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Stilbenoids in Grapes and Wine
Noélia Duarte, Cátia Ramalhete, Patrícia Rijo, Mariana Alves Reis,and Maria-José U. Ferreira
Contents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Stilbenes in Vitis vinifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Bioavailability and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Bioactivities of Stilbenoids: In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Human Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Application in Food: Wine Derivatives as Dietary Supplements and Related Products . . 207 Safety: Toxicity and Side Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Marketed Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
10 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
N. Duarte · M.-J. U. Ferreira (*)Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,Lisbon, Portugale-mail: [email protected]
C. RamalheteResearch Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,Lisbon, Portugal
ATLÂNTICA – Escola Universitária de Ciências Empresariais, Saúde, Tecnologias e Engenharia,Barcarena, Oeiras, Portugal
P. RijoResearch Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,Lisbon, Portugal
Research Center for Biosciences & Health Technologies (CBIOS), Universidade Lusófona deHumanidades e Tecnologias, Lisbon, Portugal
M. A. ReisInterdisciplinary Center of Marine and Environmental Research (CIIMAR/CIMAR),University of Porto, Matosinhos, Portugal
© Springer Nature Singapore Pte Ltd. 2020J. Xiao et al. (eds.), Handbook of Dietary Phytochemicals,https://doi.org/10.1007/978-981-13-1745-3_21-1
1
AbstractStilbenoids are naturally occurring polyphenols, found in several edible plants,such as berries, peanuts, and grapes, which have been attracting increasinginterest owing to their potential benefits for human health, namely, to preventand treat chronic diseases related with aging. They can be constitutivelyexpressed or biosynthesized as phytoalexins. Structurally characterized by aC6–C2–C6 scaffold, they can be found as monomers, dimers, trimers, or verycomplex oligomers. In this chapter, the focus will be on dietary stilbenoids foundin the Vitis vinifera, namely, in grapes and wine, which are the principal dietarysource of stilbenes. Among them, trans-resveratrol is the most extensively inves-tigated stilbene. The main bioactive constituents, bioavailability and metabolism,the in vivo biological properties of the major constituents, and their role in humanhealth, application in food, as well as issues related with safety and marketed andpatented products will be also discussed.
KeywordsStilbenes · Vitis vinifera · Wine · Polyphenols · Oligostilbenes · Trans-resveratrol · In vivo studies · Bioavailability · Metabolism · Clinical trials
1 Introduction
Stilbenes or stilbenoids are plant-derived polyphenols, occurring in several plantfamilies being particularly abundant in Gnetaceae, Pinaceae, Cyperaceae, Fabaceae,Dipterocarpaceae, and Vitaceae (Rivière et al. 2012). They have been attractingincreasing interest due to their diverse biological activities and potential benefits forhuman health, namely, cardioprotection, neuroprotection, antidiabetic properties,anti-inflammation, and cancer prevention and treatment, making them importantlead compounds in drug discovery and development. An important example ofthis is trans-resveratrol (trans-3,40,5-trihydroxystilbene, 1), commonly known asresveratrol, which has shown several biological activities, including strong antiox-idant effects, thus providing cardiovascular protection by reducing oxidative stress.The cardioprotective effects of resveratrol have been connected to the “FrenchParadox.” The term is related to the paradoxical epidemiological observation thatFrench population has a low incidence of coronary heart disease, despite the highintake of saturated fats. It was hypothesized that French paradox might be owing tothe consumption of red wine, which may contradict the negative effects of a diet richin saturated fat. This conclusion fostered the interest in the overall phenolic constit-uents of red wine and particularly in resveratrol that has been extensively investi-gated (Akinwumi et al. 2018).
In plants, stilbenoids can be constitutively expressed or can be synthetized asphytoalexins in response to fungal and bacterial pathogens and/or to other environ-mental stress factors (Xiao et al. 2008). This class of secondary metabolites derives
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from phenylpropanoid and acetate-malonate pathway with a chemical structurebased on a C6–C2–C6 backbone, where two phenyl rings are joined by an ethylenebridge. They can assume both a trans (E isomers, 1–6, Fig. 1) and a cis configuration(Z isomers, 7 and 8, Fig. 1), which elicits different pharmacological activities, beingthe trans isomer the most common and stable configuration in naturally occurringstilbenes. Structural variations in stilbenoids include different substituent patterns inthe aryl groups, which may bear different hydroxyl, methoxyl, prenyl, or geranylmoieties and may also appear as glycosides (Figs. 1 and 2). They can be foundas monomers and as very complex oligomers, which may result from oxidative
OH
HO
OH
H3CO
H3CO
H3CO
OCH3
OCH3
OCH3
OCH3
pterostilbene (2)
12
3
45
6
7
8
1'
2'3'
4'
5'6'
trans-resveratrol (1)
cis-resveratrol (7)combretastatin A4 (8)
gnetol (3)
OH
OH
OH
OH
oxyresveratrol (4)
trans-piceatannol (5)
OH
HO HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
isorhapontigenin (6)
OH
OH
OH
OH
Fig. 1 Examples of monomeric trans-stilbenes (1–6) and cis-stilbenes (7 and 8) with hydroxyl andmethoxyl substituents
Stilbenoids in Grapes and Wine 3
coupling of monomeric stilbenes through C–C or C–O–C bonds of resveratrol (1),oxyresveratrol (trans-2,30,4,50-tetrahydroxy-stilbene) (4), piceatannol (trans-3,30,4,50-tetrahydroxy-stilbene) (5) or isorhapontigenin (trans-3,40,5-trihydroxy-30-methoxystilbene) (6) (Pawlus et al. 2012; Rivière et al. 2012; Shen et al. 2009).
Vitis vinifera L. (Fig. 3) belongs to the Vitaceae family and is the main dietarysource of stilbenes, which are consumed in the form of grapes, grape juice, and wine.The therapeutic and health-promoting properties due to the consumption of grapesand related products have been attributed to the presence of a complex combinationof polyphenolic compounds, which includes not only monomeric stilbenes such asresveratrol (1) but also simple phenolics and flavonoids among others, being thelatter the largest group (Georgiev et al. 2014).
In this review, the focus will be on the beneficial health effects of the dietarystilbenoids found in the V. vinifera. The activities of the main stilbenoids, found ingrapes and wine, on relevant animal assays and clinical trials will be discussed interms of bioavailability and metabolism, principal biological activities, and their rolein human health. Application in food, as well as issues related with safety andmarketed and patented products, will be also discussed.
OH
O
OHtrans-piceid (10)
OH
HOHO
H
H
HOHH
OH
pawhuskin A (9)HO
OH
OH
OH
astringin (11)
OH
O
OH
OH
H
HOHO
H
HOHH
OHOH
Fig. 2 Examples of monomeric stilbenes with prenyl, geranyl, and glycosyl substituents
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2 Stilbenes in Vitis vinifera
Structurally characterized by a C6–C2–C6 scaffold, stilbenes are secondary metab-olites derived from a mixed biosynthetic pathway (Fig. 4). In this way, onep-coumaroyl-CoA starter molecule, derived from the shikimate pathway, is con-densed to three malonyl-CoA molecules derived from the acetate pathway, givingrise to a polyketide chain (Dewick 2009). Stilbene synthase enzyme catalyzes thefolding and subsequent cyclization through an aldol condensation reaction thatgenerates the basic skeleton of stilbenes, represented by resveratrol (1). Furthermethylation, glucosylation, and prenylation reactions give rise to a myriadof stilbenoid derivatives.
