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We have wine even in this country, for our soil factors for cardiovascular diseases, cerebrovas-
grows grapes and the sun ripens them, but this cular accidents, and cancers. Currently, the
drinks like nectar and ambrosia all in one
The Odyssey, Book IX, Homer Mediterranean dietary style is certainly the diet
most similar to the nutritional habits that deter-
mined the evolution of the genus Homo during
1. INTRODUCTION the Palaeolithic era. Nonetheless, some of the
previously mentioned risk factors, for instance
Fruits and vegetables represent important excessive red meat consumption (>7 times per
components of the Mediterranean diet, a com- week), plays an important role in the nutri-
plex of dietary habit characterized by frequent tional etiology of many malignancies [1].
consumption of whole grains, legumes, fish, As attested by Theophrastus and Hesiod,
olive oil for seasoning, low intake of red meat, viticulture and winemaking were widely prac-
yoghurt, and cheese, and associated with regu- ticed in ancient Greece, though it is widely
lar, moderate wine drinking (two glasses/day). believed that winemaking began earlier, in the
According to the modern guidelines, dietary Neolithic Period (8500-^000 BC). Besides reli-
patterns including 400-600 g (up to 8 servings gious, social, and academic (at the symposium)
of 80 g) of fruits and vegetables per day, with contexts wherein wine was introduced, its
a reduced calorie content and an increased medical uses were studied by Greek physi-
nutrient density, as well as diets deficient in cians, as reported by Hippocrates (460-370 BC).
saturated fats and refined carbohydrates and He recommended wine to cure fever and as
abounding in fiber, significantly lower the analgesic, antiseptic, diuretic, tonic, and diges-
incidence of insulin resistance (metabolic) syn- tive [2]. The Romans also attributed therapeu-
drome, type II diabetes, and obesity, risk tic properties to wine, and Galen (129-200 AD),
in particular, provided a detailed description climate, high UV irradiance, exposure to excess
on the medical uses of wine in his practice. In light (photo-oxidation), water deficit, ; :
the care of gladiators, he used wine as an anti- anthropogenic pollutants [7,8].
septic for wound healing and as an analgesic
for surgery. Other illnesses that Roman physi-
cians treated by wine included depression,
memory loss, constipation, diarrhea, gout, hali- 2.1 Phenylpropanoids
tosis, snakebites, tapeworms, urinary tract ail- The phenylpropanoid pathway starts the
ments, and vertigo [3]. aromatic amino acid phenylalanine with the
In this chapter, we first provide a brief phenylpropanoid moiety C 6-C:. -£ leads to
description of the complex grape chemistry, i.e. derivatives with one, two, or BOK aromatic
the great variety of metabolites synthesized by rings (C6), each ring with a chars:*"--istic
the plant and stored in different berry tissues; substitution pattern, and with dr: ~t
second, we describe the most relevant biologi- modifications of the propane residue ot fte
cal activities of grapevine products and grape (C3) (Figure 38.1). Hydroxycinnamic 2d&
chemicals, reported in both human and animal (C6-C3) include p-coumaric, caffeic, ferubc tai
in vivo/in vitro experimental models. sinapic acids, with different degrees of hydnat-
ylation and methylation of C 6 (Figure 3SJJ.
2. GRAPE CHEMISTRY The cleavage of a C2 fragment from A K
aliphatic side chain of p-coumaric acid to
hydroxybenzoic acids (C 6-Ci), such salicylic,
Secondary metabolites (phytochemicals) in gra- vanillic, gallic, and syringic (Figure 38.1) [9].
pevine (Vitis vinifera L.) occur in wood, leaves, The condensation of three C2 residues
stems (rachis and pedicels), and berries [4]. an activated hydroxycinnamic acid
Although the berries are consumed as fruit two classes of metabolites with a second
and, along with stems, for winemaking, the matic ring linked to the phenylproparvo*i
leaves are normally not used as an edible ety, the flavonoids (C 6-C3-C6) (Figure
vegetable by humans, with the exception of and the stilbenes (C6-C2-C5) (Figure 35 T T*
some typical Greek (dolmadakia) and Middle The basic flavonoid chemical structure
Eastern dishes of stuffed grapevine leaves rol- flavan nucleus, consisting of 15 carbon >■■
led with rice. Therefore, in this section, we will arranged in three rings: two benzene rirw? (A
emphasize the phytochemicals present in berry and B) combined by an oxygen-ecc- ^5
tissues. pyran ring (C) (Figure 38.2). The mam one
Grape chemistry is rather complex and of flavonoids in grape (flavanones. fbvmes. fe-
some thousands of compounds have been iden- vonols, flavanols, and anthocyanid:.- -m
tified in the genus Vitis, included in the three the level of oxidation and saturation ct sae C
main classes of natural products, phenylpropa- ring, while individual compounds %*':"} ir: *
noids, isoprenoids, and alakaloids, widely dis- class vary in the substitution pattern c: •* A
tributed both in plant foods and medicinal and B rings [11].
herbs [5,6], In general, secondary metabolites Among flavonoids, anthocyanidins ane fc
exert a functional role in plant/ grapevine most abundant pigments in grape be
ecology, mainly as phytoalexins, compounds skin. Their conjugated derivatives (jr.- —T>-«
involved in defense against pathogens and anthocyanins, mainly bound to sugars, r
phytophages, as well as intolerance to detri- cinnamates, or organic acids, are waier-so - ■■
mental abiotic conditions; for instance, adverse
E 5S.1 TSe simple phenols, hydroxycinnamates and hydroxybenzoates, are originated from the phenylpropanoid
*ac Bars with deamination of phenylalanine by phenylalanine ammonia-lyase (PAL), leading to frans-cinnamic
to p-coumaric acid via cinnamic acid 4-hydroxylase (CA4H).
