatment of these diseases, which makes them a major economic problem. Synthetic drugs available on the market very often have several side effects, and their long-term use very often harms organs that were not originally affected by the disease. Polyphenols isolated from the vine are not cytotoxic, and numerous studies have shown that they have a positive effect on the cardiovascular system, and neuroprotective and anticancerogenic activities. An increase in the number of studies for the treatment of these diseases is necessary to determine their effects and to become approved drugs by the competent authorities such are FDA and EMA.
Grapevine extracts and individual polyphenolic compounds isolated from grapevines show strong antimicrobial activity in terms of antibacterial and antiviral action. These properties of the above-mentioned compounds could have a strong impact on humanity. In the last ten years, an increasing number of different strains of bacteria that are resistant to existing antibiotics have been discovered. They have greatly increased mortality from certain bacterial infections. The development of new antibiotics is a very time consuming and expensive process with an uncertain outcome. Therefore, research on natural compounds with antimicrobial activity constitutes a turning point in solving problems in the treatment of diseases caused by resistant strains. Numerous studies have shown that grape polyphenols can effectively inhibit the growth of certain strains of bacteria, but also act synergistically with antibiotics on otherwise resistant bacteria. Compared to the number of effective antibiotics available on the market, the number of antiviral drugs is small. The action of certain antiviral drugs is generally limited to a small number of virus strains. Numerous studies confirm that polyphenols isolated from vines have an antiviral effect on several viruses. Due to climate change and many other factors of today's lifestyle, the emergence of new pandemic viruses is to be expected in the future. Therefore, new research on the antiviral activity of polyphenols is necessary.
Grapevine genetic resources represent a huge and mostly unexplored source of different phenolic compounds, with the majority of results being available only on a small and limited number of cultivars that are in the focus of the grape and wine industry. Given that there are more than 5000 different cultivars of grapevine still existing in grapevine repositories around the world, huge efforts are still needed in the future to obtain full insights into grapevines as a source of phenolic compounds. Taking into account that in the majority of studies a synergistic effect of individual compounds present in the grape extracts is observed, and that these contents are not very high, it can be concluded that the grape is a very potent source of polyphenolic compounds with effects on human health.