Stilbenoids, found in several edible plants, such as berries, peanuts, and grapes,are considered promising compounds to prevent and treat chronic diseases relatedwith aging (Khawand et al. 2018). More than 1000 stilbene derivatives have beenidentified (Pawlus et al. 2012), ranging from monomers to octamers and differentsubstituent patterns. In Vitaceae family, they are found in several species, includingV. vinifera, the most important species for wine production. In fact, red wine andgrapes are the main dietary source of the most known and studied stilbene resveratrol(1). Resveratrol (1) and its derivatives, with several patterns of oligomerizationand glycosylation, are also found in the whole plant, such as leaves, canes, wood,and roots of the grapevine and in larger amount than in grape berries (Gabaston et al.2017).
Fig. 3 Vitis vinifera (Pixabay 2019; Wikimedia commons 2019)
Stilbenoids in Grapes and Wine 5
Oligomeric stilbenes were previously classified into two main groups, accordingto the presence or not of at least one five-membered oxygen-containing heterocycle(Sotheeswaran and Pasupathy 1993). Owing to its limitation, there have been severalattempts to improve this classification, by dividing first oligomeric stilbenesaccording to their monomeric moieties and heterogeneous coupling and then furtherclassifying these based on the presence (group A; e.g., ε-viniferin, 12) or absence(group B; e.g., pallidol, 13) of oxygen-containing heterocycles (Fig. 5) (Shen et al.2009).
Most of the stilbenes found in Vitis species as monomers are methoxylated andpredominantly glycosylated derivatives of resveratrol (1) and piceatannol (5).The monomers can be found as trans and cis isomers such trans- and cis-piceid[trans-(10) and cis-resveratrol-3-O-β-D-glucopyranoside], pterostilbene (trans-3,5-dimethoxy-40-hydroxy-stilbene) (2), and astringin (11), the 3-β-D-glucoside ofpiceatannol (Figs. 1 and 2). In V. vinifera, the number of these stilbenes is lower inwine than in grapes (ISVV database 2017).
CO2H
NH2
O O
SCoA
SCoA
HO+ 3x
SCoAO
1. Deamination2. Hydroxylation3. CoA activation
OH
O
O
O
O
OH
OH
OH
HO
Aldol reaction(Stilbenesynthase)
Starter Unit
Extender Unit
1
Shikimate pathway
Acetate pathway
Fig. 4 Biosynthesis of stilbenes
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Concerning the dimeric forms of stilbenes found in wine, most of them bearresveratrol (1) as monomer. They are essentially derivatives of viniferin and pallidol(13), being the most frequent ε-viniferin (12) (Fig. 5), the major oligomer found inVitis species. While several trimers and tetramers (13) have been also found inVitis species, including in different plant parts of V. vinifera, only one of theseoligostilbenes, namely, the tetramer hopeaphenol (13, Fig. 6), has also been reportedin wine. Two pentamers were isolated from V. amurensis roots, but there is no reportfor the presence of this type of oligomers in V. vinifera. Conversely, more recently,the resveratrol hexamer viniphenol A (14) (Fig. 6) was isolated from the vine stalksof V. vinifera (Pawlus et al. 2012; Shen et al. 2017).
H
H
OH
HO
pallidol (13)
OHO
HO
HOHO
OH
OH
OH
OH
OH
�-viniferin (12)
Fig. 5 Oligostilbenes with(12) and without (13) oxygen-containing heterocycles
O
HO
HO
HO
OH
O
O
HOHO
OH
HO
HO
OH
OH
OH
OH
HO
H
H
HH
H
H
H
H
OH
H
H
H
H
viniphenol A (14)
OH
H
O
OH
OH
HO
OH
OH
HO
HOHO
OH H
H
H
H
OH
hopeaphenol (13)
Fig. 6 Examples of oligostilbenes found in V. vinifera
Stilbenoids in Grapes and Wine 7
3 Bioavailability and Metabolism
In the last two decades, extensive studies have been conducted in order to find outthe biological effects of stilbenes. Concomitantly, bioavailability and other pharma-cokinetic preclinical studies have been performed in animal models and humans.In this regard, the most studied stilbene is resveratrol (1), although in recent years,pterostilbene (2) and piceatannol (5) have also begun to be explored. Unfortunately,and notwithstanding all the promising biological effects reported for several diseasemodels, the pharmacokinetic properties and bioavailability of these stilbenes are notso satisfactory, a fact that may limit their application in clinical use.
Despite its low water solubility (< 0.05 mg/mL), resveratrol (1) is rapidlyabsorbed at the small intestine by passive diffusion and undergoes apromptly and extensive phase II metabolic reactions catalyzed by uridine 50-diphosphoglucoronyltransferases (UGTs) and sulfotransferases (SULTs) in the intes-tinal epithelial cells and liver. It is reported that after an oral administrationof resveratrol, its plasma half-life is about 8–14 min and consequently only about1–2% of its free form may be found in plasma (Dvorakova and Landa 2017).Resveratrol-3-O-glucoronide (15), resveratrol-40-O-glucoronide (16), their sulfateanalogues (17, 18), and resveratrol-3,40-O-disulfate (19) have been reported as themajor phase II metabolites (Fig. 7). The conjugation to sulfates and glucuronic acidincreases water solubility and allows the excretion by the kidneys, being thesemetabolites mainly eliminated in urine (Singh et al. 2015).
On the other hand, in vitro studies using human liver microsomes demonstratedthe formation of two tetrahydroxylated resveratrol metabolites, piceatannol (5) andanother one that was putatively identified as 3,4,5,40-tetrahydroxystilbene (20),which resulted from phase 1 metabolic reactions. This study suggested the majorrole of cytochrome P450 (CYP), particularly the isoform CYP1A2 in this metabolicprocess (Piver et al. 2004). Other metabolites have been identified, in fecal samples,such as 3,40-dihydroxy-trans-stilbene (21), dihydroresveratrol (22), and 3,40-dihydroxybibenzyl (lunularin, 23), highlighting the important function of gut micro-biota in resveratrol metabolism (Fig. 6) (Wang and Sang 2018). The extensivemetabolism of resveratrol decreases circulating levels of the free molecule; thus, itis suggested that biological effects could be also attributed to resveratrol metabolitesor probably to the enterohepatic recycling involving biliary secretion, which mayresult in some deconjugation by gut microflora in the small intestine andreabsorption of the original molecule (Akinwumi et al. 2018).