conferring blue, provide catechins,
dark blue, red, the monomeric units
and "ma>s to for
flowers, fruits, proanthocyanidin
and other plant biosynthesis
Anihocyanins of (Figure 38.2) (see
Vitis are later in this section).
structurally :«i Molecules
five belonging to the
aglycones/anthoc stilbene family are
yanidins - mal- also abundant in
rv2nidan, berry skin cells, and
delphinidin, possess the basic
peonidin, and chemical structure
pet-- which based on the frans-
differentiate on resveratrol skeleton
the basis of r (Figure 38.3).
and position of Stilbenes comprise
their hydroxyl piceids,
groups tear pterostilbenes, and
degree of viniferins that are
methylation glucosides,
(Figure 38.2) dimethylated
Flavonols derivatives, and
include mainly oligo-mers of
kaempferol, ~r resveratrol,
--i myricetin respectively (Figure
aglycones. 38.3) [14,15].
Apigenin is one
in grape,
whereas
flavanols
FIGURE 38.2 General flavonoid chemical structure: the different classes of flavonoids vary in the lead ^ onfl
and substitution of the C ring, while individual compounds within a class differ in the substitution pattern of A and 11
as in the case of anthocyanidins. Polymerization of flavonoid units, particularly flavanols, leads to \
Proanthocyanidins, 3 and 11 (Figure
also known as con- 38.2) [16]. In Vitis,
densed tannins or pmm* cyanidins are
simply tannins, are both mainly present in
-olig-omeric and seed. ski*, stem
polymeric compounds tissues of the bunch.
arising from flavanol In seeds, prcer
condensation. In cyanidins represent
grapes, the most com- the major fracticr. ct
mon monomers total polyphenol
include catechin extract and are
epimers [(+ )-catechin tharacssr by a
and (— )-epicatechin], lower degree of
whose polymerization polymerizstacr
degree ranges mainly -those present in
between berry skin.
However
pMMaAocyanidins are more easily extracted 2.2 Isoprenoids
d~~~~z winemaking, because of their localiza-
tt:r _■ the vacuole and cell wall, thus confer- Isoprenoids, or terpenoids, are a huge and
nng important organoleptic properties to wine, diversified group of lipidic compounds, deriving
such as astringency, bitterness, browning, tur- from acetyl-coenzyme A (CoA) via the
bidity, and color stability [17-19]. intermediate molecule mevalonate and by the
To summarize, flavonoids, stilbenes, and activity of key enzymes hydroxymethylglutaryl-
prwthocyanidins are collectively grouped in CoA (HMG-CoA) synthase and reductase
pcv.: henok (Figure 38.4), the name indicating (Figure 38.5) [20]. Several hundreds of volatile
both the compounds with a second aromatic compounds have been identified in grapes,
nng and those arising from the polymerization whose aroma is largely due to C 10 mono-
of flavonoidic/catechin units. terpenes, representatives of isoprenoids and
major components of essential oils. They include
FIGURE 38.4 Basic moieties of phenylpropanoids.
the acyclic alcohols geraniol, nerol, linalool, citro-
nellol, and homotrienol and the monocyclic o-
terpineol (Figure 38.5) [21-23]. In human
breast adenocarcinoma (MCF-7) cells, geraniol
was shown to inhibit cell cycle progression
and proliferation, via a mevalonate-independent
pathway (similarly to statins, some isoprenoids,
including monoterpenes, can inhibit HMG-CoA
reductase activity and, consequently, mevalonate
accumulation, thus inhibiting cell growth) [24].
Carotenoids are isoprenoid terraterpenes
(C40) accumulating in ripening grape berries,
whose oxidation produces volatile fragments,
the Ci3-norisoprenoids (Figure 38.5) [23]. These
are strongly odoriferous compounds, such as
/Monone (aroma of viola), /3-damascenone
(aroma of honey and exotic fruits), /3-
damascone (aroma of rose and fruits) and /?-
ionol (aroma of flowers and fruits) (Figure 38.5)
[21,25-28]. In particular, /3-ionone, a compound
with an end ring analogue of /J-carotene,
exhibits a potent anticancer activity, as reported
in both animal models and cell lines. In 2008 a
dose-dependent inhibition of carcinogenesis by
dietary /?-ionone was observed in a rat mam-
mary gland cancer model induced by 7,12-dime-
thylbenz[a]anthracene (DMBA) [29]. Moreover,
in previous studies, /?-ionone inhibited the
Polyphenols differ from simple phenols because of a second
growth of MCF-7, human gastric adenocarci-
aromatic ring, whereas proanthocyanidins are oligomeric or noma (SGC-7901), human colon adenocarci-
polymeric derivatives of flavonoids with polymerization noma (Caco-2), and human promyelocytic
degree, ranging from 3 to 11 in grape. leukemia (HL-60) cell lines through different
FIGURE 38.5 Major classes of grape isoprenoids, mevalonate being the precursor.