However, the absorption and metabolic processes appear to be dependent ondifferent factors, such as specific inter-individual variability, administration routesand doses, species, gender, disease status, and mode of consumption (fasting or withfood or wine). Several studies carried out both on animals and humans compared thepharmacokinetics of resveratrol regarding different doses and treatment regimensand administrations routes (oral and intravenous). To study the influence of theadministration routes on resveratrol pharmacokinetics in humans, 14C-labeled res-veratrol was administered orally or intravenously with doses of 25 and 0.2 mg,respectively (Walle et al. 2004). A high absorption rate was observed followed by anextensive biotransformation in both types of administration, resulting in only trace
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amounts of free resveratrol in the systemic circulation. After an oral dose of 25 mg, atotal radioactivity of 491 � 90 ng/mL (resveratrol and metabolites) was reached atabout 1 h after the dose. Curiously, at around 6 h after consumption, a second peakplasma total radioactivity was observed, which was suggested to be a result of theenterohepatic recirculation of conjugated metabolites. The urinary excretion wasassessed and indicated at least 70% of absorption and a recovery of total radioac-tivity ranging from 53.4% to 84.9% in urine after oral consumption. Similar resultswere obtained after the i.v. dose of 0.2 mg of 14C-labeled resveratrol. One hour afterthe bolus injection, a rapid decrease of total radioactivity was observed on plasma,but the second plasma peak was not detected (Walle et al. 2004).
In a study performed in healthy volunteers that took orally low doses ofresveratrol (5 or 50 mg), the main metabolites found in plasma were glucuronides.On the other hand, when high doses were administered (> 250 mg), resveratrol-3-O-sulfate (17) was the main identified metabolite. Interestingly, in plasma samplescollected 11–21 h after intake, the main identified metabolite was the derivativeconjugated with both glucuronide and sulfate moieties (Wang and Sang 2018).
In order to study the influence of different doses of resveratrol, a pharmacokineticstudy was conducted by orally administering single doses of 0.5, 1, 2.5, or 5 g, in 10healthy volunteers per dose level (Boocock et al. 2007). The rapid absorption ofresveratrol was corroborated in this study, achieving a peak plasma concentration inthe 0.83 h–1.5 h post-dose. Peak plasma levels of resveratrol at the highest dose were
Fig. 7 Resveratrol metabolites resulting from phase 1 and 2 metabolic reactions and microbialbiotransformation in gut
Stilbenoids in Grapes and Wine 9
0.54 � 0.38 μg/mL achieved in the 1.5 h post-dose. Two monoglucuronides andresveratrol-3-O-sulfate (17) were the main identified metabolites, and their concen-tration exceeds those of resveratrol by up to 20-fold. The study also concluded thatexcretion rates were highest in the 4 h post-dose, and when the lowest dose wasconsumed, 77% of all urinary species were excreted within this period. In the 24 hpost-administration of the 0.5 g dose level, the amount of resveratrol excreted in theurine was below 0.04% of the dose, whereas urinary excretion of the three resver-atrol conjugates ranged from 0.51% (one of the glucuronides) to 11.4% of the dose(resveratrol-3-O-sulfate, 17). This study proved that only traces of resveratrol couldbe found in plasma even after high-dose consumption, suggesting that ingestion ofresveratrol equivalent to the amount contained in several hundred bottles of red wineis not enough to elicit the biological effects associated with this compound (Boococket al. 2007). The concentrations used in in vitro or in vivo studies with resveratrolsupplements are too high to be reached in the organism after wine consumption.Avery interesting research was conducted aiming at study resveratrol bioavailabilityafter a moderate consumption of red wine (300–600 mL) associated with fasting anddifferent meals: a standard meal and meals with high and low amount of lipids(Vitaglione et al. 2005). It appears that resveratrol bioavailability is not influenced byfasting, the type of meal, or the amount of lipid content. Moreover, it was demon-strated that resveratrol absorption and metabolism after wine consumption are highlyvariable, being found in the serum of roughly half of the subjects in free or inglucuronidated form and in very low concentrations. Other studies were carried outto study the matrix effects on the absorption and bioavailability. Theoretically, theabsorption rate of resveratrol may increase when administered with a matrix thatpromotes its solubility (Peng et al. 2018). These researches comprised the moderateconsumption of red wine, grape juice, resveratrol tablets, and different doses ofresveratrol dissolved in different matrices, including white wine, grape juice, vege-table shakes, and diluted ethanol or whisky. Controversial results were obtainedsince some studies concluded that there were no significant differences wherever thetype of matrix, whereas other studies claimed that absorption and bioavailability ofresveratrol were higher when pure compound was administered (Peng et al. 2018).
It was also found that there is no significant difference between a single high-dosetreatment and repeated administrations of resveratrol. In a double-blind, randomized,placebo-controlled study to investigate the multiple-dose pharmacokinetics, fourgroups of 10 healthy volunteers received 25, 50, 100, or 150 mg of resveratrol, sixtimes/day (every 4 h), for 2 days (Akinwumi et al. 2018; Wang and Sang 2018).Similarly to other studies, peak plasma concentrations were reached at 0.8–1.5 hpost-dose. Despite the multiple administrations, the degree of resveratrol accumula-tion in plasma was not significant even for the highest dose. However, high inter-individual variability and circadian variations were observed, being the residualplasma concentration highest after morning administration and lowest during thenight (Akinwumi et al. 2018).
Although much less studied than resveratrol, the bioavailabilities of pterostilbene(2) and piceatannol (5) are nowadays beginning to be unraveled. In comparison withresveratrol, pterostilbene (2) has two methoxyl groups located at C-3 and C-5, which
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confer higher lipophilicity improving the cell permeability and increasing oralabsorption. Moreover, pterostilbene (2) is considered to be more metabolically stablesince it has only one free hydroxyl group available for phase 2 metabolic reactions;therefore, pterostilbene-40-O-glucuronide (24) and pterostilbene-40-O-sulfate (25)were characterized as its main metabolites (Fig. 8). Consequently, it seems thatpterostilbene is tenfold more bioavailable than resveratrol (Akinwumi et al. 2018;Peng et al. 2018; Wang and Sang 2018).
Regarding piceatannol (5), the vast majority of studies is yet rather limited to cellsand animal models. In vitro, piceatannol is considered to be a product of CYP450metabolism of resveratrol. This was corroborated by in vivo studies, wherepiceatannol was also detected in plasma and liver of athymic mice, 5 min afteroral administration of resveratrol (Kershaw and Kim 2017). Concerning piceatannolmetabolism, in vitro studies using human liver cytosol and microsomes described theexistence of sulfation and glucuronidation metabolic reactions and identifiedpiceatannol disulfate, two monosulfated, and three monoglucuronide metabolites,although the exact location of the conjugated groups was not possible to be deter-mined (Fig. 9) (Kershaw and Kim 2017).
After i.v. administration in rats, glucuronides seem to be the main metabolites.After intragastric administration, a piceatannol-monoglucuronide was found tobe the most abundant metabolite, but other ones were also observed, such assulfated and methylated metabolites, in particular isorhapontigenin (6) andrhapontigenin (26) (Fig. 9). Rhapontigenin (26) was also found in urine, as well aspiceatannol mono- and diglucuronides, O-methylpiceatannol-monoglucuronide, andO-methylpicetannol-monosulfate. In contrast to piceatannol (5), methylated com-pounds were not found as metabolites of resveratrol (Kershaw and Kim 2017).