molecular mechanisms: arrest of cell cycle, both in grape and grape products. Tetrahydro-/?-
induction of apoptosis, and inhibition of cell carbolines are tricyclic indole derivatives occur-
proliferation, invasiveness, and metastasis ring in low amounts in grapes, grape juice, and
[24,30-32]. Because of the similarity between wine. In particular, carboxylic acids of tetra-
the chemical structure of /Monone and retinoids hydro-/3-carbolines, widespread in fruits, arise
(/3-carotene, vitamin A, retinoic acids), it was from Trp via a nonenzymatic Pictet-Spengler
suggested that /3-ionone can act as a ligand condensation, i.e. the indole nucleus cyclization
agonist of the retinoid receptors, a class of with carbonyl substrates, aldehydes typically
nuclear receptors regulating cell growth, differ- (Figure 38.6). They occur at the ng/g and /zg/L
entiation, and apoptosis, as demonstrated by levels in grapes and juice/wine, respectively,
the dose-dependent up-regulation of the reti- contributing to the antioxidant capacity of these
noid receptor mRNA in the human colon can- products [34-37]. Additionally, tetrahydro-/3-car-
cer HCT-166 cell line [33]. bolines are synthesized in mammalian brain
tissues via the endogenous condensation of
Trp, or 5-hydroxytryptophan, with formalde-
2.3 Alkaloids hyde or acetaldehyde [38]. In the central nervous
Indole alkaloids, deriving from the aromatic system, they display a wide spectrum of psy-
amino acid tryptophan (Trp), have been detected choactive properties, as neurotransmitters and
FIGURE 38.6 Main indole alkaloids of grape from tryptophan.
Tetrahydro-^-carboIines arise via a non-enzymatic condensation with carbonyl substrates (aldehydes) and indole nucleus
cyclization, while melatonin is synthesized through the intermediates 5-hydroxytryptophan and serotonin.
neuromodulators: despite their
tetrahydro-/3- occurrence in many
carbolines function as commercial fermented
potent reversible and smoked
inhibitors of the foodstuffs, such as
enzyme monoamine vinegar, beer, cheese,
oxidase (MAO), yogurt, bread, and fish
besides binding to the [45,46].
benzodiazepine, Melatonin (N-
imidazoline, serotonin acetyl-5-
(5-hydroxytryptamine, methoxytryptarrune)
5-HT), and dopamine has been discovered
receptors and inhibiting in the berry skin of
5-HT (re)uptake grapes [47,48]. The
[39,40]. MAO essential amino acid
catalyzes the oxidative Trp is the precursor of
deamination of all 5-methoxyindoles,
biogenic amines, or
including indoleamines/tryptam
neurotransmitters ines, including
(dopamine, 5-HT, melatonin, through
tryptamine, the intermediate 5-HT
norepinephrine), and the activity of
vasoactive dietary hydroxyindole-O-
(tyramine) and methyltransferase
xenobiotic amines, (Figure 38.6) [49].
thus being implicated in Melatonin was long
neurological disorders, thought to be a
psychiatric conditions, neurohormone found
and depression exclusively in
[41,42]. In addition to vertebrates, until its
the reported detection in bacteria,
psychophar- protozoans, algae,
macological effects, plants, fifngi, and
tetrahydro-/3- invertebrates [50].
carbolines are potent Ever since, melatonin
antioxidant and has been found in
anticancer agents, edible plants,
active by different medicinal herbs, and
biochemical and seeds, though its
molecular mechan- physiological and
isms, such as pathophysiologi-cal
induction of apoptosis, function in planta is
inhibition of DNA still unclear.
topoisomerase I and II, Nevertheless, a
and of cyclin- hormone-like role has
dependent kinases been putatively
[35,43,44]. However, attributed to
the precise effects of melatonin in some
these compounds on plant species [51-53].
human health are still Among the examined
debated, and the grape cultivars,
amount of dietary Nebbiolo and Croatina
tetra-hydro-/3- contain the highest
carbolines effectively melatonin levels, 0.9
accumulated in and 0.8ng/g,
biological tissues and
fluids is still
unknown,
respectively, whereas the lowest concentra- health benefits widely attributed to these plant
tion has been detected in Cabernet Franc foods and beverages. However, in the last dec-
(0.005 ng/g) [47]. As expected, melatonin has ades, a lot of studies focused mainly on poly-
been found in wine too, with concentrations phenols, considered as the archetypes of
ranging from 0.05 to 0.5 ng/g, and, interest- grapevine product bioactivities. In this section,
ingly, in humans, serum melatonin level increa- we also emphasize polyphenolic properties,
ses significantly after red wine intake (1 h after treating separately the findings of grape
a single 100 mL supplementation) [54,55]. research.
Melatonin is also a powerful antioxidant. It
possesses an electron-rich aromatic indole ring
and easily acts as an electron donor for mole-
cules lacking it, thereby reducing and repairing 3.1 Oxidative Stress and Structure-
electrophilic radicals [56]. After oxidation by a Radical Scavenging Activity Relationship
free radical, for instance hydroxyl anion radical of Polyphenols
(* OH ~), melatonin generates a resonance-stabi-
lized nitrogen-centred radical, the indolyl (or At biochemical level, oxidative stress can be
melatonyl) cation radical. The latter, after a fur- denned as a disturbance in the cell oxidation/
ther quenching of superoxide anion radical reduction (redox) status. Metabolism of aerobic
(* O2~), forms the stable, nontoxic N 1-aceryl-N2- organisms unavoidably and continuously pro-
formyl-5-methoxykynuramine, itself a powerful duces partially reduced oxygen intermediates,
antioxidant and an anti-inflammatory able to more reactive than molecular oxygen in its
improve mitochondrial metabolism and to ground state, including both radical and non-
inhibit cyclooxygenase (COX) 2 [57,58]. radical forms collectively termed as reactive
Intriguingly, it was postulated that a reaction oxygen species (ROS) (Figure 38.7). During
between melatonin and peroxidases is present respiration, leakage of electrons from the mito-
in plant tissues, able to improve the production chondrial transport chain leads to the single-
of kynuramines [59]. Finally, melatonin can electron reduction of molecular oxygen and to
FIGURE 38.7 Homeostasis of the cell oxidation/reduc -
also counteract the cell oxidative burden indi-
rectly, by stimulating the production of ROS
detoxifying enzymes, specifically glutathione
peroxidase, glutathione reductase, and superox-
ide dismutase [60].