As mentioned above, oral bioavailability of resveratrol is highly compromised byits extensive metabolism and elimination rate (Peng et al. 2018). Currently, thesefactors are trying to be overcome by using drug delivery systems employingnanotechnology for resveratrol encapsulation, which comprise liposomes, polymericnanoparticles, solid lipid nanoparticles, lipospheres, and cyclodextrins (Summerlinet al. 2015). Studies performed in animal models showed that these novel nano-carriers possess several advantages, including improved solubility, physicochemicalstability, and protection from metabolic reactions thus overcoming first-pass effects.Moreover, such nanoformulations could enhance transport across the membranes,increasing concentration of resveratrol in tissues such as the brain, liver, and kidney,enabling targeted and controlled drug release (Siddiqui et al. 2015).
O
H3CO
OCH3
3
5
4' 4'
O COOHOH
OHOH
24
O
H3CO
OCH3
3
5
SO
OOH
25
Fig. 8 Main metabolites of pterostilbene (2)
Stilbenoids in Grapes and Wine 11
Recently, a clinical trial was designed by Calvo-Castro et al. (2018) in order toassess the improvement of the oral bioavailability of resveratrol from vineatrol bymicellar solubilization (registration number: NCT02944097). Vineatrol is a stan-dardized ethanolic extract of grapevine shoots that contains 33.3% resveratrol mono-mers and oligomers, including 5.8% resveratrol (1), 14.5% trans-ε-viniferin (12),and minor amounts of ampelopsin (2.4%), hopeaphenol (13) (2.2%), piceatannol (5)(0.5%), R2-viniferin (1.2%), R-viniferin (1.8%), miyabenol C (2.3%), and E-omega-viniferin (2.6%). Thus, twelve healthy volunteers (six women, six men) randomlyingested a single dose of 500 mg vineatrol (30 mg resveratrol, 75 mg ε-viniferin) asnative powder or liquid micelles (placebo uncontrolled). Oral ingestion of thesoluble vineatrol micelles improved the relative systemic bioavailability with oftotal resveratrol when compared to the native powder, without any indication ofadverse effects. The mean maximum total plasma resveratrol concentrations after theintake were 300 and 28 nmol/L, respectively. The authors conclude that a micellarmicroemulsion provides a safe delivery of resveratrol in order to reach relevantbioactive concentrations to systemic circulation (Calvo-Castro et al. 2018).
4 Bioactivities of Stilbenoids: In Vivo Studies
Considering the bioactivities of stilbenes, using in vivo studies, there are severalworks describing the large benefit effects on different human health areas, such ashypolipidemic action, obesity, type 2 diabetes, and dementia prevention and anti-inflammatory, antioxidant, antiaging, anticancer, depigmentation, and cardiovascu-lar effects. Several works, focusing on wine and grapes, have been reviewed
Fig. 9 Piceatannol metabolites
12 N. Duarte et al.
regarding stilbenoids in vivo studies, emphasizing not only the well-studied resver-atrol (1) but also pterostilbene (2), oxyresveratrol (4), piceatannol (5), piceid (10),(Figs. 1 and 2), and oligostilbenes (Table 1).
In a previous study involving animal organs, Waffo-Téguo et al. (2001) indicatedthat resveratrol (1) directly inhibits the activity of COX-2. The authors investigatedthe inhibitory effects of grape phenols on carcinogen-induced precancerous lesionsin mouse mammary grand organ cultures. In this study, 12 phenols isolated fromgrape plant cell cultures were used to evaluate the potential of inhibition of
Table 1 Bioactivities of stilbenes from wine and grapes and main conclusions of recent worksregarding animal studies
Stilbenes Animal study Main conclusions References
Resveratrol (1),pterostilbene (2),oxyresveratrol (4)piceatannol (5), and cis-piceid (27)
Bioactivitiesreview whichincludes animalstudies in mice
Stilbenoids have differentbiological properties such ascardioprotection,neuroprotection, antidiabeticproperties, depigmentation,anti-inflammation, cancerprevention and treatment
Akinwumiet al.(2018)
Resveratrol (1),pterostilbene (2), andpiceatannol (5)
Anti-inflammatoryactivity review ofanimal studies inmice
Anti-inflammatory activity ofnatural stilbenoids as apromising group of potentialanti-inflammatory drugs
Dvorakovaet al.(2017)
Oligostilbenes found ingrapes and wine
Review work inmice
Antioxidant, anti-inflammatory, anticancer,antimicrobial, antiviral,cardioprotective,neuroprotective, andhepatoprotective activities ofgrape stilbenes
Georgiev etal. (2014)
Resveratrol (1),piceatannol (5), andpterostilbene (2)
Review study inmice
Effects of stilbenoids: asantitumor, antiplatelet,antioxidant, antimicrobial,anti-inflammatory, analgesic,anesthetic, and a behavior ofmembrane-interactive drugs
Tsuchiya(2015)
Resveratrol (1) Animal study inmice and rats
Resveratrol and otherstilbenes: potential inhibitoryeffect on transient receptorpotential (TRP) channels andmodulation of TRP channelactivity
Yu et al.(2013)
Resveratrol (1),piceatannol (5), and cis-piceid (27) astringin,resveratroloside, cis-resveratroloside
Animal organstudy in mice
Stilbenoids: potentialinhibition ofcyclooxygenases andpreneoplastic lesions
Waffo-Téguo et al.(2001)
Stilbenoids in Grapes and Wine 13
cyclooxygenases and preneoplastic lesion formation in carcinogen-treated mousemammary glands in organ culture. This report showed the potential cancer-chemo-preventive activity of astringin (11), a plant stilbenoid found in wine. Astringin (11)and its aglycone piceatannol (5) revealed activity in the mouse mammary glandorgan culture assay but displayed no activity in COX-1 and COX-2 assays. Resver-atrol (1) presented bioactivity in the same bioassays indicating that astringin (11) andpiceatannol (5) may act as potential cancer-chemopreventive agents using a differentmechanism from that of resveratrol (1) (Waffo-Téguo et al. 2001).
Yu et al. (2013) investigated whether resveratrol (1) and other stilbenoidscould modulate TRP (transient receptor potential) channels in sensory neurons invitro and had analgesic effects in vivo, using whole-cell patch-clamp techniques andbehavioral analysis. This study showed that resveratrol (1) and other stilbenoidshave a potential inhibitory effect on TRP channels, exerting its activity in differentways. TRP is a family of ion channels that are activated by physical (temperature andmechanical force) and chemical stimuli. Sensory TRP channels are sensitized bypro-inflammatory agents and mediate heightened pain sensitivity.
Studies on obesity and diabetes and using animal models have concluded that, inaddition to the known anti-inflammatory and antioxidant activities, grape seedextract prevents metabolic syndrome, type 2 diabetes, and obesity (by modulatingthe metabolic endotoxemia and by improving the gut barrier integrity; Georgievet al. 2014).
In 2017, Dvorakova and Landa reviewed the anti-inflammatory activity of res-veratrol (1), pterostilbene (2), and piceatannol (5). Resveratrol (1) was described toinhibit the expression of cyclooxygenase-2 (COX-2) in vivo models using rats. Itsanalgesic properties against pain (acute or chronic) could be assessed throughalterations of the expression of serum tumor necrosis factor-alpha and whole brainnitric oxide in the diabetic rat model, reduction of expression of COX-2 in inflam-matory pain model, or inhibition of cyclin-dependent kinase 5 activity in primaryafferent neurons.