3. GRAPEVINE CHEMICAL/
PRODUCT BIO ACTIVITIES: FOCUS
ON POLYPHENOLS
UPIDS
AGEING.!
(membrane lipid
peroxidation)
FIGURE 38.9 Biochemical targets and clinical conditions mechanistically linked to oxidative stress.
targets, organisms evolved sophisticated strate- group, a catechol structure) on the B ring, and
gies, collectively termed antioxidant defenses, a double bond between C2 and C3 {A2'3) conju-
that counteract the imbalance of the cell redox gated with a keto function at C 4 of the C ring
homeostasis and keep ROS levels under the (Figure 38.2-38.10). Hydroxyl groups on the B
cytotoxic threshold (Figure 38.7) [67]. ring donate hydrogen and an electron to radi-
Antioxidant defenses also comprise vitamins cal species, stabilizing them and giving rise
and nutraceuticals, i.e. nonenzymatic sca- to a relatively stable flavonoid radical
vengers abundant in plant foodstuffs and intro- (Figure 38.10). The C2-Q double bond and the
duced by diets, including polyphenol-rich 4-keto group are responsible for electron delo-
grape products [68-71]. Any compound capa- calization from the B ring (Figure 38.10).
ble of quenching ROS, without itself undergo- Hydroxyl groups in positions 3 and 5, in com-
ing conversion to a destructive radical species, bination with 4-oxo function and C2-C3 double
can be considered as an antioxidant, as in the bond, contribute to further enhance the radical
case of dietary flavonoids [72,73]. scavenging activity (Figure 38.10) [75-78].
As already introduced, the basic flavonoid
chemical structure is the flavan nucleus, con-
sisting of 15 carbon atoms arranged in three 3.2 Antioxidant and Antimutagenic
rings (C6-C3~C6): two aromatic rings (A and B)
Activity of Polyphenols
connected by a three-carbon-atom heterocyclic
ring, an oxygen-containing pyran ring (C) Probably the most investigated biological
(Figure 38.2) [11]. Flavonoids act as anti- activity of polyphenols is their antioxidant
oxidants by donating electrons and stopping power, though they also possess a plethora of
radical chains (Figure 38.10). This activity is more or less correlated properties, such as anti-
attributed to the phenolic hydroxyls, increasing mutagenic, anti-inflammatory, antitumoral,
with the number of OH groups in A and B antihypertensive, cardio- and neuroprotective
rings (Figure 38.10) [74]. The structural require- activities [6,11].
ments considered to be essential for effective As reported above, polyphenols supplied by
radical scavenging by flavonoids are the pres- diet exert health benefits by ROS scavenging,
ence of a 3',4'-dihydroxy group (o-diphenolic and this has also been shown in studies based
FIGURE 38.10 The activity of flavonoids as radical scavengers is attributed to the phenolic hydroxyls and increases
with the number of — OH groups in A and B rings.
An — OH can react with a free radical (R •) to give a flavonoid radical (Fv-O *); afterwards three different termination reac -
tions can occur: a coupling reaction between a flavonoid radical with another radical species (A); a dimerization with
another flavonoid radical (B); a further loss of a hydrogen atom from the flavonoid radical (C).
on the the acute intake of
supplementation of a phenolic-rich
grapevine products juice (400 mL), with
[70,79]. In one study, grape as major
the antioxidant ingredient, improved
capacity of red grape the antioxidant status
was evaluated in in healthy subjects,
HepG2 (human determined both in
hepatocellular liver serum and urine by
carcinoma) cell line FRAP. In the same
and positively work, the authors
correlated to the total showed that the
phenolic content and phenolic compounds
to the oxygen radical of the juice were
absorbance capacity bioavailable, as
(ORAC) values of revealed by the
grape extracts. The increase of phe-nolics
authors concluded that that could bind the
increasing fruit con- lipid fraction of
sumption represents a serum and by their
suitable strategy to rise in the urinary
counteract oxidative excretion, with a
stress and to reduce maximum reached 2h
the risk of cancer [80]. after consumption
Dietary polyphenols [83]. In pre- and
also contribute to postmenopausal
elevate the women, the whole-
antioxidant capacity body oxidative stress
of human blood. was significantly
Daily consumption reduced after the daily
of grape juice supplementation of a
(lOmL/kg body lyophilized grape
weight) for 2 weeks powder (36 g) for 4
resulted in an weeks, by reducing the
increased resistance of levels of urinary F2-
LDL to ex vivo isoprostanes,
oxidation, biomarkers of
comparable to the oxidative stress [84].
value obtained after a- Consuming black*
tocopherol (400IU) grape (lg/kg body
supplementation [81]. weight) exerted
These results further similar effects,
confirmed the data compared with juice
previously reported and powder,
by Day and collea- significantly raising
gues, showing that the plasma
the daily intake of antioxidant potential
grape juice (125 of healthy volunteers
mL), for 1 week, 4h after ingestion [85].
significantly reduced Oxidative DNA
the LDL damage, leading to
oxidizability. They modifications of DNA
also showed an 8% bases, is related to
increase in plasma mutagene-sis,
antioxidant capacity carcinogenesis, and
measured 1 h after the aging (Figure 38.9).
grape juice sup- Daily grape juice
plementation as supplementation (480
ferric-reduced mL), for 8 weeks,
antioxidant potential reduced DNA strand
(FRAP) [82], In a breaks in
short-term study,
peripheral lymphocytes, as detected by the sin- showing a synergistic action [90]. Furthermore,
gle cell gel electrophoresis (comet assay, a pow- pretreatment with resveratrol prevented the
erful tool in mutagenesis studies), beside accumulation of DNA strand breaks induced by
decreasing the amount of released ROS [86]. tobacco smoke condensate in cell lines of differ-
Similarly, treatment of human lymphocytes ent histogenetic origin, as assessed by the comet
with a grape seed extract reduced the fre- test [91]. Accordingly, in animal cell cultures, res-
quency of micronuclei (an assay for the detec- veratrol failed to induce DNA damage, though
tion of DNA damage) by 40% and the it slightly increased chromosomal aberrations at
production of malonyldialdehyde (a biomarker the highest assayed doses [92].
of lipid peroxidation) by 30%, while increasing In conclusion, it seems that the protective
the activities of the antioxidant enzymes cata- effect against ROS-induced oxidative DNA
lase and glutathione S-transferase by 10 and damage cannot be attributed to polyphenols
15%, respectively [87]. In another study, the singly present in grape, but rather to the syner-
antimutagenic activity of both aqueous and gism among polyphenols themselves and/or
methanolic extract from two Greek grape varie- between them and other types of bioactive
ties (red and white) was assessed against the chemicals.
ROS-induced DNA damage, using the
Sa/morce/to/reversion assay and the oxidant
mutagens bleomycin and H2O2. Unexpectedly,
both polyphenol-rich fractions and single poly- 3.3 Cancer Chemoprevention
phenols (resveratrol, catechin, epicatechin, quer- Prevention has become as important as ther-
cetin, gallic and protocatechuic acids) from red apy to control cancer, in order to reduce both
grape extracts did not affect the bleomycin- cancer morbidity and mortality. Cancer chemo-
and H2O2-induced mutagenicity, either posi- prevention is the strategy of preventing, arrest-
tively or negatively, though some slight pro- ing, or reversing carcinogenesis by means of
oxidant and mutagenic effects were reported chemopreventive agents, dietary therapeutics
for resveratrol and quercetin at the assayed effective at each step of neoplastic progression.
concentrations [88]. In agreement with these Therefore, chemopreventive agents can be
results, the reported data on the antimutagenic divided into blocking agents, that arrest the initi-
activity of individual grape phytochemicals are ation stage of malignancy, and suppressing
still elusive. In purified calf thymus DNA trea- agents, that act on tumor promotion and
ted with oxidants, resveratrol exhibited a progression by inhibiting the malignant transfor-
bimodal response on the formation of 8- mation of initiated cells. Again, chemopreven-
hydroxy-2-deoxyguanosine (8-OH-dG, a bio- tive agents can avoid the time-dependent tumor
marker of oxidative DNA damage), with a resistance (chemoresistance) to chemotherapeu-
slight pro-oxidant effect, at lower concentra- tic agents, and their nonspecific toxicity toward
tions, and an antioxidant activity at higher non-target cells [93-99].
concentrations, reducing the 8-OH-dG accumula- Cancer is a multistage and multifactorial dis-
tion in a dose-dependent manner. This bio- ease, the second leading cause of death world-
marker causes G->T and A->C transversions wide after heart disease, whose risk and
during DNA replication, resulting in carcinogen- incidence augments with age. In addition to
esis [89]. Intriguingly, melatonin, besides being genetic factors, environmental and nutritional
more effective than resveratrol, reversed the pro- factors play a major role in cancer etiology. In
oxidant DNA damage induced by low concentra- industrialized developed countries, breast,
tions of resveratrol, when added in combination, prostate, and colorectal cancers predominate,
because of a diet rich in animal foods and process, including the cell redox status
refined carbohydrates and deficient in plant (Figure 38.11) [101].