Recently, Akinwumi et al. (2018) reviewed the biological propertiesof stilbenoids such as resveratrol (1), pterostilbene (2), oxyresveratrol(4), piceatannol (5), and cis-piceid (10). The biological activities covered werefrom cardioprotection, neuroprotection, antidiabetic properties, depigmentation,and anti-inflammation to cancer prevention and treatment. The properties describedare thought to be mediated by different universal signaling pathways. The hypo-lipidemic effect of the dietary resveratrol (1) was studied in hepatoma-bearing ratswhere the antitumor growth and anti-metastasis effects were also determined.In addition, resveratrol (1) was studied on isolated rat hearts from ischemia reperfu-sion injury, and its potential cardioprotective effects were attributed to its peroxylradical scavenging activity (Akinwumi et al. 2018).
14 N. Duarte et al.
Table
2Sum
maryof
clinicaltrialsusingstilb
enes
andgrapeextracts
Con
ditio
nStudy
design
Dosages
Biomarkers
Effects
References
Colon
cancer
Rando
mized
four-dosecoho
rts
(n=
8)Noplacebo
Duration14
days
120g/dayof
grape
powder(n
=2)
80g/dayof
grape
powder(n
=3)
80mg/dayof
plant-
derivedresveratrol
tablets(n
=1)
20mg/dayof
plant-
derivedresveratrol
tablets(n
=2)
Wnt
pathway:
inhibitio
nof
expression
ofcyclinD1andaxinII
Beneficialfor
preventio
nof
colon
cancer
Ngu
yenet
al.(20
09)
Cardiov
ascular
disease(CVD)
prim
ary
preventio
n
Rando
mized
threeparallelarms,
placebocontrolled(n
=75
)Duration12
mon
ths
1capsule/dayfor
6mon
ths+2capsules/
dayforthenext
6mon
ths
Group
A:grape
extractwith
8mgof
resveratrol(n
=25
)Group
B:grape
extractwith
out
resveratrol(n
=25
)Placebo
(n=
25)
Atherog
enicmarkers:redu
ced
levelsof
Apo
B,L
DLox
(group
A)
Inflam
matoryandfibrinolytic
markers:redu
cedlevelsof
TNFα,hsCRP,andPA
I1and
increasedIL-10(group
A)
Beneficialfor
preventio
nof
CVDon
patientswith
statin
medication
Tom
é-Carneiroet
al.(20
12a,
b)
Cardiov
ascular
disease
second
ary
preventio
n
Rando
mized
threeparallelarms,
placebocontrolled(n
=75
)Duration12
mon
ths
1capsule/dayfor
6mon
ths+2capsules/
dayforthenext
6mon
ths
Group
A:grape
extractwith
8mgof
resveratrol(n
=25
)Group
B:grape
extractwith
out
Inflam
matorymarkersin
PBMCs:
increasedlevelsof
adipon
ectin
anddo
wnregulationof
Krupp
el-
likefactor
2,NF- KB,activator
protein-1,
c-Jun,
activ
ating
transcriptionfactor
2,and
CREB-binding
protein(group
A)
Beneficial
Tom
é-Carneiroet
al.(20
13a)
(con
tinued)
Stilbenoids in Grapes and Wine 15
Table
2(con
tinue
d)
Con
ditio
nStudy
design
Dosages
Biomarkers
Effects
References
resveratrol(n
=25
)Placebo
(n=
25)
Diabetes
mellitus
type
2Rando
mized
threeparallelarms,
placebocontrolled(n
=35
male
type
2diabeticandhy
pertensive
medicated
patients)
Duration12
mon
ths
Sam
eas
Tom
é-Carneiroetal.
(201
3a)
Inflam
matorymarkers:alteratio
nof
miR-21,
miR-181
b,miR-663
,miR-30c2,
miR-155
,and
miR-
34aandredu
ctionof
CCL3,
IL-
1,andTNF-α
(group
A)
Beneficial
Tom
é-Carneiroet
al.(20
13b)
Cardiov
ascular
Rando
mized
2�
2blockdesign
(n=
80,p
atientswith
hypercho
lesterolem
iawith
orwith
outcholesterolmedication),
placebocontrolled.
Duration6–8weeks
Twiceaday
Group
A:50mg
pterostilbene
Group
B:125
mg
pterostilbene
Group
C:50mg
pterostilbene
+10
0mggrape
extract
Placebo
Increase
ofLDLlevels(group
sAandB)
Reduced
bloo
dpressure
(group
B)
Beneficialfor
hypertensive
popu
latio
n
Riche
etal.
(201
3)
16 N. Duarte et al.
5 Human Benefits
The potential biological activities of wine stilbenes have been the subject of severalin vitro and in vivo studies. However, the link between these preclinical studiesand the beneficial effects in humans is not totally clarified yet. At the time of thisreview, there were 152 trials registered in ClinicalTrials.gov dealing with resveratrol(1). A recent review covered some of the latest clinical trials using this stilbene(Ramírez-Garza et al. 2018). However, only a few studies deal with the effects ofresveratrol or other stilbenes from grapes or wine. Herein, examples of relevantclinical trials using stilbenes and grape extracts are reviewed (Table 2).
The first clinical trial dealing with the potential beneficial effects of grape powderin patients with colon cancer was published in 2009 by Nguyen et al. (registrationnumber NCT00256334). This pilot trial was performed as a four-dose cohorts study.Eight patients, preselected due to colon cancer diagnosis, were randomly assignedto each cohort, being treated orally every day until surgery (14 days). Thus, twopatients ingested 120 g/day of grape powder (0.114 mg of resveratrol); three patientsingested 80 g/day of grape powder (0.073 mg of resveratrol); one patient had 80 mg/day of plant-derived resveratrol tablets (15.5 mg of resveratrol); and two patientsingested 20 mg/day of plant-derived resveratrol tablets (3.9 mg of resveratrol). At thetimes of diagnostic colonoscopy and surgery, cancer and normal colonic mucosatissues were obtained. These samples were evaluated by Wnt (wingless/integrated)pathway-specific microarray and quantitative real-time polymerase chain reaction(qRT-PCR), comparing pre- and postexposure to resveratrol or grape powder.The Wnt signaling pathway activates mutations essential for the development ofcolon cancer. On the cancerous tissues, the treatment with resveratrol or grapepowder had no change in the composite Wnt target gene expression. However, onnormal colonic mucosa, the treatment with 80 g/day of grape powder inhibited theexpression of cyclinD1 and axinII (Wnt target genes). This had the lowest resveratrolcontent of all the cohorts but also had many other bioactive components (otherpolyphenols) not present in the plant-derived resveratrol capsules. This clinical trialpresented some limitations such as a small sample size and no control of the dietaryintake of the patients nor control of medication that could affect Wnt pathway.But despite the limitations, the authors suggest that low doses of resveratrol orconsumption of grapes may have a beneficial role in colon cancer prevention.