foods. Conversely, in developing countries, Chemopreventive properties of grape pro-
where diet is largely based on cereal/starchy ducts are more likely attributable to the com-
foods, esophageal, stomach, and liver cancers bined effect of their bioactive components,
have a higher incidence [100]. rather than to one or a few specific molecules,
An intricate network of signaling pathways is although resveratrol represents the most stud-
involved in cancer pathogenesis, regulating the ied example of grape biologically active com-
(im)balance between cell growth-promoting and pound [5,6]. In his seminal and pioneering
growth-inhibiting mechanisms (Figure 38.11). study, Pezzuto and his group [b] reported for
At a molecular level, the interactions with both the first time the chemopreventive potential of
transcription factors and receptors have been resveratrol in different assays representing the
proposed as putative mechanisms for the three carcinogenesis stages. They also showed
reported anticarcinogenic activitiy of (grape) that resveratrol inhibited the development of
polyphenols (Figure 38.11). Furthermore, che- preneoplastic lesions in a mouse mammary
mopreventive dietary agents can promote gland culture treated with the carcinogen
apoptosis in premalignant and malignant cells DMBA, as well as tumorigenesis in a mouse
by modifying different stages of the apoptotic skin cancer model [102]. Nevertheless, it must
FIGURE 38.11 Mechanisms of cancer prevention of polyphenols by inhibiting (|) or enhancing (T): reactive oxygen spe-
cies (ROS) production; important transcription factors (NFnB, AP-1) and enzymes involved in inflammation (cyclooxygen-
ase-2, COX-2); androgen and estrogens receptors (ARs, ERs); aryl hydrocarbon receptors (AHRs); pro-apoptotic (p53, BAX)
and antiapoptotic proteins (Bcl-2, Survivin); omithine decarboxylase (ODG) and other enzymes regulating cell growth
(DNA polymerase, ribonucleotide reductase); hypoxia inducing factor la (HIT-la) and matrix metalloproteinases (MMPs)
involved in metastasis and angiogenesis (see text for details).
be underlined that, when consuming a food or expression of genes involved in cell adaptation,
beverage, we ingest its potpourri of chemicals. differentiation, and proliferation (Figure 38.11)
Cancer and chronic inflammation are caus- [112-116].
ally linked, as demonstrated by the increased The interaction with both androgen (ARs)
gene expression and/or activity of enzymes syn- and estrogen receptors (ERs), belonging to the
thesizing arachidonate-derived proinflammatory nuclear steroid hormone receptor family,
mediators, and by the augmented production of represents another molecular mechanism
these mediators (prostanoids) in various cancers involved in resveratrol-mediated chemopreven-
[103,104]. Hence, inhibition of cyclooxygenases tion (Figure 38.11). A decrease in cell prolifera-
(COXs), particularly of indudble COX-2 iso- tion was reported in the androgen-responsive
zyme, and blockage of prostaglandin cascade prostate cancer cell lines LNCaP treated with
are relevant and effective mechanisms to coun- resveratrol and quercetin, due to the inhibition
teract multistage carcinogenesis (Figure 38.11) of both expression and function of ARs
[105]. Among polyphenols, resveratrol's cap- [117,118]. ARs represent essential mediators
ability of blocking various components of of androgen activity, controlling the transcrip-
the proinflammatory cascade has been known tion of androgen-inducible genes, such as pros-
for a long time, as reported in phorbol ester- tate-specific antigen (PSA). Therefore they are
treated human mammary epithelial cells, where implicated in the development of hormone-
resveratrol inhibited both COX-2 gene trans- responsive prostate cancer. Moreover, the
cription and enzymatic activity [106]. Anti- growth of androgen-unresponsive prostate can-
inflammatory properties of resveratrol were cer cells was also inhibited by resveratrol,
then reported in a variety of models and at dif- though to a lesser extent than that of the andro-
ferent biochemical levels. This compound signifi- gen-responsive cell lines [119]. Estrogens regu-
cantly inhibited the expression of COX-2 in: i) late the transcription of target genes by
mouse peritoneal macrophages treated with binding to different intracellular estrogen
LPS, 12-O-tetradecanoylphorbol-13-acetate (TPA) receptors (ERa and ER/3) with tissue and
or H2O2 [107]; ii) RAW 264.7 macrophages stimu- ligand specificity, influencing the growth, dif-
lated with LPS plus interferon ^ (INF-i) [108]; ferentiation, and function of target tissues and
and iii) mouse skin treated with TPA [109]. playing a pivotal role in breast cancer. As a
In vivo (F344 rat), resveratrol at concentra- phytoestrogen, resveratrol was shown to pos-
tions between 1 and 2mg/kg body weight sess either estrogen agonist and antagonist
suppressed both the number and size of N- activity, thus raising some controversy regard-
nitrosomethylbenzylamine (NMBA)-induced ing its therapeutic application against estrogen-
esophageal tumors per rat, by targeting COXs responsive breast cancers [120-122].
and prostaglandin E2 (PGE2) [110]. Resveratrol Phytoestrogens are diphenolic plant metabo-
and other stilbenes also decreased COX-2 activ- lites that exert estrogen agonist/antagonist
ity and reduced the production of PGE 2 in activity because of their structural similarities
peripheral blood leukocytes treated with LPS to natural and synthetic estrogen steroids.
plus INF-( [111]. They are either hormone-like compounds, with
Resveratrol was shown to inhibit two impor- inherent estrogenic activity, or can be converted
tant transcription factors, NFnB, involved in by intestinal flora to weakly estrogenic com-
signaling pathways mediating inflammation, pounds. Other phytoestrogens include lignans
oncogenesis (including angiogenesis and metasta- and isoflavones, present in whole cereals and
sis), apoptosis and (together with anthocyanins) legumes respectively, and classified as selective
the activator protein 1 (AP-1), regulating the estrogen receptor modulators (SERMs) [123].
Inhibition of both aromatase (estrogen syn- function, are related to more than half of
thetase) activity and expression by grape seed human cancers [101]. However, in different cell
extract represents another mechanism of breast lines, polyphenols induce apoptosis by mechan-
cancer suppression (Figure 38.11), as demon- isms other than p53 gene modulation [135,136].
strated in an aromatase-transfected MCF-7 Suppression of anti-apoptotic survivin may be
breast cancer xenograft model [124]. another pro-apoptotic mechanism promoted by
Aromatase is a cytochrome P450 enzyme, grapevine polyphenols, as reported for green tea
which converts Ci9 androgens to aromatic C 18 polyphenols, which decreased both mRNA and
estrogens, expressed at higher levels in breast protein expression of survivin (Figure 38.11)
cancer than in normal tissues. Therefore, its [137]. Survivin is a member of the inhibitor of
overexpression in breast cancerous cells can apoptosis protein (LAP) family, overexpressed in
influence the tumoral progression itself, several human neoplasms [138].
because of the major role of estrogens in breast The inhibition of ribonucleotide reductase
cancer development [125]. (the enzyme that catalyzes the reduction of ribo-
Aryl hydrocarbon receptor (AHR) is a cyto- nucleotides into deoxyribonucleotides), of DNA
solic protein that translocates to the nucleus polymerase, of ornithine decarboxylase (ODC,
upon ligand binding. Metabolic activation of a key enzyme of polyamine synthesis greatly
aryl hydrocarbons (AH) results from their bind- involved in cancer growth), and the promotion
ing to AHR that, after migration, activates the of cell cycle arrest are key processes further con-
transcription of the CYP1A1 gene, encoding for tributing to the chemopreventive potential of
the cytochrome P450 (CYP450) isozyme polyphenols (Figure 38.11) [139-146].
CYP1A1. CYP450 enzymes are involved in the Grape seed extract showed promising effi-
metabolism of a variety of xenobiotics, includ- cacy also against two important processes
ing carcinogens such as AH, and are over- involved in cancer progression, angiogenesis
expressed in a variety of tumors (Figure 38.11). and metastasis, inhibited in prostate and breast
The metabolized active forms of carcinogens carcinoma, respectively [147,148]. Because of
can subsequently interact with DNA, thus caus- increased metabolic activity and oxygen con-
ing mutations. Resveratrol was shown to exert sumption of rapidly proliferating cells, solid
a strong inhibitory effect on AH-induced tumors are likely to maintain an intratumoral
CYP1A1 expression, both at the mRNA and pro- hypoxic environment, which, in turn, induces a
tein level, as well as on other CYP450 isozymes, set of hypoxia-responsive genes in order to
such as CYP1A2 and CYP3A4 [126-128]. allow tumor cell adaptation. The expression of
Besides the above-mentioned signaling net- these genes is regulated by the hypoxia induc-
works regulated by polyphenols, the induction ible factor (HIF), a major regulator of cellular
of apoptosis is another molecular mechanism oxygen homeostasis [149]. Resveratrol was
involved in their antiproliferative effects reported to inhibit the expression of HIF-la in
(Figure 38.11) [101]. In a variety of tumor cell human ovarian cancer cells OVCAR-3, as well
lines, among them leukemia cells, it was as of the vascular endothelial growth factor
reported that resveratrol activates the mito- (VEGF), an HIF-regulated angiogenic factor
chondrial-dependent apoptotic pathway by the [150]. Angiogenic factors promote neovascular-
up-regulation of pro-apoptotic p53 and Bax ization of interstitial stroma, the tissue sur-
proteins and the down-regulation of the death rounding the primary tumor site, a process
inhibitory protein Bcl-2 [129-134]. The gene which supplies nutritional requirements to pro-
p53 is an important oncosuppressor whose liferating neoplastic cells and facilitates their
mutations, as well as the loss of p53 protein access to the vascular system (intravasation).
Before penetrating blood vessel endothelium, Moreover, the growth of human colorectal car-
from the primary tumor site, and gaining cinoma cells was inhibited by a grape seed
access to the blood stream, cancer cells must extract rich in proanthocyanidins [161], and a
invade local tissues by degrading extracellular red grape dietary fiber (obtained from seeds)
matrix (ECM) components and, ultimately, tra- induced epithelial hypoplasia with a decrease
verse the basement membrane. Once in circula- in the depth of crypts in both rat cecal and dis-
tion, these cells can form metastatic colonies at tal colonic mucosa as well as a decrease in
secondary locations. Resveratrol was reported crypt density and mucosal thickness [162].
to inhibit the invasiveness of diverse cancer Photochemoprevention by botanical agents
cells by reducing the expression and activity of may prevent skin cancer at various stages of
matrix metalloproteinase (MMP)-2 and MMP-9, carcinogenesis, as shown in different models.
involved in ECM degradation [151-153]. Topical application of resveratrol and apigenin
Apart from the beneficial effects exerted by to SKH-1 hairless mice, prior to UV exposure,
polyphenols mainly on prostate, breast, and effectively prevented radiation-induced carcino-
blood cancers, as discussed above, other types genesis [163]. Mechanisms involved in photo-
of tumors can benefit from regular, moderate chemoprevention by resveratrol include the
consumption of grape products. Carcinomas of inhibition of NFK,B signaling, as demonstrated
the digestive tract are common and their risk in UV-exposed normal human epidermal kera-
increases with age. In gastric cancer cells, res- tinocytes [164]. In SKH-1 mice, grape seed
veratrol was shown to inhibit cell proliferation proanthocyanidins also prevented UV-induced
and to induce apoptosis [154—156]. Resveratrol oxidative stress, decreased lipid peroxidation,
and red wine extracts also inhibited the and inhibited the activation of NFK,B signaling
growth of 15 clinical isolates of Helicobacter and, finally, photoearcinogenesis [165,166]. As
pylori, the primary etiological determinant of for other cell lines, resveratrol induced apopto-
gastric cancer [157]. Grape polyphenols, mainly sis in two human melanoma cell lines by acti-
quercetin, were shown to suppress the forma- vating a MAP kinase pathway [167].