Besides cancer chemopreventive activity, consumption of wine and grapes hasoften been associated with benefits for cardiovascular system. In this regard, aclinical trial was designed to investigate the effects of a dietary resveratrol-richgrape supplement in primary and secondary prevention of cardiovascular disease(registration number: NCT01449110).
For primary prevention of cardiovascular disease studies, 75 patients on statintreatment and at high risk of cardiovascular disease (diabetes mellitus or hypercho-lesterolemia plus arterial hypertension, active tobacco smoking, or overweight/obesity) took part of this trial. The subjects were randomly distributed through thethree parallel arms (triple-blinded and placebo controlled): grape extract containing8 mg of resveratrol and other resveratrol derivatives piceid (10) and viniferins intrace amounts (group A; n= 25), a grape extract with a similar polyphenolic content
Stilbenoids in Grapes and Wine 17
but lacking resveratrol (group B; n = 25), and placebo (group C; maltodextrin;n= 25). The treatments were encapsulated, and the patients consumed 1 capsule/dayfor the first 6 months and 2 capsules/day for the next 6 months to assess possibledose-response effects. Therefore, to evaluate the effects on the atherogenic markers,the authors only used data after the first 6 months of treatment (Tomé-Carneiro et al.2012a). Group A showed significant decreases in the low-density lipoprotein cho-lesterol (LDLc; �4.5%), apolipoprotein B (ApoB; �9.8%), oxidized LDL (LDLox;�20%), and LDLox/ApoB (�12.5%). On the contrary, no significant changes wereobserved for both groups B and C. In conclusion, the intake of one capsule/day ofresveratrol-rich grape supplement significantly reduced LDLox and ApoB inpatients undergoing primary prevention of cardiovascular disease with statin med-ication (Tomé-Carneiro et al. 2012a). Furthermore, for the evaluation of inflamma-tory and fibrinolytic markers, Tomé-Carneiro et al. (2012b) measured serum levelsof interleukin (IL)-6, IL-10, IL-18, tumor necrosis factor-α (TNFα), solubleintercellular adhesion molecule-1 (sICAM1), high-sensitivity C-reactive protein(hsCRP), adiponectin, and plasminogen activator inhibitor type 1 (PAI1). After 1-year treatment, the key findings of this study were that the resveratrol-rich grapesupplement (group A) significantly decreased the inflammation-related markershsCRP (�26%), TNFα (�19.8%), PAI1 (�16.8%), and IL-6/IL-10 ratio (�24%)and increased anti-inflammatory IL-10 (19.8%). No significant effects wereobserved for the placebo and grape extract without resveratrol groups. The authorsconcluded that 1-year consumption of grape supplement enriched in resveratrolimproved the inflammatory and fibrinolytic status in patients who were on statinsfor prevention of cardiovascular artery disease (Tomé-Carneiro et al. 2012b).
For the secondary prevention of cardiovascular disease studies, 75 patients whocoronary syndrome, cerebrovascular accident or peripheric arteriopathy eventoccurred at least 6 months or more, before the recruitment took part of this trial,which followed the same design as described above, using the same treatmentgroups and duration (Tomé-Carneiro et al. 2013a). After 1-year treatment, thetranscriptional profiling of inflammatory genes in peripheral blood mononuclearcells (PBMCs) was explored using microarrays and functional gene expressionanalysis. The main outcomes were observed for the group treated with resveratrol-containing grape supplement (group A). The increased levels of adiponectin levels(10%) prevented the increase of PAI1 levels, and downregulation of pro-inflamma-tory transcription factors (Kruppel-like factor 2, NF-KB, activator protein-1, c-Jun,activating transcription factor 2, and CREB-binding protein) was found to bemodulated in PBMCs. The non-high-density lipoprotein cholesterol load wasreduced in both groups A and B. In contrast, the placebo group showed significantdecrease of the levels of adiponectin and IL-10 and increase of PAI1. The authorsconcluded the presence of resveratrol in the grape supplement was essential for theobserved benefic effects and pointed toward further research on this nutraceutical asa possible safe coadjuvant food supplement in the follow-up of coronary arterydisease patients (Tomé-Carneiro et al. 2013a).
These human studies have evidenced the cardioprotective benefits of resveratroland grape extracts through the amelioration of inflammatory and atherogenicmarkers. Nevertheless, many of these biochemical markers are also involved in the
18 N. Duarte et al.
development of type 2 diabetes mellitus (T2DM). Therefore, Tomé-Carneiro etal. (2013b) further investigated the molecular changes associated to the regularintake of the resveratrol-containing grape extract in PBMCs isolated from hyperten-sive patients with T2DM. A subset of 35 male type 2 diabetic and hypertensivemedicated patients, which took part in the larger clinical trial previously described(Tomé-Carneiro et al. 2013a), were analyzed in this study. The authors evaluated thechanges in genes and microRNAs involved in the inflammatory response. Thesupplementation of these patients with resveratrol-rich grape extract during 1 yearhighly correlated with the alteration of a group of miRNAs involved in the regulationof the inflammatory response (miR-21, miR-181b, miR-663, miR-30c2, miR-155,and miR-34a) as well as with the reduction of the expression of pro-inflammatorycytokines (CCL3, IL-1, and TNF-α) and increase of the transcriptional repressorLRRFIP-1 (leucine-rich repeat flightless-interacting protein). Although the authorsconclude that long-term supplementation with a grape extract containing resveratrolhas beneficial immunomodulatory effects in circulating immune cells of T2DMhypertensive medicated patients, they do not discard the hypothesis of the combinedaction of resveratrol with other phenolic compounds present in the grape extract orwith some of the specific medication administered to these patients (Tomé-Carneiroet al. 2013b).
On the subject of benefic cardiovascular effects of stilbenes, another clinical trialconcerning the activity of pterostilbene (2) on metabolic parameters was conducted(registration number, NCT01267227). Eighty subjects with hypercholesterolemia(total cholesterol �200 mg/dL and/or baseline low-density lipoprotein cholesterol�100 mg/dL) were randomized in a 2 � 2 block design for presence of cholesterolmedication into one of four groups: 50 mg pterostilbene, 125 mg pterostilbene,50 mg pterostilbene plus 100 mg grape extract, and placebo. The treatments weretaken orally, twice a day for 6–8 weeks. The endpoints of this study were high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, bloodpressure, and weight. In both groups treated with pterostilbene (2), there was anincrease of LDL levels (17.1 mg/dL). This effect was not observed for the combi-nation of pterostilbene (2) and grape extract. The presence of a baseline cholesterolmedication appeared to attenuate LDL effects. Regarding the triglyceride levels, nosignificant results were observed for any group. Neither pterostilbene significantlyaffected HDL levels. Nevertheless, both systolic (�7.8 mmHg) and diastolic bloodpressure (�7.3 mmHg) were reduced with 125 mg dose of pterostilbene. In conclu-sion, pterostilbene was able to increase LDL levels and reduce blood pressure whenused in doses of 250 mg/day. The authors call the attention for future studies toevaluate high-dose pterostilbene (2) with grape extract in a hypertensive population(Riche et al. 2014). This clinical trial was also used to evaluate the effects on thesafety of long-term pterostilbene administration in humans (Riche et al. 2013). Sincesome of the patients that took part on the clinical trial were taking statins, it wasimportant to evaluate if there were any possible drug-drug interactions. It wasconcluded that pterostilbene did not demonstrate any biochemical hepatic adversedrug reactions nor had a direct effect of on measures of renal or glucose markers.In conclusion, pterostilbene was considered generally safe for use in humans up to250 mg/day (Riche et al. 2013).