tion of aberrant crypt foci, in animal models of The paradigm that health benefits arising
carcinogenesis, by modulating both cell prolif- from fruit consumption are due to the efficacy
eration and apoptosis [158]. The crypt is the of all the biologically active phytocomponents
fundamental unit of epithelial proliferation in has been emphasized on pancreatic cancer
the colonic mucosa, where genetically dam- cells. Pancreatic cancer is a highly aggressive
aged stem cells are removed from the epithe- malignancy, currently treated with limited suc-
lium by apoptosis, before they undergo clonal cess by conventional therapeutics and with an
expansion. Hence, increased apoptosis in the extremely poor prognosis [168]. A mixture of
proliferating zone of the colonic crypt provides isoflavone 10 nM + cucurmin 500 nM +
a protective mechanism against crypt cell epigallocatechin-3-gallate 125 nM + resveratrol
hyperproliferation and neoplasia [158]. In both 125 nM inhibited by 40%, up to 72 h, the cell
cancerous and noncancerous human colon tis- growth of BxPC-3 cells, a human pancreatic
sues, black grape extracts modified the activity cancer cell line, via a mechanism partly due to
of enzymes involved in DNA turnover, adeno- the inactivation of NFKB. The authors con-
sine deaminase, 5' nucleotidase, and xanthine cluded that a combined treatment with phyto-
oxidase, thus depriving cancer cells of nucleo- chemicals induces a greater inhibition of cell
tides for proliferation [159]. Similar results growth than that obtained after treatment with
were reported on cancerous and noncancerous single compounds [169]. Finally, in human pan-
human urinary bladder tissues [160]. creatic cancer cell line PancO2 inoculated into
C57BL/6 mice, both resveratrol and quercetin In cardiovascular diseases, an important
suppressed pancreatic cancer via different inflammatory process takes place after leuko-
mechanisms, namely apoptosis induction (by cyte mobilization. Phospholipase A2 leads to
caspase 3 and 8), cell cycle arrest at Gl phase, the hydrolysis of plasma membrane phospholi-
and inhibition of tumor cell migration (inva- pids, mainly phosphatidylcholine and phospha-
siveness) through the ECM barrier [170]. tidylethanolamine, and to the subsequent
release of arachidonic acid. This acid is the sub-
strate of COX and lipoxygenase (LOX), the
enzymes involved in the synthesis of prosta-
noids (prostaglandins and thromboxanes) and
3.4 Atherogenesis, Hypertension, and
leukotrienes, respectively, the inflammation
Cardioprotection mediators collectively grouped in eicosanoids.
Atherosclerosis is a chronic, inflammatory, Among COX-derived platelet modulators,
fibroproliferative process of large and medium- thromboxanes, mainly thromboxane A2
sized arteries. It results in the progressive for- (TXA2), potentiate platelet reactivity, whereas
mation of fibrous plaques that impair the prostacyclins help to maintain platelets in a qui-
blood flow inside the vessels. In the affected escent state [173]. Polyphenols, mainly quercetin
artery, atherosclerotic lesions resulting from an and resveratrol, were shown to inhibit COX and
eccentric thickening of the intima can either LOX activities and eicosanoid synthesis (Table
promote an occlusive thrombosis or produce a 38.1, Figure 38.12) [107,174-178].
gradual stenosis of the arterial lumen. In the A great deal of evidence suggests an inverse
first case, thrombus formation due to the dis- relationship between grape product consump-
ruption of the lesion surface can lead to infarc- tion and cardiovascular disease. Clinical trials
tion of the organ supplied by the afflicted demonstrated that grape and grape juice
vessel, such as in a heart attack, when a coro-
nary artery is suddenly blocked, or in a throm-
TABLE 38.1 Mechanisms of Grape Polyphenol-
botic stroke, when a cerebral artery is
Promoted Cardiovascular Protective Effects
damaged. In the second case, the stenosis of
Mechanisms References
the vessel limits the blood supply to local tis-
sues, leading to a progressive and gradual Inhibition of inflammation and eicosa- [84,107,174-
injury of the affected organ [171]. noid synthesis 178,207]
Endothelial dysfunction, oxidative modifica- Improvement of endothelial function [186-190]
tion of LDL, platelet aggregation, and inflam- Decrease of LDL [84,196]
mation are key factors in atherogenesis and Increase of HDL [84,196]
hypertension. Endothelial cells exert multiple Inhibition of LDL oxidation # [81,82,187]
physiological functions, maintaining the integ- Inhibition of platelet aggregation and [177,179,180,191-
rity of the vascular wall and representing a thrombosis 195]
permeable barrier through which diffusion Improvement of vasorelaxation [193,199-202]
and active transport of several substances Inhibition of ET-1 [204]
occur. Furthermore, endothelial cells consti- Improvement of fibrinolysis [209]
tute a non-thrombotic and non-adherent Inhibition of VSMC proliferation and [210-212]
surface for platelets and leukocytes; they regu- vascular hyperplasia
late the vascular tone by producing nitric LDL, low density lipoproteins; HDL, high density
oxide (NO), eicosanoids, endothelins, and lipoproteins; ET-1, endothelin-1; VSCM, vascular smooth
cytokines [172]. muscle cells.
vasoconstriction
endothelin(l)
INFLAMMATIO
T
(eicosanoid
s cytokines
NF-kB
COX, LOX