Stilbenoids in Grapes and Wine 19
6 Application in Food: Wine Derivatives as DietarySupplements and Related Products
Due to the health benefits attributed to polyphenolic compounds present in wine,during the last years, other options have been investigated. These include non-alcoholic wines, grape juices, powders, extracts, and other products obtained fromgrapevine, which show the same biological benefits but are devoid of the alcoholiccontent (Georgiev et al. 2016).
Regarding nonalcoholic wines, they can be an option for some social groups (asyoung adolescents, nondrinkers, drivers, etc.) who normally do not consume winebecause of their content in alcohol. However, due to wine dealcoholization tech-niques, the quality, and, more importantly, the sensory characteristics are modified oreven reduced. To overcome this setback, different techniques have been investigatedin order to protect the organoleptic properties of nonalcoholic wines, for example,the membrane contactor and distillation under vacuum methods (Motta et al. 2017).
Without alcoholic content, powders obtained by freeze-dried wine and encapsu-lated in a maltodextrin matrix are being used as a source of wine polyphenols thatcan be added to other foods. This new product showed to have 3.7-fold morepolyphenols than the same amount of red wine (Rocha-Parra et al. 2018).
A rich phenolic content powder can be also obtained from the pomace, the grapebiomass discarded after the vinification process. A method to produce the drypomace extract without using organic solvents was recently described. This extrac-tion, based on aqueous cyclodextrins, showed to be an effective process to recover-ing phenolic compounds from food-derived products (Georgiev et al. 2016). It is
Fig. 10 Selected applications of grape derivatives
20 N. Duarte et al.
known that pomaces obtained from red wines have higher content of phenolic andother natural compounds with antioxidant activity. Recently, Beres et al. (2017)reviewed the main applications of this powder grape pomace extract not only in foodbut also in pharmaceutical and cosmetic industries (Fig. 10).
By using a sieve, it is possible to separate the grape seeds from the pomace. Grapeseeds have become increasingly popular on markets in different countries, suchas the United States (Yamakoshi et al. 2002). From the seeds, it is possible toproduce grape seed powder or grape seed oil or even dry grape seed extracts(Georgiev et al. 2016). These products are important due to their high polyphenoliccontent ranging from 60% to 70% of total extractable compounds and attract theinterest of the pharmaceutical, cosmetic, and food industry as a cost-effective sourceof natural antioxidant compounds (Fig. 10). In market, it is possible to buy Mega-Natural®-Gold Grape Seed Extract (Polyphenolics, USA) and Citricidal® grapefruitseed extract (NutriBiotic, USA) (Teixeira et al. 2014).
From the pomace, it is also possible to separate the grape skins by vibratingsieves. The powder obtained by prior dried and ground of the skins can be usedas a nutraceutical or as a source to obtained pure antioxidant compounds, as thestilbenoid compound resveratrol (Georgiev et al. 2016). The pomace is also theorigin of the so-called antioxidant dietary fibers. These soluble fibers are obtained byextraction of grape pomace using different techniques (Beres et al. 2017). Regardingtheir application (Fig. 10), some studies have shown the use of grape pomace andseed flours in different food products such as popsicles, cereal bars, biscuits andcookies, and muffins, in order to improve their content in antioxidant compounds(Beres et al. 2017). In order to prevent alterations such as flavor changes, color,texture, and lipid oxidation during freezing storage of some food products, forexample, association of codfish or seafood with the extracted fibers obtained frompomace is being studied (Zhu et al. 2015; Beres et al. 2017). Moreover, studies inchicken breast hamburger are also being carried on (Beres et al. 2017).
7 Safety: Toxicity and Side Effects
During the last years, a high interest in natural product consumption and in particularresveratrol and related stilbenoids has been highlighted. The toxicity of resveratrol(1) has been exhaustively studied not only in animal models but also in humans.Regarding animal studies, the acute, subchronic, and chronic toxicity of resveratrol,synthesized by DSM Nutritional Products Ltd., was highly studied in multiple invitro and in vivo animal assays (Williams et al. 2009). It revealed to be well toleratedand nontoxic, being a NOAEL (no-observed-adverse-effect level) for resveratroldefined as 0.75 g/kg bw/day dose (Edwards et al. 2011). In relation to its safety inhumans, several studies have shown that at low doses, resveratrol is well tolerated;however more information is needed especially regarding its long-term use(Akinwumi et al. 2018). An important data is the acceptable daily intake (ADI) inhumans for resveratrol, which was calculated by using data from animal toxicity
Stilbenoids in Grapes and Wine 21
studies and a standard default safety factor of 100 which gives an ADI of 0.45 g/dayfor a 60 kg individual (Edwards et al. 2011).
In a recent review (Wahab et al. 2017), it was reported that there are asmall number of dose-independent adverse effects such as nephrotoxicity andgastrointestinal problems associated with administration of resveratrol in humans.It is highlighted that 0.45 g/day of resveratrol is a safe dose for a 60 kg person;however, its supplementation in higher doses could be slight toxic (Wahab et al.2017). In fact, using daily doses of 2.5 g or higher amount of resveratrol, some minorside effects may occur, for example, nausea, vomiting, diarrhea, and liver dysfunc-tion in patients with nonalcoholic fatty liver disease (Salehi et al. 2018). In anotherstudy, it is pointed that resveratrol was found to be safe and well tolerated andwithout any other new side effect when used in a long-term experiment or in dosesup to 5 g/day (Ramírez-Garza et al. 2018). It is important to note that the resultsmentioned above were obtained in healthy population; therefore, caution must betaken concerning the results obtained in people that have some pathology (Salehi etal. 2018). Some of the last clinical trials using resveratrol are summarized in a recentreview (Ramírez-Garza et al. 2018); however, further investigation is needed toevaluate the safety of resveratrol for long-term treatment.
It should be pointed that some adverse effects were also found for resveratrol.In fact, some studies have shown that resveratrol may act as a pro-oxidizing agent,contrarily to its widely known chemopreventive and antioxidant properties (Salehi etal. 2018).
It is extremely important to pay attention to possible interactions between resver-atrol and other stilbenoid derivatives and conventional medicines. In fact, resveratrolmay interact with several medical drugs (Salehi et al. 2018). In this regard, itwas observed that high doses of resveratrol (1000 mg/day or above) inhibit cyto-chrome P450 isoenzymes such as CYP3A4, CYP2C9, and CYP2D6 and can induceCYP1A2, being responsible for a number of drug interactions, by changing drugclearance and consequently their bioavailability and toxicity (Wahab et al. 2017;Salehi et al. 2018). The interaction between resveratrol and anticoagulant, anti-platelet, or even nonsteroidal anti-inflammatory drugs could be a possibility, espe-cially due to an intensification of both bruising and bleeding risk, associated to thereduction of human platelet aggregation activity showed by resveratrol when testedin in vitro assays (Salehi et al. 2018).
A recent review conducted by Zha (2018) showed also interaction betweenresveratrol and transporter proteins, especially with ABC transporter membraneproteins. In fact, it was reported that this natural compound could inhibit P-glyco-protein (P-gp), multidrug resistance-associated protein 2 (MRP2), and organic aniontransporter 1/3 (OAT1/OAT3) and consequently could enhance the exposure ofpatients to some anticancer drugs.
It is also important to emphasize the interaction of red wine polyphenols withhuman microbiota. A study conducted by Queipo-Ortuño et al. (2012) indicated thatconsumption of red wine significantly increase the growth of some importantmicrobiota bacteria identities, namely, Enterococcus, Prevotella, Bacteroides,
22 N. Duarte et al.
Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, and Blautia coccoidessuggesting its possible prebiotic benefit.
8 Marketed Products
Wine is the most popular and widely discussed nutritional grape product with provenbeneficial health effects on human body, when moderately consumed. However, it isimportant to highlight the different content of resveratrol/stilbenoids among differentwines. On the other hand, it is known that the effective concentration of resveratrol inorder to achieve the claimed biological activities may be higher than that obtainedfrom wine drinking or food consumption. These reasons led to the constant search ofnew food sources and emphasized the potential of nutritional stilbenoid supplements(Navarro et al. 2018). Nowadays, the number of new food supplements containingpure resveratrol or this in a mixture of compounds is growing. However, mainlydue to inadequate industry quality control, the majority of these supplements donot have the claimed concentration of resveratrol. This issue is highlighted by thework of Rossi et al. (2012). Resveratrol is also used by cosmetic industry. Onepromising and famous example is the cosmetic trademark Caudalie®, whose prod-ucts contain as main ingredients resveratrol and other grapeseed polyphenols andstilbenoids. Its first patent is dated from 1999 and was related with compositionswhich are essentiality resveratrol esters, in the form of monomers and/or oligomers(Vercauteren et al. 1999). However, the search for new derivatives and methods toimprove its cosmetic products is intense and constant as highlighted by a recentpatent presented by the same industry. This patent is related with a new process forstimulating hyaluronic acid synthesis using resveratrol monomers and/or oligomers(Thomas et al. 2016). As showed above, the selection of the source of resveratrolis very important in order to obtain high amount of resveratrol and related stilbenoidswith a realistic price. From the different strategies known, namely, the chemicalsynthesis and the use of by-products derived from tropical foods (mango pulpor even the guava) or from plants with little economical values or even from by-products of harvesting or wine production, the use of biotechnology and geneticengineering is increasingly attracting attention in the last years (Navarro et al. 2018).In fact, there are different engineering techniques that have been studied, namely, theproduction of resveratrol and related compounds, using microorganisms andenzymes. Kuo et al. (2017) described a successful approach to have mass productionof resveratrol by using a small portion of extract of Polygonum cuspidatum and thewine yeast Dekkera bruxellensis. In this way, a repeated fed-batch fermentationprocess was scaled up to 1200 L in order to produce 35 mg of resveratrol per hour,liter, and round (Kuo et al. 2017). To produce higher quantity of resveratrol and othernatural compounds, the genetic engineering, namely, transgenic organisms, is beingused with very good results. A recent review summarized current progresses made inresveratrol biosynthesis, using bacterial hosts (Braga et al. 2018).
Regarding biotechnological approaches, plant cell biotechnology of grapes andmainly grape cell suspensions are seen as promising and alternative methods for
Stilbenoids in Grapes and Wine 23
producing bioactive nutraceuticals. In fact, the growth of grape cells in bioreactorsfor their production displays advantages when compared to the conventionalbreeding. In order to increase yields of these phytochemicals, several optimizationstrategies have been used, as exemplified for the non-resveratrol-producing grape-vine cell suspension of a grape hybrid, which produced resveratrol in the presence ofmethyl jasmonate. When scaling up the culture to a laboratory bioreactor, it achieveda yield of 230 mg/L (Georgiev et al. 2014).
9 Patents
Resveratrol is widely known as possessing a highly variety of biologicalbenefits, including antitumor, antioxidant, anti-inflammatory, cardioprotective,neuroprotective, and other activities. Due to all beneficial activities, the number offood supplements containing resveratrol increases every day, and their study, asmedicines, is a reality. However, their therapeutic use has been hindered by a numberof factors that need to be solved. On one hand, until now, it is not known the realcellular or molecular targets of resveratrol. On the other hand, its low bioavailabilityand possible oxidative degradation during preparation for biological applicationshamper the finding of a therapeutic dose that can benefit human health. These mainreasons led to the development of different resveratrol derivatives with the objectiveof improving the therapeutic potential of resveratrol-based compounds (Li et al.2016).
In a recent review, a total of 29 patents and over 200 resveratrol derivativeswere reported. These derivatives were studied mainly to cancer, cardiac diseases,metabolic disorders, and neurologic diseases. The cited review also refers somestudies regarding the application of resveratrol derivative patents in nutraceuticalcompositions and cosmetics (Li et al. 2016). In another review of patents, made from2009 to 2012, these two classes of applications have been also discussed togetherwith findings in the main therapeutic areas referred above (Pezzuto et al. 2013).
In a search of new patents from the last year, the same areas of researchare pointed. As an example, a German patent describes the process for producinga skin care formulation containing dimers of resveratrol. This formulation couldbe used to topical application or pharmaceutical preparation of cosmetic skin care(Merryvital 2018).
10 Conclusions and Perspectives
Summarizing, stilbenoids are plant-derived polyphenols with several biologicalactivities, which have been attracting increasing interest due to their potentialbenefits for human health. They can be found as monomers or very complexoligomers resulting from oxidative coupling of monomeric stilbenes, whose struc-tural variations include different substituent patterns in the aryl rings. In human diet,
24 N. Duarte et al.
the principal sources of stilbenes are grapes and wine. Despite the beneficialevidences of stilbenes on human health, there is still some ambiguity between highbioactivity and low bioavailability, namely, for resveratrol, the most studied stilbene.Consequently, further studies are crucial for evaluating the bioavailability andother pharmacokinetic parameters of stilbenes, thus providing evidence for theirtherapeutic importance in human health. Their potential biological activities havebeen the subject of several in vitro and in vivo studies. Despite the reduced number ofregistered clinical trials addressing the human benefits of stilbenes from wine orgrapes, the ones reviewed herein point to an increasing evidence that resveratroland pterostilbene exert beneficial cardioprotective effects in medicated patients.Nevertheless, further studies are needed to unveil the underlying mechanisms bywhich this may occur and to establish correlations between the promising effectsobserved in animal models.
Due to the health benefits attributed to stilbenes in human health, the number offood supplements, medicines, and cosmetics in the market is increasing every day.However, in order to increase the number of stilbenes in health-promoting products,there are still some issues that need to be solved, namely, the source of resveratroland quality control in industry production.
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